Research Article: Layer-by-Layer Proteomic Analysis of Mytilus galloprovincialis Shell

Date Published: July 28, 2015

Publisher: Public Library of Science

Author(s): Peng Gao, Zhi Liao, Xin-xing Wang, Lin-fei Bao, Mei-hua Fan, Xiao-min Li, Chang-wen Wu, Shu-wei Xia, Senjie Lin.


Bivalve shell is a biomineralized tissue with various layers/microstructures and excellent mechanical properties. Shell matrix proteins (SMPs) pervade and envelop the mineral crystals and play essential roles in biomineralization. Despite that Mytilus is an economically important bivalve, only few proteomic studies have been performed for the shell, and current knowledge of the SMP set responsible for different shell layers of Mytilus remains largely patchy. In this study, we observed that Mytilus galloprovincialis shell contained three layers, including nacre, fibrous prism, and myostracum that is involved in shell-muscle attachment. A parallel proteomic analysis was performed for these three layers. By combining LC-MS/MS analysis with Mytilus EST database interrogations, a whole set of 113 proteins was identified, and the distribution of these proteins in different shell layers followed a mosaic pattern. For each layer, about a half of identified proteins are unique and the others are shared by two or all of three layers. This is the first description of the protein set exclusive to nacre, myostracum, and fibrous prism in Mytilus shell. Moreover, most of identified proteins in the present study are novel SMPs, which greatly extended biomineralization-related protein data of Mytilus. These results are useful, on one hand, for understanding the roles of SMPs in the deposition of different shell layers. On the other hand, the identified protein set of myostracum provides candidates for further exploring the mechanism of adductor muscle-shell attachment.

Partial Text

Mollusk shell, primarily consisting of calcium carbonate crystals together with organic matrix, has been investigated as a typical biomineralization model for dozens years [1–4]. It is well known that there are three major calcium carbonate minerals (calcite, aragonite, and vaterite) with the same principal composition, despite the different structures [5]. Of these minerals, the two most thermodynamically stable structures, calcite and aragonite, are deposited extensively as biominerals. In general, most of the adult shell of bivalves found in nature are composed of calcite and/or aragonite with different shapes and morphologies, including ‘‘nacreous”, ‘‘prismatic”, ‘‘foliated”, ‘‘cross-lamellar”, ‘‘granular”, ‘‘composite-prismatic”, and ‘‘homogeneous” structures [6, 7]. Among the most studied of them are nacre and prism. Nacre is a widespread mollusk shell texture. It is represented within the three main classes of mollusks, Bivalves, Gastropods, and Cephalopods. The “nacre” terminology refers to a well-defined type of microstructure characterized by small flat tablets of aragonite tightly packed together by organic cement [8, 9]. Prism is usually calcite needles of various lengths and diameters. The thin oblique calcite prism named “fibrous prism” had been detected in Mytilus edulis shell [10]. Myostracum is usually a very thin layer located in the attachment of the adductor muscle, commonly called the muscle scar or imprint, to the umbo of each valve. The adductor muscle scar, where the adductor muscle functions to close the shell, is the most conspicuous area on bivalve shell [11, 12].

The mollusk shell is constructed by different calcium carbonate layers and among the most studied of them are the nacro-prismatic shells of Cambrian origin, such as bivalves, gastropods and cephalopods [29]. As shown in Fig 1, the microstructure of M. galloprovincialis shell consists of nacre, fibrous prisms, and myostracum. Nacre and fibrous prism occupied the most area of the shell sectional structure, forming a typical pattern of nacro-prismatic model. Besides that, myostracum is also an important shell layer because it is strongly attached with the adductor muscle at the adductor muscle scar, indicating a function involving in shell-muscle adhesion for this layer. In the present study, we have investigated the SMPs associated with three shell layers of M. galloprovincialis in order to search for characteristic biomineral protein signatures of various shell layers. To extract the strongly mineral linked shell proteins, we used two-step strategy (acetic acid and urea, respectively) to dissolve organic matrix of each shell layer. As a strong denaturing agent, urea has been successfully used for dissolving hydrophobic and cross-linked proteins from mussel byssus [30]. We noticed that the acetic acid-insoluble matrix can partially be dissolved in urea and additional proteins were thus identified, indicating urea can be used as an effective reagent for extracting shell proteins. By combining proteomic analysis with EST database interrogations, a whole set of 113 proteins were identified from M. galloprovincialis shell. Of this set, 15 proteins are shared by all the three layers, including three Calponin-like proteins, three Collagen-like proteins, one Filament-like protein, MUSP-3, one Perlucin-like protein, one Shell Matrix Protein, and five uncharacterized proteins with unusual amino acid composition (Table 1). Of these common proteins, Perlucin is a shell protein that could accelerate the precipitation of calcium carbonate [31], and the Shell Matrix Protein also contains a carbohydrate binding domain. These results suggest that these common shell proteins may play a key role in the formation of the whole shell of M. galloprovincialis although most of these proteins are novel proteins with unknown function. Further, of the whole 113 protein identified from M. galloprovincialis shell, 23 proteins are exclusive to nacre, 34 to myostracum, and 21 to fibrous prism. In addition, eight proteins are shared by nacre/myostracum, eight by nacre/fibrous prism, and four by myostracum/fibrous prism (Fig 4 and Table 1). The mosaic pattern of distribution of these proteins in three shell layers revealed that (i) different shell layers contained different protein sets, (ii) a possible filiation may exist among three shell layers, and (iii) the myostracum contained more unique proteins than those from nacre and fibrous prism, which may result from the role of myostracum in shell-muscle attachment. Moreover, many novel proteins were identified from M. galloprovincialis shell, dramatically increasing the SMP data of Mytilus. Among these novel shell proteins are myostracum-related proteins, protein inhibitor-like proteins, EGF domain-containing protein, alveoline-like proteins, and various low-complexity domain-containing proteins.

Mytilus shell is a good model of nacro-prismatic structure for studying the shell formation and SMPs responsible for different shell microstructures. A parallel proteomics analysis was performed for three layers, nacre, myostracum, and fibrous prism, which form the shell of M. galloprovincialis. A set of 54, 61, and 48 proteins were identified from nacre, myostracum, and fibrous prism respectively. For each shell layer, about a half of identified proteins were unique and the others are shared by two or all of the three layers, which indicated a possible evolutionary relationship among the three layers. In addition, the myostracum contained the largest protein set, highlighting the important function of myostracum in attachment with adductor muscle. Moreover, many novel shell proteins were identified, including the proteins with possible or established link to biomineralization and some uncharacterized proteins with unusual amino acid composition. These data are useful for understanding the roles of SMPs associated to the formation of different shell layers (nacre vs. myostracum vs. fibrous prism) or different morphology of calcium carbonate (aragonite vs. calcite), and the identified protein set of myostracum provides candidates for further exploring the molecular mechanism of adductor muscle-shell attachment.