Like-acetyl-glucosaminyltransferase 1 (LARGE1) synthesizes and extends matriglycan on α-dystroglycan (α-DG) during skeletal muscle differentiation and regeneration; however, the mechanisms which regulate matriglycan elongation are unknown. Here, we show that Protein O-Mannose Kinase (POMK), which phosphorylates mannose of core M3  preceding matriglycan synthesis, is required for LARGE1-mediated generation of full-length matriglycan on α-DG (~150 kDa). In the absence of Pomk gene expression in mouse skeletal muscle, LARGE1 synthesizes a very short matriglycan resulting in a ~ 90 kDa α-DG which binds laminin but cannot prevent eccentric contraction-induced force loss or muscle pathology. Solution NMR spectroscopy studies demonstrate that LARGE1 directly interacts with core M3 and binds preferentially to the phosphorylated form. Collectively, our study demonstrates that phosphorylation of core M3 by POMK enables LARGE1 to elongate matriglycan on α-DG, thereby preventing muscular dystrophy.Matriglycan  The heterlecan)  and has erlecan) . Over 18erlecan) , and deferlecan) . Additioerlecan) . Crystalerlecan) , and theerlecan) . During erlecan) . Howeverin utero or within a day or two of birth (O-Mannose Kinase (POMK) is a glycosylation-specific kinase that phosphorylates mannose of the core M3 trisaccharide  during synthesis of the O-mannose-linked polysaccharide ending in matriglycan , which is lethal of birth . Proteinriglycan . Interesriglycan , suggestTo determine if matriglycan can be expressed in the absence of POMK function, and therefore better understand the role of POMK in matriglycan synthesis, we studied skeletal muscle from a patient (NH13-284) with a homozygous POMK (D204N) mutation  and congPomk using LoxP sites and Cre driven by the muscle creatine kinase (Mck) promoter, or both the Mck promoter and the paired box 7 (Pax7) promoter  quadriceps muscles revealed hallmarks of a mild muscular dystrophy  muscles of 18–20- week-old Control and M-POMK KO mice. EDL muscle mass and cross-sectional area (CSA) were reduced in M-POMK KO mice compared to control mice  for laminin-111 compared to control muscle  α-DG with anti-core DG antibody , howeverl muscle , but higCre; PomkLoxP/LoxPMck mice with two exoglycosidases, α-Xylosidase and β-Glucuronidase, which in combination digest matriglycan  for laminin-111 was reduced compared to wild-type α-DG (α-DG(WT)) but was greater than that of α-DG from LARGE1 KO HAP1 cells  of FKTN  modifies GalNAc with ribitol-phosphate for synthesis of full-length matriglycan . Overexp of FKTN , increas of FKTN . HAP1 ce binding . FKTN ov binding . These e binding , as a DGt 90 kDa . Similart 90 kDa .LARGE1 KO HAP1 cells by generating very high molecular weight matriglycan (POMK or POMK/LARGE1 KO HAP1 cells did not produce very high molecular weight matriglycan (POMK/LARGE1 KO HAP1 cells with POMK enabled LARGE1 to synthesize high molecular weight matriglycan (Overexpression of LARGE1 can rescue the defect in matriglycan synthesis in distinct forms of CMD as well as in riglycan . Howeverriglycan . Only thriglycan . These fd = 11.5 ± 1.2 µM for GGMp-MU compared to Kd >90 µM for GGM-MU)  . This inO-mannose-linked polysaccharide ending in matriglycan. LARGE1 is responsible for the synthesis of matriglycan, and addition of matriglycan enables α-DG to serve as a predominant ECM receptor in many tissues, in particular, skeletal muscle and brain. Over eighteen genes are implicated in matriglycan synthesis, and complete loss-of-function mutations in these genes abrogate synthesis of the O-mannose linked modification and preclude the addition of matriglycan, thereby leading to dystroglycanopathies, congenital and limb-girdle muscular dystrophies with or without structural brain and eye abnormalities. Here, we have used a multidisciplinary approach to show that the absence of POMK activity does not preclude addition of matriglycan. Instead, in the absence of core M3 phosphorylation by POMK, LARGE1 synthesizes a short, non-extended form of matriglycan on α-DG (~90 kDa). However, in order to generate full-length mature matriglycan on α-DG (~150 kDa), LARGE1 requires phosphorylation of core M3 by POMK  on α-DG during synthesis of the  by POMK .Our study shows that the short form of matriglycan is able to bind to laminin with high affinity and thus enables α-DG(POMK) to function as an ECM receptor. Given the very small increase in apparent MW in α-DG(POMK) compared to α-DG from cells and muscle lacking LARGE1 , the shoMuscle-specific POMK KO mice express the short, non-extended form of matriglycan on ~90 kDa α-DG and develop a mild muscular dystrophy phenotype. Muscle physiology studies demonstrate that the short matriglycan expressed in the absence of POMK can maintain specific force but cannot prevent eccentric contraction-induced force loss or skeletal muscle pathology. Interestingly, missense mutations in FKRP that cause LGMD2I also show reduced expression of matriglycan  and exhiPOMK KO HAP1 cell lines demonstrated that the synthesis of the short, non-extended form of matriglycan occurs on threonine-317 of the mucin-like domain and, like full-length matriglycan, requires LARGE1, DGN, and CDP-ribitol. Cell biological experiments demonstrated that the DGN is necessary for synthesis of the short form of matriglycan. As the binding of LARGE1 to the DGN is essential for the synthesis of full-length matriglycan on α-DG  with brain malformations.Pomk gene consists of five exons, exons 1, 2, and 3, which are non-coding and exons 4 and 5, which are coding (The e coding . We usedPomk_5P1 TTCTTTCTGTGATGTGTGCTTATTCPomk_5P2 CAGACACTCACCCTTTACCTTAGWild-type: 197 bpTargeted: 235 bpPomk_3P1 AGCCACACCTTCCTACAGTCPomk_3P2 AAGCTCTGCCCAGAGAGAAGWild-type: 123 bpTargeted: 162 bpPomk_5’_guide(601) CGTGTCCCGCCAGGAATGAAPomk_3’_guide(3P1) TCAGGAGGCGGCTCCCAGTGPomk_5’_donorTCCTCATCTTCTCCCTGTGCAGTCAATCTGCACAGCTCCCTGCACACATGGCTTATAGAGTGGTTCTCACCCCGCCCTTCATAACTTCGTATAGCATACATTATACGAAGTTATGGTACCTCCTGGCGGGACACGAATAAGCACACATCACAGAAAGAAGTCTGTTGTCTTGACTGCCCAGCCCTCCGCAGCTGCCACCCPomk_3’_donorAGTGTGAGATTCAAGTGTGGATATGCAGTGATCCTCTGGCCACACTTGTGAGCAGCCACACCTTCCTACAGTCCCTCACTATAACTTCGTATAGCATACATTATACGAAGTTATGGATCCGGGAGCCGCCTCCTGAGCCCTGCTGTGTAACCCACCTACCTTCCCTCCTTTCACACTAGAAGCTGAGAGCTCTTCTCTTCB6SJLF1/J mice were purchased from Jackson Labs . Male mice older than 8 weeks were used to breed with 3–5 week-old super-ovulated females to produce zygotes for electroporation. Female ICR ) mice were used as recipients for embryo transfer.Cre under the mouse creatine kinase (Mck) promoter, B6.FVB(129S4)-Tg(Ckmm-cre)5Khn/J (stock no. 006475) Mrc/J, (stock no. 010530) . Male F1 progeny with the genotype Cre; PomkLoxP/+Mck were bred to female LoxP/LoxPPomk mice. A Cre PCR genotyping protocol was used to genotype the Cre allele using standard Cre primers. The primers used were Sense: TGATGAGGTTCGCAAGAACC and Antisense: CCATGAGTGAACGAACCTGG.Mice expressing  006475)  and the  010530)  were purPax7-Cre and Mck-Cre. Genotyping of Cre; Pax7Cre; PomkLoxP/LoxPMck mice was performed by Transnetyx using real-time PCR.Sanger sequencing of tail DNA was performed by the University of Iowa Genome Editing Core Facility to confirm incorporation of 5’ and 3’ LoxP sites. PCR probes were developed at Transnetyx to genotype mice expressing both Cre; PomkLoxP/LoxPMck mice, N = 3 mice of each genotype  were used. For studies with Cre; Pax7Cre; PomkLoxP/LoxPMck mice, animals of varying ages were used as indicated, and N = 3 each of LoxP/LoxPPomk and Cre; Pax7Cre; PomkLoxP/LoxPMck were used. Littermate controls were employed whenever possible. The number of animals required was based on previous studies . All animals were maintained in a climate-controlled environment at 25°C and a 12/12 light/dark cycle. Animal care, ethical usage, and procedures were approved and performed in accordance with the standards set forth by the National Institutes of Health and IACUC. For studies with  studies  and expeChemically modified CRISPR-Cas9 crRNAs and CRISPR-Cas9 tracrRNAs were purchased from Integrated DNA Technologies (IDT) ). The crRNAs and tracrRNA were suspended in T10E0.1 and combined to 1 µg/µL (~29.5 µM) final concentration in a 1:2 (µg: µg) ratio. The RNAs were heated at 98°C for 2 min and allowed to cool slowly to 20°C in a thermal cycler. The annealed cr:tracrRNAs were aliquoted to single-use tubes and stored at −80°C.Cas9 nuclease was also purchased from IDT (Alt-R S.p. HiFi Cas9 Nuclease). Cr:tracr:Cas9 ribonucleoprotein complexes were made by combining Cas9 protein and each cr:tracrRNA; final concentrations: 60 ng/µL (~0.4 µM) Cas9 protein and 60 ng/µL (~1.7 µM cr:tracrRNA). The Cas9 protein and annealed RNAs were incubated at 37°C for 10 min. The two RNP mixes were combined and incubated at 37°C for an additional 5 min. The single stranded oligonucleotide donors (ssODN) were purchased from IDT as Ultramers. The ssODNs were added to the RNPs and the volume adjusted to the final concentrations in the injection mix were 10 ng/µL each ssODN; 20 ng/µL each guide RNA and 40 ng/µL Cas9 protein.2 until all zygotes were injected. Fifteen to 25 embryos were immediately implanted into the oviducts of pseudo-pregnant ICR females.Pronuclear-stage embryos were collected using previously described methods . EmbryosLoxP/+Pomk mice using primers flanking the 5’ loxP site, ACTCCAGTTGGTTTCAGGAAG and GAGGGAAGAGAAGTCAGGAAAG. For the 3’ loxP site, primers of sequence ACCGAGTGTGAGATTCAAGTG and GGTTGCTGGTAGGGTTAAGAG were used. The 5’ loxP site contains a KpnI cleavage site, and the 3’ loxP site contains a BamHI site. The screen of the 5’ loxP site gives a product of 803 base pairs for the LoxP allele when uncut. Kpnl digestion of the 5’ loxP site gives three products of 381, 355, and 67 base pairs. A screen of the 3’ loxP site gives a product of 396 base pairs for the uncut allele with loxP site, while BamHI digestion of the 3’ loxP site gives products of 273 and 123 base pairs.Insertion of loxP1 (5’) and loxP2 (3’) sites was confirmed by cloning and sequencing of genomic PCR products  from taiTTCTTTCTGTGATGTGTGCTTATTC) or loxP2 (CAGACACTCACCCTTTACCTTAG) site. The wild-type allele is 197 bp while the floxed allele is 235 bp. LoxP/+Pomk mice were backcrossed five generations onto a C57BL/6J background and backcrossed mice used whenever possible.Genotyping was carried out using primers flanking the exon five loxP1 site or the (Forelimb grip strength was measured at 1 month and 4 months of age using previously published methods . A mouseCreatine kinase levels were measured in 8-week-old mice 2 hr after mild downhill run (three meters per minute for 5 min followed by 15 m per minute for 10 min) at a 15- degree downhill incline as previously described . Blood wMice were weighed as previously described . Weights2, 2; MgSO4, 1; NaH2PO4, 1; NaHCO3, 24; glucose, 11). The bath was maintained at 25°C, and the solution was bubbled with 95% O2 and 5% CO2 to stabilize pH at 7.4. The proximal tendon was clamped to a post and the distal tendon tied to a dual mode servomotor . Optimal current and whole muscle length (L0) were determined by monitoring isometric twitch force. Optimal frequency and maximal isometric tetanic force (F0) were also determined. The muscle was then subjected to an eccentric contraction (ECC) protocol consisting of eight eccentric contractions (ECCs) at 3 min intervals. A fiber length Lf-to-L0 ratio of 0.45 was used to calculate Lf. Each ECC consisted of an initial 100 millisecond isometric contraction at optimal frequency immediately followed by a stretch of Lo to 30% of Lf beyond Lo at a velocity of 1 Lf/s at optimal frequency. The muscle was then passively returned to Lo at the same velocity. At 3, 15, 30, 45, and 60 min after the ECC protocol, isometric tetanic force was measured. After the analysis of the contractile properties, the muscle was weighed. The CSA of muscle was determined by dividing the muscle mass by the product of Lf and the density of mammalian skeletal muscle (1.06 g/cm3). The specific force was determined by dividing Fo by the CSA (kN/mm2). 