Post-translational glycosylation and proteolytic processing of a lysyl oxidase precursor.
The synthesis and post-translational processing of a lysyl oxidase precursor protein predicted by the cDNA sequence of rat aorta lysyl oxidase were investigated. In vitro transcription of the cloned lysyl oxidase cDNA and cell-free translation of its mRNA transcript yielded a 47-kDa protein in the absence and a 50-kDa glycosylated protein in the presence of pancreatic membranes, each of which were immunoprecipitated with antibody against the 32-kDa bovine enzyme. Similarly, an N-glycosylated, 50-kDa protein band was synthesized by and immunoprecipitated from cultured neonatal rat aorta smooth muscle cells labeled with [35S]methionine. Pulse-chase studies of proteins newly synthesized by these cells demonstrated that the glycosylated 50-kDa precursor is secreted and that it is processed to the 32-kDa molecular form of lysyl oxidase principally in the medium. The presence of an extracellular processing enzyme activity which converts the 50-kDa precursor to the 32-kDa species was demonstrated by incubating conditioned medium of neonatal rat aorta smooth muscle cell cultures as a source of processing activity with conditioned medium containing 35S-labeled precursor synthesized but not processed by a tumorigenic cell line transfected with a lysyl oxidase expression vector. In contrast to the 50-kDa species, the 32-kDa protein does not appear to be N-glycosylated consistent with the loss of N-linked oligosaccharide units during the processing to the smaller species. The modes of biosynthesis and secretion of lysyl oxidase are discussed in terms of other nonproteolytic activities required for activation of prolysyl oxidase.
Department of Biochemistry, Boston University School of Medicine, Massachusetts 02118.
P C
PC
Trackman
D
D
Bedell-Hogan
J
J
Tang
H M
HM
Kagan
D000818:Animals; D001011:Aorta / Q000166:cytology / Q000201:enzymology; D002478:Cells, Cultured; D004247:DNA / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors; D006031:Glycosylation; D006868:Hydrolysis; D014176:Protein Biosynthesis; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D008249:Protein-Lysine 6-Oxidase / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D014158:Transcription, Genetic
D011498:Protein Precursors; D012333:RNA, Messenger; D004247:DNA; D008249:Protein-Lysine 6-Oxidase