Moss J M, Reid G E, Mullin K A, Zawadzki J L, Simpson R J, McConville M J
Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Victoria 3052, Australia.
J Biol Chem. 1999 Mar 5;274(10):6678-88. doi: 10.1074/jbc.274.10.6678.
Protozoan parasites of the genus Leishmania secrete a number of glycoproteins and mucin-like proteoglycans that appear to be important parasite virulence factors. We have previously proposed that the polypeptide backbones of these molecules are extensively modified with a complex array of phosphoglycan chains that are linked to Ser/Thr-rich domains via a common Manalpha1-PO4-Ser linkage (Ilg, T., Overath, P., Ferguson, M. A. J., Rutherford, T., Campbell, D. G., and McConville, M. J. (1994) J. Biol. Chem. 269, 24073-24081). In this study, we show that Leishmania mexicana promastigotes contain a peptide-specific mannose-1-phosphotransferase (pep-MPT) activity that adds Manalpha1-P to serine residues in a range of defined peptides. The presence and location of the Manalpha1-PO4-Ser linkage in these peptides were determined by electrospray ionization mass spectrometry and chemical and enzymatic treatments. The pep-MPT activity was solubilized in non-ionic detergents, was dependent on Mn2+, utilized GDP-Man as the mannose donor, and was expressed in all developmental stages of the parasite. The pep-MPT activity was maximal against peptides containing Ser/Thr-rich domains of the endogenous acceptors and, based on competition assays with oligosaccharide acceptors, was distinct from other leishmanial MPTs involved in the initiation and elongation of lipid-linked phosphoglycan chains. In subcellular fractionation experiments, pep-MPT was resolved from the endoplasmic reticulum marker BiP, but had an overlapping distribution with the cis-Golgi marker Rab1. Although Man-PO4 residues in the mature secreted glycoproteins are extensively modified with mannose oligosaccharides and phosphoglycan chains, similar modifications were not added to peptide-linked Man-PO4 residues in the in vitro assays. Similarly, Man-PO4 residues on endogenous polypeptide acceptors were also poorly extended, although the elongating enzymes were still active, suggesting that the pep-MPT activity and elongating enzymes may be present in separate subcellular compartments.
利什曼原虫属的原生动物寄生虫分泌多种糖蛋白和黏蛋白样蛋白聚糖，这些似乎是重要的寄生虫毒力因子。我们之前提出，这些分子的多肽主链被一系列复杂的磷酸聚糖链广泛修饰，这些磷酸聚糖链通过共同的Manα1-PO4-Ser连接与富含丝氨酸/苏氨酸的结构域相连（伊尔格，T.，奥弗拉特，P.，弗格森，M. A. J.，卢瑟福，T.，坎贝尔，D. G.，以及麦康维尔，M. J.（1994年）《生物化学杂志》269卷，24073 - 24081页）。在本研究中，我们表明墨西哥利什曼原虫前鞭毛体含有一种肽特异性甘露糖-1-磷酸转移酶（pep-MPT）活性，该活性将Manα1-P添加到一系列特定肽中的丝氨酸残基上。通过电喷雾电离质谱以及化学和酶处理确定了这些肽中Manα1-PO4-Ser连接的存在和位置。pep-MPT活性可在非离子洗涤剂中溶解，依赖于Mn2 +，利用GDP-Man作为甘露糖供体，并且在寄生虫的所有发育阶段都有表达。pep-MPT活性对含有内源性受体富含丝氨酸/苏氨酸结构域的肽具有最大活性，并且基于与寡糖受体的竞争测定，它与参与脂质连接的磷酸聚糖链起始和延伸的其他利什曼原虫MPT不同。在亚细胞分级分离实验中，pep-MPT与内质网标志物BiP分离，但与顺式高尔基体标志物Rab1分布重叠。尽管成熟分泌糖蛋白中的Man-PO4残基被甘露糖寡糖和磷酸聚糖链广泛修饰，但在体外测定中，类似的修饰并未添加到肽连接的Man-PO4残基上。同样，内源性多肽受体上的Man-PO4残基延伸也很差，尽管延伸酶仍然有活性，这表明pep-MPT活性和延伸酶可能存在于不同的亚细胞区室中。
