Peters plus syndrome mutations affect the function and stability of human β1,3-glucosyltransferase
2021; Elsevier BV; Volume: 297; Issue: 1 Linguagem: Inglês
10.1016/j.jbc.2021.100843
ISSN1083-351X
AutoresAo Zhang, Aarya Venkat, Rahil Taujale, James L. Mull, Atsuko Ito, Natarajan Kannan, Robert S. Haltiwanger,
Tópico(s)Lysosomal Storage Disorders Research
ResumoPeters Plus Syndrome (PTRPLS OMIM #261540) is a severe congenital disorder of glycosylation where patients have multiple structural anomalies, including Peters anomaly of the eye (anterior segment dysgenesis), disproportionate short stature, brachydactyly, dysmorphic facial features, developmental delay, and variable additional abnormalities. PTRPLS patients and some Peters Plus-like (PTRPLS-like) patients (who only have a subset of PTRPLS phenotypes) have mutations in the gene encoding β1,3-glucosyltransferase (B3GLCT). B3GLCT catalyzes the transfer of glucose to O-linked fucose on thrombospondin type-1 repeats. Most B3GLCT substrate proteins belong to the ADAMTS superfamily and play critical roles in extracellular matrix. We sought to determine whether the PTRPLS or PTRPLS-like mutations abrogated B3GLCT activity. B3GLCT has two putative active sites, one in the N-terminal region and the other in the C-terminal glycosyltransferase domain. Using sequence analysis and in vitro activity assays, we demonstrated that the C-terminal domain catalyzes transfer of glucose to O-linked fucose. We also generated a homology model of B3GLCT and identified D421 as the catalytic base. PTRPLS and PTRPLS-like mutations were individually introduced into B3GLCT, and the mutated enzymes were evaluated using in vitro enzyme assays and cell-based functional assays. Our results demonstrated that PTRPLS mutations caused loss of B3GLCT enzymatic activity and/or significantly reduced protein stability. In contrast, B3GLCT with PTRPLS-like mutations retained enzymatic activity, although some showed a minor destabilizing effect. Overall, our data supports the hypothesis that loss of glucose from B3GLCT substrate proteins is responsible for the defects observed in PTRPLS patients, but not for those observed in PTRPLS-like patients. Peters Plus Syndrome (PTRPLS OMIM #261540) is a severe congenital disorder of glycosylation where patients have multiple structural anomalies, including Peters anomaly of the eye (anterior segment dysgenesis), disproportionate short stature, brachydactyly, dysmorphic facial features, developmental delay, and variable additional abnormalities. PTRPLS patients and some Peters Plus-like (PTRPLS-like) patients (who only have a subset of PTRPLS phenotypes) have mutations in the gene encoding β1,3-glucosyltransferase (B3GLCT). B3GLCT catalyzes the transfer of glucose to O-linked fucose on thrombospondin type-1 repeats. Most B3GLCT substrate proteins belong to the ADAMTS superfamily and play critical roles in extracellular matrix. We sought to determine whether the PTRPLS or PTRPLS-like mutations abrogated B3GLCT activity. B3GLCT has two putative active sites, one in the N-terminal region and the other in the C-terminal glycosyltransferase domain. Using sequence analysis and in vitro activity assays, we demonstrated that the C-terminal domain catalyzes transfer of glucose to O-linked fucose. We also generated a homology model of B3GLCT and identified D421 as the catalytic base. PTRPLS and PTRPLS-like mutations were individually introduced into B3GLCT, and the mutated enzymes were evaluated using in vitro enzyme