Jürgen Markl

Subunit heterogeneity in arthropod hemocyanins: II. Crustacea

Summary1.The hemocyanins of 10 decapod Crustacea were dissociated and their subunits analyzed by high resolution polyacrylamide electrophoresis (PAGE): 5 brachyuran crabs (Cancer pagurus, Carcinus maenas, Macropipus holsatus, Hyas araneus, Maja squinado), 3 Astacura (Astacus leptodactylus, Homarus americanus, Homarus gammarus) and the spiny lobstersPalinurus vulgaris andPanulirus interruptus2.All of the species save the spiny lobsters possess a major hemocyanin component sedimenting with 24 S. A second hemocyanin component sedimenting with ca 16 S was found inH. gammarus, M. squinado, C. pagurus andM. holsatus (about 10 per cent in each case) and inA. leptodactylus (about 25 per cent). A second, major blood protein (10–25% of the total blood protein) was observed inH. gammarus where its sedimentation coefficient was 24 S,M. squinado (16 S),H. araneus (24 S) andC. pagurus (16 S). This second protein has no respiratory function. Two such non-respiratory proteins sedimenting with 24 S and 16 S were found inH. americanus.3.Between 2 and 7 hemocyanin bands were obtained after incubation with sodium dodecylsulfate (SDS) and β-mercaptoethanol and subsequent electrophoresis in polyacrylamide gradients. The average molecular weight was about 75,000 in the crabs, 80,000 in the crayfishes and 85,000 in the spiny lobsters. The non-respiratory proteins yield between one and four chains with molecular weights ranging from 76,000 to 87,000.4.The hemocyanins were dissociated at pH 9.6 into “native” subunits, but dissociation was not quantitative in several species. By gel filtration, the products were separated into undissociated material and hemocyanin monomers (5 S). InAstacus leptodactylus a dimeric subunit (7 S) was obtained in addition; its components are linked by a disulfide bridge. The subunit mixtures were separated by PAGE into 4 to 6 distinct bands.5.To establish the total number of different polypeptide chains present in each hemocyanin, the two electrophoretic patterns were related to each other by preparative isolation of “native” subunits and subsequent analysis in SDS-PAGE. The number of different polypeptide chains ranges from four to seven in the species studied by us. In those species which contained both 24 S and 16 S hemocyanin, more different polypeptide chains were found in the 24 S hemocyanin than in the 16 S hemocyanin, the only exception beingHomarus gammarus.

THE QUATERNARY STRUCTURE OF 4 CRUSTACEAN 2-HEXAMERIC HEMOCYANINS - IMMUNOCORRELATION, STOICHIOMETRY, REASSEMBLY AND TOPOLOGY OF INDIVIDUAL SUBUNITS

SummaryTwo-hexameric (2×6) hemocyanins from the brachyuran crabsCancer pagurus andCallinectes sapidus, the freshwater crayfishAstacus leptodactylus and the lobsterHomarus americanus were isolated and dissociated into native subunits.The subunits of each hemocyanin were analyzed by electrophoresis and immunology. Three immunologically distinct subunit types, which were termedα,β andγ, could be identified in each case. They were isolated preparatively, and interspecifically correlated. Subunitα is subdivided into several electrophoretically distinct isoforms which are immunologically closely related (Astacus) or identical (other species). InAstacus andCancer one of these isoforms was shown to dimerize and to act as inter-hexamer bridge. It represents a fourth subunit type termedα′. A fifth, ‘diffuse’ component, which in PAGE migrated at the position of a dimer, was identified in the crossed immunoelectrophoretic patterns as denatured hemocyanin.A common feature of the four hemocyanins is the presence of 4 copies ofβ and 8 copies ofα/γ within the 2×6 particles. Theα:γ ratio is 4:4 in the two Astacidea and 6:2 in the two Brachyura.α′ exists in 2 copies inAstacus andCancer which means that a single dimerα′-α′ is present in a two-hexamer. This leaves 2 monomericα copies inAstacus and 4 inCancer.Every subunit from the four species except ofAstacusα′-α′ was capable to form hexamers in reassembly experiments. If subunit combinations were tested, hetero-hexamers were formed preferentially. Two-hexamers were reconstituted only in the presence of all subunit types and the native subunit stoichiometry was required to obtain twohexamers in considerable yields. Factors limiting 2×6 reassembly are discussed.Authentic 2×6 molecules ofAstacus, Homarus andCancer hemocyanin were immunolabeled with subunit-specific antibody fragments (Fab) or IgG molecules, and the resulting immuno complexes were studied in the electron microscope. A topological model of the quaternary structure of decapod 2×6 hemocyanins is derived, showing the position of each copy of the four subunit types. In this model, the inter-hexamer bridgeα′-α′ is surrounded by twoβ and twoγ subunits forming the central core of the dodecamer. Two additionalβ and two additionalγ subunits form the periphery together with oneα subunit occupying the peripheral short edges of each hexameric half structure. The model is discussed with respect to the current literature.

