Structural and Mechanistic Insights into the Catalytic-Domain-Mediated Short-Range Glycosylation Preferences of GalNAc-T4

Abstract

Mucin-type O-glycosylation is initiated by a family of polypeptide GalNAc-transferases (GalNAc-Ts) which are type-II transmembrane proteins that contain Golgi luminal catalytic and lectin domains that are connected by a flexible linker. Several GalNAc-Ts, including GalNAc-T4, show both long-range and short-range prior glycosylation specificity, governed by their lectin and catalytic domains, respectively. While the mechanism of the lectin-domain-dependent glycosylation is well-known, the molecular basis for the catalytic-domain-dependent glycosylation of glycopeptides is unclear. Herein, we report the crystal structure of GalNAc-T4 bound to the diglycopeptide GAT*GAGAGAGT*TPGPG (containing two α-GalNAc glycosylated Thr (T*), the PXP motif and a “naked” Thr acceptor site) that describes its catalytic domain glycopeptide GalNAc binding site. Kinetic studies of wild-type and GalNAc binding site mutant enzymes show the lectin domain GalNAc binding activity dominates over the catalytic domain GalNAc binding activity and that these activities can be independently eliminated. Surprisingly, a flexible loop protruding from the lectin domain was found essential for the optimal activity of the catalytic domain. This work provides the first structural basis for the short-range glycosylation preferences of a GalNAc-T.

Exploring Regulation of Protein O-Glycosylation in Isogenic Human HEK293 Cells by Differential O-Glycoproteomics

“…Past in vitro studies of GalNAc-Ts predicted both overlap in functions as well as unique functions for individual isoforms (11), and the results obtained here with the differential glycoproteomics strategy clearly recapitulates this scenario with the majority of the O-glycoproteome covered by redundancies. Only minor subsets of glycosites were distinctly controlled by the GalNAc-T1/T2/T3 isoforms and several of the glycosites were reproduced from our previous study in HepG2 SC (Fig.…”

“…The lectin domains coordinate glycosylation of distant glycosites (Ϯ 8 -10 residues of initial O-glycosites) with isoform-specific orientation (7,8), and recently the molecular basis for this has been shown to involve combined docking of the partially glycosylated substrate into the lectin and catalytic domain (9). Moreover, a subset of the GalNAc-T isoenzymes selectively or exclusively recognizes acceptor sites with GalNAc residues found immediately adjacent (10), and structural studies demonstrate that the catalytic domain accommodates the first introduced GalNAc residue (10,11). Most of our current understanding is, however, still based largely on in vitro analyses with synthetic peptide and glycopeptide substrates (12,13).…”

Structural basis for peptide substrate specificities of glycosyltransferase GalNAc-T2

“…The residues R362, K363, Q364, and H365 reside on the flexible, semi-conserved catalytic loop 41 of the enzyme. The flap-like loop can additionally contribute to the variability of the −1 pocket size across the GalNAc-T isoforms.…”

UDP-N-acetyl-α-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase from the snail Biomphalaria glabrata – structural reflections

“…The number reflects the distance from the hydroxyl amino acid). Isoenzymes of ppGalNAcTs which prefer to glycosylate acceptor substrates with a Thr/Ser-Pro-X-Pro sequence, where "X" may be any small hydrophobic amino acid, contain three highly conserved aromatic amino acids (Phe280, Trp282, Phe361 in human ppGalNAcT2) which interact with the acceptor [34,38,39]. The corresponding amino acids in the snail Bge-ppGalNAcT are Phe308, Trp310 and Phe389 (Fig.…”