NT5C2) and fucosyltransferase 8 (FUT8) genes were strongly associated with increased susceptibility to T2D. DNA hypermethylation in promoter regions reduced NT5C2 gene expression, but not FUT8 in T2D patients. NT5C2 protein expression was decreased in pancreatic β-cells from T2D mice. Transient transfection NT5C2 into RIN-m5F cells down-regulated DNA methyltransferase I (DNMT1) expression and up-regulation of the insulin receptor. Moreover, NT5C2 knockdown induced in DNMT1 overexpression and insulin receptor inhibition. Taken together, these results showed that NT5C2 epigenetically regulated insulin receptor in patients and mice with T2D, and maybe provide for T2D therapy strategy.Epigenetics alternation of non-genetic variation and genome-wide association study proven allelic variants may associate with insulin secretion in type 2 diabetes (T2D) development. We analyzed promoter DNA methylation array to evaluate the associated with increased susceptibility to T2D  and found 1,091 gene hypermethylated in promoter regions. We performed the association study of T2D and found 698 single nucleotide polymorphisms in exon and promoter sites by using 2,270 subjects . A comparison of DNA hypermethylation and gene silencing of mouse T2D results in our T2D patients’ results showed that the 5′-nucleotidase, cytosolic II ( T2D is characterized by hyperglycemia, pancreatic β-cell dysfunction, decreased insulin signaling action, and increased hepatic glucose formation5. In the pancreas, DNA methylation is involved in regulating de novo β-cell formation6. Previously studies have indicated that epigenetic factors conduce to the onset of T2D12. However, the mechanisms how DNA methyltransferase I (DNMT1) regulate T2D are unclear, and the roles of DNMT1 in T2D onset and development have not been clarified.The increasing prevalence of type 2 diabetes (T2D) is a global health problem. T2D can lead to various complications, including diabetic nephropathy, retinopathy, stroke, neuropathy, atherosclerosis, and hypertension, affecting patient quality of life and mortality. T2D is a disease with multifactor whose onset and development depend on not only genetic factors but also other factors, for example, epigenetics regulations. Epigenetic mechanisms may play an important role in T2D development13. DNA methylation, may be associated with regulation of the risk of many pathologies, such as T2D. DNA methylation can also affect the relationship between T2D and environmental exposure14.Investigation of the associations of DNA methylation in the peripheral blood may facilitate the identification of biomarkers for noninvasive early disease detection in epidemiological studiesEvaluation of epigenetics difference by Genome-wide DNA methylation arrays have become popular to assess disease progression. Currently, T2D onset and development biomarker identify from by epigenetics regulation still unclear in Taiwan Han Chinese. We using a DNA methylation array results to comparison with single nucleotide polymorphisms (SNPs) to looking for biomarker. Moreover, how DNMT1 affects the insulin signaling pathway, we examined epigenetic changes in patients with T2D, a mouse model of T2D, and in vitro transfections experiences. Our results provided the important role of DNA methylation in the T2D disease pathway.https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE81868). Analysis of DNA methylation status was carried out using model-based analysis of tiling-arrays (MAT) levels, found 1,091 genes were hypermethylation in promoter regions . The primary data are accessible via the GEO database, accession number GSE101879 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE101879). We found 260 genes showing DNA promoter hyper-methylation and gene silencing  and fucosyltransferase 8 (FUT8) genes which were associated with susceptibility to T2D not only SNPs but also DNA promoter hypermethylation in human T2D, and gene expression inhibition following by DNA methylation in T2D model mice Fig. . The pos18. Result showed that relative NT5C2 mRNA levels were lower in patients with T2D than in controls , normal weight (18.5 ≤ BMI < 24 kg/m2), overweight (24 ≤ BMI < 27 kg/m2), obesity (BMI ≥ 27 kg/m2). We analyzed those 698 SNPs associated with T2D among subjects with BMI ≧ 24. Results show that 291 SNPs, including rs12573221, were associated with T2D in the overweight subgroup   Table .www.ncbi.nlm.nih.gov/pubmed/28443625) and rs11191580 was associated with BMI in Asian population  (www.ncbi.nlm.nih.gov/pubmed/24861553). The identified SNP, rs12573221, was not highly linkage with those two SNPs (R2 < 0.2).We further analyzed the SNPs near the NT5C2 gene which is associated with BMI in Asian and European populations Table . The infWe used a mouse model of T2D to further elucidate the role of NT5C2 in T2D. In KK-Ay mice (42 weeks old) with late-stage T2D, insulin resistance was found to be associated with hypertrophy in pancreas islets and degranulation of β cells. Moreover, NT5C2 protein expression was inhibited in pancreatic, especially in β-cells, in T2D mice versus control mice Fig. .Figure 3The role of DNMT1 in modulating the effects of NT5C2 in the insulin signaling pathway is unclear. Therefore, we used an in vitro transient transfection model to analyze the effects of NT5C2 expression on insulin signaling. A plasmid containing human DNMT1 or NT5C2 was constructed and transiently transfected into RIN-m5F cells. The results indicated that DNMT1 overexpression caused NT5C2 and insulin receptor inhibition Fig. A, wherea15. Diabetic nephropathy and diabetic retinopathy include two major microvascular complications and occur with a high concordance rate in T2D. Previously study has suggested that these two complications might go through the same pathogeneses, such as glucose metabolism, angiogenesis, inflammation, and oxidative stress. The medications for T2D usually used oral hypoglycemic drugs and insulin injections. These complications may development for 5 years before the diagnosis of T2D16.There are one in three T2D patients with the microvascular complications at the time of diagnosis. The complications of T2D included cardiovascular disease, foot disease, retinopathy, and kidney disease21. In this study, we found that the NT5C2 and FUT8 genes were hypermethylated in both patients and mice with T2D, but only NT5C2 showed downregulation of mRNA expression in T2D patients. DNA methylation arrays have made better the study of DNA methylation, but little CpG methylation site are limited19. The role of epigenetics regulation in T2D pathogenesis needed to analysis, epigenetic regulations may predict to T2D onset and development21. Human, animal model, and in vitro T2D model are also needed, these models may explanation how epigenetic regulations affect T2D onset and development20.GWAS and DNA methylation array data can show markers that may explain onset and development of T2D population. Analysis of DNA methylation may also have uses as a diagnostic and therapeutic tool in patients with T2D21. DNMT1 was significantly expression higher in T2D than in controls18. DNMT1 with C allele single nucleotide polymorphism (rs78789647), promoter hypermethylated that affects insulin signaling pathway21. In this study, NT5C2 was regulated by DNMT1 and affected the insulin receptor. NT5C2 is an enzyme that dephosphorylate noncyclic nucleoside monophosphates to nucleoside and inorganic phosphate22. This enzyme is critical for control of energy balance, metabolic regulation, and cell replication23. NT5C2 is involved in maintaining cellular nucleotide and nucleoside levels by catalyzing the hydrolysis of AMP to adenosine, and hydrolysis IMP to inosine24. NT5C2 is expressed in skeletal muscle from tissue expression study23. NT5C2 in human myotubes, increases AMP-activated protein kinase (AMPK), acetyl-CoA carboxylase phosphorylation, and promotes lipid oxidation and glucose transport. The NT5C2 and AMPK activity in T2D and obesity may play an important role in the regulation of insulin action and lipid metabolism in skeletal muscle23.We reported that the DNMT1 was associated with susceptibility to T2D patients23. We checked the NT5C2 protein expression of the brain, heart, muscle, spleen, and testis by immunohistochemistry staining. Since the NT5C2 were low activity presentations in these tissues, there was no significant difference between KK and KK-Ay mice (data not showed). KK-Ay mice with the lethal yellow obese (Ay) mutation and develop diabetes of polygenic origin, showing severe obesity, hypertriglyceridemia, hyperglycemia, hyper-insulinemia, and insulin function loss by 42 weeks of age26. In our results, pancreatic β-cell mass was proliferation, and NT5C2 protein expression was inhibited in T2D mice’s pancreatic β-cell. Our previously results showed DNMT1 was significantly overexpression in T2D mice’s pancreatic β-cells21. Previously study have showed the importance of DNA methylation in pancreatic islet function10. In these studies, researchers identified the promoters of the insulin gene (INS)27; pancreatic and duodenal homeobox 1 (PDX1)28, which encodes a transcription factor important for both pancreatic development29 and the function of mature β-cells30; PPARG coactivator 1 alpha (PPARGC1A)31; and glucagon-like peptide 1 receptor (GLP1R)32, which stimulates insulin secretion and protects β-cell proliferation, as hypermethylated in islets from donors with T2D compared with those in islets from nondiabetic donors33. A hypermethylation was associated with reduced mRNA expression of the respective gene in pancreatic islets and with higher glycated HbA1c, suggesting the role in β-cell disturbs in T2D. High levels of glucose were also found to directly increase DNA methylation of Pdx1 and Ins in clonal β-cells28.NT5C2 is present in the brain, heart, skeletal muscle, erythrocytes, spleen, testis, fibroblasts, and endothelial cellsIn conclusion, the present study demonstrated the existence of a specific methylome map associated with genome-wide association studies in T2D. To the best of our knowledge, this study is the first to investigate the DNA methylation results and candidate genes from a combination of GWAS to determine NT5C2 gene is associated with T2D development.18, peripheral blood leukocytes were obtained from T2D cases and healthy control. DNA (50 ng) amplified by using a Whole Genome Amplification Kit , and 7.5 μg of reaction DNA was fragmented and labeled. Reaction was using the Affymetrix GeneChip Promoter 1.0R array. Arrays were stained with streptavidin–phycoerythrin, and scanned on an Affymetrix GeneChip Scanner 3000. The 40 samples  which pass QC according principal component analysis. The methylation regions of the T2D and the controls were compared using the Model-based Analysis of Tiling-array (MAT) calculation (P < 0.001)34.Genomic DNA extraction according to the manufacturer’s method  and our previously methods35 and were diagnosed according to the American Diabetic Association Criteria by using medical records and fasting plasma glucose levels. Ocular history data collected were from the questionnaires. For each patient, SBP, DBP and BMI were determined, and blood samples were collected by venipuncture for genomic DNA isolation and serological tests, including fasting glucose and HbA1c, at the time of participate the study at clinical office of CMUH. The healthy controls were selected from the Taiwan BioBank. All of the participating T2D cases and controls were of Han Chinese origin in Taiwan. Genomic DNA extracted from peripheral blood leukocytes with the Genomic DNA kit  was genotyped by using Affymetrix Axiom genome-wide TWB array according to standard quality control procedures. The information of SNPs on promoter region and exonic sites on the chip were extracted.A total of 2,270 subjects, including 560 T2D cases and 1,710 controls were used for GWAS in this study. T2D subjects were enrolled from China Medical University Hospital (CMUH)This study was approved by the Human Studies Committee of China Medical University Hospital (CMUH103-REC2-071) and informed consent was obtained from all participants. The study was conducted in accordance with the tenets of the Declaration of Helsinki.2. The healthy controls were comparable with respect to BMI, gender, age at study, and level of HbA1c. Total RNA was isolated from human blood using a High Pure RNA Isolation Kit  according to the