Characterization of a pseudohemocyanin gene (PtPhc1) and its immunity function in response to Vibrio parahaemolyticus infection in the swimming crab Portunus trituberculatus.

Pseudohemocyanin is a member of the hemocyanin superfamily, but little research is available on its function in immunology. In this study, a Portunus trituberculatus pseudohemocyanin gene, named PtPhc1, was obtained by gene cloning. The PtPhc1 cDNA was 2312 bp in length, encoding 684 amino acids while exhibiting a characteristic hemocyanin structural domain. Tissue expression analysis revealed ubiquitous expression of PtPhc1 across all tissues, with the highest level of expression observed in the hepatopancreas. The expression pattern of PtPhc1 in response to Vibrio parahaemolyticus infection was clarified using RT-qPCR in swimming crabs. Notably, the expression peaked at 24 h, and increased 1435-fold compared to the control group in the hepatopancreas. While the expression level reached the maximum value at 72 h, which was 3.24 times higher than that of the control group in hemocytes. Remarkably, the reduction in PtPhc1 expression led to a noteworthy 30% increase in the mortality rate of P. trituberculatus when exposed to V. parahaemolyticus. In addition, in vitro bacterial inhibition assays exhibited a dose-dependent suppression of bacterial proliferation by recombinant PtPhc1 protein, with a notable inhibition rate of 48.33% against V. parahaemolyticus at a concentration of 0.03 mg/mL. To the best of our knowledge, the results establish the function of pseudohaemocyanin in immunity for the first time, contributing to a deeper comprehension of innate immune regulatory mechanisms in aquatic organisms and advancing strategies for disease-resistant breeding.

Modulation of SREBP Expression and Fatty Acid Levels by Bacteria-Induced ER Stress Is Mediated by Hemocyanin in Penaeid Shrimp.

Many environmental and pathogenic insults induce endoplasmic reticulum (ER) stress in animals, especially in aquatic ecosystems, where these factors are crucial for life. In penaeid shrimp, pathogens and environmental stressors induce hemocyanin expression, but the involvement of hemocyanin in ER stress response is unknown. We demonstrate that in response to pathogenic bacteria (Vibrio parahaemolyticus and Streptococcus iniae), hemocyanin, ER stress proteins (Bip, Xbp1s, and Chop), and sterol regulatory element binding protein (SREBP) are induced to alter fatty acid levels in Penaeus vannamei. Interestingly, hemocyanin interacts with ER stress proteins to modulate SREBP expression, while ER stress inhibition with 4-Phenylbutyric acid or hemocyanin knockdown attenuates the expression of ER stress proteins, SREBP, and fatty acid levels. Contrarily, hemocyanin knockdown followed by tunicamycin treatment (ER stress activator) increased their expression. Thus, hemocyanin mediates ER stress during pathogen challenge, which consequently modulates SREBP to regulate the expression of downstream lipogenic genes and fatty acid levels. Our findings reveal a novel mechanism employed by penaeid shrimp to counteract pathogen-induced ER stress.

Sizing Up the Onychophoran Genome: Repeats, Introns, and Gene Family Expansion Contribute to Genome Gigantism in Epiperipatus broadwayi.

Genome assemblies are growing at an exponential rate and have proved indispensable for studying evolution but the effort has been biased toward vertebrates and arthropods with a particular focus on insects. Onychophora or velvet worms are an ancient group of cryptic, soil dwelling worms noted for their unique mode of prey capture, biogeographic patterns, and diversity of reproductive strategies. They constitute a poorly understood phylum of exclusively terrestrial animals that is sister group to arthropods. Due to this phylogenetic position, they are crucial in understanding the origin of the largest phylum of animals. Despite their significance, there is a paucity of genomic resources for the phylum with only one highly fragmented and incomplete genome publicly available. Initial attempts at sequencing an onychophoran genome proved difficult due to its large genome size and high repeat content. However, leveraging recent advances in long-read sequencing technology, we present here the first annotated draft genome for the phylum. With a total size of 5.6Gb, the gigantism of the Epiperipatus broadwayi genome arises from having high repeat content, intron size inflation, and extensive gene family expansion. Additionally, we report a previously unknown diversity of onychophoran hemocyanins that suggests the diversification of copper-mediated oxygen carriers occurred independently in Onychophora after its split from Arthropoda, parallel to the independent diversification of hemocyanins in each of the main arthropod lineages.