Immunological Correspondences Between the Hemocyanin Subunits of 86 Arthropods: Evolution of a Multigene Protein Family

One of the most striking features of arthropod hemocyanins is their remarkable subunit heterogeneity, which has been documented in a large variety of papers. Intensive studies on several hemocyanins have shown intraspecific differences between subunits in electrophoretic mobility, immunogenicity, oligomeric topology, primary structure, and oxygen binding function. Additionally, a marked interspecific diversity of the electrophoretic subunit patterns was observed. This, however, prohibits an easy comparison of data, because by no means it is obvious which subunit of a hemocyanin corresponds to a particular subunit of another hemocyanin. It has required much effort and the collaboration of several laboratories to analyse the interspecific correspondencies between the hemocyanin subunits of two xiphosura, two scorpions, and two spiders (1–4).

Hemocyanins in spiders

Summary1.The hemocyanin of the lycosid spiderCupiennius salei was separated into its hexameric (16 S) and dodecameric (24 S) components, and analyzed quantitatively. The reassociation and topologic distribution of the subunits were studied.2.There are two types of subunits. One is monomeric (5 S) and consists of 5 electrophoretically distinct bands which are, however, immunologically identical. The other is a disulphide bridged dimer (7 S) which yields 2 components upon electrophoresis or immunoelectrophoresis. The significance of this heterogeneity was not studied. The dimer is antigenically deficient with respect to the monomer.3.Whereas the 16 S hemocyanin is composed of six monomers, 24 S hemocyanin contains 10 monomers and 1 dimer.4.Alkaline dissociation of 24 S hemocyanin (dodecamer) into subunits passes through a heptameric state (18 S) which is composed of 5 monomers and the dimer. In the electron microscope, 16 S-like units with a seventh polypeptide attached can be distinguished.5.Treatment of 24 S or 18 S hemocyanin with reducing agents to cleave the disulphide bridge leads to a second type of hexamer (16 S′) which is electrophoretically distinct from native hexamers (16 S), and composed of 5 monomers and one constituent polypeptide chain of the dimer.6.Upon dialysis of a monomer/dimer mixture against neutral buffer containing 40 mM calcium, 16 S, 18 S and 24 S particles are formed. The three reconstituted hemocyanins exhibit subunit compositions identical to the native hemocyanins and the 18 S component obtained during dissociation.7.The results suggest that the 24 S hemocyanin particle consists of two identical hexamers linked by the disulphide bridge of a dimeric subunit shared by both hexamers.

