Regulation of I-branched poly-N-acetyllactosamine synthesis. Concerted actions by I-extension enzyme, I-branching enzyme, and beta1,4-galactosyltransferase I.
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 beta1, 6-N-acetylglucosaminyltransferase. I-branched poly-N-acetyllactosamine can carry bivalent functional oligosaccharides such as sialyl Lewisx, which provide much better carbohydrate ligands than monovalent functional oligosaccharides. In the present study, we first demonstrate that I-branching beta1, 6-N-acetylglucosaminyltransferase cloned from human PA-1 embryonic carcinoma cells transfers beta1,6-linked GlcNAc preferentially to galactosyl residues of N-acetyllactosamine close to nonreducing terminals. We then demonstrate that among various beta1, 4-galactosyltransferases (beta4Gal-Ts), beta4Gal-TI is most efficient in adding a galactose to linear and branched poly-N-acetyllactosamines. When a beta1,6-GlcNAc branched poly-N-acetyllactosamine was incubated with a mixture of beta4Gal-TI and i-extension beta1,3-N-acetylglucosaminyltransferase, the major product was the oligosaccharide with one N-acetyllactosamine extension on the linear Galbeta1-->4GlcNAcbeta1-->3 side chain. Only a minor product contained galactosylated I-branch without N-acetyllactosamine extension. This finding was explained by the fact that beta4Gal-TI adds a galactose poorly to beta1,6-GlcNAc attached to linear poly-N-acetyllactosamines, while beta1, 3-N-acetylglucosaminyltransferase and beta4Gal-TI efficiently add N-acetyllactosamine to linear poly-N-acetyllactosamines. 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.
Ujita
M
M
Glycobiology Program, Cancer Research Center, The Burnham Institute, La Jolla, California 92037, USA. minoru@burnham-inst.org
McAuliffe
J
J
Suzuki
M
M
Hindsgaul
O
O
Clausen
H
H
Fukuda
M N
MN
Fukuda
M
M
D000818:Animals; D004912:Erythrocytes / Q000378:metabolism; D005700:Galactosyltransferases / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008954:Models, Biological; D017351:N-Acetylglucosaminyltransferases / Q000378:metabolism*; D011134:Polysaccharides / Q000096:biosynthesis*; D011817:Rabbits; D013724:Teratoma / Q000378:metabolism; D014407:Tumor Cells, Cultured
D011134:Polysaccharides; C037199:poly-N-acetyllactosamine; C106501:N-acetylglucosaminyltransferase IGnT; D005700:Galactosyltransferases; D017351:N-Acetylglucosaminyltransferases