Molecular isolation and expression analysis of hemocyanin isoform 2 of Macrobrachium rosenbergii.

OBJECTIVE: Hemocyanin is a copper-bearing protein in the hemolymph of many arthropods and mollusks and functions as an oxygen transport and important nonspecific immune protein. METHODS: In this study, complementary DNA of hemocyanin isoform 2 of the prawn Macrobrachium rosenbergii (MrHc2) was isolated by rapid amplification of cDNA ends and mRNA expression was characterized to elucidate molecular basis of its function. RESULT: With a molecular mass of 77.3 kDa, MrHc2 contained three domains: hemocyanin-all-alpha, hemocyanin-copper-containing, and hemocyanin-immunoglobulin-like domains. Molecular phylogenetic analysis revealed that MrHc2 belongs to the γ-type subunit and is closely related to hemocyanin subunit 1 of the palaemonid shrimp Macrobrachium nipponense. In addition, MrHc2 resided in a different clade relative to hemocyanin (MrHc) of M. rosenbergii (α-type subunit) and in a different subclade relative to the hemocyanin proteins of penaeid shrimp. The messenger RNA transcript of MrHc2 was highly expressed in the hepatopancreas and weakly expressed in the gills, intestine, stomach, muscle, and hemocytes. Upon challenge with M. rosenbergii nodavirus (MrNV), the expression of MrHc2 was 1.96-, 2.93-, and 1.96-fold on days 3, 4, and 5, respectively, and then gradually declined to basal levels on day 7. CONCLUSION: This study suggests that MrHc2 plays an important role in the innate immune response of M. rosenbergii to MrNV.

The Evolution of Hemocyanin Genes in Caenogastropoda: Gene Duplications and Intron Accumulation in Highly Diverse Gastropods.

Hemocyanin is the oxygen transport protein of most molluscs and represents an important physiological factor that has to be well-adapted to their environments because of the strong influences of abiotic factors on its oxygen affinity. Multiple independent gene duplications and intron gains have been reported for hemocyanin genes of Tectipleura (Heterobranchia) and the caenogastropod species Pomacea canaliculata, which contrast with the uniform gene architectures of hemocyanins in Vetigastropoda. The goal of this study was to analyze hemocyanin gene evolution within the diverse group of Caenogastropoda in more detail. Our findings reveal multiple gene duplications and intron gains and imply that these represent general features of Apogastropoda hemocyanins. Whereas hemocyanin exon-intron structures are identical within different Tectipleura lineages, they differ strongly within Caenogastropoda among phylogenetic groups as well as between paralogous hemocyanin genes of the same species. Thus, intron accumulation took place more gradually within Caenogastropoda but finally led to a similar consequence, namely, a multitude of introns. Since both phenomena occurred independently within Heterobranchia and Caenogastropoda, the results support the hypothesis that introns may contribute to adaptive radiation by offering new opportunities for genetic variability (multiple paralogs that may evolve differently) and regulation (multiple introns). Our study indicates that adaptation of hemocyanin genes may be one of several factors that contributed to the evolution of the large diversity of Apogastropoda. While questions remain, this hypothesis is presented as a starting point for the further study of hemocyanin genes and possible correlations between hemocyanin diversity and adaptive radiation.

The evolution of hemocyanin genes in Tectipleura: a multitude of conserved introns in highly diverse gastropods.

BACKGROUND: Hemocyanin is the oxygen transporter of most molluscs. Since the oxygen affinity of hemocyanin is strongly temperature-dependent, this essential protein needs to be well-adapted to the environment. In Tectipleura, a very diverse group of gastropods with > 27,000 species living in all kinds of habitats, several hemocyanin genes have already been analyzed. Multiple independent duplications of this gene have been identified and may represent potential adaptations to different environments and lifestyles. The aim of this study is to further explore the evolution of these genes by analyzing their exon-intron architectures. RESULTS: We have reconstructed the gene architectures of ten hemocyanin genes from four Tectipleura species: Aplysia californica, Lymnaea stagnalis, Cornu aspersum and Helix pomatia. Their hemocyanin genes each contain 53 introns, significantly more than in the hemocyanin genes of Cephalopoda (9-11), Vetigastropoda (15) and Caenogastropoda (28-33). The gene structures of Tectipleura hemocyanins are identical in terms of intron number and location, with the exception of one out of two hemocyanin genes of L. stagnalis that comprises one additional intron. We found that gene structures that differ between molluscan lineages most probably evolved more recently through independent intron gains. CONCLUSIONS: The strict conservation of the large number of introns in Tectipleura hemocyanin genes over 200 million years suggests the influence of a selective pressure on this gene structure. While we could not identify conserved sequence motifs within these introns, it may be simply the great number of introns that offers increased possibilities of gene regulation relative to hemocyanin genes with less introns and thus may have facilitated habitat shifts and speciation events. This hypothesis is supported by the relatively high number of introns within the hemocyanin genes of Pomacea canaliculata that has evolved independently of the Tectipleura. Pomacea canaliculata belongs to the Caenogastropoda, the sister group of Heterobranchia (that encompass Tectipleura) which is also very diverse and comprises species living in different habitats. Our findings provide a hint to some of the molecular mechanisms that may have supported the spectacular radiation of one of Metazoa's most species rich groups.

