Characterisation of the biosynthesis and processing of the neutrophil granule membrane protein CD63 in myeloid cells.
The biosynthesis and processing of the neutrophil granule membrane protein CD63, present in azurophil granules, was investigated in four myeloid cell lines. The amount of CD63 synthesised differed, so did the amount of protein processed to high molecular weight forms, with the demonstration of a more prominent synthesis of CD63 in K562 cells. Newly synthesised CD63 was initially detected as two precursor forms with molecular weight of 32 and 35 kDa, respectively. These two initial forms were processed further to yield high molecular weight forms of CD63 with a mean molecular weight of 50 kDa. Treatment with endoglycosidase H or N-glycosidase F revealed a protein core, free from asparagine-linked carbohydrates, with a molecular weight of 23 kDa. Newly synthesised CD63 was susceptible to digestion with endoglycosidase H, and the protein was not completely resistant to endoglycosidase H until after 4 h of chase, indicating that transport through the medial and trans-Golgi complex with conversion of high-mannose carbohydrates to complex oligosaccharide side chains had occurred. This finding indicates a relatively long processing time for CD63 compared to that of soluble azurophil granule proteins. By digestion with O-glycanase, the existence of O-linked oligosaccharides on CD63 could not be demonstrated. Biosynthetic labelling of cells in the presence of brefeldin A showed the importance of a functional Golgi apparatus for processing of the protein to its high molecular weight forms.
Ageberg
M
M
Department of Haematology, Lund University, Lund, Sweden. malin.ageberg@hematologi.lu.se
Lindmark
A
A
D015703:Antigens, CD / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D001692:Biological Transport; D020126:Brefeldin A / Q000494:pharmacology; D045744:Cell Line, Tumor; D003594:Cytoplasmic Granules / Q000737:chemistry; D006026:Glycoside Hydrolases / Q000502:physiology; D006596:Hexosaminidases / Q000502:physiology; D006801:Humans; D006868:Hydrolysis; D008562:Membrane Glycoproteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D022423:Myeloid Cells / Q000737:chemistry / Q000378:metabolism*; D009504:Neutrophils / Q000737:chemistry / Q000378:metabolism; D010980:Platelet Membrane Glycoproteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D011498:Protein Precursors / Q000096:biosynthesis; D011499:Protein Processing, Post-Translational; D060149:Tetraspanin 30
D015703:Antigens, CD; C554586:CD63 protein, human; D008562:Membrane Glycoproteins; D010980:Platelet Membrane Glycoproteins; D011498:Protein Precursors; D060149:Tetraspanin 30; D020126:Brefeldin A; D006026:Glycoside Hydrolases; D006596:Hexosaminidases; C060638:glycopeptide alpha-N-acetylgalactosaminidase