Direct Mapping of Additional Modifications on Phosphorylated O-glycans of α-Dystroglycan by Mass Spectrometry Analysis in Conjunction with Knocking Out of Causative Genes for Dystroglycanopathy
2016; Elsevier BV; Volume: 15; Issue: 11 Linguagem: Inglês
10.1074/mcp.m116.062729
ISSN1535-9484
AutoresHirokazu Yagi, Chu‐Wei Kuo, Takayuki Obayashi, Satoshi Ninagawa, Kay‐Hooi Khoo, Koichi Kato,
Tópico(s)Biotin and Related Studies
ResumoDystroglycanopathy is a major class of congenital muscular dystrophy caused by a deficiency of functional glycans on α-dystroglycan (αDG) with laminin-binding activity. Recent advances have led to identification of several causative gene products of dystroglycanopathy and characterization of their in vitro enzymatic activities. However, the in vivo functional roles remain equivocal for enzymes such as ISPD, FKTN, FKRP, and TMEM5 that are supposed to be involved in post-phosphoryl modifications linking the GalNAc-β3-GlcNAc-β4-Man-6-phosphate core and the outer laminin-binding glycans. Herein, by direct nano-LC-MS2/MS3 analysis of tryptic glycopeptides derived from a truncated recombinant αDG expressed in the wild-type and a panel of mutated cells deficient in one of these enzymes, we sought to define the full extent of variable modifications on this phosphorylated core O-glycan at the functional Thr317/Thr319 sites. We showed that the most abundant glycoforms carried a phosphorylated core at each of the two sites, with and without a single ribitol phosphate (RboP) extending from terminal HexNAc. At much lower signal intensity, a novel substituent tentatively assigned as glycerol phosphate (GroP) was additionally detected. As expected, tandem RboP extended with a GlcA-Xyl unit was only identified in wild type, whereas knocking out of either ISPD or FKTN prevented formation of RboP. In the absence of FKRP, glycoforms with single but not tandem RboP accumulated, consistent with the suggested role of this enzyme in transferring the second RboP. Intriguingly, the single GroP modification also required functional FKTN whereas absence of TMEM5 significantly hindered only the addition of RboP. Our findings thus revealed additional levels of complexity associated with the core structures, suggesting functional interplay among these enzymes through their interactions. The simplified analytical workflow developed here should facilitate rapid mapping across a wider range of cell types to gain better insights into its physiological relevance. Dystroglycanopathy is a major class of congenital muscular dystrophy caused by a deficiency of functional glycans on α-dystroglycan (αDG) with laminin-binding activity. Recent advances have led to identification of several causative gene products of dystroglycanopathy and characterization of their in vitro enzymatic activities. However, the in vivo functional roles remain equivocal for enzymes such as ISPD, FKTN, FKRP, and TMEM5 that are supposed to be involved in post-phosphoryl modifications linking the GalNAc-β3-GlcNAc-β4-Man-6-phosphate core and the outer laminin-binding glycans. Herein, by direct nano-LC-MS2/MS3 analysis of tryptic glycopeptides derived from a truncated recombinant αDG expressed in the wild-type and a panel of mutated cells deficient in one of these enzymes, we sought to define the full extent of variable modifications on this phosphorylated core O-glycan at the functional Thr317/Thr319 sites. We showed that the most abundant glycoforms carried a phosphorylated core at each of the two sites, with and without a single ribitol phosphate (RboP) extending from terminal HexNAc. At much lower signal intensity, a novel substituent tentatively assigned as glycerol phosphate (GroP) was additionally detected. As expected, tandem RboP extended with a