Limulus Hemocyanin Research Articles

Sequencing the mitochondrial ATP6 synthase gene: comparing variations in Limulus and Argopecten to human SNPs

Mt‐ATP6 is a highly conserved gene belonging to the ATPase family, a group of enzymes required for cellular respiration in the electron transport chain. Human mutations in this gene result in neuropathy, ataxia, retinitis pigmentosa, and Leigh Syndrome. Target organisms: Argopecten irradians and Limulus polyphemus, are declining in Long Island Sound (LIS) respectively affecting the fishing industry and the availability of Limulus blue blood used in medicine.After extracting DNA from Limulus hemocyanin and Argopecten adductor muscle, two sets of overlapping primers were designed to encompass the approximate 700 base pairs of the ATP6 gene using the mitochondrial genome GenBank entries (NC_003057 and NC_012977) as templates. PCR protocols were constructed accordingly; subsequent electrophoresis revealed appropriate products which were then purified, diluted and quantitated using a biophotometer.At this writing, the primer sets will be further diluted to sequencing concentrations and the 38 templates of more than 400 base pairs each will be sequenced using the school's ABI Prism 310 Genetic Analyzer, a single capillary automated sequencer. Single nucleotide polymorphisms (SNPs) and translated protein sequences will be compared to the control GB entries, to each other, and to the human mt‐ATP6 gene using NCBI, ClustalW, the SNP Database, and MITOMAP data files.Women's Seamen's Friend Society of CTApplied BiosystemsWhatmanNOAA, Milford CTCarmela Cuomo, Ph.D

Leptin alters antibody isotype in the pig in vivo, but does not regulate cytokine expression or stimulate STAT3 signaling in peripheral blood monocytes in vitro.

Although leptin modulates immunological pathways in some species, the role of leptin as a regulator of immunocyte function in the pig has not been studied. Therefore, the primary objective of this study was to determine whether leptin influences specific immunocyte response variables in the pig in vivo or in vitro. Fifteen pigs (five pigs per treatment) were 1) injected with recombinant human leptin and allowed to consume feed ad libitum, 2) injected with vehicle and allowed to consume feed ad libitum, or 3) injected with vehicle and limit-fed to the intake of the leptin-injected group. All the pigs also were injected with the antigen, Limulus hemocyanin, on d 0 and 15 of the experiment. Exogenous leptin decreased (P < 0.05) daily feed intake and antigen-specific immunoglobulin (Ig) G1, but had no effect on lymphocyte proliferation or antigen-specific IgG2. In a second series of experiments, peripheral blood mononuclear cells (PBMC) were isolated from venous blood to determine the effect of stimulation with the polyclonal mitogen, concanavalin A (ConA), on the long form of the leptin receptor (Ob-Rl) mRNA abundance, and to determine whether leptin altered mitogen-induced proliferation, cytokine production, or signal transducer and activator of transcription 3 (STAT3) activation. Leptin had no effect on the proliferation of PBMC or on cytokine mRNA abundance or secretion. The abundance of Ob-Rl mRNA was decreased (P < 0.05) in response to stimulation with ConA. Constitutive STAT3 DNA binding was evident in mobility shift assays, but was not altered by either leptin or serum deprivation. These data indicate that leptin modifies antibody isotypes in the pig, and that Ob-Rl expression is downregulated in response to polyclonal mitogens in porcine PBMC. The constitutive activation of STAT3, coupled with the absence of leptin-inducible binding, indicates an alternative signaling pathway for leptin in pig PBMC.

Preliminary crystallographic studies of Limulus polyphemus hemocyanin subunits IIIa, IIIb and IV

Crystals of Limulus hemocyanin subunits IIIa, IIIb and IV are suitable for X-ray diffraction analysis. The three-dimensional structure of subunit IV is determined by molecular replacement and non-crystallographic symmetry averaging methods. A tentative model of subunit IIIa is obtained from a partial data set. Both structures, similar to subunit II, could provide primary structure segments suitable for oligonucleotide probe synthesis.

