Abbate M, Bachinsky D, Zheng G, Stamenkovic I, McLaughlin M, Niles J L, McCluskey R T, Brown D
Renal Unit, Massachusetts General Hospital, Boston.
Eur J Cell Biol. 1993 Jun;61(1):139-49.
The alpha 2-macroglobulin receptor-associated protein (alpha 2-MRAP) is a 39 to 44 kDa protein that copurifies with the alpha 2-macroglobulin receptor (alpha 2-MR/LRP) and also with gp330, a highly glycosylated protein located within kidney proximal tubules and glomerular podocytes. Both gp330 and the alpha 2-macroglobulin receptor are members of the low density lipoprotein receptor family but the physiological ligands for gp330 are unknown. In order to understand potential functions of the alpha 2-MRAP, specific anti-alpha 2-MRAP antibodies were used for immunocytochemical studies on paraformaldehyde lysine periodate (PLP)-fixed rat kidneys and on snap-frozen/acetone-fixed tissue. Conflicting results were obtained. After PLP fixation, alpha 2-MRAP was detected almost exclusively in rough endoplasmic reticulum (RER) cisternae; cell surface staining was virtually absent. In snap-frozen tissue, intense staining of the proximal tubule brush border was found, with little or no cytoplasmic staining. A series of experiments showed that during incubation of snap-frozen tissues, endogenous alpha 2-MRAP is released in soluble form from its intracellular location (i.e., the RER) and binds to gp330 on the brush border of proximal tubules. The location of binding sites for alpha 2-MRAP in rat kidney was also examined, using an alpha 2-MRAP-IgG fusion protein. In both snap-frozen and PLP-fixed tissues, this probe bound exclusively to brush borders, and not to intracellular sites. Our results demonstrate: a) that in renal proximal tubule cells, alpha 2-MRAP is located predominantly in the RER, b) that alpha 2-MRAP-binding sites are present on gp330, which is on the proximal tubule brush border, and c) that the apparent brush border localization of alpha 2-MRAP detected in snap-frozen sections is due to an artifactual redistribution of endogenous alpha 2-MRAP that occurs during tissue processing.
α2-巨球蛋白受体相关蛋白（α2-MRAP）是一种分子量为39至44 kDa的蛋白质，它与α2-巨球蛋白受体（α2-MR/LRP）以及gp330一起被纯化出来，gp330是一种高度糖基化的蛋白质，位于肾近端小管和肾小球足细胞内。gp330和α2-巨球蛋白受体都是低密度脂蛋白受体家族的成员，但gp330的生理配体尚不清楚。为了了解α2-MRAP的潜在功能，使用特异性抗α2-MRAP抗体对经多聚甲醛赖氨酸高碘酸盐（PLP）固定的大鼠肾脏以及速冻/丙酮固定的组织进行免疫细胞化学研究。结果相互矛盾。PLP固定后，α2-MRAP几乎仅在粗面内质网（RER）池中被检测到；几乎没有细胞表面染色。在速冻组织中，发现近端小管刷状缘有强烈染色，细胞质染色很少或没有。一系列实验表明，在速冻组织孵育过程中，内源性α2-MRAP以可溶形式从其细胞内位置（即RER）释放出来，并与近端小管刷状缘上的gp330结合。还使用α2-MRAP-IgG融合蛋白检测了大鼠肾脏中α2-MRAP结合位点的位置。在速冻和PLP固定的组织中，该探针仅与刷状缘结合，而不与细胞内位点结合。我们的结果表明：a）在肾近端小管细胞中，α2-MRAP主要位于RER中；b）α2-MRAP结合位点存在于近端小管刷状缘的gp330上；c）在速冻切片中检测到的α2-MRAP明显的刷状缘定位是由于组织处理过程中内源性α2-MRAP的人为重新分布所致。
