Nesfatin-1 and -2 are produced from a reaction in which the N-terminus of human Nucleobindin-2 undergoes proteolytical processing. To date, Nucleobindin-2 and/or nesfatin-1 have only been shown to act as peptide hormones. On the other hand, the purpose of nesfatin-2 remains unknown. Since Nucleobindin-2/nesfatin-1 is thought impact the control of a wide range of physiological processes, including energy homeostasis, neurodegenerative processes and carcinogenesis, its ligands/interactions deserve special studies and attention. However, there are no reports about the molecular properties of the proteolytical products of human Nucleobindin-2 in the literature. Hence, this study aimed to analyze the effect of Zn(II) and Ca(II) on human nesfatin-1, -2, and -1/2 structures. Herein, we report that human nesfatin-1 is a member of the intrinsically disordered protein family, as indicated by circular dichroism and analytical ultracentrifugation experiments. In contrast, we found that the human nesfatin-2 and nesfatin-1/2 structures were globular with intrinsically disordered regions. Under Zn(II) treatment, we observed concentration-dependent structurization and compaction of intrinsically disordered nesfatin-1 and its propensity for oligomerization, as well as destabilization of both nesfatin-2 and nesfatin-1/2. Furthermore, dissociation constants for Zn(II) binding by nesfatin-1, nesfatin-2, and nesfatin-1/2 were also reported. Moreover, structurally distinct nesfatin-1 and -2 seem to be interdependent when linked together, as indicated by the observed molecular properties of nesfatin-1/2, which in turn are not a simple sum of the properties exhibited by the former peptides. Thus, herein, we shed new light on the molecular behavior of human nesfatins, which might help to elucidate the complex function of those peptides.Video abstractThe online version contains supplementary material available at 10.1186/s12964-022-00980-7. Nucleobindin-2 (Nucb2) and/or nesfatin-1 were first discovered as central and peripheral hormones that are engaged in the regulation of energy homeostasis , circadiGallus gallus (ggNucb2) and hNucb2 homologs for intrinsic disorder [The multidomain nature of Nucb2 is partially reflected by its wide spectrum of physiological functions, as discussed below. The recently described propensity of disorder  seems todisorder , 14. IDPdisorder . Insteaddisorder . Anotherdisorder . Skorupsdisorder  revealeddisorder . N-termidisorder  to bind Hence, in this paper, we describe the molecular properties of hN1, hN2, and hN1/2 and the effect of Zn(II) and Ca(II) on their structure. Our results indicate that hN1 in its apo form displays a highly disordered structure that, upon Zn(II) treatment, undergoes disorder-to-order transition accompanied by dimerization of the peptide and formation of the hydrophobic core. We also observed that Zn(II) is bound by ordered hN2 and hN1/2; however, despite the tighter binding by hN1/2, this interaction was strongly structure-destabilizing. The ordered structure of hN2 and hN1/2 peptides along with the distinct structure of the free hN1 fragment imply that in vivo proteolytic processing of hNucb2 by PCs and further processing of hN1/2 may act as an activation mechanism, which in turn might assign the hN2 fragment a structural, yet unclear role. Thus, the results presented in this paper provide new insight into the molecular properties of hN1, hN2, and hN1/2. The structures of hN1 and hN2 appear entirely dissimilar; however, the peptides seem to be structurally interdependent, as shown by the properties of hN1/2, which in turn might have extensive physiological implications that undoubtedly necessitate further investigation.2PO4 × 2H2O, pH 7.0; 300 mM NaCl), Buffer A2 , Buffer B . Buffer C . All buffers were prepared at ambient temperature and filtered through a 0.22 μm filter.Buffer A1 , phenylmethylsulfonyl fluoride (PMSF), 2-carboxy-2′-hydroxy-5′-sulfoformazylbenzene monosodium salt  were purchased from Sigma Aldrich. Carbenicillin (R) was purchased from Roth. All remaining chemicals were of analytical grade and are commercially available.hN1F (GCGCGAGCTCGTGCCGATTGATATCGATAAA), hN1R (GCGCAAGCTTCTACAGTTCATCCAGTTTGGTAC), hN2F (GCGCGAGCTCCAAGAAGTTGGTCGTCTGC), hN2R (GCGCAAGCTTCTACTCGTGTTCTTTCATCATTTC).PD10 desalting and Superdex 75 Increase 10/300 GL columns were purchased from GE Healthcare. Ni–NTA agarose resin was purchased from Qiagen.http://pondr.com). The backbone molecular dynamics of proteins was assessed with the DynaMine tool [http://dynamine.ibsquare.be/).Prediction of intrinsically disordered regions (IDRs) of hN1, hN2, and hN1/2 was based on their amino acid sequence and the following disorder prediction tools: PONDR-VL-XT , 19  with primers that introduced SacI and BamHI restriction sites. The template was a modified recombinant pQE-80L (Qiagen) vector containing cDNA of hNucb2, which was prepared by our team previously . AmplifiE. coli competent cells (Thermo Scientific) were transformed with 4 ng of each construct and incubated overnight on LB  agar. The chosen transformants were transferred to TB (R + C) medium and incubated for 8 h at 37 °C and 200 rpm. Then, 30 ml (6% v/v) of the above inoculum was added to 0.5 l of TB (R + C) medium and incubated for 5 h at 29 °C and 200 rpm until an optical density of 0.7–0.8 at 600 nm was reached. Subsequently, IPTG was added to the culture medium to a final concentration of 0.25 mM, and the culture was incubated for the next 3 h at 29 °C and 200 rpm. The cells were harvested by centrifugation for 8 min at 4 °C and 5,500 g, and the pellet was suspended in 12 ml of A1 buffer supplemented with 20 μg/ml PMSF. All extracts were stored at -80 °C before use. Then, the extracts were thawed on ice, and PMSF (20 μg/ml), DNase I (10 μg/ml), and RNase I (10 μg/ml) were added. Cell lysis was conducted by sonication on ice followed by incubation for 1 h at 4 °C and 10 rpm on a vertical shaker to remove nucleic acids. The cell lysates were centrifuged , and the supernatant was collected, followed by the subsequent addition of 2 ml (50%) of preequilibrated (buffer A1) Ni–NTA resin. The supernatant was then incubated for 30 min at 4 °C and 10 rpm. The supernatant was transferred onto an empty Tricorn column, which was subsequently connected to an Äkta Explorer  system. The resin was washed with 20 bed volumes of buffer A1. Next, contaminant proteins were eluted with 10 bed volumes with buffer A1 supplemented with 35 mM imidazole (hN1) or 20 mM imidazole . Finally, the proteins of interest were eluted with 10 bed volumes of buffer A2, and the chosen fractions were pooled and desalted to buffer A1 on a PD10 desalting column according to the manufacturer’s protocol. Then, HRV3C protease  was added at a 1:100 w/w ratio, and the digestion solution was incubated overnight at 4 °C and 10 rpm. The above solution was then incubated with 0.6 ml of preequilibrated Ni–NTA resin for 30 min at 4 °C and 10 rpm and loaded onto the column. Flow-through was collected and concentrated on Amicon Ultra-4 centrifugal filter units (Millipore) to a 500-μl volume. Proteins of interest were further purified by size exclusion chromatography (SEC) on a Superdex 75 Increase 10/300 GL column connected to the Äkta Avant chromatography system . Protein separation was monitored by the A280 measurement, and the concentration was estimated with the following extinction coefficients: 4470 1/(M × cm) (hN1), 2980 1/(M × cm) (hN2) and 7450 1/(M × cm) (hN1/2). The chosen fractions were pooled, aliquoted, and stored at -80 °C until future use. The chemical purity and identity of the proteins were verified by SDS–PAGE analysis according to Laemmli [Bl21 (DE3) pLysS  Laemmli  and mass2 (10 mM), EDTA (5 mM) or ZnCl2 (20–500 μM). The CD spectra of an appropriate buffer were subtracted from each protein spectrum, and the CD data were converted to the mean residue ellipticity. Raw data were further smoothed with the Savitzky–Golay filter . The smoothed data from the replicates were averaged. Deconvolution of individual CD spectra was performed with the CDPro package using the CONTINLL algorithm and SDP48 (hN1) or SP43  set as a reference [Circular dichroism (CD) spectra were acquired with a Jasco J-815 CD-spectropolarimeter equipped with a Peltier-type temperature controller and quartz cuvettes with a 0.1 cm optical path length. The chosen spectral accumulation parameters were a scanning rate of 50 nm/min, a 1 nm bandwidth, and a wavelength range of 190–260 nm. The protein concentration was 0.15 mg/ml in each sample. Each spectrum was obtained at 20 °C with 3 accumulations per sample. The proteins were suspended in buffer B or buffer B supplemented with either CaCleference –25. CD d2 (10 mM), EDTA (5 mM) or ZnCl2 . The changes in tertiary and/or quaternary structures upon interaction of free hN1 and hN2 were detected by centrifuging the solution of the above peptides in the w/w ratio of 1:1, 1:2, and 1:4 (hN2:hN1) in the presence of either EDTA (10 mM) or ZnCl2 (50 μM). Analysis was performed at 20 °C and 50 000 rpm. The parameters obtained with SEDNTERP [2/CaCl2, respectively), and viscosity . Time-corrected data were analyzed with Sedfit software (version 16.1c) using the built-in continuous sedimentation coefficient distribution model, c(s). Maximum-entropy regularization of the c(s) model was set to a confidence level of 0.68 [Sedimentation-velocity analytical ultracentrifugation (SV-AUC) was conducted on a Beckmann Coulter Proteome-Lab XL-I ultracentrifuge  endowed with an An-60Ti rotor. Protein solutions were prepared with two sets of concentrations, and the concentrations for hN1/2 were 1.85, 1.32, 0.92 mg/ml. For hN1 and hN2, the concentrations were 1.3, 1.0, 0.7 mg/ml. The protein samples were suspended in buffer B supplemented with either CaClSEDNTERP  were as  of 0.68 , 28.4 (titrant) concentration was either 7 or 0.5 mM for the titration of hN1 and hN2 and hN1/2, respectively. The titrand and titrant concentrations were adjusted to obtain the best isotherms for proper analysis of equilibria. After temperature equilibration, successive injections of the titrant were made into the reaction cell in 5.22 μl increments at 300 s intervals with stirring at 200 rpm. Control experiments were performed to determine the heats of titrant dilution using identical injections of Zn(II) in the absence of protein.The binding of Zn(II) to hN1, hN2, and hN1/2 was monitored using a Nano-ITC calorimeter  at 25 °C with a cell volume of 1 ml. All experiments were performed in buffer C. The hN1 and hN2 (titrands) concentrations were 0.1 mM, whereas the ZnSOA + B ⇌ AB. The data were fitted first with constrained values obtained from prefitting in NanoAnalyze and subsequently revised in SEDPHAT with floating parameters of an incompetent fraction of protein and -logKd with the corresponding ΔHx. The error estimates for the fitting results were produced using Monte Carlo analysis, individually for each experiment, with 500 iterations and a 0.9 level of confidence.The titration data were analyzed using NanoAnalyze (version 3.3.0), NITPIC (version 1.2.7) , 30 and 4 solution in a 384-well microplate (Greiner) and a total volume of 50 μl. Final concentration of nesfatins was 10 μM, and 50 μM of the fluorophore. All samples were prepared in triplicate. The microplate was incubated for 20 min at room temperature with shaking, centrifuged , and then scanned with BMG Clariostar Plus reader in a fluorescence intensity (FI) mode. ANS was excited at 350 nm and the fluorescence emission spectra were acquired from 375 to 650 nm. The data were averaged and smoothed with Savitzky-Golay filter for spectra presentation. Unsmoothed fluorescence intensity of the samples at 486 nm versus ZnSO4 concentration was fitted against Hill1 model with the Hill parameter left unrestricted.Stock solution of 8-Anilino-1-naphtalene-sulfonic acid (ANS) was prepared in dimethyl sulfoxide (DMSO) and the