Jardim A, Funk V, Caprioli R M, Olafson R W
Department of Biochemistry and Microbiology, University of Victoria, B.C. Canada.
Biochem J. 1995 Jan 1;305 ( Pt 1)(Pt 1):307-13. doi: 10.1042/bj3050307.
A novel membrane molecule, previously observed to be co-isolated with lipophosphoglycan and called lipophosphoglycan-associated protein, has been detected in Leishmania donovani promastigotes and amastigotes. This kinetoplastid membrane protein (KMP-11) has been purified by preparative SDS/PAGE after organic solvent extraction of promastigote membranes. Isoelectric-focusing experiments indicated that this was an acidic protein with an isoelectric point of 4.8. Immunoblot analysis of subcellular fractions, together with 125I-labelling experiments, showed this molecule to be associated with the promastigote cell surface membrane. KMP-11 was expressed at a copy number similar to that of lipophosphoglycan (1 x 10(6)-2 x 10(6) molecules per cell), making this glycoprotein one of the major features on the parasite cell surface. The primary structure, less a blocked N-terminal region, was determined by automated Edman degradation of peptides derived from CNBr or enzymic fragmentation. Several post-translational modifications were also found during these studies, including an O-linked oligosaccharide and an NG-monomethylarginine functionality which was verified by m.s. Finally, a set of sequential synthetic peptides was made based on the established partial sequence allowing structural determination of two distinct antibody-binding sites for the monoclonal antibodies L98 and L157.
一种新的膜分子，先前观察到它与脂磷壁酸共分离并被称为脂磷壁酸相关蛋白，已在杜氏利什曼原虫前鞭毛体和无鞭毛体中被检测到。这种动质体膜蛋白（KMP - 11）在对前鞭毛体膜进行有机溶剂提取后，通过制备性SDS/PAGE进行了纯化。等电聚焦实验表明这是一种酸性蛋白，其等电点为4.8。对亚细胞组分的免疫印迹分析以及¹²⁵I标记实验表明，该分子与前鞭毛体细胞表面膜相关。KMP - 11的表达拷贝数与脂磷壁酸相似（每个细胞1×10⁶ - 2×10⁶个分子），这使得这种糖蛋白成为寄生虫细胞表面的主要特征之一。通过对源自溴化氰或酶切片段的肽段进行自动埃德曼降解，确定了其一级结构，但不包括封闭的N端区域。在这些研究中还发现了几种翻译后修饰，包括O - 连接寡糖和通过质谱验证的NG - 单甲基精氨酸官能团。最后，基于已确定的部分序列合成了一组连续的合成肽，从而确定了单克隆抗体L98和L157的两个不同抗体结合位点的结构。
