Structural Basis of Carbohydrate Transfer Activity by Human UDP-GalNAc: Polypeptide α-N-Acetylgalactosaminyltransferase (pp-GalNAc-T10)
Glycogene Function Team of Research Center for Glycoscience (RCG), National Institute of Advanced Industrial Science and Technology (AIST), Central 2, 1-1-1 Umezono, Tsukuba, Ibaraki 305-8568, Japan. Journal of Molecular Biology
(Impact Factor: 4.33).
07/2006; 359(3):708-27. DOI: 10.1016/j.jmb.2006.03.061
Mucin-type O-glycans are important carbohydrate chains involved in differentiation and malignant transformation. Biosynthesis of the O-glycan is initiated by the transfer of N-acetylgalactosamine (GalNAc) which is catalyzed by UDP-GalNAc:polypeptide alpha-N-acetylgalactosaminyltransferases (pp-GalNAc-Ts). Here we present crystal structures of the pp-GalNAc-T10 isozyme, which has specificity for glycosylated peptides, in complex with the hydrolyzed donor substrate UDP-GalNAc and in complex with GalNAc-serine. A structural comparison with uncomplexed pp-GalNAc-T1 suggests that substantial conformational changes occur in two loops near the catalytic center upon donor substrate binding, and that a distinct interdomain arrangement between the catalytic and lectin domains forms a narrow cleft for acceptor substrates. The distance between the catalytic center and the carbohydrate-binding site on the lectin beta sub-domain influences the position of GalNAc glycosylation on GalNAc-glycosylated peptide substrates. A chimeric enzyme in which the two domains of pp-GalNAc-T10 are connected by a linker from pp-GalNAc-T1 acquires activity toward non-glycosylated acceptors, identifying a potential mechanism for generating the various acceptor specificities in different isozymes to produce a wide range of O-glycans.
Available from: jcs.biologists.org
- "These include the human Polypeptide a-N-Acetylgalactosaminyltransferase (pp- GalNAc-T10; estimated precision 100%, E-value59.5e-12; see Fig. 5A) that transfers N-Acetylgalactosamine (Gal-NAc) from the substrate UDP-GalNAc to glycosylated peptides (Kubota et al., 2006), as well as the SpsA glycosyltransferase from Bacillus subtilis (estimated precision 100%, E-value51.4e-10; see supplementary material Fig. S3) that uses nucleotide-diphosphosugar donors to synthesise the bacterial spore coat (Charnock and Davies, 1999). "
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ABSTRACT: The chitin synthase that makes the primary septum during cell division in budding yeasts is an important therapeutic target with an unknown activation mechanism. We previously found that the C2-domain of the Saccharomyces cerevisiae Inn1 protein plays an essential but uncharacterised role at the cleavage site during cytokinesis. By combining a novel degron allele of INN1 with a point mutation in the C2-domain, we screened for mutations in other genes that suppress the resulting defect in cell division. In this way we identified 22 dominant mutations of CHS2 (Chitin Synthase II) that map to two neighbouring sites in the catalytic domain. Whereas Chs2 in isolated cell membranes is normally almost inactive, unless protease treatment is used to bypass inhibition, the dominant suppressor allele Chs2-V377I has enhanced activity in vitro. We show that Inn1 associates with Chs2 in yeast cell extracts, and interacts in the yeast two-hybrid assay with the amino-terminal 65% of Chs2 that contains the catalytic domain. In addition to compensating for mutations in the Inn1 C2-domain, the dominant CHS2 alleles also suppress cytokinesis defects produced by lack of the Cyk3 protein, and our data support a model whereby the C2-domain of Inn1 acts in conjunction with Cyk3 to regulate the catalytic domain of Chs2 during cytokinesis. These findings suggest novel approaches for developing future drugs against important fungal pathogens.
