Characterization of the structure and function of new membrane-type matrix metalloproteinase

MT-MMP的5个成员之一，MT4-MMP，最初是由Puente等人通过cDNA克隆从乳腺癌细胞系中鉴定出来的（Cancer Res. 56, 944-949, 1996）。然而，一个存在于所有MMP家族成员中的信号肽的编码区被cDNA中的未知序列所取代，不能直接在转染细胞中表达该基因产物。相反，我们分离了小鼠和人的mt4-mmp（mt4-mmp(m)）基因，该基因可以通过具有信号肽的编码区来指导基因产物的表达。Puente's序列是一个次要的转录本，仅占mt4-mmp mRNA总数的1/10，并且被发现来自于第一个内含子的异常剪接产物。因此，我们分离出了MT4-MMP的主要功能转录本。假设的MT4-MMP的跨膜结构域位于非常c端，而其他的在跨膜结构域之后有大约20个氨基酸的细胞质结构域。这些序列通常作为糖基磷脂酰肌醇（GPI）锚定信号而不是作为跨膜结构域，这表明MT4-MMP可能是GPI锚定的蛋白酶。我们的研究结果表明，可以并入GPI单元的[3H]乙醇胺以序列依赖的方式特异性标记MT4-MMP c端。此外，磷脂酰肌醇特异性磷脂酶C （PI-PLC）处理从转染细胞表面释放MT4-MMP。这些结果表明，MT4-MMP是MMP家族中第一个gpi锚定的蛋白酶。在转染细胞的培养过程中，MT4-MMP在内源性金属蛋白酶的作用下从细胞表面脱落。MT4-MMP在细胞表面的gpi锚定表明该蛋白酶具有独特的生物学功能和特性。

One of the 5 MT-MMP members, MT4-MMP, was originally identified by cDNA cloning from breast carcinoma cell line by Puente at al. (Cancer Res. 56, 944-949, 1996). However, the coding region for a signal peptide, which exists in all MMP family members, is substituted with an unknown sequence in the cDNA, and it could not direct expression of the gene product in the transfected cells. In contrast, we isolated a mouse and human mt4-mmp (mt4-mmp(m)) gene that can direct expression of the gene product by having a coding region for a signal peptide. The Puente's sequence was minor transcript representing only 1/10 of the total mt4-mmp mRNA and found to be derived from an aberrant splicing product for the first intron. Thus, we have isolated a major functional transcripts of MT4-MMP.A putative transmembrane domain of MT4-MMP locates at the very C-terminal end while others have an about 20 aminoacids cytoplasmic domain following the transmembrane domain. Such sequences often act as a glycosyl-phosphatidyl inositol (GPI) anchoring signal rather than as a transmembrane domain, suggesting the possibility that MT4-MMP is a GPI-anchored proteinase. Our result showed that [3H] ethanolamine, that can be incorporated into the GPI unit, specifically labeled the MT4-MMP C-terminal end in a sequence dependent manner. In addition, phosphatidylinositol-specific phospholipase C (PI-PLC) treatment released the MT4-MMP from the surface of transfected cells. These results indicate that MT4-MMP is the first GPI-anchored proteinase in the MMP family. During cultivation of the transfected cells, MT4-MMP appeared to be shed from the cell surface by the action of an endogenous metalloproteinase. GPI-anchoring of MT4-MMP on the cell surface indicates a unique biological function and character for this proteinase.