Identification of common and unique peptide substrate preferences for the UDP-GalNAc:polypeptide alpha-N-acetylgalactosaminyltransferases T1 and T2 derived from oriented random peptide substrates.
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.
Gerken
Thomas A
TA
W. A. Bernbaum Center for Cystic Fibrosis Research, Departments of Pediatrics and Biochemistry, Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA.
Raman
Jayalakshmi
J
Fritz
Timothy A
TA
Jamison
Oliver
O
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D002417:Cattle; D006031:Glycosylation; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D017350:N-Acetylgalactosaminyltransferases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism; D020539:Sequence Analysis, Protein; D013379:Substrate Specificity; D020080:Tandem Repeat Sequences; D014530:Uridine Diphosphate / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D000097763:Polypeptide N-acetylgalactosaminyltransferase
D000596:Amino Acids; D007527:Isoenzymes; D017350:N-Acetylgalactosaminyltransferases; D010446:Peptide Fragments; D014530:Uridine Diphosphate; D000097763:Polypeptide N-acetylgalactosaminyltransferase