Identification of Common and Unique Peptide Substrate Preferences for the UDP-GalNAc:Polypeptide α-N-acetylgalactosaminyltransferases T1 and T2 Derived from Oriented Random Peptide Substrates

Thomas A Gerken,Jayalakshmi Raman,Timothy A Fritz,Oliver Jamison

doi:10.1074/jbc.m605149200

Abstract

A large family of UDP-GalNAc:polypeptide alpha-N-acetylgalactosaminyltransferases (ppGalNAc Ts) catalyzes the first step of mucin-type protein O-glycosylation by transferring GalNAc to serine and threonine residues of acceptor polypeptides. The acceptor peptide substrate specificity and specific protein targets of the individual ppGalNAc T family members remain poorly characterized and poorly understood, despite the fact that mutations in two individual isoforms are deleterious to man and the fly. In this work a series of oriented random peptide substrate libraries, based on the GAGAXXXTXXXAGAGK sequence motif (where X = randomized positions), have been used to obtain the first comprehensive determination of the peptide substrate specificities of the mammalian ppGalNAc T1 and T2 isoforms. ppGalNAc T-glycosylated random peptides were isolated by lectin affinity chromatography, and transferase amino acid preferences were determined by Edman amino acid sequencing. The results reveal common and unique position-sensitive features for both transferases, consistent with previous reports of the preferences of ppGalNAc T1 and T2. The random peptide substrates also reveal additional specific features that have never been described before that are consistent with the x-ray crystal structures of the two transferases and furthermore are reflected in a data base analysis of in vivo O-glycosylation sites. By using the transferase-specific preferences, optimum and selective acceptor peptide substrates have been generated for each transferase. This approach represents a relatively complete, facile, and reproducible method for obtaining ppGalNAc T peptide substrate specificity. Such information will be invaluable for identifying isoform-specific peptide acceptors, creating isoform-specific substrates, and predicting O-glycosylation sites.

Highlights

UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases2 represent a large family of Golgi resident glycosyltransferases that initiate mucin-type O-glycosylation by transferring ␣-GalNAc to peptide Ser and Thr residues
These findings suggest that certain ppGalNAc T isoforms may have specific protein substrates, of presently unknown identity, whose glycosylation is integral for normal development or cellular processes
We report on an approach for obtaining a more complete determination of the peptide substrate specificities of ppGalNAc T1 and T2 by utilizing a series of oriented random peptide substrates, each containing a central Thr or Ser residue flanked by a series of randomized residues

Summary

Introduction

UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (ppGalNAc Ts) represent a large family of Golgi resident glycosyltransferases that initiate mucin-type O-glycosylation by transferring ␣-GalNAc to peptide Ser and Thr residues. Recently the role of ppGalNAc T3 in familial tumoral calcinosis has been shown to be its site-specific glycosylation of the phosphaturic factor, FGF23, which modulates it processing and secretion [24] Except for this recent example, the peptide substrate specificities and specific protein targets of the individual mammalian ppGalNAc T isoforms remain poorly characterized and poorly understood. To begin to address this deficiency, we have used as substrates, for ppGalNAc T1 and T2, the large apomucin tandem repeat domains containing multiple Ser and Thr residues from the pig and dog salivary gland mucins (PSM and CSM) [29, 30] Combined, these substrates contain over 50 unique glycosylation sites.

Methods

Results

Conclusion