Expression of lysyl oxidase from cDNA constructs in mammalian cells: the propeptide region is not essential to the folding and secretion of the functional enzyme.
Rat aortic lysyl oxidase cDNA was expressed under a metallothionein promoter in Chinese hamster ovary cells using a dihydrofolate reductase selection marker. One methotrexate-resistant cell line, LOD-06, generated by transfecting with full-length cDNA, yielded lysyl oxidase proteins consistent with the 50 kDa proenzyme and a 29 kDa mature catalyst. A second cell line, LOD32-2, was generated by transfection with a truncated cDNA lacking sequences which code for the bulk of the propeptide region. Both cell lines secreted apparently identical, 29 kDa forms of mature lysyl oxidase each of which catalyzed the deamination of human recombinant tropoelastin and alkylamines, consistent with the known specificity of lysyl oxidase. The secreted enzyme forms were inhibited by chemical inhibitors of lysyl oxidase activity, including beta-aminopropionitrile, phenylhydrazine, ethylenediamine, alpha, alpha'-dipyridyl, and diethyldithiocarbamate. Sensitivity to these agents is consistent with the presence of copper and carbonyl cofactors in the expressed enzymes, characteristic of lysyl oxidase from connective tissues. These results indicate the lack of essentiality of the deleted proprotein sequence for the proper folding, generation of catalytic function, and secretion of lysyl oxidase.
Kagan
H M
HM
Department of Biochemistry, Boston University School of Medicine, Massachusetts 02118, USA.
Reddy
V B
VB
Panchenko
M V
MV
Nagan
N
N
Boak
A M
AM
Gacheru
S N
SN
Thomas
K M
KM
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D016466:CHO Cells; D002384:Catalysis; D006224:Cricetinae; D018076:DNA, Complementary / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015870:Gene Expression; D006801:Humans; D008668:Metallothionein / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011401:Promoter Regions, Genetic; D017510:Protein Folding; D011498:Protein Precursors / Q000737:chemistry* / Q000378:metabolism; D008249:Protein-Lysine 6-Oxidase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D051381:Rats; D013329:Structure-Activity Relationship; D014162:Transfection
D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D011498:Protein Precursors; D008668:Metallothionein; D008249:Protein-Lysine 6-Oxidase