Molecular Cloning, Structure and Phylogenetic Analysis of a Hemocyanin Subunit from the Black Sea Crustacean Eriphia verrucosa (Crustacea, Malacostraca).

Hemocyanins are copper-binding proteins that play a crucial role in the physiological processes in crustaceans. In this study, the cDNA encoding hemocyanin subunit 5 from the Black sea crab Eriphia verrucosa (EvHc5) was cloned using EST analysis, RT-PCR and rapid amplification of the cDNA ends (RACE) approach. The full-length cDNA of EvHc5 was 2254 bp, consisting of a 5' and 3' untranslated regions and an open reading frame of 2022 bp, encoding a protein consisting of 674 amino acid residues. The protein has an N-terminal signal peptide of 14 amino acids as is expected for proteins synthesized in hepatopancreas tubule cells and secreted into the hemolymph. The 3D model showed the presence of three functional domains and six conserved histidine residues that participate in the formation of the copper active site in Domain 2. The EvHc5 is O-glycosylated and the glycan is exposed on the surface of the subunit similar to Panulirus interruptus. The phylogenetic analysis has shown its close grouping with γ-type of hemocyanins of other crustacean species belonging to order Decapoda, infraorder Brachyura.

Responsiveness of metallothionein and hemocyanin genes to cadmium and copper exposure in the garden snail Cornu aspersum.

Terrestrial gastropods express metal-selective metallothioneins (MTs) by which they handle metal ions such as Zn2+ , Cd2+ , and Cu+ /Cu2+ through separate metabolic pathways. At the same time, they depend on the availability of sufficient amounts of Cu as an essential constituent of their respiratory protein, hemocyanin (Hc). It was, therefore, suggested that in snails Cu-dependent MT and Hc pathways might be metabolically connected. In fact, the Cu-specific snail MT (CuMT) is exclusively expressed in rhogocytes, a particular molluscan cell type present in the hemocoel and connective tissues. Snail rhogocytes are also the sites of Hc synthesis. In the present study, possible interactions between the metal-regulatory and detoxifying activity of MTs and the Cu demand of Hc isoforms was explored in the edible snail Cornu aspersum, one of the most common European helicid land snails. This species possesses CdMT and CuMT isoforms involved in metal-selective physiological tasks. In addition, C. aspersum expresses three different Hc isoforms (CaH ɑD, CaH ɑN, CaH ß). We have examined the effect of Cd2+ and Cu2+ exposure on metal accumulation in the midgut gland and mantle of C. aspersum, testing the impact of these metals on transcriptional upregulation of CdMT, CuMT, and the three Hc genes in the two organs. We found that the CuMT and CaH ɑD genes exhibit an organ-specific transcriptional upregulation in the midgut gland of Cu-exposed snails. These results are discussed in view of possible interrelationships between the metal-selective activity of snail MT isoforms and the synthesis and metabolism of Hc isoforms.

Hemocyanin and hexamerins expression in response to hypoxia in stoneflies (Plecoptera, Insecta).