manufacturer’s instructions. cDNA was synthesized from 1 µg total RNA using a High Capacity cDNA Reverse Transcription Kit  in a 20-µL reaction volume, according to the manufacturer’s instructions. cDNA was diluted to 10 ng/L, and 1 µL cDNA was used for each qRT-PCR assay in a final reaction volume of 10 µL. For quantification of gene expression with the ABI ViiA 7 Real-Time PCR System (Applied Biosystems), FastStart Universal SYBR Green Master mix (Roche) was used16. Primer sequences were as follows: NT5C2 sense, 5′-TGCAGCATCTTTCATCAACC-3′ and antisense, 5′-TGCTCCAC CGTTGATTCAT-3′; GAPDH sense, 5′-CAGCCTCAAGATCA TCAGCA-3′ and antisense, 5′-TGTGGTCATGAGTCCTTCCA-3′.There were 192 subjects were recruited from China Medical University Hospital, Taichung, Taiwan. The 94 subjects were diagnosed as T2D according to medical records and fasting plasma glucose levels using American Diabetic Association Criteria. The criteria for 98 healthy controls were no diagnostic history of T2D. The HbA1C values were lower than 6%, and BMI less than 32 kg/mAy/J) mice were obtained from Jackson Laboratories . Forty-two-week-old mice were used for analyses. Five mice were housed in one cage and fed lab chow ad libitum . The animal room was with controlled temperature from 22 to 25 °C, the humidity was 50 to 70%, and 12-h light and 12-h dark. The experiment according to the criteria for the care and use of KK and KK-Ay mice laid out in the ‘‘Guidebook for the Care and Use of Laboratory Animals’’36. This study was approved by the Institutional Animal Care and Use Committee (IACUC) of China Medical University (IACUC: 102-217).KK and KK-Ay  according to the manufacturer’s instructions. The mix was hybridized to a GeneChip Mouse Exon 1.0 ST Array (Affymetrix) in an oven overnight. Probe intensities were detected by using an Affymetrix GeneChip Scanner 3000 7G, and probe cell intensity data (CEL) were analyzed by using Affymetrix Expression Console software version 4.0 to generate CHP files with the Robust Multichip Analysis (RMA)-sketch algorithm processes. The transcript structure levels for gene and exon level analyses, which restricts analysis to exon-level probe sets that map to BLAT sequences of mRNAs with noted coding sequence regions. Genes and exons expression difference were identified by Transcriptome Analysis Console 2.0.0.9 software (Affymetrix).Total RNA was isolated from mice liver tissue using the RNA isolation kit according to the manufacturer’s instructions  as our previously methods2O2 solution for 30 min. Then the slices were boiling in 0.01 M citrate buffer for 20 min for antigen retrieval, and washed in 50 mM Tris-HCl (pH 7.6) with 0.05% Tween for 2 min. Slices were incubated with 5% nonfat dry milk for 30 min for block nonspecific binding. The slides were then hybrid with anti-NT5C2 antibodies  for 1 h, then incubated with secondary antibody, and incubated with a peroxidase-labeled streptavidin–biotin complex and diaminobenzidine substrate to demonstrate the NT5C2 labeled cells.For paraffin tissue slice deparaffinization and rehydration, the slices were incubated in 3% H16. Total protein was isolated from the cells after 48 h reaction.RIN-m5F rat pancreatic β-cells were purchased from Food Industry Research and Development Institute . Cells were seeded at 150,000 cells/well in six-well culture plates and incubated until the culture reached 50–80% confluence. Cells were then transfected with the empty pCMV3-untagged vector, pCMV3-DNMT1 construct, or pCMV3-NT5C2 construct. Lentiviral expression system for NT5C2 or DNMT1 shRNA  using Xfect Transfection Reagent , according to the manufacturer’s instructions and methodsg for 30 min at − 20 °C. The supernatant was used for western blotting. Proteins sample of the supernatant were used by SDS PAGE using 10% acrylamide gels and transferred to PVDF membranes. PVDF membranes were cut according to molecular weight and reacted with antibodies separately. The samples drive from the same experiment and the blots were processed in parallel. Membranes were incubated in 5% skill milk with 0.1% Tween-20 in Tris-buffered saline, followed by incubation with rabbit anti-NT5C2 , anti-DNMT1 , anti-insulin receptor , or anti-GAPDH  polyclonal primary antibodies. Membranes were then incubated with HRP-conjugated goat anti-rabbit IgG  secondary antibodies. Proteins were observed using SuperSignal West Pico Chemiluminescent Substrate or SuperSignal West Femto Chemiluminescent Substrate  according our previously published methods16. The numbers in the figure indicated the Image J results of protein expression versus vector control.RIN-m5F cells were homogenized in ice-cold RIPA lysis buffer , protease inhibitor, and phosphatase inhibitor . The homogenate was incubated on ice for 1 h at 4 °C and centrifuged at 12,000×37. The significance level was set at P value less than 0.05.SPSS software 15.0 for windows  were used for data statistically analysis. The result values were showed as means ± standard deviations. The Kolmogorov–Smirnov test was used for the normality of the data. Pearson correlation matrix used for hierarchical gene analysis and heat maps for genetic association. According to the probability distribution pattern and the total number of cases and control, parametric or nonparametric were used for tests. Subgroup analyses were done by obesity level (BMI level) of subjectsSupplementary Table S1.Supplementary Table S2.Supplementary Figures and Table S3.Supplementary Table S4.


Alternative splicing is one of the most regulated processes of eukaryotic gene expression. From one gene, multiple mRNA isoforms are produced by alternative splicing decision. Alternative splicing of the 9 variable exons of human CD44 pre-mRNA produces various mRNAs that are involved in different aspects of cancer progression and development. Here we identify Tra2β and SRSF9 as regulatory proteins with opposite roles in CD44 exon v10 splicing. While Tra2β promotes v10 inclusion through interacting with GAAGACG sequence in v10, SRSF9 inhibits v10 inclusion by binding to AAGAC. Our results provide a novel insight into the regulation of CD44 splicing.CD44 is a transmembrane glycoprotein involved in cell–cell and cell–matrix interactions. Several CD44 protein isoforms are generated in human through alternative splicing regulation of nine variable exons encoding for the extracellular juxta-membrane region. While the CD44 splicing variants have been described to be involved in cancer progression and development, the regulatory mechanism(s) underlying their production remain unclear. Here, we identify Tra2β and SRSF9 as proteins with opposite roles in regulating CD44 exon v10 splicing. While Tra2β promotes v10 inclusion, SRSF9 inhibits its inclusion. Mechanistically, we found that both proteins are able to target v10 exon, with GAAGAAG sequence being the binding site for Tra2β and AAGAC that for SRSF9. Collectively, our data add a novel layer of complexity to the sequential series of events involved in the regulation of CD44 splicing. Pre-mRNA splicing is a molecular process by which a newly made precursor messenger RNA (pre-mRNA) transcript is transformed into a mature messenger RNA (mRNA). In this maturation step of RNA, introns are removed and the exons are ligated to form mRNA molecules . SplicinCD44 is a family of non-kinase transmembrane glycoproteins expressed in connective tissues, bone marrow, and embryonic stem cells . Human CAlternative splicing is controlled by a large group of ubiquitously expressed splicing regulators characterized by the presence of an arginine–serine (RS)-rich domain  . Tra2β iIn this study, we applied a minigene-based approach to determine the opposite roles of Tra2β and SRSF9 on CD44 v10 splicing. In addition, both proteins functionally target v10 exon, with Tra2β targeting the GAAGAAG sequence and SRSF9 targeting the AAGAC sequence located at CD44 exon v10. Thus, the ability of these RNA sequences to interact with Tra2β or SRSF9 proteins and the ability of these proteins to be the functional regulator of CD44 exon v10 splicing are well correlated. Collectively, our results provided new regulatory mechanisms for v10 exon of CD44.2 incubator at 37 °C in Dulbecco’s Modified Eagle Medium (DMEM) and Roswell Park Memorial Institute Medium (RPMI) medium containing 10% fetal bovine serum , 2 mM glutamine, 100 iU/mL penicillin, and 100 μg streptomycin, as described previously [HEK293T and HCT116 cells were grown in a 5% COeviously . Polyetheviously . An amouF/TR and SF/SR). All primer sequences used to generate these plasmids are listed in CD44 v10 minigenes were described previously , whereasRNA pulldown analysis was performed, as described previously . BrieflyCells were incubated with cell lysis buffer  at 4 °C for 30 min. Proteins were separated with 12% SDS-PAGE gel followed by transferring to nitrocellulose membrane. Tra2β, SRSF9, and α-tubulin were detected using anti-Tra2β , anti-SRSF9 , and anti-α-tubulin  antibodies, respectively.http://www.introni.it/spliceaid.html) [http://rbpmap.technion.ac.il) [Tra2β putative RNA-binding motifs prediction was performed using SpliceAid 2 (id.html) . Human Cn.ac.il) , with thn.ac.il) , are shop < 0.0001, *** p < 0.001, ** p < 0.01, and * p < 0.05.RT-PCR, immunoblotting, and immunoprecipitation experiments were performed in triplicate. RT-PCR products were loaded into agarose gels followed by staining with Ethidium Bromide (EtBr). Image J program was used to quantify the bands. Data are presented as means ± SD (standard deviation of the mean). Statistical differences between three groups were determined using one-way ANOVA. Statistical significance was marked as **** As shown in http://www.introni.it/spliceaid) [http://rbpmap.technion.ac.il) [We have previously demonstrated that hnRNP L inhibits CD44 v10 splicing by binding an evolutionally conserved CA-rich sequence within intron 9 . In ordeliceaid)  and RBPmn.ac.il)  bioinforn.ac.il) . In partn.ac.il) , 0.5–1.5n.ac.il) , and in n.ac.il) . Addition.ac.il) ), we fouhttps://portals.broadinstitute.org/ccle), we found that HCT116 cells express high endogenous Tra2β and SRSF9  and HCT116 cells  and inhibited v10 skipping, accordingly. Opposite to the function of Tra2β, we found that SRSF9 inhibited v10 exon inclusion significantly in both HEK293T  and HCT116 cells . However, in both experiments, control plasmid did not affect v10 exon splicing A,B. Thesnd SRSF9 . Similarnd SRSF9 C. These p-value: 4.77 × 10−4) in RBPmap prediction; (iii) identified by both SpliceAid2 and RBPmap tools. To test the possibility that Tra2β could affect v10 splicing through interacting with this sequence, we mutated the GAAGAAG  sequence into GGAGUAG  sequence in the CD44 v10 minigene (Because Tra2β is an RBP, we next sought to determine functional target sequences in CD44 v10 exon or its flanking introns. Among the predicted Tra2β binding motifs C, we focminigene A. If thiWe next wondered the relationship between binding ability of RNA motifs and its promoting activity of v10 exon. To analyze physical interaction between Tra2β and GAAGAAG, we synthesized biotin-labeled wild-type and M-Tra2β RNA to perform an RNA-pulldown assay using streptavidin followed by immunoblotting with anti-Tra2β antibody (We next asked whether SRSF9 could also target the regulatory function of v10 splicing. From eight potential binding motifs shown in 3 kinds of various mRNA alternative splicing isoforms [Different from regular skipped exons, which include only one alternative exon, human CD44 pre-mRNA contains 10 various exons instead. Thus, CD44 pre-mRNA is able to produce ~10isoforms . In the Splicing regulatory mechanisms of various exon other than v10 in CD44 have been studied previously ,44,46,47We were not able to detect the effects of Tra2β and SRSF9 on endogenous v10 splicing. shRNA effects are not always opposite to the overexpression results. The difference might be caused by the differences of experimental designs: overexpression or shRNA, minigene, or endogenous. It is also possible that, in the minigene, v10 exon is linked to C5 and C6 exons, while endogenous v10 exon is linked to v9 and C6. Another possibility is that other various exons also play important roles in the v10 selection by overexpression or shRNA. How these exons function each other needs to be determined.Tra2β and SRSF9 regulate alternative CD44 v10 splicing through interacting with GAAGAAG and AAGAC independently.


