Ujita M, McAuliffe J, Suzuki M, Hindsgaul O, Clausen H, Fukuda M N, Fukuda M
Glycobiology Program, Cancer Research Center, The Burnham Institute, La Jolla, California 92037, USA.
J Biol Chem. 1999 Apr 2;274(14):9296-304. doi: 10.1074/jbc.274.14.9296.
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.
I-分支的聚-N-乙酰乳糖胺是一种独特的碳水化合物，由连接到线性聚-N-乙酰乳糖胺上的N-乙酰乳糖胺分支组成，它由I-分支β1,6-N-乙酰葡糖胺基转移酶合成。I-分支的聚-N-乙酰乳糖胺可以携带二价功能性寡糖，如唾液酸化路易斯x，与单价功能性寡糖相比，它能提供更好的碳水化合物配体。在本研究中，我们首先证明，从人PA-1胚胎癌细胞克隆的I-分支β1,6-N-乙酰葡糖胺基转移酶优先将β1,6-连接的GlcNAc转移到靠近非还原末端的N-乙酰乳糖胺的半乳糖基残基上。然后我们证明，在各种β1,4-半乳糖基转移酶（β4Gal-Ts）中，β4Gal-TI最有效地将半乳糖添加到线性和分支的聚-N-乙酰乳糖胺上。当将β1,6-GlcNAc分支的聚-N-乙酰乳糖胺与β4Gal-TI和i-延伸β1,3-N-乙酰葡糖胺基转移酶的混合物一起孵育时，主要产物是在线性Galβ1→4GlcNAcβ1→3侧链上有一个N-乙酰乳糖胺延伸的寡糖。只有少量产物含有未延伸N-乙酰乳糖胺的半乳糖基化I-分支。这一发现可以通过以下事实来解释：β4Gal-TI很难将半乳糖添加到连接在线性聚-N-乙酰乳糖胺上的β1,6-GlcNAc上，而β1,3-N-乙酰葡糖胺基转移酶和β4Gal-TI能有效地将N-乙酰乳糖胺添加到线性聚-N-乙酰乳糖胺上。总之，这些结果强烈表明，I-分支的半乳糖基化是I-分支聚-N-乙酰乳糖胺合成中的限速步骤，使得聚-N-乙酰乳糖胺主要沿着线性聚-N-乙酰乳糖胺侧链延伸。这些发现与之前关于人红细胞、PA-1胚胎癌细胞和兔红细胞中的聚-N-乙酰乳糖胺含有多个短I-分支的发现完全一致。
