scholarly journals Lessons from Biomineralisation: the Role of Post-translational Modification

2021 ◽  
Author(s):  
◽  
Benjamin Matthewson
Keyword(s):  
Calcium Carbonate ◽  
Sea Urchin ◽  
Calcium Binding ◽  
Keratan Sulfate ◽  
Organic Matrix ◽  
Active Species ◽  
Protein Glycosylation ◽  
Post Translational Modification ◽  
Spin Filters

<p>In this thesis we present our findings following analysis of the acidic organic matrix (SMP) occluded in the calcite spines of the New Zealand sea urchin Evechinus chloroticus. The main focus involves correlation of the structure and function of the post-translational modifications (PTMs). The experimental framework developed to achieve this involved mapping the structure of the PTMs throughout SMP based on molecular weight (MW) followed by selective removal of each of the identified PTMs. The functional analysis involved the use of SMP, and its derivatives, as additives in an in vitro calcium carbonate crystallisation assay. The adoption of in vitro methods was considered appropriate as the focus of this work was to develop strategies towards programmable crystal growth in vitro. From analysis of the PTMs we have shown that there is extensive protein glycosylation, sulfation, and phosphorylation; all are involved in rendering the isoelectric point (pI) of the SMP macromolecules. The sulfates are exclusively housed on the glycan framework whereas the phosphate is protein bound. The majority of the SMP glycone is charged with O-glycosylation accounting for 80.0 +/- 4.0 wt%. The structure of the glycans includes sulfated HexNAc oligomers, and potentially mucin-like/keratan sulfate and/or carrageenan structures. Using Stains-All we have shown that the desulfated HexNAc oligomers have the ability to bind calcium which signals relevance in the formation of calcium carbonate. SMP was fractionated by MW across a series of spin-filters. Use of the various fractions in the crystallisation assay showed that the species in the greater than 30 kDa fraction held the ability to increase the number of crystals nucleated. In contrast, the macromolecules in the 10 to 30 kDa range contained the full complement of morphologically active species. The result that these functions can be isolated demonstrates that they are independently controlled. The structure-function relationships determined include: the protein and the acidic glycans are jointly sufficient to generate the nucleating function; deglycosylated SMP holds the complete morphological activity, however, the glycans contribute by increasing reproducibility presumably through regulatory influences; and the sterically hindered phosphate residues make a slight contribution to this morphological activity. These results indicate that analyses which involve characterisation of the morphological function of cloned biomineral proteins may indeed correspond to their native counterparts. The observation that the morphologically active species are phosphorylated identifies them as the calcium-binding phosphoproteins. The morphological activity of SMP stripped of all PTMs is equivalent to the proteins extracted from the aragonitic layer of Haliotis iris. Characterisation of SMP demonstrated similarities with the OMs of other sea urchin species. For example, SMP appears to include SM30. In addition, the overall structure of SMP includes abundant acidic glycosylation with a relatively neutral protein component. This structural make-up is in contrast to the highly acidic proteins which are barely post-translationally modified.</p>

Lessons from Biomineralisation: the Role of Post-translational Modification

2021 ◽  
Author(s):  
◽  
Benjamin Matthewson
Keyword(s):  
Calcium Carbonate ◽  
Sea Urchin ◽  
Calcium Binding ◽  
Keratan Sulfate ◽  
Organic Matrix ◽  
Active Species ◽  
Protein Glycosylation ◽  
Post Translational Modification ◽  
Spin Filters