Many freshwater ecosystems worldwide undergo hypoxia events that can trigger physiological, behavioral, and molecular responses in many organisms. Among such molecular responses, the regulation of the hemocyanin (Hc) protein expression which plays a major role in oxygen transportation within aquatic insects remains poorly understood. The stoneflies (Plecoptera) are aquatic insects that possess a functional Hc in the hemolymph similar to crustacean that co-occurs with a nonfunctional Hc protein, hexamerins (Hx). However, the role of both proteins during hypoxia remains undetermined. Here, we evaluated the effect of hypoxia on the expression of Hc and Hx proteins via a comparison between hypoxia and normoxia amino acid sequence variation and protein expression pattern within 23 stonefly species. We induced short-term hypoxia in wild-caught stoneflies species, sequenced the target region of Hc and Hx by complementary DNA synthesis, characterized the protein biochemistry using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, ultrafiltration, and polarographic fluorometric method, and amplified the genome region of the hypoxia-inducible factor (HIF) transcriptional response element that regulated Hc using genome walking library approach. We found a lack of Hc expression in all examined species during hypoxia conditions, despite recognition of the HIF gene region as a possible regulatory factor of Hc, suggesting that compensatory responses as metabolic changes or behavioral tracheal movements to enhance respiratory efficiency could be possible mechanics to compensate for hypoxia. A short Hc-like novel isoform was detected instead in these 23 species, possibly due to either protein degradation or alternative splicing mechanisms, suggesting that the protein could be performing a different function other than oxygen transportation. Hx during hypoxia was expressed and exhibited species-level amino acid changes, highlighting a possible role during hypoxia. Our results demonstrate that hypoxia could enable a similar potential adaptive response of multiple species regarding specific physiological requirements, thereby shedding light on community behavior in stress environments that may help us to improve conservation practices and biomonitoring.

A SNP of the hemocyanin gene (LvHc) is a marker for high growth and ammonia-tolerance in Pacific white shrimp Litopenaeus vannamei.

Expression levels of hemocyanin (LvHc), activating transcription factor 4 (LvAtf4), glutathione S-transferase (LvGst), caspase 2 (LvCasp2) and anti-lipopolysaccharide factor (LvAlf) were examined in the hepatopancreas of Pacific white shrimp Litopenaeus vannamei juveniles exposed to a lethal concentration of ammonia-N (32.15 mg/l). The expression levels of all transcripts except LvAlf were significantly greater (P < 0.05) in tolerant shrimp (Lv-AT; N = 30) that survived up to 72 h post treatment (hpt) than in susceptible shrimp (Lv-AS24 and Lv-AS72; N = 45 and 15), that died within 24 h or between 24 and 72 hpt, respectively. Subsequently, effects of non-lethal concentrations of ammonia-N (control, 10 and 20 mg/l) on the expression of LvHc in juvenile shrimp were examined. Compared to the control, expression levels of LvHc transcripts in hemocytes and the hepatopancreas of tested shrimp changed after exposure to ammonia-N. One SNP (C > T545) was found in the LvHc322 gene segment. Real-time PCR amplification of specific alleles (real-time PASA) was developed for detection of C > T545 genotypes. Juveniles in the lethal exposure test that carried a C/T545 genotype showed a greater average body weight and total length (8.46 ± 0.36 g and 10.05 ± 0.16 cm) than those with a C/C545 genotype (7.48 ± 0.31 g and 9.60 ± 0.13 cm) (P < 0.05). Similar results were found in the second generation (G2) of a growth-improved stock (3 and 4 families of BIOTEC-G2-L1 and BIOTEC-G2-L2) and in commercially farmed shrimp (2 groups). Accordingly, expression levels and SNP of LvHc can serve as markers for selection high growth performance in ammonia-tolerant L. vannamei.

The sterol regulatory element binding protein homolog of Penaeus vannamei modulates fatty acid metabolism and immune response.

The sterol regulatory element binding proteins (SREBPs) transcription factors family, which regulate the expression of genes involved in cellular lipid metabolism and homeostasis, have recently been implicated in various physiological and pathophysiological processes such as immune regulation and inflammation in vertebrates. Consistent with other invertebrates, we identified a single SREBP ortholog in Penaeus vannamei (designated PvSREBP) with transcripts ubiquitously expressed in tissues and induced by lipopolysaccharide (LPS), Vibrio parahaemolyticus and Streptococcus iniae. In vivo RNA interference (RNAi) of PvSREBP attenuated the expression of several fatty acid metabolism-related genes (i.e., cyclooxygenase (PvCOX), lipoxygenase (PvLOX), fatty acid binding protein (PvFABP) and fatty acid synthase (PvFASN)), which consequently decreased the levels of total polyunsaturated fatty acids (ΣPUFAs). In addition, PvSREBP silencing decreased transcript levels of several immune-related genes such as hemocyanin (PvHMC) and trypsin (PvTrypsin), as well as genes encoding for heat-shock proteins (i.e., PvHSP60, PvHSP70 and PvHSP90). Moreover, in silico analysis revealed the presence of SREBP binding motifs on the promoters of most of the dysregulated genes, while shrimp depleted of PvSREBP were more susceptible to V. parahaemolyticus infection. Collectively, we demonstrated the involvement of shrimp SREBP in fatty acids metabolism and immune response, and propose that PvSREBP and PvHMC modulate each other through a feedback mechanism to establish homeostasis. The current study is the first to show the dual role of SREBP in fatty acid metabolism and immune response in invertebrates and crustaceans.