Seeking to improve the access to regenerative medicine, this study investigated the structural and transcriptional effects of storage temperature on human oral mucosal epithelial cells (OMECs).Cells were stored at four different temperatures  for two weeks. Then, the morphology, cell viability and differential gene expression were examined using light and scanning electron microscopy, trypan blue exclusion test and TaqMan gene expression array cards, respectively.GADD45B) and cell proliferation inhibition (TGFB2) were upregulated at 12°C and 24°C. Upregulation was also observed in multifunctional genes responsible for morphology, growth, adhesion and motility such as EFEMP1 (12°C) and EPHA4 (4°C–24°C). Among genes used as differentiation markers, PPARA and TP53  were downregulated in all temperature conditions, whereas KRT1 and KRT10 were either unchanged (4°C) or downregulated , except for upregulation at 12°C for KRT1.Cells stored at 4°C had the most similar morphology to non-stored controls with the highest viability rate (58%), whereas the 37°C group was most dissimilar with no living cells. The genes involved in stress-induced growth arrest (Cells stored at 12°C and 24°C were stressed, although the expression levels of some adhesion-, growth- and apoptosis-related genes were favourable. Collectively, this study suggests that 4°C is the optimal storage temperature for maintenance of structure, viability and function of OMECs after two weeks. Limbal stem cell deficiency (LSCD) is a potentially painful and blinding condition caused by damage or loss of the limbal stem cells , 2. CellFor treatment of unilateral LSCD, the use of cultured autologous limbal stem cells from the healthy cornea is often the method of choice. In the event of bilateral LSCD, which is more prevalent, the application of allogeneic limbal stem cells remains an option, but is considered less favourable because it requires systemic immunosuppressive drugs with potential adverse effects and the long-term outcome is not always satisfactory. Therefore, the quest for alternative cell types was initiated. Oral mucosal epithelial cells (OMECs) were the first alternative autologous source to be studied in both rabbits  and humaThe prepared epithelial stem cells in vitro may not be used immediately for transplantation due to patient readiness and the time required for transport from laboratory units to clinics. Such a gap gives the opportunity for phenotypic investigations , steriliCryopreservation and refrigeration (4°C) are two common storage methods, especially for cultured epidermal cells. However, both suffer from the drawback of low cell viability –31. StudThe present study aimed to examine ten significantly differentially expressed genes among the studied groups by Utheim et al. , and 20 Local ethical approval and verbal informed consent were obtained before OMEC harvesting. According to guidelines, the relevant form was completed and signed by the specialist during interview with the next of kin before transferring cadaver to the Department of Pathology, Oslo University Hospital. The research was conducted in accordance with the Declaration of Helsinki. The experimental protocols were approved by The Regional Committee for Medical and Health Research Ethics, Section C, South East Norway (reference: 2017/418).The defined proprietary culture medium, CNT-Prime, was purchased from Cellntec Advanced Cell Systems AG . Dulbecco’s Modified Eagle Medium/Nutrient F-12 Ham + GlutaMAX™-I (DMEM/F12) was bought from Invitrogen Life Technologies . Membrane inserts (Transwell cat. no. 3450) and 48-well non-tissue culture polystyrene plates  were from Corning Costar  and Becton Dickinson Labware , respectively. The rest were obtained from Sigma Aldrich .2+ and Ca2+-free Hanks’ balanced salt solution containing 1.2 U/mL dispase II at 37°C overnight. The separation of epithelial cell layer from the lamina propria layer was performed under a dissecting microscope by sterile forceps and scalpel. Afterwards, the tissue was rinsed with DMEM/F12, cut into explants (1–3 mm2), put on plastic inserts and allowed to attach in DMEM/F12 supplemented with 10% fetal bovine serum and P/S. Subsequently, the cultured cells were incubated at 37°C (5% humidified CO2) for two weeks. Then, the expanded cells were harvested (0.25% trypsin-EDTA solution) as passage 1 (P1) cells.Donated lower lip biopsy was harvested from cadaver’s oral cavity within 24 h post mortem in a non-refrigerated setting. The cadaver was a 56-year-old male with heart disease and good oral hygiene. The biopsy was transported to tissue-culture laboratory within 1 h in Dulbecco’s Modified Eagle Medium/Nutrient F-12 Ham + GlutaMAX™-I (DMEM/F12) supplemented with 100 U/mL penicillin-streptomycin (P/S) at room temperature and then rinsed off using the same medium. It was cut into 1 cm × 0.5 cm pieces and then incubated in Mg2. The culture medium was changed every 2–3 days.Trypan blue exclusion test was used for P1 cell counting and viability assessment before being frozen in 5% DMSO at -80°C overnight followed by liquid nitrogen storage. The OMECs were seeded on culture vessels in serum-free CNT-Prime medium supplemented with P/S on a 48-well-plate. All the cultures were incubated at 37°C and 5% CO2 for 5–7 days. The culture medium was changed every 2–3 days. Then, cells were trypsinized and cultured on 24-well plates with (for scanning electron microscopy) or without (for other tests) 8 mm round glass coverslips to obtain a confluent monolayer under the same condition. Before random selection for storage in cabinets at four different temperatures , the storage medium containing MEM with 12.5 mM HEPES, 3.57 mM sodium bicarbonate and 50 mg/ml gentamycin was replaced and the 24-well plates were properly sealed.The frozen OMECs were thawed, seeded and expanded on culture flasks in serum-free CNT-Prime medium supplemented with P/S at 37°C with 5% COOral mucosa epithelial cells were isolated from a single donor and cultured on uncoated plastic plates to minimize confounding variation, focus on temperature-associated differences and limit variation of scaffolds in the form of biological substrates such as amniotic membrane and fibrin . Three tThe morphology of individual cells and the integrity of the complete cell layer in all four different temperature conditions were evaluated by a Leica DMIL inverted phase contrast microscope  coupled with a Canon EOS 5D mark II camera . The images were captured at random positions within each well at 4×, 10×, 20× and 40× magnification before (baseline) and after two weeks incubation time .Immediately after the incubation period, fixative  pre-adjusted to desired temperature was added in a 1:1 ratio to each well. Afterwards, the plates were returned to their respective storage cabinets for 15 min. Then, fixed cells on each coverslip were dehydrated using a graded ethanol series consisting of a 10-min treatment with 70, 80, 90, and 96% ethanol, followed by 4 x 15-min treatments with 100% ethanol. Samples were then critical point dried  and coverslips were mounted on SEM stubs using carbon tape. Finally, samples were sputter coated with 6 nm platinum using a Cressington 308R coating system, then all samples were examined and digital images recorded using a Hitachi S-4800 Field Emission Scanning Electron Microscope operated at 5.0 kV.The viability of cultured cells was evaluated by the trypan blue exclusion test .The cells were harvested directly in RLT buffer  and mixed by pipetting up and down. The lysate was then passed through a needle (0.9-mm diameter) attached to a sterile plastic syringe 10 times for achieving a homogenous lysate. RNeasy micro kit  was used for extraction and purification of total RNA. Concentration and purity of isolated RNA were assessed by spectrophotometry . Using the High Capacity cDNA RT Kit , reverse transcription (RT) was performed with 200 ng of total RNA per 20 μL RT reaction based on the manufacturer’s instruction.CCNH, CDKN2B, GADD45B, GAS5, HDAC6, HIST2H4B, HUS1, RPSA, TBRG4 and TGFB2) among the studied groups were selected, accordingly. In addition, 19 genes from various relevant pathways were included. GAPDH was also included to evaluate whether energy consumption originated from oxidative phosphorylation or glycolysis.Comparative relative quantification was performed using the TaqMan Gene Expression Array Card for 30 genes . The choThe samples were prepared for PCR by mixing 55 μL of cDNA with 55 μL of TaqMan™ Universal PCR Master Mix, no AmpErase™ UNG . Then, each fill reservoir of a TaqMan Array Card  were loaded with 100 μL of sample-specific PCR reaction mix. Three wells were allocated per gene in this experiment. The array cards were run in the QuantStudioTM 12K Flex Real-time PCR system  under the following conditions: 50°C for 2 min, 95°C for 10 min, then 40 cycles of 95°C for 15s and 60°C for 60s. All samples were run in triplicates.