Kinetics and type of immune response following intranasal and subcutaneous immunisation of calves

Protection of animals against respiratory infections has long been known to depend on respiratory mucosal immunity. However, few studies have been reported on the immune response following intranasal (i.n.) immunisation with non-living, soluble antigens. This study determined the kinetics of the humoral and cellular immune responses in calves after i.n. immunisation with Limulus haemocyanin (LH) with cholera toxin adjuvant, or subcutaneous (s.c.) immunisation withLH in incomplete Freund’s adjuvant. A proliferative response of peripheral blood mononuclear cells cultured in vitro with LH was observed in animals immunised 7–10 days after i.n. and s.c. immunisations with no significant differences between the two immunised groups. LH -specific antibody was present in the serum of animals immunised s.c. (IgM, IgG1 and IgG2) and i.n. (IgA). Although significant IgA responses were observed, i.n. immunisations in cattle with soluble protein antigens and cholera toxin as an adjuvant did not induce a strong systemic immune response.

γ-Hydroxyphosphonate inducing single-chain antibodies capable of catalyzing soman hydrolysis

A γ-hydroxyphosphonate P 6 ( O 1 -methyl- O 2 -(1, 2, 2-trimethylpropyl)-2-hydroxy-5- nitrophenyl methylphosphonic acid) which is proposed to be an analog of the transition state in hydrolysis of soman was synthesized. Artificial antigens were obtained by conjugating P 6 to the carrier proteins BSA (bovine serum albumin) and LPH ( Limulus polyphenus hemocyanin). Mice were immunized with P 6 -LPH and recombinant single-chain antibody phage display library was constructed. After 4 rounds of panning against P 6 -BSA and competitive inhibition enzyme immunoassay, more than 70 strains of phage antibodies capable of binding soman were obtained and 11 of them can accelerate the hydrolysis reaction of soman. One of them (EP 6 ) was studied further. Soluble single-chain antibody was prepared and purification was performed by gel filtration and ion exchange chromatography. The kinetic experiment was carried out showing that the turnover number k cat = 198 min -1 and the rate enhancement k cat / k uncat = 122 419. When 0.16 mg mL -1 EP 6 was preincubated in vitro with 0.132 mmol L -1 (220 mg kg -1 = 1.1×LD 95 ) of soman prior to the administration to mice by subcutaneous route, all animals (19 mice) survived whereas all the control mice (14) treated with PBS and soman died within 30 min. Furthermore, EP 6 could prolong the latent time of spasm and death when mice were passively immunized with EP 6 intravenously 15 min before 1×LD 95 of soman challenge. These results demonstrate that EP 6 is able to increase the rate of soman degradation and protect against soman's toxicity, especially in vitro.

Limulus hemocyanin, an unusual ligand for myelin basic protein from central nervous system.

Limulus polyphemus hemocyanin binds with high affinity to the 18.5 and 21.5 kD isoforms of rat central nervous system myelin basic protein.

Presence of functional domains in Limulus polyphemus hemocyanin

In an attempt to isolate structural domains of arthropod hemocyanins and possibly to investigate their functional properties, we have undertaken proteolytic digestion experiments of isolated subunits from Panulirus interruptus and Limulus polyphemus oxy-hemocyanin. Satisfactory results have been obtained using trypsin at high concentration and short digestion times. Results show that, in the case of Panulirus hemocyanin, only subunit a is susceptible to trypsin digestion, but that proteolytic cleavage is associated with the loss of the copper-oxygen band; on the other hand, in the case of Limulus hemocyanin, four subunits (I, II, III and IV) show a significant susceptibility to trypsin, and their fragmentation takes place with preservation of the oxygen-binding capacity. A more detailed study of the digestion products of subunit IV from Limulus hemocyanin reveals that the proteolytic fragments keep together in a single non-covalent complex. Attempts to separate the native fragments results in the precipitation of the digestion products. Subunit IV of Limulus with proteolytic cuts binds O2 and CO with the same affinity as the native subunit, suggesting that the copper site is still preserved structurally and is functionally active in a 37 kDa trypsin-resistent domain.

Allosteric control in Limulus polyphemus hemocyanin: functional relevance of interactions between hexamers.

