Asprich: A novel aspartic acid-rich protein family from the prismatic shell matrix of the bivalve Atrina rigida.
ABSTRACT Almost all mineralized tissues contain proteins that are unusually acidic. As they are also often intimately associated with the mineral phase, they are thought to fulfill important functions in controlling mineral formation. Relatively little is known about these important proteins, because their acidic nature causes technical difficulties during purification and characterization procedures. Much effort has been made to overcome these problems, particularly in the study of mollusk-shell formation. To date about 16 proteins from mollusk-shell organic matrices have been sequenced, but only two are unusually rich in aspartic and glutamic acids. Here we screened a cDNA library made from the mRNA of the shell-forming cells of a bivalve, Atrina rigida, using probes for short Asp-containing repeat sequences, and identified ten different proteins. Using more specific probes designed from one subgroup of conserved sequences, we obtained the full sequences of a family of seven aspartic acid-rich proteins, which we named "Asprich"; a subfamily of the unusually acidic shell-matrix proteins. Polyclonal antibodies raised against a synthetic peptide of the conserved acidic1 domain of these proteins reacted specifically with the matrix components of the calcitic prismatic layer, but not with those of the aragonitic nacreous layer. Thus the Asprich proteins are constituents of the prismatic layer shell matrix. We can identify different domains within these sequences, including a signal peptide characteristic of proteins destined for extracellular secretion, a conserved domain rich in aspartic acid that contains a sequence very similar to the calcium-binding domain of Calsequestrin, and another domain rich in aspartic acid, that varies between the seven sequences. We also identified a domain with DEAD repeats that may have Mg-binding capabilities. Although we do not know, as yet, the function of these proteins, their generally conserved sequences do indicate that they might well fulfill basic functions in shell formation.
Article: Prismin: A New Matrix Protein Family in the Japanese Pearl Oyster (Pinctada fucata) Involved in Prismatic Layer Formation[show abstract] [hide abstract]
ABSTRACT: The hard tissue of the Japanese pearl oyster, Pinctada fucata, consists of two layers, the outer prismatic layer, bearing calcite, and the inner nacreous layer, bearing aragonite. An EDTA-insoluble fraction of the prismatic layer of P. fucata was extracted with urea. In-vitro crystallization experiments showed that this urea-soluble fraction contained the factor(s) that promoted the growth of calcite crystals. We purified a protein from this fraction and deduced the internal amino acid sequences EYDFDRPDPYDP and EYDFERPD. We performed 3′ RACE using primer DPPF1, encoding EYDFDRPDPYDP, and an oligo-dT adapter primer and amplified a fragment of approximately 300 bp. We screened cDNA libraries using the 300 bp fragment and obtained two clones that we named prismin 1 and 2. Both cDNAs encode proteins of 51 amino acids. Homology searches revealed 91% amino acid identity between prismin 1 and 2. The synthetic peptide DFDRPDPYDPYDRFD, corresponding to the carboxy terminal region of prismin 1, has calcite growing activity and calcium binding capability, showing that the carboxy-terminal region is a functional domain. Prismin 1 is expressed strongly in the outer edge and in the inner part of the mantle tissue. However, immunoblot analysis revealed that prismin protein exists only in the prismatic layer, not in the nacreous layer, despite the presence of the mRNA. Therefore, we conclude that prismin is a novel prismatic layer-specific calcite growth factor.ZOOLOGICAL SCIENCE 05/2010; · 0.95 Impact Factor
Article: Insights into shell deposition in the Antarctic bivalve Laternula elliptica: gene discovery in the mantle transcriptome using 454 pyrosequencing.[show abstract] [hide abstract]
ABSTRACT: The Antarctic clam, Laternula elliptica, is an infaunal stenothermal bivalve mollusc with a circumpolar distribution. It plays a significant role in bentho-pelagic coupling and hence has been proposed as a sentinel species for climate change monitoring. Previous studies have shown that this mollusc displays a high level of plasticity with regard to shell deposition and damage repair against a background of genetic homogeneity. The Southern Ocean has amongst the lowest present-day CaCO3 saturation rate of any ocean region, and is predicted to be among the first to become undersaturated under current ocean acidification scenarios. Hence, this species presents as an ideal candidate for studies into the processes of calcium regulation and shell deposition in our changing ocean environments. 454 sequencing of L. elliptica mantle tissue generated 18,290 contigs with an average size of 535 bp (ranging between 142 bp-5.591 kb). BLAST sequence similarity searching assigned putative function to 17% of the data set, with a significant proportion of these transcripts being involved in binding and potentially of a secretory nature, as defined by GO molecular function and biological process classifications. These results indicated that the mantle is a transcriptionally active tissue which is actively proliferating. All transcripts were screened against an in-house database of genes shown to be involved in extracellular matrix formation and calcium homeostasis in metazoans. Putative identifications were made for a number of classical shell deposition genes, such as tyrosinase, carbonic anhydrase and metalloprotease 1, along with novel members of the family 2 G-Protein Coupled Receptors (GPCRs). A membrane transport protein (SEC61) was also characterised and this demonstrated the utility of the clam sequence data as a resource for examining cold adapted amino acid substitutions. The sequence data contained 46,235 microsatellites and 13,084 Single Nucleotide Polymorphisms(SNPs/INDELS), providing a resource for population and also gene function studies. This is the first 454 data from an Antarctic marine invertebrate. Sequencing of mantle tissue from this non-model species has considerably increased resources for the investigation of the processes of shell deposition and repair in molluscs in a changing environment. A number of promising candidate genes were identified for functional analyses, which will be the subject of further investigation in this species and also used in model-hopping experiments in more tractable and economically important model aquaculture species, such as Crassostrea gigas and Mytilus edulis.BMC Genomics 01/2010; 11:362. · 4.07 Impact Factor
[show abstract] [hide abstract]
ABSTRACT: Remarkable progress has been made to date in the discovery of material binding peptides and their utilization in nanotechnology, which has brought new challenges and opportunities. Nowadays phage display is a versatile tool, important for the selection of ligands for proteins and peptides. This combinatorial approach has also been adapted over the past decade to select material-specific peptides. Screening and selection of such phage displayed material binding peptides has attracted great interest, in particular because of their use in nanotechnology. Phage display selected peptides are either synthesized independently or expressed on phage coat protein. Selected phage particles are subsequently utilized in the synthesis of nanoparticles, in the assembly of nanostructures on inorganic surfaces, and oriented protein immobilization as fusion partners of proteins. In this paper, we present an overview on the research conducted on this area. In this review we not only focus on the selection process, but also on molecular binding characterization and utilization of peptides as molecular linkers, molecular assemblers and material synthesizers.Molecules 01/2011; 16(2):1426-51. · 2.39 Impact Factor