18–20-week-old male mice were used, and right and left EDL muscles from each mouse were employed whenever possible, with n = 5 to 8 muscles used for each analysis. Each data point represents an individual EDL. Statistics were calculated using GraphPad Prism eight software and Student’s unpaired t-test was used (two-sided). Differences were considered significant at a p-value less than 0.05.To compare the contractile properties of muscles, extensor digitorum longus (EDL) muscles were surgically removed as described previously . The musAB_2617216) was added to sections overnight at 4°C followed by Alexa Fluor-conjugated goat IgG against mouse IgM , for 40 min. The sections were also stained with rabbit polyclonal antibody to β-DG  followed by Alexa Fluor-conjugated 488 Goat anti-rabbit IgG (1:500). Whole sections were imaged with a VS120-S5-FL Olympus slide scanner microscope. Antibody IIH6 is a monoclonal to the glycoepitope of α-DG  for 15 min. Primary antibodies used were mouse monoclonal: α-DG IIH6 (clone IIH6C4)  according manufacturer's instructions. For immunofluorescence analysis, unfixed frozen serial sections (7 µm) were incubated with primary antibodies for 1 hr, and then with the appropriate biotinylated secondary antibodies for 30 min followed by  IIH6C4) , β-DG (L30 slices of 30 µM thickness were taken with a cryostat (Leica CM3050S Research Cryostat) from skeletal muscle or heart that had been frozen in liquid nitrogen-cooled 2-methylbutane. For biochemical analysis of murine skeletal muscle, quadriceps muscle were used.Samples were solubilized in 500 µL of 1% Triton X-100 in 50 mM Tris pH 7.6 and 150 mM NaCl with protease inhibitors (per 10 mL buffer: 67 µL each of 0.2 M phenylmethylsulfonylfluoride (PMSF), 0.1 M benzamidine and 5 µL of each of leupeptin (Sigma/Millipore) 5 mg/mL, pepstatin A (Millipore) 1 mg/mL in methanol, aprotinin (Sigma-Aldrich) 5 mg/mL, calpeptin (Fisher/EMD Millipore) 1.92 mg/mL in Dimethyl Sulfoxide (DMSO), Calpain Inhibitor 1 (Sigma-Aldrich) 1.92 mg/mL in DMSO). Samples were vortexed for 4 min and solubilized for 2.5 hr at 4°C with rotation. Samples were then spun down at 12,000 rpm for 30 min at 4°C on a Beckman Tabletop Centrifuge. The supernatant was incubated with 100 µL WGA-Agarose slurry  overnight at 4°C with rotation. The next day samples were washed three times in 50 mM Tris pH 7.6 and 150 mM NaCl with 0.1% TX-100 and protease inhibitors. 100 µL of 5X Laemmli Sample Buffer (LSB) was added, samples boiled for 10 min, and 125 µL of this was loaded in each lane of gels for western blotting.Fibroblasts used for biochemical analyses were grown in 20% Fetal Bovine Serum  and 1% penicillin/streptomycin (Invitrogen). Cells were split at 1:2 every 2 days using Trypsin-EDTA .For flow cytometry analyses, fibroblasts cultured from skin biopsies were grown in Dulbecco’s modified Eagles medium (Invitrogen) with 20% fetal bovine serum , 1% glutamax (Thermo Fisher Scientific) and 1% penicillin/streptomycin (Sigma-Aldrich). Upon approximately 90% confluence, cells were washed with PBS without Ca and Mg, detached with non-enzymatic dissociation solution (Sigma-Aldrich cat. C5914) and fixed in 2% paraformaldehyde for 10 min. Cells were subsequently incubated on ice with the following antibodies diluted in PBS/0.1% FBS: anti-α-DG IIH6 (Millipore) for 30 min, anti-mouse biotinylated IgM  for 20 min, Streptavidin-Phycoerythrin (BD Pharmingen) for 15 min. Negative controls for each fibroblast population were incubated with 0.1% FBS/PBS without the primary antibodies. Cells were washed twice and centrifuged at 1850 g for 4 min, after each incubation step. After the last wash, cell pellets were resuspended in 500 µL of PBS. A total of 10,000 event were acquired using the Cyan ADP analyser  and analysed using FlowJo software version 7.6.5 .CVCL_Y019) are a haploid human cell line with an adherent, fibroblast-like morphology, originally derived from parent cell line KBM-7 (RRID:CVCL_A426). WT C631 cells (a diploid cell line containing duplicated chromosomes of HAP1) were purchased from Horizon Discovery, and gene-specific knockout (KO) HAP1 cells were generated by Horizon Discovery. Absence of the gene was confirmed via PCR amplification and Sanger sequencing. The identity of the cells has been authenticated by the company using the STR profiling method. Mycoplasma testing of the cells was performed on a routine basis to ensure the cells are not contaminated. HAP1 KO cell lines have complete loss of gene function and are validated in the lab by performing western blot analysis before and after gene transfer with the appropriate gene. For each HAP1 KO cell line, a matched WT control parental cell line (WT C631) was provided, ensuring that phenotypes can be attributed directly to the genetic modification. These cells are cultured in Iscove’s Modified Dulbecco’s medium (IMDM) supplemented with 10% fetal calf serum and 1% Pen-Strep antibiotics.HAP1 cells  and were previously described (POMK knockout (KO) HAP1 cells lack the single copy of the wild-type POMK allele and are therefore null at the POMK locus. The sequence of the guide RNA used is TGAGACAGCTGAAGCGTGTT. Absence of the wild-type POMK allele was confirmed by Horizon Discovery via PCR amplification and Sanger sequencing. PCR primers used for DNA sequencing are POMK Forward 5’-ACTTCTTCATCGCTCCTCGACAA-3’, and POMK Backward 5’- GGATGCCACACTGCTTCCCTAA-3’. The identity of the cells has been authenticated by the company using the STR profiling method. Mycoplasma testing of the cells was performed on a routine basis to ensure the cells are not contaminated.HAP1 cells bearing a 10 bp deletion of exon 4 of escribed . POMK knPOMK and DAG1 expression (POMK/DAG1 KO HAP1 cells) were generated using CRISPR/Cas9 by Horizon Discovery. A 16 bp deletion in the DAG1 gene (exon 2) was introduced into the POMK KO HAP1 line (HZGHC001338c004). The sequence of the guide RNA is CCGACGACAGCCGTGCCATC; NM_004393. PCR primers for DNA sequencing were forward TAGCAAGACTATCGACTTGAGCAAA and reverse GCAATCAAATCTGTTGGAATGGTCA.HAP1 cells lacking both POMK and LARGE1 expression (POMK/LARGE1 KO HAP1 cells) (HZGHC007364c011) were generated using CRISPR/Cas9 by Horizon Discovery. A 43 bp deletion of exon 3 of LARGE1 was introduced into the POMK KO HAP1 line (HZGHC001338c004). The guide RNA sequence was CTCGGCGATGGGATGGGGCT and the primer sequence was PCR forward GAGGCATGGTTCATCCAGATTAAAG and PCR reverse CTTTACCTCGCATTTCTCCACGA.HAP1 cells lacking both POMK gene, generated using the CRISPR/Cas9 system, were purchased from Horizon Discovery . The mutation in POMK is predicted to lead to a frameshift. These cells also lacked expression of ISPD. The guide RNA sequence was TGAGACAGCTGAAGCGTGTT. The sequences of PCR primers were PCR forward ACTTCTTCATCGCTCCTCGACAA and PCR reverse GGATGCCACACTGCTTCCCTAA.HAP1 cells containing a 1 bp insertion of exon 4 of the LARGE. The guide RNA sequence was GCTCTCGCGCTCCCGCTGGC and the primer sequence for 122–7 was PCR forward ATGGAGTAGGTCTTGGAGTGGTT and PCR reverse GAGGCATGGTTCATCCAGAGTTAAAG.HAP1 cells  were purchased from Horizon Discovery. Cells were generated using a CRISPR/Cas9-mediated 1 bp deletion of exon 3 of FKTN. The sequence of the guide RNA was CAGAACTTGTCAGCGTTAAA and the sequences of PCR forward CAGATCAAAGAATGCCTGTGGAAAT and PCR reverse TGCAAAGAGAAGTGTGATCAGAAAA.HAP1 cells  were purchased from Horizon Discovery. CRISPR/Cas9 was used to introduce a 16 bp deletion of exon 3 of KpnI-XhoI fragments from the DGFc3 mutants corresponding to the mutant constructs  were then subcloned into pAd5RSVK-NpA  as was the XhoI-XbaI fragment from an adenovirus encoding dystroglycan WT. E1-deficient recombinant adenoviruses  were generated by the University of Iowa Viral Vector Core (VVC) using the RAPAd system  and ISPD have been described previously  was generated and described previously . DG T317d system . Assays d system . Ad-POMKeviously . Adenovieviously . DGFc340eviously . The insd system .2 in Iscove’s Modified Dulbecco's Medium  supplemented with 10% Fetal Bovine Serum (FBS) and 1% penicillin/streptomycin (Invitrogen). Cells were split every 3 days at 1:10 using Trypsin-EDTA (ThermoFisher Scientific). On day one for adenovirus transfection experiments, media was changed to 2% IMDM, and an average of 5.9 × 106POMK KO HAP1 cells were infected at the indicated multiplicity of infection (MOI) with the indicated adenovirus. On day 2, infection medium was replaced with 10% IMDM, and on day three the cells were processed for biochemical analyses.HAP1 cells were maintained at 37°C and 5% COFor western blots and laminin overlay, HAP1 cells and fibroblasts were washed twice in ice-cold Dulbecco’s phosphate-buffered saline . The second PBS wash contained the protease inhibitors (0.23 mM PMSF and 0.64 mM benzamidine). Plates were scraped, spun down for 5 min at 14, 000 rpm at 4°C, and pellets were solubilized in 1% Triton X-100 in Tris-buffered saline  with protease inhibitors (0.23 mM PMSF and 0.64 mM benzamidine) for 1 hr at 4°C. Samples were then spun down at 14,000 rpm for 5 min, and supernatants incubated in 200 µL wheat-germ agglutinin (WGA)-agarose  as previously described . The folAB_2617216) was characterized previously and used at 1:100 (AB_10891298), was used at a concentration of 1:200 for immunoblotting the core α-DG and β-DG proteins, and the secondary was a donkey anti-sheep  used at 1:2000 concentration. Anti-POMK  was used at 1:500, and the secondary was 1:2000 Goat anti-Mouse IgG1 (LI-COR Bioscience). The antibody against the Na/K ATPase  was used at 1:1000 in 5%-milk Blotto, and the secondary was 1:10,000 Goat anti-Mouse IgG1 (LI-COR Bioscience). Anti-myc  was used at 1:2000 in 2% milk and the secondary was 1:2000 Goat