Probing polypeptide GalNAc-transferase isoform substrate specificities by in vitro analysis

Abstract

N-acetylgalactosaminyltransferase (GalNAc)-type (mucin-type) O-glycosylation is an abundant and highly diverse modification of proteins. This type of O-glycosylation is initiated in the Golgi by a large family of up to 20 homologous polypeptide GalNAc-T isoenzymes that transfer GalNAc to Ser, Thr and possibly Tyr residues. These GalNAc residues are then further elongated by a large set of glycosyltransferases to build a variety of complex O-glycan structures. What determines O-glycan site occupancy is still poorly understood, although it is clear that the substrate specificities of individual isoenzymes and the repertoire of GalNAc-Ts in cells are key parameters. The GalNAc-T isoenzymes are differentially expressed in cells and tissues in principle allowing cells to produce unique O-glycoproteomes dependent on the specific subset of isoforms present. In vitro analysis of acceptor peptide substrate specificities using recombinant expressed GalNAc-Ts has been the method of choice for probing activities of individual isoforms, but these studies have been hampered by biological validation of actual O-glycosylation sites in proteins and number of substrate testable. Here, we present a systematic analysis of the activity of 10 human GalNAc-T isoenzymes with 195 peptide substrates covering known O-glycosylation sites and provide a comprehensive dataset for evaluating isoform-specific contributions to the O-glycoproteome.

Site-specific O-glycosylation of members of the low-density lipoprotein receptor superfamily enhances ligand interactions

“…Many of the other 20 GALNT genes have been identified as candidate genes for dispositions to common diseases (25,34), and we were recently able to provide extensive validation for the role of GALNT2 in dyslipidemia in man, primates and rodents identified several sitespecifically regulated O-glycoprotein targets (19). The substrate specificities and functions of GalNAc-Ts have mainly been studied by in vitro enzyme assays using short peptides as acceptors in the past (35), and this approach has provided important insights into specific functions of many individual isoenzymes including GalNAc-T11. However, although GalNAc-T11 does function with a number of short peptide substrates (21-22,35), it only functions in vitro with the LA linker regions in the context of the entire folded LAmodules (11).…”

“…Knockout of GALNT11 or the COSMC chaperone required for the core1 synthase C1GALT1 to truncate O-glycans to the initial GalNAc residue in HepG2 cells did not affect expression, trafficking or shedding of the endogenous LDLR, as evaluated by SDS-PAGE Western blot analysis of total cell lysates ( Fig. 2A) and 35 S-labelled pulse-chase experiments (Fig. 2B).…”

Influenza A Virus-Induced Expression of a GalNAc Transferase, GALNT3, via MicroRNAs Is Required for Enhanced Viral Replication

“…This type of glycosylation is initiated by the UDP-GalNAc polypeptide:N-acetylgalactosaminyltransferase (ppGalNAcT) family and formed by GalNAc attachment to the serine/threonine (Ser/Thr) side chains of protein substrates (1)(2)(3). The generation of complex O-linked glycans is involved in a variety of biological molecular processes, such as cell motility, cell growth and death, cell-to-cell communication, the immune response, and host-pathogen interactions (4)(5)(6).…”

A Contra Capture Protein Array Platform for Studying Post-translationally Modified (PTM) Auto-antigenomes

“…GalNac-T11 could specifically glycosylate POMC. The expressed NRP2 fragment displayed on the array was also glycosylated by GalNac-T2 and T3 although a corresponding synthetic peptide was glycosylated only by GalNac-T2 in earlier studies (68). This may be due to a better presentation of the NRP2 fragment on the array instead of as an isolated synthetic peptide (68).…”

“…7A, green boxes)). On these arrays, we have spotted genes encoding specific substrates for the three different GALNTs based on studies on synthetic peptides, an NRP2 fragment for GalNac-T2, a POMC fragment for GalNac-T11, an APOE and a CD55 fragment for GalNac-T3 (44,67,68). Besides wild type APOE, we have also spotted a gene encoding an APOE mutant with the glycosylation sites Thr and Ser by GALNT3 mutated to Ala that cannot be glycosylated by GalNac-T3.…”