Abstract
Using a defined acceptor substrate peptide as an affinity chromatography ligand we have developed a purification scheme for a unique human polypeptide, UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase (GalNAc-transferase) (White, T., Bennett, E.P., Takio, K., Sørensen, T., Bonding, N., and Clausen, H. (1995) J. Biol. Chem. 270, 24156-24165). Here we report detailed studies of the acceptor substrate specificity of GalNAc-transferase purified by this scheme as well as the Gal-NAc-transferase activity, which, upon repeated affinity chromatography, evaded purification by this affinity ligand. Using a panel of acceptor peptides, a qualitative difference in specificity between these separated transferase activities in four rat organs and two human organs also revealed qualitative differences in specificity. The results support the existence of multiple Gal-NAc-transferase activities and suggest that these are differentially expressed in different organs. As the number of GalNAc-transferases existing is unknown, as is the specificity of the until now cloned and expressed GalNAc-transferases (T1 and T2), it is as yet impossible to relate the results obtained to specific enzyme proteins. The identification of acceptor peptides that can be used to discriminate GalNAc-transferase activities is an important step toward understanding the molecular basis of GalNAc O-linked glycosylation in cells and organs and in pathological conditions.
Highlights
Using a defined acceptor substrate peptide as an affinity chromatography ligand we have developed a purification scheme for a unique human polypeptide, UDPGalNAc:polypeptide N-acetylgalactosaminyltransferase (GalNAc-transferase)
We present evidence that affinity chromatography using a defined synthetic acceptor substrate peptide resulted in separation of two different GalNAc-transferase activities and that these appear to be differentially expressed in organs
Since the human transferase purified by the same procedure using Triton X-100 as detergent in the affinity chromatography was found to be the soluble fragment without the hydrophobic transmembrane segment, this difference in yield could be related to a relatively lower ratio of soluble versus membrane-bound transferase
Summary
The results support the existence of multiple GalNAc-transferase activities and suggest that these are differentially expressed in different organs. In several cases characterization of glycosylation sites has identified peptide motifs that suggest the nature of the acceptor substrate specificities of transferases initiating protein glycosylation. Assigning detailed acceptor substrate specificity toward the cloned and expressed GalNAc-T1 and -T2 awaits comparative studies of recombinant transferases, and data obtained so far using purified enzyme preparations are likely to be biased by copurified mixtures of enzymes (Homa et al, 1993; White et al, 1995; Wang et al, 1993). We present evidence that affinity chromatography using a defined synthetic acceptor substrate peptide resulted in separation of two different GalNAc-transferase activities and that these appear to be differentially expressed in organs. The study identified acceptor peptides capable of discriminating different GalNAc-transferase activities, which should prove valuable for detailed studies of the specificity of identified and cloned enzymes in this family
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