anti-Mouse IgG1 (LI-COR Bioscience). Blots were developed with infrared (IR) dye-conjugated secondary antibodies (LI-COR Bioscience) and scanned using the Odyssey infrared imaging system (LI-COR Bioscience). Blot images were captured using the included Odyssey image-analysis software.The mouse monoclonal antibody against α-DG  containing 5% milk followed by incubation with mouse Engelbreth-Holm-Swarm (EHS) laminin  overnight at a concentration of 7.5 nM at 4°C in LBB containing 3% bovine serum albumin (BSA) and 2 mM CaCl2. Membranes were washed and incubated with anti-laminin antibody  followed by IRDye 800 CW dye-conjugated donkey anti-rabbit IgG  at 1:2500 dilution.Laminin overlay assays were performed as previously described . PVDF-FLEDTA treatment of laminin overlay assays was performed as described above for laminin overlays; however, calcium was excluded from all buffers made with LBB  and 10 mM EDTA was added to all LBB-based buffers, including LBB wash buffer, 5% milk-LBB, and 3% BSA-LBB buffers.HAP1 cells were washed twice in ice-cold PBS, scraped, and spun down at 14,000 rpm for 5 min at 4°C. After removing supernatant, the cell pellet was resuspended in 0.1 M MES buffer pH 6.5 with 1% Triton X-100 with Protease Inhibitors (0.23 mM PMSF and 0.64 mM Benzamidine) for 1 hr at 4°C rotating. Samples were spun down again, and the supernatant was incubated with 200 µL of WGA-agarose beads  overnight at 4°C with rotation. Samples were washed the next day three times in 0.1 M MES pH 6.5 with 0.1% Triton X-100 and protease inhibitors, and 100 µL of the beads were resuspended in 100 µL of the wash buffer.For fibroblast POMK activity measurements, cells were processed as above and solubilized in 1% TX-100 in 50 mM Tris and 150 mM NaCl pH 7.6 with protease inhibitors as described above and incubated with WGA-agarose beads. The next day, WGA beads were washed three times and resuspended in 0.1% TX-100 in 0.1 M MES pH 6.5 buffer with protease inhibitors.For measurement of mouse and human skeletal muscle POMK activity, 30 slices of 30 µM thickness were taken using a Leica 3050 s cryostat from quadriceps muscle frozen in liquid nitrogen-cooled 2-methylbutane. Samples were solubilized in 250 µL of 1% Triton X-100 in 0.1 M MES pH 6.5 with protease inhibitors  5 mg/mL, pepstatin A (Millipore) 1 mg/mL in methanol, aprotinin (Sigma-Aldrich) 5 mg/mL, calpeptin (Fisher/EMD Millipore) 1.92 mg/mL in dimethyl sulfoxide (DMSO), calpain inhibitor 1 (Sigma-Aldrich) 1.92 mg/mL in DMSO). Samples were solubilized for 2.5 hr at 4°C on a rotator. Samples were then spun down at 14,000 rpm for 30 min at 4°C on a Beckman Tabletop Centrifuge. The supernatant  was separated from the pellet, and 10 µL of this was used for POMK assays.For POMK reaction in HAP1 cells and fibroblasts, 20 µL slurry (consisting of 10 µL beads and 10 µL MES buffer) was incubated with reaction buffer for a final reaction volume of 40 µL. For POMK assay from skeletal muscle, 10 µL of total lysate was incubated with 20 µL of reaction buffer for a reaction volume of 30 µL. The final reaction concentration was 10 mM ATP, 10 mM MnCl2, 10 mM MgCl2, 10 µM GGM-MU, 0.1% TX-100 in 0.1 M MES Buffer pH 6.5. Reactions were run at 37°C for 24 hr for HAP1 cells, 48 hr for fibroblasts, or 16 hr for skeletal muscle. Experiments were done in triplicate, with each replicate representing a separate plate of cells or animal. After POMK reaction, 6 µL 0.5 M EDTA was added to 30 µL of reaction supernatant, and the mixture boiled for 5 min. 25 µL of this mixture and added to 30 µL ddH20 in HPLC vial and run on an LC18 column of a reverse-phase HPLC  with a 16% B med sensitivity gradient. The reaction was analyzed using a 4.6 × 250 mm Supelcosil LC-18 column (Supelco). Solvent A was 50 mM ammonium formate (pH 4.0), and solvent B was 80% acetonitrile in solvent A. Elution of the MU derivative was monitored by fluorescence detection  and peak area used as a measure of activity. The enzymatic activity was calculated as the peak area of the product.2, 5 mM MgCl2, and 0.05% Triton X-100  were incubated in a volume of 50 µL  for 12 hr at 37°C, with 0.4 mM Xylose-β-MU (Xyl-β-MU) and 10 mM Uridine diphosphate glucuronic acid (UDP-GlcA) in 0.1 M MES buffer, pH 6.0, at 5 mM MnClon X-100 . The rea2, 5 mM MgCl2, and 0.05% Triton X-100. The reaction was terminated by adding 25 µL of 0.1 M EDTA and boiling for 5 min, and the supernatant was analyzed using an LC-18 column. Elution of the MU derivative was monitored by fluorescence detection  and peak area used as a measure of activity. The percent product was determined by taking the product peak area and dividing by the total peak areas of substrate plus product peak. Then this number was taken and multiplied by 100 for percent conversion to product.For assessment of B4GAT1 activity in HAP1 cells, the HAP1 WGA beads were incubated in a volume of 80 µL for 26 hr at 37°C, with 0.4 mM Xyl-β-MU and 10 mM UDP-GlcA in 0.1 M MES buffer, pH 6.0, at 5 mM MnCl2, 5 mM MgCl2, and 0.5% Triton X-100. The reaction was terminated by adding 25 µL of 0.1 M EDTA and boiling for 5 min, and the supernatant was analyzed using an LC-18 column. Elution of the MU derivative was monitored by fluorescence detection  and peak area used as a measure of activity. The GlcA-T activity was assessed by subtracting the background observed in the negative control sample without donor sugar and normalized against the amount of protein measured using the DC protein assay (Bio-Rad).For the assessment of endogenous LARGE1 GlcA-T activity in skeletal muscle, Triton X-100-solubilized lysates were incubated in a volume of 25 µL for 3 hr at 37°C, with 0.4 mM Xyl-α1,3-GlcA-β-MU and 10 mM UDP-GlcA in 0.1 M MES buffer, pH 6.0, at 5 mM MnCl2, 5 mM MgCl2, and 0.05% Triton X-100. The samples were run for 46 hr at 37°C. The reaction was terminated by adding 25 µL of 0.25 M EDTA and boiling for 5 min, and the supernatant was analyzed using an LC-18 column.For assessment of LARGE1 enzymatic activity in HAP1 cells, the Triton X-100-solubilized HAP1 cells were loaded onto WGA beads and processed as described for POMK assay above. The next day after wash, beads were incubated in a volume of 90 µL with 0.4 mM Xyl-α1,3-GlcA-β-MU and 10 mM UDP-GlcA in 0.1 M MOPS buffer, pH 6.0, at 5 mM MnClFor the assessment of endogenous LARGE1 activity in fibroblasts, supernatants from Triton X-100-solubilized fibroblasts were (20 µL) directly used. Supernatants were incubated in a volume of 100 µL for 24 hr at 37°C, with 0.4 mM Xyl-α1,3-GlcA-β-MU and 10 mM UDP-GlcA in 0.1 M MES buffer, pH 6.0, at 5 mM MnCl2, 5 mM MgCl2, and 0.5% Triton X-100. The reaction was terminated by adding 25 µL of 0.1 M EDTA and boiling for 5 min, and the supernatant was analyzed using an LC-18 column.Elution of the MU derivative was monitored by fluorescence detection  and peak area used as a measure of activity. The percent product was determined by taking the product peak area and dividing by the total peak areas of substrate plus product peak. Then this number was taken and multiplied by 100 for percent conversion to product.2, 10 mM MnCl2, 0.1 M MES pH 6.5, 10 µM GM-MU, and 10 mM UDP-GalNAc. Reactions were run at 37°C for 72 hr. Experiments were done in triplicate, with each replicate representing a separate plate of cells. After B3GALNT2 reaction, 6 µL 0.5 M EDTA was added to 30 µL of reaction supernatant, and the mixture boiled for 5 min. 25 µL of this mixture and added to 30 µL ddH20 in HPLC vial and run on an LC18 column of a reverse-phase HPLC (Shimadzu Scientific) with a 16% B med sensitivity gradient. The reaction was analyzed using a 4.6 × 250 mm Supelcosil LC-18 column . Solvent A was 50 mM ammonium formate (pH 4.0), and solvent B was 80% acetonitrile in solvent A. Elution of the MU derivative was monitored by fluorescence detection  and peak area used as a measure of activity. The enzymatic activity was calculated as the peak area of the product.To assess B3GALNT2 activity in HAP1 cells, 20 µL of the WGA beads from HAP1 cells were incubated with a 20 µL volume of the reaction mix. The final volume of reaction buffer was 40 µL (30 µL reaction mixture and 10 µL WGA beads). The final concentrations were 10 mM MgClT. maritima β-glucuronidase (S. solfataricus α-xylosidase (Xylsa), both bearing a His-tag were overexpressed in E. coli, and purified using Talon metal affinity resin as described and activity determined as described (g), the crude extract was incubated with Benzonase (Novagen) for 1 hr at room temperature and then heat-fractionated for 10 min at 75°C. The supernatant was purified by using Talon metal affinity resin.Exoglycosidase treatment was carried out as described previously . T. marironidase  (Bgus) aescribed  with somSamples to be digested by Bgus and Xylsa were exchanged into 150 mM sodium acetate (pH 5.5) solution and mixed with Bgus (0.45 U) and/or Xylsa (0.09 U), or no enzymes, and incubated overnight at 65°C. Samples were then run on SDS-PAGE, transferred to PVDF-FL (Millipore), and probed with anti-α-DG core antibody (AF6868) and anti-α-DG glycan antibody (IIH6). Enriched rabbit α-DG (100 μL of the 150 mM sodium acetate (pH 5.5) solution) was mixed with Bgus (0.45 U) and/or Xylsa (0.09 U), or no enzymes, and incubated overnight at 65°C. Samples were then run on SDS-PAGE, transferred to PVDF-FL (Millipore), and subjected to immunoblotting.2O2, and reactions were stopped with 2 N H2SO4. Absorbance per well was read at 490 nm by a microplate reader.Solid-phase assays were performed as described previously . BrieflyLoxP/LoxPPomk skeletal muscle, respectively) reaction was set to 1. Percent conversion of each experimental reaction was subsequently normalized to that of the control, and statistics on normalized values were performed using GraphPad Prism 8. For analysis of POMK and LARGE1 activity in fibroblasts and mouse skeletal muscle, Student’s t-test was used (two-sided). Differences were considered significant at a p-value less than 0.05. Graph images were also created using GraphPad Prism and the data in the present study are shown as the means + / - SD unless otherwise indicated. The number of sampled units, n, upon which we report statistics for in vivo data, is the single mouse (one mouse is n = 1).The included Shimadzu post-run software was used to analyze POMK, LARGE1, and B4GAT1 activity in fibroblasts and mouse skeletal muscle, and the percent conversion to product was recorded. The means of three experimental replicates  were calculated using Microsoft Excel, and the mean percent conversion to product for the WT or control sample -MU was first calculated using the included Shimadzu analysis software. The means plus standard deviations of the percent conversion to GGM(P)-MU for three experimental replicates was calculated using GraphPad Prism 8. One-way ANOVA with the Dunnett’s Method for Multiple Comparisons was performed, and the data for the