GlcA-Xyl unit was only identified in wild type, whereas knocking out of either ISPD or FKTN prevented formation of RboP. In the absence of FKRP, glycoforms with single but not tandem RboP accumulated, consistent with the suggested role of this enzyme in transferring the second RboP. Intriguingly, the single GroP modification also required functional FKTN whereas absence of TMEM5 significantly hindered only the addition of RboP. Our findings thus revealed additional levels of complexity associated with the core structures, suggesting functional interplay among these enzymes through their interactions. The simplified analytical workflow developed here should facilitate rapid mapping across a wider range of cell types to gain better insights into its physiological relevance. Dystroglycanopathy is a group of congenital muscular dystrophies that arise from glycosylation defects of α-dystroglycan (αDG). 1The abbreviations used are:αDGα-dystroglycanB3GALNTβ-1,3-N-acetylgalactosaminyltransferaseCIDcollision-induced dissociationcore M1Gal-β4-GlcNAc-β2-Mancore M2Gal-β4-GlcNAc-β2-(Gal-β4-GlcNAc-β6-)Mancore M3GalNAc-β3-GlcNAc-β4-ManCDP-Rbocytidine diphosphate ribitolDMEMDulbecco's modified Eagle's mediumFBSfetal bovine serumFKTNfukutinFKRPfukutin-related proteinGlcAglucuronic acidGroglycerolGroPglycerol phosphateHCDhigher-energy collisional dissociationHRPhorseradish peroxidaseHexhexoseHexAhexuronic acidISPDisoprenoid synthase domain containingKOknockoutLCliquid chromatographyHexNAcN-acetyl hexosaminePOMTO-mannosyltransferasePOMKO-mannose kinasePentpentosePCRpolymerase chain reactionPOMGNTprotein O-mannose β-1,4-N-acetylglucosaminyltransferaseRboribitolRboPribitol phosphateMS/MS or MS2tandem mass spectrometryTAPtandem affinity purificationTMEM5transmembrane protein 5Xylxylose. The hallmark and cause of these diseases, which include Fukuyama-type congenital muscular dystrophy, muscle-eye-brain diseases, Walker-Warburg syndrome, and congenital muscular dystrophy type 1D, are a lack of functional O-mannosyl glycans of αDG capable of binding laminin (1.Chiba A. Matsumura K. Yamada H. Inazu T. Shimizu T. Kusunoki S. Kanazawa I. Kobata A. Endo T. Structures of sialylated O-linked oligosaccharides of bovine peripheral nerve a-dystroglycan. The role of a novel O-mannosyl-type oligosaccharide in the binding of a-dystroglycan with laminin.J. Biol. Chem. 1997; 272: 2156-2162Abstract Full Text Full Text PDF PubMed Scopus (380) Google Scholar, 2.Holt K.H. Crosbie R.H. Venzke D.P. Campbell K.P. Biosynthesis of dystroglycan: processing of a precursor propeptide.FEBS Lett. 2000; 468: 79-83Crossref PubMed Scopus (152) Google Scholar, 3.Wells L. The O-mannosylation pathway: glycosyltransferases and proteins implicated in congenital muscular dystrophy.J. Biol. Chem. 2013; 288: 6930-6935Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). To date, dozens of causative gene products of dystroglycanopathy have been identified, all of which have been demonstrated or are assumed to be involved in the synthesis of the laminin-binding glycans. A plethora of O-glycans including the normal O-GalNAc mucin types and all three core types of O-mannosyl glycans, namely, M1 (Gal-β4-GlcNAc-β2-Man), M2 [Gal-β4-GlcNAc-β2-(Gal-β4-GlcNAc-β6-)Man], and M3 (GalNAc-β3-GlcNAc-β4-Man) (4.Yoshida-Moriguchi T. Campbell K.P. Matriglycan: a novel polysaccharide that links dystroglycan to the basement membrane.Glycobiology. 