Abstract
Using 500-MHz 1H NMR spectroscopy we have investigated the branch specificity that bovine colostrum CMP-NeuAc:Gal beta 1----4GlcNAc-R alpha 2----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----4GlcNAc beta 1----2Man alpha 1----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 1----4GlcNAc beta 1----2Man alpha 1----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----4GlcNAc beta 1----6Man alpha 1----6 branch is extremely resistant to alpha 2----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----4GlcNAc beta 1----6Man alpha 1----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----4GlcNAc beta 1----4Man alpha 1----3 branch. In contrast, the presence of the Gal beta 1----4GlcNAc beta 1----4Man alpha 1----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----4GlcNAc part of the core is essential for the expression of branch specificity of the sialyltransferase. The sialylation patterns observed in vivo in glycoproteins of different origin are consistent with the in vitro preference of alpha 2----6-sialyltransferase for the Gal beta 1----4GlcNAc beta 1----2Man alpha 1----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----4GlcNAc-R.
Highlights
Using 500-MHz ‘H NMR spectroscopy we have in- tain at least two branches (N-acetyllactosamine units) that vestigated the brancshpecificity that bovine colostrum are linked@1+2 to the2 a-mannose residues of the core and CMP-NeuAc:GalBl+4GlcNAc-R a2+6-sialyltrans- can carry additional branches linkepd1-6 to M a n a 1 4 and ferase shows in its sialylation of bi, tri, and tetraantennary glycopeptides and oligosaccharides of the N-acetyllactosamine type
In order to clarify the mechanism underlying the in uiuo production of specific sialylation patterns occurring on the glycans of N-glycoproteins, we investigated the branch specificity of bovine colostrum a244alyltransferase with bi, tri, and tetraantennary substrates in uitro
We have shown that this enzyme can differentiate between the branches of bi- and triantennary glycopeptides [19, 20]
Summary
Cans of the N-acetyllactosamine typeare the result of the complementary branch specificity of the various glycosyltransferases that arespecific for the acceptor sequence Gal/31+4GlcNAc-R. The results are Complex- or N-acetyllactosamine-type glycansoccur on many mammalian N-glycoproteins [1,2]. 2-7, and Tables 11-IV) are presented in miniprint at the end of this paper. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD20814. Request Document No 86M-2314, cite the authors, and include a check or money order for $5.60 perset of photocopies. Full size photocopies are included in the microfilm edition of the Journal thaits available from Waverly Press. The denoting system used to identify the various galactose residues is indicated in Fig. 1for GP4
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