Structural Basis of Carbohydrate Transfer Activity by Human UDP-GalNAc: Polypeptide α-N-Acetylgalactosaminyltransferase (pp-GalNAc-T10)
ABSTRACT 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.
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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. "
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.PLoS ONE 12/2011; 6(12):e29390. DOI:10.1371/journal.pone.0029390 · 3.23 Impact Factor
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- "The relative positioning of the two domains has been attributed, in part, to differences in linker sequence properties. Peripheral GalNAc-T1 linker residues were shown to interact with both the catalytic and lectin domains, whereas a more stretched linker in GalNAc-T10 results in weaker interaction with the catalytic domain (Kubota et al. 2006). Therefore, linker flexibility may function to control the relative orientation of the lectin and catalytic domains and serve to drive lectin-mediated substrate specificities of GalNAc-Ts. "
ABSTRACT: Glycosylation of proteins is an essential process in all eukaryotes and a great diversity in types of protein glycosylation exists in animals, plants and microorganisms. Mucin-type O-glycosylation, consisting of glycans attached via O-linked N-acetylgalactosamine (GalNAc) to serine and threonine residues, is one of the most abundant forms of protein glycosylation in animals. Although most protein glycosylation is controlled by one or two genes encoding the enzymes responsible for the initiation of glycosylation, i.e. the step where the first glycan is attached to the relevant amino acid residue in the protein, mucin-type O-glycosylation is controlled by a large family of up to 20 homologous genes encoding UDP-GalNAc:polypeptide GalNAc-transferases (GalNAc-Ts) (EC 220.127.116.11). Therefore, mucin-type O-glycosylation has the greatest potential for differential regulation in cells and tissues. The GalNAc-T family is the largest glycosyltransferase enzyme family covering a single known glycosidic linkage and it is highly conserved throughout animal evolution, although absent in bacteria, yeast and plants. Emerging studies have shown that the large number of genes (GALNTs) in the GalNAc-T family do not provide full functional redundancy and single GalNAc-T genes have been shown to be important in both animals and human. Here, we present an overview of the GalNAc-T gene family in animals and propose a classification of the genes into subfamilies, which appear to be conserved in evolution structurally as well as functionally.Glycobiology 12/2011; 22(6):736-56. DOI:10.1093/glycob/cwr182 · 3.15 Impact Factor
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- "This loop most likely rearranges upon binding to the donor substrate (Figure 3A; Hurtado-Guerrero et al., 2010). This conformational change is a general feature observed in GTs with GT-A and GT-B fold (Boix et al., 2001; Flint et al., 2005; Qasba et al., 2005; Gordon et al., 2006; Kubota et al., 2006; Ramakrishnan et al., 2006; Ziegler et al., 2008). In the structure of Lgt1 the loop shows only sufficient electron density in the UDP–glucose bound form (closed conformation) due to high mobility of the loop without intact donor substrate. "
ABSTRACT: Legionella causes severe pneumonia in humans. The pathogen produces an array of effectors, which interfere with host cell functions. Among them are the glucosyltransferases Lgt1, Lgt2 and Lgt3 from L. pneumophila. Lgt1 and Lgt2 are produced predominately in the post-exponential phase of bacterial growth, while synthesis of Lgt3 is induced mainly in the lag-phase before intracellular replication of bacteria starts. Lgt glucosyltransferases are structurally similar to clostridial glucosylating toxins. The enzymes use UDP-glucose as a donor substrate and modify eukaryotic elongation factor eEF1A at serine-53. This modification results in inhibition of protein synthesis and death of target cells.In addition to Lgts, Legionella genomes disclose several genes, coding for effector proteins likely to possess glycosyltransferase activities, including SetA (subversion of eukaryotic vesicle trafficking A), which influences vesicular trafficking in the yeast model system and displays tropism for late endosomal/lysosomal compartments of mammalian cells. This review mainly discusses recent results on the structure-function relationship of Lgt glucosyltransferases.Frontiers in Microbiology 04/2011; 2:76. DOI:10.3389/fmicb.2011.00076 · 3.99 Impact Factor