concentration of the fluorophore was verified at 376 nm with the extinction coefficient of 9140 1/(M × cm) . ProteinKd value of 2.09 μM (pH 7.4) [4. The absorbance at 618 nm was monitored with a Jasco V-630 spectrophotometer in a 1-cm quartz cuvette. The apparent chromophore Kd, ZI was calculated by fitting the data with OriginPro 2018 software against the quadratic equation. Competition experiments were performed by incubating various amounts of hN1 and hN1/2 suspended in buffer B with 50 μM ZI previously saturated with 50 μM ZnSO4. All samples were prepared in a 384-well microplate (Greiner) with a final volume of 100 μl. The microplate was incubated for 20 min at room temperature with shaking, centrifuged , and then scanned with a BMG Clariostar Plus plate reader in absorbance spectrum mode. The Kd of nesfatins was calculated as described by Kocyła et al. [Kd, ZI determined under our conditions.The binding of Zn(II) to hN1 and hN1/2 was further verified with Zincon (ZI), which forms a 1:1 complex with Zn(II) and has a published (pH 7.4) . ZI stoca et al.  using thThe predictions for the IDRs of hN1, hN2, and hN1/2 was performed based upon their amino acid sequences. For this purpose, the PONDR VL-XT , 19 and 2 order parameter based upon a vast database of chemical shifts of proteins derived from NMR experiments. This parameter is sequence-dependent and is a measure of the N–H bond vector rotational freedom [2 < 0.69), rigid , or a region that can be viewed as an intermediate between the two  [The DynaMine algorithm is a potent backbone dynamics predictor that calculates the S freedom . The out2 < 0.8) . There aα-helical content [β-strands (23 ± 12%). The structure of hN1 is thus very distinct from that of the remaining nesfatins, as hN2 and hN1/2 contain 48 ± 10% and 45.2 ± 0.4% α-helices, respectively. However, the contribution of IDRs is also significant and totals 24 ± 6% and 30 ± 2% for hN2 and hN1/2, respectively. The striking difference in the secondary structure content between free hN1 and hN2 along with the ordered nature of hN1/2 seems to indicate a strong effect of the hN2 fragment on the structure of hN1/2, since the N-terminal fragment covering hN1 is ordered due to the fragment.CD spectra in the far-UV region 195–265 nm) were recorded to validate the in silico prediction of the IDR content. The CD spectra of the apo-nesfatins are shown in Fig. 65 nm wer content . This obTo further examine this structural interdependency of both fragments, the spectrum for the mixed solutions of hN1 and hN2 (hN1 + 2) was acquired .Next, we tested the changes in secondary structure in the presence of Zn(II) and Ca(II), as their binding to the full-length protein has been reported , 17. We The behavior of hN2 Fig. D and hN1The presence of Zn(II) induced significant changes in the secondary structure of human nesfatins, and the presence of Ca(II) did not induce significant changes, as discussed in the previous paragraph. Hence, to evaluate whether these observations are accompanied by tertiary and/or eventually quaternary structure changes, we performed SV-AUC experiments. values for these proteins oscillate approximately 0.91S for hN1, 1.2S for hN2, and 1.73S for hN1/2 in each cell (Table app) was approximately 10.9 kDa for hN1 and hN2 and 21 kDa for hN1/2, which is in very good agreement with the results obtained from the MS experiments  displays a more globular shape. Conversely, free hN1 displays an extended and cylindrical shape. Taken together, the results indicate that that proteolytic processing of hN1/2 may result in the formation of two structurally different products, in which the molecular properties are very distinct when compared to those of the conjoined hN1 and hN2.The SV-AUC data Fig.  revealedometries . Thus, tss ratio . In cont parameter for hN1  parameter from 0.9S to 1.4/1.53 and 2.1/2.4S  parameter change is accompanied by an increase in the Mapp from 10.9 kDa to approximately 14.3 and 25.5 kDa, indicative of the formation of a dimer by holo-hN1. There is also a significant reduction in the f/f0 parameter from 1.8 to 1.23 for Zn(II)-saturated hN1, which is strongly evident of structural compaction; this is possibly due to the adoption of a more globular shape, which is in good agreement with the results of CD spectroscopy (see “Secondary structure estimation and its changes under Zn(II) treatment” section). We also probed for hN1/hN2 interactions with SV-AUC experiments , we did not observe any significant changes in the c(s) distribution  concentration-dependent disorder-to-order transition. It is also worth noting that similar effects of compaction and oligomerization under Zn(II) and Ca(II) treatment were reported by Skorupska et al. for hNucb2 and ggNucb2 , 17.To accurately determine the thermodynamic parameters of Zn(II) binding by hN1, hN2, and hN1/2 and to compare them, we performed isothermal titration calorimetry experiments.Kd value of approximately 61 μM  to hN1 and hN1/2 resulted in very different thermograms. Titration of hN1 with Zn(II) is characterized by prominent endothermic heats that plateau at a very high molar ratio of Zn(II)/hN1  complexation by hN1/2 is more favorable compared to that of hN1 and is approximately − 7 kcal/mol, which exhibits a much more dominant enthalpic contribution compared with that of the hN1 reaction . The final difference between these two proteins pertains to complex stoichiometry, which in the case of hN1/2 with Zn(II) was fitted to be 2:1 (Zn(hN1/2)2), based on the incompetent protein fraction value of approximately 0.5.The hN1/2 Zn(II) binding process varies substantially compared to that of its shorter predecessor. First, the metal ion binding thermogram shows negative heats  ions” section).Previous experiments proved that hN1 undergoes major structural rearrangements under Zn(II) treatment (see “Secondary structure estimation and its changes under Zn(II) treatment” and “Quaternary structure changes of nesfatins in the presence of Zn(II) ions measured with SV-AUC” sections). In contrast, in the presence of Zn(II), hN2 and hN1/2 exhibited a tendency to precipitate (see “Secondary structure estimation and its changes under Zn(II) treatment” section), but unexpectedly, the ITC results showed that hN1/2 does bind Zn(II) . To further probe Zn(II)-induced structural changes in nesfatins, we performed 8-aniline-1-napthalene-sulfonic acid (ANS) fluorescence spectroscopy experiments. ANS is a fluorophore that is characterized by a low quantum yield in an aqueous environment. However, upon binding to exposed hydrophobic sites of the proteins, ANS exhibits increased fluorescence intensity and an observable blueshift in the spectrum . In thisKd was estimated to be 69 ± 9 μM by utilizing the fitting of the FI data at 486 nm versus Zn(II) concentration . Taken together, these results augment the effects of Zn(II) on the structure of nesfatins, their ability to bind them and especially the Zn(II) sensitivity of hN2, which seems to translate to hN1/2.As expected, free ANS excited at 350 nm had the lowest observed fluorescence intensity (FI), with a maximum at 517 nm  binding properties of hN1 and hN1/2, which also underlines their potential involvement in Zn(II) homeostasis.To further confirm the ability of hN1 and hN1/2 to bind Zn(II), we performed competitive titration experiments using Zincon (50 μM) saturated with Zn(II) (50 μM). For this purpose, we determined the apparent dissociation constant of the chromophore under our buffer conditions to obtain the value of terature . We obsea et al.  and was a et al. . As expeHerein, we present new insight into the structural characterization and interactions of the products from the proteolytical processing of hNucb2, namely, hN1, hN2, and hN1/2. To the best of our knowledge, this is also the first description of the molecular properties of these peptides. In the first stage of our studies, we performed in silico analysis of human nesfatins, which revealed their possible mosaic-like character with intertwined disordered-ordered regions; the results were similar to the results described by Skorupska et al. for hNucb2 . Moreoveβ-strands were also found to have a strong contribution (23 ± 12%) to the secondary structure of hN1. Therefore, these results prove that hN1 is indeed a member of the IDPs family, and it might perform the diverse physiological effects through similar mechanisms discussed earlier. Moreover, ubiquitous central and peripheral expression of Nucb2/N1 throughout the body further underlines its biomedical importance. The peptide was found in the central nervous system in the hypothalamic nuclei as follows: arcuate, paraventricular, supraoptic, and dorsomedial nucleus, as well as in other brain centers such as the brainstem [For this reason, CD spectroscopy was employed to gain insight into secondary structure content, and the obtained data agreed with the in silico results. The content of IDRs in hN1 was 57 ± 10%, and ion 23 ± % to the rainstem . Expressrainstem , 41, andrainstem . Other trainstem , testis rainstem , and hearainstem . The broα-helical contents of 48 ± 10% and 45.2 ± 0.4%, respectively. The content of IDRs was also noticeable and totaled 24 ± 6% and 30 ± 2% for hN2 and hN1/2, respectively. These results were complementary to the in silico analysis. Moreover, the results raise the question of whether in vivo proteolytical processing of hN1/2 could function as an activation mechanism that enables hN1 interaction with multiple targets through the transition from a mostly ordered to an unordered structure. For this reason, we decided to probe for mutual hN1 and hN2 interactions in solution with CD and SV-AUC experiments. CD spectroscopy revealed no changes in the secondary structure that could result from the interactions between hN1 and hN2, based on the difference between the spectrum of hN1/2 and the overlapping spectra of the mixed solution of hN1 + 2 and their algebraic sum. Thus, free hN1 indeed exhibits different molecular properties than those when linked covalently with hN2, and the mere presence of free hN2 in solution is insufficient to induce any structural changes. In addition, we also did not observe significant changes in the c(s) distribution of the solution of hN1 + 2 at any w/w ratio in SV-AUC experiments, both in the presence and absence of Zn(II) ions. Therefore, based on the lack of changes in the secondary, tertiary, and quaternary structures, we conclude that there is no interaction between free hN1 and hN2 in vitro under our conditions. This observation further illustrates the interdependency between the hN1 structure (and possibly function) and hN2 that translates to the properties of hN1/2 and hNucb2. Hence, hN2 could function as a molecular switch that turns the appropriate molecular character of hN1 on and off.The secondary structure of the remaining peptides was very distinct compared to that of hN1. CD experiments revealed that hN2 and hN1/2 exhibit ordered characteristics with Metal ions represent an important group of signal transducers and effectors and are essential cofactors of many proteins. Among them, one of the most biologically important metal ions is Zn(II). This trace element is the second most abundant d-block metal in living organisms after iron . Zn(II) α-helical content. Moreover, with the fitting of the data at 208 and 222 nm, the apparent Kd was estimated, which was in good agreement with the ITC and fluorescence spectroscopy results. The ITC results also showed a 1:1 binding model for hN1-Zn(II), corresponding to one bound ion per monomer. We also demonstrated that Zn(II) binding is associated with a disorder-to-order transition, a common feature of IDPs. These structural changes were also observed in SV-AUC experiments. The value of the f/f0 parameter for apo-hN1 indicates its elongated form, while holo-hN1, in the presence of Zn(II), undergoes compaction due to the adoption of a more globular structure. The