Calcium regulates tertiary structure and enzymatic activity of human endometase/matrilysin-2 and its role in promoting human breast cancer cell invasion
Calcium regulates tertiary structure and enzymatic activity of human endometase/matrilysin-2 and its role in promoting human breast cancer cell invasion
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DOI：
10.1042/bj20061390
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发表时间：
2007-04-01
影响因子：
4.1
通讯作者：
Sang, Qing-Xiang Amy
中科院分区：
文献类型：
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作者：
Lee, Seakwoo;Park, Hyun I.;Sang, Qing-Xiang Amy
人类 MMP-26（基质金属蛋白酶-26）（也称为内皮酶或基质溶素-2）是人类乳腺癌、前列腺癌和其他上皮来源癌症的假定生物标志物。钙调节蛋白质结构和功能，并可充当细胞中的分子信号或开关。 MMP 和钙之间的关系几乎没有被研究过，而且 MMP-26 也没有研究过。我们研究了 MMP-26 中的钙结合位点和钙的作用。 MMP-26 具有 1 个高亲和力和 1 个低亲和力钙结合位点。在生理低钙条件下恢复高亲和力钙结合，钙解离常数为 63 nM，而不会引起二级和三级结构变化。高亲和力钙结合可保护 MMP-26 免受热变性。该位点的突变体（D165A 或 E191A）失去酶活性。低亲和力钙结合在较高钙浓度下恢复，K-d2（低亲和力钙解离常数）值为120 μM，伴随着酶活性可逆恢复和三级结构变化，但没有二级结构重排。低亲和力钙结合位点（C3 位点）K189E 或 D114A 的突变分别诱导对 Ca2+ 离子的亲和力增强或由低亲和力钙结合引发的酶活性的不可逆损失。非钙结合位点的突变（C2 位点的 V184D）表明 C2 不是真正的钙结合位点。同源模型突变结构的观察结果与这些实验结果相关。与转染非活性形式 MMP-26 (E209A) 的对照细胞相比，转染野生型 MMP-26 cDNA 的人乳腺癌细胞系 MDA-MB-231 显示出钙依赖性侵袭潜力。在 K189E 突变 MDA-MB-231 细胞系中观察到不依赖钙的高侵袭性。
Human MMP-26 (matrix metalloproteinase-26) (also known as endometase or matrilysin-2) is a putative biomarker for human carcinomas of breast, prostate and other cancers of epithelial origin. Calcium modulates protein structure and function and may act as a molecular signal or switch in cells. The relationship between MMPs and calcium has barely been studied and is absent for MMP-26. We have investigated the calcium-binding sites and the role of calcium in MMP-26. MMP-26 has one high-affinity and one low-affinity calcium binding site. High-affinity calcium binding was restored at physiologically low calcium conditions with a calcium-dissociation constant of 63 nM without inducing secondary and tertiary structural changes. High-affinity calcium binding protects MMP-26 against thermal denaturation. Mutants of this site (D165A or E191A) lose enzymatic activity. Low-affinity calcium binding was restored at relatively high calcium concentrations and showed a K-d2 (low-affinity calcium-dissociation constant) value of 120 mu M, which was accompanied with the recovery of enzymatic activity reversibly and tertiary structural changes, but without secondary structural rearrangements. Mutations at the low-affinity calcium-binding site (C3 site), K189E or D114A, induced enhanced affinity for the Ca2+ ion or an irreversible loss of enzymatic activity triggered by low-affinity calcium binding respectively. Mutation at non-calcium-binding site (V184D at C2 site) showed that C2 is not a true calcium-binding site. Observations from homology-modelled mutant structures correlated with these experimental results. A human breast cancer cell line, MDA-MB-231, transfected with wild-type MMP-26 cDNA showed a calcium-dependent invasive potential when compared with controls that were transfected with an inactive form of MMP-26 (E209A). Calcium-independent high invasiveness was observed in the K189E mutant MDA-MB-231 cell line.