Hemocyanin from Shrimp Litopenaeus vannamei Has Antiproliferative Effect against HeLa Cell In Vitro.

Hemocyanin (HMC) has been shown to participate in multiple roles of immune defence. In this study, we investigated the antiproliferative effect and underpinning mechanism of HMC from Litopenaeus vannamei in vitro. Sulforhodamine B (SRB) assay indicated that HMC could dramatically inhibit the growth of HeLa cells, but not 293T cells under the same conditions. Moreover, typical morphological features of apoptosis in HeLa cells including the formation of apoptotic body-like vesicles, chromatin condensation and margination were observed by using 4, 6-diamidino-2- phenylindole dihydrochloride (DAPI) staining and fluorescence analysis. An apoptotic DNA ladder from 180 to 300 bp was also detected. Furthermore, 10 variation proteins associated with apoptosis pathway, viz. G3PDH isoforms 1/2 (G3PDH1/2), aldosereductase, ectodemal dysplasia receptor associated death receptor domain isoform CRA_a (EDARADD), heat shock 60kD protein 1 variant 1 (HSP60), heat shock 70kDa protein 5 precursor (HSP70), heat shock protein 90kDa beta member 1 precursor (HSP90), 14-3-3 protein ζ/δ, Ran and ubiquitin activating enzyme E1(UBE1), were identified from HMC-treated HeLa cells by the proteomic and quantitative real-time RT-PCR strategies. Importantly, the reactive oxygen species (ROS), mitochondrial membrane potential (Δψm) and caspase-9/3 activities were changed significantly in HMC-treated HeLa cells. Together, the data suggests that L. vannamei HMC mediates antiproliferative properties through the apoptosis mechanism involving the mitochondria triggered pathway.

Diverse immune functions of hemocyanins.

Substantial evidence gathered recently has revealed the multiple functionalities of hemocyanin. Contrary to previous claims that this ancient protein is involved solely in oxygen transport within the hemolymph of invertebrates, hemocyanin and hemocyanin-derived peptides have been linked to key aspects of innate immunity, in particular, antiviral and phenoloxidase-like activities. Both phenoloxidase and hemocyanin belong to the family of type-3 copper proteins and share a high degree of sequence homology. While the importance of phenoloxidase in immunity and development is well characterised, the contribution of hemocyanin to biological defence systems within invertebrates is not recognised widely. This review focusses on the conversion of hemocyanin into a phenoloxidase-like enzyme and the array of hemocyanin-derived immune responses documented to date.

The effects of light stimulation during incubation on indicators of stress susceptibility in broilers.

Providing light during incubation has been shown to decrease bilateral physical asymmetry of broilers posthatch, which may indicate that early light stimulation reduces later stress susceptibility. This experiment evaluated the effects of lighting during embryogenesis on other measures of stress responsiveness in broilers. Cobb 500 eggs (n = 1,404) were incubated under 0L:24D, 1L:23D, 6L:18D, or 12L:12D; the light level was 550 lx. The broilers were then raised in floor pens under a 12L:12D lighting regimen, and various stress parameters were measured during wk 3 to 6 of age. There was an effect of incubation lighting regimen on posthatch stress responses. Following 1 h of crating, the change in corticosterone (CORT) concentration was less in the 12L:12D (-0.06 ng/mL, P < 0.05) treatment than all other treatments (pooled mean = 0.24 ng/mL); however, there were no treatment differences in CORT response to adrenocorticotropic hormone administration (pooled mean pre- vs. 1 h postadministration = 17.5 ng/mL, P > 0.05). Anti-keyhole limpet hemocyanin titers were higher in the 12L:12D birds (92,395 units/mL; P < 0.05) than all other treatments (pooled mean = 68,407 units/mL) on d 1 postcrating. Additionally, composite asymmetry scores were lower in the 12L:12D treatment (0.92 mm) than all other treatments (pooled mean = 1.14 mm, P < 0.05). These results demonstrate that providing 12 h of light per day during incubation can reduce the stress susceptibility of broilers posthatch.

A broad genomic survey reveals multiple origins and frequent losses in the evolution of respiratory hemerythrins and hemocyanins.

