Biosynthesis of platelet glycoprotein V expressed as a single subunit or in association with GPIb-IX.
Glycoprotein (GP) V is noncovalently linked to GPIbalpha, GPIbbeta and GPIX within the platelet GPIb-V-IX complex, a receptor for von Willebrand factor and thrombin. Two functions have been ascribed to GPV, namely, the modulation of thrombin- and collagen-dependent platelet responses. The biosynthesis of this molecule was investigated in pulse-chase metabolic labelling experiments performed in CHO cell lines transfected with GPV, alone or in the presence of GPIb-IX. GPV could not be detected at the surface of cells expressing the single subunit but was found instead as a soluble form in the culture medium. In pulse-chase studies, an immature 70 kDa protein was detected in cell lysates, whereas a fully processed 80-82 kDa form was only observed in the culture supernatants at later chase times. Immature GPV was N-glycosylated and retained before the medial Golgi while the secreted molecule contained complex sialylated sugars. The mature soluble form of GPV was produced by an enzymatic cleavage which was not affected by inhibitors of proteasome, calpain or metalloproteinases. When GPV was cotransfected with GPIb-IX, the former was no longer found in the culture supernatant but was retained in the cell membrane as shown by fluorescence-activated cell sorting and confocal microscopy analyses. Surface expressed GPV was processed from an immature 70 kDa form to produce a mature 80 kDa protein, processing similar to the intracellular trafficking of GPIbalpha. These results indicate that correct biosynthesis and surface expression of GPV in platelets requires the presence of the other subunits of the GPIb-V-IX complex.
Strassel
Catherine
C
INSERM U.311, Etablissement Français du Sang-Alsace, Strasbourg, France.
Moog
Sylvie
S
Baas
Marie-Jeanne
MJ
Cazenave
Jean-Pierre
JP
Lanza
François
F
D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D001792:Blood Platelets / Q000378:metabolism*; D015153:Blotting, Western; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004591:Electrophoresis, Polyacrylamide Gel; D005434:Flow Cytometry; D006031:Glycosylation; D006801:Humans; D020014:K562 Cells; D018613:Microscopy, Confocal; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000378:metabolism*; D010980:Platelet Membrane Glycoproteins / Q000096:biosynthesis*; D011233:Precipitin Tests
D000911:Antibodies, Monoclonal; D019038:Platelet Glycoprotein GPIb-IX Complex; D010980:Platelet Membrane Glycoproteins