Ng K, Handman E, Bacic A
Walter and Eliza Hall Institute of Medical Research, Royal Melbourne Hospital, Victoria, Australia.
Glycobiology. 1994 Dec;4(6):845-53. doi: 10.1093/glycob/4.6.845.
Lipophosphoglycan (LPG) is the major cell surface molecule of promastigotes of all Leishmania species. It is comprised of three domains: a conserved GPI anchor linked to a repeating phosphorylated disaccharide (P2; PO4-6-Gal(beta 1-4)Man(alpha 1-) backbone variously substituted with galactose, glucose and arabinose residues in L.major and capped with a neutral oligosaccharide. Using a microsomal membrane preparation from L.major, we have been able to demonstrate that galactose from UDP-[14C]galactose can be transferred to an endogenous acceptor, characterized as LPG. An in vitro assay was established, based on anion-exchange HPLC, that concurrently identifies and quantitates the products of the galactosyltransferases. We show that the products formed are [14C]galactose-labelled P3 (PO4-6-[Gal(beta 1-3)]Gal(beta 1-4)Man(alpha 1-), P4b (PO4-6-[Gal(beta 1-3)Gal(beta 1-3)]Gal(beta 1-4)Man(alpha 1-) and P5b(PO4-6-[Gal(beta 1-3)Gal(beta 1-3)Gal(beta 1-3)]Gal(beta 1- 4)Man(alpha 1-). These are major galactosylated repeating units of the backbone of L.major LPG. The same products are also formed when LPG from L.donovani, which contains an unbranched backbone of P2 repeats, is used as an exogenous acceptor with L.major microsomal membranes and UDP-[14C]galactose. In addition, no formation of radioactive backbone repeats (P2) was detected in membrane incubations containing UDP-[14C]galactose with or without added unlabelled GDP-mannose, indicating that the addition of the (beta 1-3)-linked galactose branches is independent of the synthesis of the repeating disaccharide (P2) backbone. Preliminary kinetic analyses suggest that the addition of multiple (beta 1-3)-linked galactose residues may be catalysed by more than one (beta 1-3) galactosyltransferase. The (beta 1-3)galactosyltransferase(s) activity was not detected in microsomal membrane preparations from promastigotes of L.donovani.
脂磷壁酸聚糖（LPG）是所有利什曼原虫前鞭毛体的主要细胞表面分子。它由三个结构域组成：一个保守的糖基磷脂酰肌醇（GPI）锚，连接到一个重复的磷酸化二糖（P2；PO4-6-Gal（β1-4）Man（α1-）主链，在硕大利什曼原虫中被半乳糖、葡萄糖和阿拉伯糖残基以各种方式取代，并由一个中性寡糖封端。使用来自硕大利什曼原虫的微粒体膜制剂，我们已经能够证明UDP-[14C]半乳糖中的半乳糖可以转移到一种内源性受体上，该受体被鉴定为LPG。基于阴离子交换高效液相色谱建立了一种体外测定法，该方法可以同时鉴定和定量半乳糖基转移酶的产物。我们表明形成的产物是[14C]半乳糖标记的P3（PO4-6-[Gal（β1-3）]Gal（β1-4）Man（α1-）、P4b（PO4-6-[Gal（β1-3）Gal（β1-3）]Gal（β1-4）Man（α1-）和P5b（PO4-6-[Gal（β1-3）Gal（β1-3）Gal（β1-3）]Gal（β1-4）Man（α1-）。这些是硕大利什曼原虫LPG主链的主要半乳糖基化重复单元。当来自杜氏利什曼原虫的LPG（其含有P2重复的无分支主链）用作与硕大利什曼原虫微粒体膜和UDP-[14C]半乳糖的外源性受体时，也会形成相同的产物。此外，在含有UDP-[14C]半乳糖且添加或不添加未标记的GDP-甘露糖进行的膜孵育中，未检测到放射性主链重复序列（P2）的形成，这表明添加（β1-3）连接的半乳糖分支独立于重复二糖（P2）主链的合成。初步动力学分析表明，多个（β1-3）连接的半乳糖残基的添加可能由一种以上的（β1-3）半乳糖基转移酶催化。在杜氏利什曼原虫前鞭毛体的微粒体膜制剂中未检测到（β1-3）半乳糖基转移酶活性。
