Modulation of host lipid metabolism by virus infection leads to exoskeleton damage in shrimp.

The arthropod exoskeleton provides protection and support and is vital for survival and adaption. The integrity and mechanical properties of the exoskeleton are often impaired after pathogenic infection; however, the detailed mechanism by which infection affects the exoskeleton remains largely unknown. Here, we report that the damage to the shrimp exoskeleton is caused by modulation of host lipid profiles after infection with white spot syndrome virus (WSSV). WSSV infection disrupts the mechanical performance of the exoskeleton by inducing the expression of a chitinase (Chi2) in the sub-cuticle epidermis and decreasing the cuticle chitin content. The induction of Chi2 expression is mediated by a nuclear receptor that can be activated by certain enriched long-chain saturated fatty acids after infection. The damage to the exoskeleton, an aftereffect of the induction of host lipogenesis by WSSV, significantly impairs the motor ability of shrimp. Blocking the WSSV-caused lipogenesis restored the mechanical performance of the cuticle and improved the motor ability of infected shrimp. Therefore, this study reveals a mechanism by which WSSV infection modulates shrimp internal metabolism resulting in phenotypic impairment, and provides new insights into the interactions between the arthropod host and virus.

The small GTPase Cdc42 regulates shell field morphogenesis in a gastropod mollusk.

In most mollusks (conchiferans), the early tissue responsible for shell development, namely, the shell field, shows a common process of invagination during morphogenesis. Moreover, lines of evidence indicated that shell field invagination is not an independent event, but an integrated output reflecting the overall state of shell field morphogenesis. Nevertheless, the underlying mechanisms of this conserved process remain largely unknown. We previously found that actomyosin networks (regularly organized filamentous actin (F-actin) and myosin) may play essential roles in this process by revealing the evident aggregation of F-actin in the invaginated region and demonstrating that nonmuscle myosin II (NM II) is required for invagination in the gastropod Lottia peitaihoensis (= Lottia goshimai). Here, we investigated the roles of the Rho family of small GTPases (RhoA, Rac1, and Cdc42) to explore the upstream regulators of actomyosin networks. Functional assays using small molecule inhibitors suggested that Cdc42 modulates key events of shell field morphogenesis, including invagination and cell rearrangements, while the roles of RhoA and Rac1 may be nonspecific or negligible. Further investigations revealed that the Cdc42 protein was concentrated on the apical side of shell field cells and colocalized with F-actin aggregation. The aggregation of these two molecules could be prevented by treatment with Cdc42 inhibitors. These findings suggest a possible regulatory cascade of shell field morphogenesis in which Cdc42 recruits F-actin (actomyosin networks) on the apical side of shell field cells, which then generates resultant mechanical forces that mediate correct shell field morphogenesis (cell shape changes, invagination and cell rearrangement). Our results emphasize the roles of the cytoskeleton in early shell development and provide new insights into molluscan shell evolution.

Diversification of von Willebrand Factor A and Chitin-Binding Domains in Pif/BMSPs Among Mollusks.

Pif is a shell matrix protein (SMP) identified in the nacreous layer of Pinctada fucata (Pfu) comprised two proteins, Pif97 and Pif 80. Pif97 contains a von Willebrand factor A (VWA) and chitin-binding domains, whereas Pif80 can bind calcium carbonate crystals. The VWA domain is conserved in the SMPs of various mollusk species; however, their phylogenetic relationship remains obscure. Furthermore, although the VWA domain participates in protein-protein interactions, its role in shell formation has not been established. Accordingly, in the current study, we investigate the phylogenetic relationship between PfuPif and other VWA domain-containing proteins in major mollusk species. The shell-related proteins containing VWA domains formed a large clade (the Pif/BMSP family) and were classified into eight subfamilies with unique sequential features, expression patterns, and taxa diversity. Furthermore, a pull-down assay using recombinant proteins containing the VWA domain of PfuPif 97 revealed that the VWA domain interacts with five nacreous layer-related SMPs of P. fucata, including Pif 80 and nacrein. Collectively, these results suggest that the VWA domain is important in the formation of organic complexes and participates in shell mineralisation.

Structural and Functional Analysis of the Amorphous Calcium Carbonate-Binding Protein Paramyosin in the Shell of the Pearl Oyster, Pinctada fucata.

