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Characterization of the subunits of Macropipus puber hemocyanin
Comp. Biochem. Physiol. Vol. 81B, No. 4, pp. 857-862, 1985 Printed in Great Britain
0305-0491/85 $3.00 + 0.00 © 1985 Pergamon Press Ltd
CHARACTERIZATION OF THE SUBUNITS OF M A C R O P I P U S PUBER H E M O C Y A N I N W. GHIDALIA,* R. VENDRELY,* C. MONTMORY,* Y. COIRAULT,* D. ROCHUt and J. M. FINEr *Immunochimie et Srrologie des Arthropodes, Laboratoire de Zoologic, Universit6 Pierre et Marie Curie, 7 quai Saint-Bernard, 75005 Paris, France (Tel: 336-25-25); tLaboratoire d'Immunochimie des Protrines, Centre National de Transfusion Sanguine, 6 rue Alexandre Cabanel, 75015 Paris, France (Tel: 306-70-00)
(Received 3 January 1985) Almtraet--1. The hemocyanin subunits of the crab Macropipus puber are characterized with regard to their electrophoretic and antigenic heterogeneity. 2. Four antigenically distinct monomeric subunits referred to as I, II, III, IV, are identified by crossed immunoelectrophoresis of the whole hemocyanin. 3. There is no evidence for a covalent dimeric component in the hemocyanin of M. puber. 4. Comparative analysis of previously isolated hexamers and dodecamers shows that hexamers are devoid of subunit III, whereas this one is required for the assembly of dodecamers.
INTRODUCTION
scribed technique (Rochu and Fine, 1978). Hemocyanin location is ascertained by the cupric reaction according to Gould and Karolus (1975).
Still relatively little known in the 1960s, hemocyanin is now a m o n g the most investigated pigments. Hemocyanin (Hc) is a copper-containing respiratory pigment protein in the blood of Arthropods. Its minim u m functional unit is a polypeptide chain with a molecular weight close to 75,000, and the oligomeric forms are made up of 6, 12, 24 and 48 subunits in the phylogenic order of decapod Crustacea (Rochu et al., 1978). While the size heterogeneity of the subunits within a species is widely discussed (Rochu and Fine, 1984c), the analysis of their electric net charge, and the immunochemical comparison of their antigenic structure have allowed their characterization in various species of Crustacea (Larson et al., 1981; Rochu and Fine, 1984a; Van Eerd and Folkerts, 1981), and their identification in the polymeric forms (Rochu and Fine, 1984b). The portunid crab Macropipus puber (Mp) is currently under study in our laboratory (Ghidalia, 1968, 1972, 1984; Ghidalia et al., 1970, 1971, 1973), but the structure of its Hc was unknown. In the present work, we report the results of analysis of the subunit characterization of the respiratory pigment in this species, carried out by the methods successfully employed for the elucidation of Cancer pagurus Hc (Rochu and Fine, 1984a,b).
Cellulose acetate electrophoresis (CAE) CAE is performed according to Fine (1981). Cellogel strips (240 x 25 ram) are used. On each strip, 4/~I of sample are submitted to electrophoresis for 4 hr at 220 V, using a dissociating buffer, barbital-Tris-glycine-EDTA-ethanolamine pH 9.8 (sodium barbital, 0.04 M; Tris, 0.04 M; glycine, 0.01 M; sodium-EDTA, 0.001 M; ethanolamine, 0.04 M), or a non-dissociating buffer, barbital-calcium lactate pH 8.6 (sodium barbital, 0.04 M; barbital, 0.0075 M; calcium lactate, 0.0012M). Proteins are stained with Amidoschwarz 10 B in methanol-acetic acid (9:1, v:v) solution and destained in the same solvent. Analytical polyacrylamide gradient gel electrophoresis (PGGE) Analytical PGGE are performed in ultrathin-layer (0.5mm) casl as described by G6rg et al. (1981) with gradient from 3 to 15~ of polyacrylamide gel. To analyze Hc subunits according to their molecular size and electric net charge, a buffer system allowing dissociation of the whole Hc without the help of anionic detergent such as SDS, is needed. The barbital-Tris-glycine-EDTA-ethanolamine buffer mentioned above for cellulose acetate electrophoresis is successfully used for analytical PGGE. Proteins are stained with Coomassie Brilliant Blue R 250. SDS-PGGE SDS-PGGE are realized in ultrathin-layer (0.05mm) gradient gels (4-22.5~) according to the technique of Grrg et al. (1981), where as modification, a phosphate buffer system (0.1 M sodium phosphate pH 7.1) takes the place of the Tris buffer system described initially.
MATERIALS AND M E T H O D S
Preparation of hemocyanin Blood of Mp is collected with a syringe by puncture in the pereiopods sinus. After clotting at room temperature and centrifugation at 6000 rpm for 20 min, clots are discarded and hemolymph is collected. To avoid enzymatic degradation of the material, an inhibitor of proteases (Aprotinin, 1/I0,000, v/v) is added prior to the purification steps. Hemocyanin is isolated by combination of preparative ultracentrifugation of the hemolymph and preparative electrophoresis of the blue pellet, following a previously deC.B.P. 8 1 / 4 ~ D
Preparative PGGE Polymeric forms of Hc are isolated by preparative PGGE in gradients (2-15~) cast according to Lambin et al. (1976). Hexamers and dodecamers are obtained by PGGE in undissociating buffer (Tris, 0.05M; CaC12, 0.01 M; HCI pH 7.5).
857
858
W. GHIDAL1Aet al.
Preparation of ant&era Anti-whole hemocyanin sera are obtained by immunization of rabbits according to a previously described procedure (Rochu and Fine, 1978).
