Amancha Praveen Kumar, Koduru Suresh, Nadimpalli Siva Kumar
Protein Biochemistry and Molecular Biology Laboratory, Biochemistry Department, University of Hyderabad, Hyderabad, 500 046, India.
Glycoconj J. 2007 Jul;24(4-5):221-9. doi: 10.1007/s10719-007-9029-0. Epub 2007 Feb 27.
The Mannose 6-phosphate receptor (MPR's) proteins are important for transporting lysosomal enzymes from trans-golgi to the pre-lysosomal compartment. These are conserved in the vertebrates from fish to mammals. We have cloned the full length cDNA for the goat MPR 46 protein and compared its sequences to the other known vertebrate MPR 46 proteins. In the present study the full-length cDNA for the goat MPR 46 protein was expressed in MPR deficient cells. The expressed protein was purified on the multivalent phosphomannan gel in the presence of divalent metal ions. The apparent molecular mass of the expressed protein was found to be approximately 46 kDa and also exhibits oligomeric nature as observed in the other species, by using an MSC1 antibody (that recognizes the MPR 46 from molluscs to mammals) as well as with a peptide specific antibody corresponding to amino acid residues (218-237) of the cytoplasmic tail of human MPR 46 protein. Furthermore the distribution of the expressed protein was visualized by immunofluorescence using MSC1 and LAMP1 antibody. Additionally in the goat MPR 46 expressing cells, the sorting function of the expressed protein to sort cathepsin D to lysosomes was studied by confocal microscopy using cathepsin D antiserum and LAMP1 antibody. The binding of goat MPR 46 to cathepsin D was shown in far Western blotting and the mannose 6-phosphate dependent binding was shown by co-immunoprecipitation.
甘露糖6-磷酸受体（MPR's）蛋白对于将溶酶体酶从反式高尔基体转运至前溶酶体区室至关重要。这些受体在从鱼类到哺乳动物的脊椎动物中是保守的。我们已克隆了山羊MPR 46蛋白的全长cDNA，并将其序列与其他已知的脊椎动物MPR 46蛋白进行了比较。在本研究中，山羊MPR 46蛋白的全长cDNA在缺乏MPR的细胞中表达。表达的蛋白在二价金属离子存在下于多价磷酸甘露聚糖凝胶上进行纯化。通过使用MSC1抗体（可识别从软体动物到哺乳动物的MPR 46）以及与人MPR 46蛋白胞质尾氨基酸残基（218 - 237）对应的肽特异性抗体，发现表达蛋白的表观分子量约为46 kDa，并且如在其他物种中观察到的那样呈现寡聚性质。此外，使用MSC1和LAMP1抗体通过免疫荧光观察表达蛋白的分布。另外，在表达山羊MPR 46的细胞中，使用组织蛋白酶D抗血清和LAMP1抗体通过共聚焦显微镜研究了表达蛋白将组织蛋白酶D分选至溶酶体的分选功能。在远缘Western印迹中显示了山羊MPR 46与组织蛋白酶D的结合，并且通过共免疫沉淀显示了甘露糖6-磷酸依赖性结合。
