Biochemical and functional characterization of cation dependent (Mr 46,000) goat mannose 6-phosphate receptor.
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.
Amancha
Praveen Kumar
PK
Protein Biochemistry and Molecular Biology Laboratory, Biochemistry Department, University of Hyderabad, Hyderabad, 500 046, India.
Koduru
Suresh
S
Nadimpalli
Siva Kumar
SK
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002412:Cations / Q000737:chemistry / Q000378:metabolism; D018076:DNA, Complementary / Q000096:biosynthesis; D019143:Evolution, Molecular; D005347:Fibroblasts / Q000737:chemistry; D005822:Genetic Vectors; D006041:Goats; D051379:Mice; D008969:Molecular Sequence Data; D017527:Receptor, IGF Type 2 / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D014162:Transfection
D002412:Cations; D018076:DNA, Complementary; D017527:Receptor, IGF Type 2