Synthetic and immunological studies on the OCT4 immunodominant motif antigen-based anti-cancer vaccine.

Objective: Cancer stem cell is one of the important causes of tumorigenesis as well as a drug target in the treatment of malignant tumor. However, at present, there is no immune vaccine targeting these cells. Octamer-binding transcription factor 4 (OCT4), a marker of embryonic stem cells and germ cells, often highly expresses in the early stages of tumorigenesis and is therefore a good candidate for cancer vaccine development. Methods: To identify the optimal carrier and adjuvant combination, we chemically synthesized and linked three different OCT4 epitope antigens to a carrier protein, keyhole limpet hemocyanin (KLH), combined with Toll-like receptor 9 agonist (TLR9). Results: Immunization with OCT4-3 + TLR9 produced the strongest immune response in mice. In prevention assays, significant tumor growth inhibition was achieved in BABL/c mice treated with OCT4-3 + TLR9 (P < 0.01). Importantly, the results showed that cytotoxic T lymphocyte activity and the inhibition of tumor growth were enhanced in mice immunized with OCT4-3 combined with TLR9. Meanwhile, multiple cytokines [such as interferon (IFN)-γ (P < 0.05), interleukin (IL)-12 (P < 0.05), IL-2 (P < 0.01), and IL-6 (P < 0.05)] promoting cellular immune responses were shown to be greatly enhanced in mice immunized with OCT4-3 + TLR9. Moreover, we considered safety considerations in terms of the composition of the vaccines to help facilitate the development of effective next-generation vaccines. Conclusions: Collectively, these experiments demonstrated that combination therapy with TLR9 agonist induced a tumor-specific adaptive immune response, leading to the suppression of primary tumor growth in testis embryonic carcinoma.

Myriapod haemocyanin: the first three-dimensional reconstruction of Scolopendra subspinipes and preliminary structural analysis of S. viridicornis.

Haemocyanins (Hcs) are copper-containing, respiratory proteins that occur in the haemolymph of many arthropod species. Here, we report the presence of Hcs in the chilopode Myriapoda, demonstrating that these proteins are more widespread among the Arthropoda than previously thought. The analysis of transcriptome of S. subspinipes subpinipes reveals the presence of two distinct subunits of Hc, where the signal peptide is present, and six of prophenoloxidase (PPO), where the signal peptide is absent, in the 75 kDa range. Size exclusion chromatography profiles indicate different quaternary organization for Hc of both species, which was corroborated by TEM analysis: S. viridicornis Hc is a 6 × 6-mer and S. subspinipes Hc is a 3 × 6-mer, which resembles the half-structure of the 6 × 6-mer but also includes the presence of phenoloxidases, since the 1 × 6-mer quaternary organization is commonly associated with hexamers of PPO. Studies with Chelicerata showed that PPO activity are exclusively associated with the Hcs. This study indicates that Scolopendra may have different proteins playing oxygen transport (Hc) and PO function, both following the hexameric oligomerization observed in Hcs.

The krüppel-like factor of Penaeus vannamei negatively regulates transcription of the small subunit hemocyanin gene as part of shrimp immune response.

