Karen I. Miller

Structures of two molluscan hemocyanin genes: significance for gene evolution.

We present here the description of genes coding for molluscan hemocyanins. Two distantly related mollusks, Haliotis tuberculata and Octopus dofleini, were studied. The typical architecture of a molluscan hemocyanin subunit, which is a string of seven or eight globular functional units (FUs, designated a to h, about 50 kDa each), is reflected by the gene organization: a series of eight structurally related coding regions in Haliotis, corresponding to FU-a to FU-h, with seven highly variable linker introns of 174 to 3,198 bp length (all in phase 1). In Octopus seven coding regions (FU-a to FU-g) are found, separated by phase 1 introns varying in length from 100 bp to 910 bp. Both genes exhibit typical signal (export) sequences, and in both cases these are interrupted by an additional intron. Each gene also contains an intron between signal peptide and FU-a and in the 3′ untranslated region. Of special relevance for evolutionary considerations are introns interrupting those regions that encode a discrete functional unit. We found that five of the eight FUs in Haliotis each are encoded by a single exon, whereas FU-f, FU-g, and FU-a are encoded by two, three and four exons, respectively. Similarly, in Octopus four of the FUs each correspond to an uninterrupted exon, whereas FU-b, FU-e, and FU-f each contain a single intron. Although the positioning of the introns between FUs is highly conserved in the two mollusks, the introns within FUs show no relationship either in location nor phase. It is proposed that the introns between FUs were generated as the eight-unit polypeptide evolved from a monomeric precursor, and that the internal introns have been added later. A hypothesis for evolution of the ring-like quaternary structure of molluscan hemocyanins is presented.

Keyhole limpet hemocyanin: Structural and functional characterization of two different subunits and multimers☆

Keyhole limpet hemocyanin (KLH), the large respiratory glycoprotein from the primitive gastropod mollusc, Megathura crenulata, is a potent immunogen used classically as a carrier protein for haptens and more recently in human vaccines and for immunotherapy of bladder cancer. Two KLH isoforms were identified and isolated by high-performance anion exchange chromatography. Subsequent analyses disclosed that these isoforms--designated KLH-A and KLH-B--were composed of distinct subunits that differed in primary structure, molecular weight (KLH-A was 449,000 and KLH-B was 392,000), polymerization/reassociation characteristics, and O2-binding constants (KLH-A had a P50 of 7.32 and KLH-B had a P50 of 2.46). Both subunits appear to be composed of eight oxygen binding domains, and reassociate in solution only with like subunits. These results support the concept that structural and functional heterogeneity is a common feature of molluscan hemocyanins, and provide a rational basis for studying and optimizing the immunostimulatory properties of KLH.

Hemocyanin of the chambered nautilus: structure-function relationships.

Abstract The chambered nautilus, Nautilus pompilius, is a cephalopod mollusc that lives in deep water in the western Pacific. Structural and functional studies were undertaken on the high-molecular-weight copper protein that it uses as an oxygen carrier. Nautilus hemocyanin was found to resemble that of another cephalopod, the squid Loligo paelii. Unlike the hemocyanin of the gastropod molluscs, neither Nautilus nor Loligo hemocyanin exists in aggregation states with sedimentation coefficients of 100 S. The highest aggregation state in these two hemocyanins has a sedimentation coefficient of 58 S. This is significantly lower than the 64–66 S values found for half-dissociated molecules of typical gastropod hemocyanins. The dissociation of Nautilus hemocyanin was found to be sensitive to pH and divalent cations such as calcium and magnesium. At high pH, and in the absence of divalent cations, subunits that are believed to be large single polypeptide chains are found. They have sedimentation coefficients of 11 S, similar to subunit sedimentation behavior throughout the mollusc hemocyanin line. Unlike squid hemocyanin, Nautilus hemocyanin shows no intermediate dissociation products. Undissociated Nautilus hemocyanin shows a moderate oxygen affinity ( log P 1 2 = 0.8 at pH 8 ), a small Bohr effect ( log P 1 2 = 0.97 at pH 7.0 ), and a fairly low degree of cooperativity in oxygen binding ( n 1 2 = 1.5–2.0 ). Dissociation of Nautilus hemocyanin by dialysis against EDTA at high pH yields material of higher affinity, no cooperativity, and very slight pH sensitivity. The difference between the oxygen affinity of whole and dissociated Nautilus hemocyanin is most pronounced at low degrees of oxygen saturation. The question of possible subunit heterogeneity in Nautilus hemocyanin was addressed. Dissociated Nautilus hemocyanin was chromatographed at high pH in the absence of divalent cations. Three distinct peaks were found. These chromatographic zones, representing putative polypeptide chains of Nautilus hemocyanin, show significant differences in their oxygen binding characteristics. The presence of structurally and functionally diverse subunits in this cephalopod hemocyanin is reminiscent of that observed for two gastropod hemocyanins, and is thus suggestive that subunit heterogeneity may be a more general feature in the molluscan hemocyanins than previously appreciated.