assays and cell-based functional assays. Our results demonstrated that PTRPLS mutations caused loss of B3GLCT enzymatic activity and/or significantly reduced protein stability. In contrast, B3GLCT with PTRPLS-like mutations retained enzymatic activity, although some showed a minor destabilizing effect. Overall, our data supports the hypothesis that loss of glucose from B3GLCT substrate proteins is responsible for the defects observed in PTRPLS patients, but not for those observed in PTRPLS-like patients. Peters plus syndrome (PTRPLS OMIM #261540) is a rare, autosomal recessive, congenital disorder of glycosylation that is characterized by multiple structural defects, including Peters anomaly of the eye (anterior eye chamber segment dysgenesis), disproportionate short stature, brachydactyly, craniofacial defects (including cleft lip/palate and broadened forehead), developmental delay, and other systematic abnormalities at variable penetrance (1Lesnik Oberstein S.A.J. Kriek M. White S.J. Kalf M.E. Szuhai K. den Dunnen J.T. Breuning M.H. Hennekam R.C.M. Peters Plus syndrome is caused by mutations in B3GALTL, a putative glycosyltransferase.Am. J. Hum. Genet. 2006; 79: 562-566Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). These patients carry intronic and/or exonic mutations in the gene encoding β1,3-glucosyltransferase (B3GLCT, formerly B3GALTL), which can be homozygous or compound heterozygous (1Lesnik Oberstein S.A.J. Kriek M. White S.J. Kalf M.E. Szuhai K. den Dunnen J.T. Breuning M.H. Hennekam R.C.M. Peters Plus syndrome is caused by mutations in B3GALTL, a putative glycosyltransferase.Am. J. Hum. Genet. 2006; 79: 562-566Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 2Aliferis K. Marsal C. Pelletier V. Doray B. Weiss M.M. Tops C.M. Speeg-Schatz C. Lesnik S.A. Dollfus H. A novel nonsense B3GALTL mutation confirms Peters plus syndrome in a patient with multiple malformations and Peters anomaly.Ophthalmic Genet. 2010; 31: 205-208Crossref PubMed Scopus (18) Google Scholar, 3Weh E. Reis L.M. Tyler R.C. Bick D. Rhead W.J. Wallace S. McGregor T.L. Dills S.K. Chao M.C. Murray J.C. Semina E.V. Novel B3GALTL mutations in classic Peters plus syndrome and lack of mutations in a large cohort of patients with similar phenotypes.Clin. Genet. 2014; 86: 142-148Crossref PubMed Scopus (27) Google Scholar). Most mutations in PTRPLS cause splicing or frameshift defects that can disrupt the transcription and/or translation of B3GLCT and are predicted to be loss of function. However, there are also several missense and nonsense mutations in B3GLCT (Fig. 1A). The missense mutations result in amino acid (aa) changes: D349N, G393E, G394E (Fig. 1A). PTRPLS-like patients have Peters anomaly and a subset of the additional phenotypes found in PTRPLS patients (3Weh E. Reis L.M. Tyler R.C. Bick D. Rhead W.J. Wallace S. McGregor T.L. Dills S.K. Chao M.C. Murray J.C. Semina E.V. Novel B3GALTL mutations in classic Peters plus syndrome and lack of mutations in a large cohort of patients with similar phenotypes.Clin. Genet. 2014; 86: 142-148Crossref PubMed Scopus (27) Google Scholar). While most PTRPLS-like patients have no mutations in B3GLCT, suggesting that mutations in other genes are causative for the phenotypes observed, some PTRPLS-like patients have heterozygous missense mutations in B3GLCT: T179S, V245M, R337H, and Q457R (personal communication, Dr Elena V. Semina, Medical College of Wisconsin) (Fig. 1A) (3Weh E. Reis L.M. Tyler R.C. Bick D. Rhead W.J. Wallace S. McGregor T.L. Dills S.K. Chao M.C. Murray J.C. Semina E.V. Novel B3GALTL mutations in classic Peters plus syndrome and lack of mutations in a large cohort of patients with similar phenotypes.Clin. Genet. 