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).     

An oxygen transporter hemocyanin can act on the late pathway of melanin synthesis

5,6-Dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA) are precursors of eumelanin. The effects of crustacean hemolymph proteins on these eumelanin-related metabolites were investigated. Zymogram analysis indicated that polymers of hemocyanin (Hc) subunits converted DHI into black pigment while no effects were observed using DHICA as a substrate. Spectrum changes for mixtures of purified Hc and DHI showed a profile similar to oxidized DHI by mushroom tyrosinase while Hc had only slight effects on DHICA. Typical inhibitors of tyrosinase and phenoloxidase severely hampered the production of oxidized DHI. Taken together with previous results, these data indicate that Hc plays a crucial role in the conversion of DHI in the hemolymph of crustaceans, which promotes late reactions in the melanin synthetic pathway as well as early reactions (oxidation of tyrosine and DOPA to dopaquinone).     

Subunit compositions of crustacean haemocyanins are species-specific: evidence from non-decapod species

Electrophoretic examination of dissociated haemocyanin subunits from a number of amphipod, decapod and isopod crustaceans supports the hypothesis that subunit composition is species-specific, despite marked within-species variation in many species. General patterns of heterogeneity on native PAGE gels were also evident between groupings within the Amphipoda. Gammarid amphipods could be split into two groups; one characterised by a high degree of heterogeneity and the other by a low degree of heterogeneity. The talitrid amphipods generally displayed a low degree of heterogeneity similar to, although still distinct from, the second gammarid category. Haemocyanin from the Hyalidae, a family allied to the talitrids was highly heterogeneous, similar to the first gammarid group and unlike the talitrids. Isopod haemocyanin banding patterns were more similar to one another than to any of the amphipod or decapod species examined. In general, the molecular weights of the amphipod Hcs tended to be greater than those of the isopods, with the decapods being lowest of all. It is suggested that Hc subunit heterogeneity may be a useful tool for investigating speciation and speciation events, and for reliably separating very closely-related species (e.g. Gammarus spp.), purely on the basis of their Hc subunit compositions.     

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)).     

Identification and characterization of a hemocyanin-derived phenoloxidase from the crab Charybdis japonica

To determine effective activators of crab hemocyanin (Hc) and the properties of Hc-derived phenoloxidase (HdPO), Hc, for the first time, was purified from hemolymph of Charybdis japonica, and the properties of activated HdPO were studied by using L-DOPA as a substrate. Three distinct subunits were isolated, and each had a molecular mass of about 80, 75 and 70 kDa, respectively. SDS and HLS were much effective in conversion of Hc into HdPO whose PO activity was optimal at pH 7.0 and temperature of 40 °C. The Km value of the HdPO was 2.90 mM for L-DOPA and 7.33 mM for tyrosine. The PO activity of HdPO was most sensitive to 1-phenyl-2-thiourea, cysteine and ascorbic acid, and much sensitive to thio urea and sodium sulfite. Based on its inhibition characteristics and the substrate specificity, this HdPO could be classified as a kind of tyrosinase-type phenoloxidase. The PO activity of HdPO was also strongly inhibited by Cu²⁺, Zn²⁺, ethylenediaminetetraacetic acid (EDTA) and diethyldithiocarbamate (DETC). The results with EDTA, DETC, and some metal ions, combined with the perfect recovery effect of Cu²⁺ on DETC-inhibited PO activity, indicate that the HdPO is a kind of copper-containing metalloenzyme. All these imply that the Hc, as an oxygen carrier, can be activated to have PO activities by SDS or HLS, and the activated HdPO has the properties of a tyrosinase-type copper-containing phenoloxidase. This study makes us to understand more easily the multifunctions of crustacean Hc in oxygen carrier and melaninization at certain stresses in host defence as well.     

