Iida Joji, Wilhelmson Krista L, Ng Janet, Lee Peter, Morrison Charlotte, Tam Eric, Overall Christopher M, McCarthy James B
Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, MN 55455, USA.
Biochem J. 2007 May 1;403(3):553-63. doi: 10.1042/BJ20061176.
We previously reported that CS (chondroitin sulfate) GAG (glycosaminoglycan), expressed on MCSP (melanoma-specific CS proteoglycan), is important for regulating MT3-MMP [membrane-type 3 MMP (matrix metalloproteinase)]-mediated human melanoma invasion and gelatinolytic activity in vitro. In the present study, we sought to determine if CS can directly enhance MT3-MMP-mediated activation of pro-MMP-2. Co-immunoprecipitation studies suggest that MCSP forms a complex with MT3-MMP and MMP-2 on melanoma cell surface. When melanoma cells were treated with betaDX (p-nitro-beta-D-xylopyranoside) to inhibit coupling of CS on the core protein, both active form and proform of MMP-2 were no longer co-immunoprecipitated with either MCSP or MT3-MMP, suggesting a model in which CS directly binds to MMP-2 and presents the gelatinase to MT3-MMP to be activated. By using recombinant proteins, we determined that MT3-MMP directly activates pro-MMP-2 and that this activation requires the interaction of the C-terminal domain of pro-MMP-2 with MT3-MMP. Activation of pro-MMP-2 by suboptimal concentrations of MT3-MMP is also significantly enhanced in the presence of excess C4S (chondroitin 4-sulfate), whereas C6S (chondroitin 6-sulfate) or low-molecular-mass hyaluronan was ineffective. Affinity chromatography studies using CS isolated from aggrecan indicate that the catalytic domain of MT3-MMP and the C-terminal domain of MMP-2 directly bind to the GAG. Thus the direct binding of pro-MMP-2 with CS through the C-domain would present the catalytic domain of pro-MMP-2 to MT3-MMP, which facilitates the generation of the active form of MMP-2. These results suggest that C4S, which is expressed on tumour cell surface, can function to bind to pro-MMP-2 and facilitate its activation by MT3-MMP-expressing tumour cells to enhance invasion and metastasis.
我们之前报道过，表达于黑素瘤特异性硫酸软骨素蛋白聚糖（MCSP）上的硫酸软骨素（CS）糖胺聚糖（GAG），对于在体外调节MT3-基质金属蛋白酶（MMP）[膜型3 MMP]介导的人黑素瘤侵袭和明胶溶解活性很重要。在本研究中，我们试图确定CS是否能直接增强MT3-MMP介导的前MMP-2激活。免疫共沉淀研究表明，MCSP在黑素瘤细胞表面与MT3-MMP和MMP-2形成复合物。当用对硝基-β-D-吡喃木糖苷（βDX）处理黑素瘤细胞以抑制CS与核心蛋白的偶联时，MMP-2的活性形式和前体形式均不再与MCSP或MT3-MMP共免疫沉淀，这提示了一种模型，即CS直接与MMP-2结合，并将明胶酶呈递给MT3-MMP以被激活。通过使用重组蛋白，我们确定MT3-MMP直接激活前MMP-2，且这种激活需要前MMP-2的C末端结构域与MT3-MMP相互作用。在存在过量硫酸软骨素4-硫酸酯（C4S）的情况下，次优浓度的MT3-MMP对前MMP-2的激活也显著增强，而硫酸软骨素6-硫酸酯（C6S）或低分子量透明质酸则无效。使用从聚集蛋白聚糖中分离的CS进行的亲和层析研究表明，MT3-MMP的催化结构域和MMP-2的C末端结构域直接与GAG结合。因此，前MMP-2通过C结构域与CS的直接结合会将前MMP-2的催化结构域呈递给MT3-MMP，这有助于产生活性形式的MMP-2。这些结果表明，在肿瘤细胞表面表达的C4S可以发挥作用，与前MMP-2结合，并促进其被表达MT3-MMP的肿瘤细胞激活，从而增强侵袭和转移。