To measure POMK activity in control and NH13-284 skeletal muscle, we only performed one experimental replicate due to the limited amount of sample available. To measure B4GAT1 activity, two technical replicates were performed from skeletal muscle. Protein concentration from control and NH13-284 skeletal muscle was also measured using two technical replicates. The percent conversion to product for the B4GAT1 reaction was divided by the protein concentration, and the values for these two technical replicates graphed using GraphPad Prism 8. The graph reported is shown as the mean + / - SD.For flow cytometry analyses, six experimental replicates were performed, and the mean fluorescence intensity (MFI) reported. Statistics were performed using the Student’s unpaired t-test, two-sided in GraphPad Prism eight and the values reported as mean + / - SD.1H NMR spectra of the core M3 trisaccharides GGM-MU and GGMp-MU in the absence and presence of POMK or LARGE1 were acquired at 25°C on a Bruker Avance II 800 MHz NMR spectrometer equipped with a sensitive cryoprobe by using a 50 ms T2 filter consisting of a train of spin-lock pulses to eliminate the broad resonances from the protein (Danio rerio POMK titrations were performed in 25 mM Tris (pH 8.0), 180 mM NaCl, and 10 mM MgCl2 in 98% D2O. LARGE1 titrations were performed in 20 mM HEPES, 150 mM NaCl, pH 7.3 in 90% H2O/10% D2O. The 13C and 1H resonances of the trisaccharides were reported previously , and cells infected at high MOI (250–1000) of adenovirus expressing DG390TEVHis. Three days later, the medium was harvested and stored at 4°C until samples were ready for MS analysis.Reductive elimination. Glycans were reductively eliminated from DG390 proteins and puriﬁed on a 50WS8 Dowex column, and the puriﬁed glycans were subjected to permethylation and puriﬁed according to published methods . Brieﬂy,Permethylation. For the permethylation reaction, three to ﬁve pellets per sample of sodium hydroxide were crushed in 3 mL dry dimethyl sulfoxide. Methyl Iodine (500 µL) as well as the resulting slurry (0.75 mL) were added to the sample. The mixture was agitated for 15 min and quenched by adding 2 mL ultrapure water with shaking. The glycans were extracted with chloroform (2 mL) and washed twice with ultrapure water. Chloroform was removed under a stream of nitrogen. The permethylated glycans were loaded on a C18 Sep-pak column, washed with 5 mL ultrapure water and successively eluted with 3 mL each of 15, 35, 50, and 75% aq. acetonitrile. The solutions were collected and lyophilized. The lyophilized 35 and 50% fractions were dissolved in 50% aqueous solution of methanol and combined for MALDI analysis.Mass spectrometry. A Bruker Autoflex III MALDI-TOF/TOF was used for acquisition of all MALDI MS data. An in-house made BSA digest was used to calibrate the MS mode. 3,4-diaminobenzophenone was used as the matrix. Permethylated samples were dissolved in 10 mL of methanol, and 1 µL of this solution was premixed with 1 µL matrix. 1 µL of this mixture was spotted on the plate. In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:The study beautifully demonstrates that POMK is required for the synthesis of full- length and high-molecular weight forms of matriglycan, and that LARGE1 requires DGN to synthesize the short, non-extended form of matriglycan but needs both the DGN and the phosphorylated core M3 to generate full-length matriglycan.Decision letter after peer review:eLife. Your article has been reviewed by two peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Michael Marletta as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Adnan Halim (Reviewer #1); James Paulson (Reviewer #2).Thank you for submitting your article "POMK regulates dystroglycan function via LARGE-mediated elongation of matriglycan" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.eLife but that some conclusions require a modest amount of additional new data, as they do with your paper, we are asking that the manuscript be revised to either limit claims to those supported by data in hand, or to explicitly state that the relevant conclusions require additional supporting data.We would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). Specifically, when editors judge that a submitted work as a whole belongs in eLife, either of which would be linked to the original paper.Our expectation is that authors will eventually carry out the additional experiments and report on how they affect the relevant conclusions either in a preprint on bioRxiv or medRxiv, or if appropriate, as a Research Advance in Both reviewers comment that the work is elegant and convincing in demonstrating the role of POMK in synthesis of matriglycan, the glycan polymer of α-dystroglycan (α-DG), and how its role in muscular dystrophy. Given that the number of comments/suggestions from reviewers is relatively few, we have opted to include them all in this decision letter, so that these can be addressed directly in revision, should you chose to revise the paper.Summary:O-Man glycans found on α-dystroglycan. Using cell lines, muscle specific Pomk KO mice, and patient muscle samples, the authors demonstrate convincingly that POMK driven mannose influences length of [GlcA-Xyl]n repeats, as shown by Western blotting techniques. Furthermore, the authors characterize muscle physiology in mice with muscle specific KO of Pomk. Finally, binding affinities of Man/Man-6-P variants of core M3 to POMK and LARGE enzymes are determined, providing mechanistic details to support of their data. Collectively, this elegant study provides important insight into the role of POMK in matriglycan biosynthesis.Walimbe et al. aims to investigate the role of POMK in relation to matriglycan biosynthesis on core M3 The synthesis the extended glycan polymer on α-DG, matriglycan, is controlled by over a dozen genes, and deficiencies in each lead to muscular dystrophies, illustrating the importance of the glycan as a scaffold in the muscle extracellular matrix, and the highly coordinated biosynthetic machinery involving each of these gene products needed to produce fully active matriglycan.While the role of POMK was previously known to attach phosphate to the mannose of the trisaccharide core that bridges matriglycan to α-DG , why the absence of this phosphate impacted the maturation of matriglycan was not known.n. Here they clearly show that without POMK the matriglycan chains are short, resulting in α-DG of ~90 kDa, and with functional POMK, the extended matriglycan chains results in α-DG of ~150 kDa. A critical experiment is showing by NMR titration that LARGE binds to the tetrasaccharide core to initiate synthesis of the polymer, and that the phosphate product of POMK increases affinity of LARGE by an order of magnitude.Through characterization of α-DG in a patient with POMK deficiency, and detailed biochemical and genetic experiments the authors show that the P-Man product is required for polymerization of matriglycan, even though the P-ManαThr/Ser product is remote from the action of the polymerase (LARGE) that adds the repeating disaccharide of matriglycan (-GlcAβ1-3Xylα1-3-)Overall the results are compelling, and the design of the experiments, and clear presentation of results and conclusions are very well done. The synthesis of matriglycan is arguably the most intricate post-translational modification in human biology, and the additional insights in the role of POMK represent a significant advance to the understanding of its role, and how its deficiency produces a mild form of muscular dystrophy.Essential revisions:Reviewers did not have specific required revisions.Minor points:n repeats.1) The authors consistently observe a band at ~250 kDa in their laminin overlays  but do not explain or comment as to why this reactivity is observed. It would be of general interest to know if this is unspecific binding, perhaps with a reference to a study or data to support this claim. Alternatively, if the authors consider the reactivity as specific, they could perhaps comment on the possibility that proteins other than α-dystroglycan could harbor laminin binding [GlcA-Xyl]LoxP/LoxPPomk  speaks for itself, however, it is difficult to understand what e.g. POMK/ISPD KO and KO + Ad-ISPD .2) Annotation of genetic background of engineered cells is difficult to understand unless main text and figure legends are read in parallel. The authors should consider editing the annotation to make it clearer and uniform so that figures can be understood without necessarily having to read main text. For example, 3) There appears to be an inconsistency for the size of α-dystroglycan/matriglycan reactivity in laminin overlays. For example, comparing Fig 5B and 5D, the majority of reactivity in 5B appears to be slightly below the 150 kDa marker - however, in 5D, the majority of reactivity is seen at-and-slightly-above 150 kDa. For HAP1 cells, reactivity is clearly below 150 kDa.4) It is unclear why α-DG reactivity is observed above 150 kDa in Fig 5A when IIH6 detects α-aDG at <150 kDa.O-glycans. The authors should also annotate all peaks (at-and-above the intensity of m/z 873.5) with their m/z values, including the abundant peaks that have been cropped. MALDI-TOF does not allow the authors to determine anomeric or epimeric configuration of annotated O-glycans (color coded), the authors should therefore consider including a sentence in figure legend to clarify this limitation to the reader.5) Fig 6D-E, mass spectrometry: The authors should include Y-axis to allow the reader to see relative abundances of annotated 6) Subsection “POMK assay”: It is unclear to me why the authors perform WGA enrichment of POMK from HAP1 and fibroblast cells, but not from mouse and human skeletal muscle.7) Subsection “Mass Spectrometry” – Permethylation: Methyl Iodine (500 mL) seems excessive?8) Subsection “Mass Spectrometry” – –Permethylation: Permethylated glycans where purified on C18 Sep-pak columns and eluted in different fractions, however, it is unclear which fractions where analyzed by MALDI-TOF.9) Subsection “Data Availability”: Raw mass spectrometry data is not made available for readership. Will authors deposit their MALDI-TOF data in a public repository?10) There should be a better discussion of the binding of LARGE to DGN and the P-Man core. For example, how far is the DGN binding site from the glycan chains in the middle of α-DG. Do the authors envision that LARGE forms a complex with simultaneous binding to both DGN and a and P-Man, or that DGN binding recruits LARGE, and it dissociates to bind P-Man and while bound to extend matriglycan? At a minimum, it would be helpful to the reader to better describe the physical relationships between DGN and the sites of attachment of the glycan cores to α-DG. Since there were only minor comments/suggestions for the author to address in revision there is no accompanying author response.


