Abstract
Mucin-type O-glycosylation is initiated by UDP-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyltransferases (GalNAc-transferases). The role each GalNAc-transferase plays in O-glycosylation is unclear. In this report we characterized the specificity and kinetic properties of three purified recombinant GalNAc-transferases. GalNAc-T1, -T2, and -T3 were expressed as soluble proteins in insect cells and purified to near homogeneity. The enzymes have distinct but partly overlapping specificities with short peptide acceptor substrates. Peptides specifically utilized by GalNAc-T2 or -T3, or preferentially by GalNAc-T1 were identified. GalNAc-T1 and -T3 showed strict donor substrate specificities for UDP-GalNAc, whereas GalNAc-T2 also utilized UDP-Gal with one peptide acceptor substrate. Glycosylation of peptides based on MUC1 tandem repeat showed that three of five potential sites in the tandem repeat were glycosylated by all three enzymes when one or five repeat peptides were analyzed. However, analysis of enzyme kinetics by capillary electrophoresis and mass spectrometry demonstrated that the three enzymes react at different rates with individual sites in the MUC1 repeat. The results demonstrate that individual GalNAc-transferases have distinct activities and the initiation of O-glycosylation in a cell is regulated by a repertoire of GalNAc-transferases.
Highlights
Hennet et al [5] recently addressed this question by analyzing mice rendered deficient in a close homologue of GalNAc-T1 by gene targeting
Glycosylation of peptides based on MUC1 tandem repeat showed that three of five potential sites in the tandem repeat were glycosylated by all three enzymes when one or five repeat peptides were analyzed
The results demonstrate that individual GalNActransferases have distinct activities and the initiation of O-glycosylation in a cell is regulated by a repertoire of GalNAc-transferases
Summary
Sf9 medium Step 2: DEAE Step 3: S-Sepharose Step 4: dialysis concentration Step 5: Mono-S Step 6: S-12 ml. Due to variable expression of a number of different GalNActransferases, which may show distinct specificities for acceptor substrates. Some GalNAc-transferases may compete for acceptor substrate sites, even if they do not glycosylate the acceptor substrate site [17]. In the present study we investigated the in vitro specificity and kinetic properties of purified recombinant GalNAc-transferases, GalNAc-T1, -T2, and -T3. The results demonstrate unique but partly overlapping acceptor substrate specificities among the three enzymes. Specific sites on peptides were glycosylated, there were differences in kinetic properties at these sites. Selective specificity of GalNAc-T3 for a 6-mer sequence in fibronectin was maintained with the intact fibronectin molecule. The results indicate that the acceptor substrate specificities of the GalNAc-transferases is largely dependent on the primary sequence of the acceptor substrate
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