SV-AUC results also showed that Zn(II)-directed dimerization of hN1 occurs, which in turn could be a prerequisite for interactions with specific ligands in vivo. Surprisingly, the ITC results revealed a 2:1 binding stoichiometry (hN1/2:Zn(II)) of hN1/2, but we did not observe oligomerization of this protein under SV-AUC conditions. This could be the result of the different buffer conditions and/or precipitation during the ITC experiment.Since the binding of Zn(II) by the N-terminal fragment of hNucb2  and Ca. It is also worth noting that the highly cooperative character of the binding curve of hN1/2 might result from the antagonistic mechanisms of hN1 and hN2 for Zn(II) binding and precipitation, respectively. This raises the question of whether Zn(II)-driven destabilization of hN1/2 and full-length hNucb2 [Kd was lower than expected, possibly due to a more complicated stoichiometry in the actual model. However, the data obtained for hN1/2 correspond well to the previous experiments.The above observations were complemented by the Zn(II)-induced hydrophobic surface exposure observed in the presence of ANS. It is also worth noting that hN1, in the absence of Zn(II), displayed the lowest ANS binding compared to that of the remaining peptides, resulting from its unordered structure, which lacks defined hydrophobic pockets that are suitable for fluorophore binding. However, structurization under Zn(II) treatment leads to the gradual formation of hydrophobic sites, resulting in increased ANS fluorescence. Zn(II) had a dual influence on the hN2 and hN1/2 structures. We first observed gradual exposure of the hydrophobic sites for both peptides followed by a decrease in fluorescence intensity, which resulted from the precipitation that was also observed after exceeding the Zn(II) threshold concentration in the CD studies. This experiment allowed for the apparent h hNucb2  might beh hNucb2 . ZI compCarassius auratus) [Danio rerio) [Microhyla ornata) [Kd and in relation to Zn(II) cellular levels. It appears that proteolytical processing does not substantially influence the Kd value, which is still in the 10–5-10–6 M range, as reported for hNucb2 [Nucleobindins are a highly conserved protein family that is also expressed in non-mammals, e.g. goldfish (auratus) , 60, zebo rerio) , frog (M ornata) , in hypo ornata) , 64, G p ornata) , and nesr hNucb2 . Zn(II) r hNucb2 . Interesr hNucb2 . Indeed,r hNucb2 .Above findings, along with the distinct mechanism of Zn(II) binding by hN1/2 (proprotein) presented here, indicates that the physiological effects of Nucb2/N1/2 and Nucb1/NLP might also be evoked by their subcellular localization. Moreover, since PC1/3 undergoes maturation in GA , its proKd value of hN1 observed here in vitro. Moreover, the signals induced and/or modulated by N1 in the amygdala could affect the functions of Nucb1/Nucb2 and NLP/nesfatins in the hypothalamus via the stria terminalis. However, there are no reports on the interactions of the apo- and holo-N1 in the synaptic cleft and its physiological implications that could shed new light on the properties of this unique peptide. Another question is whether N1, N2, and N1/2 display amyloidogenic properties that are more akin to other Zn(II)-dependent amyloid proteins, such as tau [β protein [α2-macroglobulin [The brain regions that constitute the limbic system include the amygdala, hypothalamus, limbic cortex, hippocampal formation, and septal area . The limh as tau , amyloid protein , and α2-globulin , or whetglobulin .In summary, we demonstrated the Zn(II)-binding ability of nesfatins and described the influence of these ions on the structure of the above peptides. We conclude that in vivo proteolytical processing of the full-length protein could be an activation mechanism that facilitates hN1-cellular trafficking and multiple-partner interactions through the order-to-disorder transition. The observed hN1 oligomerization under Zn(II) might be a prerequisite for interaction with specific ligands and be a part of its complex regulatory framework. We have also demonstrated that the destabilization of hN1/2/hNucb2 in the presence of Zn(II) might arise from the properties of hN2, which presumably also has a structural role. Nonetheless, further studies are necessary to elucidate the role of Zn(II) in nesfatins sensing and its molecular mechanism as well as physiological purpose.Additional file 1. Supplementary figures and tables.


ILP2), with air-modulation, and high-concentration CO2 stress, in the larvae of Tribolium castaneum. We assessed the changes in carbohydrate-related content, trehalase activity, and the expression levels of trehalose pathway genes. And pupation, adult emergence, pupation rate, and mortality were assessed. There was a significant change in the expression of ILPs in T. castaneum, at a certain concentration of CO2. ILP2 RNAi did not alter the trehalose content significantly, however, the glycogen and glucose content increased significantly. High-concentration CO2 stress altered the trehalose content and reduced the glycogen and glucose content. The expression levels of TPS and TRE2 were up-regulated by hypoxia/hypercapnia and dsILP2 combination, with the increase of CO2 concentration, other trehalase genes begin to respond successively. ILP2 knockout raised the mortality and reduced the pupation rate and eclosion rate in CO2. Understanding the insulin pathway responses to hypoxic stress induced by a high concentration of CO2 would further elucidate the mechanisms underlying trehalose metabolism in insects.RNAi was used to downregulate the expression of insulin-like peptides ( Tribolium castaneum (Herbst) is a coleopteran belonging to the family, Tenebrionidae. It is distributed in the warmer regions and is an extremely important universal pest of stored grains . The T. castaneum were treated with 25% CO2 + 75% air, 50% CO2 + 50% air, 75% CO2 + 25% air and 95% CO2 + 5% air. The group treated with normal concentration of CO2 was the control group (CK). T. castaneum on the first day of eighth instar larvae were cultured under different levels of CO2 for 48 h and assay ILP genes  relative expression  was injected into the soft part between the third and fourth abdominal segments of T. castaneum on the first day of eighth instar larvae. The larvae were collected 48 h after dsRNA (dsILP2 or dsGFP) injection combined with different concentration CO2 treatment, and stored at −80°C. Fifteen insects each were used for the analysis of interferent-effect of dsRNA, gene expression, sugar content, and activity of the enzymes in the glucose metabolism pathways. All assays were performed in triplicate.Premier 5.0 software was used to design specific dsRNA primers for The mortality observed within 24 h of the injection was recorded as mechanical death. The mortality after 48 h of processing, the pupal rate, and the emergence rate were recorded.Total RNA was isolated from each sample by using the MiniBEST Universal Extraction Kit , following the manufacturer’s instructions. The RNA integrity was verified by 1% agarose gel electrophoresis, and the RNA concentration was measured by a NanoDrop2000 spectrophotometer . First-strand complementary DNA (cDNA) synthesis was performed using the PrimeScript® RT Reagent Kit  following manufacturer’s instructions.TcILP2 were assessed using qRT-PCR. The insects from each treatment were collected 48 h after dsRNA injection, for assessing the efficiency of the RNAi, using qPCR. Following TcILP2 knockdown under different concentrations of CO2, the transcript levels of six genes pertaining to the trehalose metabolic pathway, including five trehalase genes  and trehalose-6-phosphate synthase gene (TcTPS), were analyzed. The qPCR was carried out on a CFX-96 real-time detection system  in a 20 µL reaction containing 1 µL (100 ng/μL) cDNA, 1 µL (10 µM) of each primer, 7 µL nuclease-free water, and 10 µL of GoTaq qPCR MasterMix (Promega). The reaction was performed under the following conditions: pre-incubation at 95°C for 2 min, followed by 40 cycles of 95°C for 30 s and annealing at 60°C for 30 s, with melting curves obtained at 65–95°C. Ribosomal protein L13a (RPL13a) was the internal control. Specific primers shown in −△△CT method . Trehalose was quantified using the anthrone-sulfuric acid method . Bars represent mean ± standard error (SE). Data were statistically evaluated using the unpaired two-tailed Student’s t test for two groups, and values were considered statistically significant when 2 stress influenced the expression of the insulin pathway genes. The expression of TcILP1 was upregulated in 25% CO2 + 75% air group and 95% CO2 + 5% air, TcILP2 was upregulated in 25% CO2 + 75% air group, and TcILP3 was upregulated in 25% CO2 + 75% air group, while that of TcILP4 was downregulated in 50% CO2 + 50% air group  in CK group (TcTRE1-1 was downregulated significantly (p < 0.05)   (dsILP2 combined with 25% CO2 + 75% air. The relative expression level of TcTRE1-2 and TcTRE1-4 were significant decrease (p < 0.05)     increased significantly (p < 0.05)  , express < 0.01) , the rel < 0.01)  after tr < 0.05) , and TcT < 0.05)  when cul < 0.05) , on the  < 0.05) . As for  < 0.05) .dsGFP and the dsILP2 treatments 48 h with the increase in the CO2 concentration, the mortality rate had been reached 100% at 50% CO2 + 50% air concentration  genes in T. castaneum  and pesticides  in Apis cerana cerana . Those results revealed, T. castaneum tried to strengthen the trehalose or glucose content at the transcript levels of the trehalose pathway genes, but in high CO2 concentrations, fat body consumes too much energy bring on the content of trehalose or glucose extremely lower than the normal.The addition of hypercapnia made a complex alteration on the hypoxia response of  changed ; 2019. Fastaneum . Further hypoxia . It was fat body . Then we2 + 75% air mix, the T. castaneum can still survive partially, and the mortality rate reaches 100% at 50% CO2 + 50% air, 75% CO2 + 25% air, and 95% CO2 + 5% air. The matching situation was 25% under CO2, dsILP2 not only has a lower mortality rate than dsGFP, but also a significant increase in pupation rate and emergence rate—may be attributed to the increase in TcILP2 expression—which is similar to Xu’s research results (2 stress causes hypoxia, which directly limits energy supply and free radical damage and other processes, and ultimately leads to insect death (ILP2 knockout and hypoxia/hypercapnia could accelerate the carbohydrates consumption and affect survival and development of insects. Thus, we hypothesized that the TcILP2 could participate in the regulation of the trehalose metabolism during hypoxia to maintain insects alive.We found that under 25% CO results . Insulin results . Moreovect death . Combina