Table 1. Correlationbetweenthe Mp Hc fractionsobservedin inert and sievingmedia Electrophoresis medium
Inert medium (cellulose acetate)
Sieving medium (polyacrylamide gradient gel)
a b c
2+3 1 4
Crossed immunoelectrophoresis Two-dimensional immunoelectrophoresis is performed according to the technique of Weeke (1973), with modifications (Rochu and Fine, 1984a). The first step is performed on cellulose acetate strips as described above. After electrophoresis in the first dimension, a narrow strip (5 mm) of cellulose acetate is cut and applied on the agarose gel. The second step is realized in antiserum-containing agarose gel with various dilutions of antiserum. Precipitation peaks are stained with Amidoschwarz 10 B. RESULTS
Charge heterogeneity of the Mp hemocyanin subunits The experimental conditions allowing the dissociation of a protein with quaternary structure once defined, zone electrophoresis in an inert medium is the easiest technique for studying the charge heterogeneity of its subunits. In such a medium, in the present case cellulose acetate, the number of the evidenced hemocyanin fractions is buffer dependent. While non-dissociating buffers demonstrate only a single fraction corresponding to the whole molecule of the pigment (Fig. IA), dissociating buffers, on the contrary, share hemocyanin among three fractions referred to as a, b, c, implying at least three distinct components in the whole molecule of Mp hemocyanin (Fig. 1B). The use of a sieving medium increases the number of these components. Mp hemocyanin, when analyzed in dissociating conditions, through PGGE, gives rise to four fractions referred to, from the anode towards the cathode, as 1 4 (Fig. 1C).
Fractions
To parallel the various fractions evidenced in both kinds of media, inert and sieving ones, a preparative electrophoresis of Mp hemocyanin is carried out in cellulose acetate. The three fractions a, b, c, once recovered are then separately analyzed through PGGE, giving the correlations shown in Table 1. Thus, in the most discriminating conditions, that is: sieving medium plus dissociating buffer, the whole molecule of Mp Hc appears to be made of four kinds of subunits with quite different electrophoretical mobilities.
Size homogeneity of the Mp Hc subunits SDS-PGGE, when performed in ultrathin layer and with experimental conditions which allow a normal binding of SDS to the hemocyanin subunits (Rochu and Fine, 1984c), exhibits only a single fraction which corresponds to all the SDS-Mp Hc subunits complexes. Due to the high resolving power of this analytical method, this shows a size homogeneity of the various Mp hemocyanin subunits and close molecular weights. In the case with M. puber, a SDS-hemocyanin complex with the mobility of a dimer has never been observed. This agrees with the results of PGGE carried out in non-denaturing conditions. No braking of any Mp hemocyanin fraction through the sieving
wHc
A
//iii/¸ ab c
B
L
i~i!~i~i~i~'i~i~i~~/~'/~ii i :~ ~iii~'/ ~ .... 123
4
h
C Fig. 1. Electrophoretic pattern of Mp Hc. (A) Cellulose acetate electrophoresis in undissociating buffer. (B) Cellulose acetate electrophoresis in dissociating buffer. (C) Ultrathin layer polyacrylamide gradient gel electrophoresis in dissociating buffer, w Hc: whole hemocyanin, a,b,c: components of dissociated Hc observed in CAE. 1,2,3,4: components of dissociated Hc observed in PGGE. h: undissociated hexamer.
Characterization of the subunits of Macropipus puber hemocyanin
A
859
B 4-
i i ili:/
iii!iiii!
+
! i
iii
i i ¸i/~/i!iiiii/i!
¸
2d
i
(
Id
Fig. 2. Crossed immunoelectrophoresis of the whole Mp Hc. First dimension (ld): cellulose acetate. Second dimension (2d): agarose gel containing anti-Mp Hc serum. (A) First dimension in undissociating buffer. (B) First dimension in dissociating buffer. I, II, III, IV: monomeric subunits of Mp Hc. effect of the gel, suggesting the occurrence of a dimeric component, has so far been observed.
Antigenic heterogeneity of the Mp hemocyanin subunits Crossed immunoelectrophoresis in anti-Mp Hc serum-containing agarose allows us to analyze the
A
subunit composition of Mp Hc. Despite the fact that cellulose acetate exhibits three components only, this medium provides four precipitates due to the subunits of Mp Hc. These four peaks showed no reaction of partial nor total identity with each other, which suggests the occurrence in the whole molecule of Mp Hc, of at least four sorts of subunits. With reference
B 4-
2d +
(
Id
Fig. 3. Crossed immunoelectrophoresis of the Mp Hc polymeric forms. First dimension (td): cellulose acetate. Second dimension (2): agarose gel containing anti-Mp Hc serum. (A) Hexamers. (B) Dodecamers. I, II, III, IV: monomeric subunits of Mp Hc.
W. GI-I/DALIA et al.
860
to the terminology already used in the case with the Cancer pagurus hemocyanin (Rochu and Fine, 1984a), the subunits have been referred to as subunits I, II, III and IV. Figure 2 shows CIE of Mp Hc after its subunits have been submitted in the first dimension, to undissociating (Fig. 2A) and to dissociating conditions (Fig. 2B).
Subunit composition of hexamers and dodecamers The Mp hemocyanin, as that of most other decapods, shows when occurring in situ two main polymeric states which are respectively, made of 6 (hexamers) and 12 (dodecamers) polypeptide chains. Both kinds of polymers have been isolated through a preparative P G G E performed with a nondissociating buffer, and then analyzed by CIE, using an anti-Mp Hc immunserum, the hemocyanin subunits being previously separated through CAE. The hexamers are composed of only three of the subunits (I, II and IV); subunit III is not present (Fig. 3A). The dodecamers appear made of the four antigenically distinct subunits I, II, III and IV, already observed (cf. supra) in the whole Mp Hc (Fig. 3B). These observations point to a possible linking function played by subunit III in the formation of dodecamers.
DISCUSSION
Size homogeneity of Mp Hc subunits In specified experimental conditions, S D S - P G G E can be a valuable technique for the evaluation of the molecular size of Hc subunits. In the present work, by using P G G E in ultrathin layer, providing a highly resolutive separation, a size homogeneity about Mp Hc subunits is observed. This homogeneity agrees with our previous results which showed that Mp Hc only gives a single fraction at about 77,000 daltons, and such a homogeneity was also noted as regards the various subunits of the hemocyanin of other species of decapod (Rochu et al., 1978). Hemocyanins made up of subunits with markedly differing molecular weights have been reported in several species of Crustacea, but reconsideration of this heterogeneity in the light of recent observations (Rochu and Fine, 1984c) would be advisable before concluding on the matter. S D S - P A G E or SDSPGGE, when carried out with Tris-containing buffers would lead to misestimated molecular weight, for when SDS and Tris are simultaneously present; the intrinsic net charge in the polypeptide chain is not completely cancelled out. Thus, separation is not entirely according to molecular weight but may also have a component of charge accounting for the observed size heterogeneity. In support of this opinion is the case with Panulirus interruptus. Five distinct subunits with molecular weights ranging from 80,000 to 94,000 have been reported (Van Eerd and Folkerts, 1981), but more recent papers mention only two subunits (Van Holde, 1984), of which the molecular weight as inferred from the respective number of their amino acid residues, is about 75,000 (Gaykema et al., 1984). This value agrees with the value of 76,000 which we have
ourselves proposed for Panulirus regius, a related species from the same genus (Rochu et al., 1978). It is noteworthy that the various polypeptide chains which are assigned to aggregate together into hexameric and dodecameric forms have the same molecular weight. A size homogeneity is probably required for the obtention of an hexameric (or multihexameric) edifice which is a natural step for the self-association of globular structures submitted to certain physicochemical coertions.
