Liou Benjamin, Haffey Wendy D, Greis Kenneth D, Grabowski Gregory A
From the Division of Human Genetics, Cincinnati Children's Hospital Medical Center, and the Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, Ohio 45229 and.
the Department of Cancer Biology, Vontz Center for Molecular Studies, University of Cincinnati Medical Center, Cincinnati, Ohio 45229.
J Biol Chem. 2014 Oct 24;289(43):30063-74. doi: 10.1074/jbc.M114.593616. Epub 2014 Sep 8.
The acid β-glucosidase (glucocerbrosidase (GCase)) binding sequence to LIMP-2 (lysosomal integral membrane protein 2), the receptor for intracellular GCase trafficking to the lysosome, has been identified. Heterologous expression of deletion constructs, the available GCase crystal structures, and binding and co-localization of identified peptides or mutant GCases were used to identify and characterize a highly conserved 11-amino acid sequence, DSPIIVDITKD, within human GCase. The binding to LIMP-2 is not dependent upon a single amino acid, but the interactions of GCase with LIMP-2 are heavily influenced by Asp(399) and the di-isoleucines, Ile(402) and Ile(403). A single alanine substitution at any of these decreases GCase binding to LIMP-2 and alters its pH-dependent binding as well as diminishing the trafficking of GCase to the lysosome and significantly increasing GCase secretion. Enterovirus 71 also binds to LIMP-2 (also known as SCARB2) on the external surface of the plasma membrane. However, the LIMP-2/SCARB2 binding sequences for enterovirus 71 and GCase are not similar, indicating that LIMP-2/SCARB2 may have multiple or overlapping binding sites with differing specificities. These findings have therapeutic implications for the production of GCase and the distribution of this enzyme that is delivered to various organs.
已确定酸性β-葡萄糖苷酶(葡糖脑苷脂酶(GCase))与LIMP-2(溶酶体整合膜蛋白2)的结合序列,LIMP-2是细胞内GCase转运至溶酶体的受体。利用缺失构建体的异源表达、现有的GCase晶体结构以及已鉴定肽段或突变型GCase的结合与共定位,来鉴定和表征人GCase中一个高度保守的11个氨基酸序列DSPIIVDITKD。与LIMP-2的结合并不依赖于单个氨基酸,但GCase与LIMP-2的相互作用受到天冬氨酸(Asp399)以及两个异亮氨酸Ile402和Ile403的严重影响。这些位点中任何一个发生单个丙氨酸取代都会降低GCase与LIMP-2的结合,改变其pH依赖性结合,同时减少GCase向溶酶体的转运,并显著增加GCase的分泌。肠道病毒71也与质膜外表面的LIMP-2(也称为SCARB2)结合。然而,肠道病毒71和GCase的LIMP-2/SCARB2结合序列并不相似,这表明LIMP-2/SCARB2可能具有多个或重叠的、具有不同特异性的结合位点。这些发现对GCase的生产以及该酶向各个器官的分布具有治疗意义。