Poly-N-acetyllactosamine synthesis in branched N-glycans is controlled by complemental branch specificity of i-extension enzyme and beta 1,4-galactosyltransferase I

Poly-N-acetyllactosamine is a unique carbohydrate that can carry various fu nctional oligosaccharides, such as sialyl Lewis X. It has been shown that t he amount of poly-N-acetyllactosamine is increased in N-glycans, when they contain Gal beta 1 --> 4GlcNAc beta 1 --> 6(Gal beta 1 --> 4GlcNAc beta 1 - -> 2)Man alpha 1 --> branched structure. To determine how this increased sy nthesis of poly-N-acetyllactosamines takes place, the branched acceptor was incubated with a mixture of i-extension enzyme (iGnT) and beta 1,4-galacto syltransferase I(beta 4Gal-TI), First, N-acetyllactosamine repeats were mor e readily added to the branched acceptor than the summation of poly-N-acety llactosamines formed individually on each unbranched acceptor. Surprisingly , poly-N-acetyllactosamine was more efficiently formed on Gal beta 1 --> 4G lcNAc beta 1 --> 2Man alpha --> R side chain than in Gal beta 1 --> 4GlcNAc beta 1 --> 6Man alpha --> R, due to preferential action of iGnT on Gal bet a 1 --> 4GlcNAc beta 1 --> 2Man alpha --> R side chain. On the other hand, galactosylation was much more efficient on pl,G-linked GlcNAc than beta 1,a -linked GlcNAc, preferentially forming Gal beta 1 --> 4GlcNAc beta 1 --> 6( GlcNA beta 1 --> 2)Man alpha 1 --> 6Man beta --> R. Starting with this pref ormed acceptor, N-acetyllactosamine repeats were added almost equally to Ga l beta 1 --> 4GlcNAc beta 1 --> 6Man alpha --> R and Gal beta 1 --> 4GlcNA beta 1 --> 2Man alpha --> R side chains. Taken together, these results indi cate that the complemental branch specificity of iGnT and beta 4Gal-TI lead s to efficient and equal addition of N-acetyllactosamine repeats on both si de chains of GlcNAc beta 1 --> 6(GlcNAc beta 1 --> 2)Man alpha 1 --> 6Man b eta --> R structure, which is consistent with the structures found in natur e. The results also suggest that the addition of Gal beta 1 --> 4GlcNA beta 1 --> 6 side chain on Gal beta 1 --> 4GlcNAc beta 1 --> 2Man --> R side ch ain converts the acceptor to one that is much more favorable for iGnT and b eta 4Gal-TI.