2015; 25: 702-713Crossref PubMed Scopus (133) Google Scholar), have been identified on various sites of αDG. Remarkably, the functional laminin-binding glycans were found to be exclusively carried on the core M3 at specific Thr317/Thr319 sites (5.Hara Y. Kanagawa M. Kunz S. Yoshida-Moriguchi T. Satz J.S. Kobayashi Y.M. Zhu Z. Burden S.J. Oldstone M.B. Campbell K.P. Like-acetylglucosaminyltransferase (LARGE)-dependent modification of dystroglycan at Thr-317/319 is required for laminin binding and arenavirus infection.Proc. Natl. Acad. Sci. U.S.A. 2011; 108: 17426-17431Crossref PubMed Scopus (84) Google Scholar). The formation of this innermost base structure involves at least three enzymes, namely, protein O-mannosyltransferase 1/2 heterodimer (POMT1/2), protein O-mannose β-1,4-N-acetylglucosaminyltransferase 2 (POMGNT2/AGO61), and β-1,3-N-acetylgalactosaminyltransferase 2 (B3GALNT2) (6.Yoshida-Moriguchi T. Willer T. Anderson M.E. Venzke D. Whyte T. Muntoni F. Lee H. Nelson S.F. Yu L. Campbell K.P. SGK196 Is a glycosylation-specific O-mannose kinase required for dystroglycan function.Science. 2013; 341: 896-899Crossref PubMed Scopus (160) Google Scholar, 7.Yagi H. Nakagawa N. Saito T. Kiyonari H. Abe T. Toda T. Wu S.W. Khoo K.H. Oka S. Kato K. AGO61-dependent GlcNAc modification primes the formation of functional glycans on a-dystroglycan.Sci. Rep. 2013; 3: 3288Crossref PubMed Scopus (33) Google Scholar, 8.Stevens E. Carss K.J. Cirak S. Foley A.R. Torelli S. Willer T. Tambunan D.E. Yau S. Brodd L. Sewry C.A. Feng L. Haliloglu G. Orhan D. Dobyns W.B. Enns G.M. Manning M. Krause A. Salih M.A. Walsh C.A. Hurles M. Campbell K.P. Manzini M.C. Stemple D. Lin Y.Y. Muntoni F. Mutations in B3GALNT2 cause congenital muscular dystrophy and hypoglycosylation of a-dystroglycan.Am. J. Hum. Genet. 2013; 92: 354-365Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). Notably, this trisaccharide core can be further phosphorylated at the 6-position of O-mannose by protein-O-mannose kinase (POMK/SGK196) (6.Yoshida-Moriguchi T. Willer T. Anderson M.E. Venzke D. Whyte T. Muntoni F. Lee H. Nelson S.F. Yu L. Campbell K.P. SGK196 Is a glycosylation-specific O-mannose kinase required for dystroglycan function.Science. 2013; 341: 896-899Crossref PubMed Scopus (160) Google Scholar). On the other hand, functional laminin binding is known to require a polymeric Xyl-GlcA repeat sequence, the elongation of which is catalyzed by LARGE (9.Inamori K. Yoshida-Moriguchi T. Hara Y. Anderson M.E. Yu L. Campbell K.P. Dystroglycan function requires xylosyl- and glucuronyltransferase activities of LARGE.Science. 2012; 335: 93-96Crossref PubMed Scopus (223) Google Scholar). Because the Xyl-GlcA repeat is released from the core upon chemical hydrolysis of phosphoester linkages by hydrogen fluoride treatment, the laminin-binding glycan synthesized by LARGE was inferred to be extended from the phosphorylated core M3 via the phosphate added by POMK. However, the exact structural element bridging this missing link is lost by such chemical treatment and thus remains undescribed until very recently (10.Praissman J.L. Willer T. Sheikh M.O. Toi A. Chitayat D. Lin Y.Y. Lee H. Stalnaker S.H. Wang S. Prabhakar P.K. Nelson S.F. Stemple D.L. Moore S.A. Moremen K.W. Campbell K.P. Wells L. The functional O-mannose glycan on a-dystroglycan contains a phospho-ribitol primed for matriglycan addition.Elife. 2016; 5: e14473Crossref PubMed Scopus (72) Google Scholar, 11.Kanagawa M. Kobayashi K. Tajiri M. Manya H. Kuga A. Yamaguchi Y. Akasaka-Manya K. Furukawa J. Mizuno M. Kawakami H. Shinohara Y. Wada Y. Endo T. Toda T. Identification of a post-translational modification with ribitol-phosphate and its defect in muscular dystrophy.Cell Rep. 2016; 14: 2209-2223Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 12.Gerin I. Ury B. Breloy I. Bouchet-Seraphin C. Bolsee J. Halbout M. Graff J. Vertommen D. Muccioli G.G. Seta N. Cuisset J.M. Dabaj I. Quijano-Roy S. Grahn A. Van Schaftingen E. Bommer G.T. ISPD produces CDP-ribitol used by FKTN and FKRP to transfer ribitol phosphate onto a-dystroglycan.Nat. Commun. 