Amorphous calcium carbonate (ACC) is an important precursor phase for the formation of aragonite crystals in the shells of Pinctada fucata. To identify the ACC-binding protein in the inner aragonite layer of the shell, extracts from the shell were used in the ACC-binding experiments. Semiquantitative analyses using liquid chromatography-mass spectrometry revealed that paramyosin was strongly associated with ACC in the shell. We discovered that paramyosin, a major component of the adductor muscle, was included in the myostracum, which is the microstructure of the shell attached to the adductor muscle. Purified paramyosin accumulates calcium carbonate and induces the prism structure of aragonite crystals, which is related to the morphology of prism aragonite crystals in the myostracum. Nuclear magnetic resonance measurements revealed that the Glu-rich region was bound to ACC. Activity of the Glu-rich region was stronger than that of the Asp-rich region. These results suggest that paramyosin in the adductor muscle is involved in the formation of aragonite prisms in the myostracum.

Hyperspectral interference tomography of nacre.

Structural characterization of biologically formed materials is essential for understanding biological phenomena and their enviro-nment, and for generating new bio-inspired engineering concepts. For example, nacre-the inner lining of some mollusk shells-encodes local environmental conditions throughout its formation and has exceptional strength due to its nanoscale brick-and-mortar structure. This layered structure, comprising alternating transparent aragonite (CaCO3) tablets and thinner organic polymer layers, also results in stunning interference colors. Existing methods of structural characterization of nacre rely on some form of cross-sectional analysis, such as scanning or transmission electron microscopy or polarization-dependent imaging contrast (PIC) mapping. However, these techniques are destructive and too time- and resource-intensive to analyze large sample areas. Here, we present an all-optical, rapid, and nondestructive imaging technique-hyperspectral interference tomography (HIT)-to spatially map the structural parameters of nacre and other disordered layered materials. We combined hyperspectral imaging with optical-interference modeling to infer the mean tablet thickness and its disorder in nacre across entire mollusk shells from red and rainbow abalone (Haliotis rufescens and Haliotis iris) at various stages of development. We observed that in red abalone, unexpectedly, nacre tablet thickness decreases with age of the mollusk, despite roughly similar appearance of nacre at all ages and positions in the shell. Our rapid, inexpensive, and nondestructive method can be readily applied to in-field studies.

A modern scleractinian coral with a two-component calcite-aragonite skeleton.

One of the most conserved traits in the evolution of biomineralizing organisms is the taxon-specific selection of skeletal minerals. All modern scleractinian corals are thought to produce skeletons exclusively of the calcium-carbonate polymorph aragonite. Despite strong fluctuations in ocean chemistry (notably the Mg/Ca ratio), this feature is believed to be conserved throughout the coral fossil record, spanning more than 240 million years. Only one example, the Cretaceous scleractinian coral Coelosmilia (ca. 70 to 65 Ma), is thought to have produced a calcitic skeleton. Here, we report that the modern asymbiotic scleractinian coral Paraconotrochus antarcticus living in the Southern Ocean forms a two-component carbonate skeleton, with an inner structure made of high-Mg calcite and an outer structure composed of aragonite. P. antarcticus and Cretaceous Coelosmilia skeletons share a unique microstructure indicating a close phylogenetic relationship, consistent with the early divergence of P. antarcticus within the Vacatina (i.e., Robusta) clade, estimated to have occurred in the Mesozoic (ca. 116 Mya). Scleractinian corals thus join the group of marine organisms capable of forming bimineralic structures, which requires a highly controlled biomineralization mechanism; this capability dates back at least 100 My. Due to its relatively prolonged isolation, the Southern Ocean stands out as a repository for extant marine organisms with ancient traits.

Direct control of shell regeneration by the mantle tissue in the pearl oyster Pinctada fucata.

Molluscs rapidly repair the damaged shells to prevent further injury, which is vital for their survival after physical or biological aggression. However, it remains unclear how this process is precisely controlled. In this study, we applied scanning electronic microscope and histochemical analysis to examine the detailed shell regeneration process in the pearl oyster Pinctada fucata. It was found that the shell damage caused the mantle tissue to retract, which resulted in relocation of the partitioned mantle zones with respect to their correspondingly secreting shell layers. As a result, the relocated mantle tissue dramatically altered the shell morphology by initiating de novo precipitation of prismatic layers on the former nacreous layers, leading to the formation of sandwich-like "prism-nacre-prism-nacre" structure. Real-time PCR revealed the up-regulation of the shell matrix protein genes, which was confirmed by the thermal gravimetric analysis of the newly formed shell. The increased matrix secretion might have led to the change of CaCO3 precipitation dynamics which altered the mineral morphology and promoted shell formation. Taken together, our study revealed the close relationship between the physiological activities of the mantle tissue and the morphological change of the regenerated shells.

