Immunotherapeutic Potential of Mollusk Hemocyanins in Murine Model of Melanoma.

The development of antitumor drugs and therapy requires new approaches and molecules, and products of natural origin provide intriguing alternatives for antitumor research. Gastropodan hemocyanins-multimeric copper-containing glycoproteins have been used in therapeutic vaccines and antitumor agents in many cancer models. MATERIALS AND METHODS: We established a murine model of melanoma by challenging C57BL/6 mice with a B16F10 cell line for solid tumor formation in experimental animals. The anticancer properties of hemocyanins isolated from the marine snail Rapana thomasiana (RtH) and the terrestrial snail Helix aspersa (HaH) were evaluated in this melanoma model using various schemes of therapy. Flow cytometry, ELISA, proliferation, and cytotoxicity assays, as well as histology investigations, were also performed. RESULTS: Beneficial effects on tumor growth, tumor incidence, and survival of tumor-bearing C57BL/6 mice after administration of the RtH or HaH were observed. The generation of high titers of melanoma-specific IgM antibodies, pro-inflammatory cytokines, and tumor-specific CTLs, and high levels of tumor-infiltrated M1 macrophages enhanced the immune reaction and tumor suppression. DISCUSSION: Both RtH and HaH exhibited promising properties for applications as antitumor therapeutic agents and future experiments with humans.

A comprehensive review on hemocyanin from marine products: Structure, functions, its implications for the food industry and beyond.

Hemocyanin, an oxygen-transport protein, is widely distributed in the hemolymph of marine arthropods and mollusks, playing an important role in their physiological processes. Recently, hemocyanin has been recognized as a multifunctional glycoprotein involved in the immunological responses of aquatic invertebrates. Consequently, the link between hemocyanin functions and their potential applications has garnered increased attention. This review offers an integrated overview of hemocyanin's structure, physicochemical characteristics, and bioactivities to further promote the utilization of hemocyanin derived from marine products. Specifically, we review its implication in two aspects of food and aquaculture industries: quality and health. Hemocyanin's inducible phenoloxidase activity is thought to be an inducer of melanosis in crustaceans. New anti-melanosis agents targeted to hemocyanin need to be explored. The red-color change observed in shrimp shells is related to hemocyanin, affecting consumer preferences. Hemocyanin's adaptive modification in response to the aquatic environment is available as a biomarker. Additionally, hemocyanin is endowed with bioactivities encompassing anti-microbial, antiviral, and therapeutic activities. Hemocyanin is also a novel allergen and its allergenic features remain incompletely characterized.

Monophosphoryl Lipid A-Rhamnose Conjugates as a New Class of Vaccine Adjuvants.

Adjuvant is an integral part of all vaccine formulations but only a few adjuvants with limited efficacies or application scopes are available. Thus, developing more robust and diverse adjuvants is necessary. To this end, a new class of adjuvants having α- and ß-rhamnose (Rha) attached to the 1- and 6'-positions of monophosphoryl lipid A (MPLA) was designed, synthesized, and immunologically evaluated in mice. The results indicated a synergistic effect of MPLA and Rha, two immunostimulators that function via interacting with toll-like receptor 4 and recruiting endogenous anti-Rha antibodies, respectively. All the tested MPLA-Rha conjugates exhibited potent adjuvant activities to promote antibody production against both protein and carbohydrate antigens. Overall, MPLA-α-Rha exhibited better activities than MPLA-ß-Rha, and 6'-linked conjugates were slightly better than 1-linked ones. Particularly, MPLA-1-α-Rha and MPLA-6'-α-Rha were the most effective adjuvants in promoting IgG antibody responses against protein antigen keyhole limpet hemocyanin and carbohydrate antigen sTn, respectively.

Purification and characterization of a hemocyanin with lectin-like activity isolated from the hemolymph of speckled shrimp, Metapenaeusmonoceros.

