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Identification, cloning and characterization of several new MMP's

We have cloned a new member of the membrane-type subfamily of matrix metalloproteinases (MT-MMPs).The cloned cDNA encodes a protein of 562 amino acids with a domain organization similar to that of other MT-MMPs, and is called MT6-MMP. Expression plasmids encoding MT6-MMP and progelatinase A resulted in activation of COS-7-secreted progelatinase A.

MT6-MMP is predominantly expressed in leukocytes, lung, and spleen and was also detected at high levels in SW480 colon carcinoma cells as well as in some anaplastic astrocytomas and glioblastomas, but not in normal colon or brain or in meningiomas, indicating that MT6-MMP may facilitate tumor progression through its ability to activate progelatinase A at the membrane of cells from colon carcinomas or brain tumors. Furthermore, a human placenta cDNA encodes a polypeptide of 261 amino acids, the smallest MMP identified to date, which contains several structural features of MMPs including the signal sequence, the prodomain involved in enzyme latency, and the catalytic domain with the zinc-binding site. However, it lacks the hinge region and hemopexin-domain present in most MMPs. According to these structural characteristics, the human MMP described herein has been called matrilysin-2 (MMP-26), because it exclusively shares with matrilysin this minimal domain organization. The amino acid sequence of matrilysin-2 also contains a threonine residue adjacent to the Zn-binding site that has been defined as a specific feature of matrilysin. The matrilysin-2 gene maps to the short arm of chromosome 11, a location distinct to that of other MMP genes. Recombinant matrilysin-2 was found to degrade type IV collagen, fibronectin, fibrinogen, and gelatin and to activate progelatinase B. Matrilysin-2 is detected not only in placenta and uterus but is widely expressed in malignant tumors from different sources.

We have identified and cloned a human fetal lung cDNA encoding ADAM-TS12. Analysis of intracellular processing of ADAM-TS12 revealed that it is synthesized as a precursor molecule that is first activated by cleavage of the prodomain in a furin-mediated process and subsequently processed into two fragments of different size: a 120-kDa N-terminal proteolytically active fragment containing the metalloproteinase and disintegrin domains, and a 83-kDa C-terminal fragment containing most of the TS-1 repeats. ADAM-TS12 gene maps to 5q35. ADAM-TS12 transcripts are only detected at significant levels in fetal lung but not in any other analyzed tissues. In addition, ADAM-TS12 transcripts were detected in gastric carcinomas.

We have also identified and cloned cDNAs encoding seven new human ADAMTSs. These novel enzymes have been called ADAMTS-13, -14, -15, -16, -17, -18, and -19. All of them show a domain organization similar to that of previously characterized family members. Expression analysis revealed that these ADAMTS genes are mainly expressed in fetal tissues, especially in lung (ADAMTS14, ADAMTS16, ADAMTS17, ADAMTS18, and ADAMTS19), kidney (ADAMTS14, ADAMTS15, and ADAMTS16), and liver (ADAMTS13, ADAMTS15 and ADAMTS18). Reverse transcriptase--polymerase chain reaction analysis also revealed the expression of some of these new ADAMTSs in different human adult tissues, such as prostate (ADAMTS13, ADAMTS17, and ADAMTS18), and brain (ADAMTS13, ADAMTS16, ADAMTS17, and ADAMTS18). High levels of ADAMTSs transcripts were also observed in some tumor biopsies and cells lines, including osteosarcomas (ADAMTS19), melanoma and colon carcinoma cells (ADAMTS13).

Chromosomal location analysis indicated that the seven identified ADAMTS genes are dispersed in the human genome mapping to 9q34, 10q21, 11q25, 5p15, 15q24, 16q23, and 5q31, respectively. We have cloned a mouse brain cDNA encoding a new protein of the ADAMTS family called ADAMTS-20. However, the C-terminal TS module is more complex than that of other

ADAMTSs, being composed of a total of 14 repeats. The structural complexity of ADAMTS-20 is further increased by the presence of an additional domain 200 residues long and located immediately adjacent to the TS module. This domain has been tentatively called GON domain and can also be recognized in some ADAMTSs such as gon-1 from Caenorhabditis elegans and human and mouse ADAMTS-9. The presence of this domain is a hallmark of a novel subfamily of structurally and evolutionarily related ADAMTSs, called GON-ADAMTSs. Expression analysis demonstrated that ADAMTS-20 transcripts can be detected at low levels in several human and mouse tissues, especially in testis. This gene is also overexpressed in some human malignant tumors, including brain, colon, and breast carcinomas.

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Keld DANØ, (Head of Finsen Laboratory)
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