Cloning of a 72 kDa matrix metalloproteinase (gelatinase) from chicken embryo fibroblasts using gene family PCR: expression of the gelatinase increases upon malignant transformation.
Chicken embryo fibroblasts secrete a 72 kDa progelatinase that displays all of the characteristics of a matrix metalloproteinase. Employing reverse-transcription PCR and degenerate oligonucleotide primers that are specific for two highly conserved sequences found in all matrix metalloproteinases, a DNA fragment specific for the chicken gelatinase was generated. Using this PCR product as a probe, cDNA clones were isolated from a chicken embryo cDNA library and the entire protein coding sequence was determined. The chicken progelatinase is 84% identical, at the amino acid level, with human and mouse 72 kDa progelatinase/type-IV procollagenase, with the greatest degree of similarity occurring in the propeptide and catalytic domains. The avian and mammalian proteinases diverge significantly in the C-terminal, hemopexin-like domain. The last 100 residues of the chicken gelatinase are only 66% identical with mammalian gelatinases. Mouse 72 kDa progelatinase, however, does not diverge significantly (> 98% identity) from human progelatinase in the hemopexin-like domain. The divergence in this domain of the chicken progelatinase may explain some of the distinct catalytic and inhibitory properties of the 72 kDa chicken progelatinase. Northern-blot analysis reveals that steady-state levels of the chicken progelatinase mRNA are increased 5-fold upon malignant transformation of chicken embryo fibroblasts with Rous sarcoma virus (RSV) and 3-fold by treatment with the tumour-promoting phorbol ester, phorbol 12-myristate 13-acetate (PMA). This represents the first reported cloning of an avian matrix metalloproteinase. The increased expression of the chicken progelatinase by RSV transformation and the tumour promoter PMA suggests that the progelatinase is regulated differently in chicken cells.
Department of Biochemistry and Cell Biology, State University of New York at Stony Brook 11794.
R T
RT
Aimes
D L
DL
French
J P
JP
Quigley
D000595:Amino Acid Sequence; D000818:Animals; D001358:Avian Sarcoma Viruses; D001483:Base Sequence; D002472:Cell Transformation, Viral; D002645:Chickens; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000235:genetics; D005347:Fibroblasts; D018093:Gelatinases / Q000235:genetics*; D015870:Gene Expression / Q000187:drug effects; D020778:Matrix Metalloproteinase 2; D008666:Metalloendopeptidases / Q000235:genetics*; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000235:genetics; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013755:Tetradecanoylphorbol Acetate
D017931:DNA Primers; D018076:DNA, Complementary; D012333:RNA, Messenger; D018093:Gelatinases; D008666:Metalloendopeptidases; D020778:Matrix Metalloproteinase 2; D013755:Tetradecanoylphorbol Acetate