<p>In this thesis we present our findings following analysis of the acidic organic matrix (SMP) occluded in the calcite spines of the New Zealand sea urchin Evechinus chloroticus. The main focus involves correlation of the structure and function of the post-translational modifications (PTMs). The experimental framework developed to achieve this involved mapping the structure of the PTMs throughout SMP based on molecular weight (MW) followed by selective removal of each of the identified PTMs. The functional analysis involved the use of SMP, and its derivatives, as additives in an in vitro calcium carbonate crystallisation assay. The adoption of in vitro methods was considered appropriate as the focus of this work was to develop strategies towards programmable crystal growth in vitro. From analysis of the PTMs we have shown that there is extensive protein glycosylation, sulfation, and phosphorylation; all are involved in rendering the isoelectric point (pI) of the SMP macromolecules. The sulfates are exclusively housed on the glycan framework whereas the phosphate is protein bound. The majority of the SMP glycone is charged with O-glycosylation accounting for 80.0 +/- 4.0 wt%. The structure of the glycans includes sulfated HexNAc oligomers, and potentially mucin-like/keratan sulfate and/or carrageenan structures. Using Stains-All we have shown that the desulfated HexNAc oligomers have the ability to bind calcium which signals relevance in the formation of calcium carbonate. SMP was fractionated by MW across a series of spin-filters. Use of the various fractions in the crystallisation assay showed that the species in the greater than 30 kDa fraction held the ability to increase the number of crystals nucleated. In contrast, the macromolecules in the 10 to 30 kDa range contained the full complement of morphologically active species. The result that these functions can be isolated demonstrates that they are independently controlled. The structure-function relationships determined include: the protein and the acidic glycans are jointly sufficient to generate the nucleating function; deglycosylated SMP holds the complete morphological activity, however, the glycans contribute by increasing reproducibility presumably through regulatory influences; and the sterically hindered phosphate residues make a slight contribution to this morphological activity. These results indicate that analyses which involve characterisation of the morphological function of cloned biomineral proteins may indeed correspond to their native counterparts. The observation that the morphologically active species are phosphorylated identifies them as the calcium-binding phosphoproteins. The morphological activity of SMP stripped of all PTMs is equivalent to the proteins extracted from the aragonitic layer of Haliotis iris. Characterisation of SMP demonstrated similarities with the OMs of other sea urchin species. For example, SMP appears to include SM30. In addition, the overall structure of SMP includes abundant acidic glycosylation with a relatively neutral protein component. This structural make-up is in contrast to the highly acidic proteins which are barely post-translationally modified.</p>

scholarly journals Roles of larval sea urchin spicule SM50 domains in organic matrix self-assembly and calcium carbonate mineralization

2013 ◽  
Vol 183 (2) ◽  
pp. 205-215 ◽  
Author(s):  
Ashit Rao ◽  
Jong Seto ◽  
John K. Berg ◽  
Stefan G. Kreft ◽  
Martin Scheffner ◽  
...  
Keyword(s):  
Calcium Carbonate ◽  
Sea Urchin ◽  
Self Assembly ◽  
Organic Matrix

scholarly journals Tyrosine nitration on calmodulin enhances calcium-dependent association and activation of nitric-oxide synthase

2019 ◽  
Vol 295 (8) ◽  
pp. 2203-2211 ◽  
Author(s):  
Joseph J. Porter ◽  
Hyo Sang Jang ◽  
Mohammad Mahfuzul Haque ◽  
Dennis J. Stuehr ◽  
Ryan A. Mehl
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Calcium Binding ◽  
Vascular Dysfunction ◽  
Post Translational Modification ◽  
Calcium Dependent ◽  
Genetic Code Expansion ◽  
Oxide Synthase ◽  
Endothelial Nitric Oxide

Production of reactive oxygen species caused by dysregulated endothelial nitric-oxide synthase (eNOS) activity is linked to vascular dysfunction. eNOS is a major target protein of the primary calcium-sensing protein calmodulin. Calmodulin is often modified by the main biomarker of nitroxidative stress, 3-nitrotyrosine (nitroTyr). Despite nitroTyr being an abundant post-translational modification on calmodulin, the mechanistic role of this modification in altering calmodulin function and eNOS activation has not been investigated. Here, using genetic code expansion to site-specifically nitrate calmodulin at its two tyrosine residues, we assessed the effects of these alterations on calcium binding by calmodulin and on binding and activation of eNOS. We found that nitroTyr–calmodulin retains affinity for eNOS under resting physiological calcium concentrations. Results from in vitro eNOS assays with calmodulin nitrated at Tyr-99 revealed that this nitration reduces nitric-oxide production and increases eNOS decoupling compared with WT calmodulin. In contrast, calmodulin nitrated at Tyr-138 produced more nitric oxide and did so more efficiently than WT calmodulin. These results indicate that the nitroTyr post-translational modification, like tyrosine phosphorylation, can impact calmodulin sensitivity for calcium and reveal Tyr site-specific gain or loss of functions for calmodulin-induced eNOS activation.