2016; 7: 11534Crossref PubMed Scopus (84) Google Scholar, 13.Riemersma M. Froese D.S. van Tol W. Engelke U.F. Kopec J. van Scherpenzeel M. Ashikov A. Krojer T. von Delft F. Tessari M. Buczkowska A. Swiezewska E. Jae L.T. Brummelkamp T.R. Manya H. Endo T. van Bokhoven H. Yue W.W. Lefeber D.J. Human ISPD is a cytidyltransferase required for dystroglycan O-mannosylation.Chem. Biol. 2015; 22: 1643-1652Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). α-dystroglycan β-1,3-N-acetylgalactosaminyltransferase collision-induced dissociation Gal-β4-GlcNAc-β2-Man Gal-β4-GlcNAc-β2-(Gal-β4-GlcNAc-β6-)Man GalNAc-β3-GlcNAc-β4-Man cytidine diphosphate ribitol Dulbecco's modified Eagle's medium fetal bovine serum fukutin fukutin-related protein glucuronic acid glycerol glycerol phosphate higher-energy collisional dissociation horseradish peroxidase hexose hexuronic acid isoprenoid synthase domain containing knockout liquid chromatography N-acetyl hexosamine O-mannosyltransferase O-mannose kinase pentose polymerase chain reaction protein O-mannose β-1,4-N-acetylglucosaminyltransferase ribitol ribitol phosphate tandem mass spectrometry tandem affinity purification transmembrane protein 5 xylose. A major technical problem in identifying the elusive structural module linking the phosphorylated core M3 and the polymeric GlcA-Xyl units is the limiting mass spectrometry (MS) sensitivity in detecting a very large glycan or glycopeptide carrying multiple negative charges in the form of HexA and phosphate. Most of the analytical work to date has used recombinant αDG truncated at the C terminus to different extents but all containing at least the first 10 amino acid residues following the putative endogenous furin cleavage site R312 that releases the N-terminal domain (5.Hara Y. Kanagawa M. Kunz S. Yoshida-Moriguchi T. Satz J.S. Kobayashi Y.M. Zhu Z. Burden S.J. Oldstone M.B. Campbell K.P. Like-acetylglucosaminyltransferase (LARGE)-dependent modification of dystroglycan at Thr-317/319 is required for laminin binding and arenavirus infection.Proc. Natl. Acad. Sci. U.S.A. 2011; 108: 17426-17431Crossref PubMed Scopus (84) Google Scholar, 14.Kanagawa M. Saito F. Kunz S. Yoshida-Moriguchi T. Barresi R. Kobayashi Y.M. Muschler J. Dumanski J.P. Michele D.E. Oldstone M.B. Campbell K.P. Molecular recognition by LARGE is essential for expression of functional dystroglycan.Cell. 2004; 117: 953-964Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). For convenience in the purification, the truncated C terminus is also commonly fused to antibody Fc or other tags, which may or may not be further removed during sample preparation. Such a strategy typically yielded very large and heterogeneous tryptic glycopeptides that contain additional O-glycosylated serine/threonine downstream of the critical Thr317/Thr319 attachment sites (Fig. 1), which hampered high-quality MS/MS sequencing and unambiguous data interpretation (10.Praissman J.L. Willer T. Sheikh M.O. Toi A. Chitayat D. Lin Y.Y. Lee H. Stalnaker S.H. Wang S. Prabhakar P.K. Nelson S.F. Stemple D.L. Moore S.A. Moremen K.W. Campbell K.P. Wells L. The functional O-mannose glycan on a-dystroglycan contains a phospho-ribitol primed for matriglycan addition.Elife. 2016; 5: e14473Crossref PubMed Scopus (72) Google Scholar). Alternatively, a tryptic site can be engineered by converting the first threonine after T319 to lysine (T322K), which would not only further truncate the target tryptic glycopeptide carrying Thr317/Thr319, but at the same time abolish the extra O-glycosylation. After further glycosidase treatment to remove mucin-type O-glycans, a recent study using such truncated αDG constructs expressed in and purified from mouse NIH 3T3 cells succeeded for the first time in detecting the further modified phosphorylated