HP G0 and G1/G2-Vs exhibit a comparable decrease in lactosylceramide and an increase in the globotriaosylceramide content. An analysis of the main glycohydrolases activity involved in glycosphingolipid catabolism showed an overall decrease in the activeness of the tested enzymes, irrespective of the type of APOL1-Vs expression. Similarly, the high throughput cell live-based assay showed a comparable increased action of the plasma membrane glycosphingolipid-glycohydrolases in living cells independent of the genetic APOL1 expression profile. Importantly, the most significative modification of the sphingolipid pattern induced by APOL1-Vs occurred in DRM resulted with a drastic reduction of radioactivity associated with sphingolipids. G1/G2-Vs present a decrease amount of globotriaosylceramide and globopentaosylceramide compared to G0. Additionally, ceramide at the DRM site and lactosylceramide in general, showed a greatest fall in G1/G2 in comparison with G0. Additionally, the levels of glucosylceramide decreased only in the DRM of human podocytes overexpressing G1/G2-Vs. These findings suggest that altered sphingolipidsprofiles may contribute to the deranged functionality of the plasma membrane in APOL1 risk milieu.Apolipoprotein L1 (APOL1) wild type (G0) plays a role in the metabolism of sphingolipids, glycosphingolipids, sphingomyelin and ceramide, which constitute bioactive components of the lipid rafts (DRM). We asked whether APOL1 variants (APOL1-Vs) G1 and G2 carry the potential to alter the metabolism of sphingolipids in human podocytes. The sphingolipid pattern in HPs overexpressing either APOL1G0 or APOL1-Vs was analysed by using a thin mono- and bi-dimensional layer chromatography, mass-spectrometry and metabolic labelling with [1-The online version of this article (10.1007/s10719-020-09944-w) contains supplementary material, which is available to authorized users. Trypanosoma brucei responsible for African sleeping sickness. sphingosine ([1-3H]Sph). As expected, the SL pattern of HPs identified by HPTLC and MS analysis covers both neutral and polar SL species. Among analyzed complex SLs, the most abundant chain lengths of fatty acids present in HPs and their unsaturation degree were C16:0, C22:0, C24:1 and C24:0. The list and structure of all identified SLs in HPs is reported in supplementary Table One of the main challenges in studying primary HPs is due to their terminally differentiated phenotype that has been overcame in part, by the y of HPs –35; therate with -3Hsphing3, covering near 5% of total SLs. Moreover, we identified GM1b and GD1α, and the mono and disialylated forms of globopentaosylceramide (Gb5Cer). Quantification of the single SL types carried out on both endogenous and metabolically tritium labeled SLs resulted in comparable results. Indeed, as is shown in supplementary Fig. The endogenous and the metabolically tritium labeled SL pattern is reported in supplementary Fig. 3 between ganglioside species (5.2% ± 0.3%). In total SLs we also found globopentaosylceramide (4.9% ± 0.9%) and its two sialylated forms, monosialosyl-globopentaosylceramide and disialosyl-globopentaosylceramide, that represent 4.5% ± 0.3% and 0.4% ± 0.2%, respectively. These structures do not belong to the isogloboseries due to the lack of expression in HPs of A3GALT2 gene encoding for this enzyme. In fact, gene expression level of A3GALT2 was undetectable in whole-transcriptome analysis generated in human immortalized podocytes (HPs) and downloaded from the NCBI SRA database . On the other hand, a high expression level of A4GALT gene was revealed, thus indicating that detected globopentaosylceramides in HPs belong to the globoseries (data not shown). The GA1 reaches 2.4% ± 0.4% of all SLs, and the gangliosides GM1b and GD1α, constitute respectively 3.4% ± 0.4% and 1% ± 0.3% among all SLs. In addition, whole transcriptional data analysis in HPs evidenced gene expression of B4GALNT1 enzyme, thus confirming the presence of 0-series gangliosides. The distribution of neutral SLs in HPs represents the following percentages listed in raising order: ceramide 5.7% ± 1%; glucosylceramide 12.5% ± 2.1%; lactosylceramide 15.6% ± 1.7%, and globotriaosylceramide 18% ± 2.6%.Common to other human cell types, also in HPs the main SL among non polar compounds are sphingomyelin  and GMThe mass spectra (MS1) with its corresponding fingerprints (MS2) shown in supplementary Fig.m/z 1626.90, 1335.79, 1173.75, 970.67, 808.62 and 646.56, suggests a Neu5Ac-Hex-HexNHAc-Hex-Hex-Cer structure characteristic of GM1, with the lack of the fragment ion at m/z 1261.78 that is characteristic for the GM3 structure Neu5Ac-Hex-Hex-Cer. Moreover, when this ganglioside was treated with V. Cholera sialidase, GM1 converted to gangliotetraosylceramide (data not shown). The V. Cholera sialidase in vitro does not act on the inner sialic acid, confirming the position of the residue of sialic acid, bound to the terminal Gal. Taken all together, these results indicate that the sialidase-labile GM1 is GM1b, as also suggested by others sphingosine dissolved in methanol is transferred into a sterile glass tube and dried under a nitrogen stream and the residue then dissolved in an appropriate volume of pre-warmed (37 °C) cell culture medium to obtain a final concentration of (3 × 10)−8 M. Cells are incubated for a 2 h pulse and, after that, the medium was removed and replaced with fresh medium without radioactive sphingosine for the 48 h. At the end of the chase periods, cells were collected for the SLs analyses and DRM preparation [[1-paration .3VO4, 1 mM phenylmethylsulfonyl fluoride, and 75 milliunits/ml aprotinin, 3–4 mg of cell protein/ml) and Dounce homogenized . Cell lysate is centrifuged  to remove nuclei and cellular debris. The postnuclear fraction is mixed with an equal volume of 85% sucrose (w/v) in 10 mM Tris buffer (pH 7.5), placed at the bottom of a discontinuous sucrose concentration gradient (30–5%) in the same buffer, and centrifuged  at 4 °C. After ultracentrifugation, eleven fractions are collected starting from the top of the tube [Cells were lysed in lysis buffer  using glass plates silica gel 60 (Merck) as stationary phase and the solvent systems used as mobile phase were: first run in chloroform-methanol 2:1, followed by a second run with the solvent systems chloroform-methanol-0.2% aqueous CaCl2, 50:42:11 (v:v:v), for the polar sphingolipids; a run with the solvent systems chloroform-methanol-water 110:40:6 (v:v:v) for the neutral lipids. Polar sphingolipids are also separated bi-dimensional thin layer chromatography using: a first run with the solvent systems chloroform-methanol-0.2% aqueous CaCl2, 50:42:11 (v:v) followed by a second orthogonal run in chloroform-methanol-0.2% aqueous CaCl2, 50:42:11 (v:v). Radioactive lipids were visualized by digital autoradiography  and quantified using BioSpace Lab’s M3 Vision software. Identification of lipids after separation was assessed by comigration with radioactive lipid standards.Radioactive lipids associated with total cell lysates, DRM and high density (HD) fractions were extracted twice with chloroform/methanol 2:1 (v:v) and chloroform/methanol/water 20:10:1 (v:v); the total lipid extracts were subjected to a two-phase partitioning by adding 20% water to the lipid extract accordingly to , 53. TheFor endogenous sphingolipid pattern, total lipid extract is subjected to a two-phase partitioning as previously described  resultinIn addition, part of the samples was used for mass analyses.MS analyses are carried out using a Thermo Quest Finnigan LCQDeca ion trap mass spectrometer  equipped with an ESI ion source and an Xcalibur data system and a TSP P4000 quaternary pump HPLC. Separations of all SLs (sphingolipids) are obtained on a 5 μm, 250x4mm LiChrospher 100 RP8 column (Merck).5Cer and its sialylated forms, and gangliosides molecular species is carried out, at a flow rate of 0.5 ml/min, using a gradient formed by the solvent system A, consisting of CH3CN/5 mM ammonium acetate buffer, pH 7 (15:85 by volume) and solvent B containing CH3CN/H2O (85:15 by volume). The gradient is linear from 30:70 to 20:80, by volume, of A:B, over 25 min, followed by a 5 min gradient from 20:80 to 0:100, by volume, of A:B, followed by 15 min of isocratic condition at 100% of B.Elution of GbElution of SM, Cer and other neutral SLs molecular species is carried out, at a flow rate of 0.5 ml/min, with a gradient formed by the solvent system A, consisting methanol/water (90:10 by vol), and solvent system B, consisting of methanol, both containing 5 mM ammonium acetate. The gradient elution program is as follows: 5 min with solvent A; 5 min with a linear gradient from 100% solvent A to 100% solvent B; 15 min with 100% solvent B; 5 min with a linear gradient from 100% solvent B to 100% methanol. Methanol is also used to wash the column for 10 min, followed by equilibration procedure with solvent A for 15 min.5Cer and its sialylated forms, and ganglioside molecular species MS analyses include sheath gas flow of 70 arbitrary units, auxiliary gas flow of 10 arbitrary units, spray voltage of 4 kV, capillary voltage of −42 V, capillary temperature of 260 °C, fragmentation voltage (used for collision induced dissociation) of 40–80%. Mass spectra are acquired over a range m/z 200–2000.Optimum conditions for GbOptimum conditions for SM, Cer and other neutral SLs molecular species MS analyses include sheath gas flow of 50 arbitrary units, spray voltage of 4 kV, capillary voltage of −47 V, capillary temperature of 260 °C, fragmentation voltage (used for collision induced dissociation) of 40–60%. Mass spectra are acquired over a m/z range 200–1600.For all experiments, source ion optics are adjusted to accomplish desolvation of ions while minimizing fragmentation.Vibrio cholera sialidase; then the samples were dried and used for HPTLC.For sialidase treatment, aliquots of polar sphingolipids, in a total volume of 0,1 ml of aqueous solution, were incubated for 20 h at 37 °C with 0,02 unit of The enzymatic activities associated with total cell lysates were determined using a previously described method based on 4-Methylumbelliferone (MUB)-derived fluorogenic substrates , 55: 4-m6 cells per hour.Cells were plated in 96-well microplates at a density of 20,000 cells/well, and plasm membrane-associated activities of GCase, NLGase, α-galactosidase, β-galactosidase, and β-hexosaminidase were assessed in these cells by a high throughput cell lived-based assay as previously described , 55. To http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Raw reads were mapped to the human reference genome (Gencode GRCh38 v24 primary assembly) using STAR aligner (v2.5.2a) [p values in order to select the differentially expressed genes (DEGs) to use in downstream analysis. Exploration data analysis  was performed using built-in functions of the DESeq2 package.Raw sequencing single-end reads were processed for quality check using FASTQC (v0.11.5) (v2.5.2a) . Gencodev2.5.2a) . Gene exv2.5.2a)  using dev2.5.2a) , testingt test or one-way ANOVA using GraphPad Prism 8 software .All the experiments were performed in triplicate and repeated ≥3 times. Data are presented as the means ± S.D. and were tested for significance with Student’s ESM 1(PDF 643 kb)