2014; 86: 142-148Crossref PubMed Scopus (27) Google Scholar). It is not clear whether these PTRPLS-like mutations contribute to the phenotypes observed in these patients. This raises a question regarding whether these PTRPLS or PTRPLS-like mutations affect B3GLCT function and/or stability. Two nonsense mutations were also identified from PTRPLS patients—Y366∗ (2Aliferis K. Marsal C. Pelletier V. Doray B. Weiss M.M. Tops C.M. Speeg-Schatz C. Lesnik S.A. Dollfus H. A novel nonsense B3GALTL mutation confirms Peters plus syndrome in a patient with multiple malformations and Peters anomaly.Ophthalmic Genet. 2010; 31: 205-208Crossref PubMed Scopus (18) Google Scholar) and R412∗ (3Weh E. Reis L.M. Tyler R.C. Bick D. Rhead W.J. Wallace S. McGregor T.L. Dills S.K. Chao M.C. Murray J.C. Semina E.V. Novel B3GALTL mutations in classic Peters plus syndrome and lack of mutations in a large cohort of patients with similar phenotypes.Clin. Genet. 2014; 86: 142-148Crossref PubMed Scopus (27) Google Scholar) (Fig. 1A). B3GLCT contains an REEL motif at the C-terminus of the protein (Fig. 1A) that serves as a KDEL-like motif, retaining B3GLCT within the endoplasmic reticulum (ER) (4Heinonen T.Y. Pasternack L. Lindfors K. Breton C. Gastinel L.N. Maki M. Kainulainen H. A novel human glycosyltransferase: Primary structure and characterization of the gene and transcripts.Biochem. Biophys. Res. Commun. 2003; 309: 166-174Crossref PubMed Scopus (19) Google Scholar, 5Sato T. Sato M. Kiyohara K. Sogabe M. Shikanai T. Kikuchi N. Togayachi A. Ishida H. Ito H. Kameyama A. Gotoh M. Narimatsu H. Molecular cloning and characterization of a novel human beta1,3-glucosyltransferase, which is localized at the endoplasmic reticulum and glucosylates O-linked fucosylglycan on thrombospondin type 1 repeat domain.Glycobiology. 2006; 16: 1194-1206Crossref PubMed Scopus (57) Google Scholar). The premature termination from Y366∗ and R412∗ results in deletion of the REEL motif, raising the question of whether these two mutations cause mislocalization of the truncated B3GLCT. B3GLCT belongs to the glycosyltransferase (GT)-31 family based on the Carbohydrate Active enZYmes (CAZy) classification (7Lombard V. Golaconda Ramulu H. Drula E. Coutinho P.M. Henrissat B. The carbohydrate-active enzymes database (CAZy) in 2013.Nucleic Acids Res. 2014; 42: D490-D495Crossref PubMed Scopus (3461) Google Scholar). Currently, there are over 30 vertebrate subfamilies in the GT31 family (8Petit D. Teppa R.E. Harduin-Lepers A. A phylogenetic view and functional annotation of the animal beta1,3-glycosyltransferases of the GT31 CAZy family.Glycobiology. 2021; 31: 243-259Crossref PubMed Scopus (4) Google Scholar), but structures of only two have been solved—mouse Manic Fringe (MFNG) (9Jinek M. Chen Y.-W. Clausen H. Cohen S.M. Conti E. Structural insights into the Notch-modifying glycosyltransferase Fringe.Nat. Struct. Mol. Biol. 2006; 13: 945-946Crossref PubMed Scopus (29) Google Scholar) and human β1,3-N-acetylglucosaminyltransfease 2 (B3GNT2) (10Hao Y. Crequer-Grandhomme A. Javier N. Singh A. Chen H. Manzanillo P. Lo M.C. Huang X. Structures and mechanism of human glycosyltransferase beta1,3-N-acetylglucosaminyltransferase 2 (B3GNT2), an important player in immune homeostasis.J. Biol. Chem. 2020; 296100042Abstract Full Text Full Text PDF PubMed