ΔNp63α, TJP1, CLND1 and OCLN)  system (Applied Biosystems) and Taqman Gene Expression assays following protocols from the manufacturer (Applied Biosystems) for 4 genes (nd OCLN) . All the©, Relative Expression Software Tool, Weihenstephan, Germany). REST implements the Pair Wise Fixed Reallocation Randomization Test© to investigate the significance of changes in gene expression [p value of ≤ 0.05 was considered to be significant. The data are presented as the mean ± standard error.The analysis of gene expression data was performed using the Relative Expression Software Tool .The viability of OMECs incubated for two weeks was decreased with increasing storage temperature compared to non-stored baseline control cells . The 4°CCDKN1A, TBRG4, PPARA and TP53) were downregulated in all four different temperature conditions when compared to non-stored baseline control (p ≤ 0.05). Four other genes were also downregulated at 4°C and 37°C, with upregulation at 12°C (EFEMP1), 24°C (CCNH and CASP4), or both 12°C and 24°C (GADD45B). Similarly, significant downregulation for all 4 temperature conditions, excluding insignificant changes at 4°C  and 12°C (RPSA and IKBKE), were also observed. In contrast, EPHA4 showed significant upregulation at 4°C, 12°C and 24°C. Six genes  presented a combination of significant (both downregulation and upregulation) as well as insignificant changes in expression in studied groups of each different temperature conditions.This study considered a log2-fold change significant at thresholds above 1 and below -1 . Moreover, this approach was only applied to 4°C, 12°C and 24°C groups as all cells died upon storage at 37°C (Supplementary). Thus, altered expression at this temperature was deemed irrelevant. We identified 18 differentially expressed genes upon storage using the TaqMan Gene Expression Array . Four ofΔNp63α, TJP1 and CLND1 were significantly upregulated at 4°C but OCLN remained unchanged. At 12°C, three of genes  were significantly downregulated, except unchanged TJP1. At 24°C, significant downregulation was observed for ΔNp63α and TJP1, whereas upregulation for CLND1 and OCLN.Results from RT-qPCR  indicateTo establish optimal storage conditions for OMECs to maximize transplantation success and extend access to regenerative medicine, this study aimed to assess the morphology, cell viability and expression of selected genes of cultured OMECs stored at four different temperatures  for two weeks. The overall morphology and ultrastructure of cells stored at 4°C was most similar to non-stored baseline controls, whereas the 37°C group was the most dissimilar ones with only dead cells. The presence of cell separation between some adjacent cells at 4°C group and prominent morphological deformation, shrinkage, and membrane blebbing at higher temperatures in our study were also observed by others, Islam et al  and JackCells stored at 4°C had the highest viability rate (58%) compared to other three experimental groups with higher temperatures, whereas the 37°C group presented no living cells. The former is in the viability range of suspended human oral keratinocyte (~63%) in DMEM, assessed using trypan blue exclusion test, after three days storage at 4°C  showed tOther cell types have also been studied for the effect of storage temperature. For example, human retinal pigment epithelium , epidermGAPDH, an important enzyme for energy metabolism, was affected by temperature. Significant GAPDH downregulation was only observed at 4°C (excluding 37°C with no viability). Similar response has also been demonstrated for corneal cultures [GAPDH is widely used as a housekeeping gene for RT-qPCR or loading control for western blot. Therefore, researchers should be mindful in selecting appropriate endogenous controls in molecular research associated with temperature. In the present study, ERBB3 was used as reference gene because its expression level was unaffected by experimental factors.Our gene analysis revealed that cultures  and oralcultures . Based ocultures . The temcultures . Additiocultures , 44–46. cultures  suggesteHIST1H4B, one of the histone cluster genes, was highly upregulated at 4°C. HIST2H4A/B, another member of the same family, was not significantly different at 4°C when compared to non-stored baseline control. In accordance with our finding, Raeder et al. [HIST1H4B in culture of human limbal epithelial cells stored at 4°C for 2–7 days. Low temperature may induce expression of this histone H4 encoding gene in order to acclimatize the cells to hypothermia by stabilizing nucleosomes and repressing transcription, even after 2 weeks storage.Histones are positively charged basic nuclear proteins that play important structural and functional roles in the transition between active and inactive chromatin states through compacting DNA . Hence, r et al. , have reEFEMP1 was upregulated at 12°C. As a member of the fibulin family of extracellular glycoproteins, it is associated with multiple structural and functional roles including morphology, growth, adhesion and motility [RPSA, another multifunctional gene , remained unchanged at 12°C but was downregulated at all other temperature conditions. Finally, EPHA4 was significantly upregulated at 4°C, 12°C and 24°C. Its key functions are the maintenance of the basement membrane and the integrity of the extracellular matrix [PPARA and TP53 were downregulated at all four temperature conditions. CDKN1A, alias p21, responded in the same fashion. This gene has been previously shown to be under the transcriptional control of p53 [KRT1 and KRT10, were either unchanged (4°C) or downregulated , except an upregulation at 12°C for KRT1. The 37°C group was not evaluated due to a majority of dead cells. The expression pattern of differentiation markers following storage have been previously reported by our group in other cultured epithelial cells [The effect of temperature on stored OMECs was also observed in the expression of genes involved in cell adhesion, migration, motility and differentiation. motility . RPSA, ar matrix . Among gl of p53 . Two othal cells , 38, 53.al cells  presenteΔNp63α, a stemness marker for OMECs [ΔNp63α expression in transplantation of cultured LSC sheets [ΔNp63α. Another important factor in transplantation is the maintenance of barrier function of cultured sheet [TJP1  on cell’s plasma membrane face for anchoring them to the actin cytoskeleton [TJP1 and CLND1) or unchanged (OCLN) at 4°C. Additionally, the culture of OMECs presented upregulation of CLND1 and OCLN at 24°C. Although 37°C led to highest expression level of tight junction-associated genes, storage of OMECs for two weeks is disadvantageous due to the dominance of dead cells, downregulation of stemness gene (ΔNp63α) and overexpression of keratinization markers (KRT1 and KRT10).The expression level of or OMECs , 55, wasC sheets . The sated sheet , which ied sheet . For exaskeleton . These mskeleton . Our stuGADD45B, stress-induced growth arrest gene, is indicative of the high level of stress imposed on OMECs after two weeks of storage at 12°C and 24°C. GADD45B is considered a crucial factor in DNA repair, cell survival, growth arrest, apoptosis and DNA demethylation [TGFB2 showed upregulated levels at the same storage temperatures, whereas CCNH was only upregulated at 12°C. TGFB2 belongs to a group of proteins that regulate several cellular functions, especially inhibition of cell proliferation through cell cycle arrest in G1 phase [CCNH) [TGFB2 [The upregulation of hylation –61. SimiG1 phase , 63. Itse [CCNH) , 65. The) [TGFB2 , 67.TAF1D, a histone-relevant transcriptional regulation gene, at 4°C and 24°C. It is in contrast to downregulation of TBRG4, also termed cell cycle progression restoration protein 2, in all four experimental groups. TAF1D, the largest subunit of TFIID, is an essential component of transcription machinery which acts through harboring bromodomains and histone acetyltransferase activity [TAF1D and genotoxic/oxidative stress-induced apoptosis [GADD45B) at 24°C. Moreover, the apoptosis-associated gene CASP4 was also upregulated at 24°C, in contrast to other downregulated groups. This was supported by our photomicrographs illustrating cells with membrane blebbing, indicative of apoptosis [One unanticipated finding was the upregulation of activity , 69. It poptosis . In thispoptosis . Howeverpoptosis  and epitpoptosis .https://david.ncifcrf.gov/) to test for over-representation in functional pathways. Unsurprisingly, Cell Cycle came out as the most significantly affected in the KEGG PATHWAY Database, and the only relevant, sustaining significance after adjustment for multiple testing by the Benjamini–Hochberg method [p = 3.4E-3, and the 5 genes: CCNH, CDKN1A, GADD45B, TGFB2 and TP53). If more experiments had been performed, the statistical power would increase, and we would probably find more affected genes as well as signaling pathways giving further insight in the cellular processes affected upon storage.The list of 22 regulated genes was subjected to DAVID Functional Annotation Tool , at the expense of increasing the likelihood of including false positives . Another limitation was the use of cells from a single donor, at the possible risk of systemic factors’ influence  on extrapolation of the results [Our study had some limitations. We decided not to adjust  results –75.The results of morphology, cell viability and gene expression assays in the current study showed that 4°C is the most appropriate temperature to store OMECs for up two weeks. Similarly, Utheim et al  found thGADD45B) and cell proliferation inhibition genes (TGFB2). However, some genes involved in cell adhesion, migration, motility and differentiation maintained transcription levels beneficial for cultured epithelial cells at 4°C–24°C. Taken together, these results suggest that storing OMECs at 4°C for two-week is the most appropriate temperature with the highest viability rate and morphology rather similar to pre-stored OME cells. Therefore, it is most likely to be the best storage conditions for clinical transplantation.This study found that the morphology of cultured human OMECs stored at 4°C for two weeks was most similar to non-stored baseline controls. Additionally, cells stored at 4°C had the highest viability rate (58%) when compared to other temperatures experimental groups. Gene analysis also revealed some levels of interruption at 12°C and/or 24°C through e.g., stress-induced growth arrest . This study considered a log2-fold change significant at thresholds over 1 and under -1 . Additionally, this approach was only applied to 4°C, 12°C and 24°C groups as all cells died upon storage at 37°C.(XLSX)Click here for additional data file.S2 TableAmong 30 studied genes, 12 were omitted (non-bolded genes). This study considered a log2-fold change significant at thresholds over 1 and under -1 . Additionally, this approach was only applied to 4°C, 12°C and 24°C groups as all cells died upon storage at 37°C.(XLSX)Click here for additional data file.


