Functional Analyses of MMPs for Aragonite Crystal Formation in the Ligament of Pinctada fucata

Abstract

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.

Natural gradient composite made of aragonite nanofibers: the ligament of bivalveAcesta marissinica

“…It should be noted that all bivalve FLs invariably consist of two phases: aragonite nanobers and proteinaceous organic materials. [3][4][5][6][7][8] The same is true for the FL of A. marissinica, as conrmed by the XRD (X-ray diffraction) and FTIR (Fourier transform infrared) analysis (Fig. S1 †).…”

“…[2][3][4][5][6][7][8] Although both the valve and FL are mineralized with CaCO 3 , the former commonly consists of calcite, aragonite, or both, with various structures [9][10][11][12][13] and low content of organic matrix (oen < 5% by weight). 13 In contrast, the latter always consists of aragonite nanobers [3][4][5][6][7][8] (simplied as nanobers hereaer) with higher content of organic materials (mainly proteins) (oen >25% by weight). 4 To date, the valve structure has been extensively studied due to its applications in bio-inspired design and fabrication of advanced materials.…”

“…2 In addition, some works focus on the organic materials assumed to play a key role in the formation of the FL structure. 7,8,22,23 Comparatively, less work focuses on the FL structure. To my knowledge, Trueman rstly studied this subject by optical microscopy (OM) 24 and proposed a structural model, which was subsequently conrmed and rened by Kahler et al by transmission electron microscopy (TEM) and OM ( Fig.…”

“…formation mechanism of organic materials, 7 and Suzuki et al studied in detail the structure and function of the fusion interphase which connects the ligament to the shell valve. 8 These above works greatly advance our understanding of the ligament.…”