In vitro synthesis of the active tissue inhibitor of metalloproteinases encoded by a complementary DNA from virus-infected murine fibroblasts.
We have recently described the characterization and expression of a murine gene highly homologous to the human tissue inhibitor of metalloproteinases/erythroid potentiating activity (TIMP/EPA) gene. We have also reported that expression of this gene is regulated in response to virus infection. In the present report we describe the use of a cDNA clone derived from mRNA isolated from Newcastle disease virus-induced murine cells to direct in vitro synthesis of proteins encoded by this murine TIMP/EPA gene. This approach was used to analyze structural and functional parameters of the TIMP/EPA protein. Translation experiments using microsomes revealed a murine protein similar in size to that of human TIMP: Mr of approximately 22,000 for the core protein and 28,000 for the processed protein. Processing in microsomes involved N-glycosylation and cleavage of the signal peptide. Both the processed and unprocessed proteins were able to inhibit degradation of collagen by collagenase but unable to inhibit virus replication. Synthesis of truncated TIMP proteins showed that the collagenase-inhibiting activity was not encoded within a delimited portion of the molecule. This result suggests that conformation is probably essential for TIMP activity.
D000818:Animals; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000096:biosynthesis / Q000235:genetics*; D005347:Fibroblasts / Q000201:enzymology; D007372:Interferons / Q000378:metabolism; D051379:Mice; D003012:Microbial Collagenase / Q000037:antagonists & inhibitors; D009522:Newcastle disease virus; D014176:Protein Biosynthesis; D011817:Rabbits; D019714:Tissue Inhibitor of Metalloproteinases; D014158:Transcription, Genetic
D004791:Enzyme Inhibitors; D019714:Tissue Inhibitor of Metalloproteinases; D004247:DNA; D007372:Interferons; D003012:Microbial Collagenase