Evolution of Epidermal Growth Factor (EGF)-like and Zona Pellucida Domains Containing Shell Matrix Proteins in Mollusks.

Several types of shell matrix proteins (SMPs) have been identified in molluskan shells. Their diversity is the consequence of various molecular processes, including domain shuffling and gene duplication. However, the evolutionary origin of most SMPs remains unclear. In this study, we investigated the evolutionary process EGF-like and zona pellucida (ZP) domains containing SMPs. Two types of the proteins (EGF-like protein (EGFL) and EGF-like and ZP domains containing protein (EGFZP)) were found in the pearl oyster, Pinctada fucata. In contrast, only EGFZP was identified in the gastropods. Phylogenetic analysis and genomic arrangement studies showed that EGFL and EGFZP formed a clade in bivalves, and their encoding genes were localized in tandem repeats on the same scaffold. In P. fucata, EGFL genes were expressed in the outer part of mantle epithelial cells are related to the calcitic shell formation. However, in both P. fucata and the limpet Nipponacmea fuscoviridis, EGFZP genes were expressed in the inner part of the mantle epithelial cells are related to aragonitic shell formation. Furthermore, our analysis showed that in P. fucata, the ZP domain interacts with eight SMPs that have various functions in the nacreous shell mineralization. The data suggest that the ZP domain can interact with other SMPs, and EGFL evolution in pterimorph bivalves represents an example of neo-functionalization that involves the acquisition of a novel protein through gene duplication.

A Bivalve Biomineralization Toolbox.

Mollusc shells are a result of the deposition of crystalline and amorphous calcite catalyzed by enzymes and shell matrix proteins (SMP). Developing a detailed understanding of bivalve mollusc biomineralization pathways is complicated not only by the multiplicity of shell forms and microstructures in this class, but also by the evolution of associated proteins by domain co-option and domain shuffling. In spite of this, a minimal biomineralization toolbox comprising proteins and protein domains critical for shell production across species has been identified. Using a matched pair design to reduce experimental noise from inter-individual variation, combined with damage-repair experiments and a database of biomineralization SMPs derived from published works, proteins were identified that are likely to be involved in shell calcification. Eighteen new, shared proteins likely to be involved in the processes related to the calcification of shells were identified by the analysis of genes expressed during repair in Crassostrea gigas, Mytilus edulis, and Pecten maximus. Genes involved in ion transport were also identified as potentially involved in calcification either via the maintenance of cell acid-base balance or transport of critical ions to the extrapallial space, the site of shell assembly. These data expand the number of candidate biomineralization proteins in bivalve molluscs for future functional studies and define a minimal functional protein domain set required to produce solid microstructures from soluble calcium carbonate. This is important for understanding molluscan shell evolution, the likely impacts of environmental change on biomineralization processes, materials science, and biomimicry research.

Identification and characterization of a mosquito-specific eggshell organizing factor in Aedes aegypti mosquitoes.

Mosquito-borne diseases are responsible for several million human deaths annually around the world. One approach to controlling mosquito populations is to disrupt molecular processes or antagonize novel metabolic targets required for the production of viable eggs. To this end, we focused our efforts on identifying proteins required for completion of embryonic development that are mosquito selective and represent potential targets for vector control. We performed bioinformatic analyses to identify putative protein-coding sequences that are specific to mosquito genomes. Systematic RNA interference (RNAi) screening of 40 mosquito-specific genes was performed by injecting double-stranded RNA (dsRNA) into female Aedes aegypti mosquitoes. This experimental approach led to the identification of eggshell organizing factor 1 (EOF1, AAEL012336), which plays an essential role in the formation and melanization of the eggshell. Eggs deposited by EOF1-deficient mosquitoes have nonmelanized fragile eggshells, and all embryos are nonviable. Scanning electron microscopy (SEM) analysis identified that exochorionic eggshell structures are strongly affected in EOF1-deficient mosquitoes. EOF1 is a potential novel target, to our knowledge, for exploring the identification and development of mosquito-selective and biosafe small-molecule inhibitors.

Biomineralization by particle attachment in early animals.

