An investigation into the significance of the N-linked oligosaccharides of Leishmania gp63.
Leishmania major promastigotes, when grown in the presence of tunicamycin (TM), produced a plasma membrane-bound, proteolytically active gp63 with a lower molecular weight than the native glycoprotein. However, this lower molecular weight form of gp63 continued to be recognized by concanavalin A (Con A), suggesting that inhibition of N-linked glycosylation was not complete. Metabolic labeling of gp63, using [35S]methionine, demonstrated that in the range of 5-10 micrograms ml-1 TM, only the lower molecular weight form was synthesized, suggesting that inhibition was complete and that lectin binding was likely due to the GPI anchored sugars. Removal of the oligosaccharides from L. major and L. mexicana amazonensis promastigotes using endoglycosidase F, caused the gp63 molecular weight to decrease to the same value observed in the presence of TM, once again without affecting the proteolytic activity. However, this deglycosylated enzyme continued to bind Con A until subsequently treated with periodate. The latter oxidation reaction resulted in complete loss of Con A binding without inhibiting the protease activity or the substrate specificity of gp63. Further investigations revealed that both glycosylated and deglycosylated gp63 were resistant to proteolytic digestion by either autolysis or cathepsin D. These findings indicate that the N-linked oligosaccharides of gp63 are not essential for folding, transport, maintenance of enzyme activity or resistance to proteolysis.
Funk
V A
VA
Department of Biochemistry and Microbiology, University of Victoria, Canada.
Jardim
A
A
Olafson
R W
RW
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002402:Cathepsin D / Q000494:pharmacology; D003208:Concanavalin A / Q000378:metabolism; D004795:Enzyme Stability; D006031:Glycosylation; D018320:Leishmania major / Q000187:drug effects / Q000378:metabolism*; D007894:Leishmania mexicana / Q000187:drug effects / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000378:metabolism; D008666:Metalloendopeptidases / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism*; D010084:Oxidation-Reduction; D010455:Peptides / Q000737:chemistry; D017510:Protein Folding; D015800:Protozoan Proteins / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity; D014415:Tunicamycin / Q000494:pharmacology
D008562:Membrane Glycoproteins; D009844:Oligosaccharides; D010455:Peptides; D015800:Protozoan Proteins; D003208:Concanavalin A; D014415:Tunicamycin; D002402:Cathepsin D; D008666:Metalloendopeptidases; C055146:glycoprotein gp63, Leishmania