core by MS analysis in negative ion mode (11.Kanagawa M. Kobayashi K. Tajiri M. Manya H. Kuga A. Yamaguchi Y. Akasaka-Manya K. Furukawa J. Mizuno M. Kawakami H. Shinohara Y. Wada Y. Endo T. Toda T. Identification of a post-translational modification with ribitol-phosphate and its defect in muscular dystrophy.Cell Rep. 2016; 14: 2209-2223Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). It was shown that a tandem ribitol phosphate unit (RboP-RboP) can be attached to the terminal GalNAc of phosphorylated core M3, which can then be extended further by repeating HexA-Pent units, consistent with the proposed model that is supported by in vitro enzymatic assays of the involved causative gene products including isoprenoid synthase domain containing (ISPD), fukutin (FKTN), and fukutin-related protein (FKRP) (11.Kanagawa M. Kobayashi K. Tajiri M. Manya H. Kuga A. Yamaguchi Y. Akasaka-Manya K. Furukawa J. Mizuno M. Kawakami H. Shinohara Y. Wada Y. Endo T. Toda T. Identification of a post-translational modification with ribitol-phosphate and its defect in muscular dystrophy.Cell Rep. 2016; 14: 2209-2223Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). It also indicated that ISPD is a cytidine diphosphate ribitol (CDP-Rbo) synthase and FKTN transfers a Rbo5P from CDP-Rbo to phosphorylated core M3, thereby providing an acceptor site for FKRP to form the RboP-RboP tandem repeat. The possibility has also been raised that TMEM5 serves as a xylosyltransferase using the RboP-RboP structure as an acceptor site to initiate the first step of Xyl-GlcA repeat sequence synthesis (10.Praissman J.L. Willer T. Sheikh M.O. Toi A. Chitayat D. Lin Y.Y. Lee H. Stalnaker S.H. Wang S. Prabhakar P.K. Nelson S.F. Stemple D.L. Moore S.A. Moremen K.W. Campbell K.P. Wells L. The functional O-mannose glycan on a-dystroglycan contains a phospho-ribitol primed for matriglycan addition.Elife. 2016; 5: e14473Crossref PubMed Scopus (72) Google Scholar). However, this rather unique glycosylation structural motif has yet to be detected intact on αDG expressed in other cells, including the widely used HEK293T cells or indeed any human cell type, owing to the aforementioned technical difficulties. It is not known whether there is structural variation associated with core M3 modifications that contributes to the tissue-specific glycosylation status of αDG (4.Yoshida-Moriguchi T. Campbell K.P. Matriglycan: a novel polysaccharide that links dystroglycan to the basement membrane.Glycobiology. 2015; 25: 702-713Crossref PubMed Scopus (133) Google Scholar) and impacts on its functions as an extracellular matrix receptor in the brain, heart, skeletal muscle, and kidney, or correlates with its reduced expression in human breast and colon cancers in relation to tumor progression (15.Sgambato A. Migaldi M. Montanari M. Camerini A. Brancaccio A. Rossi G. Cangiano R. Losasso C. Capelli G. Trentini G.P. Cittadini A. Dystroglycan expression is frequently reduced in human breast and colon cancers and is associated with tumor progression.Am. J. Pathol. 2003; 162: 849-860Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). By adopting similar truncation and tryptic site engineering of recombinant αDG, we demonstrate a highly sensitive analytical workflow from in-gel digestion to direct nano-LC-MS2/MS3 analysis without additional chemo-enzymatic treatments, for unambiguous identification of the target glycopeptides carrying a further modified phosphorylated core M3. Diagnostic fragment ions were afforded by complementary modes of MS2 in positive ion mode, which can be programmed for target MS3 and/or used for rapid filtering of large spectral data sets to allow meaningful manual data interpretation in anticipation of novel substituents. We provide the requisite MS