Charge and antigenic heterogeneity ofMp Hc subunits Whereas S D S - P G G E indicates the existence of a subunits size homogeneity, in the absence of SDS, because the medium is an inert one or a sieving one, the electrophoretic separation of the subunits is due to their respective electric net charge only, or to the addition of their electric net charge, their molecular size and their spatial configuration. In these conditions, heterogeneous band patterns are observed. Three and four fractions are seen when the Mp Hc molecule is respectively, submitted to CAE and PGGE. Partially distinct amino acid sequences responsible for electric net charge differences can be reflected in the structure of the antigenic determinants of the various subunits. Indeed, 'the immunochemical investigation carried out by CIE leads to the following pattern: four different kinds of subunits (I, I, II and IV) with distinct electric net charge and antigenic structure occur inside the Mp Hc. Owing to the concomitant existence of hexamers and dodecamers in the respiratory pigment of M. puber, the subunit composition of these polymeric forms must be analyzed. While the four types of subunits (I, II, III and IV) occur in dodecamers, only three of them (I, II, IV) are present in hexamers. The occurrence of subunit III exclusively in dodecameric forms, indicates that this subunit plays a role in the building of dodecamers, whereas the hexamers are devoid of subunit III.
Structural comparison of hemocyanins from other species The result of the analysis of the structure of Mp Hc can be compared with that observed in Cancer pagurus (Cp) (Rochu and Fine, 1984a,b). First, in both species, one kind of subunit referred to as subunit III is only present inside dodecamers. Second, there is no evidence for a dimeric component in hexamers nor in dodecamers of Mp, whereas in the case with Cp a homodimer takes part in the constitution of hexamers and dodecamers. Finally, while the various subunits of a definite species show no reaction of partial nor total identity with each other, there are crossreactivities between subunits from Mp and Cp evoking the existence of an evolutive process from a common primitive ancestor (Rochu and Fine, 1984b). Although this dimeric component present in hexamers is not involved in a linkage role, there is, on the other hand, a strong presumption that in both species the presence of subunit III is required for the assembly of dodecamers from the polypeptide chains. The concomitant existence of hexamers and dodecamers, added to the inability for hexamers to promote dodecamers, were also observed in Cancer
Characterization of the subunits of Macropipus puber hemocyanin
rnagister (Ellerton et al., 1970), in Panulirus interruptus (Van den Berg et al. 1977) and in Cherax destructor (Jeffrey et al., 1978). Moreover, in Cp, the difficulty for dodecamers to acquire and store their architectural cohesion was pointed out (Rochu and Fine, 1980). In Cherax destructor, a dimeric component, which in this case too is probably a homodimer, has been proved essential for the reassembly of dodecamers from isolated monomers (Murray and Jeffrey, 1974; Jeffrey et al., 1978). To reconcile these facts with our data concerning Mp and Cp, the following evolutionary pattern can be evoked for arthropod hemocyanin. The linking of two hexamers through a dimer would be a primitive feature and is consequently found in the species which appeared early in the course of evolution. Thus, in Cherax destructor, of which the family has been known in Australia since the Pleiostocene but which belongs to an infraorder, the Astacidae appeared with its main features since Permotrias, hexamers join up through a dimer (Jeffrey et al., 1978). The same is true for Astacus leptodactylus (Pilz et al., 1980). In three species of primitive arthropods as different as a tarantula, Eurypelma californicum (Markl et al., 1981), a scorpion, Androctonus australis (Lamy et al., 1979a) and a merostomate, which is a living fossil, Limulus polyphemus (Lamy et al., 1979b; Brenowitz et al., 1981), a heterodimer seems to play a similar role. In Mp and Cp, on the contrary, in which the respective subfamilies, the Portuninae and the Cancrinae appeared about the middle of the Tertiary, the aggregation into dodecamers is only possible when a monomeric component referred to as subunit III is present. Moreover, the absence of a dimer in Mp and its occurrence inside Cp hexamers show that dimers are not required in this species for the dodecamer formation. Thus, this dimer which is inter-hexameric and still functions as a joining piece in primitive species becomes intra-hexameric and no more functional in more evolved species as in Cp in which it may be considered as a vestige, and it completely disappears in others such as Mp, the cohesion of dodecamers being then secured without the involvement of disulfide bridges. Hemocyanin, in which hexamers are linked together through disulfide bridges, splits less easily than one in which such covalent linkages do not take part in the inter-hexameric binding that makes it more labile. When analyzed by PGGE with alkaline buffers pH 10, Astacus sp. hemocyanin still shows high polymeric forms, whereas that of Mp and Cp is quite dissociated into subunits, the pigment of a more primitive crab Mai'a squinado displaying an intermediate pattern (Ghidalia et al., unpublished data). Evolution of hemocyanin developed throughout the arthropoda by a process in which the transition in the nature of the connection into dodecamers, changing from covalent to non-covalent, may account for the increased lability of the molecular structure of the pigment, as evolution goes on, making hemocyanin components of the lowest rank more common in the more evolved species than in the more primitive one. In the light of this example, applying similar ways of analysis to the hemocyanin of many other species of Crustacea would allow us to understand the key
861
structural features of their respiratory pigment, and also the structural modifications it underwent in the course of evolution. Such an investigation is presently at hand in our laboratories.