Circular RNA (circ) AFF4 was documented to regulate osteogenesis but the underlying mechanism remains to be elucidated. The preliminary study showed that circ_AFF4 may promote osteogenesis via FNDC5/Irisin. Furthermore, the online prediction tool indicated the interaction of circ_AFF4, insulin-like growth factor-2 mRNA-binding protein 3 (IGF2BP3), FNDC5 and lysine (K)-specific demethylase 1 A (KDM1A). Therefore, this study aims to elucidate the relationships of KDM1A, circ_AFF4, IGF2BP3 and FNDC5/Irisin during osteogenesis.in vivo model was utilized to further confirm the regulatory effect on bone formation.The alkaline phosphatase (ALP) activities and osteogenic-related factors were determined using ALP and alizarin red S (ARS) staining, real-time quantitative PCR(RT-qPCR) and western blot. Immunoprecipitation (RIP), pull-down assay and fluorescence in situ hybridization (FISH) were used to examine the interactions among circ_AFF4/IGF2BP3/FNDC5. A mouse in vitro data was confirmed with in vivo model.Circ_AFF4 and KDM1A expression levels were increased during osteoinduction of BM-MSCs. Knockdown of circ_AFF4 and KDM1A significantly suppressed BM-MSC osteogenesis. We also proved that KDM1A directly bound to circ_AFF4 and FNDC5 promoter and induced circ_AFF4 and FNDC5 expression. Furthermore, circ_AFF4 enhanced the stability of FNDC5 by generating a circ_AFF4, IGF2BP3 and FNDC5 RNA-protein complex, and thereby induced Irisin and osteogenesis. The These findings elucidate that KDM1A induces circ_AFF4, which promotes promote osteogenesis via IGF2BP3. This study indicates that circ_AFF4 may potentially represent a critical therapeutic target for the diseases.The online version contains supplementary material available at 10.1186/s10020-022-00557-7. Irisin, a myokine derived from the cleavage of fibronectin III domain-containing protein 5 (FNDC5), is released into the bloodstream in response to muscle contraction , we identified that IGF2BP3 may bind to FNDC5. Considering the critical role of FNDC5 in circ_AFF4-mediated osteogenesis, we speculated that IGF2BP3 is potentially also involved in osteogenesis.Circular RNAs (circRNAs), which is a class of small noncoding RNAs, form a covalently closed loop . The combination regulatory effects of KDM1A, circ_AFF4 and IGF2BP3 remain to be further elucidated.Histone modification is crucial in controlling the proliferation, migration and differentiation of MSCs  were obtained from Procell  and cultured in Dulbecco´s Modified Eagle´s Medium  with 10% fetal bovine serum  and 100 U/mL penicillin . Only BM-MSCs from passages 3–5 were used for this study.Flow cytometry was used to characterize BM-MSCs using antibodies against CD45, CD14, CD90, CD73, HLA-DR and CD105 .6 cells per well) using Lipofectamine 2000  in 20 µL lipofectamine. After 6 h, the culture medium was changed to the normal medium. After 48 h, cells were used for the in vitro experiments. For lentivirus infection, lentiviruses carrying sh-KDM1A, circ_AFF4 overexpression lentiviruses, FNDC5 overexpression lentiviruses and correspondence control vectors were obtained from Genepharma and were used to infect BM-MSCs. Cells were selected with 2.5 µg/mL puromycin after 2 days post-transduction. 4–5 days later, puromycin was removed and cells were continuously cultured in the normal medium until fully recovered for animal studies.Based on previous studies, cell transfections were performed  was added. Cells cultured in normal DMEM were utilized as controls. The medium was changed every 3 days until 14 days. The cells were harvested for further analyses.Osteogenic Differentiation Medium BulletKit  was used to induce BM-MSC osteogenic differentiation followed by manufacturer instructions. In brief, 1 × 10Adult C57BL/6J male mice that were between 12 and 14 weeks and weighing 25–30 g were given by Hunan SJA laboratory animal Co., Ltd  and were kept in an animal facility . The Ethics Committee of the First Affiliated Hospital of Nanhua University (registration number: ChiCTR1900021723) has approved all the experiments.Sixty mice in total were separated into six groups, (1) control, (2) sh-KDM1A, (3) circ_AFF4, (4) shKDM1A + circ_AFF4, (5) FNDC5, and (6) shKDM1A + FNDC5. BM-MSCs that infected with sh-NC and lentiviruses, sh-KDM1A, circ_AFF4 overexpression lentiviruses, sh-KDM1A plus circ_AFF4 overexpression lentiviruses, FNDC5 overexpression lentiviruses and sh-KDM1A plus FNDC5 overexpression lentiviruses cultured in OM for 1 week and seeded on bioceramic scaffolds with β-tricalcium phosphate and hydroxyapatite mixture  based on a previously published protocol . In brief, after being washed with PBS three times, cells were fixed with a fixing solution. Afterward, cells were incubated with ALP staining solution for 30 min in the dark followed by washing with PBS 3 times and visualized with a light microscope .After being fixed with 4% PFA, cells were subsequently incubated with Alizarin Red S solution that was freshly prepared (Sigma-Aldrich) for about 30 min at room temperature. After 3 times wash with PBS the staining was imaged with a microscope (Zeiss).BM-MSCs were seeded into six-well plates and maintained until they reach 60% confluency. Afterward, Actinomycin D (5 µg/mL) or DMSO (Sigma-Aldrich) was used to treat cells for 0, 12 and 24 h and then collected. 2 µg of total RNA was treated with RNase R  for 15 min. After treatment, the RNA expression levels were analyzed by RT-qPCR.After being washed 3 times with PBS, cells were treated with RNase R (Sigma-Aldrich) for 15 min followed by fixation with 4% PFA. The cells were placed onto glass slides and then dehydrated with ethanol. After hybridization, the slides were cleaned three times in 50% formamide/2 × SSC fand and incubated with circ_AFF4 and IGF2BP3 specific probes  at 37 °C in standard hybridization buffer . Afterward, cells treated with the DAPI  for 15 min. The images were taken utilizing fluorescence microscopy with a 60X objective .The cells were fixed with 4% PFA for about 15 min followed by blocked with the goat serum and incubated with primary antibodies against Anti-RUNX2  at 4 °C overnight. After the secondary antibody  incubation, the cells were further incubated with DAPI. The images were captured under a confocal microscope .https://blast.ncbi.nlm.nih.gov/Blast.cgi), was utilized to predict the binding sequence of circ_AFF4 with FNDC5, and AnimalTFDB  was utilized to predict the interaction of KDM1A with circ_AFF4 and FNDC5.The online bioinformatic tools, BLAST (in vitro transcription using TranscriptAid T7 High Yield Transcription Kit (Thermo Scientific) and purified utilizing GeneJET RNA Purification Kit (Thermo Scientific). The cell lysates that were extracted from BM-MSCs were treated with purified IGF2BP3 or mutants and with streptavidin-coated magnetic beads (Invitrogen) in order to pull down the biotin-coupled RNA complex following the user´s instructions. Magnetic beads were washed extensively. The enrichment of circ_AFF4 or IGF2BP3 was examined by RT-qPCR analysis. The bound proteins were eluted and analyzed by SDS-PAGE. The proteins were analyzed by western blot.Biotinylated IGF2BP3 and the mutants with the mutated putative binding sites for circ_AFF4 were generated by BM-MSCs were collected, and RIP assay was carried out utilizing an EZ-Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Merck Millipore). Briefly, RIP lysis buffer was added to the cells that centrifuged at 2500 g for 10 min and then 13,000 g for 10 min. The pellets were incubated with magnetic beads conjugated to either human anti-IGF2BP3 antibody , or the nonspecific anti-IgG antibody  at 4 °C overnight. Afterward, protein K buffer was added for another 2 h at 4 °C. After centrifuging five times, the co-immunoprecipitated RNA was isolated and used for further RT-qPCR analysis.The Irisin levels in BM-MSCs were measured using ELISA kits  based on the manufacturer’s protocol.RNA EMSA assay was performed utilizing a LightShift™ Chemiluminescent RNA EMSA Kit (Thermo Fisher Scientific), based on the users´ instructions. In brief, a biotin-labeled oligonucleotide probe was purchased from Sangon Biotech  and was used to treat purified protein in a binding buffer for about 30 min. Electrophoresis on 6% nondenaturing polyacrylamide gel in 0.5× TBE buffer was used to resolve the complexes at 100 V for 1 h, which was then transferred to a nylon membrane for 30 min at 400 mA. The membrane was crosslinked, blocked and incubated with HRP-linked streptavidin for 15 min. For supershipft assay, recombinant GST-IGF2BP3 proteins were preincubated with ant-IGF2BP3 antibody  for 20 min at 0 °C and then incubated with labeled probe for supershift assays.The assay was performed based on a reported protocol  and their sequences were listed in Table The total RNA was extracted from cells or tissues utilizing the Trizol reagent . 2 µg of total RNA was reversely transcribed utilizing SYBR Premix Ex Taq  based on the manufacturer’s instructions. qPCR was carried out utilizing Taqman® Universal PCR mixture Kit (Thermo Fisher Scientific) based on the user’s instruction. The relative mRNA levels were quantified using the 2Femur bone tissue from mice was fixed in 4% PFA, subsequently embedded in paraffin and cut into 5 μm sections. As previously reported  was used for Masson staining based on the manufacturer’s protocol. The images were taken using a light microscope.Total cellular protein was isolated from the cells using the radio-immunoprecipitation assay (RIPA) buffer (Beyotime) containing protease inhibitors at 4 ºC for 30 min. Protein concentrations were measured with BCA protein, separated by SDS-PAGE (30 µg/well), and transferred to polyvinylidene fluoride membranes and blocked. The membranes were incubated with primary antibodies including ALP , OPN , RUNX2 , Colla1 , and KDM1A . Afterward, these membranes were washed and followed by incubation with a secondary antibody . Eventually, an ECL kit (Millipore) was utilized to visualize the protein bands.The EZ-ChIP kit  was used to perform the ChIP assay based on a previously published protocol  followed by the Turkey post hoc test was applied in cases of comparison among multiple groups. As shown by FACS, the protein expression levels of surface markers CD90, CD73 and CD105 were detected in the BM-MSCs while CD45, HLA-DR and CD14 protein expression levels were not examined in BM-MSCs , we identified that the AAAGA site in circ_AFF4 may directly bind to the 3´UTR of FNDC5 with AU-Rich Elements (Fig. It was shown by previous studies that IGF2BP3 or IGF2BP3/circRNA complex stabilizes mRNA (Wang et al. To thoroughly understand the role of circ_AFF4 and IGF2BP3 in BM-MSC osteogenic, we firstly transfected BM-MSCs with lentiviruses harboring circ_AFF4 or shIGF2BP3 for a stable overexpression or knockdown followed by and osteogenesis capacity evaluation after culturing in OM for 14 days. Transfection of BM-MSC with shIGF2BP3-1#, shIGF2BP3-2#, shIGF2BP3-3#, shIGF2BP3-4# resulted in a decrease in IGF2BP3 expression (Fig. Collectively, Circ_AFF4 and IGF2BP3 regulate BM-MSC osteogenic differentiation via FNDC5/Irisin.A recent study showed that KDM1A mediated osteogenic differentiation of MSCs (Wang et al. Taken together, knockdown of KDM1A inhibits the osteogenic differentiation capacity of BM-MSCs.The computational prediction tool, AnimalTFDB, predicted that there might be five binding sites between KDM1A and circ_AFF4 promoter Fig.  A. ChIP To further confirm the role of KDM1A in circ_AFF4 or FNDC5 mediated osteogenic differentiation, BM-MSCs were transfected with shKDM1A, lentiviruses harboring circ_AFF4, lentiviruses harboring circ_AFF4 plus shKDM1A, lentiviruses harboring FNDC5 or lentiviruses harboring FNDC5 plus shKDM1A to knockdown KDM1A and overexpress circ_AFF4 or FNDC5. The transfection was performed successfully as shown in Fig. These data suggest that KDM1A locates at upstream of circ_AFF4 and FNDC5 and binds to circ_AFF4 and FNDC5 to promote Irisin expression.in vivo role of KDM1A, circ_AFF4 and FNDC5 in bone formation, we seeded BM-MSCs transfected with lentiviruses harboring circ_AFF4, lentiviruses harboring FNDC5 or shKDM1A or their correspondence controls on scaffold material circ_AFF4 and then implanted in mice. As observed in ALP and ARS staining images, bone samples from mice with circ_AFF4 overexpression or FNDC5 overexpression displayed increased bone cell differentiation and calcium deposits (Supplementary Fig. To identify the in vivo. Nevertheless, this promotion effect is reversed by KDM1A depletion, indicating that KDM1A regulates bone formation via circ_AFF4 and FNDC5.Altogether, circ_AFF4 or FNDC5 induces new bone formation in vitro and in vivo. We showed that circ_AFF4 was increased during osteoinduction of BM-MSCs. Increased circ_AFF4 expression promotes the stability of FNDC5 mRNA by generating a circ_AFF4/IGF2BP3/FNDC5 ternary complex to enhance osteogenic differentiation of BM-MSCs. This study further revealed that KDM1A locates at upstream of circ_AFF4 and induces the expression of circ_AFF4, which enhances the downstream protein IGF2BP3/FNDC5 and further increases KDM1A expression. As far as we know, this is the first time demonstrating that the KDM1A/circ_AFF4/FNDC5 positive axis promotes osteogenesis via FNDC5/Irisin. This in vitro data was further confirmed with in vivo model.In this study, we demonstrated the importance of circ_AFF4 in osteogenesis both in vitro and promoted fracture healing in vivo (Mi et al. Circ_AFF4 was demonstrated to be involved in mediating osteogenesis (Lin et al. in vivo. Nevertheless, a publication showed that KDM1A knockdown suppressed ALP activity and mineralization in vitro but promoted osteogenesis-related factors in vitro and in vivo (Wang et al. This study also revealed the crucial role of KDM1A in circ_AFF4 mediating BM-MSC osteogenesis. KDM1A was elevated in BM-MSCs that were treated with OM for 14 days and KDM1A promoted bone formation in vitro experiments, were relatively limited since most of the in vitro study data were collected from only three independent studies.This study has two limitations. First, the animal experiments in this study did not include any controls for exercise level in mice but physical activity is an important factor of Irisin expression. Secondly, the sample sizes for this study, especially In conclusion, for the first time, we reported the KDM1A/circ_AFF4/FNDC5 axis during BM-MSC osteoinduction. We further uncovered the critical role of circ_AFF4 in stabilizing FNDC5 mRNA stability by generating a circ_AFF4/IGF2BP3/FNDC5 complex to promote osteogenesis (Supplementary Fig. Below is the link to the electronic supplementary material.Supplementary Material 1Supplementary Material 2


