Folian-cv1 Is a Member of a Highly Acidic Phosphoprotein Class Derived From the Foliated Layer of the Eastern Oyster (Crassostrea virginica) Shell and Identified in Hemocytes and Mantle

Abstract

The proteins derived from the foliated shell layer of the oyster, Crassostrea virginica, are unusually acidic and highly phosphorylated. Here we report the identification of a gene encoding a member of this class of phosphoproteins that we collectively refer to as folian. Using an in silico approach, a virtual probe was constructed from an N-terminal sequence (DEADAGD) determined for a 48 kDa folian phosphoprotein and used to screen an oyster EST databank. A sequence that matched the N-terminus of the 48 kDa protein was found and used to identify the full-length gene from a C. virginica BAC library. The molecular weight of the deduced gene product is 32 kDa and was named folian-cv1. Genomic Southern analysis revealed two variants of the gene. The mature protein is composed of 43.3% Asp, 32.6% Ser, and 9.1% Glu with 37.5% of the amino acids of the protein potentially phosphorylated. The primary sequence of folian-cv1 is organized in blocks, with a short relatively hydrophobic block at the N-terminus and with the remainder containing low complexity regions largely dominated by aspartic acid and serine. Overall, the protein is predicted to be highly disordered. PCR and sequence analyses identified folian-cv1 expression in the mantle and hemocytes. Immuno-histochemical staining of mantle tissue reveals that cells of the shell-facing epithelium and in the periostracal groove secrete a continuous layer of folian-positive material and that folian-positive hemocytes move through the mantle epithelium. The function in shell formation of folian proteins including folian-cv1 is not known. However, based on the complexity of this class of proteins and the two methods of their delivery to the region of shell formation, it is possible they are involved in diverse ways in this process.

Identification and Characterization of the Larval Settlement Pheromone Protein Components in Adult Shells of Crassostrea gigas: A Novel Function of Shell Matrix Proteins

“…The overall SDS-PAGE profiles in Figure 4 suggest that aspartic acid-rich proteins were the major components of the EDTA-soluble organic matrix in this study as is reported in other bivalves [ 40 , 55 ]. This implies that these proteins may also bind to calcium [ 55 ] and that they are intrinsically disordered proteins.…”

“…This indicates that CgSE, which is predominantly composed of Stains-all-stainable acidic proteins, was rich in aspartic acid. This is typical for proteins found in shells [ 40 , 52 ] and which may exhibit properties common to intrinsically disordered proteins [ 52 ]. Moreover, TFMS-treated CgSE showed no distinguishable bands; instead, a diffused and scaling smeared pattern from 20 kDa to >250 kDa appeared.…”

“…They have also demonstrated in another study the involvement of a WGA-binding sugar chain from the shell extract that could mediate conspecific cue-settlement induction [ 24 ]. Previous studies, therefore, tried to pursue the investigation of the nature and properties of this chemical cue by using a different chemical solution that has been widely used in extracting molluscan shell matrix proteins, i.e., Ethylenediaminetetraacetic acid (EDTA) [ 39 , 40 ]. A previous study has demonstrated that a chemical cue from adult shells ( C. gigas EDTA-soluble shell extract, CgSE) was isolated that could induce larval settlement in a positive-concentration-dependent manner and that GlcNAc and Sialic acid sugars from the chemical cue were found to interact with a WGA lectin-like receptor from the oyster larva [ 26 ].…”

Transcriptome Dynamics of an Oyster Larval Response to a Conspecific Cue-Mediated Settlement Induction in the Pacific Oyster Crassostrea gigas

“…Interestingly, gigasin-6 isoforms X1 and X2, as well as dentin sialophosphoprotein-like, were upregulated in the conspecific cue-induced PL batch. Gigasin-6 and its isoforms have been reported as a component in the adult shell matrix in C. gigas [60,61], while our sequence homology search on dentin sialophosphoproteinlike showed close homology to folian-cv1 in C. viriginica [62]. Others were related to signal regulation processes such as calmodulin A (LOC105327998), Wnt inhibitory factor 1/EGF-like domain containing protein 2 (Wif-1) (LOC105335286), tyrosine-protein kinase receptor Tie (LOC105338560), beta-2 adrenergic receptor (LOC105336903), nuclear receptors (LOC105338842, LOC105345246), and FMFRamide receptor (LOC105343238).…”

Regulatory Role of Sugars on the Settlement Inducing Activity of a Conspecific Cue in Pacific Oyster Crassostrea gigas

“…Additionally, the presence of endogenous WGA-binding sugar found on the larval foot and mantle, in the present study, further confirms one of the hypotheses of Vasquez et al (2014). Their hypothesis explains that, during exposure of pediveliger larvae with WGA lectin treatment, this lectin may have to bound to shell matrix glycoproteins in the internal organs and mucopolysaccharides on the foot of the larvae [ 50 , 52 , 53 , 54 , 55 ], and thus, somehow interfered with C. gigas larval settlement behavior [ 9 ]. Shell matrix proteins, which are made by the mantle, interact with each other or with polysaccharides or chitin to produce the shell framework [ 56 ].…”