Functional Analyses of MMPs for Aragonite Crystal Formation in the Ligament of Pinctada fucata
Functional Analyses of MMPs for Aragonite Crystal Formation in the Ligament of Pinctada fucata
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DOI：
10.3389/fmars.2018.00373
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发表时间：
2018-10-16
影响因子：
3.7
通讯作者：
Suzuki, Michio
中科院分区：
文献类型：
--
作者：
Kubota, Kazuki;Kintsu, Hiroyuki;Suzuki, Michio
双壳纲软体动物在海洋中碳酸钙的形成中起着重要作用。双瓣铰链韧带是一种坚硬但柔韧的组织，可以抵抗在打开和关闭时施加在铰链上的应力。韧带包括由致密的有机基质包围的纤维文石晶体的微结构。基质由与纤维文石晶体排列在一起的有机纤维组成。先前的研究在fucata韧带的有机纤维中发现了一种金属蛋白酶组织抑制剂（Pf-TIMP: Pinctada fucata- tissue inhibitor of metalloproteinase）。该酶在体外表现出对基质金属蛋白酶（MMP）活性的强烈抑制，表明MMP也在韧带有机纤维的形成中发挥作用。利用转录组数据，我们从套峡（附着在韧带上的软组织）中鉴定了MMP基因。为了研究MMP基因在体内的功能，我们进行了RNA干扰实验。注射dsRNA后，MMP14973和MMP07860基因的表达均受到抑制。累积注射MMP07860 dsRNA诱导文石纤维聚集取向，而注射MMP14973对文石纤维聚集取向影响较小。当脱钙韧带在饱和碳酸钙溶液中孵育时，文石纤维沿着表面排列。当重组人（hr） MMP-13处理脱钙韧带时，碳酸钙的沉淀被抑制。为了详细研究MMP在碳酸钙结晶中的一般功能，我们在含有或不含重组人（hr） MMP-1的胶原凝胶中沉淀文石晶体。用hrmp -1治疗增加了胶原凝胶和碳酸钙之间的相互作用。这些结果表明，fucata Pinctada (Pf)-MMPs降解韧带中的细胞外基质，产生精细的有机纤维，调节纤维文石晶体的取向。
The mollusk class, Bivalvia, plays an important role in the formation of calcium carbonate in the ocean. The bivalve hinge ligament is a hard but pliant tissue that resists the stress placed on the hinge during opening and closing. The ligament comprises a fine microstructure of fibrous aragonite crystals surrounded by a dense organic matrix. The matrix consists of organic fibers that are aligned with the fibrous aragonite crystals. Previous studies identified a tissue inhibitor of metalloproteinase (Pf-TIMP: Pinctada fucata-Tissue Inhibitor of Metalloproteinase) in the organic fibers of P. fucata ligaments. This enzyme exhibited strong inhibition of matrix metalloproteinase (MMP) activity in vitro, suggesting that MMPs also play a role in formation of the organic fibers of the ligament. Using transcriptome data, we identified MMP genes from the mantle isthmus, which is a soft tissue attached to the ligament. To investigate the function of MMP genes in vivo, we performed RNA interference experiments. The expression of MMP14973 and MMP07860 genes was inhibited after injection of each dsRNA. Cumulative injection of MMP07860 dsRNA induced aggregated aragonite fiber orientation, whereas the injection of MMP14973 showed minor effects. When the decalcified ligament was incubated in a solution saturated with calcium carbonate, aragonite fibers aligned along the surface. When the decalcified ligament was treated with recombinant human (hr) MMP-13, the precipitation of calcium carbonate was inhibited. To investigate general MMP functions in calcium carbonate crystallization in detail, we precipitated aragonite crystals in collagen gels treated with or without recombinant human (hr) MMP-1. Treatment with hrMMP-1 increased the interaction between collagen gels and calcium carbonate. These results imply that Pinctada fucata (Pf)-MMPs degrade extracellular matrices in the ligament to produce the fine organic fibers that regulate the orientation of fibrous aragonite crystals.