The PRDM9-dependent histone methylation H3K4me3 and H3K36me3 function in assuring accurate homologous recombination at recombination hotspots in mammals. Beyond histone methylation, H3 lysine 9 acetylation (H3K9ac) is also greatly enriched at recombination hotspots. Previous work has indicated the potential cross-talk between H3K4me3 and H3K9ac at recombination hotspots, but it is still unknown what molecular mechanisms mediate the cross-talk between the two histone modifications at hotspots or how the cross-talk regulates homologous recombination in meiosis.Here, we find that the histone methylation reader ZCWPW1 is essential for maintaining H3K9ac by antagonizing HDAC proteins’ deacetylation activity and further promotes chromatin openness at recombination hotspots thus preparing the way for homologous recombination during meiotic double-strand break repair. Interestingly, ectopic expression of the germ-cell-specific protein ZCWPW1 in human somatic cells enhances double-strand break repair via homologous recombination.Taken together, our findings provide new insights into how histone modifications and their associated regulatory proteins collectively regulate meiotic homologous recombination.The online version contains supplementary material available at 10.1186/s13059-022-02758-z. Meiotic homologous recombination during the first meiotic prophase, a process that increases genome diversity in gametes, and thus, is a hallmark not only for sexual reproduction, but also evolution –4. RemarMeiotic DSBs and crossovers are distributed non-randomly on chromosomes and are regarded as recombination hotspots –30, whicBeyond the known enrichment for H3K4me3 and H3K36me3 at recombination hotspots, H3 lysine 9 acetylation (H3K9ac) is also enriched at recombination hotspots , 42. It In addition to histone modifications, chromatin accessibility is believed to be essential for homologous recombination –55. In bIn this study, we found that the germ-cell-specific protein ZCWPW1, the first histone methylation reader at meiotic recombination hotspots in mammals, functions to prevent histone deacetylases from removing H3K9ac, thus promoting chromatin openness at recombination hotspots. Interestingly, ectopic expression of ZCWPW1 in human somatic cells is able to promote DSB repair via homologous recombination.Zcwpw1 in male mice results in the complete failure of meiosis prophase I, which leads to spermatocytes arrested at the zygotene stage (with incomplete DSB repair) . A. A59]. AWe subsequently performed Co-immunoprecipitation (Co-IP) assays to confirm the HDAC-ZCWPW1 interactions in testes. The three examined HDACs  were pulled down using a ZCWPW1 antibody Fig. e and S3fZcwpw1–/– testes. However, the levels of the candidate proteins were comparable between WT and Zcwpw1–/– testes  in HEK293T cells co-overexpressing PRDM9 and ZCWPW1 . When weZcwpw1−/− testes. We found that 67% (2676/4015) (group A1) of the ZCWPW1 binding sites lost the ATAC signal in Zcwpw1−/− mouse testes  of the group A1 ZCWPW1 binding sites showed simultaneous reductions in chromatin accessibility and H3K9ac signal intensity  in HEK293T cells ectopically expressing PRDM9 and ZCWPW1 [The histone modification reader ZCWPW1 specifically recognizes dual histone methylation marks deposited by PRDM9 at hotspots in testes , 49, 50.In mammals, meiotic homologous recombination hotspots are marked by a special combination of histone modifications, such as H3K4me3, H3K36me3 and H3K9ac , 46. It It is well-established that H3K4me3 and H3K9ac at promoter regions are regarded as active histone modification marks, and these usually recruit chromatin remodeler proteins to make the promoter regions more accessible and thus regulate gene transcription , 58. BotZcwpw1 [Both histone modification and chromatin remodeling are critical for DSB repair , 69–71, Zcwpw1 –50, and Zcwpw1 –76. The Zcwpw1−/− testes, while these same H3K4me3 peaks are lost in Prdm9−/− testes  . These fIn summary, our study has shown that the histone methylation reader ZCWPW1 is able to mediate the cross-talk between histone methylation and histone acetylation and that it functions to promote chromatin openness at DSB hotspots, and together these roles of ZCWPW1 pave the way for homologous recombination during meiotic DSB repair Fig. .Zcwpw1 knockout mice and Prdm9 knockout mice were generated in our previous studies [Spo11 gene  is located on mouse chromosome 2 and comprises 13 exons, with the ATG start codon in exon 1 and the TAA stop codon in exon 13. Exon 2 to exon 12 were selected as the target site, which covered 79.12% of the coding region. The Spo11 knockout mice were generated by deleting exon 2 to exon 12 using the CRISPR/Cas9-mediated genome editing system in the C57BL/6 genetic background .The  studies , 48. TheSpo11 knockout founders were genotyped by PCR followed by DNA sequencing analysis. The homozygous mutant mice were generated by intercrossing of heterozygous mutants, and genotyping was performed by PCR amplification of genomic DNA extracted from mouse tails. PCR primers for the Spo11 mutant allele were Forward: 5′-TAT CAA CTG GGG CCT GTG GTC AG-3′ and Reverse: 5′-GTG TGC AGG CTT GCG ACA GTG-3′, yielding a 440 bp fragment. PCR primers for the Spo11 WT allele were Forward: 5′-TCC GTT CAG TGT GGT TCT CC-3′ and Reverse: 5′-GTG TGC AGG CTT GCG ACA GTG-3′, yielding a 222 bp fragment.The All mice were maintained with free access to water and food in a specific pathogen-free facility under a 12-h light/dark cycle. All experimental protocols were approved by the regional ethics committee of Shandong University.The primary antibodies used for ChIP-seq and CUT&TAG were rabbit anti-ZCWPW1   and rabbZcwpw1 and Smchd1 was synthesized by Wuhan GeneCreate Biological Engineering. The Zcwpw1 was cloned into pCAG-GFP  with a C-terminal fusion EGFP tag or pcDNA3.1(+) with a C-terminal fusion HA-V5 tag for transient expression in cell lines. Mouse Hdac1 (#P10549), Hdac2 (#P5693), and Hdac6 (#P10426) expression plasmids were purchased from Wuhan Miaoling Biological Technology. The I-SceI expression plasmid (pCBASce) used in the homologous recombination reporter assay was kindly provided by Professor Jun Huang from Zhejiang University. All constructs used in this study were confirmed by DNA sequencing.Complementary DNA (cDNA) for mouse 2 incubator. For transient transfection experiments, cells were transfected with expression vectors using the X-tremeGENE HP DNA Transfection Reagent  following the manufacturer’s instructions. HeLa and HEK293T cells were purchased from the National Collection of Authenticated Cell Cultures, and U2OS cells were kindly provided by Professor Qiang Chen from Wuhan University. U2OS DR-GFP cell lines were kindly provided by Professor Jun Huang from Zhejiang University [HeLa, HEK293T, and U2OS cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10%(v/v) fetal bovine serum and 1% penicillin streptomycin at 37°C in a 5% COiversity , 80.Homologous recombination reporter assays were performed as described previously . Briefly4 cells in 10 μl PBS were mixed with 100 μl 0.8% low-gelling agarose and were layered as microgels on microscope slides. Slides were then immersed in lysis solution at 4°C for 1–2 h in the dark. After lysis, the slides were incubated in alkaline unwinding solution for 20 minutes at room temperature. Electrophoresis was performed at neutral pH at 1 V/cm for 10–15 min. After neutralization, samples were stained with ethidium bromide for 10 min and imaged with an Olympus fluorescence microscope. DNA damage was quantified by measuring the comet tail length using Comet Assay Software Pect (CASP 1.2.3 beta 1).The neutral comet assay was performed using the Comet Assay Kit  according to the manufacturer’s instructions. Briefly, HEK293T cells were cultured in 6-well plates and transfected with pCAG  or ZCWPW1-pCAG using Lipofectamin 3000 Transfection Reagent  following the manufacturer’s protocol. After 24 h, cells were treated with 2 mM hydroxyurea for 16 h. A total of 1 × 10g for 15 min at 4°C. The supernatant was transferred into a new tube and incubated with primary antibody or control IgG with rotation overnight at 4°C. Then, the antibodies were isolated by adsorption to Pierce protein A/G beads  for 2 h. After washing, SDS loading buffer was added to the beads and boiled. Samples were separated by SDS-PAGE and immunoblotted with primary and secondary antibodies. Immunoreactive bands were detected and analyzed with a BIO-RAD ChemiDoc MP Imaging System and Image Lab Software .Testes or transfected cells were lysed with Pierce IP lysis buffer  with protease inhibitors  on ice for 30 min, then centrifuged at 12,000×Zcwpw1 cDNA was subcloned into the pGBKT7 vector as bait. Full length mouse Hdac1, Hdac2, Hdac6, and Smchd1 cDNAs were subcloned into the pGADT7 vector as prey. The bait and prey plasmids were co-transformed into the yeast two-hybrid gold strain yeast, and the positive transformants were selected on nutrition-restricted plates (SD/-Leu/-Trp/-His).The yeast two-hybrid assay was performed by GeneCreate Biological Engineering, Wuhan, China. Briefly, full length mouse HeLa cells were cultured on coverslips transfected with pCAG  or ZCWPW1-pCAG using X-tremeGENE HP DNA Transfection Reagent following the manufacturer protocol. After 24 h, cells were treated with 2mM Hydroxyurea for 16 h, 1 μM Camptothecin for 3 h, or 50 μM ETO for 6 h. Transfected cells on glass coverslips in 6-well plates were fixed in 4% paraformaldehyde in PBS pH 7.4 for 10 min at room temperature. The samples were incubated for 10 min with PBS containing 0.3% Triton X-100. After washing in PBS three times, the cells were blocked with 1% BSA and 22.52 mg/mL glycine in PBST (PBS+ 0.1% Tween 20) for 30 min at room temperature. Then, the samples were incubated with diluted primary antibody in 1% BSA in PBST in a humidified chamber for 2 h at room temperature or overnight at 4°C. After washing in PBS three times, the cells were incubated with secondary antibodies in 1% BSA for 1 h at room temperature in the dark. The coverslips were mounted with a drop of mounting medium containing DAPI. The coverslips were sealed with nail polish to prevent drying and movement under the microscope.Immunostained slides were imaged by confocal microscopy . Projection images were