Subunit heterogeneity in crustacean hemocyanins as deduced by two-dimensional immunoelectrophoresis

Summary1.Multiple subunits of hemocyanins from 3 brachyuran crabs (Carcinus maenas, Cancer pagurus, Hyas araneus), a spiny lobster (Palinurus vulgaris), a freshwater crayfish (Astacus leptodactylus), and a lobster (Homarus americanus) were isolated by preparative polyacrylamide gel electrophoresis (PAGE), and compared by two-dimensional immunoelectrophoresis.2.In the 24 S hemocyanin isolated from each of the 3 crabs, two of the four subunits separated are immunologically identical; the third subunit is antigenically deficient compared to the first two. The fourth chain is immunologically unrelated to the other three.3.In the 16 S hemocyanin ofPalinurus two of the four subunits are immunologically identical; the third is closely related. The fourth chain is partially identical with the other three, but is antigenically deficient.4.The 16 S hemocyanin ofAstacus is composed of two immunologically unrelated subunits. The 24 S hemocyanin of this species contains, in addition, a dimeric subunit which is partially identical with one of the former, but not related to the other.5.In the 24 S hemocyanin fromHomarus, five subunits were separated; a group of 2, and a group of 3 subunits which are immunologically identical, but which are not related to each other.6.At the level of quaternary structure, a common principle is suggested for crustacean hemocyanins: 24 S hemocyanins are composed of three types of subunits, while 16 S hemocyanins contain only two types.

On the Role of Individual Subunits in the Quaternary Structure of Crustacean Hemocyanins

Among crustaceans, a widespread hemocyanin aggregate is the dodecamer (24S), composed of two hexameric halfs. According to computer correspondence analysis data (Bijlholt, unpublished), a one-point contact between the two hexamers is probable. A special subunit is required acting as inter-hexamer linker. In the dodecameric 24S hemocyanins of the crayfishes Cherax destructor and Astacus leptodactylus, a disulfide bridged dimer plays this role (1,2). Accordingly, heptameric intermediates were observed when Astacus dodecamers were stepwise dissociated into subunits. In contrast, dodecamers from the crab Cancer pagurus and the lobster Homarus americanus pass hexameric dissociation intermediates, and no dimers were found (3). One particular subunit, designated as alpha’, is absent in native 16S hemocyanin of Cancer, and forms dimers under reassociation conditions (4). Apart from the bridging unit, additional subunit types form the two basic hexamers in 24S crustacean hemocyanins. Although X-ray data on the spiny lobster, Panulirus interruptus hemocyanin afforded a deep insight into the conformation of a 16S particle (5), the question remains whether or not those “hexamer-formers” are structurally equivalent and interchangeable. Recently, new data on subunit correspondencies of crustacean hemocyanins became available, resulting in a definition of the immunological subunit types alpha, beta, and gamma (6). This now enables a better interspecific comparison of the results of our earlier (4) and the more recent experiments about reassembly and immuno labeling. They were performed with hemocyanin subunits from the brachyuran crabs Cancerpagurus and Callinectessapidus , and the astacuran crayfishes Homarus americanus and Astacus leptodactylus .

Mercury Ions — A Tool to Study the Specific Role of Individual Subunits in the Allosteric Interaction of Arthropod Hemocyanins

Native 37S hemocyanin of the tarantula Eurypelma californicum shows a complex oxygen binding behavior with low oxygen affinity, strong normal Bohr effect, and high cooperativity (Hill coefficient beyond 7). All three effects are created by an interaction of the 24 constituent subunits, because isolated subunits have neither Bohr effect nor cooperativity, and their oxygen affinity is high (1). The oxygen binding behavior of the native 24-mer establishes itself by an interaction of two dodecamers which, on their part, consist of two interacting hexamers (2).

The Spatial Range of Allosteric Interaction in A 24-Meric Arthropod Hemocyanin

In the 37 S hemocyanin (Hc) of the tarantula, Eurypelma californicum all the specific oxygen binding features arise by subunit interaction. Native tarantula Hc shows a relatively low oxygen affinity (P50 = 25 Torr at pH 7.5 and 25 iaC), a high Bohr effect and extremely high cooperativity (n50 running up to > 9) which is also pH dependent (1). Isolated subunits have high oxygen affinity and no Bohr effect (2). The quaternary structure of this Hc is well known (3). It can be described as an assembly of two pairs of unequal hexamers. Each hexamer comprises five distinct monomers, a, d, e, f and g, and one half of a heterodimer, bc. So, while the whole molecule is symmetric, the half-molecule is not, strictly speaking. It may be regarded as symmetric only in so far as it is composed of two hexamers.