Lectins or agglutinins are mainly proteins or glycoproteins, reported to uphold an ability to agglutinate the red blood cells (RBCs) with a known sugar specificity in a diverse group of organisms. In the present study, we purified a hemocyanin (named as MmHc) from a shrimp, Metapenaeus monoceros by size-exclusion chromatography. Further characterization revealed that the purified MmHc showed hemagglutination activity that was found to be specifically inhibited by Lewis B and Lewis Y tetrasaccharides. The MmHc displayed two oligomers of molecular weight approximately ∼78 and ∼85 kDa in SDS-PAGE. The native molecular mass of MmHc was found to be ∼457 kDa as determined by size-exclusion chromatography which indicated that the purified MmHc is an oligomeric protein. MmHc showed a maximum activity within pH 7.0-8.0, while a wide range of temperature stability was observed between 4 to 55 °C, however, it did not show any dependency on metal ions for binding. Subsequently, the analysis of the peptides by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) identified the purified MmHc as shrimp hemocyanin showing significant similarity to the hemocyanin of Penaeus vannamei. The results of multiple sequence alignment and detailed analysis of the molecular interactions predicted by AutoDock suggested that besides the oxygen carrier function, this MmHc may have multiple roles and can interact well with the Lewis Y antigen through a typical sugar binding motif containing the similar hydrophilic amino acids as the conserved residues.

Embryonic and post-embryonic development of the spider Polybetes pythagoricus (Sparassidae): A biochemical point of view.

Analysis of energy expense during development has achieved special interest through time on account of the crucial role of the consumption of resources required for offspring survival. Spider eggs have a fixed composition as well as some initial energy that is supplied by mothers. These resources are necessary to support the metabolic expense not only through the embryonic period but also during the post-embryonic period, as well as for post emerging activities before spiderlings become self-sustaining. Depletion of these resources would be critical for spiders since it could give rise to prey competition as well as filial cannibalism. Even though spiders represent a megadiverse order, information regarding the metabolic requirements during spiders development is very scarce. In this study, we analyse the changes in protein, lipid and carbohydrate content as well as the variation in lipovitellin reserves and hemocyanin content during Polybetes pythagoricus development. Our results show that lipovitellins and phospholipids represent the major energy source throughout embryonic and post-embryonic development. Lipovitellin apolipoproteins are gradually consumed but are later depleted after dispersion. Phosphatidylethanolamine is mainly consumed during the post-embryonic period, while triacylglycerides are consumed after juveniles' dispersion. Finally, hemocyanin concentration starts to increase in postembryonic stages.

New putative phenol oxidase in ascidian blood cells.

The phenol oxidase system is ancient and ubiquitously distributed in all living organisms. In various groups it serves for the biosynthesis of pigments and neurotransmitters (dopamine), defence reactions and tissue hardening. Ascidians belong to subphylum Tunicata, which is considered the closest living relative to Vertebrates. Two phenol oxidases previously described for ascidians are vertebrate-like and arthropod-like phenol oxidases. In our present study, we described a new ascidian protein, Tuphoxin, with putative phenol oxidase function, which bears no sequence similarity with two enzymes described previously. The closest related proteins to Tuphoxin are mollusc haemocyanins. Unlike haemocyanins, which are oxygen transporting plasma proteins, Tuphoxin is synthesised in ascidian blood cells and secreted in the extracellular matrix of the tunic-ascidian outer coverings. Single mature transcript coding for this phenol oxidase can give several protein products of different sizes. Thus limited proteolysis of the initial protein is suggested. A unique feature of Tuphoxins and their homologues among Tunicata is the presence of thrombospondin first type repeats (TSP1) domain in their sequence which is supposed to provide interaction with extracellular matrix. The finding of TSP1 in the structure of phenol oxidases is new and we consider this to be an innovation of Tunicata evolutionary lineage.

Selective Cytotoxicity and Changes in Protein Expression of T24 Bladder Carcinoma Permanent Cell Line after Treatment with Hemocyanins.

