Regulation of I-Branched Poly-N-Acetyllactosamine Synthesis

Abstract

I-branched poly-N-acetyllactosamine is a unique carbohydrate composed of N-acetyllactosamine branches attached to linear poly-N-acetyllactosamine, which is synthesized by I-branching ␤1,6-N-acetylglucosaminyltransferase. I-branched poly-N-acetyllactosamine can carry bivalent functional oligosaccharides such as sialyl Lewis x , which provide much better carbohydrate ligands than monovalent functional oligosaccharides. In the present study, we first demonstrate that I-branching ␤1,6-N-acetylglucosaminyltransferase cloned from human PA-1 embryonic carcinoma cells transfers ␤1,6-linked GlcNAc preferentially to galactosyl residues of N-acetyllactosamine close to nonreducing terminals. We then demonstrate that among various ␤1,4-galactosyltransferases (␤4Gal-Ts), ␤4Gal-TI is most efficient in adding a galactose to linear and branched poly-Nacetyllactosamines. When a ␤1,6-GlcNAc branched poly-N-acetyllactosamine was incubated with a mixture of ␤4Gal-TI and i-extension ␤1,3-N-acetylglucosaminyltransferase, the major product was the oligosaccharide with one N-acetyllactosamine extension on the linear Gal␤134GlcNAc␤133 side chain. Only a minor product contained galactosylated I-branch without N-acetyllactosamine extension. This finding was explained by the fact that ␤4Gal-TI adds a galactose poorly to ␤1,6-GlcNAc attached to linear poly-N-acetyllactosamines, while ␤1,3-N-acetylglucosaminyltransferase and ␤4Gal-TI efficiently add N-acetyllactosamine to linear poly-Nacetyllactosamines. Together, these results strongly suggest that galactosylation of I-branch is a rate-limiting step in I-branched poly-N-acetyllactosamine synthesis, allowing poly-N-acetyllactosamine extension mostly along the linear poly-N-acetyllactosamine side chain. These findings are entirely consistent with previous findings that poly-N-acetyllactosamines in human erythrocytes, PA-1 embryonic carcinoma cells, and rabbit erythrocytes contain multiple, short I-branches.

Poly-N-acetyllactosamine Extension inN-Glycans and Core 2- and Core 4-branchedO-Glycans Is Differentially Controlled by i-Extension Enzyme and Different Members of the β1,4-Galactosyltransferase Gene Family

“…We have also found that ␤4Gal-TI, iGnT, and I-branching enzyme (IGnT) are involved in the synthesis of I-branched poly-Nacetyllactosamines (30). In the same study, we found that the addition of N-acetyllactosamine repeats to linear N-acetyllactosamine is preferred over the extension of N-acetyllactosamine to an I branch, mainly because galactosylation of a GlcNAc␤136 branch is inefficient (30). Third, we demonstrated that similar size and similar amounts of poly-Nacetyllactosamine are synthesized in both side chains of GlcNAc␤136(GlcNAc␤132)Man␣136Man␤13 R, which was preformed by the action of N-acetylglucosaminyltransferase V. We found that this equal distribution of poly-N-acetyllactosamines is achieved because ␤4Gal-TI and iGnT have complementary branch specificity toward two side chains (31).…”

“…In I branch formation (30), the GlcNAc␤136 branch in the Gal␤134GlcNAc␤133(GlcNAc␤-136)Gal␤13 R structure is very inefficient for galactosylation, resulting in short I branches as seen in human erythrocytes (57). This inefficiency can be also explained by competition between the Gal terminal in the linear side chain and UDP-Gal for ␤4Gal-TI.…”

“…In a similar fashion, Gal␤134GlcNAc␤136(Gal␤133)GalNAc␣-13octyl (compound 6) was synthesized by coupling of compound 3 with Poly-N-acetyllactosamine Synthesis by iGnT and ␤4Gal-Ts-To assay poly-N-acetyllactosamine formation, 0.5 mM acceptor was incubated with different ␤4Gal-Ts (760.0 nmol/h/ml) and iGnT (380.0 nmol/h/ml) under the conditions described previously (27,31). The incubation mixture was purified by a C 18 -reverse phase Sep-Pak cartridge column (Waters), and the product was analyzed by HPLC using an NH 2 -bonded silica column (Varian Micropak AX-5) as described previously (27,30,31). The radioactivity of aliquots in the effluent was determined.…”

“…To this end, we have previously demonstrated (27) that poly-N-acetyllactosamines in core 2-branched O-glycans are synthesized with a newly discovered member of ␤1,4-galactosyltransferase, ␤4Gal-TIV (28) and a newly cloned i-extension enzyme (iGnT) (29). We have also found that ␤4Gal-TI, iGnT, and I-branching enzyme (IGnT) are involved in the synthesis of I-branched poly-Nacetyllactosamines (30). In the same study, we found that the addition of N-acetyllactosamine repeats to linear N-acetyllactosamine is preferred over the extension of N-acetyllactosamine to an I branch, mainly because galactosylation of a GlcNAc␤136 branch is inefficient (30).…”

Identification and Characterization of Three Novel β1,3-N-Acetylglucosaminyltransferases Structurally Related to the β1,3-Galactosyltransferase Family

“…For example, poly-N-acetyllactosamines are preferentially formed in the specific branch in complex type N-glycans, which are formed by ␤1,6-N-acetylglucosaminyltransferase V (102). In addition, core 2 ␤1,6-N-acetylglucosaminyltransferases 1 and 2 and large I ␤1,6-N-acetylglucosaminyltransferases are branching enzymes critical for elongation with poly-N-acetyllactosamines (48,80,81,(103)(104)(105). Discovery of the multiple ␤3Gn-Ts in addition to other multiple glycosyltransferases involved in the biosynthesis of poly-N-acetyllactosamines (e.g.…”