Localization and processing of glycosylphosphatidylinositol anchored cathepsin D.
We have investigated the effect of a glycosylphosphatidylinositol anchor on the distribution of the soluble lysosomal enzyme cathepsin D. Only 10% of the chimeric protein (CD-GPI) could be detected on the plasma membrane after transfection in CHO cells. Similarly to endogenous cathepsin D, intracellular CD-GPI was detected in vesicular structures, suggesting that CD-GPI is targeted to lysosomes. CD-GPI is present as three forms with M(r) 55, 50 and 37 kD which could correspond to the precursor, intermediate and mature forms of cathepsin D, respectively. CD-GPI was shown to be GPI anchored by differential extractability with Triton X-114 before and after phosphatidylinositol phospholipase C hydrolysis. Intracellular CD-GPI is mainly substituted with oligosaccharides containing uncovered mannose 6-phosphate residues whereas these residues are covered in the cell surface precursor form of CD-GPI. Ammonium chloride treatment reduces the lysosomal delivery of CD-GPI and increases the cell surface expression of its precursor form.
Ogier-Denis
E
E
INSERM U410, Faculté de Médecine Xavier Bichat, Paris, France.
Bauvy
C
C
Couvineau
A
A
De Stefanis
D
D
Isidoro
C
C
Codogno
P
P
D000818:Animals; D016466:CHO Cells; D002402:Cathepsin D / Q000235:genetics / Q000378:metabolism*; D002451:Cell Compartmentation; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D005455:Fluorescent Antibody Technique; D017261:Glycosylphosphatidylinositols / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008247:Lysosomes / Q000201:enzymology*; D008358:Mannose / Q000378:metabolism; D008856:Microscopy, Fluorescence; D009844:Oligosaccharides / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014162:Transfection
D017261:Glycosylphosphatidylinositols; D009844:Oligosaccharides; D011993:Recombinant Fusion Proteins; D002402:Cathepsin D; D008358:Mannose