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Anaïs Jensen

p76 studie, a new mannose-6-phospahte protein: Biochemical characterizations, lysosomal localization and fonctional approach

Published on 26 April 2007
Thesis presented April 26, 2007

Abstract:
The protein « p76 » (« hypothetical protein LOC196463 ») was identified some years ago in our laboratory during the course of a proteomic analysis of mannose-6-phosphate proteins purified from human cell lines. The present thesis describes the biochemical characterization and the intracellular localization of p76, as well as some functional tests carried out with recombinant p76. We demonstrated that human p76 sequence bears 6 N-glycosylations and that mannose-6-phosphate sugars were present. Proteolytic maturation of human and murine p76 precursors was observed; a few intermediate forms were partially characterized thanks to anti-p76 antibodies which were developed in the laboratory. Most importantly, we were able to demonstrate the lysosomal localization​ of this protein by both immunofluorescence and sub-cellular fractionation of mouse liver homogenates. As p76 shows a significant sequence homology to a recently cloned phospholipase B from Dictyostelium discoideum, some functional tests were performed, but no phospholipase activity could be detected with recombinant hp76-myc. However, a fat blot experiment showed that hp76-myc binds cardiolipin, a particular phospholipid enriched in mitochondrial and bacterial membranes.

Keywords:
Lysosomal localization, mannose-6-phosphate, post-traditional​ modification, sub-cellular fractionation, immunofluorescence, proteomic

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