then prepared using Photoshop (Adobe) software packages or Bitplane Imaris (version 8.1) software. For live cell laser micro-point irradiation, U2OS cells were seeded in a glass-bottom dish . After attachment, the indicated expression plasmids were transfected into U2OS cells. Laser micro-point irradiation was conducted 24 h after transfection, and images were captured at the indicated time point. Laser micro point irradiation was performed under an inverted confocal microscope  with a micro-point laser workstation (Andor).The HDAC activity assay was performed using the Histone Deacetylase Activity Assay Kit  according to the manufacturer’s instructions . The HDARIME is a technique ideally suited for the identification of transcriptional co-factors and chromatin-associated proteins . ZCWPW1-RIME analysis was performed according to published protocols  using ZC6 cells from PD13–PD14 testes were cross-linked in 100 μl of 1% formaldehyde in PBS at room temperature for 10 min and then quenched with 25 μl 1.25M glycine solution and washed with PBS. The cells were then incubated in 150 μl lysis buffer  for 20 min on ice then sonicated using a Diagenode Bioruptor sonication device for 23 cycles (30s ON and 30s OFF). A total of 150 μl 300mM SDS-free RIPA buffer  and 200 μl 140mM SDS-free RIPA buffer was added to the samples. After centrifugation at 13,000×g for 10 min at 4°C, 40 μl of the supernatant was removed and used as the sample input. The remaining supernatant was transferred to a 1 ml tube containing suspended antibody-coated Protein A beads, followed by incubation on a tube rotator overnight at 4°C. Next, the incubated Protein A beads were washed once with RIPA buffer containing 250 mM NaCl, washed three times with RIPA buffer containing 500 mM NaCl, and washed once with TE buffer . Next, the beads were transferred to a new 0.5 ml tube, followed by incubation in 100 μl ChIP elution buffer  containing 5 μl proteinase K  at 55°C for 2 h and then at 65°C for 4 h. The eluate was transferred to a fresh 0.5 mL tube, and the enriched DNA was purified by 1.8X SPRIselect beads, followed by dissolution in 50 μl TE buffer. Finally, the NEBNext Ultra II DNA Library Prep Kit for Illumina  was used for library construction according to the product instructions. Libraries were sequenced using the Illumina X-ten and NovaSeq 6000 platform in PE150 mode .The ChIP-seq libraries were prepared as previously described  with furg for 5 min at room temperature. Cell pellets were resuspended with 100 μl wash buffer. A total of 10 μl concanavalin A-coated magnetic beads were washed twice with 100 μl binding buffer and then added to the cell tubes and incubated at room temperature for 10–15 min. After removing the supernatant, the bead-bound cells were resuspended with 50 μl antibody buffer containing 3 μg rabbit anti-ZCWPW1 antibody or 2 μg rabbit anti-H3K9ac antibody. After incubation at room temperature for 2 h or overnight at 4°C, the primary antibody was carefully discarded and 0.5 μl Guinea pig anti-rabbit IgG  diluted with 50 μl Dig-wash buffer was added to the cells. The cells were then incubated with rotation at room temperature for 1 h. The cells were then washed gently with 200 μl Dig-wash buffer three times, and 2 μl pA/G–Tnp together with 98 μl Dig-300 buffer was added to the samples. After incubating at room temperature for 1 h, the samples were washed gently with 200 μl Dig-300 buffer three times. Then, 10 μl 5 × TTBL mixed with 40 μl Dig-300 buffer was added to each sample and the samples were incubated at 37 °C for 1 h. The interactions were quenched by adding 5 μl 20 mg/ml Proteinase K, 100 μl Buffer L/B, and 20 μl DNA extraction beads and incubating the samples at 55°C for 10 min. The supernatant was discarded and the beads were washed once with 200 μl Buffer WA and twice with 200 μl Buffer WB and resuspended with 22 μl nuclease free water. For library amplification, 15 μl of purified DNA was mixed with 25 μl of 2× CAM, along with 5 μl of uniquely barcoded i5 and i7 primers from the TruePrep Index Kit V2 for Illumina . A total volume of 50 μl of sample was placed in a thermal cycler  using the following program: 72 °C for 3 min; 98 °C for 3 min; 12–16 cycles of 98 °C for 10 s, 60 °C for 5 s, and 72°C for 1 min; and holding at 12 °C. To purify the PCR products, 2× volumes of VAHTS DNA Clean Beads  were added and incubated at room temperature for 5 min. The beads were washed twice with 200 μl fresh 80% ethanol and eluted in 22 μl ddH2O. All CUT&Tag libraries were sequenced by Novogene using the Illumina NovaSeq 6000 platform in PE150 mode .The CUT&Tag assay was performed as previously described  with mod5 cells were washed with 500 μl PBS and centrifuged at 600×g for 5 min at room temperature. Cell pellets were lysed in 50 μl lysis buffer for 5 min on ice to prepare the nuclei. Immediately after lysis, 950 μl wash buffer was added and mixed thoroughly, and the tubes were centrifuged at 500×g at 4°C for 5 min to remove the supernatant. Nuclei were then incubated with the 40 μl Tn5 transposome and tagmentation buffer at 37°C for 30min. To end the tagmentation, 10 μl stop buffer was added directly into the reaction. To purify the DNA, 2× volumes of Novoprotein Tagment DNA extraction beads  were added and incubated at room temperature for 5 min. The beads were washed twice with 200 μl fresh 80% ethanol and eluted in 37 μl elution buffer. For library amplification, 35 μl of purified DNA was mixed with 10 μl 5× Amplimix along with 2.5 μl of uniquely barcoded i5 and i7 primers from NovoNGS Index Kit for Illumina . A total volume of 50 μl of sample was placed in a Thermal Cycler  with the following program: 72 °C for 3 min; 98 °C for 30 s; 12–15 cycles of 98°C for 15 s, 60°C for 15 s, and 72°C for 8 s; 72°C for 2 min; and holding at 12°C. To purify the PCR products, 1.2× volumes of Novoprotein DNA Clean Beads  were added and incubated at room temperature for 5 min. The beads were washed twice with 200 μl fresh 80% ethanol and eluted in 20 μl elution buffer. All ATAC-seq libraries were sequenced using the Illumina NovaSeq 6000 platform in PE150 mode.The ATAC-seq libraries of mouse testes were prepared as previously described , 85 withThe ChIP-seq raw reads were cropped to 100 bp, and the low-quality reads were removed using Trimmomatic v0.32 . Paired The CUT&Tag raw reads were cropped to 40 bp, and the low-quality reads were removed using Trimmomatic v0.32 . Paired The ATAC-seq raw reads were cropped to 40 bp, and the low-quality reads were removed using Trimmomatic v0.32 . Paired t tests were used to analyze differences between two groups. All tests and p-values are provided in the corresponding legends and/or figures.Statistical analysis was carried out with GraphPad Prism 6. Unpaired Zcwpw1−/− testes. Figure S9. Uncropped western blot gel images in Fig. Additional file 1: Figure S1. ZCWPW1 binds at hotspot sites prior to DSB formation. Figure S2. Genome-wide properties of ZCWPW1-associated H3K9ac signal. Figure S3. Confirmation of ZCWPW1 binding proteins in mouse testes by Co-IP and yeast two hybrid. Figure S4. ZCWPW1 preserves the H3K9ac signal. Figure S5. Genome-wide properties of the ZCWPW1-associated ATAC signal. Figure S6. Ectopic expression of ZCWPW1 did not affect the cell cycle or doubling time. Figure S7. Ectopic expression of ZCWPW1 promotes DSB repair in somatic cells. Figure S8. H3K4me3 peaks at hotspot regions could be detected in Additional file 2: Table S1. Proteins that putatively interact with ZCWPW1 were detected in RIME assay.Additional file 3: Table S2. Summary of all ChIP-seq, ATAC-seq and CUT&TAG experiments performed, indicating target, sample, genotype and data source.Additional file 4. Review History.


Plasmodium falciparum VAR2CSA protein was identified as a probe targeting cancer-associated chondroitin sulfate A-like epitopes. In this study, we found that rVAR2 could also bind to heparin (Hep) and chondroitin sulfate E. Therefore, we used rVAR2 as a model to establish a method based on random mutagenesis of the GAG-binding protein and phage display to identify and optimize probes targeting tumor GAGs. We identified a new probe, VAR2HP, which selectively recognized Hep by interacting with unique epitopes consisting of a decasaccharide structure that contains at least three HexA2S(1–4)GlcNS6S disaccharides. Moreover, we found that these Hep-like epitopes were overexpressed in various cancer cells. Most importantly, our in vivo experiments showed that VAR2HP had good biocompatibility and preferentially localizes to tumors, which indicates that VAR2HP has great application potential in tumor diagnosis and targeted therapy. In conclusion, this study provides a strategy for the discovery of novel tumor-associated GAG epitopes and their specific probes.The high heterogeneity and mutation rate of cancer cells often lead to the failure of targeted therapy, and therefore, new targets for multitarget therapy of tumors are urgently needed. Aberrantly expressed glycosaminoglycans (GAGs) have been shown to be involved in tumorigenesis and are promising new targets. Recently, the GAG-binding domain rVAR2 of the  Targeted therapies have been recognized as the most promising therapeutic strategy for cancers because they can directly alter cell signaling events through the use of inhibitors and indirectly deliver antineoplastic drugs to cancer cells through tumor-targeting molecules , 2, 3. CGlycosaminoglycans (GAGs), linear negatively charged heteropolysaccharides composed of repeating hexosamine-containing disaccharide units, are ubiquitously expressed in all mammalian tissues. GAGs are classified into four categories according to the type of disaccharide composition and linkage between disaccharide units: chondroitin sulfate (CS)/dermatan sulfate (DS), heparin (Hep)/heparan sulfate (HS), hyaluronan, and keratan sulfate. GAGs participate in various physiological and pathological processes including tumorigenesis , 13, 14.3 recognized by the single-chain variable fragment (scFv) antibody NS4F5 is upregulated in ovarian cancer  has been detected in the mouse Lewis lung carcinoma cell line LM66-H11 with high metastatic potential n cancer . Another GlcNS6S , 19, 20.otential , murine otential  and ovarotential , which hotential , 25. Nototential . ConsideO-sulfate), which is exclusively expressed in the placenta, has been found in a various of malignant cells and can be specifically targeted by a recombinant fragment ID1-DBL2X-ID2a (rVAR2) of the Plasmodium falciparum VAR2CSA protein (E. coli BL21(DE3) could interact with not only CS-A but also Dosidicus gigas cartilage-derived CS-E . The recombinant VAR2 (rVAR2) was purified to >95% purity by nickel affinity chromatography coupled with gel filtration (A) and tested for its binding ability to various GAGs using a binding assay. The results showed that the obtained rVAR2 could not only strongly interact with CS-A but also significantly interact with CS-E and Hep (A and S2C), and especially a surface plasmon resonance binding analysis showed that rVAR2 bond to Hep with a high affinity (KD ∼ 61 nM) (B). Notably, the interaction of rVAR2 with Hep and CS-E has not been investigated in previous studies, which may limit knowledge about the selectivity of rVAR2 binding to GAGs. The binding of rVAR2 to CS-E may be due to the high proportion (>80%) of 4-O-sulfated GalNAc residues in CS-E  containing 95.88% of Hep/HS and 4.12% of CS/DS  with significant binding capacity to HepG2-GAGs were obtained (B), among which GAG-4 with the highest binding ability to HepG2-GAGs, named VAR2HP, was selected for further study.The gene of ID1-ID2a (VAR2) domain was codon-optimized and synthesized based on the previously reported protein sequence , cloned ltration A and tes and Hep A and S2C∼ 61 nM) B. Notabl in CS-E . In cont in CS-E , 32, whiof CS/DS  were bioobtained B, among 292C293Y294T295D296K297L298E299L300 N301 and an N312 residue (A). Then, the above