Hemerythrins and hemocyanins are respiratory proteins present in some of the most ecologically diverse animal lineages; however, the precise evolutionary history of their enzymatic domains (hemerythrin, hemocyanin M, and tyrosinase) is still not well understood. We survey a wide dataset of prokaryote and eukaryote genomes and RNAseq data to reconstruct the phylogenetic origins of these proteins. We identify new species with hemerythrin, hemocyanin M, and tyrosinase domains in their genomes, particularly within animals, and demonstrate that the current distribution of respiratory proteins is due to several events of lateral gene transfer and/or massive gene loss. We conclude that the last common metazoan ancestor had at least two hemerythrin domains, one hemocyanin M domain, and six tyrosinase domains. The patchy distribution of these proteins among animal lineages can be partially explained by physiological adaptations, making these genes good targets for investigations into the interplay between genomic evolution and physiological constraints.

Influence of temperature, hypercapnia, and development on the relative expression of different hemocyanin isoforms in the common cuttlefish Sepia officinalis.

The cuttlefish Sepia officinalis expresses several hemocyanin isoforms with potentially different pH optima, indicating their reliance on efficient pH regulation in the blood. Ongoing ocean warming and acidification could influence the oxygen-binding properties of respiratory pigments in ectothermic marine invertebrates. This study examined whether S. officinalis differentially expresses individual hemocyanin isoforms to maintain optimal oxygen transport during development and acclimation to elevated seawater pCO(2) and temperature. Using quantitative PCR, we measured relative mRNA expression levels of three different hemocyanin isoforms in several ontogenetic stages (embryos, hatchlings, juveniles, and adults), under different temperatures and elevated seawater pCO(2). Our results indicate moderately altered hemocyanin expression in all embryonic stages acclimated to higher pCO(2), while hemocyanin expression in hatchlings and juveniles remained unaffected. During the course of development, total hemocyanin expression increased independently of pCO(2) or thermal acclimation status. Expression of isoform 3 is reported for the first time in a cephalopod in this study and was found to be generally low but highest in the embryonic stages (0.2% of total expression). Despite variable hemocyanin expression, hemolymph total protein concentrations remained constant in the experimental groups. Our data provide first evidence that ontogeny has a stronger influence on hemocyanin isoform expression than the environmental conditions chosen, and they suggest that hemocyanin protein abundance in response to thermal acclimation is regulated by post-transcriptional/translational rather than by transcriptional modifications.

Functional properties of hemocyanin from Oncomelania hupensis, the intermediate host of Schistosoma japonicum.

The gastropod mollusc, Oncomelania hupensis is a unique intermediate host for the human parasite Schistosoma japonicum. It is a primary factor for the epidemic of schistosomiasis and its distribution is consistent with the epidemic area of schistosomiasis. Here we report the functional properties of hemocyanin of O. hupensis (OhH), a copper-containing respiratory protein which was isolated from its hemolymph and purified by ammonium sulfate fractionation and ultracentrifugation. We identified the protein characters including UV absorption at 340 nm, copper content and quaternary structure. Furthermore, by induction of phenoloxidase and enzyme-linked immunosorbent assay we show that OhH exhibited o-diphenoloxidase activity after limited proteolysis, and shared carbohydrate epitopes with glycoconjugates of S. japonicum.

Structural and functional characterization of haemocyanin from the anemone hermit crab Dardanus calidus.

Oxygen-binding to haemocyanin (Hc) is generally an exothermic process, with overall enthalphy of oxygenation varying from species to species. A number of crustacean Hcs showed a null or reduced enthalphy of oxygenation, among others, the anomuran Pagurus bernhardus and Paralithodes camtscaticae possess a completely temperature-independent oxygen-binding in a wide range of temperature and pH. Functional analysis performed on purified native, hexameric and dodecameric Hc forms of the anemone hermit crab Dardanus calidus allowed to calculate the enthalphy of oxygenation values that resulted equal to -36.2, -33.8 and -26.8 kJ/mol, respectively. Thus, the temperature sensitivity of oxygen binding of D. calidus Hc is in contrast with the temperature independence reported for P. bernhardus and P. camtscaticae, suggesting a high Hc functional heterogeneity within Anomura. Functional characterization also evidenced a strong oxygen affinity modulation by protons (DeltalogP(50)/DeltapH = -0.97) and lactate [DeltalogP(50)/Deltalog(lactate) = -0.38], and a significant decrease in cooperativity by physiological concentration of lactate (n(50) from 2.8 to 1.7 at pH 7.5).