Hemocyanin of the horseshoe crab Limulus polyphemus is composed of 48 oxygen-binding subunits, which are arranged in eight hexameric building blocks. Allosteric interactions in this oligomeric protein have been examined by measurement of high-precision oxygen-equilibrium curves, using an automated Imai cell. Several models were compared in numerical analysis of the data. A number of conclusions can be drawn with confidence. (1) Oxygen binding by Limulus hemocyanin cannot satisfactorily be described by the two-state MWC model [Monod, J., Wyman, J., & Changeux, J.P. (1965) J. Mol. Biol. 12, 88-118] for allosteric transitions with either the hexamer or dodecamer as the allosteric unit. (2) Of the models tested, the data sets can be best described by an extended MWC model that allows for an equilibrium, within the 48-subunit ensemble, between cooperative hexamers and cooperative dodecamers. The model invokes T and R states for both hexamers (T6 and R6) and dodecamers (T12 and R12). Allosteric effectors modulate oxygen affinity and cooperativity by affecting the R to T equilibria within hexamers and dodecamers and by shifting the equilibria between hexamers and dodecamers. (3) The fitted model parameters show that under most conditions the intersubunit contacts within T-state hexamers are more constrained than those within T-state dodecamers. (4) The oxygen affinities of the hexameric and dodecameric R states are the same, but under all conditions examined the conformation of the fully oxygenated molecule is that of the dodecameric R state. (5) Between pH 7.4 and pH 8.5 the dodecameric T state has a higher affinity for oxygen than the hexameric T state, allowing for "T-state cooperativity".(ABSTRACT TRUNCATED AT 250 WORDS)

Fluorescence polarization of Limulus hemocyanin

The polarization of the native fluorescence of Limulus hemocyanin (Hc) as a function of pH (4–10) has been measured. At pH 7, the polarization decreases in the order oxyHc, deoxyHc, stripped Hc, apoHc. The observed changes in polarization are explained in terms of conformational changes and the association-dissociation behavior of the 48-mer (60S), 24-mer (37S), and 12-mer (24S) aggregates and the monomer (5S) subunits. For oxyHc (pH 5.0–7.5), polarization as function of pH is reversible and independent of time, whereas at higher pH (7.8–8.4) the polarization decreases as a function of time. The polarization-time plots show a first rapid fall in the polarization followed by a phase of much slower change, with the rate of initial depolarization increasing with increasing pH. At pH 8.4, the depolarization is ascribed to dissociation from the 48-mer and 24-mer to the monomer species. In the pH range 4.5–6.5, the reaction scheme is proposed as follows: deprotonation of P 12(H +) 2i to form P 12H +, aggregation of two P 12H + into P 24(H +) 2, deprotonation of two amino acid residues of P 24(H +) 2 to form P 24, and aggregation of two P 24 into P 48 (60S).

Genetic control of eosinophilia in parasitic infections: Responses of mouse strains to treatment with cyclophosphamide and parasite antigen

Lammas D.A., Mitchell L.A. and Wakelin D. 1988. Genetic control of eosinophilia in parasitic infections: responses of mouse strains to treatment with cyclophosphamide and parasite antigen. International Journal for Parasitology 18:1077–1085. Strain-dependent variation in the capacity of inbred and congenic mice to mount an eosinophilia in response to inoculation with the antigens of Mesocestoides corti, Trichinella spiralis or with Limulus haemocyanin (LCH), following pretreatment with cyclophosphamide (CY), is described. SWR, NIH, BALB/c, C3H and SJL mice were eosinophil high responder strains whereas C57 BL/10 and CBA mice were eosinophil low responder strains. Congenic strains with the B10 background (B10.S, B10.G and B10.BR) were all low eosinophil responders, although B10.G mice showed a level of response consistently above the other B10 congenic strains. Some of the gene(s) for high responsiveness appeared to be dominant, because F(In1)hybrids between high and low eosinophil response parental strains were intermediate to high responders. The strain-dependent pattern of eosinophil responsiveness to LHC or to M. corti and T. spiralis antigens, following CY pretreatment, was similar to that obtained previously following infection with either M. corti or T. spiralis, suggesting that heterogeneity in capacity to produce eosinophils operates independently of the nature of the eliciting stimulus.