REFERENCES
Brenowitz M., Bonaventura C., Bonaventura J. and Gianazza E. (1981) Subunit composition of a high molecular weight oligomer: Limulus polyphemus hemocyanin..4rchs Biochem. Biophys. 210, 748-761. Ellerton H. D., Carpenter D. E. and Van Holde K. E. (1970) Physical studies of hemocyanins V. Characterization and subunit structure of the hemocyanin of Cancer magister. Biochemistry 9, 2225-2232. Fine J. M. (1981) Electrophor~seet Immunoelectropho~:eseen Pratique Courante. pp. 15-32. Maloine S.A., Paris. Gaykema W. P. J., Hol W. G. J., VereijkenJ. M., Soeter N. M., Bak H. J. and Beintema J. J. (1984) 3.2./~ structure of the copper-containing oxygen-carrying protein Panulirus interruptus haemocyanin. Nature, Lond. 3119,23-29. Ghidalia W. (1968) Etude 61ectrophorrtique et immunochimique du srrum d'un Crustac6 Drcapode: Macropipus puber (Linnr). Application ~ l'immunochimie systrmatique des Crustacrs. Th/~seprrsent~e ~. la Facult6 des Sciences de Paris. Ghidalia W. (1972) Electrophoretical analysis of Macropipus puber (L.) male serum: spectrophoretical study of the fractions. Comp. Biochem. Physiol. 43A, 969-973. Ghidalia W. (1984) Structural and biological aspects of pigments. In Biology of Crustacea, Vol. 9 (Edited by Mantel L. H.), pp. 301-394. Academic Press, New York. Ghidalia W., Vendrely R. and De Monty De Reze M. (1970) Analyse electrophorrtique de la variation des fractions protriques du srrum de Macropipus puber (L.) mille au cours du cycle d'intermue. Archs Zool. exp. g~n. I l l , 77-92. Ghidalia W., Vendrely R. and De Monty De Reze M. (1971) Electrophoretic analysis of Macropipus puber Crustacea male serum: the copper fractions. Comp. Biochem. Physiol. 40A, 479-485. Ghidalia W., Vicomte M. and King Bien Tan (1973) Immunoelectrophoretic analysis of Macropipus puber male serum. Comp. Biochem. Physiol. 44B, 715-724. Grrg A., Postel W., Westermeier R., Gianazza E. and Righetti P. G. (1981) SDS-gel gradient electrophoresis, isoelectric focusing and high-resolution two-dimensional electrophoresis in horizontal, ultrathin layer polyacrylamide gels. In Electrophoresis 1981 (Edited by Allen R. C. and Arnaud P.), pp. 259-270. De Gruyter, Berlin. Gould E. and Karolus J. J. (1975) A new stain for copper protein complexes: its use with Crustacea hemocyanins. Analyt. Biochem. 67, 515-519. Jeffrey P. D., Shaw K. and Treacy G. B. (1978) Hemocyanin from the Australian freshwater crayfish Cherax destructor. Characterization of a dimeric subunit and its involvement in the formation of the 25 S component. Biochemistry 17, 3078-3084. Lambin P., Rochu D. and Fine J. M. (1976) A new method for determination of molecular weights of proteins by electrophoresis across a sodium dodecyl sulfate (SDS)polyacrylamide gradient gel. Analyt. Biochem. 74, 567-575. Lamy J., Lamy J. and Weill J. (1979a) Arthropod hemocyanin structure: isolation of eight subunits in the scorpion. Archs Biochem. Biophys. 193, 140-149. Lamy J., Lamy J. and Weill J., Bonaventura J., Bonaventura C. and Brenowitz M. (1979b) Immunological correlates between the multiple hemocyanin subunits of Limulus polyphemus and Tachypleus tridentatus. Archs Biochem. Biophys. 196, 324-339. Larson B. A., TerwilligerN. B. and Terwilliger R. C. (1981)
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Subunit heterogeneity of Cancer magister hemocyanin. Biochim. biophys. Acta 667, 294-302. Markl J., Savel A. and Linzen B. (1981) Subunit composition of dissociation intermediates and its bearing in quaternary structure of Eurypelma hemocyanin. HoppeSeyler's Z. Physiol. Chem. 362, 1255-1262. Murray A. C. and Jeffrey P. D. (1974) Hemocyanin from the Australian freshwater crayfish Cherax destructor. Subunit heterogeneity. Biochemistry 13, 3667-3671. Pilz I., Goral K., Hoylaerts M., Witter R. and Lontie R. (1980) Studies by small angle X-ray scattering of the quaternary structure of the 24 S component of the haemocyanin of Astacus leptodactylus in solution. Eur. J. Bioehem, 105, 539-543. Rochu D. and Fine J. M. (1978) Antigenic structure of the hemocyanin in six species of decapod Crustacea. Comp. Biochem. Physiol. 59A, 145-150. Rochu D. and Fine J. M. (1980) Cancer pagurus hemocyanin: electrophoretic and antigenic heterogeneity of the monomeric subunits. Comp. Biochem. Physiol. 66B, 273-278. Rochu D. and Fine J. M. (1984a) Characterization of the subunits of Cancer pagurus hemocyanin. Comp. Biochem. Physiol. 77B, 333-336. Rochu D. and Fine J. M. (1984b) Cancer pagurus hemocyanin: subunit arrangement and subunit evolution in
functional polymeric forms. Comp. Biochem. Physiol. 78B, 67-74. Rochu D. and Fine J. M. (1984c) The molecular weights of arthropod hemocyanin subunits: influence of Tris buffer in SDS-PAGE estimations. Comp. Biochem. Physiol. 79, 41~,5. Rochu D., Lambin P., Ghidalia W. and Fine J. M. (1978) Hemocyanin subunits and their polymeric forms in some decapod Crustacea. Comp. Biochem. Physiol. 59B, 117-122. Van den Berg A. A., Gaastra W. and Kuiper J. A. (1977) Heterogeneity of Panulirus interruptus hemocyanin. In Structure and Function of Haemocyanin (Edited by Bannister J. V.), pp. 22-30. Springer-Verlag, Berlin. Van Eerd J. P. and Folkerts A. (1981) Isolation and characterization of five subunits of the hemocyanin from the spiny lobster Panulirus interruptus. In Invertebrate Oxygen-Binding Proteins. Structure, Active Site and Function (Edited by Lamy J. and Lamy J.), pp. 139-149. Marcel Dekker, New York. Van Holde K. E. (1984) Oxygen carriers of a third-kind, Nature, Lond. 309, 18. Weeke B. (1973) Crossed immunoelectrophoresis. In A Manual of Quantitative Immunoelectrophoresis. Methods and Applications (Edited by Axelsen N. H., Kroll J. and Weeke B.), Scand. J. Immun. 2, Suppl. 1, 46-56.
