Regulation of I-branched poly-N-acetyllactosamine synthesis - Concerted actions by i-extension enzyme, I-branching enzyme, and beta 1,4-galactosyltransferase I

I-branched poly-N-acetyllactosamine is a unique carbohydrate composed of N- acetyllactosamine branches attached to linear poly-N-acetyllactosamine, whi ch is synthesized by I-branching beta 1,6-N-acetylglucosaminyl-transferase. I-branched poly-N-acetyllactosamine can carry bivalent functional oligosac charides such as sialyl Lewis(x), which provide much better carbohydrate li gands than monovalent functional oligosaccharides. In the present study, we first demonstrate that I-branching beta 1,6-N-acetylglucosaminyltransferas e cloned from human PA-1 embryonic carcinoma cells transfers beta 1,6-linke d GlcNAc preferentially to galactosyl residues of N-acetyllactosamine close to nonreducing terminals, We then demonstrate that among various beta 1,4- galactosyltransferases (beta 4Gal-Ts), beta 4Gal-TI is most efficient in ad ding a galactose to linear and branched poly-N-acetyllactosamines. When a b eta 1,6-GlcNAc branched poly-N-acetyllactosamine was incubated with a mixtu re of beta 4Gal-TI and i-extension beta 1,3-N-acetylglucosaminyltransferase , the major product was the oligosaccharide with one N-acetyllactosamine ex tension on the linear Gal beta 1-->4GlcNAc beta 1-->3 side chain, Only a mi nor product contained galactosylated I-branch without N-acetyllactosamine e xtension. This finding was explained by the fact that beta 4Gal-TI adds a g alactose poorly to beta 1,6-GlcNAc attached to linear poly-N-acetyllactosam ines, while beta 1,3-N-acetylglucosaminyltransferase and beta 4Gal-TI effic iently add N-acetyllactosamine to linear poly-N-acetyllactosamines. Togethe r, these results strongly suggest that galactosylation of I-branch is a rat e-limiting step in I-branched poly-N-acetyllactosamine synthesis, allowing poly-N-acetyllactosamine extension mostly along the linear poly-N-acetyllac tosamine side chain. These findings are entirely consistent with previous f indings that poly-N-acetyllactosamines in human erythrocytes, PA-1 embryoni c carcinoma cells, and rabbit erythrocytes contain multiple, short I-branch es.