Biochemical characterization and lysosomal localization of the mannose-6-phosphate protein p76 (hypothetical protein LOC196463)
Résumé
Most soluble lysosomal proteins carry mannose-6-phosphate (Man6P), a specific carbohydrate marker that enables their binding to cellular Man6P receptors (MPRs) and their subsequent targeting towards the lysosome. This characteristic was exploited to identify novel soluble lysosomal proteins by proteomic analysis of Man6P proteins purified from a human cell line. Among the proteins identified during the course of this study (Journet, Chapel, Kieffer, Roux, and Garin (2002) Proteomics, 2, 1026-1040), some had not been previously described as lysosomal proteins. We focused on a protein detected at 76 kDa using SDS-PAGE. We named this protein "p76" and it appeared later in the NCBI protein database as the "hypothetical protein LOC196463". In the present report, we describe p76 identification by mass spectrometry and we analyse several of its biochemical characteristics. The presence of Man6P sugars was confirmed by an MPR overlay experiment, showing the direct and Man6P dependent interaction between p76 and the MPR. The presence of 6 N-glycosylation sites was validated by progressive peptide-N-glycosidase F (PNGase F) deglycosylation. Experiments using N-terminus and C-terminus directed anti-p76 antibodies provided insights into p76 maturation. Most importantly, we were able to demonstrate the lysosomal localisation of this protein, initially suggested by its Man6P tags, by both immunofluorescence and sub-cellular fractionation of mouse liver homogenates.
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