Both onco-suppressor PREP1 and the oncogene MEIS1 bind to PBX1. This interaction stabilizes the two proteins and allows their translocation into the nucleus and thus their transcriptional activity. Here, we have combined cross-linking mass-spectrometry and systematic mutagenesis to detail the binding geometry of the PBX1-PREP1 (and PBX1-MEIS1) complexes, under native in vivo conditions. The data confirm the existence of two distinct interaction sites within the PBC domain of PBX1 and unravel differences among the highly similar binding sites of MEIS1 and PREP1. The HR2 domain has a fundamental role in binding the PBC-B domain of PBX1 in both PREP1 and MEIS1. The HR1 domain of MEIS1, however, seem to play a less stringent role in PBX1 interaction with respect to that of PREP1. This difference is also reflected by the different binding affinity of the two proteins to PBX1. Although partial, this analysis provides for the first time some ideas on the tertiary structure of the complexes not available before. Moreover, the extensive mutagenic analysis of PREP1 identifies the role of individual hydrophobic HR1 and HR2 residues, both in vitro and in vivo. PREP1 or MEIS1 dimerize with PBX1 and direct its DNA binding to specific and only partially overlapping sequences8. Unveiling the PBX-interaction surface of PREP or MEIS would allow not only to better understand their molecular function but would also represent the basis for searching inactivating therapeutic molecules.PREP1 , and MEIS1 (Myeloid ecotropic insertion site 1) are transcription factors belonging to the three amino acids loop extension (TALE) homeodomain family10 and in the adult functions as a tumour-suppressor11. The tumour-suppressor role of PREP1 is associated with the maintenance of genomic stability12, control of DNA replication timing13, and protection of the nuclear envelope structure . Prep1 hypomorphic (Prep1i/i) mouse fibroblasts accumulate DNA damage, chromosomal abnormalities and display increased basal and genotoxicity-induced apoptosis12. Indeed, down-regulation of PREP1 induces DNA damage and consents oncogenes like Ras or Meis1 to evade senescence and transform12. Prep1i/i mice develop spontaneous tumours also at the heterozygous state and Prep1 haplo-insufficient mice accelerate the development of oncogenes-dependent tumours14. Human tumours are low in PREP115, while PREP1 overexpression slows-down the growth of tumour cells in vitro and in vivo4. Because of these properties PREP1 is considered a tumour suppressor. In particular, overexpression of PREP1 inhibits the growth of MEIS1-dependent tumour cells, competing in vivo for the MEIS1 DNA-binding sites5. All known functions of PREP1 depend on the association with PBX.PREP1 is essential in embryonic development17, and is involved in leukemogenesis in children and adults. When overexpressed, MEIS1 is able to transform tumour suppressor-deficient mouse cells in the absence of other oncogenes4 and to accelerate the occurrence of HoxA9-dependent Acute Myeloid Leukemia (AML)20. In human, the overexpression of MEIS1 is critical in Mixed Lineage Leukemia (MLL) caused by the fusion of the MLL to several other genes19. Thus MEIS1 inhibition represents a potentially good therapeutic target for MLL treatment22. MEIS1 overexpression has been also implicated in promotion of cell proliferation, resistance to chemotherapy of leukemic cells23 and in skin cancer24. In ovarian cancer25 and in neuroblastomas26 MEIS1 is appreciably overexpressed compared to other cancer types.MEIS1 has a crucial role in embryonic hematopoiesis4, which thus acts as an oncogene or as a tumour-suppressor, depending on its partner, MEIS1 or PREP1. In fact, PREP1 and MEIS1 compete molecularly and functionally5. PREP1 and MEIS1 bind PBX1 in the cytoplasm, therefore in the absence of DNA; this interaction allows the nuclear localization of the complexes27.Both the tumorigenic activity of MEIS1 and the anti-tumorigenic activity of PREP1 require the interaction with the cofactor PBX29, to the NMR studies of the PREP1 homeodomain30 and the crystal structure of MEIS1 homeodomain31. No crystallographic information is available for the PREP-PBX or MEIS-PBX interaction site. Therefore, we have tried to better define the minimal PREP1-PBX1 and MEIS1-PBX1 interaction interface using biochemical, cellular and genetic techniques.Structural knowledge of target transcription factors is potentially a tool for drug design, but in TALE proteins this is currently limited to the crystallographic and NMR structures of the HOX-PBX1 DNA-binding motif (the homeodomain) and short flanking regions32 which is divided into two subdomains, separated by a flexible linker. The MEINOX domains of MEIS1,2,3 and of PREP1,2 are split into two motifs called homology region 1  and homology region 2 , whose secondary structure predictions suggest a α-helical structure1. Besides the homeodomain and HR1/HR2 domains, PREP1 and MEIS1 proteins do not share high sequence similarity33. However, in HR1 and HR2, both PREP and MEIS present very highly conserved heptad leucine/isoleucine-rich motifs34, previously described as leucine zippers35. The almost-identity makes it difficult to discriminate between PREP1 vs. MEIS1 binding to PBX, but on the other side it allows to transfer to MEIS1 the information obtained from PREP1.N-terminal to the homeodomain of MEIS1 and PREP1, is present a highly conserved MEINOX region37 as it abolishes the formation of the PREP1-PBX1 complex and prevents nuclear localization4. Mutating L63 and L67 (HR1 domain of PREP1) into alanine decreases the binding to PBX136. In MEIS1, band shift experiments have shown that deletions within the HR1 region (ΔL71-L96) impair dimerization with PBX138. Deletion of 3 or 4 residues in the heptad repeats of HR1 or HR2 domains of MEIS1 (HR1ΔLFPLL or HR2ΔLLEL) has been reported to be insufficient to disrupt the interaction with PBX, while their combination (HR1ΔLFPLL/HR2ΔLLEL) or deletion of more residues within the heptad repeat of the HR2 domain (HR2ΔLRF/ΔLLEL or HR2ΔIQVL/ΔLLEL) completely destroys the interaction with PBX and prevents leukemogenesis, without altering MEIS1 expression39. In addition, mutations of 5 hydrophobic residues into alanine in the HR2 151-VLRFHLLELE-160 stretch of MEIS120 abolish the co-immunoprecipitation with PBX.The PBX proteins contain at their amino terminus two motifs, PBC-A and PBC-B, which together are known as the PBC domain. Deletion of the HR1-HR2 domains in PREP1 indicates that they are required for the dimerization to the PBC domain of PBX2.In PBX proteins the highly conserved 75-residues-long PBC-A and 88-residues long PBC-B are identified as PREP or MEIS binding regions. However, so far only the direct involvement in heterodimerization of the PBC-A has been demonstrated4. In this paper we have reanalysed the PREP1-PBX1 interaction using more detailed and alternative approaches and better defined the specific interaction surface. In addition, we have performed experiments on MEIS1 that have allowed us to identify differences in the interaction mechanisms between PREP1 and MEIS1.As PREP1 and MEIS1 heterodimerize with the same surface of PBX, association with PBX is mutually exclusive. Therefore, PREP1 competes with MEIS in the oncogenic function46 enables visualization of the interacting regions by bridging two proteins with a cross-linker and creating hybrid peptides that are identified by mass-spectrometry following protein dissection by enzymatic proteolysis. The chemical cross-linker covalently connects two functional groups (i.e. lysines) in close proximity and exposed on the protein’s outer surface. Since distant or buried residues are not able to react with the cross-linker, XLMS allows to map protein complex interfaces.Cross-linking coupled with mass-spectrometry (XLMS)4 and very prone to degradation in solution47. On the contrary, we used the full-length MEIS1 for studying the MEIS1-PBX1 complex. As cross-linker, we used the commercial BS3, a homobifunctional cross-linker containing a 11.3 Å spacer, able to link two lysine residues  was cross-linked with the PBC-A (K65 and K87), the PBC-B (K153), the homeodomain (K242) and with the C-terminal region (K297 and K308) of PBX1. No MEIS1 residue in the HR1 cross-linked with PBX1, suggesting that the HR1 domain of MEIS1 is not or less involved in the heterodimerization. We thereby conclude that in the MEIS1-PBX1 complex, the HR2 of MEIS1 and the PBC-B of PBX1 are the domains mostly involved in the formation of the heterodimer. It must be noticed that K195 in the PBC-B of PBX1 was not found in any inter cross-linked peptide with MEIS1, while it was very reactive in the complex with PREP1, in particular with the PREP1 homeodomain (K268). The lack of reactivity of the homeodomain of MEIS1 might be due to an inherent limit of the technology. Indeed, our XLMS analysis revealed that K270, K271, K305 and K306 of MEIS1 were present in the looped cross-linked peptides  into alanine. Among all the mutants, L129A and L132A which both sit in the second turn of the helix, most affected PBX1 binding; in particular, L132A reduced the binding to 40%.We also mutated four hydrophobic residues within the HR3 domain, but none of them affected significantly the binding of PREP1 to PBX1 Fig. C.To evaluate the effect of mutating the entire set of residues in HR1 and in HR2 that reside on the same side of the α-helix, we generated the quadruple mutants HR1m and HR2m bearing the L63A/L66A/L67A/L70A and I122A/L125A/L129A/L132A mutations, respectively. As shown in Fig. D of PBX1 for PREP1 was estimated to be 18 ± 4 nM, the KD of MEIS1 was almost fivefold higher (80 ± 17 nM), in agreement with the results of XLMS that indicate two major sites of interaction for PREP1 and only one for MEIS1. Importantly, in line with the mutagenesis data, PREP1 HR1m and HR2m mutants displayed no measurable binding for PBX1 under the experimental conditions used here.On the basis of these observations, we next measured the affinity for PBX1 of recombinant wild-type PREP1 and mutants HR1m and HR2m. For comparison, also the affinity of wild-type MEIS1 was determined under the same experimental conditions. Affinity was measured by dose-dependent ELISAs, by coating the different proteins on plate wells and probing the interaction with soluble PBX1. As shown in Fig. 48. Similarly, the conformational analysis of HR1m and HR2m quadruple mutants of PREP1 indicated a persistence of the α-helical structures with minima at ~ 208 and ~ 222 nm, and a positive peak at ~ 195 nm. Data thus show that the conformation of the three proteins, was globally preserved also in the presence of four mutations, in agreement with previous studies showing that PREP1 is endowed with a remarkable structural stability48 and possesses a high tolerance to conformational changes.We used CD spectroscopy on the wild-type protein and on the HR1m and HR2m mutants to test whether the mutations had grossly modified the global protein fold. Differences were observed in the CD spectra of HR1m and HR2m compared to the wild-type protein  reduced the binding to PREP1 only moderately (to about 65%). Substitution of both 204 and 211 residues had a stronger effect (reduction to about 50%).In conclusion, the interaction of PBX1 with PREP1/MEIS1 occurs not only through PBC-A (43–55 and 77–84 stretches), but also through PBC-B (residues 197–204). However, we can hypothesize that the most important interaction of the PBC-B region 197–207 occurs with the homeodomain of PREP1 and MEIS1 that in MEIS1 is invisible by XLMS due to the intrinsic limit of the technique.Structural data obtained by CD spectroscopy on these proteins again show that mutations do not alter the overall protein fold and that the reduced affinity derives mostly from side chain replacements and not from impairment of the global protein folding . This assay allows the in situ detection of target molecules which are in close proximity (< 40 nm) to each other. The method consists in a first step in which single-stranded oligonucleotides are conjugated to specific antibodies to recognize the target proteins; subsequently the signal is amplified by PCR and then DNA is hybridized with complementary oligonucleotides labeled with a fluorophore. Therefore if the target molecules are in sufficient proximity to each other, the DNA signal is amplified and PLA will produce a robust, specific, and visible fluorescence signalAltogether, these data corroborate and strengthen the view that the hydrophobic residues substituted in HR1m and HR2m mutants are required for the binding to PBX1.The results presented in this work confirm the overall type of interaction of PBX1 with PREP1 or MEIS1. Furthermore, the present data provide novel information on the three-dimensional organization of the domains in the heterocomplexes, highlight the relative importance of individual domains and residues involved in the interactions and suggest protein areas that might be targeted to specifically inhibit MEIS1 or PREP1 heterodimerization with PBX1.55. In addition, it provides an interesting, although very superficial, information on how the individual domains must be organized in space in the heterodimeric molecules . For example, HR1 and HR2 of PREP1 must be located in proximity of PBC-A and PBC-B, respectively; in addition, the homeodomains of PREP1 and PBX1 must be close in space