Caenorhabditis elegans intestine consists of 20 cells harboring a dense intermediate filament network positioned below the apical plasma membrane that forms a junction-anchored sheath around the intestinal lumen. This evolutionarily conserved arrangement provides mechanical and overall stress-protection, and it serves as an important model for deciphering the role of intestinal architecture in metazoan biology. We recently reported that the loss-of-function mutation of the intestinal intermediate filament organizer IFO-1 perturbs this architecture, leading to reduced body size and reproduction. Here, we demonstrate that the IFO-1 mutation dramatically affects cholesterol metabolism. Mutants showed an increased sensitivity to cholesterol depletion, reduced cholesterol uptake, and cholesterol transfer to the gonads, which is also observed in worms completely lacking an intermediate filament network. Accordingly, we found striking similarities to transcriptome and lipidome profiles of a nuclear hormone receptor (NHR)-8 mutant. NHR-8 is homologous to mammalian LXR (liver X receptor) that serves as a sterol sensor and transcriptional regulator of lipid metabolism. Remarkably, increasing exogenous cholesterol partially rescues the developmental retardation in IFO-1 mutants. Our results uncover a novel link of the intestinal intermediate filament cytoskeleton to cholesterol metabolism that contributes to compromised growth and reproduction.The intestine is an organ essential to organismal nutrient absorption, metabolic control, barrier function and immunoprotection. The  Caenorhabditis elegans intestine provides an ideal in vivo system to study intestinal structure–function relationships. The adult intestine consists of only 20 cells that are arranged as nine twisted rings (from int1 to int9), consisting of four (int1) or two cells (from int2 to int9) ::cfp] *kcIs6]IV strain has been described in , 1 mM MgSO4, and 0.5 mM CaCl2) were prepared containing either 5 mg/L cholesterol in ethanol (control condition) or an equal amount of pure ethanol. The plates were inoculated with an overnight culture of OP50 E. coli, which was washed two times with the M9 buffer and re-suspended in 1/5 of its volume. Synchronized L1 larvae, which had hatched overnight on NGM plates without food and cholesterol, were transferred onto the test plates and incubated at 20 °C. To prevent overcrowding, a chunk of agar containing F2 larvae (if any) was transferred onto a fresh plate at day 3. Representative pictures were taken at days 4 and 6 using an SMZ1500 stereoscopic zoom microscope equipped with an µEye USB camera and a Plan Apo WD70 1× objective .For cholesterol deprivation experiments, 10 cm agarose plates  and incubated at 20 °C. The developmental stage was monitored daily, and gravid adults were transferred to a fresh plate each day to count the total offspring from each animal.w/v) agarose, 52 mM NaCl) with and without cholesterol, was poured into 24-well plates and inoculated with an overnight culture of OP50 E. coli. The experiment was started by placing several embryos of bleached animals into the wells (see above). Early larvae (L1/L2) and total offspring were counted after 63 and 72 h at 20 °C.For the measurement of larval arrest, 1 mL of agarose (1.75% (w/v) and 52 mM NaCl), inoculated with 800 µL of an overnight culture of OP50 E. coli, supplemented with 0.5 µCi of [4-14C]-cholesterol . Synchronized L1 larvae, which had hatched overnight on NGM-plates without food and cholesterol, were placed on the feeding plates and allowed to grow to adulthood at 20 °C. The fed animals were washed two times with an M9 buffer, and groups of 20 animals each were either transferred to normal NGM plates or collected for lysis. The animals on the NGM plates were allowed to lay eggs for 3 days at 15 °C, after which they were removed and collected for lysis. After 2 days at 20 °C, the F1 generation was flushed off the plates, washed two times with an M9 buffer, and subjected to lysis. Lysis was carried out for 1 h at 65 °C in a single-worm lysis buffer, containing 0.5 µg/µL proteinase K. Afterwards, all samples were mixed with 5 mL of scintillation liquid Irgasafe plus , and radioactivity was measured in a Hidex 600 SL liquid scintillation counter .Cholesterol feeding experiments were carried out at 20 °C on 10 cm agarose plates  were previously published in [ifo-1(kc2) were published in [Transcriptomic data for ished in , and traished in . Both liished in .7-dafachronic acid and Δ4-dafachronic acid were from Biomol .The following materials were used for lipid extraction and analysis: UHPLC methanol, butylated hydroxytoluene, ammonium formate and ethyl acetate from Sigma-Aldrich ; UHPLC grade water, UHPLC grade acetonitrile, and formic acid from Biosolve ; C4 ceramide (d18:1/4:0), C10 ceramide (d18:1/10:0), C12 GluCer (d18:1/12:0), and C17 Cer (d18:1/17:0) from Avanti Polar Lipids ; isopropanol from Carl Roth ; and chloroform from Merck . In addition, the following standards were obtained: 7 dehydrocholesterol and 7 ketocholesterol (d7) were from Sigma-Aldrich , and ΔC. elegans: Worms were collected at young adult stage, and homogenates were obtained by beads beating for 30 min at 4 °C using a Qiagen TissueLyser ; 0.1% (w/v) of butylated hydroxytoluene (BHT) was added to prevent autoxidation reactions. Protein concentrations were measured using the BCA assay kit  prior to lipid analyses. A volume of homogenate corresponding to 600 µg of protein was used for ceramide analysis, a volume of homogenate corresponding to 200 µg of protein was used for fatty acid analysis (FAME), and a volume of homogenate corresponding to 200 µg of protein underwent solid phase extraction (SPE) for steroid enrichment (see corresponding sections).Lipid extraction from Fatty acid analysis (FAME): GC–MS analyses were carried out on a GC/MS  interfaced with a robotic auto sampler PAL 1 system (PAL LHXxt). One microliter of the derivatized worm sample was injected into the liner  by the pulsed-split mode at 200 °C. Analytes were separated using a DB Ultra Inert column . The initial oven temperature was set to 180 °C and held for 1 min, and then the temperature was increased by 30 °C/min up to 300 °C and held at that temperature for 1 min. At the end, the temperature was increased up to 325 °C by 30 °C/min. The temperature of the transfer line was set to 325 °C, and helium was used as the carrier gas at a flow rate of 1.4 mL/min in a constant flow mode. Ions were generated using an electron impact ion source (70 eV and 300°C) and analyzed by a triple quadrupole. C13:0 fatty acid was used as internal standard. Data were analyzed using MassHunter Workstation Software, Qualitative Analysis, Version B.06.00 . The relative response was calculated by dividing the peak area of the analyte to the internal standard peak area and further normalized to protein concentration.v/v) mixture of methanol and chloroform and incubated for 1 h at 4 °C. Samples were then centrifuged for 5 min at 3000 rcf at 4 °C, and the liquid phase was transferred and dried in a speed vac. Samples were reconstituted with 60:40 (v/v) methanol:water, and 5 µL were injected into the LC–MS system. The identification of ceramides and glucosylated ceramides was performed on a Q Exactive Mass Spectrometer  coupled with a binary pump system . Lipid species were separated using an RP column  using solvents A (60:40 v/v acetonitrile: water with 10 mmol/L ammonium formate) and B (90:10 v/v isopropanol:water with 10 mmol/L ammonium formate), as previously reported [v/v) isopropanol water with 0.1% formic acid.Ceramide targeted analysis: Ceramides were extracted by adding a 2:1  and C12 GluCer d18:1/12:0 were used as internal standards. Data were analyzed using Xcalibur version 4.0 and Trace Finder version 4.1 . The relative response for each steroid species was calculated by dividing the peak area of the analyte to the internal standard peak area, and the result was further normalized to protein concentration.Ceramides and glycosylated ceramides were detected in negative ion mode using an H-ESI ion source with the following parameters: sheath gas of 10, auxiliary gas of 2, spray voltage of 3 kV, and ion transfer tube temperature of 250 °C. Data were acquired using a targeted-selected ion monitoring chromatogram mode (t-SIM) with a resolution of 70.000, AGC 5 ev/v) formic acid before adding the sample. The homogenate was directly applied to the column, and phospholipids were removed by adding 1 mL of hexane. Steroids were eluted using 500 µL of ethyl acetate, as previously described [v/v) methanol/water, and 5 µL were injected into the LC–MS/MS system.Steroid analysis: HLB prime cartridges  were pre conditioned with 1 mL of methanol and 1 mL of water with 1% (escribed . The orgv/v) formic acid and eluent B as acetonitrile with 0.1% (v/v) formic acid; 2 µL of the reconstituted sample were injected. The flow rate was 0.1 mL/min for the first 5 min and then increased to 0.150 mL/min thereafter. The column temperature was set at 30 °C. The ESI ionization parameters were as follows: 3.5 kV, 25 a.u. sheath gas, 5 a.u. auxiliary, and 350 °C transfer ion capillary. All the spectra were acquired in positive ion mode.LC–MS analysis was carried out on a triple quadrupole mass spectrometer (QqQMS) . Prior to the measurements, gradient optimization was performed using authentic standards. Steroids were separated with a reverse phase column  using a binary pump system  with solvent A as water with 0.1%  was used. Data were analyzed using Xcalibur version 4.0 and Trace Finder version 4.1. The relative response for each steroid species was calculated by dividing the peak area of the analyte to the internal standard peak area, and the result was further normalized to protein concentration.