Google Scholar, 11Kadirvelraj R. Yang J.Y. Kim H.W. Sanders J.H. Moremen K.W. Wood Z.A. Comparison of human poly-N-acetyl-lactosamine synthase structure with GT-A fold glycosyltransferases supports a modular assembly of catalytic subsites.J. Biol. Chem. 2020; 296100110Abstract Full Text Full Text PDF PubMed Scopus (3) Google Scholar). A recent phylogenetic study demonstrated that B3GLCT is a unique member of the GT31 family because it harbors two putative GT domains, which are more similar to each other than the rest of the members in the GT31 family (8Petit D. Teppa R.E. Harduin-Lepers A. A phylogenetic view and functional annotation of the animal beta1,3-glycosyltransferases of the GT31 CAZy family.Glycobiology. 2021; 31: 243-259Crossref PubMed Scopus (4) Google Scholar). Heinonen et al. (4Heinonen T.Y. Pasternack L. Lindfors K. Breton C. Gastinel L.N. Maki M. Kainulainen H. A novel human glycosyltransferase: Primary structure and characterization of the gene and transcripts.Biochem. Biophys. Res. Commun. 2003; 309: 166-174Crossref PubMed Scopus (19) Google Scholar) initially discussed that there are two "DxD" motifs in human B3GLCT: 132EEE134 in the N-terminal region (originally annotated as a stem region) and 349DDD351 in the C-terminal GT domain (C-GT) (Fig. 1A). The "DxD" motif marks the active site of GT-A fold glycosyltransferases and is responsible for the binding of nucleotide sugar donors and the chelation of divalent cation cofactors for catalysis (6Taujale R. Venkat A. Huang L.C. Zhou Z. Yeung W. Rasheed K.M. Li S. Edison A.S. Moremen K.W. Kannan N. Deep evolutionary analysis reveals the design principles of fold A glycosyltransferases.Elife. 2020; 9e54532Crossref PubMed Scopus (16) Google Scholar). Kozma et al. (12Kozma K. Keusch J.J. Hegemann B. Luther K.B. Klein D. Hess D. Haltiwanger R.S. Hofsteenge J. Identification and characterization of abeta1,3-glucosyltransferase that synthesizes the Glc-beta1,3-Fuc disaccharide on thrombospondin type 1 repeats.J. Biol. Chem. 2006; 281: 36742-36751Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar) demonstrated that mutation of 349DDD351 to 349ADD351 or 349ADA351 significantly reduced in vitro B3GLCT enzymatic activity, but whether the N-terminal domain containing 132EEE134 is catalytically active is unknown. Nonetheless, the presence of two distinct domains in B3GLCT raises the question of whether PTRPLS is caused by loss of function of the N-terminal or C-terminal domains. B3GLCT catalyzes the transfer of glucose (Glc) to O-linked fucose (O-Fuc) on thrombospondin type-1 repeats (TSRs), forming a unique glucose-β1,3-fucose disaccharide (GlcFuc) (5Sato T. Sato M. Kiyohara K. Sogabe M. Shikanai T. Kikuchi N. Togayachi A. Ishida H. Ito H. Kameyama A. Gotoh M. Narimatsu H. Molecular cloning and characterization of a novel human beta1,3-glucosyltransferase, which is localized at the endoplasmic reticulum and glucosylates O-linked fucosylglycan on thrombospondin type 1 repeat domain.Glycobiology. 2006; 16: 1194-1206Crossref PubMed Scopus (57) Google Scholar, 12Kozma K. Keusch J.J. Hegemann B. Luther K.B. Klein D. Hess D. Haltiwanger R.S. Hofsteenge J. Identification and characterization of abeta1,3-glucosyltransferase that synthesizes the Glc-beta1,3-Fuc disaccharide on thrombospondin type 1 repeats.J. Biol. Chem. 2006; 281: 36742-36751Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). 