Allostery in very large molecular assemblies

In contrast to small allosteric systems (like hemoglobin) those containing very large numbers (n) of binding sites never exhibit cooperativity (as measured by the Hill coefficient, nH) even approaching the potential limit, n. The reason for this appears to be that in such macromolecules the cooperative unit always represents some sub-structure of the entire structure. On the other hand, it is frequently observed that such sub-structures, when isolated, do not exhibit cooperativity at all. This paper describes studies of some molluscan hemocyanins that explore this apparent anomaly. It is concluded that it is the higher order structure of the molecule that provides a framework within which the sub-structures may exhibit their allosteric behavior.

Structure and function of hemocyanin from thalassinid shrimp

SummaryThe subunit structure, association equilibria, and oxygen binding of the hemocyanins of three thalassinid shrimp have been investigated. The organismsCallianassa californiensis, Callianassa gigas andUpogebia pugettensis are found in similar but distinct habitats in a limited region. All of the hemocyanins exhibit a common pattern of subunit structure, but differ in details of the response of that structure to variations in ionic environment. All three are heterogeneous in polypeptide chain composition, but have quite different distributions of components. The oxygen binding of the twoCallianassa hemocyanins is virtually identical under conditions approximating physiological; that ofUpogebia, which shows lower tolerance to anoxia, is significantly different. These similarities and differences are discussed in terms of the physiological requirements of the species.

BIVALVE HEMOCYANIN: STRUCTURAL, FUNCTIONAL, AND PHYLOGENETIC RELATIONSHIPS

The hemocyanin-like molecule found in the blood of the most primitive bivalves (protobranchs) reversibly binds O2. Its respiratory properties and its sedimentation behavior are both distinctive. Although its electron-dense image looks like that of the gastropod hemocyanins, its molecular weight differs from those of all other molluscan Hcs and is more consistent with the concept of bivalve hemocyanin as a pair of octopod hemocyanins. Bivalve hemocyanin occurs in the solemyoids as well as the nuculoids, which argues for the integrity of the Protobranchia as a natural taxon. The ancestral bivalve O2 carrier was previously believed to be a simple intracellular hemoglobin, which is found in the less primitive Pteriomorpha. The most obvious interpretation of the present results, however, is that hemocyanin is the primitive bivalve O2 cannier and that it was replaced by the red blood cell, which originated at least twice: once in the pteriomorph bivalves and at least once in other taxa.

Assembly of Octopus dofleini hemocyanin : a study of the kinetics by sedimentation, light scattering and electron microscopy

The kinetics of association of Octopus dofleini hemocyanin subunits to form the native decameric molecule have been studied with a combination of sedimentation, light scattering and electron microscopy. The reaction, initiated by addition of magnesium, is relatively slow, requiring hours to reach completion, with monomer and decamer as predominant molecular species throughout. Analysis of the light-scattering data, including stopped-flow studies, reveals an initial lag period in the reaction, followed by a second-order process that is rate limiting. The lag period depends on both protein and magnesium ion concentration. Electron microscope studies reveal intermediates in the process, and support a model of assembly in which nucleation begins at the dimer level. Theoretical models for the process are compared.

Molluscan Hemocyanins: Structure and Function

Hemocyanins are copper proteins that serve oxygen transport in two, and only two, phyla: Arthopoda and Mollusca. Although molluscan and arthropod hemocyanins share certain general features (for example, each has a binuclear copper site for oxygen binding) and exhibit much similarity in function, it is now becoming apparent that the structural differences between the hemocyanins from these two phyla are much more significant than their similarities. For example, size and arrangements of subunits are wholly different, and comparison of amino acid sequences indicates little homology except in one limited region. It is now generally held by workers in the field that the molluscan and arthropod hemocyanins are at best very distantly related, and probably arose in their separate phyla through independent evolutionary events (Mangum 1985; Drexel et al. 1987).

The structure of Octopus dofleini hemocyanin

Abstract 1. 1. Octopus dofleini hemocyanin has a molecular weight of 3,580,000 determined by sedimentation equilibrium and a sedimentation coefficient, S 20,w 0 , of 51.0S. 2. 2. The 51S hemocyanin is stable in 50 mM Mg 2+ and 10 mM Ca 2+ from pH 7.0 to pH above 9.5, but dissociates in 10 mM EDTA at pH 8.0 to a single subunit. This dissociation is fully reversible. 3. 3. The subunit has a molecular weight of 359,000 and a sedimentation coefficient, S 20 ,w 0 , of 11.1S. 4. 4. At pH 7.0 the 51S hemocyanin did not appear to be fully oxygen-saturated in the ultracentrifuge. Physiological implications of this observation are considered.

An investigation of the nature of Bohr, Root, and haldane effects inOctopus dofleini hemocyanin

Summary1.The pH dependence ofOctopus dofleini hemocyanin oxygenation is so great that below pH 7.0 the molecule does not become fully oxygenated, even in pure O2 at 1 atm pressure. However, the curves describing percent oxygenation as a function of