Silk-based fibres vs collagen gel: major organic components of bivalve shells
thesisposted on 28.03.2022, 16:13 by Oluwatoosin Bunmi Adebayo Agbaje
Biominerals are biogenic hierarchical nanocomposite materials consisting of mineral phases, such as calcite, aragonite or vaterite, and intimately intergrown with the organic macromolecules, including proteins, polysaccharides and lipids. There is a large diversity among biominerals, each with its structural motifs, inorganic crystal formation - shape, and micro- or macroscopic properties. For instance, shells of mollusc are formed with different ratios of inorganic-organic materials and different structural motifs resulting in a large variety of calcareous biocomposites with material properties outperforming those of their synthetic counterparts. Amongst all different shell microstructures, nacre is the most studied to date, while comparable knowledge is lacking for non-nacre shell structures such as homogeneous and crossed-lamellar structures. In this work both, nacre and non-nacre structures are investigated and compared for commonalities and differences. The aims are to characterise inorganic-associated macromolecules in shells of different bivalve species with different microstructures and to study the interface and the interactions between mineral and biomolecules. To achieve these aims, shells of 12 species from 7 molluscan families are studied. The calcareous shells are grouped into nacroprismatic (Hyriopsis cumingii, Cucumerunio novaehollandiae, Alathyria jacksoni, Pinctada maxima, Pinctada fucata martensii, Diplodon chilensis patagonicus), homogeneous (Arctica islandica) and crossed-lamellar (Tridacna gigas, Tridacna derasa, Fulvia tenuicostata, Callista disrupta, Callista kingii) based on their shell microstructures. Except for Pinctada shells that consist both calcitic and aragonitic layers, all shells are entirely aragonitic. A common phenomenon exists between the compositions of organics present in the nacreous layer of the studied nacroprismatic shells. Thermogravimetric analysis exhibits a total amount of organics in the range of 3.14 - 4.13 wt%. The amino acid composition reveals a high amount of glycine and alanine (ca 54% in total) and consists of moderate polar amino acids - aspartate and glutamate (~ 16% in total). Solid-State Nuclear Magnetic Resonance (NMR) spectroscopy using Cross-Polarization Magic-Angle Spinning (CP-MAS) was employed and revealed silk-based fibres as the primary organic framework in nacre. As shown by electron microscopy, extensive hydrogen-bonded β-sheet nanocrystals are well-organised along and within a semi-amorphous protein (in analogy to the spider dragline silk and Bombyx mori (cocoon) silk). While it may certainly be beneficial to explore available sugar moieties in the organic macromolecules, these appear to be present in concentrations not necessarily significantly higher than, for instance, phosphates post-translationally intercalated into the silk fibroins as discovered in this thesis. Moreover, the sugar moiety is probably N-glycosylated and is significantly different from that found in non-nacre microstructures. The composition of shells with homogeneous and crossed lamellar structures are significantly distinct from that of nacre. Though the calcareous microstructures of non-nacre shells are divergent, the amounts of organics are comparable (1.6 wt% by TGA compared to 3-4 wt% for nacreous shells). Glycine, aspartate and proline (~40% in total) are the prominent residues. Different to nacreous and homogeneous shells, the decalcification method failed to extract and concentrate the insoluble organic moiety in crossed lamellar shells. The macromolecular content of homogeneous shells is not suggestive of a silk-like composition as in nacreous shells, instead, consists mainly of collagen gel. A remarkable structural feature of the biopolymer is a three-dimensional polygonal meshwork of type IV collagen gel aggregates, which, in turn, probably constitutes an O-glycosylated sugar moiety. The ultrastructure of crossed lamellar shells was characterised with high-resolution electron microscopy. The imaging displays the fine structure of nanogranular particles that are composites of inorganic and organic matrices, arranged either in nanometer-sized laths or a polycrystalline fibre-like fabric. In the freshwater and marine bivalve shells studied in this project, the major biological scaffolding in nacre consist of silk-based fibres, while homogeneous (and probably crossed lamellar) shows proteinaceous content that is different from a silk-like biopolymer, instead, consists primarily of collagen gel. These findings are new and contrast with the long-standing view that chitin is the most important ingredient of the organic matrix in mollusc shells. Here, glucosamine (the monomer which upon acetylation is the basis of chitin) was found to represent only a very minor part of the organic moiety. Furthermore, the distinctly different biopolymers in nacre versus non-nacre shells found in this work suggests that the composition of the insoluble organic matrix differs depending on species, similarly to that of the soluble organic matrix, which is well-known in the literature. As the chemical palette available to form a silk-based fibre and collagen gel include many building blocks, namely the 20 naturally occurring amino acids, this work presumes that it is worth reconsidering the relative importance of the available biopolymers that assist in providing templating for inorganic minerals. While the insoluble organic moiety in nacre (Hyriopsis cumingii, Cucumerunio novaehollandiae, Alathyria jacksoni, Pinctada maxima) consists primarily of silk-based fibres, and thus serve as structural proteins, that of homogeneous (and probably crossed lamellar) shells constitute mainly of collagen IV gel. Moreover, the presence and the structures of sugar moieties glucosamine and mannose for nacre; glucosamine, galactosamine and galactose for non-nacre shells support the importance of saccharides in composite biominerals, however, make an interesting contrast to the abundance of insoluble saccharides proposed in the literature.