The present study was conducted to examine the adaptability of 11 sugar beet cultivars grown under drought stress in the controlled glasshouse. The treatment was initiated on 30-day-old sugar beet plants where drought stress was made withholding water supply for consecutive 10 days while control was done with providing water as per requirement. It was observed that drought stress expressively reduced plant growth, photosynthetic pigments, and photosynthetic quantum yield in all the cultivars but comparative better results were observed in S1 (MAXIMELLA), S2 (HELENIKA), S6 (RECODDINA), S8 (SV2347), and S11 (BSRI Sugarbeet 2) cultivars. Besides, osmolytes like proline, glycine betaine, total soluble carbohydrate, total soluble sugar, total polyphenol, total flavonoid, and DPPH free radical scavenging activity were remarkably increased under drought condition in MAXIMELLA, HELENIKA, TERRANOVA, GREGOIA, SV2348, and BSRI Sugar beet 2 cultivars. In contrast, activities of enzymes like superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) were significantly decreased in all, while the cultivars SV2347, BSRI Sugar beet 1 and BSRI Sugar beet 2 were found with increased ascorbate peroxidase (APX) activity under drought condition. In parallel, polyphenol oxidase (PPO) was increased in all cultivars except HELENIKA. Overall, the cultivars HELENIKA, RECODDINA, GREGOIA, SV2347, SV2348, BSRI Sugar beet 1, and BSRI Sugar beet 2 were found best fitted to the given drought condition. These findings would help further for the improvement of stress adaptive sugar beet cultivars development in the breeding program for drought-prone regions. To overcome the threats on the agriculture and ecosystem, top-most priority has been given to evaluate the plant response under hostile environments, such as heat, drought, cold, toxic metals, and nutrient deficiency ,2. TheseDrought tolerant or resurrection plants are quite rare in nature due to the complexity of the adaptation process for the plant. It down-regulates cell enlargement (near about four times higher than cell division)  as well Different morphological traits like relative water content (RWC), chlorophyll fluorescence, grain yield, plant biomass, and plant height have been used to evaluate drought stress ,15,16,172O2) and free radical  species, which are persistently detrimental to plants cells  and placed in the 16 cells hole growing pot (27 × 27 × 6 cm) containing commercial horticultural soil . The seedlings were irrigated daily using tap water to the field capacity level up to 30 days. Two treatments were imposed on morphologically uniformed and healthy seedlings, i.e., (i) seedlings were grown under normal growth condition with regular watering and (ii) seedlings were subjected to water deficit by withholding the water supply for 10 days. The sample (youngest completely formed leaves) was collected from each cultivar and immerged immediately in liquid nitrogen followed by freeze-drying and subjected to further analysis.Sugar beet seedlings were grown in a semi-controlled greenhouse in the department of Bio-Health Convergence, Kangwon National University, Chuncheon, Korea. The environmental condition such as temperature, relative humidity (RH) and average photoperiod was recorded at 30/25 °C (day/night), 60–70% RH and 12 h respectively. Seeds of sugar beet  leaves were extracted (10 mL of 80% acetone) and placed at room temperature for 15 min. The collected extract was transferred into a tube and centrifuged at 4000 rpm for 10 min. The absorbance was taken at 647, 663 and 470 nm, respectively using a spectrophotometer . Chlorophyll a, Chlorophyll b, Total chlorophyll and Carotenoid were determined according to the formula proposed by Lichtenthaler (1987)  and exprThe photosynthetic quantum yield (Fv/Fm) of photosystem II (PSII) was measured using the Fluor Pen FP 100  by measuring OJIP transient under the dark-adapted condition at least for 20 min .Leaves from three seedlings of each genotype from control and water stress treatment were collected and immediately weighted (FW). Later, the leaves were put in deionized water and weighed (g) again after 48 h to determine the leaf turgid weight (TW). Finally, dry weight (DW) was determined by placing in an oven at 60 °C for 48 h. The relative water content (%) of the leaves was calculated according to the following formula :RWC (%) The drought tolerance index given by Fernandez  and adju1) using a conductivity meter . The second set was also placed in hot water bath (100 °C for 15 min) to record its conductivity (C2) again. The membrane stability index (MSI) was measured as: MSI = [1 − (C1/ C2)] × 100.The membrane stability index (MSI) was taken by the methodology developed by Sairam et al. (2002) . Leaf sag for 5 min. After that, 4 mL of Trichloroacetic acid  containing thiobarbituric acid (0.5%) was added to 1 mL aliquot of supernatant in a test tube. The test tube was placed in a water bath and warmed at 95 °C for 30 min followed by cooled quickly on an ice bath. The resulting mixture was centrifuged again at 10,000× g for 15 min and the absorbance was taken at 532 nm. To avoid unspecific turbidity absorbance at 600 nm was subtracted from 532 nm and an extinction coefficient of 155 mM−1∙cm−1 was used to calculate the concentration. Lipid peroxidation was determined by estimating the malondialdehyde (MDA) content in leaves of sugar beet seedlings. MDA was calculated by following the method of Heath and Packer (1968)  with sliw/v) TCA and centrifuged at 12,000× g for 15 min. For every 0.5 mL of the aliquot of the supernatant, 0.5 mL of 10 mM potassium phosphate buffer (pH 7.0) and 1 mL of 1 M KI were added followed by incubation in darkness for 1 h. The absorbance was measured at 390 nm where 0.1% TCA was used as blank prove in place of leaf extract. A standard H2O2 curve was prepared to calculate the concentration of H2O2 in the sample.The estimation was done according to Singh et al. (2006) . Freeze-Proline concentration was determined by following the method of Bates et al. (1973) . Approxi−1, respectively. An improved HPLC method developed by Xu et al. (2018) [v/v) mixed with 0.2% phosphoric acid. Sample (2 µL) was injected with a flow rate of 0.5 mL∙min−1 where oven temperature was 35 °C and the detection wavelength was 195 nm.A freeze-dried sample (25 mg) was extracted with methanol (80%) and sonicated for 2 h at room temperature. The solution was filtered through a syringe filter . The stock solution of betaine was prepared in ultrapure water (10 mg/mL) and went under serial dilution with the mobile phase to prepare 10, 8, 6, 4, 2, and 1 mg∙mL. (2018)  was usedEach sample  was homogenized in 5 mL of ethanol (95%). The insoluble fraction of the extracts were washed with 5 mL of ethanol (70%) followed by centrifuging at 3500 rpm for 10 min and the supernatant was kept in a refrigerator (4 °C) for the determination of TSC and Sucrose content.−1 dry weight.TSC content was determined according to Khoyerdi et al. (2016) . Briefly−1 dry weight [In the case of sucrose content, 0.2 mL of the supernatant was mixed with 0.1 mL of KOH (30%) and heated at 100 °C for 10 min. After cooling at room temperature 3 mL of anthrone (150 mg anthrone mixed with 100 mL 70% sulfuric acid) was added. Ten minutes later, the samples were cooled, and absorbance was read at 620 nm. Sucrose concentration was calculated using the standard curve and the results were expressed as μg∙gy weight .g for 20 min at 4 °C. The enzyme extract was stored at 4 °C for analysis [2CO3, and 100 µL enzyme extract, and in case of the blank reaction mixture, 100 µL buffer was used instead of enzyme extract. After that, 200 µL of 0.1 mM Riboflavin was added to each mixture. The tubes were shaken and irradiated under the fluorescent light (15 W) for 15 min. The absorbance of each solution was measured at 560 nm. One unit of SOD represented as the quantity of enzyme that caused 50% inhibition of NBT reduction under the experimental conditions. Leaves samples were collected and immersed immediately in liquid nitrogen followed by freeze-drying and stored at −80 °C until use. A 25 mg sample was homogenized in 5 mL 50 mM sodium phosphate buffer (pH 7.8) using a pre-chilled mortar and pestle, then centrifuged at 15,000× analysis . The actanalysis  with sliv/v), 100 µL H2O2 (300 mM), and 2.7 mL 25 mM potassium phosphate buffer with 2 mM EDTA (pH 7.0). The increased rate of absorbance was measured spectrophotometrically at 470 nm (ε = 26.6 mM∙cm−1). The assay mixture for CAT contained 100 µL of enzyme extract, 100 µL of H2O2 (300 mM), and 2.8 mL of 50 mM potassium phosphate buffer with 2 mM EDTA (pH 7.0). CAT activity was assayed by observing decline absorbance reading at 240 nm (ε = 39.4 mM∙cm−1).The peroxidase  and catalase  activities were determined according to Zhang (1992) . The 3 m2O2 (300 mM), and 2.775 mL of 25 mM potassium phosphate buffer with 2 mM EDTA (pH 7.0). The oxidation of ascorbate was determined by the decrease of absorbance at 290 nm (ε = 2.8 mM∙cm−1). The activity of Ascorbate peroxidase  was assayed by the method developed by Nakano and Asada (1981) . A 3 mL The polyphenol oxidase  activity was determined following the method described by Tagele et al. (2019) . The reav/v in water) followed by sonication at 35 °C for 60 min. Afterward, the extracts were filtered  and kept in a refrigerator .The freeze-dried (25 mg) sample was dissolved in 10 mL of ethanol  was added. After that, 600 µL of distilled water was added to get the final volume (4 mL) and left for 1 h incubation at room temperature. The absorbance was taken at 725 nm, and the phenolic acid was calculated from a standard calibration curve of Gallic acid and expressed as µg∙g−1 dry weight.Folin–Ciocalteu method was performed to estimate the total phenolic content (TPC) of the sample . A react3)3  and 100 µL of potassium acetate (1 M) solution, and finally, 3.3 mL of distilled water was added to adjust the volume up to 4 mL. The reaction mixture was vortexed and left at room temperature for incubation for 40 min and the absorbance was measured at 415 nm by a UV-Vis spectrophotometer. The total flavonoid was measured as mg/g of Quercetin equivalent on a dry weight basis.The total flavonoid content (TFC) was estimated following the method described by Adnan et al. (2019) with modifications . In brieDPPH  was used to assess the antioxidant capacity of sugar beet leaf extract following the method described by Braca et al. (2003) . FirstlyAll results were expressed as mean ± SD (standard deviation) and mean ± SE (standard error) in tables and graphs respectively with 3 replications. All graphs were prepared by GraphPad prism 5 . A two-factor analysis followed by Duncan’s multiple range test (DMRT) was done by Statistix 10 . The correlation test was done by SPSS statistical software package . The least significant differences (LSD) were calculated to compare the means of different treatments with 5% level of probability.p < 0.05) under drought compared to the control condition in most of the genotypes  in drought compared to control condition (−1 dry weight), chlorophyll b (10.22 mg∙g−1 dry weight), and total chlorophyll content (33.28 mg∙g−1 dry weight) while the genotype S11 was detected