0305-0491/85 $3.00 + 0.00 © 1985 Pergamon Press Ltd
CHARACTERIZATION OF THE SUBUNITS OF M A C R O P I P U S PUBER H E M O C Y A N I N W. GHIDALIA,* R. VENDRELY,* C. MONTMORY,* Y. COIRAULT,* D. ROCHUt and J. M. FINEr *Immunochimie et Srrologie des Arthropodes, Laboratoire de Zoologic, Universit6 Pierre et Marie Curie, 7 quai Saint-Bernard, 75005 Paris, France (Tel: 336-25-25); tLaboratoire d'Immunochimie des Protrines, Centre National de Transfusion Sanguine, 6 rue Alexandre Cabanel, 75015 Paris, France (Tel: 306-70-00)
(Received 3 January 1985) Almtraet--1. The hemocyanin subunits of the crab Macropipus puber are characterized with regard to their electrophoretic and antigenic heterogeneity. 2. Four antigenically distinct monomeric subunits referred to as I, II, III, IV, are identified by crossed immunoelectrophoresis of the whole hemocyanin. 3. There is no evidence for a covalent dimeric component in the hemocyanin of M. puber. 4. Comparative analysis of previously isolated hexamers and dodecamers shows that hexamers are devoid of subunit III, whereas this one is required for the assembly of dodecamers.
INTRODUCTION
scribed technique (Rochu and Fine, 1978). Hemocyanin location is ascertained by the cupric reaction according to Gould and Karolus (1975).
Still relatively little known in the 1960s, hemocyanin is now a m o n g the most investigated pigments. Hemocyanin (Hc) is a copper-containing respiratory pigment protein in the blood of Arthropods. Its minim u m functional unit is a polypeptide chain with a molecular weight close to 75,000, and the oligomeric forms are made up of 6, 12, 24 and 48 subunits in the phylogenic order of decapod Crustacea (Rochu et al., 1978). While the size heterogeneity of the subunits within a species is widely discussed (Rochu and Fine, 1984c), the analysis of their electric net charge, and the immunochemical comparison of their antigenic structure have allowed their characterization in various species of Crustacea (Larson et al., 1981; Rochu and Fine, 1984a; Van Eerd and Folkerts, 1981), and their identification in the polymeric forms (Rochu and Fine, 1984b). The portunid crab Macropipus puber (Mp) is currently under study in our laboratory (Ghidalia, 1968, 1972, 1984; Ghidalia et al., 1970, 1971, 1973), but the structure of its Hc was unknown. In the present work, we report the results of analysis of the subunit characterization of the respiratory pigment in this species, carried out by the methods successfully employed for the elucidation of Cancer pagurus Hc (Rochu and Fine, 1984a,b).
Cellulose acetate electrophoresis (CAE) CAE is performed according to Fine (1981). Cellogel strips (240 x 25 ram) are used. On each strip, 4/~I of sample are submitted to electrophoresis for 4 hr at 220 V, using a dissociating buffer, barbital-Tris-glycine-EDTA-ethanolamine pH 9.8 (sodium barbital, 0.04 M; Tris, 0.04 M; glycine, 0.01 M; sodium-EDTA, 0.001 M; ethanolamine, 0.04 M), or a non-dissociating buffer, barbital-calcium lactate pH 8.6 (sodium barbital, 0.04 M; barbital, 0.0075 M; calcium lactate, 0.0012M). Proteins are stained with Amidoschwarz 10 B in methanol-acetic acid (9:1, v:v) solution and destained in the same solvent. Analytical polyacrylamide gradient gel electrophoresis (PGGE) Analytical PGGE are performed in ultrathin-layer (0.5mm) casl as described by G6rg et al. (1981) with gradient from 3 to 15~ of polyacrylamide gel. To analyze Hc subunits according to their molecular size and electric net charge, a buffer system allowing dissociation of the whole Hc without the help of anionic detergent such as SDS, is needed. The barbital-Tris-glycine-EDTA-ethanolamine buffer mentioned above for cellulose acetate electrophoresis is successfully used for analytical PGGE. Proteins are stained with Coomassie Brilliant Blue R 250. SDS-PGGE SDS-PGGE are realized in ultrathin-layer (0.05mm) gradient gels (4-22.5~) according to the technique of Grrg et al. (1981), where as modification, a phosphate buffer system (0.1 M sodium phosphate pH 7.1) takes the place of the Tris buffer system described initially.
MATERIALS AND M E T H O D S
Preparation of hemocyanin Blood of Mp is collected with a syringe by puncture in the pereiopods sinus. After clotting at room temperature and centrifugation at 6000 rpm for 20 min, clots are discarded and hemolymph is collected. To avoid enzymatic degradation of the material, an inhibitor of proteases (Aprotinin, 1/I0,000, v/v) is added prior to the purification steps. Hemocyanin is isolated by combination of preparative ultracentrifugation of the hemolymph and preparative electrophoresis of the blue pellet, following a previously deC.B.P. 8 1 / 4 ~ D
Preparative PGGE Polymeric forms of Hc are isolated by preparative PGGE in gradients (2-15~) cast according to Lambin et al. (1976). Hexamers and dodecamers are obtained by PGGE in undissociating buffer (Tris, 0.05M; CaC12, 0.01 M; HCI pH 7.5).
857
858
W. GHIDAL1Aet al.
Preparation of ant&era Anti-whole hemocyanin sera are obtained by immunization of rabbits according to a previously described procedure (Rochu and Fine, 1978).
Table 1. Correlationbetweenthe Mp Hc fractionsobservedin inert and sievingmedia Electrophoresis medium
Inert medium (cellulose acetate)
Sieving medium (polyacrylamide gradient gel)
a b c
2+3 1 4
Crossed immunoelectrophoresis Two-dimensional immunoelectrophoresis is performed according to the technique of Weeke (1973), with modifications (Rochu and Fine, 1984a). The first step is performed on cellulose acetate strips as described above. After electrophoresis in the first dimension, a narrow strip (5 mm) of cellulose acetate is cut and applied on the agarose gel. The second step is realized in antiserum-containing agarose gel with various dilutions of antiserum. Precipitation peaks are stained with Amidoschwarz 10 B. RESULTS
Charge heterogeneity of the Mp hemocyanin subunits The experimental conditions allowing the dissociation of a protein with quaternary structure once defined, zone electrophoresis in an inert medium is the easiest technique for studying the charge heterogeneity of its subunits. In such a medium, in the present case cellulose acetate, the number of the evidenced hemocyanin fractions is buffer dependent. While non-dissociating buffers demonstrate only a single fraction corresponding to the whole molecule of the pigment (Fig. IA), dissociating buffers, on the contrary, share hemocyanin among three fractions referred to as a, b, c, implying at least three distinct components in the whole molecule of Mp hemocyanin (Fig. 1B). The use of a sieving medium increases the number of these components. Mp hemocyanin, when analyzed in dissociating conditions, through PGGE, gives rise to four fractions referred to, from the anode towards the cathode, as 1 4 (Fig. 1C).