BACKGROUND: Some molluscan hemocyanins (Hcs) have significant immunological and antitumor potential, enabling their application in oncology. The antitumor activity of Hcs from marine snails Rapana venosa (RvH), giant keyhole limpet Megathura crenulata (KLH) and garden snails Helix lucorum (HlH), as well as their different derivatives, were studied in vitro on a permanent T24 cell line of bladder cancer and normal urothelial cell line HL 10/29 compared to doxorubicin. METHODS: The antiproliferative activity of the tested Hcs was determined using the WST-1 assay and BrdU ELISA assay. Morphological changes in both urothelial cell lines were confirmed by fluorescence microscopy. The proteomic analysis of a bladder cancer cell line before and after treatment with functional unit (FU) ßc-HlH-h using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry revealed differences in the expression of some proteins. RESULTS: Studies prove that the T24 tumor cell line is dose- and time-dependent, sensitive to the action of the tested isoforms, and it glycosylated FU of these hemocyanins. Selective inhibition of T24 cell growth was observed after incubation with structural subunits (ßc-HlH, RvHI and RvHII) and FUs (ßc-HlH-h and RvHII-e). Additionally, fluorescent microphotographs did not show apoptotic or necrotic alterations in the normal urothelial cell line HL 10/29. The FU ßc-HlH-h demonstrated the highest antiproliferative effect (similarly to doxorubicin), in which predominantly apoptotic and less late apoptotic or necrotic changes in the tumor cells were observed. Several downand up-regulated proteins identified by proteome analysis may be associated with the apoptosis pathway. CONCLUSION: The present study illustrated the selectivity of the cytotoxic effect of Hcs against the Т24 cancer cell line. This is the first report of protein expression in T24 human bladder cancer cells under the influence of FU ßc-HlH-h. That is probably due to the specific oligosaccharide structures rich in methylated hexoses exposed on the surface of ßc-HlH-h.

Functional Characterization, Antimicrobial Effects, and Potential Antibacterial Mechanisms of NpHM4, a Derived Peptide of Nautilus pompilius Hemocyanin.

Hemocyanins present in the hemolymph of invertebrates are multifunctional proteins that are responsible for oxygen transport and play crucial roles in the immune system. They have also been identified as a source of antimicrobial peptides during infection in mollusks. Hemocyanin has also been identified in the cephalopod ancestor Nautilus, but antimicrobial peptides derived from the hemocyanin of Nautilus pompilius have not been reported. Here, the bactericidal activity of six predicted peptides from N. pompilius hemocyanin and seven mutant peptides was analyzed. Among those peptides, a mutant peptide with 15 amino acids (1RVFAGFLRHGIKRSR15), NpHM4, showed relatively high antibacterial activity. NpHM4 was determined to have typical antimicrobial peptide characteristics, including a positive charge (+5.25) and a high hydrophobic residue ratio (40%), and it was predicted to form an alpha-helical structure. In addition, NpHM4 exhibited significant antibacterial activity against Gram-negative bacteria (MBC = 30 µM for Vibrio alginolyticus), with no cytotoxicity to mammalian cells even at a high concentration of 180 µM. Upon contact with V. alginolyticus cells, we confirmed that the bactericidal activity of NpHM4 was coupled with membrane permeabilization, which was further confirmed via ultrastructural images using a scanning electron microscope. Therefore, our study provides a rationalization for the development and optimization of antimicrobial peptide from the cephalopod ancestor Nautilus, paving the way for future novel AMP development with broad applications.

Antitumor Properties of Epitope-Specific Engineered Vaccine in Murine Model of Melanoma.

Finding new effective compounds of natural origin for composing anti-tumor vaccines is one of the main goals of antitumor research. Promising anti-cancer agents are the gastropodan hemocyanins-multimeric copper-containing glycoproteins used so far for therapy of different tumors. The properties of hemocyanins isolated from the marine snail Rapana thomasiana (RtH) and the terrestrial snail Helix aspersa (HaH) upon their use as carrier-proteins in conjugated vaccines, containing ganglioside mimotope GD3P4 peptide, were studied in the developed murine melanoma model. Murine melanoma cell line B16F10 was used for solid tumor establishment in C57BL/6 mice using various schemes of therapy. Protein engineering, flow cytometry, and cytotoxicity assays were also performed. The administration of the protein-engineered vaccines RtH-GD3P4 or HaH-GD3P4 under the three different regimens of therapy in the B16F10 murine melanoma model suppressed tumor growth, decreased tumor incidence, and prolonged the survival of treated animals. The immunization of experimental mice induced an infiltration of immunocompetent cells into the tumors and generated cytotoxic tumor-specific T cells in the spleen. The treatment also generates significantly higher levels of tumor-infiltrated M1 macrophages, compared to untreated tumor-bearing control mice. This study demonstrated a promising approach for cancer therapy having potential applications for cancer vaccine research.