evidence for similar occurrence of tandem RboP-Xyl-GlcA substituent and its incompletely, extended forms on the phosphorylated core M3 in HEK293T cells. Moreover, we performed CRISPR/Cas9 genome editing to create a panel of mutated HCT116 colon cancer cells lacking ISPD, FKTN, FKRP, or TMEM5 and mapped the glycosylation variants of recombinant αDG expressed by these cells to address the in vivo functions of these causative gene products. The following antibodies were used in this study: anti-Flag mAb (Sigma-Aldrich, St. Louis, MO); anti-myc (Wako, Osaka, Japan); anti-FKTN (Proteintech, Chicago, IL); IIH6 mAb (Millipore, Billerica, MA); anti-β-DG mAb (Novocastra, Newcastle, UK); horseradish peroxidase (HRP)-conjugated anti-human IgG-Fc goat Ab (Invitrogen, Carlsbad, CA); HRP-conjugated anti-mouse IgM mAb (Thermo Fisher Scientific, Waltham, MA); and HRP-conjugated anti-mouse IgG mAb (Invitrogen). For the expression of Fc-fused αDG recombinant proteins, amino acid substitutions and deletion mutants of αDG (αDG373(T322R)) (Fig. 1) were made by standard PCR and genetic engineering techniques using the expression plasmids as constructed previously (7.Yagi H. Nakagawa N. Saito T. Kiyonari H. Abe T. Toda T. Wu S.W. Khoo K.H. Oka S. Kato K. AGO61-dependent GlcNAc modification primes the formation of functional glycans on a-dystroglycan.Sci. Rep. 2013; 3: 3288Crossref PubMed Scopus (33) Google Scholar, 16.Nakagawa N. Manya H. Toda T. Endo T. Oka S. Human natural killer-1 sulfotransferase (HNK-1ST)-induced sulfate transfer regulates laminin-binding glycans on alpha-dystroglycan.J. Biol. Chem. 2012; 287: 30823-30832Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). For Flag-tagged expression vectors of human FKTN, FKRP, TMEM5, and ISPD, the encoding sequence of individual open reading frames was amplified by PCR and cloned into pCMV14–3×FLAG vector (Sigma). For the human FKTN-myc expression vector, the FKTN coding sequence amplified by PCR was cloned into pSecTag2 (Invitrogen). The construction of vector (pKO) for TMEM5-KO cells was performed basically in accordance with previous reports (17.Ninagawa S. Okada T. Sumitomo Y. Horimoto S. Sugimoto T. Ishikawa T. Takeda S. Yamamoto T. Suzuki T. Kamiya Y. Kato K. Mori K. Forcible destruction of severely misfolded mammalian glycoproteins by the non-glycoprotein ERAD pathway.J. Cell Biol. 2015; 211: 775-784Crossref PubMed Scopus (30) Google Scholar, 18.Fu Y. Sander J.D. Reyon D. Cascio V.M. Joung J.K. Improving CRISPR-Cas nuclease specificity using truncated guide RNAs.Nat. Biotechnol. 2014; 32: 279-284Crossref PubMed Scopus (1386) Google Scholar). The 1.5-kb fragment of the human TMEM5 gene used for the 5′ arm was amplified by PCR from HCT116 cell genomic DNA using the primers 5′-ggggtacCAAATTATGCAGTCATTTGC-3′ and 5′-ctagctagcGATAAGAAACGAGCAGAGCC-3′, and then subcloned between the KpnI and NheI sites to create the pKO 5′ arm (hTMEM5). The 1.5-kb fragment of the TMEM5 gene used for the 3′ arm was amplified similarly using the primers 5′-ataagaatgcggccGCCCTGTACTGCCTATTCTC-3′ and 5′-ataagaatgcggccgcGTTGACATATGCATTGCAATC-3′, and then subcloned into the NotI site of the pKO 5′ arm (hTMEM5) to create pKO-hTMEM5. The target DNA sequence of the guide RNA (gRNA) was as follows: GCCCGAAGAAGACGTGGTAGG, which was inserted into the pX330 vector (Addgene) to create pX330-guide-TMEM5. To generate the KO cells of TMEM5, pX330-guide-TMEM5 and pKO-TMEM5 were transiently transfected into HCT116 cells in a six-well plate by Lipofectamine 2000 (Invitrogen). At 96 h post-transfection, cells were incubated in puromycin (0.5 μg/ml). Approximately 40 colonies were obtained 20 days later. Homologous recombination in HCT116 cells was confirmed by genomic PCR and sequencing. For the preparation of