obtained E. coli BL21 (DE3) cells harboring pET22b-var2hp were expanded and induced with isopropyl 1-thio-β-D-galactopyranoside, and after sonication and centrifugation, the VAR2HP protein was purified from the resulting lysis supernatant by a nickel affinity chromatography. SDS-PAGE showed VAR2HP was purified as a single band at approximately 70 KDa (B), which was consistent with the theoretical calculated value. The VAR2HP protein was preserved in PBS buffer for the following study.After sequencing, the var2hp gene was shown to consist of 1953 bp with a GC content of 51.56% and to encode a protein containing 651 amino acids with an isoelectric point (PI) of 8.10, and a calculated molecular weight of 73.07 kDa using the peptide mass tool on the ExPASy server of the Swiss Institute of Bioinformatics. Compared with the sequence of rVAR2, VAR2HP has eleven extra amino acids, including a fragment L residue A. Then, y 70 KDa B, which A) and to Hep at a level comparable to that of rVAR2 to CS-A (C). Additionally, no obvious binding to CS-A or HS was detected with increasing concentrations of VAR2HP  and N312 in VAR2HP binding to Hep, three mutants were recombinantly constructed and expressed using specifically designed primers (226 to Asn395) containing the two mutation sites; VAR2HP-noAsn, which was a mutant of VAR2HP with N312 deleted; and VAR2-Asn, which was a mutant of VAR2 with an inserted Asn in the position corresponding to N312 of VAR2HP (B–D and C). Binding assays showed that VAR2HP-T bound to no GAGs (B), indicating that the inserted fragment L292C293Y294T295D296K297L298E299L300 N301 and residue N312 together do not determine the binding capacity of VAR2HP to Hep. On the other hand, compared with VAR2HP, the mutant VAR2HP-NoAsn showed decreased binding to Hep and weak but significant binding to other GAGs (C), suggesting that the inserted N312 residue plays a key role in the selectivity of VAR2HP to Hep. However, the assay showing the binding of VAR2-Asn to GAGs indicated that the insertion of Asn312 alone did not meaningfully change the GAG-binding features of rVAR2, although a slight increase in binding activity to DS was observed (D). Taken together, these results suggested that the insertions of both fragment L292C293Y294T295D296K297L298E299L300 N301 and residue N312 resulted in a dramatic change in template rVAR2 GAG-binding properties and endowed the mutant VAR2HP with strict selectivity for Hep. Surface plasmon resonance showed that VAR2HP strongly interacts with Hep , although the affinity is slightly lower than that (KD ∼ 30 nM) with HepG2-GAGs (E and F).To further investigate the roles of two mutation sites . These results showed that decasaccharide is the essential motif with binding affinity for VAR2HP, similar to Hep polysaccharide.Although VAR2HP was selected by using HepG2-GAGs as targets, it is usually very difficult to prepare enough GAGs from tumor cells for detailed structural analyses. Therefore, as an alternative, commercially available Hep was used to investigate the structural characteristics of epitopes recognized by VAR2HP. First, a series of unsaturated even-numbered oligosaccharides from disaccharides to hexadecasaccharides (UDP2 to UDP16) with various disaccharide compositions  were preFigs. 4C and Fig. 5C), indicating the subtle influence of oligosaccharide structure on the interaction. Moreover, in contrast to the decasaccharides P10-1 to P10-3 with low activity levels, all the highly active decasaccharides (P10-4 to P10-11), contained at least three trisulfated disaccharides on average , was repurified through gel filtration, and the main fraction was preliminarily sequenced with an enzymatic method that we recently developed , which differed from the HS motif (GlcNS6S-IdoA2S)3 recognized by the scFv antibody NS4F5 . Similar results were observed in the binding assay of VAR2HP-Bio to HepG2 cells, human cervical adenocarcinoma HeLa cells and 4T1 murine mammary carcinoma cells (A–C). These results indicated that other cancer cells can also express Hep-like epitopes recognized by VAR2HP.To investigate whether the Hep-like epitopes recognized by VAR2HP are expressed on cells other than HepG2 cells, VAR2HP was first biotinylated (VAR2HP-Bio) as described previously . After vFigs. 5ma cells , A–C. ThB). In contrast, normal HEK 293T cells, TC-1 cells, HaCaT cells, and human colon adenocarcinoma HT29 cells were very weakly stained by VAR2HP-FITC , suggesting that they likely express very low levels of VAR2HP-targeted epitopes compared with the aforementioned highly stained cancer cells. Unsurprisingly, the staining of various cells by VAR2HP-FITC was strongly inhibited by pretreatment with Hepases but not with CSase ABC , confirming the expression of Hep-like epitopes on various cells.To quantitatively analyze the expression of these Hep-like epitopes on various cells, fluorescein isothiocyanate (FITC)-labeled VAR2HP (VAR2HP-FITC), which had been shown to retain selective binding activity to Hep , HuH-7 cells have the highest percentage (12.34%) and content (208.43 pmol) of HexA2S(1–4)GlcNS6S in 1 mg of dry cells. In contrast, HCC Li-7 cells express much lower amount of HexA2S(1–4)GlcNS6S (30.69 pmol) than HCC HuH-7 cells (208.43 pmol) and HepG2 cells (173.90 pmol) . Clearly, the amount of trisulfated HexA2S(1–4)GlcNAcS6S expressed on cells is one of important factors in determining the VAR2HP-binding capacity of cells. However, it should be noted that 4T1 cells express a lower content (28.23 pmol) and proportion (4.81%) of HexA2S(1–4)GlcNAcS6S than Li-7 cells  but show a higher VAR2HP-binding capacity, indicating that the content of HexA2S(1–4)GlcNAcS6S expressed on cells is not the only key factor for VAR2HP-binding. Undoubtedly, the most important thing is that these trisulfated disaccharides can efficiently form the VAR2HP-binding epitopes in the HS chains.Based on the structural properties of the Hep epitopes targeted by VAR2HP, we speculate that the content of HexA2S(1–4)GlcNS6S in HS chains on the cell surface is one of key factors for the binding of VAR2HP. Disaccharide composition analysis showed that HS chains extracted from all test cells contained HexA2S(1–4)GlcNS6S residues   and S6, C), further confirming that VAR2HP bound to HS/Hep but not to CS/DS chains on the cell surface.Finally, to investigate the localization of VAR2HP on cells, HuH-7 cells were stained with VAR2HP-Bio and visualized by confocal microscopy. The results showed that VAR2HP-Bio (green) clearly bound to the cell membrane, and the staining was eliminated by treatment with Hepases but not CSase ABC C, furthein vivo, the targeting of VAR2HP to organs in vivo was first investigated by injecting VAR2HP-FITC into mice through the tail vein. VAR2HP-FITC mainly targeted the intestine, stomach, and liver and could remain in these organs for at least 6 h (A), indicating that VAR2HP has good biocompatibility, which led to delayed immune clearance, similar to its template protein rVAR2 GlcNS6S disaccharides than the spleen, lung, or heart  and lung area (black circle) (C), which can likely be attributed to the targeting of VAR2HP-FITC to tumors in the lungs. To confirm this speculation, the lungs were evaluated by fluorescence imaging analysis, and tumor nodules (white arrow) showed much higher fluorescence intensity than the surrounding tissue (D). These results suggested that VAR2HP can be a promising carrier for selectively delivering drugs to tumors overexpressing Hep-like epitopes in vivo.To study whether VAR2HP targets tumors  circle) C, which g tissue D. These e.g., rVAR2), phage display, and screening using tumor-derived GAGs. The protein rVAR2, which is the ID1-DBL2X-ID2a region of VAR2CSA that mediates the binding of P. falciparum infected erythrocytes to placental syncytiotrophoblasts (C). Based on their sequences, VAR2HP is a mutant of rVAR2 with insertion of fragment L292C293Y294T295D296K297L298E299L300N301 and residue N312. Binding assays with various mutants of VAR2HP have shown that both the fragment L292C293Y294T295D296K297L298E299L300N301 and residue N312 play a key role but not the only role in the selectivity of VAR2HP for Hep (B–D). Previous studies have shown scFv antibodies interact with Hep/HS occurs via some basic residue-containing sequences, such as XBBXBX, XBBBXXBX, and XBBBXXBBBXXBBX, where ‘B’ is a basic residue and ‘X’ is a random amino acid residue .GAGs aberrantly expressed by tumor cells have the potential to be epitopes for the diagnosis and targeted therapy of cancers, and several scFv antibodies against GAGs with unique structures, such as HS20  and GD3Ghoblasts , has beehoblasts . As expehoblasts C. Based  for Hep , B–D. Pr residue . This ki residue  nor does residue , so the  residue . TherefoS3A, and  and in t in vivo , C and DC and D and Fig. 4C and S3A and The structural characteristics of epitopes recognized by VAR2HP were investigated by using oligosaccharides prepared from commercial Hep instead of rare HS extracted from cancer cells. The results have shown that the structure with similar affinity for VAR2HP as Hep polysaccharide is decasaccharides containing at least three HexA2S(1–4)GlcNS6S disaccharides , which suggests that Hep-like HS on the cell surface is involved in the interaction with VAR2HP. However, it is worth noting that the content (6.56%) of HexA2S(1–4)GlcNS6S in HS from HepG2 cells is much lower than that (65.34%) in Hep but the affinity (KD ∼ 30 nM) of VAR2HP with HepG2-GAGs is higher than that (KD ∼ 38 nM) with Hep (E and F). These results indicate that the VAR2HP epitopes on cancer cells have some unique structural features compared with Hep, which needs to be further investigated. Among the cells used in this study, HCC cells such as HuH-7 and HepG2 were strongly stained in vitro, and mouse liver was targeted in vivo by VAR2HP, indicating that HCC cells and normal liver tissues are rich in the VAR2HP epitopes (B and 6A). As an exception, HCC Li-7 cells were very weakly stained by VAR2HP, which may be due to the overexpression of CSPG CD44 on their cell surface rather than HSPG glypican-3 like other HCC cells . These properties of VAR2HP are very similar to the case of its parental template rVAR2 targeting cells with CS-A-like epitopes (A and S2C), the high selectivity of VAR2HP for Hep-like epitopes may limit its binding to more cell types but could increase its selectivity for cancer cells to reduce damage to normal cells in targeted therapy. In vivo, VAR2HP can target the liver, stomach, kidney, and intestine due to the relatively high content of HexA2S(1–4)GlcNS6S in their HS/Hep , indicating that some other factors may be involved in the binding or interfere with enzyme action in the intestine. Obviously, this organ-targeting issue will raise concerns that tumor-targeted therapy with VAR2HP may cause harm to these organs. In fact, since tumor antigens are always more or less expressed in normal tissues and organs, the side effects of tumor-targeted therapy are inevitable, but it can promote anticancer drug accumulation in tumor at reduced doses, thereby minimizing or avoiding undesired side effects , suggesting that VAR2HP has a greater affinity for the Hep-like epitopes overexpressed in tumors and has potential for targeted therapy.than 6 h A. These epitopes . Compareepitopes A and S2C effects , 50. In In conclusion, this study provides a common platform for purposefully screening and optimizing GAG-specific probes by choosing appropriate GAG targets and GAG-binding templates including scFv antibodies against GAGs, which are urgently needed for not only tumor targeted therapy but also other structural and functional studies of various GAGs.E. coli. DH5α cells, and E. coli. BL21(DE3) cells were purchased from Vazyme Biotech, Co Ltd. ProteinRuler II was purchased from TransGen Biotech, Inc. The expression plasmid pET-22b (+) was purchased from Invitrogen. The plasmid vector pComb3XSS was obtained from the Barbas Laboratory, TSRI. The VCSM13 helper phage and host bacteria E. coli. XL-blue cells were purchased from Bio-view Shine, Inc. CSase ABC (EC 4.2.2.4), Hepases I (EC 4.2.2.7), II (EC 4.2.2.