Constitutive and induced CD44 shedding by ADAM-like proteases and membrane-type 1 matrix metalloproteinase
Constitutive and induced CD44 shedding by ADAM-like proteases and membrane-type 1 matrix metalloproteinase
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DOI：
10.1158/0008-5472.can-03-3502
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发表时间：
2004-02-01
期刊：
影响因子：
11.2
通讯作者：
Seiki, M
中科院分区：
文献类型：
--
作者：
Nakamura, H;Suenaga, N;Seiki, M
CD 44是透明质酸的受体，介导调节复杂细胞行为（包括癌细胞迁移和侵袭）的信号传导。CD 44的胞外部分的脱落是配体和细胞之间分子释放相互作用调节的最后一步。然而，高度糖基化形式的CD 44阻碍了脱落的确切切割位点和负责蛋白酶的鉴定。在这项研究中，我们发现，膜型1基质金属蛋白酶（MT 1-MMP）的表达增加脱落的65-70 kDa的CD 44 H（标准形式）片段，并产生两个额外的较小的片段。我们纯化了脱落的片段并通过质谱法鉴定了切割位点。制备通过切割识别新暴露的COOH末端的特异性抗体，并用于分析每个位点的脱落。65-70 kDa片段的脱落受到金属蛋白酶组织抑制剂3（TIMP-3）的抑制，但不受TIMP-1和TIMP-2的抑制，表明涉及去整合素和金属蛋白酶（ADAM）样蛋白酶，尽管脱落受MT 1-MMP的影响。相反，两个较小片段的脱落被TIMP-2和TIMP-3抑制，但不被TIMP-1抑制，表明MT 1-MMP本身参与。在这些位点切割的脱落片段也在人肿瘤组织中检测到。与周围正常组织相比，在肿瘤中观察到MT 1-MMP敏感位点之一的脱落增加。然而，没有观察到与脱落的ADAM样蛋白酶的显着差异。因此，CD 44 H脱落的切割位点是第一次确定，结果提供了一个基础，探索未知的生物学作用的脱落在不同的网站。
CD44 is a receptor for hyaluronan and mediates signaling that regulates complex cell behavior including cancer cell migration and invasion. Shedding of the extracellular portion of CD44 is the last step in the regulation of the molecule-releasing interaction between the ligand and cell. However, highly glycosylated forms of CD44 have hampered the identification of the exact cleavage sites for shedding and the responsible proteases. In this study, we found that expression of membrane-type 1 matrix metalloproteinase (MT1-MMP) increased shedding of the 65-70 kDa CD44H (standard form) fragments and generated two additional smaller fragments. We purified the shed fragments and identified the cleaved sites by mass spectrometry. Specific antibodies that recognize the newly exposed COOH terminus by cleavage were prepared and used to analyze shedding at each site. Shedding of the 65-70 kDa fragments was inhibited by tissue inhibitor of metalloproteinase 3 (TIMP-3) but not by TIMP-1 and TIMP-2, suggesting involvement of a disintegrin and metalloproteinase (ADAM)-like proteases, although shedding is affected by MT1-MMP. Conversely, shedding of the two smaller fragments was inhibited by TIMP-2 and TIMP-3 but not TIMP-1, suggesting involvement of MT1-MMP itself. Shed fragments cleaved at these sites were also detected in human tumor tissues. Increased shedding at one of the MT1-MMP-sensitive sites was observed in the tumor compared with the surrounding normal tissue. However, no significant difference was observed with shedding by ADAM-like proteases. Thus, the cleavage sites for the shedding of CD44H were identified for the first time, and the results provide a basis for exploring the unknown biologic roles of shedding at different sites.