Effect of Sulfate Content of Biomacromolecules on the Crystallization of Calcium Carbonate

2001 ◽  
Vol 711 ◽  
Author(s):  
José I. Arias ◽  
Carolina Jure ◽  
Juan P. Wiff ◽  
María S. Fernández ◽  
Víctor Fuenzalida ◽  
...  
Keyword(s):  
Calcium Carbonate ◽  
Dermatan Sulfate ◽  
Keratan Sulfate ◽  
Organic Matrix ◽  
Sulfate Proteoglycan ◽  
Sulfate Content ◽  
Dermatan Sulfate Proteoglycan ◽  
Chicken Eggshell ◽  
Natural Composite

ABSTRACTNatural composite bioceramics such as bone, teeth, carapaces and shells contain organic and inorganic moieties, with the organic matrix components directly involved in the precise formation of these structures. We have previously shown that chicken eggshell contains two main sulfated polymers (proteoglycans), referred to as mammillan and ovoglycan which are involved in nucleation and growth of the eggshell calcite crystals. They differ on their anionic properties due to the carboxylate and sulfate content of their glycosaminoglycan component. Based on biological and biochemical evidences, the putative role of mammillan, a keratan sulfate proteoglycan, is in the nucleation of the first calcite crystals, while that of ovoglycan, a dermatan sulfate proteoglycan, is to regulate the growth and orientation of the later forming crystals of the chicken eggshell. In this communication, a systematic study of the influence of variable concentrations of glycosaminoglycans differing in their sulfation status on the morphology, size and number of calcium carbonate crystals after crystallization on microbridges from a calcium chloride solution under an atmosphere of ammonium carbonate at different pH is presented. Depending on the pH and concentration, the variation of sulfation status drastically changed the morphology, size and number of calcite crystals. The produced calcite particles with various morphologies are promising candidates for some novel materials with desirable shape- and texture-depending properties.

Calcium carbonate bio-precipitation in counter-diffusion systems using the soluble organic matrix from nacre and sea-urchin spine

2014 ◽  
Vol 26 (4) ◽  
pp. 523-535 ◽  
Author(s):  
Maria Sancho-Tomás ◽  
Simona Fermani ◽  
Jaime Gómez-Morales ◽  
Giuseppe Falini ◽  
Juan Manuel García-Ruiz
Keyword(s):  
Calcium Carbonate ◽  
Sea Urchin ◽  
Organic Matrix ◽  
Diffusion Systems ◽  
Sea Urchin Spine ◽  
Counter Diffusion

scholarly journals A pipeline for interrogating and engineering single-subunit oligosaccharyltransferases

2017 ◽  
Author(s):  
Thapakorn Jaroentomeechai ◽  
Xiaolu Zheng ◽  
Jasmine Hershewe ◽  
Jessica C. Stark ◽  
Michael C. Jewett ◽  
...  
Keyword(s):  
Protein Glycosylation ◽  
Covalent Attachment ◽  
Post Translational Modification ◽  
Characterization Methods ◽  
In Vitro Characterization ◽  
Domains Of Life ◽  
Higher Eukaryotes ◽  
Single Subunit

Asparagine-linked (N-linked) protein glycosylation is one of the most abundant types of post-translational modification, occurring in all domains of life. The central enzyme in N-linked glycosylation is the oligosaccharyltransferase (OST), which catalyzes the covalent attachment of preassembled glycans to specific asparagine residues in target proteins. Whereas in higher eukaryotes the OST is comprised of eight different membrane proteins of which the catalytic subunit is STT3, in kinetoplastids and prokaryotes the OST is a monomeric enzyme bearing homology to STT3. Given their relative simplicity, these single-subunit OSTs (ssOSTs) have emerged as important targets for mechanistic dissection of poorly understood aspects of N-glycosylation and at the same time hold great potential for the biosynthesis of custom glycoproteins. To take advantage of this utility, this chapter describes a multipronged approach for studying and engineering ssOSTs that integrates in vivo screening technology with in vitro characterization methods, thereby creating a versatile and readily-adaptable pipeline for virtually any ssOST of interest.