Active-site heterogeneity in Limulus hemocyanin as revealed by reaction with peroxides

Previously reported differences in the reactivities toward active-site ligands such as hydrogen peroxide indicate that the active-site geometries of the arthropod and mollusc hemocyanins are significantly different. Results are presented which demonstrate that the purified subunits composing the native hemocyanin of an arthropod show comparable active-site heterogeneity. Neither whole nor stripped samples of Limulus oxyhemocyanin are completely oxidized by reaction with hydrogen peroxide. Moreover, the addition of hydrogen peroxide to deoxy or completely oxidized samples causes partial regeneration to the oxy form. This behavior is most pronounced for one of the five major chromatographic fractions, Zone III, which retains greater than 60% of its copper-oxygen absorbance band when treated with hydrogen peroxide. Zone III is composed of three subunits (IIIA, IIIB, and IIIB′). With excess hydrogen peroxide, the deoxy and oxidized forms of Subunit IIIA can be completely regenerated to a fully functional oxy state, while Subunits IIIB and IIIB′ are less than 30% regenerated. Upon long-term storage, Subunit IIIA slowly loses 340 nm absorbance, an aging process that can be reversed by hydrogen peroxide. These features of Subunit IIIA, that set it apart from other subunits of the 48-member ensemble of intact Limulus hemocyanin, are typically exhibited by mollusc hemocyanins rather than arthropod hemocyanins. These findings clearly illustrate that significantly different active-site geometries can occur within a single type of metalloprotein and suggest that comparisons between the various Limulus subunits and Subunit IIIA may prove valuable in unraveling the structural basis for the disparity in the chemical reactivity exhibited by hemocyanins of different species.

Dimerization of native Limulus polyphemus hemocyanin in the presence of calcium ions and sucrose

In the presence of both sucrose and millimolar concentrations of calcium, native Limulus polyphemus hemocyanin, having a sedimentation coefficient of ~ 60S, forms a dimer with a sedimentation coefficient of ~ 90S. Dimer formation was observed by sucrose gradient ultracentrifugation and gel filtration. It occurs at pH 7.0 but not at pH 8.5 or 6.0, and it is dependent on the calcium concentration. Treatment with EDTA to remove calcium reverses the dimerization process. The effect of sucrose may be due to its known preferential exclusion from the vicinity of proteins [15]. Such preferential exclusion would favor the formation of dimers if the excluded volume of the protein were to decrease on dimerization. Formation of dimer is accompanied by the binding of calcium to hemocyanin, as determined by analysis of gradient fractions by atomic absorption spectroscopy. In addition, stoichiometric amounts of europium (III), known to occupy the calcium binding sites of hemocyanin, also promotes dimer formation. Dimer formation is not induced by 100 mM sodium chloride. The observation of a 90S form of Limulus hemocyanin indicates that there is nothing inherent in the structure of the 60S molecule that prevents further aggregation.

THE CO2SENSITIVITY OF THE HEMOCYANINS AND ITS RELATIONSHIP TO Cl-SENSITIVITY

The effect of CO2 on hemocyanin-oxygen binding is not generally related to the effect of Cl-. Some hemocyanins respond to both and some to either one alone. The direction of the responses of O2 affinity of the various hemocyanins to CO2 is poorly correlated with the direction of responses to other effectors. The influence of CO2 on Busycon and Limulus hemocyanins reaches its maximum at high pH. Since the effect can be abolished by restoring divalent cation activities to the control levels prior to the addition of CO2, we suggest that the effect is not specific but rather indirect, by the pairing of the allosteric effectors Ca+2 and Mg+2 with the CO2 anions. In contrast the effect of CO2 on crustacean hemocyanins is greaterat low pH and it can be enhanced by maintaining HCO3- levels within narrow limits and permitting PCO2 to vary by a large factor. This finding suggests that the effective species is molecular CO2. While a influences only oxygen affinity, CO2 may influence cooperativity as well. Indifferen...

