Expression analysis of recombinant lysyl oxidase (LOX) in myofibroblastlike cells.
Lysyl oxidase (LOX), originally known as the enzyme required for initiation of covalent cross-linking in collagens and elastin, is now known to be a member of a family of genetically related proteins. LOX, or a related protein, has also been localized intracellularly, both in association with the cytoskeleton and in the cell nucleus. To determine the structural requirements for secretion, maturation, and nuclear location of LOX in a cellular context, we have devised an homologous cell model for expression of the recombinant protein. Murine recombinant LOX was expressed in 3T6-5 myofibroblast-like cells as a 51-kD precursor, which was observed in the cytoplasm but not in the nucleus. To investigate whether potential alternative translation initiation sites were involved in specifying a nuclear form of LOX, constructs mutated or deleted for ATG(+1) were used, but alternative initiation at CTG(-315) or ATG(+418) did not lead to the expression of intranuclear forms. Residues 23 to 157 of the proregion were essential for export of the precursor, while mutation of the putative site for maturation by procollagen C-proteinase abolished processing to the mature form of the enzyme. Cross-linking of collagen, as measured by pyridinoline analysis, increased twofold with the recombinant cells, compared to non-transfected controls. This shows the specific contribution of LOX, as opposed to other genetic forms of the enzyme, to cross-linking in a cellular context.
Seve
Sophie
S
Institut de Biologie et Chimie des Protéines, CNRS UMR-5086, 7 passage du Vercors, 69367 Lyon Cedex 07, France.
Decitre
Marie
M
Gleyzal
Claudine
C
Farjanel
Jean
J
Sergeant
Alain
A
Ricard-Blum
Sylvie
S
Sommer
Pascal
P
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D055395:Bone Morphogenetic Protein 1; D019485:Bone Morphogenetic Proteins / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D002999:Clone Cells; D003094:Collagen / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology* / Q000201:enzymology; D017353:Gene Deletion; D008666:Metalloendopeptidases / Q000235:genetics / Q000378:metabolism; D051379:Mice; D016297:Mutagenesis, Site-Directed; D032446:Myoblasts / Q000166:cytology* / Q000201:enzymology; D008249:Protein-Lysine 6-Oxidase / Q000096:biosynthesis* / Q000235:genetics; D011995:Recombination, Genetic; D014162:Transfection
D000596:Amino Acids; D019485:Bone Morphogenetic Proteins; C015484:pyridinoline; D003094:Collagen; D008249:Protein-Lysine 6-Oxidase; D008666:Metalloendopeptidases; C525707:Bmp1 protein, mouse; D055395:Bone Morphogenetic Protein 1