M. Azizur Rahman

Carbonic Anhydrase in Calcified Endoskeleton: Novel Activity in Biocalcification in Alcyonarian

Carbonic anhydrase (CA) is a key enzyme in the chemical reaction of living organisms and has been found to be associated with calcification in a number of invertebrates including calcareous sponges, but until now no direct evidence has been advanced to show CA activity in alcyonarian corals. However, it is essential to understand the role of CA in the process of biocalcification in alcyonarian. Here we describe the novel activity of CA and its relationship to the formation of calcified hard tissues in alcyonarian coral, Lobophytum crassum. We find that two CA proteins, which were partially purified by electro-elution treatment, can control the morphology of CaCO3 crystals and one of them is potentially involved in the process of biocalcification. Previously, we isolated CA from the total extract of alcyonarian, and further, we report here a single protein, which has both calcium-binding and CA activities and is responsible for CaCO3 nucleation and crystal growth. This matrix protein inhibited the precipitation of CaCO3 from a saturated solution containing CaCl2 and NaHCO3, indicating that it can act as a negative regulator for calcification in the sclerites of alcyonarians. The effect of an inhibitor on the enzyme activity was also examined. These findings strongly support the idea that carbonic anhydrase domain in alcyonarian is involved in the calcification process. Our observations strongly suggest that the matrix protein in alcyonarian coral is not only a structural protein but also a catalyst.

First evidence of chitin in calcified coralline algae: new insights into the calcification process of Clathromorphum compactum

Interest in calcifying coralline algae has been increasing over the past years due to the discovery of extensive coralline algal dominated ecosystems in Arctic and Subarctic latitudes, their projected sensitivity to ocean acidification and their utility as palaeoenvironmental proxies. Thus, it is crucial to obtain a detailed understanding of their calcification process. We here extracted calcified skeletal organic matrix components including soluble and insoluble fractions from the widely-distributed Subarctic and Arctic coralline alga Clathromorphum compactum. The lyophilized skeletal organic matrix fractions showed comparatively high concentrations of soluble and insoluble organic matrices comprising 0.9% and 4.5% of skeletal weight, respectively. This is significantly higher than in other skeletal marine calcifiers. Attenuated Total Reflection-Fourier Transform Infrared Spectroscopy (ATR-FTIR) and X-Ray Diffraction (XRD) results indicate that chitin is present in the skeletal organic matrices of C. compactum. This polymer exhibits similar hierarchical structural organizations with collagen present in the matrix and serves as a template for nucleation and controls the location and orientation of mineral phases. Chitin contributes to significantly increasing skeletal strength, making C. compactum highly adapted for living in a shallow high-latitude benthic environment. Furthermore, chitin containing polysaccharides can increase resistance of calcifiers to negative effects of ocean acidification.

Studies on Two Closely Related Species of Octocorallians: Biochemical and Molecular Characteristics of the Organic Matrices of Endoskeletal Sclerites

Two species of alcyonarian corals, Lobophytum crassum and Sinularia polydactyla, are closely related to each other. It is reported that the calcified organic substances in the skeletons of both contain a protein–polysaccharide complex playing a key role in the regulation of biocalcification. However, information on the matrix proteins of endoskeletal sclerite has been lacking. Hence we studied the proteinaceous organic matrices of sclerites for both species, to analyze the sequences and the functional properties of the proteins present. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the preparations showed four bands of proteins with apparent molecular masses of 102, 67, 48, and 37xa0kDa for L. crassum and seven bands of 109, 83, 70, 63, 41, 30, and 22xa0kDa for S. polydactyla. A major protein band of about 67xa0kDa in L. crassum and two bands of proteins of about 70 and 63xa0kDa in S. polydactyla yielded N-terminal amino acid sequences. Periodic acid-Schiff staining indicated that the 67-kDa protein in L. crassum, and 83- and 63-kDa proteins in S. polydactyla were glycosylated. For detection of calcium binding proteins, a Ca2+ overlay analysis was conducted in the extract via 45Ca autoradiography. The 102- and 67-kDa calcium binding proteins in L. crassum, and the 109- and 63-kDa Ca2+ binding proteins in S. polydactyla were found to be radioactive. An assay for carbonic anhydrase (CA), which is thought to play an important role in the process of calcification, revealed specific activities. Newly derived protein sequences were subjected to standard sequence analysis involving identification of similarities to other proteins in databases. The significantly different protein expressions and compositional analysis of sequences between two species were demonstrated.