Respiratory protein-generated reactive oxygen species as an antimicrobial strategy.

The evolution of the host-pathogen relationship comprises a series of invasive-defensive tactics elicited by both participants. The stereotype is that the antimicrobial immune response requires multistep processes. Little is known about the primordial immunosurveillance system, which probably has components that directly link sensors and effectors. Here we found that the respiratory proteins of both the horseshoe crab and human were directly activated by microbial proteases and were enhanced by pathogen-associated molecular patterns, resulting in the production of more reactive oxygen species. Hemolytic virulent pathogens, which produce proteases as invasive factors, are more susceptible to this killing mechanism. This 'shortcut' antimicrobial strategy represents a fundamental and universal mode of immunosurveillance, which has been in existence since before the split of protostomes and deuterostomes and still persists today.

The hemocyanin of the shamefaced crab Calappa granulata: structural-functional characterization.

Arthropod hemocyanins (Hcs) transport and store oxygen and are composed of six subunits, or multiples thereof depending on the species. Calappa granulata Hc is found as a mixture of dodecamers (95%) and hexamers (5%). Removal of calcium ions and alkaline pH induce an incomplete partially reversible dissociation of dodecameric Hc. Two-dimensional electrophoretic pattern of dissociated Hc indicated a large heterogeneity in Hc subunit: most differences are likely to be explained by post-translational modifications. Dodecameric Hc showed a large Bohr effect (Deltalog P50/DeltapH = -0.95) and a normal cooperativity (h50 values = 2.7 +/- 0.2) in the presence of 10 mM CaCl2. The hexameric molecule displayed lower Bohr effect and cooperativity than the dodecamer. Lactate effect on the oxygen affinity (Deltalog P50 = 0.55) and the increase of lactate concentrations in animals kept in emersion were related to the increased oxygen requirements that occur during hypoxia in vivo. Calcium affects oxygen affinity only at high concentrations: this Hc appeared to lack the calcium high-affinity binding sites found in other species. The effect of temperature on both oxygen affinity and cooperativity was measured in the absence and presence of 10 mM lactate, allowing calculation of the exothermic contribution of lactate binding (DeltaH = -25 kJ mol(-1)).

Hemocyanin of the molluscan Concholepas concholepas exhibits an unusual heterodecameric array of subunits.

We describe here the structure of the hemocyanin from the Chilean gastropod Concholepas concholepas (CCH), emphasizing some attributes that make it interesting among molluscan hemocyanins. CCH exhibits a predominant didecameric structure as revealed by electron microscopy and a size of 8 MDa by gel filtration, and, in contrast with other mollusc hemocyanins, its stabilization does not require additional Ca(2+) and/or Mg(2+) in the medium. Polyacrylamide gel electrophoresis studies, analyses by a MonoQ FPLC column, and Western blots with specific monoclonal antibodies showed that CCH is made by two subunits noncovalently linked, named CCH-A and CCH-B, with molecular masses of 405 and 350 kDa, respectively. Interestingly, one of the subunits undergoes changes within the macromolecule; we demonstrated that CCH-A has an autocleavage site that under reducing conditions is cleaved to yield two polypeptides, CCH-A1 (300 kDa) and CCH-A2 (108 kDa), whereas CCH-B remains unchanged. The CCH-A nick occurs at 4 degrees C, increases at 37 degrees C, and is not inhibited by the addition of protease inhibitors and/or divalent cations. Since the CCH structure is a heterodimer, we investigated whether subunits would be either intermingled, forming heterodecamers, or assembled as two homogeneous decamers. Light scattering and electron microscope studies of the in vitro reassociation of purified CCH subunits demonstrated that the sole addition of Mg(2+) is needed for its reassembly into the native decameric molecule; no homodecamer reorganization was found with either CCH-A or CCH-B subunits alone. Our evidence showed that C. concholepas hemocyanin is an unusual example of heterodecameric organization.

Respiratory adaptations to the deep-sea hydrothermal vent environment: the case of Segonzacia mesatlantica, a crab from the Mid-Atlantic Ridge.

