Title : Biochemical characterization and lysosomal localization of
the mannose-6-phosphate protein p76 (hypothetical
protein LOC196463 )
Abstract :
- Most soluble lysosomal proteins carry Man6P (mannose 6-phosphate), a specific carbohydrate marker that enables their binding to cellular MPRs (Man6P receptors) 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 the latter 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 by SDS /PAGE
- We named this protein ' p76 ' and it appeared later in the NCBI protein database as the 'hypothetical protein LOC196463 '
- In the present paper, we describe the identification of p76 by MS and we analyse several of its biochemical characteristics
- The presence of Man6P sugars was confirmed by an MPR overlay experiment, which showed the direct and Man6P-dependent interaction between p76 and the MPR
- The presence of six N-glycosylation sites was validated by progressive peptide-N-glycosidase F deglycosylation
- Experiments using N- and C-termini directed anti- p76 antibodies provided insights into p76 maturation
- Most importantly, we were able to demonstrate the lysosomal localization of this protein , which was initially suggested by its Man6P tags, by both immunofluorescence and sub-cellular fractionation of mouse liver homogenates