Self-association and oxygen-binding characteristics of the isolated subunits of Limulus polyphemus hemocyanin

The hemocyanin of the horseshoe crab Limulus polyphemus is characteristic of arthropod hemocyanins in that it is a high-molecular-weight oligomer composed of functionally and structurally distinct subunits. The protein forms a 48-subunit complex, the largest form of arthropod hemocyanin, whose oxygen-binding characteristics are modulated by subunit interaction within the oligomer. It has previously been shown that a number of electrophoretic isozymes, which are identical immunochemically, are present in dissociated Limulus hemocyanin. In this study it is demonstrated that the electrophoretic differences in the antigenically identical subunits are not reflected in their oxygen-binding and self-assembly properties or in the roles they play in reassembly and function of the 48-subunit native molecule. The chloride-dependent modulation of the oxygen-binding properties of those Limulus subunits which do not self-assemble, as documented here, illustrates that this allosteric effect may be operable at the tertiary level. For each of the purified subunits the effects of pH and calcium ions on oxygen-binding characteristics and self-assembly reactions are reported, and the roles of specific subunits in reassembly of distinct aggregation states are further documented.

Assembly and calcium-induced cooperativity of Limulus IV hemocyanin: a model system for analysis of structure-function relationships in the absence of subunit heterogeneity

Hemocyanins, the high molecular weight copper proteins which serve as oxygen carriers in many arthropods and molluscs, are representative of multisubunit complexes which are capable of reversible dissociation and assembly. Although reversible, in many hemocyanins these processes are not in true thermodynamic equilibria, and it has been suggested that there is "microheterogeneity" among the molecules in solution. An alternative explanation is that their complex behavior is due to the existence of quaternary interactions between structurally distinct types of subunits within the native molecule which have varying pH and ionic strength sensitivity. Limulus IV hemocyanin was used as a model system to examine structure-function relationships in the absence of subunit heterogeneity. Purified subunit IV of Limulus polyphemus hemocyanin is homogeneous by a number of electrophoretic and immunological criteria and is capable of undergoing pH-dependent self-assembly into hexamers. The monomer-hexamer transition was found to be an equilibrium whose rate is dependent on the presence or absence of calcium ions. The observation that the assembly of this homopolymer behaves as a true equilibrium suggests that the nonequilibrium dissociation profiles observed for native Limulus hemocyanin are related to the extensive subunit heterogeneity of the native protein. In calcium-containing buffers, the monomer-hexamer transitions of Limulus IV hemocyanin can be described by a cooperative mechanism with approximately six protons per hexamer lost on assembly from acid pH and three protons gained on assembly from alkaline pH. Increased ionic strength or increased temperature favors dissociation. Like the native molecule, Limulus IV hemocyanin behaves as an allosteric protein.(ABSTRACT TRUNCATED AT 250 WORDS)

The role of multivariate image analysis in solving the architecture of the limulus hemocyanin molecule

The detailed distribution of stain grains in contact with the stain-excluding portions of a protein molecule is random: inspection of a single image of a macromolecule reveals little below the resolution level of gross morphology (Fig 1). However, new insights can be gained by analyzing a large set of molecule images resulting from the same experiment.Firstly, the noise reduction due to averaging allows higher-resolution detail to be retrieved. Secondly, the analysis of such a set reveals new information that is qualitatively different from information contained in a single molecule image: the patterns of variation in local stain distribution. The stain variation at any point, and the way in which stain variations at different points of the structure are correlated, reflect certain constraints that have to do with the local binding characteristics of the molecule (e.g. accessibility of charged sites) and with its detailed topography. In the past, few observations were made relating to these phenomena because they are very difficult to quantify.

Spectroscopy of (carbon monoxy)hemocyanins. Phosphorescence of the binuclear carbonylated copper centers.

The luminescence of CO-hemocyanin has been studied in several hemocyanins from both arthropods and molluscs. All of them show an emission in the region 550-560 nm with quantum yield = 0.2-0.4, though individual differences are apparent. Solvent composition (as well as the presence of calcium and lanthanides) has no effect on the emission. Lifetimes of this luminescence are in the range 60-140 microseconds, indicating that the emitting state may be a triplet. The presence of both copper atoms (Cu+) per CO binding site is required for the luminescent state since half-met and half-apo derivatives do not show such an emission. Though not clearly related to any of the functional properties, systematic differences in luminescence parameters are apparent between arthropodal and molluscan hemocyanins. Limulus hemocyanin appears to be very different from both types of hemocyanin.