Segonzacia mesatlantica (Crustacea; Decapoda; Brachyura) is the only endemic crab species known from the Mid-Atlantic Ridge (MAR) hydrothermal vents. Known from all explored sites in the Atlantic, its wide distribution makes this species a model to study physiological adaptation, and specifically respiratory strategies. Native haemocyanin (Hc) comprises four non-covalent associations in equilibrium formed by monomers, hexamers, dodecamers and octadecamers made up of approximately 75 kDa polypeptide chains. Four different amino acid chains are observed with a molecular mass ranging from 75,234 to 75,972 Da. Experiments carried-out under pressure suggested that the percentage of monomer increased in the haemolymph under hypoxic condition. We have also observed a shift of the proportion of the two dodecamer series, suggesting a rapid modification of the Hc phenotype between hypoxic and hyperoxic conditions. Native Hc possesses a high oxygen affinity ( P50 = 2.2 Torr at 15 degrees C and pH 7.5), a large Bohr effect (Deltalog P50 / DeltapH approximately -2.7) and a slightly reverse temperature effect (DeltaH = +17.19 kJ mol(-1). The composition of Segonzacia haemolymph is similar to that of other littoral species except for the large enrichment in free copper and zinc. As for other species from hydrothermal vent sites, Segonzacia haemolymph possesses a higher buffer capacity than littoral species. Moreover, species from the hydrothermal vent decapods from Pacific hydrothermal vent that encounter higher CO2 content in their environment have a higher buffer capacity than Atlantic vent species. The results presented are discussed in relation with the physico-chemical characteristics of the hydrothermal vent environment.

Resistance to stress and Hc functional modulation in Liocarcinus sp.

This study is included in a project aimed to study the alterations on the structure of the Northern Adriatic Sea ecosystem produced by fishing activity. The indirect or secondary effects of fishery such as the changes of the structure and trophic relationships of the ecosystem are under investigation and we have particularly considered the effects on species such as Liocarcinus depurator that are captured and then rejected because devoid of commercial value. The objective of this study is the Liocarcinus sp. adaptative resistance to stress and the effects of biochemical parameters (allosteric effectors) on Hc functional modulation.

The murine myeloid cell line 32Dcl3 as a model system for studying neutrophil functions.

The murine myeloid cell line 32Dcl3 is one of the few cell lines that can terminally differentiate into neutrophils. Granulocyte colony-stimulating factor (G-CSF) drives the differentiation of these cells; therefore, G-CSF receptor signaling for neutrophil proliferation and differentiation has been studied extensively using this cell line as a model. Differentiated 32Dcl3 cells exhibit a striking morphologic similarity to normal neutrophils; however, the degree to which differentiated 32Dcl3 cells are functionally similar to normal neutrophils remains unknown. In this study, we compared the function of differentiated 32Dcl3 cells with mouse neutrophils. Our results demonstrate that a subclone of differentiated 32Dcl3 cells (32Dcl3C) exhibits normal neutrophil functions of phagocytosis, degranulation, adhesion and shape change in response to appropriate stimuli. These observations suggest that this cell line can serve as an effective model system to study similar mature neutrophil functions. However, 32Dcl3C cells fail to produce superoxide in response proper stimuli.

Structural basis for salinity-induced alteration in oxygen binding by haemocyanin from the estuarine amphipod Chaetogammarus marinus (L.).

In the estuarine amphipod Chaetogammarus marinus, differences in O(2) binding by haemocyanins (Hc) could be related to natural and salinity-related quantitative variation in just one polypeptide subunit (band 2) and not to variations in any of the other seven bands present. Band 2 was always present, irrespective of salinity treatment, and naturally makes up 6%-36% of the total Hc. However, low salinity exposure (S=4 per thousand for 48-49 h, T=15 degrees C) was accompanied by an increase in the prevalence of greater concentrations of band 2 (i.e., range, 20%-36% of total Hc present) and a concomitant increase in Hc-O(2) affinity (half saturation [P(50)] decreased from 1.38 to 1.12 kPa at pH=7.81, T=15 degrees C). A similar salinity-related mechanism (that is, one band altering) has been shown previously for the blue crab Callinectes sapidus, although the functional consequences were different. In contrast with C. marinus, an increase in the proportion of one polypeptide subunit in C. sapidus resulted in a decrease in Hc-O(2) affinity. This study has confirmed that between-individual variation (quantitative rather than qualitative) in just one Hc subunit may have functional consequences, although the significance of such variation is difficult to interpret.

