METH-1 and METH-2 are novel proteins that contain the anti angiogenic domain of thrombospondin 1

Thrombospondin-1 (TSP-1) is a secreted trimeric glycoprotem with demonsirated anti-angiogenic activity in vivo and in vitro. The anti-angiogenic activity of TSP-1 has been mapped to a repeated sequence in the gene, referred to as a type-1 repeat. In a search for additional inhibitors of angiogenesis, we screened a number of human cDNA libraries with the anti-angiogenic region of TSP-1 and found several cDNAs that encode such sequences. Two novel cDNAs, representing two apparently distinct genes, contained both a type I domain, and a potential metalloproteinase/Zn++-binding domain. Due to their deduced protein structure, the cDNAs were named METH-1 and -2 (ME= metalloproteinase and TH= thrombospondin). METH-1 and METH-2 share 49% amino acid sequence identity yet showed no overall homology to previously published genes. Therefore, METH genes appear to represent founding members of a potentially new family of proteins. Northern blot analysis revealed that transcripts for both genes are expressed widely. METH-1 is highly expressed in the ovary, heart, placenta and, to a lesser degree, in most other tissues tested. METH-2 is most predominant in lung and brain. In situ hybridization assays revealed that METH-1 was expressed predominantly by epithelial cells, although muscle cells were also positive. METH-2 was expressed in both epithelial and endothelial cells. Both METH-1 and -2 were highly expressed in the mammary gland. Northern blot analysis of non transformed and malignant mammary epithelial cell lines indicated that METH-1 expression was lost in transformed cells, indicating that METH-1 might have tumor suppressor activity. Based on sequence analysis, we postulated that both METH-1 and -2 would possess anti-angiogenic properties and have tested synthetic peptides from within the TSP-1 homologous region for their ability to inhibit angiogenesis in CAM assays. In addition, we have generated recombinant METH-1 and a truncated version of this protein to test further the effect of this novel protein on angiogenesis and other processes.