ISPD-, FKTN-, or FKRP-KO cells, the gRNAs flanking the target enhancer regions were designed by ToolGen (Seoul, South Korea) dRGEN synthesis services. The target DNA sequences of the gRNAs used in this study were as follows: for ISPD deletion, ATTGAAAATTGACCTGTGGCGGG; for FKTN deletion, GAGTAGAATCAATAAGAACGTGG; and for FKRP deletion, CATGCGGCTCACCCGCTGCCAGG. The gRNA expression plasmids (pRGEN-U6-sgRNA) were purchased from ToolGen. To generate the KO cells lacking ISPD, FKTN, or FKRP, individual gRNA plasmids and a plasmid expressing Cas9-GFP (Addgene Cambridge, MA, catalog #44719) were transiently transfected into HCT116 cells in a six-well plate with a Cas9:gRNA molar ratio of 1:5 using Polyethylenimine Max (Polysciences, Inc., Warrington, PA), in accordance with a previous report (19.Longo P.A. Kavran J.M. Kim M.S. Leahy D.J. Transient mammalian cell transfection with polyethylenimine (PEI).Methods Enzymol. 2013; 529: 227-240Crossref PubMed Scopus (312) Google Scholar). At 48 h post-transfection, cells with high green fluorescent protein (GFP) expression were identified using fluorescence-activated cell sorting with the Aria II cell sorter (BD Biosciences). Sorted cells were plated into individual wells of a 96-well plate and then re-plated as single cells in 6-cm dishes. The deletions in the clones were confirmed by sequencing. HEK293T as well as HCT116 and its mutated cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) in 5% CO2 at 37 °C. For the expression of αDG373(T322R)-Fc, cells were grown overnight and transfected using Polyethylenimine Max (Polysciences, Inc.), in accordance with a previous report (19.Longo P.A. Kavran J.M. Kim M.S. Leahy D.J. Transient mammalian cell transfection with polyethylenimine (PEI).Methods Enzymol. 2013; 529: 227-240Crossref PubMed Scopus (312) Google Scholar). Protein expression was performed in DMEM supplemented with 10% FBS treated with a Protein G affinity column (GE Healthcare) to remove immunoglobulin G. The secreted Fc-fused αDG fragments in culture medium were purified using a Protein G affinity column. Western blots were performed as described previously (16.Nakagawa N. Manya H. Toda T. Endo T. Oka S. Human natural killer-1 sulfotransferase (HNK-1ST)-induced sulfate transfer regulates laminin-binding glycans on alpha-dystroglycan.J. Biol. Chem. 2012; 287: 30823-30832Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 20.Yagi H. Yanagisawa M. Suzuki Y. Nakatani Y. Ariga T. Kato K. Yu R.K. HNK-1 epitope-carrying tenascin-C spliced variant regulates the proliferation of mouse embryonic neural stem cells.J. Biol. Chem. 2010; 285: 37293-37301Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Immunoprecipitation was performed in accordance with procedures described previously (17.Ninagawa S. Okada T. Sumitomo Y. Horimoto S. Sugimoto T. Ishikawa T. Takeda S. Yamamoto T. Suzuki T. Kamiya Y. Kato K. Mori K. Forcible destruction of severely misfolded mammalian glycoproteins by the non-glycoprotein ERAD pathway.J. Cell Biol. 2015; 211: 775-784Crossref PubMed Scopus (30) Google Scholar). At 24 h after transfection, cells were lysed in buffer A (50 mm Tris/Cl, pH 8.0, containing 1% Nonidet P-40, 150 mm NaCl, protease inhibitor mixture (Nacalai Tesque, Kyoto, Japan), 20 μm MG132 (Peptide Institute, Osaka, Japan), and 2 μm Z-VAD-fmk (Promega, Madison, WI)). Anti-Flag, anti-myc, and anti-FKTN antibodies and protein G-Sepharose beads (GE Healthcare) were used for the precipitation of FKRP-flag, TMEM5-flag, FKTN-myc and TMEM5-TAP. Beads were washed with high salt buffer (50 mm Tris/Cl, pH 8.0, containing 1% Nonidet P-40 and 150 mm NaCl) twice, washed with PBS, and then heated at 65 °C in Laemmli's sample buffer. Immunoprecipitates were subjected to SDS-PAGE and immunoblotting. The gel