BRANCH SPECIFICITY OF BOVINE COLOSTRUM CMP-SIALIC ACID - GAL-BETA-1-]4GLCNAC-R ALPHA-2-]6-SIALYLTRANSFERASE - SIALYLATION OF BIANTENNARY, TRIANTENNARY, AND TETRAANTENNARY OLIGOSACCHARIDES AND GLYCOPEPTIDES OF THE N-ACETYLLACTOSAMINE TYPE

Abstract

Using 500-MHz 1H NMR spectroscopy we have investigated the branch specificity that bovine colostrum CMP-NeuAc:Gal.beta.1.fwdarw.4GlcNAc-R .alpha.2.fwdarw.6-sialyltransferase shows in its sialylation of bi-, tri-, and tetraantennary glycopeptides and oligosaccharides of the N-acetyllactosamine type. The enzyme appears to highly prefer the galactose residue at the Gal.beta.1.fwdarw.4GlcNAc.beta.1.fwdarw.2Man.alpha.1.fwdarw.3 branch for attachment of the 1st mol of sialic acid in all the acceptors tested. The 2nd mol of sialic acid becomes linked mainly to the Gal.beta.l.fwdarw.4GlcNAc.beta.1.fwdarw.2Man.alpha.1.fwdarw.6 branch in bi- and triantennary substrates, but this reaction invariably proceeds at a much lower rate. Under the conditions employed, the Gal.beta.1.fwdarw.4GlcNAc.beta.1.fwdarw.6Man.alpha.1.fwdarw.6 branch is extremely resistant to .alpha.2.fwdarw.6-sialylation. A higher degree of branching of the acceptors leads to a decrease in the rate of sialylation. In particular, the presence of the Gal.beta.1.fwdarw.4 GlcNAc.beta.1.fwdarw.6Man.alpha.1.fwdarw.6 branch strongly inhibits the rate of transfer of both the 1st and the 2nd mol of sialic acid. In addition, it directs the incorporation of the 2nd mol into tetraantennary structures toward the Gal.beta.1.fwdarw.4GlcNAc.beta.1.fwdarw.4Man.alpha.1.fwdarw.3 branch. In contrast, the presence of the Gal.beta.1.fwdarw.4GlcNAc.beta.1.fwdarw.4Man.alpha.1.fwdarw.3 branch has only minor effects on the rates of sialylation and, consequently, on the branch preference of sialic acid attachment. Results obtained with partial structures of tetraantennary acceptors indicate that the Man.beta.1.fwdarw.4GlcNAc part of the core is essential for the expression of branch specificity of the sialytransferase. The sialylation patterns observed in vivo in glycoproteins of different origin are consistent with the in vitro preference of .alpha.2.fwdarw.6-sialyltransferase for the Gal.beta.1.fwdarw.4GlcNAc.beta.1.fwdarw.2Man.alpha.1.fwdarw.3 branch. Our findings suggest that the terminal structures of branched glycans of the N-acetyllactosamine type are the result of the complementary branch specificity of the various glycosyltransferases that are specific for the acceptor sequence Gal.beta.1.fwdarw.4GlcNAc-R.