Available from: glycob.oxfordjournals.org
- "To elucidate the function of the lectin domain, here we report an adaptable strategy for the GalNAc-T assay using glycopeptides modified with α-GalNAc or other sugars [β-GalNAc, α-Fuc and β-GlcNAc]. As described above, most of previous studies utilized α-GalNAc glycopeptides as substrates for GalNAc-T assays to reveal the effect of the lectin domain in the reactions (Hanisch et al. 2001; Hassan and Reis et al. 2000; Tenno et al. 2002; Fritz et al. 2006; Kubota et al. 2006; Wandall et al. 2007; Raman et al. 2008; Pedersen et al. 2011). Here, we have used a peptide sequence derived from the MUC5AC mucin (GTTPSPVPTTSTTSAP; Guyonnet Duperat et al. 1995) and human recombinant GalNAc-T3 (Bennett et al. 1996). "
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ABSTRACT: Mucin-type glycosylation [α-N-acetyl-d-galactosamine (α-GalNAc)-O-Ser/Thr] on proteins is initiated biosynthetically by 16 homologous isoforms of GalNAc-Ts (uridine diphosphate-GalNAc:polypeptide
N-acetylgalactosaminyltransferases). All the GalNAc-Ts consist of a catalytic domain and a lectin domain. Previous reports
of GalNAc-T assays toward peptides and α-GalNAc glycopeptides showed that the lectin domain recognized the sugar on the substrates
and affected the reaction; however, the details are not clear. Here, we report a new strategy to give insight on the sugar
recognition ability and the function of the GalNAc-T3 lectin domain using chemically synthesized natural-type (α-GalNAc-O-Thr) and unnatural-type [β-GalNAc-O-Thr, α-Fuc-O-Thr and β-GlcNAc-O-Thr] MUC5AC glycopeptides. GalNAc-T3 is one of isoforms expressed in various organs, its substrate specificity extensively
characterized and its anomalous expression has been identified in several types of cancer (e.g. pancreas and stomach). The
glycopeptides used in this study were designed based on a preliminary peptide assay with a sequence derived from the MUC5AC
tandem repeat. Through GalNAc-T3 and lectin-inactivated GalNAc-T3, competition assays between the glycopeptide substrates
and product analyses (MALDI-TOF MS, RP-HPLC and ETD-MS/MS), we show that the lectin domain strictly recognized GalNAc on the
substrate and this specificity controlled the glycosylation pathway.
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- "Since the N-terminal domain of CEECAM1 could yield an active chimeric protein when combined with GLT25D1, the experiment showed that both proteins are structurally closely related and that such chimeric constructs are neither instable nor inactive per se. The N-terminal domain of GLT25D1, GLT25D2 and CEECAM1 also shared structural similarity with glycosyltransferases like the polypeptide N-acetylgalactosaminyltransferases-2, -10 (CAZY family GT27) and the α1,4 N-acetylhexosaminyltransferase EXTL2 (CAZY family GT64) , , , indicating that the sequence context around the first DXD motif is suitable for an interaction with Mn2+ and with the diphosphate moiety of the donor substrate. "
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ABSTRACT: Collagen is modified by hydroxylation and glycosylation of hydroxylysine residues. This glycosylation is initiated by the β1,O galactosyltransferases GLT25D1 and GLT25D2. The structurally similar protein cerebral endothelial cell adhesion molecule CEECAM1 was previously reported to be inactive when assayed for collagen glycosyltransferase activity. To address the cause of the absent galactosyltransferase activity, we have generated several chimeric constructs between the active human GLT25D1 and inactive human CEECAM1 proteins. The assay of these chimeric constructs pointed to a short central region and a large C-terminal region of CEECAM1 leading to the loss of collagen galactosyltransferase activity. Examination of the three DXD motifs of the active GLT25D1 by site-directed mutagenesis confirmed the importance of the first (amino acids 166-168) and second motif (amino acids 461-463) for enzymatic activity, whereas the third one was dispensable. Since the second DXD motif is incomplete in CEECAM1, we have restored the motif by introducing the substitution S461D. This change did not restore the activity of the C-terminal region, thereby showing that additional amino acids were required in this C-terminal region to confer enzymatic activity. Finally, we have introduced the substitution Q471R-V472M-N473Q-P474V in the CEECAM1-C-terminal construct, which is found in most animal GLT25D1 and GLT25D2 isoforms but not in CEECAM1. This substitution was shown to partially restore collagen galactosyltransferase activity, underlining its importance for catalytic activity in the C-terminal domain. Because multiple mutations in different regions of CEECAM1 contribute to the lack of galactosyltransferase activity, we deduced that CEECAM1 is functionally different from the related GLT25D1 protein.
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