Reversible heat inactivation of copper sites precedes thermal unfolding of molluscan (Rapana thomasiana) hemocyanin

Hemocyanin (Hc) is a type-3 copper protein, containing dioxygen-binding active sites consisting of paired copper atoms. In the present study the thermal unfolding of the Hc from the marine mollusc Rapana thomasiana (RtH) has been investigated by combining differential scanning calorimetry, Fourier transform infrared (FTIR) and UV-vis absorption spectroscopy. Two important stages in the unfolding pathway of the Hc molecule were discerned. A first event, with nonmeasurable heat absorption, occurring around 60°C, lowers the binding of dioxygen to the type-3 copper groups. This pretransition is reversible and is ascribed to a slight change in the tertiary structure. In a second stage, with midpoint around 80°C, the protein irreversibly unfolds with a loss of secondary structure and formation of amorphous aggregates. Experiments with the monomeric structural subunits, RtH1 and RtH2, indicated that the heterogeneity in the process of thermal denaturation can be attributed to the presence of multiple 50kDa functional units with different stability. In accordance, the irreversible unfolding of a purified functional unit (RtH2-e) occurred at a single transition temperature. At slightly alkaline pH (Tris buffer) the C-terminal β-sheet rich domain of the functional unit starts to unfold before the α-helix-rich N-terminal (copper containing) domain, triggering the collapse of the global protein structure. Even around 90°C some secondary structure is preserved as shown by the FTIR spectra of all investigated samples, confirming the high thermostability of molluscan Hc.     

Reversible heat inactivation of copper sites precedes thermal unfolding of molluscan (Rapana thomasiana) hemocyanin

Hemocyanin (Hc) is a type-3 copper protein, containing dioxygen-binding active sites consisting of paired copper atoms. In the present study the thermal unfolding of the Hc from the marine mollusc Rapana thomasiana (RtH) has been investigated by combining differential scanning calorimetry, Fourier transform infrared (FTIR) and UV–vis absorption spectroscopy. Two important stages in the unfolding pathway of the Hc molecule were discerned. A first event, with nonmeasurable heat absorption, occurring around 60 °C, lowers the binding of dioxygen to the type-3 copper groups. This pretransition is reversible and is ascribed to a slight change in the tertiary structure. In a second stage, with midpoint around 80 °C, the protein irreversibly unfolds with a loss of secondary structure and formation of amorphous aggregates. Experiments with the monomeric structural subunits, RtH1 and RtH2, indicated that the heterogeneity in the process of thermal denaturation can be attributed to the presence of multiple 50 kDa functional units with different stability. In accordance, the irreversible unfolding of a purified functional unit (RtH2-e) occurred at a single transition temperature. At slightly alkaline pH (Tris buffer) the C-terminal β-sheet rich domain of the functional unit starts to unfold before the α-helix-rich N-terminal (copper containing) domain, triggering the collapse of the global protein structure. Even around 90 °C some secondary structure is preserved as shown by the FTIR spectra of all investigated samples, confirming the high thermostability of molluscan Hc.     

Regulation of the Chlamydia trachomatis histone H1-like protein Hc2 is IspE dependent and IhtA independent

The chlamydial histone-like proteins, Hc1 and Hc2, function as global regulators of chromatin structure and gene expression. Hc1 and Hc2 expression and activity are developmentally regulated. A small metabolite that disrupts Hc1 interaction with DNA also disrupts Hc2 interactions; however, the small regulatory RNA that inhibits Hc1 translation is specific to Hc1.     

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.     

Identification of heat shock protein 60 as the ligand on Histoplasma capsulatum that mediates binding to CD18 receptors on human macrophages

Histoplasma capsulatum (Hc), is a facultative intracellular fungus that binds to CD11/CD18 receptors on macrophages (Mphi). To identify the ligand(s) on Hc yeasts that is recognized by Mphi, purified human complement receptor type 3 (CR3, CD11b/CD18) was used to probe a Far Western blot of a detergent extract of Hc cell wall and cell membrane. CR3 recognized a single 60-kDa protein, which was identified as heat shock protein 60 (hsp60). Biotinylation of viable yeasts, followed by precipitation with streptavidin-coated beads, and Western blotting with anti-hsp60 demonstrated that hsp60 was on the surface of Hc yeasts. Electron and confocal microscopy revealed that hsp60 resided on the yeast cell wall in discrete clusters. Recombinant hsp60 (rhsp60) inhibited attachment of Hc yeasts to Mphi. Recombinant hsp60 and Abs to CD11b and CD18 inhibited binding of yeasts to Chinese hamster ovary cells transfected with CR3 (CHO3). Polystyrene beads coated with rhsp60 bound to Mphi, and attachment was inhibited by Abs to CD11 and CD18. Freeze/thaw extract (F/TE), a preparation of Hc yeast surface proteins that contained hsp60, inhibited the attachment of Hc yeasts to Mphi. Depletion of hsp60 from F/TE removed the capacity of F/TE to block binding of Hc to Mphi. Interestingly, rhsp60 did not inhibit binding of Hc yeasts to dendritic cells (DC), which recognize Hc via very late Ag 5. Moreover, F/TE inhibited attachment of Hc to DC even when depleted of hsp60. Thus, Hc hsp60 appears to be a major ligand that mediates attachment of Hc to Mphi CD11/CD18, whereas DC recognize Hc via a different ligand(s).     