Biotechnological Mineral Composites via Vaterite Precursors

2012 ◽  
Vol 1465 ◽  
Author(s):  
E. Weber ◽  
C. Guth ◽  
M. Eder ◽  
P. Bauer ◽  
E. Arzt ◽  
...  
Keyword(s):  
Calcium Carbonate ◽  
Organic Molecules ◽  
Organic Matrix ◽  
Stable Form ◽  
Ambient Conditions ◽  
Calcium Carbonate Precipitation ◽  
Mineral Phases ◽  
Mineralization Processes ◽  
Co Precipitation

ABSTRACTVaterite is one of the thermodynamically less stable polymorphs of calcium carbonate. Under ambient conditions it transforms into calcite, the most stable form of calcium carbonate. Organisms are able to stabilize minerals such as vaterite by means of organic molecules. The exact mechanisms how biomineralization proteins interact with metastable mineral phases are, however, less well understood. Many in vitro studies were performed using calcite as a model system. A deeper understanding of the interaction of organic molecules with metastable mineral phases would make them useful as a tool to control mineralization processes in vitro. In this study, we report on the co-precipitation of a natively soluble histidine-tagged GFP (green fluorecent protein) with a metastable vaterite phase and the subsequent insolubility of the fluorescent organic matrix in a 30μl calcium carbonate precipitation assay. The intrinsic fluorescence of GFP is conserved during the interaction with the mineral phase, indicating proper folding even in the insoluble state. This experiment can be extended to obtain deeper insights into some mechanistic models of biomineralization proteins by tracking native and modified GFP proteins microscopically during various stages of mineral precipitation and dissolution.

scholarly journals A calcium-binding, asparagine-linked oligosaccharide is involved in skeleton formation in the sea urchin embryo.

1989 ◽  
Vol 109 (3) ◽  
pp. 1289-1299 ◽  
Author(s):  
M C Farach-Carson ◽  
D D Carson ◽  
J L Collier ◽  
W J Lennarz ◽  
H R Park ◽  
...  
Keyword(s):  
Sea Urchin ◽  
Calcium Binding ◽  
Divalent Cations ◽  
Surface Protein ◽  
Oligosaccharide Chain ◽  
Oligosaccharide Moiety ◽  
Mesenchyme Cells ◽  
Skeleton Formation ◽  
Primary Mesenchyme Cells

We have previously identified a 130-kD cell surface protein that is involved in calcium uptake and skeleton formation by gastrula stage embryos of the sea urchin Strongylocentrotus purpuratus (Carson et al., 1985. Cell. 41:639-648). A monoclonal antibody designated mAb 1223 specifically recognizes the 130-kD protein and inhibits Ca+2 uptake and growth of the CaCO3 spicules produced by embryonic primary mesenchyme cells cultured in vitro. In this report, we demonstrate that the epitope recognized by mAb 1223 is located on an anionic, asparagine-linked oligosaccharide chain on the 130-kD protein. Combined enzymatic and chemical treatments indicate that the 1223 oligosaccharide contains fucose and sialic acid that is likely to be O-acetylated. Moreover, we show that the oligosaccharide chain containing the 1223 epitope specifically binds divalent cations, including Ca+2. We propose that one function of this negatively charged oligosaccharide moiety on the surfaces of primary mesenchyme cells is to facilitate binding and sequestration of Ca+2 ions from the blastocoelic fluid before internalization and subsequent deposition into the growing CaCO3 skeleton.

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