Novel Molluskan Biomineralization Proteins Retrieved from Proteomics: A Case Study with Upsalin

Abstract

The formation of the molluskan shell is regulated by an array of extracellular proteins secreted by the calcifying epithelial cells of the mantle. These proteins remain occluded within the recently formed biominerals. To date, many shell proteins have been retrieved, but only a few of them, such as nacreins, have clearly identified functions. In this particular case, by combining molecular biology and biochemical approaches, we performed the molecular characterization of a novel protein that we named Upsalin, associated with the nacreous shell of the freshwater mussel Unio pictorum. The full sequence of the upsalin transcript was obtained by RT‐PCR and 5′/3′ RACE, and the expression pattern of the transcript was studied by PCR and qPCR. Upsalin is a 12 kDa protein with a basic theoretical pI. The presence of Upsalin in the shell was demonstrated by extraction of the acetic‐acid‐soluble nacre matrix, purification of a shell protein fraction by mono‐dimensional preparative SDS‐PAGE, and by submitting this fraction, after trypsic digestion, to nano‐LC‐MS/MS. In vitro experiments with the purified protein showed that it interferes poorly with the precipitation of calcium carbonate. Homology searches also could not affiliate Upsalin to any other protein of known function, leaving open the question of its exact role in shell formation. An antibody raised against an immunogenic peptide of Upsalin was found to be specific to this protein and was subsequently assayed for immunogold localization of the target protein in the shell, revealing the ubiquitous presence of Upsalin in the nacreous and prismatic layers. Recently, with the application of high‐throughput proteomic studies to shells, the number of candidate proteins without clear functions has been increasing exponentially. The Upsalin example highlights the crucial need, for the scientific community dealing with biomineralization in general, to dedicate the coming years to designing experimental approaches, such as gene silencing, that focus on the functions of mineral‐associated proteins.

The Skeletal Proteome of the Coral Acropora millepora: The Evolution of Calcification by Co-Option and Domain Shuffling

“…High-throughput approaches such as proteomics and transcriptomics have often revealed completely novel proteins that lack conserved domains or significant database matches that would allow to classify them into families and to hypothesize about their functions (Ramos-Silva et al 2012). Galaxin exemplifies the case of such “orphans” because its function remains unknown (Reyes-Bermudez et al 2009).…”

The Biomineralization Proteome: Protein Complexity for a Complex Bioceramic Assembly Process

“…The transcriptome or RNA expression levels in these organisms are also being explored with the goal of understanding the timing of transcription, translation, and protein secretion as it pertains to the events occurring in the biomineralizing matrix . What has emerged from these studies…”

“…The mollusk, of the phyla Lophotrochozoa, is one of the most diverse groups on Earth (>50 000 species) that creates a protective outer shell that has a high degree of organization, much more so than bone, and this system has been intensely studied for several decades . In most bivalve mollusks, the shell consists of three layers (Figure ): 1) the periostracum, which is a thin organic coating or “skin” comprising the outermost layer of the shell; 2) the prismatic layer, the outermost mineral layer of the shell that comprises organized parallel rods of single crystal calcite, a calcium carbonate polymorph; 3) nacre layer, or the innermost mineral layer of the shell that comprises hexagonal tablets of single crystal aragonite, a calcium carbonate polymorph, that are arranged in a “brick‐and‐mortar” organization . Both the prismatic and nacre mineral particles are coated with a protein‐containing film or layer that is termed the framework layer and this will be discussed in more detail below .…”

“…The outer epithelial layer of the mantle secretes biomineralization proteins and the calcium carbonate mineral phase into a fluid‐filled space termed the extrapallial space. This space separates the mantle epithelium from the nacre layer . Note that Figure applies to mollusks which possess a bilayer prismatic–nacre shell; however, in some mollusks, there is only a single mineral layer, either a prismatic or a nacre one…”

“…Within these genomes lurk mineralization‐associated genes. Since many biomineral‐associated proteins are extracellular and thus subjected to post‐translational modifications, these genomic studies have been augmented by proteomic studies that identify gene products that are phosphorylated, sulfated, glycosylated, enzymatically cleaved, or otherwise chemically modified at side‐chain groups . The challenge that we are now facing is to identify these mineralization‐associated proteins, their structure, synthesis, secretion, and temporal/regional localization, and most importantly, their purpose or function.…”