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Cell and Tissue Research May 2022Rhogocyte is a unique molluscan cell that synthesises a supramolecular respiratory protein known as hemocyanin. Its ability to synthesise the protein has eluded the...
Rhogocyte is a unique molluscan cell that synthesises a supramolecular respiratory protein known as hemocyanin. Its ability to synthesise the protein has eluded the scientists despite hemocyanin's importance as a carrier protein and complex molecule with anti-viral activity. Although a hypothetical model of hemocyanin release from the rhogocytes lacunae was proposed based on colloid-osmotic pressure mechanism, lack of in vitro studies limits further validation of this model. In this study, we aim to investigate the impact of cell culture conditions and nature of hemocyanin biosynthesis of rhogocyte cells dissociated from Haliotis laevigata mantle tissue. Population of cells with different hemocyanin expression levels was profiled using flow cytometry, while hemocyanin concentrations in the media were elucidated by ELISA assay. We demonstrated that addition of lipoprotein supplement into the media resulted in a burst secretion of hemocyanin into the culture media. Over 7 days of culture, the population of cells tagged with hemocyanin antibody increased steadily while hemocyanin release in the media decreased significantly. Variation of culture medium, temperature, growth supplement type and concentration also impacted the cell growth and hemocyanin biosynthesis. These results indicated the possibility of an active process triggered by the addition of supplement to synthesise the protein at the highest amount during the first hour. The current study provides a glimpse of the hemocyanin biosynthesis by rhogocyte that may be significant to understand the cell ability to synthesise supramolecular protein and secretion through lacunae.
Topics: Animals; Flow Cytometry; Gastropoda; Hemocyanins; Lipoproteins
PubMed: 35088179
DOI: 10.1007/s00441-022-03577-1 -
Ecotoxicology and Environmental Safety Aug 2022Anthropogenic factors and climate change have serious effects on the aquatic ecosystem and aquaculture. Among water pollutants, ammonia has the greatest impact on...
Anthropogenic factors and climate change have serious effects on the aquatic ecosystem and aquaculture. Among water pollutants, ammonia has the greatest impact on aquaculture organisms such as penaeid shrimp because it makes them more susceptible to infections. In this study, we explored the effects of ammonia stress (0, 50, 100, and 150 mg/L) on the molecular structure and functions of the multifunctional respiratory protein hemocyanin (HMC) in Penaeus vannamei. While the mRNA expression of Penaeus vannamei hemocyanin (PvHMC) was up-regulated after ammonia stress, both plasma hemocyanin protein and oxyhemocyanin (OxyHMC) levels decreased. Moreover, ammonia stress changed the molecular structure of hemocyanin, modulated the expression of protein phosphatase 2 A (PP2A) and casein kinase 2α (CK2α) to regulate the phosphorylation modification of hemocyanin, and enhanced its degradation into fragments by trypsin. Under moderate ammonia stress conditions, hemocyanin also undergoes glycosylation to improve its in vitro antibacterial activity and binding with Gram-negative (Vibrio parahaemolyticus) and Gram-positive (Staphylococcus aureus) bacteria, albeit differently. The current findings indicate that P. vannamei hemocyanin undergoes adaptive molecular modifications under ammonia stress enabling the shrimp to survive and counteract the consequences of the stress.
Topics: Ammonia; Animals; Ecosystem; Hemocyanins; Penaeidae; Vibrio parahaemolyticus
PubMed: 36068754
DOI: 10.1016/j.ecoenv.2022.113827 -
Cellular and Molecular Life Sciences :... Jan 2017It is now well documented that peptides with enhanced or alternative functionality (termed cryptides) can be liberated from larger, and sometimes inactive, proteins. A... (Review)
Review
It is now well documented that peptides with enhanced or alternative functionality (termed cryptides) can be liberated from larger, and sometimes inactive, proteins. A primary example of this phenomenon is the oxygen-transport protein hemoglobin. Aside from respiration, hemoglobin and hemoglobin-derived peptides have been associated with immune modulation, hematopoiesis, signal transduction and microbicidal activities in metazoans. Likewise, the functional equivalents to hemoglobin in invertebrates, namely hemocyanin and hemerythrin, act as potent immune effectors under certain physiological conditions. The purpose of this review is to evaluate the true extent of oxygen-transport protein dynamics in innate immunity, and to impress upon the reader the multi-functionality of these ancient proteins on the basis of their structures. In this context, erythrocyte-pathogen antibiosis and the immune competences of various erythroid cells are compared across diverse taxa.
