Structure-Function Analysis of the UDP-N-acetyl-d-galactosamine:PolypeptideN-acetylgalactosaminyltransferase

Abstract

Mucin-type O-glycosylation is initiated by a family of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (ppGaNTases). Based on sequence relationships with divergent proteins, the ppGaNTases can be subdivided into three putative domains: each putative domain contains a characteristic sequence motif. The 112-amino acid glycosyltransferase 1 (GT1) motif represents the first half of the catalytic unit and contains a short aspartate-any residue-histidine (DXH) or aspartate-any residue-aspartate (DXD)-like sequence. Secondary structure predictions and structural threading suggest that the GT1 motif forms a 5-stranded parallel ␤-sheet flanked by 4 ␣-helices, which resembles the first domain of the lactose repressor. Four invariant carboxylates and a histidine residue are predicted to lie at the Cterminal end of three ␤-strands and line the active site cleft. Site-directed mutagenesis of murine ppGaNTase-T1 reveals that conservative mutations at these 5 positions result in products with no detectable enzyme activity (D156Q, D209N, and H211D) or <1% activity (E127Q and E213Q). The second half of the catalytic unit contains a DXXXXXWGGENXE motif (positions 310 -322) which is also found in ␤1,4-galactosyltransferases (termed the Gal/GalNAc-T motif). Mutants of carboxylates within this motif express either no detectable activity, 1% or 2% activity (E319Q, E322Q, and D310N, respectively). Mutagenesis of highly conserved (but not invariant) carboxylates produces only modest alterations in enzyme activity. Mutations in the C-terminal 128-amino acid ricin-like lectin motif do not alter the enzyme's catalytic properties.

The Lectin Domain of UDP-N-acetyl-d-galactosamine:PolypeptideN-acetylgalactosaminyltransferase-T4 Directs Its Glycopeptide Specificities

“…Previous experiments involving site-specific mutagenesis of selected conserved residues confirmed that mutations in the catalytic domain eliminated catalytic activity. In contrast, mutations in the lectin domain had no or only little effects on catalytic activity of at least one GalNAc-transferase isoform, GalNAc-T1 (14). However, recent evidence demonstrates that some GalNAc-transferases in vitro exhibit unique activities with partially GalNAc-glycosylated glycopeptides.…”

“…However, it is conceivable that GalNAc glycosylation of Ser 20 precedes Thr 14 , and that initial attachment of GalNAc at Ser 20 indeed induces GalNAc-T4 to utilize Thr 14 . Surprisingly, GalNAc transfer to Ser 10 in -VTSA-with the GalNAc 2 TAP25 (TAPPAHGV T*S 10 APDTRPAPGST*APPA) substrate was poor compared with GalNAc 4 TAP24.…”

“…Tandem Repeat Sequence-GalNAc-T4 transfers two GalNAc residues to the GalNAc 4 TAP24 substrate (T* APPAHGVT*S 10 APDT 14 RPAPGS*T*APP) at Ser 10 in -VTSAand Thr 14 in -PDTR- (7). The kinetics of the reaction of GalNAc-T4 with GalNAc 4 TAP24, GalNAc 2 TAP25 (TAP-PAHGV T*SAPDTRPAPGST*APPA), and TAP24, as monitored by CE analysis, is shown in Fig.…”

“…These results show that GalNAc-T4 can glycosylate Ser 20 in -GSTA-efficiently, in addition to the two sites previously identified (7), provided the substrate has GalNAc residues at Thr 9 and/or Thr 21 . The kinetics of the reaction is such that it is not possible to identify which of the two sites, Thr 14 or Ser 20 , is first glyco- (13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)ϩ3GalNAc. The identified degradation products are labeled in the spectra according to TAP25 peptide sequence.…”

The Components of the Saccharomyces cerevisiaeMannosyltransferase Complex M-Pol I Have Distinct Functions in Mannan Synthesis

“…Structural studies on a number of enzymes have shown that these aspartate residues coordinate the Mn 2ϩ ion that plays a central role in catalysis (31)(32)(33)(34). Moreover, the aspartates have been shown to necessary for the activity of a wide range of glycosyltransferases (29,(35)(36)(37)(38). In this paper we determine the stoichiometry of Mnn9p and Van1p in M-Pol I, and use mutations in their DXD motifs to investigate in vivo and in vitro the contribution the two proteins make to the activity of the complex.…”