Fractions
To parallel the various fractions evidenced in both kinds of media, inert and sieving ones, a preparative electrophoresis of Mp hemocyanin is carried out in cellulose acetate. The three fractions a, b, c, once recovered are then separately analyzed through PGGE, giving the correlations shown in Table 1. Thus, in the most discriminating conditions, that is: sieving medium plus dissociating buffer, the whole molecule of Mp Hc appears to be made of four kinds of subunits with quite different electrophoretical mobilities.
Size homogeneity of the Mp Hc subunits SDS-PGGE, when performed in ultrathin layer and with experimental conditions which allow a normal binding of SDS to the hemocyanin subunits (Rochu and Fine, 1984c), exhibits only a single fraction which corresponds to all the SDS-Mp Hc subunits complexes. Due to the high resolving power of this analytical method, this shows a size homogeneity of the various Mp hemocyanin subunits and close molecular weights. In the case with M. puber, a SDS-hemocyanin complex with the mobility of a dimer has never been observed. This agrees with the results of PGGE carried out in non-denaturing conditions. No braking of any Mp hemocyanin fraction through the sieving
wHc
A
//iii/¸ ab c
B
L
i~i!~i~i~i~'i~i~i~~/~'/~ii i :~ ~iii~'/ ~ .... 123
4
h
C Fig. 1. Electrophoretic pattern of Mp Hc. (A) Cellulose acetate electrophoresis in undissociating buffer. (B) Cellulose acetate electrophoresis in dissociating buffer. (C) Ultrathin layer polyacrylamide gradient gel electrophoresis in dissociating buffer, w Hc: whole hemocyanin, a,b,c: components of dissociated Hc observed in CAE. 1,2,3,4: components of dissociated Hc observed in PGGE. h: undissociated hexamer.
Characterization of the subunits of Macropipus puber hemocyanin
A
859
B 4-
i i ili:/
iii!iiii!
+
! i
iii
i i ¸i/~/i!iiiii/i!
¸
2d
i
(
Id
Fig. 2. Crossed immunoelectrophoresis of the whole Mp Hc. First dimension (ld): cellulose acetate. Second dimension (2d): agarose gel containing anti-Mp Hc serum. (A) First dimension in undissociating buffer. (B) First dimension in dissociating buffer. I, II, III, IV: monomeric subunits of Mp Hc. effect of the gel, suggesting the occurrence of a dimeric component, has so far been observed.
Antigenic heterogeneity of the Mp hemocyanin subunits Crossed immunoelectrophoresis in anti-Mp Hc serum-containing agarose allows us to analyze the
A
subunit composition of Mp Hc. Despite the fact that cellulose acetate exhibits three components only, this medium provides four precipitates due to the subunits of Mp Hc. These four peaks showed no reaction of partial nor total identity with each other, which suggests the occurrence in the whole molecule of Mp Hc, of at least four sorts of subunits. With reference
B 4-
2d +
(
Id
Fig. 3. Crossed immunoelectrophoresis of the Mp Hc polymeric forms. First dimension (td): cellulose acetate. Second dimension (2): agarose gel containing anti-Mp Hc serum. (A) Hexamers. (B) Dodecamers. I, II, III, IV: monomeric subunits of Mp Hc.
W. GI-I/DALIA et al.
860
to the terminology already used in the case with the Cancer pagurus hemocyanin (Rochu and Fine, 1984a), the subunits have been referred to as subunits I, II, III and IV. Figure 2 shows CIE of Mp Hc after its subunits have been submitted in the first dimension, to undissociating (Fig. 2A) and to dissociating conditions (Fig. 2B).
Subunit composition of hexamers and dodecamers The Mp hemocyanin, as that of most other decapods, shows when occurring in situ two main polymeric states which are respectively, made of 6 (hexamers) and 12 (dodecamers) polypeptide chains. Both kinds of polymers have been isolated through a preparative P G G E performed with a nondissociating buffer, and then analyzed by CIE, using an anti-Mp Hc immunserum, the hemocyanin subunits being previously separated through CAE. The hexamers are composed of only three of the subunits (I, II and IV); subunit III is not present (Fig. 3A). The dodecamers appear made of the four antigenically distinct subunits I, II, III and IV, already observed (cf. supra) in the whole Mp Hc (Fig. 3B). These observations point to a possible linking function played by subunit III in the formation of dodecamers.
DISCUSSION
Size homogeneity of Mp Hc subunits In specified experimental conditions, S D S - P G G E can be a valuable technique for the evaluation of the molecular size of Hc subunits. In the present work, by using P G G E in ultrathin layer, providing a highly resolutive separation, a size homogeneity about Mp Hc subunits is observed. This homogeneity agrees with our previous results which showed that Mp Hc only gives a single fraction at about 77,000 daltons, and such a homogeneity was also noted as regards the various subunits of the hemocyanin of other species of decapod (Rochu et al., 1978). Hemocyanins made up of subunits with markedly differing molecular weights have been reported in several species of Crustacea, but reconsideration of this heterogeneity in the light of recent observations (Rochu and Fine, 1984c) would be advisable before concluding on the matter. S D S - P A G E or SDSPGGE, when carried out with Tris-containing buffers would lead to misestimated molecular weight, for when SDS and Tris are simultaneously present; the intrinsic net charge in the polypeptide chain is not completely cancelled out. Thus, separation is not entirely according to molecular weight but may also have a component of charge accounting for the observed size heterogeneity. In support of this opinion is the case with Panulirus interruptus. Five distinct subunits with molecular weights ranging from 80,000 to 94,000 have been reported (Van Eerd and Folkerts, 1981), but more recent papers mention only two subunits (Van Holde, 1984), of which the molecular weight as inferred from the respective number of their amino acid residues, is about 75,000 (Gaykema et al., 1984). This value agrees with the value of 76,000 which we have
ourselves proposed for Panulirus regius, a related species from the same genus (Rochu et al., 1978). It is noteworthy that the various polypeptide chains which are assigned to aggregate together into hexameric and dodecameric forms have the same molecular weight. A size homogeneity is probably required for the obtention of an hexameric (or multihexameric) edifice which is a natural step for the self-association of globular structures submitted to certain physicochemical coertions.