Origin and evolution of arthropod hemocyanins and related proteins.

Arthropod hemocyanins are large, multimeric, (n x 6) copper-containing proteins that deliver oxygen in the haemolymph of many chelicerate, crustacean, myriapod, and also possibly some insect species. The arthropod hemocyanins belong to a large protein superfamily that also includes the arthropod phenoloxidases, certain crustacean and insect storage proteins (pseudo-hemocyanins and hexamerins), and the insect hexamerin receptors. Here I summarise the present knowledge of the origin, functional adaptations, and evolution of these proteins. Arthropod and mollusc hemocyanins are, if at all, only distantly related. As early as in the arthropod stem line, the hemocyanins emerged from a phenoloxidase-like enzyme. The evolution of distinct hemocyanin subunits, as well as the formation of multi-hexamers occurred independently within the arthropod subphyla. Hemocyanin subunit evolution is strikingly different in the Chelicerata, Myriapoda and Crustacea. Hemocyanins individually gave rise to two distinct copper-less storage proteins, the insect hexamerins and the crustacean pseudo-hemocyanins (cryptocyanins). The receptor responsible for the uptake of hexamerin by the larval fat body of the insects emerged from a hexamerin-precursor. Molecular phylogenetic analyses show a close relationship of the crustacean and insect proteins, providing strong support for a pancrustacean taxon, while structural data suggest a myriapod-chelicerate clade.

Structural and functional properties of hemocyanin from Cyanagraea praedator, a deep-sea hydrothermal vent crab.

Cyanagraea praedator (Crustacea: Decapoda: Brachyura) is an endemic species of the East Pacific Rise hydrothermal vents, living in the upper part of black smoker chimneys. Because we were seeking species that have made respiratory adaptations to the hydrothermal environment, we looked at Cyanograea hemocyanin (Hc) and determined its quaternary structure and the oxygen-binding properties in relation to temperature, pH, and lactate. C. praedator Hc is composed of dodecamers and hexamers, with dodecamers formed by the perpendicular association of two hexamers. The composition of these polymers was determined by electrophoresis and, for the first time, by electrospray mass spectrometry. Dodecamers and hexamers are composed of six subunits common to the two forms, with molecular mass ranging from 75,008 Da to 75,534 Da. In addition, we found two dodecamer-specific subunits, at 75,419 Da and 75,629 Da. The native hemocyanin possesses a high oxygen affinity (P(50) varies between 4 and 10 Torr at pH 7.5, 15 degrees C) and a large Bohr coefficient (Delta log P(50)/DeltapH approximately -1.8). Oxygen affinity is not affected by lactate or, surprisingly, temperature between 5 degrees C and 35 degrees C (DeltaH = 1.16 kJ/mol(1) 5-35 degrees C). Dialysis of native hemolymph elicited a significant increase in Hc-O(2) affinity (DeltaP(50) = 2.5 Torr at pH 7.5), an effect opposite the usual trend observed for crustacean hemocyanins. In this article these functional properties are interpreted in relation to characteristics of the environment.

Respiratory and circulatory compensation to hypoxia in crustaceans.

Crustaceans are often tolerant of hypoxic exposure and many regulate O(2) consumption at low ambient O(2). In acute hypoxia, most increase branchial water flow, and many also increase branchial haemolymph flow, both by an increase in cardiac output and by shunting flow away from the viscera. The O(2)-binding affinity of crustacean O(2) carriers increases in hypoxic conditions, as a result of hyperventilation induced alkalosis. In chronic hypoxic exposure some crustaceans do not sustain high ventilatory pumping levels but increased effectiveness of O(2)-uptake across the gills is maintained as a result of the build up of metabolites such as lactate and urate which also function to increase the haemocyanin O(2)-binding affinity. Chronic exposure to hypoxia also may increase O(2)-binding capacity and promote the synthesis of new high O(2)-affinity carrier molecules. Exposure to untenable rates or levels of O(2) depletion causes many decapodan crustaceans to surface and ventilate the gills with air. Burrowing crayfish provide an example of animals, which excel in all these mechanisms. Control mechanisms involved in compensatory responses to hypoxia are discussed.