Crystallization by particle attachment (CPA) of amorphous precursors has been demonstrated in modern biomineralized skeletons across a broad phylogenetic range of animals. Precisely the same precursors, hydrated (ACC-H2O) and anhydrous calcium carbonate (ACC), have been observed spectromicroscopically in echinoderms, mollusks, and cnidarians, phyla drawn from the 3 major clades of eumetazoans. Scanning electron microscopy (SEM) here also shows evidence of CPA in tunicate chordates. This is surprising, as species in these clades have no common ancestor that formed a mineralized skeleton and appear to have evolved carbonate biomineralization independently millions of years after their late Neoproterozoic divergence. Here we correlate the occurrence of CPA from ACC precursor particles with nanoparticulate fabric and then use the latter to investigate the antiquity of the former. SEM images of early biominerals from Ediacaran and Cambrian shelly fossils show that these early calcifiers used attachment of ACC particles to form their biominerals. The convergent evolution of biomineral CPA may have been dictated by the same thermodynamics and kinetics as we observe today.

An acidic protein, Hf15, from Haliotis fulgens involved in biomineralization.

Biomineralization leads to the hardening of mineralized materials, such as the shell of Mollusk, to fulfill a wide range of functions, such as (but not limited to) skeletal support, protection of the soft tissues, navigation, etc. The study of the proteins responsible for this process, shell matrix proteins (SMPs), allows addressing questions related to structure-function relationship and to the mechanism of mineral formation, which is limited in gastropod species. In this study, a low molecular weight protein was isolated from the insoluble fraction after decalcification with acetic acid of the shell of Haliotis fulgens and, named Hf15. The unglycosylated protein has a theoretical molecular weight of 15 kDa, it possesses calcium and chiting binding properties. Hf15 can precipitate calcium carbonate in vitro in presence of different salts. Analysis by LC-MS of the five peptide sequences of Hf15 generated by trypsinization revealed that two peptides displayed homology to an uncharacterized protein 3-like from Haliotis rufescens, Haliotis asinia and H. sorenseni. The results obtained indicated that Hf15 is a novel SMP involved in shell mineralization in Haliotis fulgens.

Identification and Characterization of the Larval Settlement Pheromone Protein Components in Adult Shells of Crassostrea gigas: A Novel Function of Shell Matrix Proteins.

The global decline of natural oyster populations emphasizes the need to improve our understanding of their biology. Understanding the role of chemical cues from conspecifics on how oysters occupy appropriate substrata is crucial to learning about their evolution, population dynamics, and chemical communication. Here, a novel role of a macromolecular assembly of shell matrix proteins which act as Crassostrea gigas Settlement Pheromone Protein Components in adult shells is demonstrated as the biological cue responsible for gregarious settlement on conspecifics. A bioassay-guided fractionation approach aided by biochemical and molecular analyses reveals that Gigasin-6 isoform X1 and/or X2 isolated from adult shells is the major inducing cue for larval settlement and may also play a role in postlarva-larva settlement interactions. Other isolated Stains-all-stainable acidic proteins may function as a co-factor and a scaffold/structural framework for other matrix proteins to anchor within this assembly and provide protection. Notably, conspecific cue-mediated larval settlement induction in C. gigas presents a complex system that requires an interplay of different glycans, disulfide bonds, amino acid groups, and phosphorylation crosstalk for recognition. These results may find application in the development of oyster aquacultures which could help recover declining marine species and as targets of anti-fouling agents.

Evolution and biomineralization of pteropod shells.

Shelled pteropods, known as sea butterflies, are a group of small gastropods that spend their entire lives swimming and drifting in the open ocean. They build thin shells of aragonite, a metastable polymorph of calcium carbonate. Pteropod shells have been shown to experience dissolution and reduced thickness with a decrease in pH and therefore represent valuable bioindicators to monitor the impacts of ocean acidification. Over the past decades, several studies have highlighted the striking diversity of shell microstructures in pteropods, with exceptional mechanical properties, but their evolution and future in acidified waters remains uncertain. Here, we revisit the body-of-work on pteropod biomineralization, focusing on shell microstructures and their evolution. The evolutionary history of pteropods was recently resolved, and thus it is timely to examine their shell microstructures in such context. We analyse new images of shells from fossils and recent species providing a comprehensive overview of their structural diversity. Pteropod shells are made of the crossed lamellar and prismatic microstructures common in molluscs, but also of curved nanofibers which are proposed to form a helical three-dimensional structure. Our analyses suggest that the curved fibres emerged before the split between coiled and uncoiled pteropods and that they form incomplete to multiple helical turns. The curved fibres are seen as an important trait in the adaptation to a planktonic lifestyle, giving maximum strength and flexibility to the pteropod thin and lightweight shells. Finally, we also elucidate on the candidate biomineralization genes underpinning the shell diversity in these important indicators of ocean health.

Illumination enhances the protein abundance of sarcoplasmic reticulum Ca2+-ATPases-like transporter in the ctenidium and whitish inner mantle of the giant clam, Tridacna squamosa, to augment exogenous Ca2+ uptake and shell formation, respectively.

The fluted giant clam, Tridacna squamosa, can perform light-enhanced shell formation, aided by its symbiotic dinoflagellates (Symbiodinium, Cladocopium, Durusdinium), which are able to donate organic nutrients to the host. During light-enhanced shell formation, increased Ca2+ transport from the hemolymph through the shell-facing epithelium of the inner mantle to the extrapallial fluid, where calcification occurs, is necessary. Additionally, there must be increased absorption of exogenous Ca2+ from the surrounding seawater, across the epithelial cells of the ctenidium (gill) into the hemolymph, to supply sufficient Ca2+ for light-enhanced shell formation. When Ca2+ moves across these epithelial cells, the low intracellular Ca2+ concentration must be maintained. Sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) regulates the intracellular Ca2+ concentration by pumping Ca2+ into the sarcoplasmic/endoplasmic reticulum (SR/ER) and Golgi apparatus. Indeed, the ctenidium and inner mantle of T. squamosa, expressed a homolog of SERCA (SERCA-like transporter) that consists of 3009 bp, encoding 1002 amino acids of 110.6 kDa. SERCA-like-immunolabeling was non-uniform in the cytoplasm of epithelial cells of ctenidial filaments, and that of the shell-facing epithelial cells of the inner mantle. Importantly, the protein abundance of SERCA-like increased significantly in the ctenidium and the inner mantle of T. squamosa after 12 h and 6 h, respectively, of light exposure. This would increase the capacity of pumping Ca2+ into the endoplasmic reticulum and avert a possible surge in the cytosolic Ca2+ concentration in epithelial cells of the ctenidial filaments during light-enhanced Ca2+ absorption, and in cells of the shell-facing epithelium of the inner mantle during light-enhanced shell formation.

Transcriptome analysis reveals the pigmentation related genes in four different shell color strains of the Manila clam Ruditapes philippinarum.

Ruditapes philippinarum is an important marine bivalve species. In this study, we conducted the RNA-seq of four different shell color strains of the R. philippinarum and investigated the analysis of the differential expression patterns of specific genes associated with pigmentation. The maximum different genes was 13 between WZ vs O, WZ vs W and WZ vs O have same numbers of different genes, was 5, Z vs W has 4 genes of 18 DEGs, W vs O just have two DEGs, while there is no DEGs between WZ vs Z. The synthesis of melanin plays important roles in the pigmentation of the shell and is closely related to the formation of the surface pattern. We speculate the possible involvement of porphyrin and chlorophyll metabolism combined with calcium signaling pathway in shell color determination. This study sheds light on the pigmentation and coloration mechanism of the Manila clam.

Global Analysis of Transcriptome and Translatome Revealed That Coordinated WNT and FGF Regulate the Carapacial Ridge Development of Chinese Soft-Shell Turtle.

The turtle carapace is composed of severely deformed fused dorsal vertebrae, ribs, and bone plates. In particular, the lateral growth in the superficial layer of turtle ribs in the dorsal trunk causes an encapsulation of the scapula and pelvis. The recent study suggested that the carapacial ridge (CR) is a new model of epithelial-mesenchymal transition which is essential for the arrangement of the ribs. Therefore, it is necessary to explore the regulatory mechanism of carapacial ridge development to analyze the formation of the turtle shell. However, the current understanding of the regulatory network underlying turtle carapacial ridge development is poor due to the lack of both systematic gene screening at different carapacial ridge development stages and gene function verification studies. In this study, we obtained genome-wide gene transcription and gene translation profiles using RNA sequencing and ribosome nascent-chain complex mRNA sequencing from carapacial ridge tissues of Chinese soft-shell turtle at different development stages. A correlation analysis of the transcriptome and translatome revealed that there were 129, 670, and 135 codifferentially expressed genes, including homodirection and opposite-direction differentially expressed genes, among three comparison groups, respectively. The pathway enrichment analysis of codifferentially expressed genes from the Kyoto Encyclopedia of Genes and Genomes showed dynamic changes in signaling pathways involved in carapacial ridge development. Especially, the results revealed that the Wnt signaling pathway and MAPK signaling pathway may play important roles in turtle carapacial ridge development. In addition, Wnt and Fgf were expressed during the carapacial ridge development. Furthermore, we discovered that Wnt5a regulated carapacial ridge development through the Wnt5a/JNK pathway. Therefore, our studies uncover that the morphogenesis of the turtle carapace might function through the co-operation between conserved WNT and FGF signaling pathways. Consequently, our findings revealed the dynamic signaling pathways acting on the carapacial ridge development of Chinese soft-shell turtle and provided new insights into uncover the molecular mechanism underlying turtle shell morphogenesis.

Porous Biphasic Calcium Phosphate Granules from Oyster Shell Promote the Differentiation of Induced Pluripotent Stem Cells.

Oyster shells are rich in calcium, and thus, the potential use of waste shells is in the production of calcium phosphate (CaP) minerals for osteopathic biomedical applications, such as scaffolds for bone regeneration. Implanted scaffolds should stimulate the differentiation of induced pluripotent stem cells (iPSCs) into osteoblasts. In this study, oyster shells were used to produce nano-grade hydroxyapatite (HA) powder by the liquid-phase precipitation. Then, biphasic CaP (BCP) bioceramics with two different phase ratios were obtained by the foaming of HA nanopowders and sintering by two different two-stage heat treatment processes. The different sintering conditions yielded differences in structure and morphology of the BCPs, as determined by scanning electron microscopy (SEM), X-ray diffraction (XRD), and Brunauer-Emmett-Teller (BET) surface area analysis. We then set out to determine which of these materials were most biocompatible, by co-culturing with iPSCs and examining the gene expression in molecular pathways involved in self-renewal and differentiation of iPSCs. We found that sintering for a shorter time at higher temperatures gave higher expression levels of markers for proliferation and (early) differentiation of the osteoblast. The differences in biocompatibility may be related to a more hierarchical pore structure (micropores within macropores) obtained with briefer, high-temperature sintering.

Application of Nano-Hydroxyapatite Derived from Oyster Shell in Fabricating Superhydrophobic Sponge for Efficient Oil/Water Separation.

The exploration of nonhazardous nanoparticles to fabricate a template-driven superhydrophobic surface is of great ecological importance for oil/water separation in practice. In this work, nano-hydroxyapatite (nano-HAp) with good biocompatibility was easily developed from discarded oyster shells and well incorporated with polydimethylsiloxane (PDMS) to create a superhydrophobic surface on a polyurethane (PU) sponge using a facile solution-immersion method. The obtained nano-HAp coated PU (nano-HAp/PU) sponge exhibited both excellent oil/water selectivity with water contact angles of over 150° and higher absorption capacity for various organic solvents and oils than the original PU sponge, which can be assigned to the nano-HAp coating surface with rough microstructures. Moreover, the superhydrophobic nano-HAp/PU sponge was found to be mechanically stable with no obvious decrease of oil recovery capacity from water in 10 cycles. This work presented that the oyster shell could be a promising alternative to superhydrophobic coatings, which was not only beneficial to oil-containing wastewater treatment, but also favorable for sustainable aquaculture.

A Complicated Relationship: Glycosylation, Ca(II), and Primary Sequence Affect the Interactions and Kinetics between Two Model Mollusk Shell Intracrystalline Nacre Proteins.

The formation of the mollusk shell requires the participation of proteins, many of which may be interactive with one another. We examined a model protein pair system from the mollusk Haliotis rufescens, wherein we probed the interactions between recombinant forms of two major nacre layer proteins, AP7, and the glycoprotein, AP24. Here, the focus was on the impact that the AP24 glycosylation and primary sequence had on AP24-AP7 binding. We find that both the glycosylated and nonglycosylated variants of AP24 bound to AP7 but with different quantities, kinetics, and internal rearrangements. Moreover, the binding of AP7 with nonglycosylated and glycosylated AP24 was found to be Ca(II)-dependent and -independent, respectively. Yet both variants of AP24 combine with AP7 to form hybrid hydrogel particles that are similar in their physical properties. Thus, AP7 and AP24 protein sequences are interactive and form hydrogels, but the interactions are tuned by glycosylation and Ca(II). These features may have an impact on the nacre matrix formation.