Leppanen A, Salminen H, Zhu Y, Maaheimo H, Helin J, Costello C E, Renkonen O
Institute of Biotechnology and Department of Biosciences, University of Helsinki, Finland.
Biochemistry. 1997 Jun 10;36(23):7026-36. doi: 10.1021/bi9627673.
Multiply branched polylactosaminoglycans are expressed in glycoproteins and glycolipids of many cells. Interest in their biology stems from their abundant expression in early embryonal cells and from their ability to carry multiple lectin-binding determinants, which makes them prominent ligands and antagonists of cell adhesion proteins. A prototype of their backbones is represented by the decasaccharide LacNAc beta1-3'(LacNAc beta1-6')LacNAc beta1-3'(LacNAc beta1-6')LacNAc (5), where LacNAc is the disaccharide Gal beta1-4GlcNAc. Here, we describe in vitro biosynthesis of glycan 5. Incubation of the linear hexasaccharide LacNAc beta1-3'LacNAc beta1-3'LacNAc (1) with UDP-GlcNAc and alpha midchain beta1,6-GlcNAc transferase activity (GlcNAc to Gal), present in rat serum [Gu, J., Nishikawa, A., Fujii, S., Gasa, S., & Taniguchi, N. (1992) J. Biol. Chem. 267, 2994-2999], gave the doubly branched octasaccharide LacNAc beta1-3'(GlcNAc beta1-6')LacNAc beta1-3'(GlcNAc beta1-6')LacNAc (4). The latter was converted to 5 by enzymatic beta1,4-galactosylation. In the initial branching reaction of 1, two isomeric heptasaccharide intermediates, LacNAc beta1-3'LacNAc beta1-3'(GlcNAc beta1-6')LacNAc (2) and LacNAc beta1-3'(GlcNAc beta1-6')LacNAc beta1-3'LacNAc (3), were formed first at comparable rates. Later, both intermediates were converted to 4, revealing two distinct pathways of the reaction: 1 --> 2 --> 4 and 1 --> 3 --> 4. These data suggest that, regardless of their chain length, linear polylactosamines similar to 1 contain potential branching sites at each of the internal galactoses. The enzyme-binding epitope of 1 is probably LacNAc beta1-3'LacNAc, because the trisaccharides GlcNAc beta1-3'LacNAc and LacNAc beta1-3Gal as well as the tetrasaccharide GlcNAc beta1-3'LacNAc beta1-3Gal were poor acceptors, while LacNAc beta1-3'LacNAc was a good one. Midchain beta1,6-GlcNAc transferase activities present in serum of several mammalian species, including man, resembled closely the rat serum activity in their mode of action and in their acceptor specificity. We suggest that analogous membrane-bound Golgi enzymes are involved in the biosynthesis of multiply branched polylactosamines in vivo.
多分支聚乳糖胺聚糖在许多细胞的糖蛋白和糖脂中表达。对其生物学特性的关注源于它们在早期胚胎细胞中的大量表达以及它们携带多个凝集素结合决定簇的能力，这使得它们成为细胞粘附蛋白的重要配体和拮抗剂。其主链的一个原型由十糖LacNAcβ1-3′(LacNAcβ1-6′)LacNAcβ1-3′(LacNAcβ1-6′)LacNAc（5）表示，其中LacNAc是二糖Galβ1-4GlcNAc。在此，我们描述了聚糖5的体外生物合成。将线性六糖LacNAcβ1-3′LacNAcβ1-3′LacNAc（1）与UDP-GlcNAc以及大鼠血清中存在的α中链β1,6-GlcNAc转移酶活性（GlcNAc转移至Gal）[顾，J.，西川，A.，藤井，S.，加萨，S.，&谷口，N.（1992）《生物化学杂志》267，2994 - 2999]一起孵育，得到双分支八糖LacNAcβ1-3′(GlcNAcβ1-6′)LacNAcβ1-3′(GlcNAcβ1-6′)LacNAc（4）。后者通过酶促β1,4-半乳糖基化转化为5。在1的初始分支反应中，首先以相当的速率形成了两种异构的七糖中间体，LacNAcβ1-3′LacNAcβ1-3′(GlcNAcβ1-6′)LacNAc（2）和LacNAcβ1-3′(GlcNAcβ1-6′)LacNAcβ1-3′LacNAc（3）。随后，两种中间体都转化为4，揭示了该反应的两条不同途径：1→2→4和1→3→4。这些数据表明，与1类似的线性聚乳糖胺，无论其链长如何，在每个内部半乳糖处都含有潜在的分支位点。1的酶结合表位可能是LacNAcβ1-3′LacNAc，因为三糖GlcNAcβ1-3′LacNAc和LacNAcβ1-3Gal以及四糖GlcNAcβ1-3′LacNAcβ1-3Gal都是不良受体，而LacNAcβ1-3′LacNAc是良好受体。包括人类在内的几种哺乳动物血清中存在的中链β1,6-GlcNAc转移酶活性在其作用方式和受体特异性方面与大鼠血清活性非常相似。我们认为，类似的膜结合高尔基体酶参与了体内多分支聚乳糖胺的生物合成。