band containing αDG was excised and subjected to in-gel digestion by sequential steps of reduction with 10 mm dithiothreitol at 37 °C for 1 h, alkylation with 50 mm iodoacetamide in 25 mm ammonium bicarbonate buffer for 1 h in the dark at room temperature, destaining with 50% acetonitrile in 25 mm ammonium bicarbonate buffer, and then overnight digestion with sequencing-grade trypsin (Promega) at 37 °C. The digested products were sequentially extracted with distilled water, 1% formic acid, and 50% acetonitrile/1% formic acid, dried down, and then redissolved in 0.1% formic acid for further cleaned up by ZipTip C18 (Millipore) before analysis. The peptide mixtures were analyzed by nanospray LC-MS/MS on an Orbitrap Fusion Tribrid (Thermo Scientific) coupled to an UltiMate 3000 RSLCnano System (Dionex, Sunnyvale, CA). Peptide mixtures were loaded onto an Acclaim PepMap RSLC 25 cm × 75 μm i.d. column (Dionex) and separated at a flow rate of 500 nL/min using a gradient of 5% to 35% solvent B (100% acetonitrile with 0.1% formic acid) in 60 min. Solvent A was 0.1% formic acid in water. The parameters used for MS and MS/MS data acquisition under the HCD product ion trigger CID mode were: top speed mode with 3-s cycle time; FTMS: scan range (m/z) = 550–2000; resolution = 120 K; AGC target = 2 × 105; maximum injection time = 60 ms; monoisotopic precursor selection on; including charge state 2–6; dynamic exclusion after two times within 10 s and then exclusion for 40 s with 10-ppm tolerance. FTMSn (HCD): isolation mode = quadrupole; isolation window = 1.6; collision energy 28% with stepped collision energy 5%; resolution = 30 K; AGC target = 1 × 105; maximum injection time = 75 ms; HCD production ions m/z 204.0867 or 366.1396 (z = 1) within top 20 product ions were used to trigger CID; ITMSn (CID): isolation mode = quadrupole; isolation window = 1.6; collision energy = 30%; AGC target = 1 × 104; ion trap scan rate = rapid. HCD and CID MS2 data sets were filtered for candidate glycopeptide spectra based on the presence of MS2 ions corresponding to the expected tryptic peptide core containing the target O-glycosylation sites and/or peptide fragment ions, and then manually interpreted and assigned as described in the Results. Further rounds of searching through the acquired data set were based on thus identified diagnostic RboP/GroP-HexNAc+ oxonium ions. For MS3 analysis, HCD MS2 ions at m/z 358.0895 and 418.1105 were targeted for CID MS3 using the inclusion list feature. The parameters used for FTMS3 (CID) were: isolation mode = iontrap; MS isolation window = 1.6; MS2 isolation window = 3.0; scan range mode: auto normal; collision energy = 30%; detector type: orbitrap; resolution = 30 K; AGC target = 1 × 105; maximum injection time = 120 ms. To facilitate direct detection and sequencing of the αDG glycopeptides carrying the target O-glycans, we have opted for an analytical strategy that would preserve as much of the native O-glycosylation and other modifications as possible without introducing any chemical cleavages or glycosidase digestions prior to the MS analysis. Our choice as minimum manipulation was truncation of αDG at R373 and fusion with Fc for secretion and ease of purification (Fig. 1 and supplemental Fig. S1). The resulting Fc-tagged and purified proteins appeared as two major bands on SDS-PAGE at positions corresponding to ∼35 kDa and just above 50 kDa (Fig. 1), neither of which was stained by IIH6, an anti-αDG antibody recognizing laminin-binding glycans, as would be expected from the apparent size. Following in-gel tryptic digestion and recovery of peptides/glycopeptides from the gel, we initially searched for the target glycopeptide Q313-R337, but were unsucce
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