A Vaccine against Benzimidazole-Derived New Psychoactive Substances That Are More Potent Than Fentanyl.

New psychoactive substance (NPS) opioids have proliferated within the international drug market. While synthetic opioids are traditionally composed of fentanyl analogues, benzimidazole-derived isotonitazene and its derivatives are the current NPS opioids of concern. Hence, in this study, we implement immunopharmacotherapy wherein antibodies are produced with high titers and nanomolar affinity to multiple benzimidazole-derived NPS opioids (BNO). Notably, these antibodies blunt psychoactive and physiological repercussions from BNO exposure, which was observed through antinociception, whole-body plethysmography, and blood-brain biodistribution studies. Moreover, we detail previously unreported pharmacokinetics of these drugs, which explains the struggle of traditional pharmaceutical opioid antagonists against BNO substances. These findings provide further insight into the in vivo effects of BNO drugs and the development of effective broad-spectrum therapeutics against NPS opioids.

Structural, Thermal, and Storage Stability of Rapana Thomasiana Hemocyanin in the Presence of Cholinium-Amino Acid-Based Ionic Liquids.

Novel biocompatible compounds that stabilize proteins in solution are in demand for biomedical and/or biotechnological applications. Here, we evaluated the effect of six ionic liquids, containing mono- or dicholinium [Chol]1or2 cation and anions of charged amino acids such as lysine [Lys], arginine [Arg], aspartic acid [Asp], or glutamic acid [Glu], on the structure, thermal, and storage stability of the Rapana thomasiana hemocyanin (RtH). RtH is a protein with huge biomedicinal potential due to its therapeutic, drug carrier, and adjuvant properties. Overall, the ionic liquids (ILs) induce changes in the secondary structure of RtH. However, the structure near the Cu-active site seems unaltered and the oxygen-binding capacity of the protein is preserved. The ILs showed weak antibacterial activity when tested against three Gram-negative and three Gram-positive bacterial strains. On the contrary, [Chol][Arg] and [Chol][Lys] exhibited high anti-biofilm activity against E. coli 25213 and S. aureus 29213 strains. In addition, the two ILs were able to protect RtH from chemical and microbiological degradation. Maintained or enhanced thermal stability of RtH was observed in the presence of all ILs tested, except for RtH-[Chol]2[Glu].

Therapeutic and Prophylactic Vaccines to Counteract Fentanyl Use Disorders and Toxicity.

The incidence of fatal overdoses has increased worldwide due to the widespread access to illicit fentanyl and its potent analogues. Vaccines offer a promising strategy to reduce the prevalence of opioid use disorders (OUDs) and to prevent toxicity from accidental and deliberate exposure to fentanyl and its derivatives. This study describes the development and characterization of vaccine formulations consisting of novel fentanyl-based haptens conjugated to carrier proteins. Vaccine efficacy was tested against opioid-induced behavior and toxicity in mice and rats challenged with fentanyl and its analogues. Prophylactic vaccination reduced fentanyl- and sufentanil-induced antinociception, respiratory depression, and bradycardia in mice and rats. Therapeutic vaccination also reduced fentanyl intravenous self-administration in rats. Because of their selectivity, vaccines did not interfere with the pharmacological effects of commonly used anesthetics nor with methadone, naloxone, oxycodone, or heroin. These preclinical data support the translation of vaccines as a viable strategy to counteract fentanyl use disorders and toxicity.

De Novo Structural Determination of the Oligosaccharide Structure of Hemocyanins from Molluscs.

