“…However, two faint bands are visible at about 14 and 20 kDa in the EDTA-soluble matrix. They suggested that the 14 kDa band is similar to the acidic N14 protein (pI 4.8) described by Kono et al [27] in P. maxima. P14 is known in the acetic acid-soluble matrix [28].…”
Section: Comparison With the Known Proteins In Nacresupporting
Mollusk shells, especially the nacre, are of commercial interest as well as palaeoenvironmental proxies. They are also investigated as biomaterials for medical purposes and biomimetics. Although the mineralogy is well-known and unique (aragonite tablets), the organic components are various. However, determination of the precise composition of the soluble organic matrix (SOM) of the nacreous layer is difficult. Among the range of possible techniques, 1D electrophoresis and High-performance liquid chromatography (HPLC) have previously been applied separately to differentiate pI and molecular weights. To date, no clear correlation has been established between the two parameters obtained in such conditions. Here, we report the use of preparative electrophoresis, coupled with HPLC, to determine the molecular weights of the pI fractions. The results are compared with 2D gel electrophoresis. It is shown that both methods have drawbacks and advantages, and are not redundant. The complexity of the composition of the nacreous tablet shown by scanning electron microscope (SEM) and Atomic Force Microscope (AFM) observations is also evidenced by electrophoresis and HPLC.
“…However, two faint bands are visible at about 14 and 20 kDa in the EDTA-soluble matrix. They suggested that the 14 kDa band is similar to the acidic N14 protein (pI 4.8) described by Kono et al [27] in P. maxima. P14 is known in the acetic acid-soluble matrix [28].…”
Section: Comparison With the Known Proteins In Nacresupporting
Mollusk shells, especially the nacre, are of commercial interest as well as palaeoenvironmental proxies. They are also investigated as biomaterials for medical purposes and biomimetics. Although the mineralogy is well-known and unique (aragonite tablets), the organic components are various. However, determination of the precise composition of the soluble organic matrix (SOM) of the nacreous layer is difficult. Among the range of possible techniques, 1D electrophoresis and High-performance liquid chromatography (HPLC) have previously been applied separately to differentiate pI and molecular weights. To date, no clear correlation has been established between the two parameters obtained in such conditions. Here, we report the use of preparative electrophoresis, coupled with HPLC, to determine the molecular weights of the pI fractions. The results are compared with 2D gel electrophoresis. It is shown that both methods have drawbacks and advantages, and are not redundant. The complexity of the composition of the nacreous tablet shown by scanning electron microscope (SEM) and Atomic Force Microscope (AFM) observations is also evidenced by electrophoresis and HPLC.
“…The water-soluble organic matrix dictates which calcium carbonate crystal structure is formed and when it is deposited (Cariolou & Morse, 1988). The bulk of the watersoluble biopolymer is thought to consist of a complex mixture of proteins and peptides (Kono et al, 2000;Weiss et al, 2000). Nacre possess in its organic matrix molecular signals that have the ability to trigger bone cell commitment.…”
Section: Biological Action Of Nacre Extractsmentioning
“…An antibody elicited against an immunogenic peptide of Upsalin showed that it is present in both layers (nacreous and prismatic) of the shell of U. pictorum. Upsalin, together with prismalin-14, [13] mucoperlin, [32] N14/N16/ pearlin, [33][34][35] and BMSP, thus represents one of the few examples of a shell protein to be characterized both at the transcriptional and the protein levels, and for which in vitro characterization has been performed after purification from the shell.…”
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.
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