Marin Frédéric, Pokroy Boaz, Luquet Gilles, Layrolle Pierre, De Groot Klaas
UMR CNRS 5561 Biogéosciences, Université de Bourgogne, 6 Boulevard Gabriel 21000 DIJON, France.
Biomaterials. 2007 May;28(14):2368-77. doi: 10.1016/j.biomaterials.2007.01.029. Epub 2007 Feb 2.
The construction of metazoan calcium carbonate skeletons is finely regulated by a proteinaceous extracellular matrix, which remains embedded within the exoskeleton. In spite of numerous biochemical studies, the precise localization of skeletal proteins has remained for a long time as an elusive goal. In this paper, we describe a technique for visualizing shell matrix proteins on the surface of calcium carbonate crystals or within the biominerals. The technique is as follows: freshly broken pieces of biominerals or NaOCl then EDTA-etched polished surfaces are incubated with an antibody elicited against one matrix protein, then with a secondary gold-coupled antibody. After silver enhancement, the samples are subsequently observed with scanning electron microscopy by using back-scattered electron mode. In the present case, the technique is applied to a particular example, the calcitic prisms that compose the outer shell layer of the mediterranean fan mussel Pinna nobilis. One major soluble protein, caspartin, which was identified recently, was partly de novo sequenced after enzymatic digestions. A polyclonal antibody raised against caspartin was used for its localization within and on the prisms. The immunogold localization indicated that caspartin surrounds the calcitic prisms, but is also dispersed within the biominerals. This example illustrates the deep impact of the technique on the definition of intracrystalline versus intercrystalline matrix proteins. Furthermore, it is an important tool for assigning a putative function to a matrix protein of interest.
后生动物碳酸钙骨骼的构建受到一种蛋白质细胞外基质的精细调控，该基质会一直嵌入外骨骼中。尽管进行了大量的生化研究，但骨骼蛋白的精确定位长期以来一直是一个难以实现的目标。在本文中，我们描述了一种用于可视化碳酸钙晶体表面或生物矿体内壳基质蛋白的技术。该技术如下：将新鲜破碎的生物矿片或先用次氯酸钠然后用乙二胺四乙酸蚀刻的抛光表面与针对一种基质蛋白产生的抗体孵育，然后与二级金偶联抗体孵育。银增强后，随后使用背散射电子模式通过扫描电子显微镜观察样品。在当前案例中，该技术应用于一个特定实例，即构成地中海扇形贻贝（Pinna nobilis）外壳层的方解石棱柱。一种最近鉴定出的主要可溶性蛋白——壳天冬氨酸蛋白酶，在酶消化后进行了部分从头测序。针对壳天冬氨酸蛋白酶产生的多克隆抗体用于其在棱柱内部和表面的定位。免疫金定位表明壳天冬氨酸蛋白酶围绕着方解石棱柱，但也分散在生物矿体内。这个例子说明了该技术对区分晶内与晶间基质蛋白的深刻影响。此外，它是为感兴趣的基质蛋白赋予假定功能的重要工具。