as notable for higher Carotenoid content (5.25 mg∙g−1 dry weight). However, under drought condition, S10 genotype demonstrated remarkable tolerant efficiency with the highest pigments content, such as chlorophyll a (5.27 mg∙g−1 dry weight), chlorophyll b (1.88 mg∙g−1 dry weight), total chlorophyll (7.15 mg∙g−1 dry weight), and carotenoid (1.48 mg∙g−1 dry weight). Also, other significant cultivars including S2, S5, S7, S10, and S11 for control whereas S1, S2, and S9 genotypes were noteworthy for the drought stress condition. In the case of photosynthetic quantum yield analysis, no significant differences were observed among the genotypes, while it was varied significantly in drought conditions (p < 0.05) where S11, S7, S6, S4, S2, and S5 performed better than others.Chlorophyll a, chlorophyll b, total chlorophyll, and carotenoid significantly decreased  genotypes were noted for S5, S10, and S11. Overall, S9 genotype was recorded as higher MSI (%) in the case of both control (92.24) and drought condition (88.17) (Drought tolerance index (DTI) varied among the sugar beet genotypes ranging from 1.55 to 0.69 . The hig (88.17) .Evidence found that the cultivars with higher DTI under drought have a better carbon assimilation rate than that of lower DTI, but they may grow slowly and their productivity also hampered . But in RWC is one of the most important and reliable physiological indices by which assist to evaluate a particular genotype whether it is drought tolerant or not ,82. As s2O2) in drought compare to control (p < 0.05) (−1 dry weight) and H2O2 (2.48 µmol∙g−1 dry weight). Moreover, genotypes S9 (36.38 µmol∙g−1 dry weight), S3 (44.34 µmol∙g−1 dry weight), and S1 (43.91 µmol∙g−1 dry weight) in MDA as well as S1 (2.10 µmol∙g−1 dry weight), S2 (2.6 µmol∙g−1 dry weight), and S8 (3.1 µmol∙g−1 dry weight) in H2O2 demonstrated considerable drought tolerance in compare to control.In this experiment, almost all the experimental genotypes contained higher malondialdehyde (MDA) and hydrogen peroxide (H < 0.05) . During 2O2 was recorded from S6 (−8.22%) followed by S8 (8.3%), S1 (8.7%), and S9 (13.15%). It was stated that oxidative burst induced by drought stimulate to increase MDA level, has been regarded as an index of lipid peroxidation, membrane permeability, and finally cell injury [2O2 may cause cell damage, but depend on the strong ROS scavenging mechanisms; it may act as a signaling molecule [Amaranthus tricolor [2O2 increased in drought conditions and they augmented progressively with the increment of drought stress. Thus, a higher generation of MDA and H2O2 can boost up the damage of cell membranes more in sensitive genotypes than tolerant genotypes. In our study, MDA and H2O2 showed a positive correlation with each other  in all the genotypes in drought than the control condition. A similar result was also observed for glycine betaine (GB), total soluble carbohydrate (TSC), and sucrose in most of the case. It was observed that S4, S5, S7, and S11 produced higher proline than others (p < 0.05), while S1, S2, S4, S6, and S11 produced higher GB and S1, S5, and S10 produced both higher TSC and sucrose under drought condition.The sugar beet genotypes varied concerning individual osmolytes in their leaves . ProlineThe accumulation of proline is a useful indicator of stress in sugar beet which can act as a signaling molecule for modulation of mitochondrial functions, influencing cell proliferation and expression of specific stress tolerant genes ,95. FromGlycine betaine also important osmoprotectants produced in response to abiotic stress which can contribute to metabolic pathways like osmotic adjustments and osmoregulation consequently reduce the negative impact ,98. UndeThe production and accumulation of TSC and sucrose in the plant can be used to avoid the adverse effect of water stress . In a pr2O2) were significantly increased under drought stress. Generally, the plant creates its defensive mechanism to scavenge the ROS which in this regard positively produces a higher level of osmolytes. All the osmolytes maintained a positive correlation among them  and 2237.5 to 1636.9 (units∙g−1 dry weight) in control and drought, respectively. Higher SOD was recorded from the genotypes S10 followed by S11, S4, S8, S9, and S7 in control, while genotype S9 was followed by S11, S10, S6, S8, and S4 in that of drought condition. SOD increased in S9 (8.38%) but decreased in all other genotypes after 10 days of water rescission. However, the minimum reduction of SOD under drought condition was recorded by 4.14%, 4.53%, 6.37%, and 9.57% from the genotypes S11, S5, S6, and S8, respectively.The activities of antioxidant enzymes varied within the variety and treatments . Superox−1 dry weight) and 1.13 to 0.28 (mM∙g−1 dry weight) in control and drought treatment, respectively. Higher CAT was recorded from S10 followed by S11, S1, S8, and S7 in control, while S7 was followed by S9, S8 in that of drought condition. The activity of CAT was reduced in all genotypes under drought stress compared to the control condition. However, the minimum reduction was recorded at 9.89%, 12.83%, 21.52%, 24.75%, and 36.5% from the genotypes S5, S7, S9, S3, and S6 respectively.Catalase (CAT) ranged from 1.79 to 0.57 (mM∙g−1 dry weight) and 6.12 to 1.39 (mM∙ g−1 dry weight) in control and drought condition, respectively. Higher APX was recorded from S9 followed by S6, S7, S3, S10, and S2 in control, while S10 followed by S9, S8, S7, and S11 in that of drought condition. It was also observed that, only S8, S10, and S11 genotypes were found with increased APX in drought treatment compared to the control condition, while the opposite result was observed from others. Also, during peroxidase (POD) analysis, S3 (1.11 mM∙g−1 dry weight) followed by S6, S7, S9, S4, and S8 demonstrated higher enzyme activity in control whereas S2 (0.43 mM∙g−1 dry weight) followed by S3, S10, S8, and S6 in drought condition.Ascorbate peroxidase (APX) ranged from 6.68 to 3.63 (mM∙g−1 dry weight) and 3.89 to 0.59 (µM∙g−1 dry weight) in control and drought conditions, respectively. Among all the genotypes, higher PPO was recorded for S3 followed by S4 and S8 in control, while S5 followed by S4, S8, S1, S3, and S6 in that of drought condition. Maximum augmentation in drought was observed 471.5% followed by 191.2%, 189.2%, and 138% from S5 followed by S6, S1, and S4 respectively.In the case of polyphenol oxidase (PPO), higher PPO was observed in all the genotypes in drought compared to the control condition. PPO ranged from 1.5 to 0.34 (µM∙g2−•) into oxygen and H2O2 [Antioxidant enzyme defense activities had a high positive relation with increased abiotic stress . Under dand H2O2 ,107. It and H2O2 . In our and H2O2  that canand H2O2 . This hy2O2 (produced through the dismutation of O2−• in peroxisomes and chloroplasts) to H2O and O2 [2O2 and MDA accumulation, resulting in excitation of enzyme activities. A similar response was also observed in previous research on cotton [2O2 generation under drought stress which also stimulates to produce high antioxidant enzyme activity. Subsequently, this enzyme activity may minimize the adverse effect of abiotic stresses either by acting as a bio-signal and/or modulating resistant gene expression [2O, but it can regenerate o-diphenol to o-quinones by providing a source of reducing power with the close association of photosystems during photosynthesis [Besides, CAT, APX, and POD are also the major enzymes involved in the detoxification of HO and O2 ,110,111.n cotton . Usuallypression . Moreovepression . In a prpression . Howeverpression . In our pression ,117,118.ynthesis ,120.In general, under stress conditions plant physiological activities and performance decline with a decrease in antioxidant enzymes activities and an increase in ROS chemicals. These phenomena indicating an inability of plant cells against ROS chemicals. The activities and involvement of different antioxidant enzymes in ROS scavenging vary with some factors including plant species, stress severity and duration . Sairam p < 0.05) in the present experiment (−1 as GAE) and 690.37 to 340.74 (µg∙g−1 as GAE) in control and drought, respectively. Higher TPC was recorded from S4 followed by S3, S10, S8, S9, and S11 in control, while S3 was followed by S5, S6, S1, S2, and S7 in that of drought condition.Total phenolic content (TPC) was augmented in all the genotypes under drought stress (periment . TPC ranp < 0.05) (−1 as QE) and 5.93 to 4.11 (mg∙g−1 as QE) in control and drought treatment. Higher TFC was recorded from S6 followed by S9, S7, and S8 in control, while S3 was followed by S1, S2, S6, S9, S8, and S4 in that of drought condition.Total flavonoid content (TFC) is also influenced by drought stress in all the genotypes compared to control ( < 0.05) . TFC ranp < 0.05) in most of the genotypes  (Antioxidant capacity (DPPH) was observed higher under drought treatment than the control condition (and S11) . DPPH ra2O2-scavenging in the phenolic/ASA/POD system [Both TPC and TFC were found higher under drought stress compared to control in all the cultivars. In a previous experiment, it was observed that TPC, TFC, and total antioxidant activity were significantly increased with the severity of drought stress in Amaranthus leafy vegetables . In our D system .Achillea species [Generally, total antioxidant activity is the combined activities of both enzymatic and non-enzymatic antioxidants in plants. In a previous experiment, total antioxidant activity along with total polyphenol and total flavonoid content significantly increased in  species  and also species  by droug species . A plant species . Since d species . Antioxi species . In our Under drought conditions, seedlings growth and development, photosynthetic pigments and photosynthetic quantum yield (Fv/Fm) expressively reduced compare to control in all sugar beet genotypes. Significant reductions were also observed in membrane stability index, leaf relative water content, and activity of antioxidant enzymes. Positive correlations were noticed between growth parameters and photosynthetic traits, physiological parameters and antioxidant enzyme activities respectively. On the other hand, drought significantly increased the reactive oxygen species and osmotic adjusting molecules in sugar beet seedlings. A remarkable increment of secondary metabolites and antioxidant capacity was also observed in drought conditions. Although enzymes have a negative correlation with osmolytes and secondary metabolites, the reactive oxygen species maintained a positive correlation with them. Based on the results, higher activities of antioxidant enzymes, osmolytes, membrane stability index and relative water content with lower reactive oxygen species accumulation were the most important factors considering drought-tolerant sugar beet seedlings. After analyzing all the data, the cultivars S2 (HELENIKA), S6 (RECODDINA), S7 (GREGOIA), S8 (SV2347), S9 (SV2348), S10 (BSRI Sugarbeet 1), and S11 (BSRI Sugarbeet 2) were found best fitted under drought condition. The findings of the experiment can help further for better understanding of different mechanisms related to the adaptability of sugar beet in drought conditions to improve drought tolerance cultivars.