Hemocyanin is a multifunctional respiratory glycoprotein, which has also been implicated in other biological functions in shrimp. Moreover, recent studies have revealed that hemocyanin is also involved in a broad range of immune-related activities in shrimp. However, in spite of the considerable interest in unraveling the reasons behind the multiple immune-related functions of hemocyanin, little is known about its transcriptional regulation. Here, DNA pull-down and Liquid Chromatography - Tandem Mass Spectrometry (LC-MS/MS) analyses were used to isolate and identify the putative transcription factor(s) that are involved in the transcriptional regulation of the small subunit hemocyanin gene of Penaeus vannamei (PvHMCs). Krüppel-like factor (designated PvKruppel), a zinc finger transcription factor homolog in P. vannamei, was identified among the putative transcription factors, while bioinformatics analysis revealed the presence of Krüppel-like factor binding site (KLF motif) on the core promoter region of PvHMCs. Mutational analysis and electrophoretic mobility shift assay (EMSA) confirmed that PvKruppel could bind to the KLF motif on the core promoter region of PvHMCs. Moreover, in response to lipopolysaccharide (LPS), Vibrio parahaemolyticus and white spot syndrome virus (WSSV) challenge, transcript levels of PvKruppel and PvHMCs were negatively correlated. Furthermore, overexpression of PvKruppel significantly reduced the promoter activity of PvHMCs, while PvKruppel knockdown by RNA interference or lipopolysaccharides (LPS) stimulation resulted in a significant increase in the transcript level of PvHMCs. Taken together, our present study provides mechanistic insights into the transcriptional regulation of PvHMCs by PvKruppel during shrimp immune response to pathogens.

Nuclear receptor E75 is a transcription suppressor of the Litopenaeus vannamei small subunit hemocyanin gene.

Hemocyanin is a respiratory protein that possesses multiple physiological and immunological functions in shrimp. However, the transcriptional regulation of the hemocyanin gene is still poorly understood. Here, the nuclear receptor E75 of Litopenaeus vannamei (LvE75) was identified as one of the transcriptional regulators that modulates the transcription of the small molecular weight hemocyanin gene of L. vannamei (LvHMCs) by inhibiting its core promoter activity in a Dual-luciferase assay. In silico analysis revealed that the core promoter (designated HsP3), which is located at +1517/+1849 bp of LvHMCs contained a putative E75 binding motif ("ACGGAAT", spanning +1812/+1818 bp). Further, LvE75 was shown to inhibit the core promoter activity by direct binding. Importantly, in vivo silencing of LvE75 resulted in a significant upregulation in the mRNA and protein expression of LvHMCs gene. Taken together, our present results provide direct evidence that LvE75 is a transcriptional suppressor of the LvHMCs gene expression.

microRNA-589-5p modulates the expression of hemocyanin as part of the anti-WSSV immune response in Litopenaeus vannamei.

The respiratory glycoprotein, hemocyanin (HMC) has multiple immune-related functions, including antiviral activity. In this study, in silico methods were used to predict seven miRNAs targeting Litopenaeus vannamei HMC (LvHMC), out of which miR-589-5p was selected for further investigation because of its role in immune response. Transcript levels of miR-589-5p were ubiquitously distributed in all shrimp tissues examined, and significantly induced in hemocytes and hepatopancreas upon challenge with white-spot syndrome virus (WSSV) as well as by marine bacterial pathogens, which suggest that miR-589-5p is involved in shrimp immune response to pathogens. Morever, using Drosophila S2 cells stably overexpressing EGFP-LvHMC, flow cytometry and dual luciferase reporter assays, miR-589-5p was shown to significantly inhibit the in vitro expression of LvHMC. In addition, in vivo knockdown of miR-589-5p using antagomir-589-5p resulted in significant down-regulation in LvHMC expression, while overexpression of miR-589-5p using agomir-589-5p decreased the level of LvHMC expression in shrimp hemocytes and hepatopancreas. Further, the increased expression of miR-589-5p resulted in high shrimp mortality following WSSV challenge, coupled with an increase in the number of WSSV copies in hemocytes and hepatopancreas. These results suggest that miR-589-5p is involved in shrimp immune response to WSSV by negatively regulating the expression of LvHMC.

Hemocyanin of the caenogastropod Pomacea canaliculata exhibits evolutionary differences among gastropod clades.

Structural knowledge of gastropod hemocyanins is scarce. To better understand their evolution and diversity we studied the hemocyanin of a caenogastropod, Pomacea canaliculata (PcH). Through a proteomic and genomic approach, we identified 4 PcH subunit isoforms, in contrast with other gastropods that usually have 2 or 3. Each isoform has the typical Keyhole limpet-type hemocyanin architecture, comprising a string of eight globular functional units (FUs). Correspondingly, genes are organized in eight FUs coding regions. All FUs in the 4 genes are encoded by more than one exon, a feature not found in non- caenogastropods. Transmission electron microscopy images of PcH showed a cylindrical structure organized in di, tri and tetra-decamers with an internal collar structure, being the di and tri-decameric cylinders the most abundant ones. PcH is N-glycosylated with high mannose and hybrid-type structures, and complex-type N-linked glycans, with absence of sialic acid. Terminal ß-N-GlcNAc residues and nonreducing terminal α-GalNAc are also present. The molecule lacks O-linked glycosylation but presents the T-antigen (Gal-ß1,3-GalNAc). Using an anti-PcH polyclonal antibody, no cross-immunoreactivity was observed against other gastropod hemocyanins, highlighting the presence of clade-specific structural differences among gastropod hemocyanins. This is, to the best of our knowledge, the first gene structure study of a Caenogastropoda hemocyanin.