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This suggests that B3GLCT modifies any TSR that is modified with O-fucose. The ADAMTS and noncatalytic ADAMTS-like proteins are localized in the extracellular matrix (ECM) and have critical roles in organogenesis, tissue organization, and cell signaling during developmental processes (29Dubail J. Apte S.S. Insights on ADAMTS proteases and ADAMTS-like proteins from mammalian genetics.Matrix Biol. 2015; 44-46: 24-37Crossref PubMed Scopus (88) Google Scholar). Mutations in several ADAMTS/ADAMTS-like proteins cause congenital disorders in humans and developmental defects in mice. For instance, mutations in ADAMTSL2 cause geleophysic dysplasia, where patients display short stature, short tubular bones, thick skin, and cardiopulmonary abnormalities (30Le Goff C. Morice-Picard F. Dagoneau N. Wang L.W. Perrot C. Crow Y.J. Bauer F. Flori E. Prost-Squarcioni C. Krakow D. Ge G. Greenspan D.S. Bonnet D. Le Merrer M. Munnich A. et al.ADAMTSL2 mutations in geleophysic dysplasia demonstrate a role for ADAMTS-like proteins in TGF-beta bioavailability regulation.Nat. Genet. 2008; 40: 1119-1123Crossref PubMed Scopus (155) Google Scholar). Elimination of Adamts9 in mice results in early embryonic lethality in mice. Interestingly, Pofut2 knockout mice display the same phenotype, suggesting that ADAMTS9 requires addition of O-fucose to its TSRs for proper function (31Benz B.A. Nandadasa S. Takeuchi M. Grady R.C. Takeuchi H. LoPilato R.K. Kakuda S. Somerville R.P.T. Apte S.S. Haltiwanger R.S. Holdener B.C. Genetic and biochemical evidence that gastrulation defects in Pofut2 mutants result from defects in ADAMTS9 secretion.Dev. Biol. 2016; 416: 111-122Crossref PubMed Scopus (25) Google Scholar). Heterozygous Adamts9 mice show an anterior segmentation dysgenesis in the eyes, as seen in PTRPLS and PTRPLS-like patients (24Dubail J. Vasudevan D. Wang L.W. Earp S.E. Jenkins M.W. Haltiwanger R.S. Apte S.S. Impaired ADAMTS9 secretion: A potential mechanism for eye defects in Peters plus syndrome.Sci. Rep. 2016; 6: 33974Crossref PubMed Scopus (19) Google Scholar). Adamts20-null mice show a white spotting defect and a high degree of hydrocephalus (26Holdener B.C. Percival C.J. Grady R.C. Cameron D.C. Berardinelli S.J. Zhang A. Neupane S. Takeuchi M. Jimenez-Vega J.C. Uddin S.M.Z. Komatsu D.E. Honkanen R. Dubail J. Apte S.S. Sato T. et al.ADAMTS9 and ADAMTS20 are differentially affected by loss of B3GLCT in mouse model of Peters plus syndrome.Hum. Mol. Genet. 2019; 28: 4053-4066Crossref PubMed Scopus (7) Google Scholar). Significantly, B3glct knockout mice phenocopy several defects seen in PTRPLS patients (craniofacial abnormalities, bone growth defects) but also show white spotting and hydrocephalus, indicating that ADAMTS20 is a biologically relevant B3GLCT substrate that requires addition of glucose to be fully functional (26Holdener B.C. Percival C.J. Grady R.C. Cameron D.C. Berardinelli S.J. Zhang A. Neupane S. Takeuchi M. Jimenez-Vega J.C. Uddin S.M.Z. Komatsu D.E. Honkanen R. Dubail J. Apte S.S. Sato T. et al.ADAMTS9 and ADAMTS20 are differentially affected by loss of B3GLCT in mouse model of Peters plus syndrome.Hum. Mol. Genet. 