Charge and antigenic heterogeneity ofMp Hc subunits Whereas S D S - P G G E indicates the existence of a subunits size homogeneity, in the absence of SDS, because the medium is an inert one or a sieving one, the electrophoretic separation of the subunits is due to their respective electric net charge only, or to the addition of their electric net charge, their molecular size and their spatial configuration. In these conditions, heterogeneous band patterns are observed. Three and four fractions are seen when the Mp Hc molecule is respectively, submitted to CAE and PGGE. Partially distinct amino acid sequences responsible for electric net charge differences can be reflected in the structure of the antigenic determinants of the various subunits. Indeed, 'the immunochemical investigation carried out by CIE leads to the following pattern: four different kinds of subunits (I, I, II and IV) with distinct electric net charge and antigenic structure occur inside the Mp Hc. Owing to the concomitant existence of hexamers and dodecamers in the respiratory pigment of M. puber, the subunit composition of these polymeric forms must be analyzed. While the four types of subunits (I, II, III and IV) occur in dodecamers, only three of them (I, II, IV) are present in hexamers. The occurrence of subunit III exclusively in dodecameric forms, indicates that this subunit plays a role in the building of dodecamers, whereas the hexamers are devoid of subunit III.
Structural comparison of hemocyanins from other species The result of the analysis of the structure of Mp Hc can be compared with that observed in Cancer pagurus (Cp) (Rochu and Fine, 1984a,b). First, in both species, one kind of subunit referred to as subunit III is only present inside dodecamers. Second, there is no evidence for a dimeric component in hexamers nor in dodecamers of Mp, whereas in the case with Cp a homodimer takes part in the constitution of hexamers and dodecamers. Finally, while the various subunits of a definite species show no reaction of partial nor total identity with each other, there are crossreactivities between subunits from Mp and Cp evoking the existence of an evolutive process from a common primitive ancestor (Rochu and Fine, 1984b). Although this dimeric component present in hexamers is not involved in a linkage role, there is, on the other hand, a strong presumption that in both species the presence of subunit III is required for the assembly of dodecamers from the polypeptide chains. The concomitant existence of hexamers and dodecamers, added to the inability for hexamers to promote dodecamers, were also observed in Cancer
Characterization of the subunits of Macropipus puber hemocyanin
rnagister (Ellerton et al., 1970), in Panulirus interruptus (Van den Berg et al. 1977) and in Cherax destructor (Jeffrey et al., 1978). Moreover, in Cp, the difficulty for dodecamers to acquire and store their architectural cohesion was pointed out (Rochu and Fine, 1980). In Cherax destructor, a dimeric component, which in this case too is probably a homodimer, has been proved essential for the reassembly of dodecamers from isolated monomers (Murray and Jeffrey, 1974; Jeffrey et al., 1978). To reconcile these facts with our data concerning Mp and Cp, the following evolutionary pattern can be evoked for arthropod hemocyanin. The linking of two hexamers through a dimer would be a primitive feature and is consequently found in the species which appeared early in the course of evolution. Thus, in Cherax destructor, of which the family has been known in Australia since the Pleiostocene but which belongs to an infraorder, the Astacidae appeared with its main features since Permotrias, hexamers join up through a dimer (Jeffrey et al., 1978). The same is true for Astacus leptodactylus (Pilz et al., 1980). In three species of primitive arthropods as different as a tarantula, Eurypelma californicum (Markl et al., 1981), a scorpion, Androctonus australis (Lamy et al., 1979a) and a merostomate, which is a living fossil, Limulus polyphemus (Lamy et al., 1979b; Brenowitz et al., 1981), a heterodimer seems to play a similar role. In Mp and Cp, on the contrary, in which the respective subfamilies, the Portuninae and the Cancrinae appeared about the middle of the Tertiary, the aggregation into dodecamers is only possible when a monomeric component referred to as subunit III is present. Moreover, the absence of a dimer in Mp and its occurrence inside Cp hexamers show that dimers are not required in this species for the dodecamer formation. Thus, this dimer which is inter-hexameric and still functions as a joining piece in primitive species becomes intra-hexameric and no more functional in more evolved species as in Cp in which it may be considered as a vestige, and it completely disappears in others such as Mp, the cohesion of dodecamers being then secured without the involvement of disulfide bridges. Hemocyanin, in which hexamers are linked together through disulfide bridges, splits less easily than one in which such covalent linkages do not take part in the inter-hexameric binding that makes it more labile. When analyzed by PGGE with alkaline buffers pH 10, Astacus sp. hemocyanin still shows high polymeric forms, whereas that of Mp and Cp is quite dissociated into subunits, the pigment of a more primitive crab Mai'a squinado displaying an intermediate pattern (Ghidalia et al., unpublished data). Evolution of hemocyanin developed throughout the arthropoda by a process in which the transition in the nature of the connection into dodecamers, changing from covalent to non-covalent, may account for the increased lability of the molecular structure of the pigment, as evolution goes on, making hemocyanin components of the lowest rank more common in the more evolved species than in the more primitive one. In the light of this example, applying similar ways of analysis to the hemocyanin of many other species of Crustacea would allow us to understand the key
861
structural features of their respiratory pigment, and also the structural modifications it underwent in the course of evolution. Such an investigation is presently at hand in our laboratories.