N-terminal diversity of Litopenaeus vannamei hemocyanin and immunity.

Hemocyanin is primarily a respiratory copper-containing glycoprotein present in the hemolymph of mollusks and arthropods. Recently, hemocyanin has attracted huge research interest due to its multifunctionality and polymorphism. Most previous immune-related studies on shrimp hemocyanin have focused on the C-terminal. Moreover, we previously reported that the C-terminal domain of Litopenaeus vannamei hemocyanin possesses single nucleotide polymorphisms (SNPs), but little is known about the molecular diversity of the N-terminal domain. In the current study, diversity within the N-terminal domain of L. vannamei hemocyanin (LvHMC-N) was explored using bioinformatics and molecular biology techniques as well as immune challenge. Twenty-five LvHMC-N variants were identified using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and DNA sequencing, with multiple sequence alignment showing that the 25 variants shared 87%-99 % sequence homology with LvHMC (AJ250830.1). In different shrimp individuals and different shrimp tissues (i.e., hemocytes, stomach, muscle and hepatopancreas), the LvHMC-N variants were expressed differently. Pathogen challenge could modulate the molecular diversity of LvHMC-N, as three LvHMC-Nr variants (LvHMC-Nr1, LvHMC-Nr2 and LvHMC-Nr3) were identified by sequencing following Vibrio parahaemolyticus challenge. Most importantly, recombinant proteins of these three variants (rLvHMC-Nr1, rLvHMC-Nr2 and rLvHMC- Nr3) had relatively high in vitro agglutinative activities against V. parahaemolyticus, Vibrio alginolyticus and Streptoccocus iniae. Our present data indicates that the N-terminus of L. vannamei hemocyanin also possess molecular diversity, which seems to be associated with immune resistance to pathogenic infections.

Developmental expression and evolution of hexamerin and haemocyanin from Folsomia candida (Collembola).

Haemocyanins constitute a group of copper-containing respiratory proteins, and hexamerins were derived from hexapod haemocyanin but lost the ability to transport oxygen and serve as storage proteins. Although hexamerins have been reported in most insect species, none of them has been identified in Collembola, one of the most primitive hexapod lineages, thereby preventing us from exploring relevant evolutionary scenarios regarding the origin and evolution of hexamerins in hexapods. Here we report on collembolan hexamerins for the first time, and investigated the temporal expression profiles of hexamerin and haemocyanin in the collembolan Folsomia candida. Haemocyanin was expressed over the entire life cycle, with higher expression at the embryonic stage than at other stages, whereas hexamerin expression was restricted to embryos, unlike insect hexamerins, which are generally expressed from larval to adult stages. A phylogenetic analysis and molecular clock estimation suggested that all investigated hexapod hexamerins have a single and ancient origin (~423 Ma), coincident with the rise of atmospheric oxygen levels in the Silurian-Devonian period, indicating a physiological link between molecular evolution and Palaeozoic oxygen changes.

Identification and characterization of glycosylation sites on Litopenaeus vannamei hemocyanin.

The respiratory glycoprotein hemocyanin has been implicated in immune-related functions. Using lectin blotting, we show that the binding of shrimp (Litopenaeus vannamei) hemocyanin to concanavalin A decreases markedly with O-glycosidase treatment but not with PNGase F. Twelve O-glycosylation sites, three on the large hemocyanin subunit and nine on the small hemocyanin subunit (HMCs), were identified by LC-MS/MS. Importantly, when the glycosylation sites at Thr-537, Ser-539, and Thr-542 on the C terminus of HMCs were replaced with alanine, the resultant mutant hemocyanin had reduced carbohydrate content, coupled with a fourfold reduction in bacterial agglutination and 0.2-fold reduction in antibacterial activities toward Vibrio parahaemolyticus and Staphylococcus aureus. These results suggest that the glycosylation sites on shrimp hemocyanin are closely related to its immunological functions.