Glycosylation of procathepsin L does not account for species molecular-mass differences and is not required for proteolytic activity.
Cathepsin L is a major lysosomal cysteine proteinase in mouse and human cells. Despite similar predicted molecular masses, procathepsin L in these two species migrates on SDS/polyacrylamide gels with apparent molecular masses of 39 kDa and 42 kDa respectively. To determine if glycosylation differences account for this discrepancy, and to ascertain whether glycosylation is essential for enzymic activity, mouse and human procathepsins L were expressed at high concentrations in mouse NIH 3T3 cells or in human A431 cells after DNA-mediated transfection of cloned DNAs for these enzymes. In pulse-chase studies, human procathepsin L transfectants synthesized and secreted large amounts of enzymically active 42 kDa proenzyme and processed it into 34 kDa and 26 kDa intracellular peptides, a pattern of secretion and processing similar to that seen with endogenous or transfected mouse procathepsin L. Both translation of cloned procathepsin L cDNAs in vitro and Endoglycosidase H treatment of 39 kDa mouse and 42 kDa human procathepsin L resulted in non-glycosylated proteins 2 kDa lower in molecular mass than the untreated proteins for both species. This suggests that glycosylation differences are not responsible for the molecular-mass disparity between the two species. Moreover, Endoglycosidase H-treated mouse enzyme retained full proteolytic activity, indicating that glycosylation of cathepsin L is not essential for enzymic function.
Smith
S M
SM
Howard Hughes Medical Institute, Bethesda, MD 20892.
Kane
S E
SE
Gal
S
S
Mason
R W
RW
Gottesman
M M
MM
D000118:Acetylglucosaminidase; D000818:Animals; D056668:Cathepsin L; D002403:Cathepsins / Q000378:metabolism*; D002478:Cells, Cultured; D003546:Cysteine Endopeptidases; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism; D004792:Enzyme Precursors / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D007700:Kinetics; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D051379:Mice; D008815:Mice, Inbred Strains; D008970:Molecular Weight; D012313:RNA / Q000096:biosynthesis; D014162:Transfection
D004792:Enzyme Precursors; D012313:RNA; D000118:Acetylglucosaminidase; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D002403:Cathepsins; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; C534281:CTSL protein, human; D056668:Cathepsin L; C534278:Ctsl protein, mouse