Topics: Animals; Biological Transport; Hemerythrin; Hemocyanins; Hemoglobins; Humans; Immunity, Innate; Oxygen
PubMed: 27518203
DOI: 10.1007/s00018-016-2326-7 -
Biochimica Et Biophysica Acta Sep 2013Hemocyanin transports oxygen in the hemolymph of many molluscs and arthropods and is therefore a central physiological factor in these animals. Molluscan hemocyanin... (Review)
Review
Hemocyanin transports oxygen in the hemolymph of many molluscs and arthropods and is therefore a central physiological factor in these animals. Molluscan hemocyanin molecules are oligomers composed of many protein subunits that in turn encompass subsets of distinct functional units. The structure and evolution of molluscan hemocyanin have been studied for decades, but it required the recent progress in DNA sequencing, X-ray crystallography and 3D electron microscopy to produce a detailed view of their structure and evolution. The basic quaternary structure is a cylindrical decamer 35nm in diameter, consisting of wall and collar (typically at one end of the cylinder). Depending on the animal species, decamers, didecamers and multidecamers occur in the hemolymph. Whereas the wall architecture of the decamer seems to be invariant, four different types of collar have been identified in different molluscan taxa. Correspondingly, there exist four subunit types that differ in their collar functional units and range from 350 to 550kDa. Thus, molluscan hemocyanin subunits are among the largest polypeptides in nature. In this report, recent 3D reconstructions are used to explain and visualize the different functional units, subunits and quaternary structures of molluscan hemocyanins. Moreover, on the basis of DNA analyses and structural considerations, their possible evolution is traced. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.
Topics: Animals; Biological Evolution; Crystallography, X-Ray; Hemocyanins; Models, Molecular; Mollusca; Protein Conformation
PubMed: 23454609
DOI: 10.1016/j.bbapap.2013.02.020 -
Journal of Molecular Evolution Dec 2021Hemocyanin 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...
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.
Topics: Animals; Gastropoda; Gene Duplication; Hemocyanins; Introns; Phylogeny
PubMed: 34757470
DOI: 10.1007/s00239-021-10036-y -
Clinical and Translational Science Mar 2023Experimental exposure of healthy volunteers to the T-cell dependent neoantigen keyhole limpet hemocyanin (KLH) permits the evaluation of immunomodulatory investigational... (Review)
Review
Experimental exposure of healthy volunteers to the T-cell dependent neoantigen keyhole limpet hemocyanin (KLH) permits the evaluation of immunomodulatory investigational medicinal product (IMP) pharmacology prior to the recruitment of patient populations. Despite widespread use, no standardized approach to the design and conduct of such studies has been agreed. The objective of this systematic review was to survey the published literature where KLH was used as a challenge agent, describing methodology, therapeutic targets addressed, and pharmacodynamic outcome measures. We searched MEDLINE, EMBASE, clinicaltrials.gov, and Cochrane CENTRAL for studies using KLH challenge in humans between January 1, 1994, and April 1, 2022. We described key study features, including KLH formulation, dose, use of adjuvants, route of administration, co-administered IMPs, and end points. Of 2421 titles and abstracts screened, 46 met the inclusion criteria, including 14 (31%) early phase trials of IMP, of which 10 (71%) targeted T-cell co-stimulation. IMPs with diverse mechanisms demonstrated modulation of the humoral response to KLH, suggesting limited specificity of this end point. Two early phase IMP studies (14%) described the response to intradermal re-challenge (delayed type hypersensitivity). Challenge regimens for IMP assessment were often incompletely described, and exhibited marked heterogeneity, including primary KLH dose (25-fold variation: 100-2500 mcg), KLH formulation, and co-administration with adjuvants. Methodological heterogeneity and failure to exploit the access to tissue-level mechanism-relevant end points afforded by KLH challenge has impaired the translational utility of this paradigm to date. Future standardization, characterization, and methodological development is required to permit tailored, appropriately powered, mechanism-dependent study design to optimize drug development decisions.
Topics: Humans; Pharmaceutical Preparations; T-Lymphocytes; Hemocyanins; Adjuvants, Immunologic
PubMed: 36420645
DOI: 10.1111/cts.13457 -
Revista Medica de Chile Feb 2011Hemocyanins, the giant oxygen transporter glycoproteins of diverse mollusks, are xenogenic to the mammalian immune system and they display a remarkable immuno-genicity.... (Review)
Review
Hemocyanins, the giant oxygen transporter glycoproteins of diverse mollusks, are xenogenic to the mammalian immune system and they display a remarkable immuno-genicity. Therefore they are ideal non-specific immunostimulants to treat some types of cancer. They are used as an alternative therapy for superficial urinary bladder cancer (SBC), that has been traditionally treated with Bacillus Calmette-Guerin (BCG). In contrast to BCG, hemocyanins do not cause side-effects, making them ideal for long-term repetitive treatments. Hemocyanins have also been exploited as carriers to develop antibodies against hapten molecules and peptides, as carrier-adjuvants for cutting-edge vaccines against cancer, drug addiction, and infectious diseases and in the diagnosis of parasitic diseases, such as Schistosomiasis. The hemocyanin from Megathura crenulata, also known as keyhole limpet hemocyanin (KLH), has been used for over thirty years for the purposes described above. More recently, hemoc yanin from the Chilean mollusk Concholepas concholepas (CCH) has proved to be a reliable alternative to KLH, either as carrier protein, and as a likely alternative for the immunotherapy of SBC. Despite KLH and CCH differ significantly in their origin and structure, we have demonstrated that both hemocyanins stimulate the immune system of mammals in a similar way by inducing a potent Thl-polarized cellular and humoral response.