REFERENCES
Brenowitz M., Bonaventura C., Bonaventura J. and Gianazza E. (1981) Subunit composition of a high molecular weight oligomer: Limulus polyphemus hemocyanin..4rchs Biochem. Biophys. 210, 748-761. Ellerton H. D., Carpenter D. E. and Van Holde K. E. (1970) Physical studies of hemocyanins V. Characterization and subunit structure of the hemocyanin of Cancer magister. Biochemistry 9, 2225-2232. Fine J. M. (1981) Electrophor~seet Immunoelectropho~:eseen Pratique Courante. pp. 15-32. Maloine S.A., Paris. Gaykema W. P. J., Hol W. G. J., VereijkenJ. M., Soeter N. M., Bak H. J. and Beintema J. J. (1984) 3.2./~ structure of the copper-containing oxygen-carrying protein Panulirus interruptus haemocyanin. Nature, Lond. 3119,23-29. Ghidalia W. (1968) Etude 61ectrophorrtique et immunochimique du srrum d'un Crustac6 Drcapode: Macropipus puber (Linnr). Application ~ l'immunochimie systrmatique des Crustacrs. Th/~seprrsent~e ~. la Facult6 des Sciences de Paris. Ghidalia W. (1972) Electrophoretical analysis of Macropipus puber (L.) male serum: spectrophoretical study of the fractions. Comp. Biochem. Physiol. 43A, 969-973. Ghidalia W. (1984) Structural and biological aspects of pigments. In Biology of Crustacea, Vol. 9 (Edited by Mantel L. H.), pp. 301-394. Academic Press, New York. Ghidalia W., Vendrely R. and De Monty De Reze M. (1970) Analyse electrophorrtique de la variation des fractions protriques du srrum de Macropipus puber (L.) mille au cours du cycle d'intermue. Archs Zool. exp. g~n. I l l , 77-92. Ghidalia W., Vendrely R. and De Monty De Reze M. (1971) Electrophoretic analysis of Macropipus puber Crustacea male serum: the copper fractions. Comp. Biochem. Physiol. 40A, 479-485. Ghidalia W., Vicomte M. and King Bien Tan (1973) Immunoelectrophoretic analysis of Macropipus puber male serum. Comp. Biochem. Physiol. 44B, 715-724. Grrg A., Postel W., Westermeier R., Gianazza E. and Righetti P. G. (1981) SDS-gel gradient electrophoresis, isoelectric focusing and high-resolution two-dimensional electrophoresis in horizontal, ultrathin layer polyacrylamide gels. In Electrophoresis 1981 (Edited by Allen R. C. and Arnaud P.), pp. 259-270. De Gruyter, Berlin. Gould E. and Karolus J. J. (1975) A new stain for copper protein complexes: its use with Crustacea hemocyanins. Analyt. Biochem. 67, 515-519. Jeffrey P. D., Shaw K. and Treacy G. B. (1978) Hemocyanin from the Australian freshwater crayfish Cherax destructor. Characterization of a dimeric subunit and its involvement in the formation of the 25 S component. Biochemistry 17, 3078-3084. Lambin P., Rochu D. and Fine J. M. (1976) A new method for determination of molecular weights of proteins by electrophoresis across a sodium dodecyl sulfate (SDS)polyacrylamide gradient gel. Analyt. Biochem. 74, 567-575. Lamy J., Lamy J. and Weill J. (1979a) Arthropod hemocyanin structure: isolation of eight subunits in the scorpion. Archs Biochem. Biophys. 193, 140-149. Lamy J., Lamy J. and Weill J., Bonaventura J., Bonaventura C. and Brenowitz M. (1979b) Immunological correlates between the multiple hemocyanin subunits of Limulus polyphemus and Tachypleus tridentatus. Archs Biochem. Biophys. 196, 324-339. Larson B. A., TerwilligerN. B. and Terwilliger R. C. (1981)
862
W. GHIDALIA et al.
Subunit heterogeneity of Cancer magister hemocyanin. Biochim. biophys. Acta 667, 294-302. Markl J., Savel A. and Linzen B. (1981) Subunit composition of dissociation intermediates and its bearing in quaternary structure of Eurypelma hemocyanin. HoppeSeyler's Z. Physiol. Chem. 362, 1255-1262. Murray A. C. and Jeffrey P. D. (1974) Hemocyanin from the Australian freshwater crayfish Cherax destructor. Subunit heterogeneity. Biochemistry 13, 3667-3671. Pilz I., Goral K., Hoylaerts M., Witter R. and Lontie R. (1980) Studies by small angle X-ray scattering of the quaternary structure of the 24 S component of the haemocyanin of Astacus leptodactylus in solution. Eur. J. Bioehem, 105, 539-543. Rochu D. and Fine J. M. (1978) Antigenic structure of the hemocyanin in six species of decapod Crustacea. Comp. Biochem. Physiol. 59A, 145-150. Rochu D. and Fine J. M. (1980) Cancer pagurus hemocyanin: electrophoretic and antigenic heterogeneity of the monomeric subunits. Comp. Biochem. Physiol. 66B, 273-278. Rochu D. and Fine J. M. (1984a) Characterization of the subunits of Cancer pagurus hemocyanin. Comp. Biochem. Physiol. 77B, 333-336. Rochu D. and Fine J. M. (1984b) Cancer pagurus hemocyanin: subunit arrangement and subunit evolution in
functional polymeric forms. Comp. Biochem. Physiol. 78B, 67-74. Rochu D. and Fine J. M. (1984c) The molecular weights of arthropod hemocyanin subunits: influence of Tris buffer in SDS-PAGE estimations. Comp. Biochem. Physiol. 79, 41~,5. Rochu D., Lambin P., Ghidalia W. and Fine J. M. (1978) Hemocyanin subunits and their polymeric forms in some decapod Crustacea. Comp. Biochem. Physiol. 59B, 117-122. Van den Berg A. A., Gaastra W. and Kuiper J. A. (1977) Heterogeneity of Panulirus interruptus hemocyanin. In Structure and Function of Haemocyanin (Edited by Bannister J. V.), pp. 22-30. Springer-Verlag, Berlin. Van Eerd J. P. and Folkerts A. (1981) Isolation and characterization of five subunits of the hemocyanin from the spiny lobster Panulirus interruptus. In Invertebrate Oxygen-Binding Proteins. Structure, Active Site and Function (Edited by Lamy J. and Lamy J.), pp. 139-149. Marcel Dekker, New York. Van Holde K. E. (1984) Oxygen carriers of a third-kind, Nature, Lond. 309, 18. Weeke B. (1973) Crossed immunoelectrophoresis. In A Manual of Quantitative Immunoelectrophoresis. Methods and Applications (Edited by Axelsen N. H., Kroll J. and Weeke B.), Scand. J. Immun. 2, Suppl. 1, 46-56.