2019; 28: 4053-4066Crossref PubMed Scopus (7) Google Scholar). A growing body of data supports the concept that POFUT2 and B3GLCT participate in a quality control pathway for the folding of TSRs (16Holdener B.C. Haltiwanger R.S. Protein O-fucosylation: Structure and function.Curr. Opin. Struct. Biol. 2019; 56: 78-86Crossref PubMed Scopus (52) Google Scholar). Both of these enzymes are located in the endoplasmic reticulum (ER), and deleting them results in secretion defects in a protein-specific manner. For instance, secretion of a portion of ADAMTS9 (TSRs2-8) is completely inhibited in POFUT2−/− HEK293T cells, but only reduced by 20% in B3GLCT−/− cells compared with wild type (WT) (26Holdener B.C. Percival C.J. Grady R.C. Cameron D.C. Berardinelli S.J. Zhang A. Neupane S. Takeuchi M. Jimenez-Vega J.C. Uddin S.M.Z. Komatsu D.E. Honkanen R. Dubail J. Apte S.S. Sato T. et al.ADAMTS9 and ADAMTS20 are differentially affected by loss of B3GLCT in mouse model of Peters plus syndrome.Hum. Mol. Genet. 2019; 28: 4053-4066Crossref PubMed Scopus (7) Google Scholar). Thus, the embryonic lethality of Pofut2 knockout mice can be explained by loss of secretion of ADAMTS9. In contrast, ADAMTS20 TSR2-8, with almost identical TSR domain structures compared with ADAMTS9, is completely dependent on B3GLCT for secretion, implying that the white spotting and hydrocephalus observed in B3glct knockout mice are due at least in part to loss of ADAMTS20 secretion (26Holdener B.C. Percival C.J. Grady R.C. Cameron D.C. Berardinelli S.J. Zhang A. Neupane S. Takeuchi M. Jimenez-Vega J.C. Uddin S.M.Z. Komatsu D.E. Honkanen R. Dubail J. Apte S.S. Sato T. et al.ADAMTS9 and ADAMTS20 are differentially affected by loss of B3GLCT in mouse model of Peters plus syndrome.Hum. Mol. Genet. 2019; 28: 4053-4066Crossref PubMed Scopus (7) Google Scholar). Taken together, these data raise the question of whether PTRPLS/PTRPLS-like mutations in B3GLCT will affect the secretion of a subset of POFUT2/B3GLCT substrate proteins by affecting the function and/or stability of B3GLCT. To address the questions raised above, we analyzed the domain structure of B3GLCT in more detail and demonstrated that the C-GT domain of B3GLCT is responsible for the transfer of Glc to O-Fuc on TSRs. We then introduced PTRPLS/PTRPLS-like mutations individually to human B3GLCT and performed kinetic analysis of the WT and mutant forms of the enzyme using in vitro enzyme assays. We also investigated the function of the mutant enzymes in vivo with cell-based assays by investigating the ability of the mutants to rescue the secretion of ADAMTS20 TSR2-8 from B3GLCT−/− cells. Our data suggested that PTRPLS mutations eliminated the function of B3GLCT, but PTRPLS-like mutants did not. We also generated a homology model of B3GLCT to predict the structural impact of these PTRPLS/PTRPLS-like mutations. Overall, our studies advanced our understanding of the molecular mechanisms that result in PTRPLS. We began by analyzing the domain distribution of human B3GLCT as illustrated in Figure 1A, including signal peptide (aa 1–68), N-terminal GT-like (N-GT-like) domain (aa 69–240), cysteine-rich linker domain (aa 241–263), C-terminal GT domain (C-GT) (aa 264–468), and C-terminus with ER retention signal REEL (aa 469–498). Both the N- and C-terminal domains have residues analogous to those found in GT-A fold enzymes, including a DxD motif, 132EEE134 and 349DDD351, respectively. The DxD motifs were conserved in vertebrates, but the N-terminal DxD motif was not conserved in fruit flies (Figs. 1B and S1). Sequence analysis showed that the C-GT domain also has a highly conserved G-loop (393GGG395), xED motif (419PDD421), and C-terminal Histidine (C-His, H463), all common GT-A fold elements (6Taujale R. Venkat A. Huang L.C. Zhou Z. Yeung W. Rasheed K.M. Li S. Edison A.S. Moremen K.W. Kannan N. Deep evolutionary analysis reveals the design principles of fold A glycosyltransferases.Elife. 