A number of studies have shown that glycosylation of proteins plays diverse functions in the lives of organisms, has crucial biological and physiological roles in pathogen-host interactions, and is involved in a large number of biological events in the immune system, and in virus and bacteria recognition. The large amount of scientific interest in glycoproteins of molluscan hemocyanins is due not only to their complex quaternary structures, but also to the great diversity of their oligosaccharide structures with a high carbohydrate content (2-9%). This great variety is due to their specific monosaccharide composition and different side chain composition. The determination of glycans and glycopeptides was performed with the most commonly used methods for the analysis of biomolecules, including peptides and proteins, including Matrix Assisted Laser Desorption/Ionisation-Time of Flight (MALDI-TOF-TOF), Liquid Chromatography - Electrospray Ionization-Mass Spectrometry (LC/ESI-MS), Liquid Chromatography (LC-Q-trap-MS/MS) or Nano- Electrospray Ionization-Mass Spectrometry (nano-ESI-MS) and others. The molluscan hemocyanins have complex carbohydrate structures with predominant N-linked glycans. Of interest are identified structures with methylated hexoses and xyloses arranged at different positions in the carbohydrate moieties of molluscan hemocyanins. Novel acidic glycan structures with specific glycosylation positions, e.g., hemocyanins that enable a deeper insight into the glycosylation process, were observed in Rapana venosa, Helix lucorum, and Haliotis tuberculata. Recent studies demonstrate that glycosylation plays a crucial physiological role in the immunostimulatory and therapeutic effect of glycoproteins. The remarkable diversity of hemocyanin glycan content is an important feature of their immune function and provides a new concept in the antibody-antigen interaction through clustered carbohydrate epitopes.

Hemocyanin Modification of Chitosan Scaffolds with Calcium Phosphate Phases Increase the Osteoblast/Osteoclast Activity Ratio-A Co-Culture Study.

The ongoing research on biomaterials that support bone regeneration led to the quest for materials or material modifications that can actively influence the activity or balance of bone tissue cells. The bone biocompatibility of porous chitosan scaffolds was modified in the present study by the addition of calcium phosphates or hemocyanin. The first strategy comprised the incorporation of calcium phosphates into chitosan to create a biomimetic chitosan-mineral phase composite. The second strategy comprised dip-coating of chitosan scaffolds with hemocyanin extracted from crayfish hemolymph. The cytocompatibility was assessed in a mono-culture of human bone marrow stromal cells (hBMSCs) and their differentiation to osteoblasts; in a mono-culture of human monocytes (hMs) and their maturation to osteoclasts; and in a co-culture of hBMSC/osteoblasts-hM/osteoclasts. Mineral incorporation caused an increase in scaffold bioactivity, as shown by reduced calcium concentration in the cell culture medium, delayed differentiation of hBMSCs, and reduced osteoclastic maturation of hMs in mono-culture. Dip-coating with hemocyanin led to increased proliferation of hBMSCs and equivalent osteoclast maturation in mono-culture, while in co-culture, both an inhibitory effect of mineral incorporation on osteoblastogenesis and stimulatory effects of hemocyanin were observed. It was concluded that highly bioactive scaffolds (containing mineral phases) restrain osteoblast and osteoclast development, while hemocyanin coating significantly supports osteoblastogenesis. These influences on the osteoblasts/osteoclasts activity ratio may support scaffold-driven bone healing in the future.

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.

Lectin-Like Activity of Hemocyanin in Freshwater Prawn, Macrobrachium rosenbergii.