意大利蝗卵发育过程中血蓝蛋白基因表达分析

【目的】意大利蝗Calliptamus italicus是新疆草原的主要优势危害种,以卵在土壤中越冬。呼吸代谢可反映蝗卵的生理状态,呼吸蛋白对于呼吸系统不完善的蝗卵尤为重要。本研究旨在明确意大利蝗卵发育过程中血蓝蛋白基因的表达情况。【方法】采用实时荧光定量PCR方法检测不同发育阶段的蝗卵以及1龄蝗蝻的血蓝蛋白2个亚基基因Hc1和Hc2的表达量。【结果】根据解剖形态观察,将意大利蝗越冬卵的整个发育过程分为10个阶段,包括9个卵发育阶段(C-Ⅰ-C-Ⅹ)和1龄蝗蝻阶段(C-Ⅹ)。Hc1和Hc2在越冬蝗卵各发育阶段以及1龄蝗蝻中均有表达。其中,在蝗卵早期发育阶段(C-Ⅰ, C-Ⅱ和C-Ⅲ),Hc1表达量逐渐增加,C-Ⅲ阶段表达量显著高于C-Ⅰ和C-Ⅱ阶段;滞育阶段(C-Ⅳ, C-Ⅴ和C-Ⅵ),胚胎发育停滞,Hc1表达量较C-Ⅲ,C-Ⅶ和C-Ⅷ阶段低;滞育后发育阶段(C-Ⅶ和C-Ⅷ),蝗卵解除滞育,快速发育,Hc1表达量较早期发育阶段和滞育阶段高,其中,C-Ⅷ阶段Hc1表达量最高(212.3156±10.5470),显著高于其他所有阶段;1龄蝗蝻(C-Ⅹ)的Hc1表达量最低,为0.4017±0.1010。Hc2表达量在C-Ⅴ阶段最高(679.7511±54.5719),显著高于其他所有阶段;除C-Ⅴ阶段外,其他各阶段之间Hc2表达量差异均不显著。Hc1在蝗卵滞育后阶段高表达,而Hc2在蝗卵滞育阶段高表达。【结论】血蓝蛋白亚基基因Hc1和Hc2在整个意大利蝗卵发育过程均有表达,且具有阶段特异性。Hc1与Hc2协同作用为蝗卵发育供氧,其中,Hc1主要负责蝗卵滞育后发育期间的氧气运载,而Hc2主要维持滞育期间的氧气运载,且载氧效率较低。研究结果可为进一步探讨意大利蝗卵的抗逆机制提供科学依据。     

Molecular cloning and expression of hctB encoding a strain-variant chlamydial histone-like protein with DNA-binding activity.

Two DNA-binding proteins with similarity to eukaryotic histone H1 have been described in Chlamydia trachomatis. In addition to the 18-kDa histone H1 homolog Hc1, elementary bodies of C. trachomatis possess an antigenically related histone H1 homolog, which we have termed Hc2, that varies in apparent molecular mass among strains. We report the molecular cloning, expression, and nucleotide sequence of the hctB gene encoding Hc2 and present evidence for in vivo DNA-binding activity of the expressed product. Expression of Hc2 in Escherichia coli induces a compaction of bacterial chromatin that is distinct from that observed upon Hc1 expression. Moreover, isolated nucleoids from Hc2-expressing E. coli exhibit markedly reduced sensitivity to DNase I. These properties of Hc2 are consistent with a postulated role in establishing the nucleoid structure of elementary bodies.     