to HR2 and PBC-B  and MEIS1 (80 ± 17 nM).The experiments presented allow to identify the important residues involved in the binding of PREP1 to PBX1, as shown by the mutations HR1m and HR2m. We have excluded by CD analysis that the strong effect we observe in quadruple mutant of the HR2 of PREP1 is due to a grossly altered conformational change. In addition, mutational analysis of PREP1 excludes any contribution of the “HR3” domain (stretch 242–252) in the binding to PBX1.Experimental evidences in the literature demonstrate a contribution only of PBC-A in the PREP1 and MEIS1 interactions. Even though the present work is mostly focused on PREP1, we also derive information demonstrating for the first time a direct involvement of PBC-B in the interaction. Indeed, XLMS underlines a solid connection between PBC-B and the HR2 domains of both PREP1 and MEIS1 through K153 of PBX1  and protein complexes  were all subcloned in pGEX-6P-2RBS (GenBank: KM817768), that support di-cistronic expression as described previously47, with PBX1 in the first cassette and PREP1 in the second cassette. DNA encoding full length MEIS1 was amplified by PCR with primers containing BglII/XhoI (New England Biolabs) restrictions sites.Single proteins . The sequence of the insert was validated by sequencing of DNA clones.76. All constructs were verified by sequencing them in both directions.Single‐point mutants and multiple mutants were prepared by PCR using primers listed in Supplementary Information Tables 77. The initial templates were: the pGEX-PREP1 vectors described above for PREP1 wild-type and the quadruple mutants in the HR1 and HR2 regions, and pMSCV vectors previously described4 for the deletion mutants ΔHD and ΔHR1 + 2. To generate the control vector pBABE-puro-GFP, pBABE-puro-PREP1-GFP was amplified with oligos GFPSnF/GFPSaR and the PCR product was inserted in pBABE-puro using SnaBI/SalI restriction sites. Primers used to subclone PREP1-GFP into pBABE retroviral vector are listed in Supplementary Information Table pBABE-puro was a gift from Hartmut Land & Jay Morgenstern & Bob Weinberg (Addgene plasmid #1764). pBABE-puro-PREP1-GFP wild-type and mutants were obtained by first amplifying the PREP1 sequences with primers PREP1FX/PREP1RK and cloning them using XhoI/KpnI (New England Biolabs) in pEGFP-N1 vector. Retroviral vectors were generated by amplification with primers PREP1SnF/GFPSaR of the sequences corresponding to the fusion proteins and cloning using SnaBI/SalI (New England Biolabs) into the pBABE-puro vector (Addgene#1764)A549 cells were grown in DMEM (Lonza) + 10% FBS + 2 mM L-glutamine. For the retroviral transduction, Phoenix-Ampho cell line was used. Briefly, Phoenix-Ampho cells were transiently transfected with the retroviral vectors pBABE-puro described above by standard calcium-phosphate protocol. Cell supernatants containing retroviruses were filtered using 0.45 µm filters and employed to perform two rounds of infection. Two days after infection, cells were selected by addition of puromycin  to the culture medium. To check for the expression of the PREP1-GFP-constructs, 5 days post-infection A549 cell lines were assayed by western blot analysis of cell lysate using anti-GFP antibody (AbCam #Ab290)  complex was run on a Resource S  cation exchange column. In both cases the proteins were eluted using a 0.1–1.0 M NaCl gradient. Proteins used for cross-linking and proteolysis analysis were further purified by size exclusion chromatography on a Superose 6 10/300 column  equilibrated in 20 mM Tris pH 7.4, 0.3 M NaCl, 5% glycerol, 0.5 mM EDTA and 1 mM DTT and eluted at a flow rate of 0.2 ml/min.Expression and purification of PREP1, MEIS1 and PBX1 were carried out as described previously3) (Sigma-Aldrich) was added. Cross-linking reaction was performed for 1 h at room temperature with shaking at 1000 rpm as described in78 . Reaction was stopped by adding 100 mM sodium bicarbonate  and incubated at room temperature for 10 min. Cross-linked proteins were separated by SDS-PAGE electrophoresis and stained by coomassie. Gel slices were excised from the gel, reduced with 10 mM DTT, alkylated with 55 mM Iodoacetamide (IAA) and finally digested overnight with Glu-C and then with Trypsin79, before peptides were desalted on StageTip C1880.Purified PBX1-PREP1 complex at 1 mg/ml was dialyzed 2 h at room temperature in 50 mM borate-buffered saline. After dialysis, 1 mM Bis(sulfosuccinimidyl) suberate sodium salt . Peptides separation was achieved on a linear gradient from 95% solvent A  to 60% solvent B  over 48 min, and from 60 to 100% solvent B in 2 min at flow rate of 0.25 µL/min on a UHPLC Easy-nLC 1000 (Thermo Fischer Scientific) connected to a 23 cm fused-silica emitter of 75 µm inner diameter , packed in-house with ReproSil-Pur C18-AQ 1.9 µm beads . MS data were acquired using a data-dependent top 15 method for HCD fragmentation. Survey full scan MS spectra (300–1800 Th) were acquired in the Orbitrap with 60,000 resolution, AGC target 3e6, IT 20 ms. For HCD spectra, resolution was set to 15,000 at m/z 200, AGC target 1e5, IT 160 ms; NCE 28% and isolation width 1.4 m/z.Samples were analyzed as described in82 searching against the database containing the sequences of PREP11–344-PBX1–308, and MEIS11–390-PBX11–308; Glu-C and trypsin specificity was indicated and up to five missed cleavages were allowed. BS3 was selected as cross linker, cysteine carbamidomethyl was used as fixed modification and methionine oxidation as variable. Mass deviation for MS/MS peaks was set at 20 ppm. Filter tolerance was set at 10 ppm, while the FDR was set to 0.05, corresponding to an E-value of 0.001. For manually curated analysis only those spectra with at least 4 consecutive b or y ions for each cross-linked peptide were considered as confidently identified83. The full-list of cross-linked peptides and relatives E-values are available in supplementary materials.For identification, raw data were processed with pLink 2 ver. 2.3E-value < 0.001 (corresponding to FDR < 0.05) were considered confidently identified; moreover, we required the majority of the ions of the spectrum assigned and 4 consecutive b or y ions for each cross-linked peptide observed83. The full-list of cross-linked peptides and relatives E-Values are available in Supplementary Information Tables In manually curated analysis, only those spectra with 48, introducing slight modifications. Briefly, PBX1 (wild-type) was coated on 96-well plates at 0.1 µM in 100 mM Tris–HCl, pH 7.5, for 16 h at 4 °C. After coating, the plates were washed three times with PBS-T buffer  and blocked with 300 µL of PBS containing 3% BSA for 90 min at 37 °C. After washing, solutions of GST-fused PREP1 and MEIS1 wild-type proteins, mutant variants (HR1m and HR2m) and the GST protein, used as control, at concentrations ranging between 0.23 nM and 0.5 µM, were added to coated wells and incubated for 1 h at 37 °C, in 100 mM Tris–HCl pH 7.5. After washing 100 µL of anti-GST antibody (Bio-Rad #MCA1352) diluted 1:250 with 100 mM Tris–HCl pH 7.5 was added. After 1 h incubation at 37 °C, the plates were washed again with PBS-T, and 100 µL of anti-mouse IgG-HRP  (Bio-Rad #AAC10P) diluted 1:1000 in PBS was added. Finally, after 1 h incubation and subsequent washing, 100 µL of chromogenic substrate  was added and the reaction stopped by adding 50 µL of 2.5 M H2SO4 to each well. The absorbance was measured at 495 nm. Results were measured in triplicate from three independent assays. Results are reported as subtraction between signals obtained with the proteins and that with the GST. Data were fitted with GraphPad Prism, version 5.0, GraphPad Software Inc, San Diego, California. Binding curves were all best fitted with a one-site binding model.ELISAs were performed as previously reported48. Peptide and protein samples were prepared at 1 µM in 10 mM phosphate buffer pH 7.4. Spectra were acquired in triplicate under the following experimental conditions: 1 mm path length (quartz cuvette), scan speed 20 nm/min, band width 1.0 nm, resolution 0.2 nm, sensitivity mdeg, response time 4 s. Spectra were averaged, blank subtracted and the CD signal converted to residue ellipticity expressed in deg × cm2 × dmol−1 × res−1.CD spectra were recorded in the range 190–260 nm on a J-810 spectropolarimeter  as reported in3+-conjugated anti-rabbit antibody  were added to the wells containing the anti-PREP1 antibody  diluted 1:5000 in 1% BSA-PBS; in the case of MEIS1 detection Eu3+-conjugated anti-streptavidin antibody  was added diluted 1:10,000 in 1% BSA-PBS. After 1 h, the plate was washed 8 times with PBS-T before incubating for 15 min with 100 µL of DELFIA Inducer . The Eu3+-label was detected by measuring time-resolved fluorescence intensity using an Envision Xcite plate reader (Perkin Elmer Corp.) employing the DELFIA label protocol. Monoclonal anti-PBX1 antibody was purchased from Sigma Aldrich (#SAB1404170), polyclonal anti-PREP1 antibody from Santa Cruz Biotechnology (#sc6245), polyclonal C17 anti-MEIS1/2 antibody (Santa Cruz Biotechnology) was conjugated  to the NHS-LC-biotin (Thermo-Scientific). The values reported represent the means of four independent experiments each conducted in triplicate. Means and standard deviations were calculated in Excel, which was also used to plot the graphs . The data are reported as normalized to wild-type protein binding (taken as 1). Type 2 (homoscedastic)T-TEST was performed using one-tail function, to determine if there was a significant difference between each mutant binding datasets against the wild-types binding datasets. We assigned * for values below 0.05 (90% significance), ** for values below 0.025 (95% significance), *** for values below 0.01 (significance 98%), **** for values below 0.005 (99% significance), and ***** for values below 0.001 (> 99% significance).OptiPlate-96 F HB, black 96-well  were coated over night at 4 °C with 100 µl of 1 µg/mL monoclonal anti-PBX1 in coating buffer (0.1 M Na Carbonate pH 9.8). After the overnight, the wells were washed 3 times with PBS-T, and then non-specific binding sites were blocked for 2 h at room temperature with 200 µL of The Blocking Solution . After washing 3 times with PBS-T, 100 µL of standards  wild-type or mutant samples in combination with wild-type proteins diluted to 10 µg/mL in 1% BSA PBS were added and incubated. For the PBX1 mutants screening, MEIS1 or PREP1 wild-type were incubated with PBX1 mutants for 1 h on ice before being added to the TR-FIA plate. In the same way, for PREP1 mutant screening, PBX1 wild-type was incubated with PREP1 mutants. After 1 h at room temperature, wells were washed 3 times before adding 100 µL of rabbit polyclonal anti-PREP1, or biotinylated goat polyclonal anti-MEIS1/2 antibody (at 0.5 µg/mL). After 1 h, the plate was washed 5 times with PBS-T and 100 µL of detection Eu54. Briefly, A549 cells expressing PREP1-GFP wild-type or PREP1-GFP mutants were fixed in PFA for 10 min at room temperature. After incubation with primary antibodies , Anti-GFP (AbCam #Ab290)), appropriate PLA probes (secondary antibodies conjugated with oligonucleotides) were added to the samples. Ligation of the oligonucleotide probes that were in proximity (less than 40 nm) was then performed, after which fluorescently labeled oligonucleotides were added together with a DNA polymerase to generate a signal detectable by a fluorescence microscope. In order to find the number of foci inside and outside nuclei, a custom Fiji84 plugin was developed. The plugin identifies foci using the ImageJ’s Find Maxima85 algorithm on the red channel image after background removal . The nuclei are recognized on the DAPI channel image, enhanced with a gaussian blur filter, applying the Huang’s threshold method. Then, the plugin counts the foci inside and outside the nuclei. The values reported represent the count of two independent experiments and for each condition we quantified 125–175 cells.For in situ proximity ligation assay , cells were labeled according to the manufacturer's instructions, and as described in84 plugin was developed. The plugin finds the nuclei on the DAPI channel using the Otsu’s thresholding algorithm87, after background removal88 and the application of a gaussian filter. Then, for each nucleus found the mean intensity of the GFP signal is extracted after background removal. For each cell, the cytoplasmic mean intensity is extracted from a drawn by hand region. The plugin reports the ratio between the nuclear and the cytoplasmic signal for each cell. To mark the cell perimeter, we stained the cytoskeleton using Alexa Fluor 647 phalloidin (Thermo Fisher Scientific). Type 2 (homoscedastic) T-TEST was performed using one-tail function, to determine if there was a significant difference between each mutant binding datasets against the wild-types binding datasets. We assigned * for values below 0.05 (90% significance), ** for values below 0.025 (95% significance), *** for values below 0.01 (significance 98%), **** for values below 0.005 (99% significance), and ***** for values below 0.001 (> 99% significance).In order to evaluate the nuclear localization of GFP-tagged PREP1 a Fiji2000 µg of total lysate were immunoprecipitated using anti-GFP magnetic beads  following the manufacture’s protocol. For the western blot, we employed anti PBX1 , anti-GFP (AbCam #Ab290), anti-vinculin (Sigma Aldrich #V4505) antibodies.Supplementary information.