Lectins are proteins that bind to the carbohydrate moieties on surface of bacteria, erythrocytes and other cells of invertebrates causing agglutination and mediate in recognition of foreign substances. In the present study, we isolated and characterized a lectin molecule present in the hemolymph of Macrobrachium rosenbergii, an important cultured freshwater prawn. Lectin in serum samples of adult prawns was assessed through hemagglutination (HA) test using rabbit RBC that showed a titre ranging from 16 to 64. This serum hemagglutinin was confirmed as a C-type lectin based on its dependency on calcium ions towards binding to rabbit RBCs. The hemagglutinin was also found to be stable at the pH range of 5.0-10.0 and temperature range of 10-40 °C. Of various sugars and glycoproteins tested in hemagglutination inhibition assay, the serum lectin was found specific only to N-acetylneuraminic acid and fetuin with respective minimum inhibitory concentrations at 50 mM and 0.31 mg/ml. Further, the lectin was purified by affinity chromatography on rabbit erythrocyte stroma, which showed hemagglutination with rabbit RBC. In electrophoretic analyses, the purified lectin showed one band with molecular weight of ~ 427 kDa in native gradient PAGE, and its two constituent polypeptide chains of ~ 81 and ~ 73 kDa in SDS-PAGE. These polypeptides were analysed in MALDI-TOF/TOF mass spectrometry and identified as hemocyanins. It was hence, concluded that hemocyanin in M. rosenbergii possesses lectin-like activity.

The high-resolution crystal structure of lobster hemocyanin shows its enzymatic capability as a phenoloxidase.

Hemocyanin (Hc) and phenoloxidase (PO) are members of the type 3 copper protein family. Although arthropod Hc and PO exhibit similar three-dimensional structures of the copper-containing active site, Hc functions as an oxygen transport protein, showing minimal or no phenoloxidase activity. Here, we present the crystal structure of the oxy form of Hc from Panulirus japonicus (PjHc) at 1.58 Å resolution. The structure of the di-copper active site of PjHc was found to be almost identical to that of PO. Although conserved amino acids and the water molecule crucial for the enzymatic activity were observed in PjHc at almost the same positions as those in PO, PjHc showed no enzymatic activity under our experimental conditions. One striking difference between PjHc and arthropod PO was the presence of a "blocker residue" near the binuclear copper site of PjHc. This blocker residue comprised a phenylalanine residue tightly stacked with an imidazole ring of a CuA coordinated histidine and hindered substrates from accessing the active site. Our results suggest that the blocker residue is also a determining factor of the catalytic activity of type 3 copper proteins.

Red color-related proteins from the shell of red swamp crayfish (Procambarus clarkii): Isolation, identification and bioinformatic analysis.

Two water-soluble red color-related proteins with the molecular masses of 24 and 73 kDa were purified from the shell of Procambarus clarkii. Initial color changes of these two proteins were detected at 30 °C and the large amount of red precipitate were obtained at 80 °C. PAGE analysis showed that the 24 kDa protein was the monomer, while the 73 kDa protein was the trimer. Identification revealed that these two proteins belonged to the hemocyanin subunit 2 family. With respect to the amino acid sequence similarity, the red color-related proteins shared the highest sequence identity with the hemocyanin derived from giant freshwater prawn (Macrobrachium rosenbergii). The phylogenetic tree analysis also clearly supported this finding. The shell-derived red color-related proteins show potential use as the edible thermal-sensitive indicator in food processing field.

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.

Molluscan Hemocyanins.

Instead of the red blood of vertebrates, most molluscs have blue hemolymph containing hemocyanin, a type-3 copper-containing protein. The hemoglobin of vertebrate blood is replaced in most molluscs with hemocyanin, which plays the role of an  oxygen transporter. Oxygen-binding in hemocyanin changes its hue from colorless deoxygenated hemocyanin into blue oxygenated hemocyanin. Molecules of molluscan hemocyanin are huge, cylindrical multimeric proteins-one of the largest protein molecules in the natural world. Their huge molecular weight (from 3.3 MDa to more than 10 MDa) are the defining characteristic of molluscan hemocyanin, a property that has complicated structural analysis of the molecules for a long time. Recently, the structural analysis of a cephalopod (squid) hemocyanin has succeeded using a hybrid method employing both X-ray crystallography and cryo-EM. In a biochemical breakthrough for molluscan hemocyanin, the first quaternary structure with atomic resolution is on the verge of solving the mystery of molluscan hemocyanin. Here we describe the latest information about the molecular structure, classification and evolution of the molecule, and the physiology of molluscan hemocyanin.