Topics: Adjuvants, Immunologic; Animals; Cancer Vaccines; Hemocyanins; Mollusca; Vaccines
PubMed: 21773663
DOI: No ID Found -
BMC Evolutionary Biology Jul 2018Hemocyanin transports O in the hemolymph of many arthropod species. Such respiratory proteins have long been considered unnecessary in Myriapoda. As a result, the...
BACKGROUND
Hemocyanin transports O in the hemolymph of many arthropod species. Such respiratory proteins have long been considered unnecessary in Myriapoda. As a result, the presence of hemocyanin in Myriapoda has long been overlooked. We analyzed transcriptome and genome sequences from all major myriapod taxa - Chilopoda, Diplopoda, Symphyla, and Pauropoda - with the aim of identifying hemocyanin-like proteins.
RESULTS
We investigated the genomes and transcriptomes of 56 myriapod species and identified 46 novel full-length hemocyanin subunit sequences in 20 species of Chilopoda, Diplopoda, and Symphyla, but not Pauropoda. We found in Cleidogona sp. (Diplopoda, Chordeumatida) a hemocyanin-like sequence with mutated copper-binding centers, which cannot bind O. An RNA-seq approach showed markedly different hemocyanin mRNA levels from ~ 6 to 25,000 reads per kilobase per million reads. To evaluate the contribution of hemocyanin to O transport, we specifically studied the hemocyanin of the centipede Scolopendra dehaani. This species harbors two distinct hemocyanin subunits with low expression levels. We showed cooperative O binding in the S. dehaani hemolymph, indicating that hemocyanin supports O transport even at low concentration. Further, we demonstrated that hemocyanin is > 1500-fold more highly expressed in the fertilized egg than in the adult.
CONCLUSION
Hemocyanin was most likely the respiratory protein in the myriapod stem-lineage, but multiple taxa may have independently lost hemocyanin and thus the ability of efficient O transport. In myriapods, hemocyanin is much more widespread than initially appreciated. Some myriapods express hemocyanin only at low levels, which are, nevertheless, sufficient for O supply. Notably, also in myriapods, a non-respiratory protein similar to insect storage hexamerins evolved from the hemocyanin.
Topics: Amino Acid Sequence; Animals; Arthropods; Base Sequence; Binding Sites; Copper; Evolution, Molecular; Genetic Variation; Hemocyanins; Monophenol Monooxygenase; Oxygen; Phylogeny; Protein Subunits; RNA, Messenger
PubMed: 29976142
DOI: 10.1186/s12862-018-1221-2 -
Journal of Experimental Zoology. Part... Feb 2021Terrestrial gastropods express metal-selective metallothioneins (MTs) by which they handle metal ions such as Zn , Cd , and Cu /Cu through separate metabolic pathways....
Terrestrial gastropods express metal-selective metallothioneins (MTs) by which they handle metal ions such as Zn , Cd , and Cu /Cu 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 β). We have examined the effect of Cd and Cu 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.
Topics: Animals; Base Sequence; Cadmium; Copper; DNA, Complementary; Gene Expression Regulation; Hemocyanins; Metallothionein; Metals; Snails
PubMed: 33146003
DOI: 10.1002/jez.2425 -
Frontiers in Immunology 2021Hemocyanin is an important non-specific innate immune defense molecule with phenoloxidase, antiviral, antibacterial, hemolytic, and antitumor activities. To better...
Hemocyanin is an important non-specific innate immune defense molecule with phenoloxidase, antiviral, antibacterial, hemolytic, and antitumor activities. To better understand the mechanism of functional diversity, proteomics approach was applied to characterize hemocyanin (HMC) expression profiles from . At first, hemocyanin was purified by Sephadex G-100 and DEAE-cellulose (DE-52) columns from shrimp serum, and 34 protein spots were identified as HMC on the 2-DE gels. Furthermore, we found that 9 HMC spots about 75 or 77 kDa were regulated by and infection at 6, 12, and 24 h. In addition, 6 different pathogen-binding HMC fractions, viz., HMC-Mix, HMC-Vp, HMC-Va, HMC-Vf, HMC-Ec, and HMC-Sa, showed different agglutinative and antibacterial activities. Moreover, lectin-blotting analysis showed significant differences in glycosylation level among HMC isomers and bacteria-binding HMC fractions. Particularly, the agglutinative activities of the HMC fractions were almost completely abolished when HMC was deglycosylated by O-glycosidase, which suggest that O-linked sugar chains of HMC played important roles in the innate immune recognition. Our findings demonstrated for the first time that HMC had molecular diversity in protein level, which is closely associated with its ability to recognize diverse pathogens, whereas glycan modification probably contributed to HMC's diversity and multiple immune activities.
Topics: Agglutination; Animals; Bacterial Infections; Glycosylation; Hemocyanins; Lectins; Penaeidae
PubMed: 34950141
DOI: 10.3389/fimmu.2021.772091