Abstract
Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) play a critical role in extracellular matrix homeostasis. We have previously cloned human and mouse TIMP-3 cDNAs and mapped their chromosomal loci (Apte, S. S., Mattei, M-G., and Olsen, B. R. (1994) Genomics 19, 86-90; Apte, S. S., Hayashi, K., Seldin, M. F., Mattei, M-G., Hayashi, M., and Olsen, B. R. (1994) Dev. Dynam. 200, 177-197); the identification of TIMP3 mutations in Sorsby's fundus dystrophy has underscored the functional importance of TIMP-3. We now report that TIMP-3 is encoded by five exons spanning over 30 kilobase pairs of mouse genomic DNA. In the attribution of protein domains to specific exons, as well as exon structures, the Timp-3 and Timp-1 genes are similar, confirming the common evolutionary origin of the TIMPs and defining a distinct gene family. We have expressed human and mouse TIMP-3 in mouse NSO myeloma cells. In each case, an N-glycosylated 27-kDa protein was generated, that, like TIMP-1 and TIMP-2, inhibited collagenase-1, stromelysin-1, and gelatinases A and B. TIMP-3 and TIMP-1 inhibition were quantitatively similar, implying that all TIMPs are equally efficient in MMP inhibition. Instead, differential regulation of the TIMP genes or divergent C-terminal protein sequences may underlie distinct biological functions for each TIMP.
Highlights
Remodeling of the extracellular matrix (ECM)l is an important process during normal development, and deregulated remodeling may have a role in the etiology of diseases such as cancer and arthritis (Pelletier et al 1990; Alexander and Werb, 1991; Liotta and Stetler-Stevenson, 1990), The matrix metalloproteinases (MMPs), a family of secreted, zinc-containing neutral proteases, are believed to play a significant role in this process
MMPs, and to find out whether there are any functional differences compared to the activities of tissue inhibitors of metalloproteinases (TIMPs)-1, we have expressed mouse and human TIMP-3 cDNAs in a myeloma cell line. rTIMP-3 is expressed as a glycosylated 24-29-kDa protein with inhibitory activities against interstitial collagenase, stromelysin-1, and gelatinase A and B. Both the spectrum of inhibition and the specific activity are comparable to those of TIMP-l. Based on these genetic and biochemical data, we have identified characteristics shared by TIMP-1, TIMP-2, and TIMP-3 and proposed that these be considered as the hallmarks of the TIMP family
Characterization of the Timp-3 Gene-Six genomic clones were isolated that spanned a distance of approximately 50 kbp (Fig. 1)
Summary
TIMP-3, TIMP-2, and TIMP-l designate the proteins, TIMP3 and TIMP2 are Human Genome Mapping Workshops approved symbols for the human TIMP-3 and TIMP-2 genes, respectively, and Timp-S and Timp-L are the approved symbols for the respective mouse genes. rTIMP-3 denotes recombinant TIMP-3. RTIMP-3 is expressed as a glycosylated 24-29-kDa protein with inhibitory activities against interstitial collagenase, stromelysin-1, and gelatinase A and B. Both the spectrum of inhibition and the specific activity are comparable to those of TIMP-l. Based on these genetic and biochemical data, we have identified characteristics shared by TIMP-1, TIMP-2, and TIMP-3 and proposed that these be considered as the hallmarks of the TIMP family
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