2020; 9e54532Crossref PubMed Scopus (16) Google Scholar) (Figs. 1B and S1). The G-loop confers flexibility, and the xED-Asp functions as a catalytic base in inverting GT-A fold enzymes (6Taujale R. Venkat A. Huang L.C. Zhou Z. Yeung W. Rasheed K.M. Li S. Edison A.S. Moremen K.W. Kannan N. Deep evolutionary analysis reveals the design principles of fold A glycosyltransferases.Elife. 2020; 9e54532Crossref PubMed Scopus (16) Google Scholar). The putative N-terminal glycosyltransferase domain has some of these features including a potential G-loop (187AAG189) (Figs. 1B and S1). Other conserved features are difficult to predict, including the catalytic base (possibly D212 or D216) and the C-terminal His (Figs. 1B and S1). To evaluate which domain is responsible for the glucosyltransferase activity, we first subcloned human B3GLCT with the REEL motif removed (B3GLCTΔREEL) to allow secretion to the medium for purification. We then mutated both DxD motifs (E132A and D349N) to abolish the 132EEE134 and 349DDD351 individually in B3GLCTΔREEL to further evaluate which of the two putative GT domains is responsible for catalytic activity. The D349N is also a PTRPLS mutation (3Weh E. Reis L.M. Tyler R.C. Bick D. Rhead W.J. Wallace S. McGregor T.L. Dills S.K. Chao M.C. Murray J.C. Semina E.V. Novel B3GALTL mutations in classic Peters plus syndrome and lack of mutations in a large cohort of patients with similar phenotypes.Clin. Genet. 2014; 86: 142-148Crossref PubMed Scopus (27) Google Scholar). We first used in vitro enzyme assays to analyze whether the E132A and D349N mutants could transfer glucose to O-fucosylated TSR3 from human thrombospondin-1 (Fuc-O-TSR3). After a 20 min transfer reaction (within the linear phase of the assay, Fig. S2), the D349N mutant completely lost catalytic activity, but E132A catalyzed the reaction at a similar level to the WT B3GLCTΔREEL (Fig. 2A). Since the N-terminal GT domain is not involved in transfer of glucose, and identification of some GT-A elements needed for catalysis is not clear, we termed it as "N-GT-like" in our domain map of human B3GLCT (Fig. 1A). To further analyze the function of the domain mutants under physiological conditions, we tested the ability of the E132A and D349N mutants to rescue the secretion of ADAMTS20 TSR2-8 in B3GLCT−/− cells. For these assays we used full-length human B3GLCT with the REEL motif intact (B3GLCT-FL) to retain transfected B3GLCT within the ER. ADAMTS20 TSR2-8 was secreted into the media from WT HEK293T cells but lost its secretion in B3GLCT−/− cells (Fig. 2, B and C, media), as shown previously (26Holdener B.C. Percival C.J. Grady R.C. Cameron D.C. Berardinelli S.J. Zhang A. Neupane S. Takeuchi M. Jimenez-Vega J.C. Uddin S.M.Z. Komatsu D.E. Honkanen R. Dubail J. Apte S.S. Sato T. et al.ADAMTS9 and ADAMTS20 are differentially affected by loss of B3GLCT in mouse model of Peters plus syndrome.Hum. Mol. Genet. 2019; 28: 4053-4066Crossref PubMed Scopus (7) Google Scholar). The E132A mutant rescued the secretion of ADAMTS20 TSR2-8 similar to WT B3GLCT-FL (Fig. 2B, media), whereas D349N completely lost the ability to rescue (Fig. 2C, media). Both the E132A and D349N mutants were detected in cell lysate at comparable levels to transfected WT B3GLCT. Serial dilution of cotransfected enzyme plasmids allowed us to mimic the endogenous B3GLCT cellular level in WT HEK293T cells for better analysis (Fig. 2, B and C, cell lysate). Together, these data confirmed tha
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