大陈岛礁海域甲壳类群落结构时空格局及其环境驱动

为探究局地尺度下近岸岛礁海域之多变水体环境、极高空间异质性对生物群落的影响,于2020—2021年4个季度在大陈岛礁海域底拖网采样,利用等级聚类(cluster)、非度量多维标度(NMDS)等多元统计方法分析甲壳类群落结构的时空格局,并整合温度、盐度、深度等环境因子进行冗余分析(RDA)以解析影响因素,同时利用丰度生物量曲线(ABC)及W统计量评估群落稳态。结果显示：全年共采集甲壳类53种,隶属2目13科29属,虾类占总种类数的56.60%,种类数秋季(43种)>冬季(41种)>春季(31种)>夏季(26种)。优势种的季节更替明显,夏秋季以哈氏仿对虾(Parapenaeopsis hardwickii)、中华管鞭虾(Solenocera crassicornis)和三疣梭子蟹(Portunus trituberculatus)为代表的中大型经济种为主,冬春季则以双斑蟳(Charybdis bimaculata)和细巧仿对虾(Parapenaeopsis tenella)为代表的小个体饵料种为主,口虾蛄(Oratosquilla oratotria)在各个季节皆为优势种...     

Hemocyte components in crustaceans convert hemocyanin into a phenoloxidase-like enzyme

The functional conversion of hemocyanin (Hc), an oxygen transporter, into an enzyme was investigated in crustaceans. Hc is converted into a phenoloxidase-like enzyme by hemocyte components, which is triggered by beta-1,3-glucan. This activation is severely hampered with leupeptin and E-64 treatment, indicating that the serine/cysteine proteases in the hemocytes are involved in the activation. In a SDS-PAGE-analysis, no change was observed between normal and activated Hc under reduced conditions. However, under non-reduced condition of normal Hc, several minor bands were observed at oligomeric position of Hc subunit, which disappeared upon activation. These results indicate that a split of the reductive bond, such as the disulfide bond between subunits, is essential for Hc activation. This is the first report to show the enzymatic conversion of Hc and the presence of the covalent bond in the Hc subunit of crustaceans.     

Interaction between the two subdomains of the C-terminal part of the botulinum neurotoxin A is essential for the generation of protective antibodies

The botulinum neurotoxin A C-terminal fragment (Hc), which mediates the binding of the toxin to neuronal cell surface receptors, comprises two subdomains, Hc-N (amino acids 873-1095) and Hc-C (amino acids 1096-1296). In order to define the minimal fragment of Hc carrying protective antigenic properties, Hc, Hc-N and Hc-C have been produced as recombinant proteins in Escherichia coli, and have been tested for their antigenicity in mouse protection assays. Hc, Hc-N and Hc-C induced similar antibody levels as shown by ELISA. However, a single immunization with Hc (10 microg) fully protected mice challenged with 10(3) mouse lethal dose 50 of toxin, whereas Hc-N, Hc-C, or Hc-N plus Hc-C did not give any protection. Triple immunizations with Hc-N or Hc-C were necessary to induce a higher level of protection. Circular dichroism and fluorescence studies showed that the isolated subdomains were folded and stable. However, an intense near-UV dichroic signal was only observed in the Hc spectrum, revealing a highly structured interface between both subdomains. Taken together, the results show that the generation of protective antibodies requires the whole Hc domain and especially the native structure of the interfacial region between Hc-N and Hc-C.     

Histoplasma capsulatum cyclophilin A mediates attachment to dendritic cell VLA-5

Histoplasma capsulatum (Hc) is a pathogenic fungus that replicates in macrophages (Mphi). In dendritic cells (DC), Hc is killed and fungal Ags are processed and presented to T cells. DC recognize Hc yeasts via the VLA-5 receptor, whereas Mphi recognize yeasts via CD18. To identify ligand(s) on Hc recognized by DC, VLA-5 was used to probe a Far Western blot of a yeast freeze/thaw extract (F/TE) that inhibited Hc binding to DC. VLA-5 recognized a 20-kDa protein, identified as cyclophilin A (CypA), and CypA was present on the surface of Hc yeasts. rCypA inhibited the attachment of Hc to DC, but not to Mphi. Silencing of Hc CypA by RNA interference reduced yeast binding to DC by 65-85%, but had no effect on binding to Mphi. However, F/TE from CypA-silenced yeasts still inhibited binding of wild-type Hc to DC, and F/TE from wild-type yeasts depleted of CypA also inhibited yeast binding to DC. rCypA did not further inhibit the binding of CypA-silenced yeasts to DC. Polystyrene beads coated with rCypA or fibronectin bound to DC and Mphi and to Chinese hamster ovary cells transfected with VLA-5. Binding of rCypA-coated beads, but not fibronectin-coated beads, was inhibited by rCypA. These data demonstrate that CypA serves as a ligand for DC VLA-5, that binding of CypA to VLA-5 is at a site different from FN, and that there is at least one other ligand on the surface of Hc yeasts that mediates binding of Hc to DC.