Characterization of C-reactive protein from the eggs of the marine teleost, cyclopterus lumpus L.

44

Biochimica et Biophysica Acta, 671 (1981) 44-49

Elsevier/North-HollandBiomedicalPress BBA 38775 CHARACTERIZATION OF C-REACTIVE PROTEIN FROM THE EGGS OF THE MARINE TELEOST, CYCLOPTER US L UMPUS L.

THELMAC. FLETCHERa,*, ANN WHITEa, ARTHURYOUNGSONa, ARPADPUSZTAIb and BRIAN A. BALDO c a N.E.R.C. Institute o f Marine Biochemistry, St Fittick's Road, Aberdeen, AB1 3RA, b The Rowett Research Institute, Bucksburn, Aberdeen {U.K.) and c Roche Research Institute of Marine Pharmacology, Dee Why, N.S.W. (Australia}

(Received Feburary 26th, 1981) (Revised manuscriptreceivedAugust 3rd, 1981) Key words: C-reactive protein; Amino acid composition; Teleost egg; (Cyclopterus lumpus)

Further evidence is presented for the existence in teleost fish of proteins homologous with mammalian C-reactive protein. The amino acid composition is given for a C-reactive protein isolated from the eggs of a marine teleost, Cyclopterus lumpus, by extractionwith lecithin in the presence of Ca2+, followed by electrofocusing. A molecular weight of 150000 was calculated from gel filtration and electrophoresis at different polyacrylamide gel concentrations, while the S2o,w was 7.4 S. The 1.5-S subunits had an apparent Mr of 20 000 by SDS-polyacrylamide gel electrophoresis and 21 000 by computer analysis based on amino acid composition. Comparison is made with the physicochemical properties of mammalian C-reactive protein.

Introduction

A C-reactive protein-like precipitin, exhibiting Ca~+-dependent precipitation with pneumococcal Cpolysaccharide, was first described in the eggs of the lumpsucker, Cyclopterus lumpus, by Fletcher and Baldo [1] and isolated by White et al. [2]. The presence of proteins analogous to mammalian Creactive protein had earlier been reported in the serum of plaice and other marine teleosts [3]. C-reactive protein is the classic acute-phase protein of man with elevated levels during inflammatory processes [4-6]. An acute-phase response has also been observed in plaice exposed to bacterial endotoxins [7]. The Ca2*-dependent binding of mammalian Creactive protein to phosphocholine residues of pneumococcal C-polysaccharide [8] can initiate activation of the classical complement pathway through C1 [9]. Phosphocholine residues are widely distributed

in phospholipids [9,10] and the C-polysaccharide (C substance) from fungi [11 ] and parasites [12]. From in vitro studies, mammalian C-reactive protein has been implicated in many immunological reactions [13] but a deffmitive physiological role in vivo has not been established. The physicochemical properties of mammalian C-reactive protein have been the subject of several studies (reviewed by White et al. [2]) and chemical analyses have culminated in the complete amino acid sequencing of human C-reactive protein [14,15]. The evolutionary persistence of C-reactive protein would indicate its probable importance and information on the chemistry of C-reactive protein from lower vertebrates will contribute to comparative studies on possible functionally significant chemical structures. To this end, structural studies on plaice C-reactive protein have been reported [16], while this paper describes further properties of lumpsucker C-reactive protein.

* To whom correspondenceshouldbe addressed. Abbreviation;SDS, sodiumdodecyl sulphate. 0005-2795/0000-0000/$02.50 © 1981 Elsevier/North-HollandBiomedicalPress

45 Materials and Methods

Tryptophan was determined spectrophotometricaUy

[201. Mature female lumpsuckers were caught in salmon nets off the Aberdeen coast during their shoreward breeding migration between Feburary and June 1979. Fish were killed by a blow on the head and their eggs removed for immediate storage at -20°C. Purification of the C-reactive protein from the eggs was as described [2] based on lecithin extraction [10], followed by electrofocusing. Immunodiffusion and SDS-polyacrylamide gel electrophoresis (50 mM imidazole buffer, pH 7.0) were as previously descn"oed [2]. Gel filtration with Sephadex G-75 (Pharmacia) in 0.15 M NaC1/0.05 M sodium citrate/0.01 M EDTA and G-200 in 0.15 M NaC1/0.005 M CaC12 was performed as in Ref. 2. Electrophoresis. Conventional polyacrylamide gel electrophoresis was carried out as described by Fehrnstr6m and Moberg [17] with Tris-glycine buffer (pH 8.9). Using gels of 4, 6, 8 and 10% acrylamide concentration, the whole molecular weight of the lumpsucker C-reactive protein was calculated according to the method of Hedrick and Smith [18]. Computer analysis. A theoretical estimation of the subunit molecular weight of both human and lumpsucker C-reactive protein, based on amino acid composition, was made by the method of Delaage [19] with a Fortran program on a DEC-20 computer. Ultracentrifugation. The protein samples were examined by sedimentation velocity experiments in a Spinco Model E analytical ultracentrifuge at 20°C. For the evaluation of the sedimentation coefficients a 'Sevel' IBM-Fortran computer program was used. The program included adjustments of the slopes for temperature, viscosity and density of solutions. A close leakage tolerance of 0.0025 cm was automatically used. The sedimentation coefficients were obtained from an unconstrained linear least-squares fit of the position of the maximum ordinate on the Schlieren photographs against time, or from a quadratic least-squares fit, when this was better. The approximate proportions of the individual sedimenting boundaries were measured by planimetry on X10 enlargements of the Schlieren patterns. Amino adds. Hydrolysis was carried out in 6 M HC1 for 24 h at 110°C and the amino acids analysed with a JEOL JLC-6AH analyser. Half-cystine was determined as cysteic acid after performic oxidation.