Analysis of Proteinaceous Components of the Organic Matrix of Endoskeletal Sclerites from the Alcyonarian Lobophytum crassum

The mesoglea of alcyonarians is occupied by an abundance of minute calcitic sclerites. The sclerites of the alcyonarian Lobophytum crassum contain a water-soluble organic matrix comprising 0.48% of the sclerite weight and a water-insoluble fraction comprising 1.15% of the sclerite weight. Analysis of proteinaceous components in the soluble fraction shows a particularly high content of aspartic acid, followed by alanine, glycine, and glutamate. Aspartic acid, glycine, alanine, and glutamate are the most abundant residues in the insoluble fraction. In both cases, the fractions show the highest concentration of aspartic acid from the total proteins. In an in vitro assay, we show that the matrix proteins extracted from the calcitic sclerites induce the formation of amorphous calcium carbonate prior to its transformation into the calcitic crystalline form. We also show scanning electron micrographs of the rhombohedral calcite crystals used as template, the protein imprinted with these crystals. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of both matrices shows the protein fractions at 67 and 48 kDa. The soluble matrix shows two additional faint bands. Both fractions stain for a carbohydrate at 67 kDa, indicating a glycoprotein at this molecular weight. A newly derived protein sequence was subjected to bioinformatics analysis involving identification of similarities to other acidic proteins. The identification of these proteins in alcyonarian endoskeletal sclerites emphasizes the fundamental importance of such acidic proteins and sheds more light on the functions of these proteins in the processes of biocalcification.

Calcite Formation in Soft Coral Sclerites Is Determined by a Single Reactive Extracellular Protein

Calcium carbonate exists in two main forms, calcite and aragonite, in the skeletons of marine organisms. The primary mineralogy of marine carbonates has changed over the history of the earth depending on the magnesium/calcium ratio in seawater during the periods of the so-called “calcite and aragonite seas.” Organisms that prefer certain mineralogy appear to flourish when their preferred mineralogy is favored by seawater chemistry. However, this rule is not without exceptions. For example, some octocorals produce calcite despite living in an aragonite sea. Here, we address the unresolved question of how organisms such as soft corals are able to form calcitic skeletal elements in an aragonite sea. We show that an extracellular protein called ECMP-67 isolated from soft coral sclerites induces calcite formation in vitro even when the composition of the calcifying solution favors aragonite precipitation. Structural details of both the surface and the interior of single crystals generated upon interaction with ECMP-67 were analyzed with an apertureless-type near-field IR microscope with high spatial resolution. The results show that this protein is the main determining factor for driving the production of calcite instead of aragonite in the biocalcification process and that –OH, secondary structures (e.g. α-helices and amides), and other necessary chemical groups are distributed over the center of the calcite crystals. Using an atomic force microscope, we also explored how this extracellular protein significantly affects the molecular-scale kinetics of crystal formation. We anticipate that a more thorough investigation of the proteinaceous skeleton content of different calcite-producing marine organisms will reveal similar components that determine the mineralogy of the organisms. These findings have significant implications for future models of the crystal structure of calcite in nature.

In Vitro Regulation of CaCO3 Crystal Growth by the Highly Acidic Proteins of Calcitic Sclerites in Soft Coral, Sinularia Polydactyla

Acidic proteins are generally thought to control mineral formation and growth in biocalcification. Analysis of proteinaceous components in the soluble and insoluble matrix fractions of sclerites in Sinularia polydactyla indicates that aspartic acid composes about 60% of the insoluble and 29% of the soluble matrix fractions. We previously analyzed aspartic acids in the matrix fractions (insoluble = 17 mol%; soluble = 38 mol%) of sclerites from a different type of soft coral, Lobophytum crassum, which showed comparatively lower aspartic acid-rich proteins than S. polydactyla. Thus, characterization of highly acidic proteins in the organic matrix of present species is an important first step toward linking function to individual proteins in soft coral. Here, we show that aspartic-acid rich proteins can control the CaCO3 polymorph in vitro. The CaCO3 precipitates in vitro in the presence of aspartic acid-rich proteins and 50 mM Mg2+ was verified by Raman microprobe analysis. The matrix proteins of sclerites demonstrated that the aspartic-acid rich domain is crucial for the calcite precipitation in soft corals. The crystalline form of CaCO3 in the presence of aspartic acid-rich proteins in vitro was identified by X-ray diffraction and, revealed calcitic polymorphisms with a strong (104) reflection. The structure of soft coral organic matrices containing aspartate-rich proteins and polysaccharides was assessed by Fourier transform infrared spectroscopy. These results strongly suggest that the aspartic acid-rich proteins within the organic matrix of soft corals play a key role in biomineralization regulation.

Analysis of the proteinaceous components of the organic matrix of calcitic sclerites from the soft coral Sinularia sp.