-), III (EC 4.2.2.8), hyaluronan from streptococcus equi, CS-A from bovine trachea, CS-C from shark cartilage, avidin from egg whites, and FITC were purchased from Sigma-Aldrich, Inc. DS from porcine skin were obtained from Seikagaku Crop. D. gigas cartilage-derived CS-E was prepared in our laboratory  database as published , codon-oboratory . Porcineboratory . EZ-LinkHT29, 4T1, A549 cells were incubated in RPMI 1640 supplemented with 10% fetal bovine serum (FBS). HEK 293T, TC-1, HaCaT, HuH-7, HepG2, and HeLa cells were cultured in Dulbecco’s modified eagle medium supplemented with 10% FBS. Hep3B2.1-7 (Hep3B) cells were incubated in MEM supplemented with 10% FBS, 1% 100 mM L-Glutamine, 1% nonessential amino acid, and 1% sodium pyruvate. Li-7 cells were incubated in RPMI 1640 supplemented with 10% FBS, 1% 100 mM L-Glutamine, and 1% sodium pyruvate. Cell culture mediums were purchased from Sparkjade Scientific Instruments Co, Ltd. The Balb/c mice weighting 18.0 to 20.0 g were raised in comfortable environment, fed standard food, and allowed to drink freely. All animal experiments were approved by the Animal Care and Use Committee of Shandong University (Document number SYXK (LU) 2019005).HepG2-GAGs were extracted from HepG2 cells as described under “GAGCTCCATATGAATTACATCAAAG-3, R’: 5′-ACTAGTGTGGTGGTGGTGGTGGTGCTCGAGATCCAGTTTGCTGC-3′, the underline in primers were restriction endonuclease recognition sites) shown in E. coli XL-Blue cells. Phage helper VCSM13 infected E. coli XL-Blue cells to express recombinant plasmid which packaged of the pComb3XSS-mutVAR2 DNA and assembled of VCSM13 phage particles, yielding a library of infectious phages displaying the variant binding proteins. The library was added to the HepG2-GAGs-modified magnetic beads. The bound phages were eluted by PBS supplemented with 0.15 mM NaCl and then were used to produce more phages by re-infected E. coli XL-Blue cells with addition of helper phage VCSM13. The phages library was subjected to five rounds of above panning.To build the phage mutation library, the plasmid mutation library of VAR2CSA ID1-DBL2X-ID2a with 6∗his tag at C terminal was amplified by primer pairs and low-fidelity Taq polymerase (Vazyme Biotech). The primer pairs  using above enriched phages as templates. The sequences were constructed onto pET22b (+) vector (Novagen) and then transformed into E. coli BL21(DE3) cells. E. coli BL21(DE3) cells harboring different recombinant plasmids were selected by monoclonalization for following expression. Monoclonal bacteria were cultured in LB broth at 37 °C until cell density reached an A600 of 0.8 to 1.0 and then were added a final concentration of 50 μM isopropyl 1-thio-β-D-galactopyranoside to induce the expression of target protein. Cells were cultured at 16 °C for an additional 24 h and harvested by centrifugation at 8000g for 5 min. The cells were washed twice by ice-cold buffer A , resuspended in buffer A, and disrupted by sonication with 60 repetitions per 5 s in ice-water bath environment. The cell lysate was centrifugated at 12,000g for 30 min to collect the supernatant. The capacities of the supernatant-contained recombinant proteins binding to GAGs were evaluated by “Binding assay” as following mentioned. The genes of proteins with high binding activity to GAGs were sequenced by Sangon Biotech, and compared by BioEdit, version 5.0, to search the differences from that of the template rVAR2. The PI of the proteins were predicted by ExPASy Compute pI/Mw tool.The target sequences were amplified by primer pairs  and eluted with a gradient concentration of imidazole in buffer A ranging from 0 to 500 mM. The fractions containing target protein were further purified by HPLC on a Superdex G-200 column using 25 mM Tris-Cl buffer containing 100 mM NaCl (pH 7.4) at monitoring 280 nm. Then the target protein was concentrated by Amicon Ultra 0.5-ml 10K unit (Millipore). The molecular weight of the purified protein was analyzed by 13.2% SDS-PAGE under reducing conditions, based on the method of Sambrook and Russell , and the2HPO4-NaH2PO4, pH 6.0–8.0; 10 mM Tris-HCl, pH 7.0–10.0), VAR2HP concentration (0–4 μg/ml), blocking agents , and incubation time with target proteins (0–180 min), each well was coated with 0.4 μg avidin, blocked with 3% skim milk for 2 h, and then added 0.1 μg/well GAGs-Bio. After washed with PBS, the wells were added supernatant containing recombinant protein or purified recombinant protein (0.2 μg/well) in 3% skim milk for 2 h at room temperature and then rinsed three times with PBST (PBS supplemented with 0.05% tween-20) for removing nonspecific binding. Finally, the bound protein was detected by 6∗His Rabbit polyclone antibody, HRP-conjugated Goat anti-Rabbit (H + L), and 3,3′,5,5′-tetramethyl benzidine as a substrate. The wells were analyzed at 450 nm using microplate reader (BioTEK). Each experiment was performed in triplicates. The wells without GAG-Bio were used as the negative controls, and the wells with GAG-Bio but without adding recombinant proteins were used as the blanks.The binding assay was performed in 96-well plates according to previously published . After oTo determine the binding capacity of VAR2HP to Hep oligosaccharides from di- to hexadecasaccharides (UDP2-UDP16) , a compeThe binding kinetic analysis between VAR2HP/rVAR2 and Hep/HepG2-GAGs was performed by using a BIAcore T200 system . Hep and HepG2-GAGs purified as described above were immobilized on a senor chip as previously reported, respectively . BrieflyGAGs from cells were extracted as previously described  with sli2PO4 over 60 min at a flow rate of 1.0 ml/min at room temperature using a fluorescence detector with excitation and emission wavelengths of 330 and 420 nm, respectively. In the case of CS/DS disaccharide assay, CS/DS sample was digested with chondroitinase ABC (CSase ABC) followed by 2-aminobenzamide labeling and analyzed by anion exchange HPLC on a YMC Pack PA-G column as described above. The percentage of each component was calculated based on its peak area.Disaccharide compositions of various GAGs were analyzed by high HPLC as described previously . Briefly4HCO3 on HPLC detected at 232 nm, respectively, and then were freeze-dried three times to remove NH4HCO3. The disaccharide compositions of the partial digests and P10-7 were individually determined by Hepases digestion combined with HPLC analysis as described above, and the sequence of P10-7 was deduced based these results.Size-defined unsaturated Hep decasaccharide (UDP10) was further subfractionated using anion exchange HPLC on a Propac PA1 column with a gradient from 1 M to 1.5 M NaCl in 80 min by monitoring at 232 nm. The main peaks (P10-1 to P10-11) were collected and desalted through HPLC on a Superdex Peptide 10/300 GL column at monitoring at 232 nm. The purified components were performed competitive inhibition binding assay by “binding assay” to compare the binding ability with VAR2HP. Furthermore, a fraction P10-7, which has the lowest anion strength among the subfractions with similar binding ability to VAR2HP as Hep polysaccharide, was sequenced by an exo-type Hepase (BIexoHep) as reported previously . BrieflyIn this assay, VAR2HP was first biotinylated with EZ-Link Biotin-LC-Hydrazide using the method reported previously , and theTo further verify VAR2HP bind to Hep on the cell surface, the cells was treated with or without 5 mU CSase ABC or/and Hepases before added VAR2HP-Bio and then performed the cell binding assay.To conveniently detect the binding of VAR2HP to living cells by flow cytometry, according to product manual (Sigma) VAR2HP and BSA (as negative control) were labeled with FITC in a molar dye-to-protein (F/P) about 1.0, which was confirmed by analyzing the absorbances at 280 nm and 495 nm by NanoDrop (ThermoFisher). The selectivity of the FITC-labeled VAR2HP (VAR2HP-FITC) was determined by binding assay with a minor modification. Briefly, wells coated with various GAGs were incubated with 50 μl VAR2HP-FITC (0.01 μg/μl) for 30 min in the dark. After washing, the fluorescence intensities of wells were detected by a microplate reader (BioTEK) at excitation 480 nm and emission 520 nm.To detect and quantify the binding of VAR2HP to living cells, different types of cells were collected after detached with 0.02% EDTA in PBS solution and blocked by 1% BSA for 30 min at room temperature. Then, the cells were incubated with 100 μl VAR2HP-FITC or BSA-FITC (5 μg) in PBS, incubated on ice for 20 min, washed three times by PBS, and then analyzed on a FACS Cytometer (BD Biosciences). To investigate whether VAR2HP selectively binds to Hep/HS chains on cell surface, cells were pretreated with CSase ABC (5 mU) or Hepase  at 37 °C for 30 min before adding VAR2HP-FITC.To determine the fluorescence spectral characteristics of VAR2HP conjugated with fluorescent molecule FITC, emission wavelength scanning was performed for FITC (0.01 μg/ml) and VAR2HP-FITC (2 μg/ml) under excitation at 480 nm by using a fluorescence measurement .To visualize the binding of VAR2HP to cell surface, cells were seeded on poly-L-lysine–treated coverslips until cells covered 80% of coverslip surface and then immobilized with 4% paraformaldehyde, washed by PBS, and treated with or without CSase ABC (5 mU) or Hepases  at 37 °C for 30 min. The coverslips were blocked with 1% BSA in PBS for 2 h at room temperature and incubated with 200 μl VAR2HP-Bio (0.1 μg/μl) in PBS for 1.5 h. After washing three times with PBS, the bound VAR2HP and nucleus were stained with streptavidin protein (FITC) and DAPI, respectively, and visualized by a scanning laser microscope LSM700 (Car Zeiss Inc) with a Zeiss LSM image Browser.in vivo, VAR2HP-FITC (20 mg/kg) was injected into 6 weeks Balb/c female mice via tail vain. The mice were euthanized at time intervals ranging from 10 min to 8 h. BSA-FITC (20 mg/kg) was injected as negative group. The organs of the mice were removed and photographed by a plant living imagine system .To detect the biocompatibility and localization of VAR2HP in vivo, Hepases  or CSase ABC (200 mU/mouse) were injected into mice via tail veins with PBS as negative control. After 1 h, each mouse was injected VAR2HP-FITC at dose of 20 mg/kg. After another 30 min, the organs were taken out and photographed as above described.To further verify the selectivity of VAR2HP to HS/Hep in vivo. Tumor-bearing mouse were first prepared as reported previously (6 cells per mouse). After about 20 days, VAR2HP-FITC was injected to the tumor-bearing mice in vivo by tail veins at dose of 20 mg/Kg. PBS and BSA-FITC were injected as blank and negative controls, respectively. After 30 min, the mice were photographed using an IVIS Spectrum System and Living Image Software (PerkinElmer) to assess fluorescence intensity distribution of VAR2HP-FITC in tissue. Finally, the mice were euthanized, and their tumorigenic sites were photographed by a gel imaging system . Each experiment was done three times in parallel at least.To evaluate the targeting of VAR2HP-FITC to tumors eviously . BrieflyStatistical analyses were performed using Origin 8.0. Each experiment was done at least three times by triplicates and quintuplicates for cell binding experiments. For comparison of the statistical differences between tween groups, Student’s t-text (two-text) was carried out.All data supporting the findings of this study are available within the paper (and its Supplementary Information files). All relevant data generated during this study or analyzed in this published article (and its Supplement files) are available from the corresponding author on reasonable request. Source data are provided with this paper.This article contains The authors declare they have no conflicts of interest with the contents of this article.