Kounnas M Z, Loukinova E B, Stefansson S, Harmony J A, Brewer B H, Strickland D K, Argraves W S
J. H. Holland Laboratory, Biochemistry Department American Red Cross, Rockville, Maryland 20855, USA.
J Biol Chem. 1995 Jun 2;270(22):13070-5. doi: 10.1074/jbc.270.22.13070.
Glycoprotein 330 (gp330) is a member of a family of endocytic receptors related to the low density lipoprotein receptor. gp330 has previously been shown to bind a number of ligands in common with its family member, the low density lipoprotein receptor-related protein (LRP). To identify ligands specific for gp330 and relevant to its localization on epithelia such as in the mammary gland, gp330-Sepharose affinity chromatography was performed. As a result, a 70-kDa protein was selected from human milk and identified by protein sequencing to be apolipoprotein J/clusterin (apoJ). Solid-phase binding assays confirmed that gp330 bound to apoJ with high affinity (Kd = 14.2 nM). Similarly, gp330 bound to apoJ transferred to nitrocellulose after SDS-polyacrylamide gel electrophoresis. LRP, however, showed no binding to apoJ in either type of assay. The binding of gp330 to apoJ could be competitively inhibited with excess apoJ as well as with the gp330 ligands apolipoprotein E, lipoprotein lipase, and the receptor-associated protein, a 39-kDa protein that acts to antagonize binding of all known ligands for gp330 and LRP. Several cultured cell lines that express gp330 and ones that do not express the receptor were examined for their ability to bind and internalize 125I-apoJ. Only cells that expressed gp330 endocytosed and degraded radiolabeled apoJ. Furthermore, F9 cells treated with retinoic acid and dibutyryl cyclic AMP to increase expression levels of gp330 displayed an increased capacity to internalize and degrade apoJ. Cellular internalization and degradation of radiolabeled apoJ could be inhibited with unlabeled apoJ, receptor-associated protein, and gp330 antibodies. The results indicate that gp330 but not LRP can bind to apoJ in vitro and that gp330 expressed by cells can mediate apoJ endocytosis leading to lysosomal degradation.
糖蛋白330（gp330）是一种与低密度脂蛋白受体相关的内吞受体家族成员。此前已表明，gp330与其家族成员低密度脂蛋白受体相关蛋白（LRP）能结合多种共同的配体。为了鉴定gp330特有的、与其在上皮组织（如乳腺）中的定位相关的配体，进行了gp330-琼脂糖亲和层析。结果，从人乳中筛选出一种70 kDa的蛋白质，经蛋白质测序鉴定为载脂蛋白J/簇集蛋白（apoJ）。固相结合试验证实，gp330与apoJ具有高亲和力结合（解离常数Kd = 14.2 nM）。同样，在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳后转移至硝酸纤维素膜上的apoJ也能与gp330结合。然而，在这两种试验中，LRP均未显示与apoJ结合。gp330与apoJ的结合可被过量的apoJ以及gp330配体载脂蛋白E、脂蛋白脂肪酶和受体相关蛋白竞争性抑制，受体相关蛋白是一种39 kDa的蛋白质，可拮抗gp330和LRP所有已知配体的结合。检测了几种表达gp330的培养细胞系和不表达该受体的细胞系结合并内化125I-apoJ的能力。只有表达gp330的细胞能内吞并降解放射性标记的apoJ。此外，用视黄酸和二丁酰环磷腺苷处理以增加gp330表达水平的F9细胞，其内化和降解apoJ的能力增强。未标记的apoJ、受体相关蛋白和gp330抗体可抑制放射性标记的apoJ的细胞内化和降解。结果表明，gp330而非LRP在体外能与apoJ结合，细胞表达的gp330可介导apoJ的内吞作用，导致其溶酶体降解。