An organic matrix consisting of a protein-polysaccharide complex is generally accepted as an important medium for the calcification process. While the role this calcified organic matrix plays in the calcification process has long been appreciated, the complex mixture of proteins that is induced and assembled during the mineral phase of calcification remains uncharacterized in many organisms. Thus, we investigated organic matrices from the calcitic sclerites of a soft coral, Sinularia sp., and used a proteomic approach to identify the functional matrix proteins that might be involved in the biocalcification process. We purified eight organic matrix proteins and performed in-gel digestion using trypsin. The tryptic peptides were separated by nano-liquid chromatography (nano-LC) and analyzed by tandem mass spectrometry (MS/MS) using a matrix-assisted laser desorption/ionization (MALDI) - time-of-flight-time-of-flight (TOF-TOF) mass spectrometer. Periodic acid Schiff staining of an SDS-PAGE gel indicated that four proteins were glycosylated. We identified several proteins, including a form of actin, from which we identified a total of 183 potential peptides. Our findings suggest that many of those peptides may contribute to biocalcification in soft corals.

Aspartic Acid-rich Proteins in Insoluble Organic Matrix Play a Key Role in the Growth of Calcitic Sclerites in Alcyonarian Coral

Acidic proteins are generally believed to control mineral formation and growth. Thus, characterization of acidic proteins in the insoluble organic matrix is an important first step toward linking function to individual proteins in alcyonarian coral. Analysis of proteinaceous components in the soluble and insoluble matrix fractions of Sinularia polydactyla indicates that aspartic acid composes about 61% of the insoluble and 29% of the soluble matrix fractions. Using an in vitro assay, we show that matrix proteins induced formation of amorphous CaCO3 precipitates prior to their transformation into the calcitic crystalline form. The crystalline form of CaCO3 in the sclerites was also identified by X-ray diffraction, revealing calcitic polymorphisms with a strong (104) reflection. The structure of alcyonarian organic matrices containing aspartate-rich proteins and polysaccharides was assessed by Fourier transform infrared spectroscopy (FT-IR). Calcium-binding analysis of components in the insoluble matrix fraction indicated that a protein of 109 kD can bind Ca2+, which is important for sclerite formation. An assay for carbonic anhydrase (CA) enzyme, which is believed to play an important role in the process of biocalcification revealed novel activity. These results strongly suggest that the aspartic acid-rich proteins within the insoluble matrix of alcyonarians play a key role in biomineralization regulation.

Characterization of the proteinaceous skeletal organic matrix from the precious coral Corallium konojoi

The Japanese red and pink corals are known to be precious because of their commercial value resulting from their use in ornaments, jewelry, and medicine. Precious corals are very interesting models for biomineralization studies and possess two different skeletal structures: an axial skeleton and an endoskeleton (sclerites). Although it has long been known that the organic matrix proteins existing in coral skeletons are critical for the oriented precipitation of CaCO3 crystals, these proteins in moderate deep‐sea Japanese precious corals remain uncharacterized. Therefore, in this study, we performed skeletal whole proteome analyses using 1D and 2D electrophoresis, nano‐LC, and MALDI‐TOF‐TOF MS. We identified a total of 147 functional coral skeletal organic matrix proteins (120 from the sclerites and 36 from the axial skeleton), including two calcium‐binding calmodulin. Among the organic matrix proteins identified, nine key proteins are highly typical and common in both skeletons. Strong glycosylation activity, which is essential for skeletal formation in calcifying organisms, was detected in both skeletons. This work demonstrates unique biomineralization‐related proteins in precious corals and provides the first description of the major proteinaceous components of CaCO3 minerals in precious corals, enabling the comparative investigation of biocalcification in other octocorals.

Control of CaCO3 Crystal Growth by the Acidic Proteinaceous Fraction of Calcifying Marine Organisms: AnInVitroStudyofBiomineralization

Only little is known about the early stages of CaCO3 crystallization (Pouget et al. 2009; Gebauer, Volkel, and Colfen 2008), though this mineral has been studied for more than a century now. Identified precursor phases are amorphous calcium carbonate (ACC) in bio(Addadi, Raz, and Weiner 2003; Weiner et al. 2003) and biomimetic mineralization. Crystal nucleation and biomineralization processes in organisms occur through a sophisticated regulation of internal chemistry that departs significantly from the “constant ionic medium” of seawater (Falini et al. 1996; Addadi et al. 2006; Rahman and Oomori 2009). Magnesium ions are mainly responsible for controlling the kinetics and thermodynamics of calcium carbonate precipitation, especially inhibition of the calcite formation (Davis, Dove, and De Yoreo 2000). The precipitation of calcite at ambient temperature is both thermodynamically and kinetically favored in solutions containing low amounts of magnesium ions. Very recently, it is reported that although Mg2+ is influential in producing aragonite in the crystallization process, acidic macromolecules produced calcite crystals in soft corals even in the presence of high Mg2+ ions (Rahman, Oomori, and Worheide 2011; Rahman and Oomori 2009).