C1qNb75 binds to the globular head modules of human C1q with sub-nanomolar affinity and impedes classical pathway mediated hemolysis by IgG and IgM. Crystal structure analysis revealed that C1qNb75 recognizes an epitope primarily located in the C1q B-chain that overlaps with the binding sites of IgG and IgM. Thus, C1qNb75 competitively prevents C1q from binding to IgG and IgM causing blockade of complement activation by the classical pathway. Overall, C1qNb75 represents a high-affinity nanobody-based inhibitor of IgG- and IgM-mediated activation of the classical pathway and may serve as a valuable reagent in mechanistic and functional studies of complement, and as an efficient inhibitor of complement under conditions of excessive CP activation.The classical pathway of complement is important for protection against pathogens and in maintaining tissue homeostasis, but excessive or aberrant activation is directly linked to numerous pathologies. We describe the development and  The complement system plays an important role in maintaining homeostasis by removal of apoptotic cells and pathogens . The claThe gC1q mediates the C1q interaction with numerous ligands including the Fc-region of antigen-bound IgG and IgM –7. RecogThe best characterized ligands for C1q are immune complexes containing IgG and IgM. The affinity between C1q and a single IgG molecule is low . HoweverInitiation of the CP can lead to opsonization, cell lysis and may induce a potent inflammatory response, and as a consequence, undesirable CP activation can lead to pathology. CP activation can result in allograft rejection following transplantation  but can With the realization that complement is involved in numerous disease states, therapeutic strategies to modulate specific complement components are gaining increased attention . Despitein vitro selection, we obtained a number of clones, which were subsequently cloned into a bacterial expression vector, expressed and purified. After initial characterization of the nanobodies's ability to inhibit IgG mediated complement activation (D = 0.3 nM) and an off-rate of 1.7 × 10−4 s−1 corresponding to a t1/2 = 4.1 × 103 s for a first-order dissociation of the monomeric interaction. Comparison of the dissociation phases for C1q and gC1q confirms that C1q binds with avidity to immobilized C1qNb75 since the C1q dissociation is characterized by an extremely slow dissociation phase as compared to the monovalent interaction (To isolate nanobodies (Nbs) against C1q we immunized a llama with a mixture of human C1q and gC1q. Peripheral blood mononuclear cells were isolated and a phage display library presenting the Nbs as fusion proteins fused to the PIII phage coat protein, was generated. After two rounds of tivation  we identtivation . Immobiltivation . Increastivation . The Nb eraction .The CP can be activated by binding of C1q to immune complexes containing complement activating antibodies. We assayed the ability of C1q in NHS to bind to surface immobilized human IgG in the presence of increasing concentrations of C1qNb75 with a solid-phase immunoassay . Human I4 terminal galactose-α-1,3-galactose residues per cell  as source of C1q, affinity purified human IgG antibodies recognizing terminal galactose-α-1,3-galactose  and pig red blood cells (RBCs), which express the relevant carbohydrate structure. These RBCs present ~6 × 10per cell  and effiper cell . Using fper cell , supportthe RBCs . Moreovethe RBCs , in accothe RBCs . This suD = 0.29 nM observed by BLI is applicable in this serum-based experiment, and binding of the nanobody to its six binding sites on C1q is non-cooperative, it appears that C1qNb75 must bind at least four gC1q's to efficiently inhibit IgG mediated CP activation.The classical pathway is activated by both IgG and IgM, and we first evaluated the effect of C1qNb75 on CP activation by IgG. CP activation was assayed by measuring C4 and C3 fragment deposition in IgG coated microtiter wells with NHS as a source of complement. C1qNb75 potently inhibited C4 deposition in a concentration-dependent manner . ConsistDepending on the level of complement regulation, activation of CP may result in TP activation and MAC formation. As an indirect assessment of MAC formation in the presence of C1qNb75, we performed hemolysis assays with IgG antibody sensitized sheep RBCs and NHS as source of complement. C1qNb75 blocked IgG mediated hemolysis in a concentration-dependent manner while no effect was observed when incubating with a control Nb . We next2 on C1q, which is in the low end for such complexes, whereas the observed shape complementarity of 0.74 is in the average range  . CDR2 reAs demonstrated in With respect to IgM binding, C1q is suggested to interact with the IgM-Fc Cμ3 domains using residues Arg108B, Arg109B, Arg150B, and Phe178B  (17), anC1q is the pattern recognition molecule of the C1 complex that initiates the CP of the complement cascade. Recognition of a plethora of molecular patterns by C1q induces C1r and C1s activation resulting in C4 cleavage and assembly of the CP C3 convertase –37. StruOur functional data show that C1qNb75 is a potent inhibitor of antibody-mediated CP activation. Thus, C1qNb75 could represent an initial candidate molecule for further development of a therapeutic agent for treatment of autoantibody-mediated conditions, like CAD, NMO, and gMG, where complement contributes to disease pathogenesis. Targeting complement at the site of C1 activation would inhibit MAC-induced membrane damage, but also C3b deposition and associated effects. In the case of CAD this includes C3b mediated extravascular hemolysis. However, complement also plays a role in stimulating adaptive immunity. Antigens coated with deposited C3 fragments  can lower the threshold for B-cell activation via complement receptor 2 (CR2) and also increase the time antigens are presented by CR2-expressing follicular dendritic cells to B-cells –41. ThusSince the C1q has a role both in pathogen clearance and in tissue homeostasis by facilitating clearance of apoptotic cells through phagocytosis, long term systemic C1q inhibition needs to be carefully evaluated . GeneticC1qNb75 does not bind to mouse C1q (data not shown). This prevents application of the C1qNb75 in murine animal models of complement associated pathogenesis unless mouse C1q is fully or partially replaced by human C1q as described in . SequencThe biological functions of C1q extend beyond activation of the classical pathway by immunoglobulins and reach the contexts of tumor growth, neurobiology, pregnancy, hemostasis and immune tolerance as reviewed in . AlthougC1q as well as other components of the CP are critical in the developing brain, and mice deficient in C1q, C4, C3, or CR3 have defects in synaptic connectivity . During in vitro applications, C1qNb75 may become a valuable tool for both functional studies and diagnostics. Due to the very high affinity of immobilized C1qNb75 for C1q, the Nb appears suited for capture and quantitation of C1q in biological samples like serum and cerebrospinal fluid. Our experiments also provide evidence for its application in flow cytometry studies to investigate competition with cell bound C1q binders. Finally, C1qNb75 specifically blocks the CP in functional assays and may thus allow investigators to unravel the specific contributions to complement activation from the CP and LP without depending on protein depletion or genetic knockouts.With respect to in vivo under conditions of excessive antibody-mediated CP activation.In conclusion, we present a potent and high affinity inhibitor of the classical pathway of complement, that is based on the small and versatile Nb scaffold, and define its mode of inhibition through a combination of functional assays and structural biology. C1qNb75 stands out as a powerful tool for investigating CP-mediated complement activation in different contexts, which could possibly help determine binding modes on activators for which the interacting regions on gC1q are unknown. We further suggest possible applications of C1qNb75 as a diagnostic tool, and as a molecule that will efficiently inhibit C1q TM beads (Bio-Rad) equilibrated in 50 mM Na2HPO4, 82 mM NaCl, 2 mM EDTA pH 7.4 (buffer A). The beads were washed extensively with buffer A and packed on a XK16  column. The column was washed with buffer A until baseline and eluted with a 300 mL linear gradient from 82 to 300 mM NaCl. The fractions containing C1q were pooled and a 1:1 v/v ratio of 66% saturated (NH4)2SO4 solution was dripped into the sample while stirring at 4°C. After 15 h, the precipitate was pelleted by centrifugation at 3,000 × g for 30 min and purified C1q was resuspended in 50 mM Tris-HCl, 500 mM NaCl pH 7.2 (storage buffer) and dialyzed three times for at least 6 h at 4°C against 2.5 L of storage buffer before flash freezing at −80°C.C1q was purified from human outdated plasma using a procedure adapted from . Three bImmunization, library generation and selection was performed essentially as described in . A llamaThe gene encoding C1qNb75 was cloned into a pET22b+ vector and transformed into LOBSTR cells . Cells w2, 1.76 mM MgCl2 pH 7.28-7.6)  placed on ice and added NHS to a final concentration of 0.2%. The diluted Nb/NHS (100 μL) mixture was transferred to IgG coated wells and incubated for 90 min at 37°C in a humidity chamber. The wells were subsequently washed three times with TBS/Tw (TBS with 0.1% (v/v) Tween 20). Hundred microliter biotinylated rabbit anti-C3c antibody  or biotinylated rabbit anti-C4c antibody  diluted to 0.5 μg/mL in TBS/Tw was added and the plates were incubated for 1 h at RT. The wells were washed three times in TBS/Tw and each well was added 100 μL of 1 μg/mL europium-labeled streptavidin  diluted in TBS/Tw containing 25 μM EDTA and incubated for 1 h at RT. The wells were washed three times in TBS/Tw and then 200 μL enhancement buffer  was added to each well. Time-resolved fluorescence was subsequently measured using a VICTOR3 Multilabel Plate counter (PerkinElmer).Each well in a 96-well Maxisorp plate  was coated with 100 μL solution of heat aggregated IgG at 15 μg/mL in 50 mM sodium carbonate (pH 