Mouse gelatinase B. cDNA cloning, regulation of expression and glycosylation in WEHI-3 macrophages and gene organisation.
Gelatinase B is a regulated matrix metalloproteinase with an important role in the remodelling of extracellular matrices and of basement membranes. To study the structure and function of gelatinase B in the mouse, the cDNA was cloned from a macrophage cell line (WEHI-3). Using this cDNA, a cosmid clone with the mouse gene was isolated. The complete gene (8 kbp) was sequenced and compared with the human gene structure. There was 78% similarity at the cDNA level and the exon/intron structure of the murine gene was similar to the human counterpart. At the 5' untranslated side, 1200 bp of the promoter/enhancer region were sequenced and found to contain several transacting-factor-binding sites. The mRNA transcription-initiation site was determined by non-isotopic primer-extension analysis. Polymerase-chain-reaction amplification of cDNAs yielded indirect evidence for a reverse-transcription stop in WEHI-3 cell mRNA. The DNA-derived mouse-protein structure exhibited 82% similarity with the human one. This similarity was functionally reflected by cross-reactivity of the mouse protein with an antiserum against human gelatinase B. The production of murine gelatinase B was studied at the protein level by zymography and at the mRNA level by Northern blot analysis. In WEHI-3 cells the gelatinase B protein is induced by bacterial lipopolysaccharide, phorbol ester, double-stranded RNA and the cytokine interleukin-1. Regulation of activity and structural heterogeneity of gelatinase B in WEHI-3 cells were shown to occur at the gene regulatory level, by expression of the matrix metalloproteinase inhibitor TIMP-1, and by glycosylation of the secreted protein.
Masure
S
S
Rega Institute for Medical Research, University of Leuven, Belgium.
Nys
G
G
Fiten
P
P
Van Damme
J
J
Opdenakker
G
G
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002460:Cell Line; D003001:Cloning, Molecular; D017364:Collagenases / Q000235:genetics* / Q000378:metabolism; D018076:DNA, Complementary; D015971:Gene Expression Regulation, Enzymologic; D006023:Glycoproteins / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D008264:Macrophages / Q000201:enzymology*; D020780:Matrix Metalloproteinase 9; D051379:Mice; D008969:Molecular Sequence Data; D011233:Precipitin Tests; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid; D017386:Sequence Homology, Amino Acid; D019714:Tissue Inhibitor of Metalloproteinases; D014158:Transcription, Genetic
D018076:DNA, Complementary; D006023:Glycoproteins; D012333:RNA, Messenger; D019714:Tissue Inhibitor of Metalloproteinases; D017364:Collagenases; D020780:Matrix Metalloproteinase 9