Subunit composition of a high molecular weight oligomer: Limulus polyphemus hemocyanin

The hemocyanin of Limulus polyphemus is a 48-subunit aggregate. This 3.3 × 10 6-dalton oligomer is composed of structurally and functionally heterogeneous subunits. Using polyacrylamide electrophoresis J. Markl, A. Markl, W. Schartau, and B. Linzen ( J. Comp. Physiol. Ser. B 130,283–292, 1979) observed 12 bands; while using immunoelectrophoresis, M. Hoylaerts, G. Preaux, R. Witters, and R. Lontie ( Arch. Int. Physiol. Biochem. 87, 417–418, 1979) and J. Lamy, J. Lamy, J. Weill, J. Bonaventura, C. Bonaventura, and M. Brenowitz. ( Arch. Biochem. Biophys. 196, 324–339, 1979) observed 8 subunits. To proceed with an analysis of subunit roles in assembly it is first necessary to determine the number of distinct subunits. Refinement of the chromatographic separation procedures has led to the isolation of 8 immunologically distinct subunits as well as additional charge isomers which cannot be distinguished immunologically. Alkaline electrophoresis revealed 15 bands and isoelectric focusing up to 17. On the basis of extensive control experiments, including composit acrylamide-agarose immunoelectrophoresis and checks for conformational isomers, aggregation, proteolysis, and other types of degradation, we conclude that the electrophoretic heterogeneity of immunologically identical subunits is not artifactual. We have extended the nomenclature used by Lamy et al. (1979) to include the electrophoretic heterogeneity by using primes (′) to denote electrophoretically distinguishable subunits which are immunologically identical. A number of patterns have become apparent by correlating the results obtained by the different techniques. For example, immunologically pure subunit II, which shows 3 bands on alkaline electrophoresis, is in fact a mixture of electrophoretically distinct subunits II, II′, II″. Except for subunits II, II′, and II″ immunoelectrophoretically identical subunits are typically homogeneous on sodium dodecyl sulfate-gels. However, slight differences in the apparent molecular weight are observed on high-resolution gels between immunologically unrelated subunits. The immunological identity and electrophoretic differences suggest that the charge isomers which are immunologically identical have similar antigenic surfaces. If a charge substitution is not in a critical location, we would expect the electrophoretically distinct but immunologically identical subunits to have identical assembly roles. Comparison of the results for Limulus hemocyanin with the hemocyanin of related species Eurypelma californicum and Androctanus australis, which have 7 and 8 immunologically distinct subunits, respectively, suggests that the calcium-mediated aggregation from 24 to 48 subunits of Limulus does not require more extensive subunit complexity.

Terbium ion binding to [formula omitted] hemocyanin

Terbium ion binds to calcium-free Limulus hemocyanin at pH 7.0 and 8.9, and promotes the aggregation of hemocyanin subunits, a phenomenon associated with calcium binding. An excitation maximum for the bound terbium at 293 nm and the results of treating the hemocyanin with N-bromosuccinimide indicate that energy transfer from tryptophan to the bound terbium is responsible for the enhancement of terbium fluorescence. At pH 8.9, addition of calcium to hemocyanin containing bound terbium results in only a partial loss of terbium fluorescence, suggesting heterogeneity in the terbium binding sites. Titration of hemocyanin with terbium also indicates multiple binding sites.

Identification of Limulus polyphemus haemocyanin messenger RNA.

Total RNA was isolated from cyanoblast-containing tissue taken from behind the compound eye of the horseshoe crab, Limulus polyphemus. Poly(A)-containing RNA separated from this by affinity chromatography on oligo(dT)-cellulose was translated in the rabbit reticulocyte haemolysate system in the presence of L-[35S]methionine. By using an antiserum to Limulus haemocyanin, polypeptides were isolated from the translation products which had a similar mobility to the authentic Limulus haemocyanin polypeptides as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis.