Perlwapin, an abalone nacre protein with three four-disulfide core (whey acidic protein) domains, inhibits the growth of calcium carbonate crystals

We have isolated a new protein from the nacreous layer of the shell of the sea snail Haliotis laevigata (abalone). Amino acid sequence analysis showed the protein to consist of 134 amino acids and to contain three sequence repeats of approximately 40 amino acids which were very similar to the well-known whey acidic protein domains of other proteins. The new protein was therefore named perlwapin. In addition to the major sequence, we identified several minor variants. Atomic force microscopy was used to explore the interaction of perlwapin with calcite crystals. Monomolecular layers of calcite crystals dissolve very slowly in deionized water and recrystallize in supersaturated calcium carbonate solution. When perlwapin was dissolved in the supersaturated calcium carbonate solution, growth of the crystal was inhibited immediately. Perlwapin molecules bound tightly to distinct step edges, preventing the crystal layers from growing. Using lower concentrations of perlwapin in a saturated calcium carbonate solution, we could distinguish native, active perlwapin molecules from denaturated ones. These observations showed that perlwapin can act as a growth inhibitor for calcium carbonate crystals in saturated calcium carbonate solution. The function of perlwapin in nacre growth may be to inhibit the growth of certain crystallographic planes in the mineral phase of the polymer/mineral composite nacre.     

Eggshell matrix protein mimics: designer peptides to induce the nucleation of calcite crystal aggregates in solution

Ansocalcin is a novel goose eggshell matrix protein with 132 amino acid residues, which induces the formation of polycrystalline calcite aggregates in in vitro crystallization experiments. The central region of ansocalcin is characterized by the presence of multiplets of charged amino acids. To investigate the specific role of charged amino acid multiplets in the crystal nucleation, three short peptides REWD-16, REWDP-17 (containing charged doublets), and RADA-16 (alternating charged residues) were synthesized and characterized. The aggregation of these peptides in solution was investigated using circular dichroism, intrinsic tryptophan fluorescence, and dynamic light scattering experiments. The peptides REWD-16 and REWDP-17 induced the polycrystalline calcite crystal aggregates, whereas RADA-16 did not induce significant changes in calcite crystal morphology or aggregate formation in in vitro crystallization experiments. The lattice and morphology of the calcite crystals were characterized using X-ray diffraction and scanning electron microscope. The results discussed in this paper reveal the importance of multiplets of charged amino acid residues toward the nucleation of polycrystalline calcite crystal aggregates in solution.     

Mechanism of calcite crystal growth inhibition by the N-terminal undecapeptide of lithostathine

Pancreatic juice is supersaturated with calcium carbonate. Calcite crystals therefore may occur, obstruct pancreatic ducts, and finally cause a lithiasis. Human lithostathine, a protein synthesized by the pancreas, inhibits the growth of calcite crystals by inducing a habit modification: the rhombohedral (10 14) usual habit is transformed into a needle-like habit through the (11 0) crystal form. A similar observation was made with the N-terminal undecapeptide (pE(1)R(11)) of lithostathine. We therefore aimed at discovering how peptides inhibit calcium salt crystal growth. We solved the complete x-ray structure of lithostathine, including the flexible N-terminal domain, at 1.3 A. Docking studies of pE(1)R(11) with the (10 14) and (11 0) faces through molecular dynamics simulation resulted in three successive steps. First, the undecapeptide progressively unfolded as it approached the calcite surface. Second, mobile lateral chains of amino acids made hydrogen bonds with the calcite surface. Last, electrostatic bonds between calcium ions and peptide bonds stabilized and anchored pE(1)R(11) on the crystal surface. pE(1)R(11)-calcite interaction was stronger with the (11 0) face than with the (10 14) face, confirming earlier experimental observations. Energy contributions showed that the peptide backbone governed the binding more than did the lateral chains. The ability of peptides to inhibit crystal growth is therefore essentially based on backbone flexibility.     

Investigating the parameters affecting the adsorption of amino acids onto AgCl nanoparticles with different surface charges

In this paper, adsorption behaviors of typical neutral (alanine), acidic (glutamic acid) and basic (lysine) amino acids onto the surfaces of neutral as well as positively and negatively charged silver chloride nanoparticles were examined. Silver chloride nanoparticles with different charges and different water content were synthesized by reverse micelle method. The adsorptions of the above mentioned amino acids onto the surfaces of differently charged silver chloride nanoparticles were found to depend strongly on various parameters including pH of the aqueous solution, type of amino acid, water to surfactant mole ratio, and type of charges on the surfaces of silver chloride nanoparticles. It was found that the interaction of –NH₃ ⁺ groups of the amino acids with silver ion could be a driving force for adsorption of amino acids. Alanine and Glutamic acid showed almost similar trend for being adsorbed on the surface of silver chloride nanoparticles. Electrostatic interaction, hydrophobicity of both nanoparticle and amino acid, complex formation between amine group and silver ion, interaction between protonated amine and silver ion as well as the number of nanoparticles per unit volume of solution were considered for interpreting the observed results.     

A novel extrapallial fluid protein controls the morphology of nacre lamellae in the pearl oyster, Pinctada fucata

Mollusk shell nacre is known for its superior mechanical properties and precisely controlled biomineralization process. However, the question of how mollusks control the morphology of nacre lamellae remains unresolved. Here, a novel 38-kDa extrapallial fluid (EPF) protein, named amorphous calcium carbonate-binding protein (ACCBP), may partially answer this question. Although sequence analysis indicated ACCBP is a member of the acetylcholine-binding protein family, it is actively involved in the shell mineralization process. In vitro, ACCBP can inhibit the growth of calcite and induce the formation of amorphous calcium carbonate. When ACCBP functions were restrained in vivo, the nacre lamellae grew in a screw-dislocation pattern, and low crystallinity CaCO(3) precipitated from the EPF. Crystal binding experiments further revealed that ACCBP could recognize different CaCO(3) crystal phases and crystal faces. With this capacity, ACCBP could modify the morphology of nacre lamellae by inhibiting the growth of undesired aragonite crystal faces and meanwhile maintain the stability of CaCO(3)-supersaturated body fluid by ceasing the nucleation and growth of calcite. Furthermore, the crystal growth inhibition capacity of ACCBP was proved to be directly related to its acetylcholine-binding site. Our results suggest that a "safeguard mechanism" of undesired crystal growth is necessary for shell microstructure formation.     

厚壳贻贝whirlin类贝壳基质蛋白的重组表达与功能分析

贝壳历来是生物工程和材料学研究的重要对象。贝壳中的贝壳基质蛋白质在贝壳的形成与发育过程中具有重要的调控作用。Whirlin类蛋白质(Whirlin-like protein,WLP)是一种从厚壳贻贝(Mytilus coruscus)中鉴定的新型贝壳基质蛋白质。序列分析结果显示,该蛋白质含有PDZ(postsynaptic density/Discs large/Zonula occludens)结构域,而该结构域对贝壳生物矿化的影响目前尚无报道。为深入了解WLP在贝壳形成中对碳酸钙晶体的影响,在序列分析基础上,采用密码子优化结合原核重组表达,获得其重组表达产物后,开展了重组WLP对碳酸钙晶体形貌及晶型的影响研究,结晶速度抑制以及碳酸钙晶体结合分析。分析结果表明,重组WLP能诱导文石型碳酸钙晶体的形貌和方解石型碳酸钙晶体的晶型发生改变;同时重组WLP对碳酸钙晶体具有结合作用,且能抑制碳酸钙晶体的结晶速度。上述结果表明,WLP对贝壳的形成及发育具有重要影响,并可能在贝壳肌棱柱层的形成中发挥了重要作用。     

Purification and functional analysis of a 40 kD protein extracted from the Strombus decorus persicus mollusk shells

A 40 kD protein has been extracted from the biomineral matrix of the calcium carbonate gastropod shell of Strombus decorus persicus. The protein was isolated by decalcification and ion exchange HPLC. We have named this protein ACLS40, i.e., aragonite crossed-lamellar structure protein. A partial sequence of the isolated ACLS40 and amino acid analysis both indicate that it does not belong to the family of very acidic proteins, i.e., rich in aspartic and glutamic residues. The shell-extracted protein shows the ability to stabilize calcium carbonate in vitro, in the form of thermodynamically unstable vaterite polymorph, and to inhibit the growth of calcite.     

Direct observation of the transition from calcite to aragonite growth as induced by abalone shell proteins

The mixture of EDTA-soluble proteins found in abalone nacre are known to cause the nucleation and growth of aragonite on calcite seed crystals in supersaturated solutions of calcium carbonate. Past atomic force microscope studies of the interaction of these proteins with calcite crystals did not observe this transition because no information about the crystal polymorph on the surface was obtained. Here we have used the atomic force microscope to directly observe changes in the atomic lattice on a calcite seed crystal after the introduction of abalone shell proteins. The observed changes are consistent with a transition to (001) aragonite growth on a (1014) calcite surface.     

Effect of various chloride salts on the utilization of phosphorus, calcium, and magnesium

Only part of the effect of dietary protein on urinary calcium excretion can be ascribed to sulfur amino acids. We hypothesized that chloride, another factor often associated with isolated proteins, and another amino acid, lysine, affect utilization of calcium. The effects of supplemental dietary chloride, inorganic or organic, on calcium, phosphorus, and magnesium utilization were studied in two rat studies. Weanling Sprague-Dawley rats were fed semi-purified diets that contained moderate (1.8 mg Cl/g diet) or supplemental (15.5 mg Cl/g diet) chloride as sodium chloride, potassium chloride, or lysine monohydrochloride with or without calcium carbonate for 56 or 119 days. Rats fed supplemental sodium chloride or potassium chloride had higher urinary phosphorus excretion, more efficient phosphorus absorption, but unchanged tissue phosphorus levels after 7 and 16 weeks of dietary treatment as compared to rats fed moderate chloride. Rats fed supplemental sodium chloride or potassium chloride excreted more calcium in urine at 7 weeks and absorbed calcium less efficiently at 16 weeks. Tissue calcium concentrations were unaffected, but total tibia magnesium and plasma magnesium concentrations were lower in rats fed supplemental sodium chloride or potassium chloride than those fed moderate chloride. Lysine chloride with or without additional calcium elevated urinary calcium excretion even more than sodium chloride and potassium chloride ingestion. Rats fed lysine chloride with supplemental calcium had smaller apparent absorption and urinary losses of phosphorus and magnesium after 16 weeks and lower tibia and plasma magnesium concentrations than rats fed lysine chloride.     

Colloidal-gold immunocytochemical localization of osteopontin in avian eggshell gland and eggshell.

During mineralization of the avian eggshell, there is a sequential and orderly deposition of both matrix and mineral phases. Therefore, the eggshell is an excellent model for studying matrix-mineral relationships and the regulation of mineralization. Osteopontin, as an inhibitor of crystal growth, potently influences the formation of calcium phosphate and calcium carbonate biominerals. The purpose of this study was to characterize matrix-mineral relationships, specifically for osteopontin, in the avian eggshell using high-resolution transmission (TEM) and scanning (SEM) electron microscopy to gain insight into how calcite crystal growth is structured and compartmentalized during eggshell mineralization. Osteopontin was localized at the ultrastructural level by colloidal-gold immunocytochemistry. In EDTA-decalcified eggshell, an extensive matrix network was observed by TEM and SEM throughout all regions and included interconnected fibrous sheets, irregularly shaped aggregates, vesicular structures, protein films, and isolated protein fibers. Osteopontin was associated with protein sheets in the highly mineralized palisades region; some of these features defined boundaries that compartmentalized different eggshell structural units. In fractured and undecalcified eggshell, osteopontin was immunolocalized on the {104} crystallographic faces of calcite-its natural cleavage plane. The specific occlusion of osteopontin into calcite during mineralization may influence eggshell structure to modify its fracture resistance.     

Characterization of two molluscan crystal-modulating biomineralization proteins and identification of putative mineral binding domains

Ethylenediamine-tetraacetic acid extracted water-soluble matrix proteins in molluscan shells secreted from the mantle epithelia are believed to control crystal nucleation, morphology, orientation, and phase of the deposited mineral. Previously, atomic force microscopy demonstrated that abalone nacre proteins bind to growing step edges and to specific crystallographic faces of calcite, suggesting that inhibition of calcite growth may be one of the molecular processes required for growth of the less thermodynamically stable aragonite phase. Previous experiments were done with protein mixtures. To elucidate the role of single proteins, we have characterized two proteins isolated from the aragonitic component of nacre of the red abalone, Haliotis rufescens. These proteins, purified by hydrophobic interaction chromatography, are designated AP7 and AP24 (aragonitic protein of molecular weight 7 kDa and 24 kDa, respectively). Degenerate oligonucleotide primers corresponding to N-terminal and internal peptide sequences were used to amplify cDNA clones by a polymerase chain reaction from a mantle cDNA library; the deduced primary amino acid sequences are presented. Preliminary crystal growth experiments demonstrate that protein fractions enriched in AP7 and AP24 produced CaCO(3) crystals with morphology distinct from crystals grown in the presence of the total mixture of soluble aragonite-specific proteins. Peptides corresponding to the first 30 residues of the N-terminal sequences of both AP7 and AP24 were generated. The synthetic peptides frustrate the progression of step edges of a growing calcite surface, indicating that sequence features within the N-termini of AP7 and AP24 include domains that interact with CaCO(3). CD analyses demonstrate that the N-terminal peptide sequences do not possess significant percentages of alpha-helix or beta-strand secondary structure in solution. Instead, in both the presence and absence of Ca(II), the peptides retain unfolded conformations that may facilitate protein-mineral interaction.     

Increasing of Soil Urease Activity by Stimulation of Indigenous Bacteria and Investigation of Their Role on Shear Strength

Abstract

Recent studies have shown that the use of biostimulation is an effective technique to eliminate the environmental side effects of traditional soil improvement methods. The use of indigenous bacteria of soil is a new method through which indigenous bacteria produce carbonate calcium by their urease activity. Stimulation of soil indigenous bacteria with the aim of calcite precipitation can considerably increase the soil shear strength. In this study, indigenous ureolytic bacteria are stimulated by adding nutrients to the soil. Subsequently urease activity of these bacteria in the presence of calcium chloride and nickel chloride causes calcium carbonate to precipitate between the sand particles. The analysis showed that the stimulated soil compared to the control soil was significantly different in terms of the soil engineering properties and the amount of precipitated calcite. Further, the treated and untreated samples were examined using direct shear test, scanning electron microscope (SEM), and energy dispersive X-ray (EDX) analysis. The results showed an increase of 30–67% in ultimate shear strength, 4–18.8% in residual shear strength, 190% in the cohesion intercept, and 16.8% in the angle of internal friction. In addition, imaging and analysis of SEM-EDX indicated the production of large amounts of calcite precipitates on surfaces of soil particles and between them.     

Inability to demonstrate calcite precipitation by bacterial isolates from travertine

Bacteria were isolated from 10 actively depositing travertine sites in Europe and North America. Forty‐four isolates were characterized and 33 (75%) were identified as Pseudomonas. Thirty isolates produced base on glucose‐peptone media and 32 isolates produced ammonia from amino acids. Because of the low bacterial biomass in the travertines, the formation of bases, including ammonia was not considered to be significant in carbonate precipitation. None of the bacteria was observed to precipitate calcite from supersaturated calcium carbonate solutions in vitro.     

Effect of calcium chloride on the conformation of proteins. Thermodynamic studies of some model compounds

Partial molar heat capacities (CP2 degrees) and volumes (V2 degrees) for some amino acids and peptides were measured in 1 M aqueous calcium chloride solutions at 298.15 degrees K using a Picker flow microcalorimeter and an oscillating-tube digital density meter. Using the data for these amino acids and peptides in water, the corresponding partial molar heat capacities of transfer (CP2,tr degree) and volumes (V2,tr degree) from water to 1 M aqueous calcium chloride were deduced. These thermodynamic parameters are significantly positive, indicating that strong interactions occur between the ions of calcium chloride and the charged centres of these amino acids and peptides. A comparison has been made with a similar transfer of these compounds to sodium chloride solutions. The thermodynamic parameters of the transfer of peptide group (-CONH) are much more positive in calcium chloride than in sodium chloride solutions. The implication of this result for the ability of calcium chloride to act as a stronger destabilizer of protein conformation is discussed.     

Novel basic protein, PfN23, functions as key macromolecule during nacre formation

The fine microstructure of nacre (mother of pearl) illustrates the beauty of nature. Proteins found in nacre were believed to be "natural hands" that control nacre formation. In the classical view of nacre formation, nucleation of the main minerals, calcium carbonate, is induced on and by the acidic proteins in nacre. However, the basic proteins were not expected to be components of nacre. Here, we reported that a novel basic protein, PfN23, was a key accelerator in the control over crystal growth in nacre. The expression profile, in situ immunostaining, and in vitro immunodetection assays showed that PfN23 was localized within calcium carbonate crystals in the nacre. Knocking down the expression of PfN23 in adults via double-stranded RNA injection led to a disordered nacre surface in adults. Blocking the translation of PfN23 in embryos using morpholino oligomers led to the arrest of larval development. The in vitro crystallization assay showed that PfN23 increases the rate of calcium carbonate deposition and induced the formation of aragonite crystals with characteristics close to nacre. In addition, we constructed the peptides and truncations of different regions of this protein and found that the positively charged C-terminal region was a key region for the function of PfN23 Taken together, the basic protein PfN23 may be a key accelerator in the control of crystal growth in nacre. This provides a valuable balance to the classic view that acidic proteins control calcium carbonate deposition in nacre.     

Use of attenuated total reflectance Fourier transform infrared spectroscopy to identify microbial metabolic products on carbonate mineral surfaces

This paper demonstrates the use of attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy to detect microbial metabolic products on carbonate mineral surfaces. By creating an ATR-FTIR spectral database for specific organic acids using ATR-FTIR spectroscopy we were able to distinguish metabolic acids on calcite surfaces following Escherichia coli growth. The production of these acids by E. coli was verified using high-performance liquid chromatography with refractive index detection. The development of this technique has allowed us to identify microbial metabolic products on carbonate surfaces in nutrient-limited cave environments.     

Assessment of cell-signaling pathways in the regulation of mammalian target of rapamycin (mTOR) by amino acids in rat adipocytes

Enhanced phosphorylation of the ribosomal protein s6 kinase, p70(s6k), and the translational repressor, 4E-BP1, are associated with either insulin-induced or amino acid-induced protein synthesis. Hyperphosphorylation of p70(s6k) and 4E-BP1 in response to insulin or amino acids is mediated through the mammalian target of rapamycin (mTOR). In several cell lines, mTOR or its downstream targets can be regulated by phosphatidylinositol (PI) 3-kinase; protein kinases A, B, and C; heterotrimeric G-proteins; a PD98059-sensitive kinase or calcium; as well as by amino acids. Regulation by amino acids appears to involve detection of levels of charged t-RNA or t-RNA synthetase activity and is sensitive to inhibition by amino acid alcohols. In the present article, however, we show that the rapamycin-sensitive regulation of 4E-BP1 and p70(s6k) in freshly isolated rat adipocytes is not inhibited by either L-leucinol or L-histidinol. This finding is in agreement with other recent studies from our laboratory suggesting that the mechanism by which amino acids regulate mTOR in freshly isolated adipocytes may be different than the mechanism found in a number of cell lines. Therefore we investigated the possible role of growth factor-regulated and G-protein-regulated signaling pathways in the rapamycin-sensitive, amino acid alcohol-insensitive actions of amino acids on 4E-BP1 phosphorylation. We found, in contrast to previously published results using 3T3-L1 adipocytes or other cell lines, that the increase in 4E-BP1 phosphorylation promoted by amino acids was insensitive to agents that regulate protein kinase A, mobilize calcium, or inhibit protein kinase C. Furthermore, amino acid-induced 4E-BP1 phosphorylation was not blocked by pertussis toxin nor was it mimicked by the G-protein agonists fluoroaluminate or MAS-7. However, amino acids failed to activate either PI 3-kinase, protein kinase B, or mitogen-activated protein kinase and failed to promote tyrosine phosphorylation of cellular proteins, similar to observations made using cell lines. In summary, amino acids appear to use an amino acid alcohol-insensitive mechanism to regulate mTOR in freshly isolated adipocytes. This mechanism is independent of cell-signaling pathways implicated in the regulation of mTOR or its downstream targets in other cells. Overall, our study emphasizes the need for caution when extending results obtained using established cell lines to the differentiated nondividing cells found in most tissues.     

Purification,characterization, and in vitro mineralization studies of a novel goose eggshell matrix protein,ansocalcin

Biomineralization is an important process in which hard tissues are generated through mineral deposition, often assisted by biomacromolecules. Eggshells, because of their rapid formation via mineralization, are chosen as a model for understanding the fundamentals of biomineralization. This report discusses purification and characterization of various proteins and peptides from goose eggshell matrix. A novel 15-kDa protein (ansocalcin) was extracted from the eggshell matrix, purified, and identified and its role in mineralization evaluated using in vitro crystal growth experiments. The complete amino acid sequence of ansocalcin showed high homology to ovocleidin-17, a chicken eggshell protein, and to C-type lectins from snake venom. The amino acid sequence of ansocalcin was characterized by the presence of acidic and basic amino acid multiplets. In vitro crystallization experiments showed that ansocalcin induced pits on the rhombohedral faces at lower concentrations (<50 microg/ml). At higher concentrations, the nucleation of calcite crystal aggregates was observed. Molecular weight determinations by size exclusion chromatography and sodium dodecyl sulfate -polyacrylamide gel electrophoresis showed reversible concentration-dependent aggregation of ansocalcin in solution. We propose that such aggregated structures may act as a template for the nucleation of calcite crystal aggregates. Similar aggregation of calcite crystals was also observed when crystallizations were performed in the presence of whole goose eggshell extract. These results show that ansocalcin plays a significant role in goose eggshell calcification.     

A microbial role in the construction of Mono Lake carbonate chimneys?

Lacustrine carbonate chimneys are striking, metre‐scale constructions. If these were bioinfluenced constructions, they could be priority targets in the search for early and extraterrestrial microbial life. However, there are questions over whether such chimneys are built on a geobiological framework or are solely abiotic geomorphological features produced by mixing of lake and spring waters. Here, we use correlative microscopy to show that microbes were living around Pleistocene Mono Lake carbonate chimneys during their growth. A plausible interpretation, in line with some recent works by others on other lacustrine carbonates, is that benthic cyanobacteria and their associated extracellular organic material (EOM) formed tubular biofilms around rising sublacustrine spring vent waters, binding calcium ions and trapping and binding detrital silicate sediment. Decay of these biofilms would locally have increased calcium and carbonate ion activity, inducing calcite precipitation on and around the biofilms. Early manganese carbonate mineralisation was directly associated with cell walls, potentially related to microbial activity though the precise mechanism remains to be elucidated. Much of the calcite crystal growth was likely abiotic, and no strong evidence for either authigenic silicate growth or a clay mineral precursor framework was observed. Nevertheless, it seems likely that the biofilms provided initial sites for calcite nucleation and encouraged the primary organised crystal growth. We suggest that the nano‐, micro‐ and macroscale fabrics of these Pleistocene Mono Lake chimneys were affected by the presence of centimetre‐thick tubular and vertically stacked calcifying microbial mats. Such carbonate chimneys represent a promising macroscale target in the exploration for ancient or extraterrestrial life.