9.6) (AMPLIQON Laboratory Reagents) and incubated ON at 4°C. The wells were blocked by addition of 250 μL TBS/Tw  supplemented with 1 mg/mL human serum albumin (HSA) for 1 h at RT. The wells were washed three times with 300 μL 20 mM HEPES-NaOH, 150 mM NaCl, pH 7.5. Nanobodies were diluted in veronal buffer 2 was prepared by pepsin treatment of rabbit anti-C1q antibody  at 10 μg/mL in PBS and incubated ON at 4 °C. The wells were blocked by addition of 250 μL TBS supplemented with 1 mg/mL HSA for 1 h at RT. The wells were washed three times with TBS/Tween. Serum or purified C1q were diluted in TBS/Tw/Ca (TBS/Tw containing 5 mM CaCl2) and 100 μL added per well. Following incubation for 1 h the wells were washed three times with TBS/Tw and 100 μL of biotinylated rabbit anti-C1q antibody  diluted to 0.25 μg/mL in TBS/Tw and the plates were incubated for 1 h at RT. The wells were washed three times in TBS/Tw and each well the received 100 μL of 1 μg/mL europium-labeled streptavidin diluted in TBS/Tw containing 25 μM EDTA and incubated for 1 h at RT. The wells were washed three times in TBS/Tw and then 200 μL enhancement buffer was added to each well and time-resolved fluorescence was measured using a VICTOR3 Multilabel Plate counter (PerkinElmer).Wells of Maxisorp plates were coated with 100 μL solution of nanobody or with rabbit F(ab′)Wells of Maxisorp plates were coated with 100 μL of normal human IgG at 15 μg/mL in in 50 mM sodium carbonate (pH 9.6) and incubated ON at 4°C. The wells were then blocked by addition of 250 μL TBS supplemented with 1 mg/mL HSA for 1 h at RT. The wells were then washed three times with TBS/Tw. Purified C1q , diluted in TBS/Tw/Ca, was mixed with nanobodies or with rabbit anti-C1q at several dilutions and 100 μL added per well. Following incubation for 1 h, the wells were washed three times with TBS/Tw and then 100 μL of biotinylated rabbit anti-C1q antibody diluted to 0.25 μg/mL in TBS/Tw were added, and the plates were incubated for 1 h at RT. The wells were washed three times in TBS/Tw and each well then received 100 μL of 1 μg/mL europium-labeled streptavidin diluted in TBS/Tw containing 25 μM EDTA and incubated for 1 h at RT. The wells were washed three times in TBS/Tw and then 200 μL enhancement buffer was added to each well and time-resolved fluorescence was measured using a VICTOR3 Multilabel Plate counter (PerkinElmer).Serum samples with nanobody at different concentrations were incubated at 37°C for 18 h to initiate autoactivation of complement fragments. The samples were subsequently diluted 1/15 and tested for C5a content. The concentration of complement fragment C5a was measured using an ELISA kit from Hycult Biotech  as described by the manufacturer. It uses incubation of samples in microtiter wells coated with a monoclonal antibody that recognizes a neo-epitope on C5a. This is followed by detection of bound C5a with a biotinylated antibody and subsequently addition of enzyme-labeled streptavidin and enzyme substrate.BLI experiments were performed on an Octet Red96 (ForteBio) at 30°C, 1,000 rpm. C1qNb75 were immobilized on amine reactive second-generation (AR2G) biosensors at 20 μg/mL in 20 mM sodium acetate, 100 mM NaCl pH 5.0. The sensors were quenched with 1 M ethanol amine and equilibrated in kinetics buffer (PBS supplemented with 1 mg/mL BSA and 0.05% Tween 20). Association and dissociation were monitored with gC1q concentrations ranging from 0.3 to 10. 9 nM and C1q concentrations ranging from 0.7 to 5.4 nM in kinetics buffer. For gC1q, the data were globally fitted with a 1:1 binding model using the Octet Data Analysis software.Venous EDTA stabilized blood was sampled from pigs undergoing experimental surgery at the Institute of Clinical Medicine, Aarhus University, Denmark and fixed with glutaraldehyde as described . Antibod2) supplemented with 1 mg/mL gelatin and diluted to 6% v/v. Lyophilized rabbit anti-sRBC stroma antibody (Sigma-Aldrich S1389) was reconstituted in 2 mL PBS and 50 μL was added to 10 mL BI buffer. The antibody was mixed with 10 mL 6% sRBCs in BI buffer and incubated at RT for 30 min. Cells were pelleted by centrifugation at 1,000 g for 6 min and the supernatant was removed. The cells were resuspended in 10 mL BI buffer. For each Nb concentration, 15 μL of NHS was added to 185 μL Nb diluted in BI buffer and incubated for 15 min on ice. Sixty microliter from each reaction were then transferred to a V-shaped 96-well plate in triplicates and placed on wet ice before 30 μL 6% sRBCs were added. The plate was vortexed and incubated at 37°C in a humid chamber at 250 rpm. After 30 min, 100 μL stop solution  was added to each well. The plate was vortexed and subsequently centrifuged at 450 g for 6 min. Fifty microliter of sample was transferred to a flat-bottom microtiter plate containing 50 μL stop solution and vortexed. The absorbance was measured at 405 nm using a VICTOR3 Multilabel Plate counter. % lysis was calculated with the formula: % lysis = ((OD405test-OD405blank)/)*100, with OD405blank being incubation with buffer and OD405totallysis incubation with water.Sheep red blood cells (sRBCs) in Alsever's solution  were washed with BI buffer  to 5 × 108 cells/mL. For each reaction 20 μL Nb was mixed with 53 μL assay buffer and 100 μL NHS diluted 1/50 in assay buffer. After 15 min incubation on ice, 27 μL sRBCs was added and 90 μL from each reaction was pipetted into a V-shaped 96-well plate in duplicates. The plate was incubated in a water bath for 60 min at 37°C with shaking at 1,400 rpm every 10 min. After 60 min, the plate was centrifuged at 1,000 g for 3 min and 80 μL form each well was pipetted into a flat bottom 96-well plate and absorbance was measured at 541 nm. % lysis was calculated by using the following formula: % lysis =((OD541test-OD541blank)/)*100, with OD541blank being sRBCs incubated with buffer and OD541totallysis being sRBCs incubated with water. IC50 was calculated with GraphPad by fitting the data with a variable slope model.sRBCs coated with rabbit anti-sheep erythrocyte IgM  were pelleted by centrifugation at 1,000 g. Supernatant was removed and sRBCs were resuspended in veronal buffer (Lonza) containing 4.9 mM barbital, 145 mM NaCl, 0.25 mM CaCl6 cells/mL in serum-free FreeStyle 293 medium (Gibco) and incubated ON at 37°C at 8% CO2 with shaking. The following day, cells were collected by centrifugation and resuspended in fresh medium to give 2 × 106 cells/mL. The cells were incubated for 1 h with shaking and transfected using 1 μg DNA (gC1q in pcDNA3.1) per 1 mL final culture volume and a 2:1(w:w) ratio of polyethylenimine  to DNA. After 4 h, the cells were diluted to 106 cells/mL in FreeStyle 293 medium and incubated ON at 37°C at 8% CO2 with shaking. The next day, 0.25 × 106 viable cells from each of the transfected cell cultures were transferred to 6-well plates containing FreeStyle 293 medium supplemented with 10% (v/v) fetal bovine serum (FBS) and incubated statically ON at 37°C at 8% CO2 to allow the cells to adhere. Selection pressure was applied by the addition of fresh medium containing 10% FBS and 200–300 μg/mL Geneticin (Gibco). The medium was changed every 2–3 days to remove dying cells and selection pressure was maintained for 11–18 days until foci could be identified. Single colonies were picked to a 24-well plate and left to proliferate in FreeStyle 293 medium supplemented with FBS and Geneticin, before being transferred to 6-well plates and adapted to 0.1% FBS. Finally, the cells were resuspended in 10 mL FreeStyle 293 medium supplemented with 250 μg/mL Geneticin and transferred into 125 mL flasks. The cells were cultured as recommended by the manufacturer until a cell viability of >90% was achieved. A western blot was performed on 20 μL media to identify clones with high gC1q expression. The western blot was performed using THETM His-Tag Antibody, (Genscript)  and Goat anti-mouse IgG HRP (Thermo Fisher Scientific) . Four clones with high gC1q expression were selected and used for small scale expression and pulldown experiment using Ni Excel beads . The best clone yielded ~2–4 mg protein/L culture.A transfected HEK293 stable cell line for expression of the single-chain form of gC1q  with a C2. Cells were harvested by centrifugation and gC1q was purified from culture media adjusted to pH 8.0 using a 5 mL Histrap excel column . The column was washed to baseline with PBS containing 500 mM NaCl and 10 mM Imidazole pH 8.0. The protein was eluted with PBS containing 500 mM NaCl and 400 mM Imidazole pH 8.0 and analyzed by SDS-PAGE. Fractions containing gC1q were concentered and loaded on a Superdex 200 Increase 10/300 column  in 20 mM HEPES-NaOH, 150 mM NaCl, pH 7.5.For large-scale expression, gC1q expressing cells were grown in FreeStyle 293 media for 7 days at 37°C, 150 rpm and 8 % COFor complex formation gC1q was mixed with molar excess of C1qNb75 and purified by size exclusion chromatography in 20 mM HEPES-NaOH, 150 mM NaCl, pH 7.5 using a BioRad ENrich SEC 70 10/300 column. Complex containing fractions were pooled and concentrated to 8 mg/mL. The complex was crystallized at 19°C by vapor diffusion against a reservoir containing 0.2 M ammonium acetate, 0.1 M BIS-Tris pH 5.5, 25% w/v PEG 3350 after mixing protein and reservoir in a 1:1 ratio. Crystals were cryoprotected in 0.1 M BIS-Tris pH 5.5, 37% w/v PEG 3350 and cryocooled in liquid nitrogen.Diffraction data were collected to 2.19 Å at the EMBL P14 beam line, PETRA III. Diffraction data from a single crystal were processed with XDS . The strThe C1-IgM model was built in the deposited cryo-ET 3D reconstruction EMDB-4878)  using Py  using P6Z6V.Diffraction data and the atomic model are deposited at the protein data bank PDBe under the entry NL, DP, HG, AZ, JB, and AH generated C1qNb75, expressed and purified proteins, and determined the structure or conducted functional assays. ST and GA supervised research. NL and GA wrote the manuscript with inputs from all authors. All authors contributed to the article and approved the submitted version.NL, AZ, DP, ST, and GA are listed as inventors on a patent describing the use of C1qNb75. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.