Proteomics of Shell Matrix Proteins from the Cuttlefish Bone Reveals Unique Evolution for Cephalopod Biomineralization

In contrast to the external shells in bivalves and gastropods, most cephalopods are missing this external protection. The cuttlefish, belonging to class cephalopod, has an internal biomineralized structure made of mainly calcium carbonate for controlling buoyancy. However, the macromolecules, especially proteins that control cuttlebone mineral formation, are not sufficiently understood, limiting our understanding of the evolution of this internal shell. In this study, we extracted proteins from the cuttlebone of pharaoh cuttlefish Sepia pharaonis and performed liquid chromatography-tandem mass spectrometry to identify the shell matrix proteins (SMPs). In total, 41 SMPs were identified. Among them, hemocyanin, an oxygen-carrying protein, was the most abundant SMP. By comparison with SMPs of other marine biominerals, hemocyanin, apolipophorin, soul domain proteins, transferrin, FL-rich, and enolase were found to be unique to the cuttlebone. In contrast, typical SMPs of external shells such as carbonic anhydrase complement control protein, fibronectin type III, and G/A-rich proteins were lacking from the cuttlebone. Furthermore, the cluster analysis of biomineral SMPs suggests that the SMP repertoire of the cuttlebone does not resemble that of other species with external shells. Taken together, this study implies a potential relationship of the cuttlefish internal shell with other internal biominerals, which highlights a unique shell evolutionary pathway in invertebrates.与双壳类（bivalves）和腹足类（gastropods）的外贝壳相比，绝大多数头足类（cephalopods）缺乏此类外部保护结构。隶属于头足纲的乌贼（cuttlefish）拥有一种内部生物矿化结构，其主要成分为碳酸钙，用于调控浮力。然而，目前学界对调控乌贼骨（cuttlebone）矿化形成的大分子，尤其是相关蛋白质的认知仍不够充分，这限制了我们对该内壳演化过程的理解。本研究从曼氏无针乌贼（Sepia pharaonis）的乌贼骨中提取蛋白质，并通过液相色谱-串联质谱法鉴定壳基质蛋白（shell matrix proteins, SMPs）。最终共鉴定出41种壳基质蛋白，其中携氧蛋白血蓝蛋白（hemocyanin）是丰度最高的壳基质蛋白。通过与其他海洋生物矿化结构的壳基质蛋白进行比对，本研究发现血蓝蛋白、载脂蛋白（apolipophorin）、soul结构域蛋白（soul domain proteins）、转铁蛋白（transferrin）、富FL蛋白（FL-rich）以及烯醇化酶（enolase）为乌贼骨所特有。与之相反，外贝壳的典型壳基质蛋白，如碳酸酐酶（carbonic anhydrase）、补体控制蛋白（complement control protein）、Ⅲ型纤连蛋白（fibronectin type III）以及富G/A蛋白（G/A-rich），均未在乌贼骨中被检测到。进一步的生物矿化壳基质蛋白聚类分析显示，乌贼骨的壳基质蛋白组成与其他具有外贝壳的物种存在显著差异。综上，本研究表明乌贼内壳与其他内部生物矿化结构存在潜在关联，这一发现凸显了无脊椎动物中独特的贝壳演化路径。