ResuRs Preparations of lumpsucker C-reactive protein giving a single band on SDS-polyacrylamide gel electrophoresis (Fig. 1) were hydrolysed and their amino acid composition is given in Table I. The compositions of human [14], rabbit and mouse [21] and a recently described invertebrate [31] C-reactive protein, are given for comparison. We did not differentiate asparagine and glutamine from aspartic and glutamic acids, respectively, but the most noticeable difference from the other C-reactive proteins is the ilack of any detectable cystine in the lumpsucker preparations. The amino acid composition was used as the basis for a computer calculation of the minimum molecular weight of both human and lumpsucker C-reactive protein. This was 21 100 (Fig. 2) for both species.

a

b

c

Fig. 1. SDS-polyacrylamidegel electrophoresis (10% gel). Subunits of C-reactive protein: (a) lumpsucker; (b) plaice and (d) human; subunits of plaice serum amyloid P-component (c).

46 0.06 /x

0.05

0.04

y

---1

0.03

Fig. 3. Sedimentation diagrams of preparations of lumpsucker C-reactive protein. Substances were dissolved in 50 mM Tris/25 mM acetic acid buffer containing 100 mM NaC1 and 1 mM EDTA, pH 8.3 (a, left) or in 5 M guanidinium hydrochloride (b) and were centrifuged at 59 780 rev./min and 20°C. Photographs were taken at 52 rain (a) and 128 min (b) after reaching full speed. The direction of the centrifugal field is from right to left.

0.02

0.01

I

I

I

I

19

20

21

22

I

23

M. W. X10-3

Fig. 2. Estimation of minimum molecular weight of human (A) and lumpsucker (o) C-reactive protein based on amino acid composition. Y is a residual obtained by the method of Delaage [19].

Electrophoresis of dissociated subunits of lumpsucker C-reactive protein in the presence of SDS gave a molecular weight of 20 400 _+500, which represents the mean of 53 runs ±S.E. In two runs, purified human C-reactive protein was available and results normalised to the known molecular weight of human C-reactive protein [22], giving a corrected weight of 19 300. Gel t'dtration on calibrated Sephadex G-75 columns, of six samples of freeze-dried lumpsucker C-reactive protein dissolved in citrate-EDTA saline, yielded units of 14300 -+ 400. These derived from some natural dissociation [2] of the whole molecule, which eluted in the void volume. The molecular weight estimations of the whole C-reactive protein gave consistent results using different methods. Calibrated Sephadex G-200 columns gave a weight of 148000-+ 3000 for 23 different samples of lumpsucker protein. Electrophoretic

methods [18] gave a weight of 150000 +_5500 for 31 samples. Lumpsucker C-reactive protein at about 1% (w/v) concentration was examined by sedimentation velocity experiments (Fig. 3). The main component had an s20,w value of 1.5 S at pH 8.3 in the presence of 1 mM EDTA. For a globular protein this value would indicate a molecular size of about 1 5 0 0 0 - 2 0 0 0 0 . Some preparations also contained a 7.4 S component (Fig. 3a). The most likely molecular size value for this component is about 150000. In 5 M guanidinium HC1, the main component had an S2o,w value of 1.3 S. This probably corresponds to a main component of 1.5 S in non-dissociating buffers. In addition, this preparation also contained small amounts of a 0.7 S component. It was not clear from these experiments whether the high or low molecular weight components in these preparations were related as aggregates or dissociated species of the main component of about 15 0 0 0 - 2 0 000 molecular size. An antiserum prepared to the lumpsucker egg Creactive protein precipitates identically with both egg and lumpsucker serum [2]. There is a strong crossreaction between this antiserum and the C-reactive protein isolated from another marine teleost, the plaice [22]. Similarly, an antiserum to plaice Creactive protein cross-reacts with lumpsucker Creactive protein (Fig. 4). These results indicate anti-

47

Fig. 4. Immunodiffusion in 1% agarose in 0.15 M NaC1. (1) Plaice C-reactive protein (300 gg/ml water); (2) rabbit antiserum to plaice C-reactive protein; (3) male lumpsucker serum; (4) rabbit antiserum to lumpsucker egg C-reactive protein.

genic similarity between C.reactive protein from distantly related fishes. There was no precipitin formed with either plaice or lumpsucker C-reactive protein against goat anti-human C-reactive protein (ICL Scientific: batch P2901) and rabbit anti-human C-reactive protein (Behring: batch 2406x). Discussion

Our results from amino acid analyses show overall similarity with published analyses of mammalian Creactive proteins (Table I). There are some differences, however, most notably the lack of detectable halfcystine in the lumpsucker. Bach et al. [23] also

reported a similar condition in rabbit C-reactive protein but the more recent analyses of Oliveira et al. [21] clearly indicate that there are two half-cystines present per mole of C-reactive protein in the rabbit, as in the mouse. Unlike in the mammals, in the lumpsucker C-reactive protein tryptophan is present in greater amounts than is tyrosine. The amino acid composition of C-reactive proteins from other lower vertebrates has still to be published but a partial sequence from the N-terminal of plaice C-reactive protein shows 41% homology with the first 35 amino acids of human C-reactive protein [16]. There is a close antigenic relationship between lumpsucker and plaice C-reactive protein but on the basis of the two antisera tested, there is none apparent between human and fish C-reactive proteins. Immunological cross-reactivity and amino acid composition can, however, prove of use in phylogenetic studies where sequence data are not available [24]. The amino acid composition of lumpsucker C-reactive protein proved suitable for computer analysis of the minimum molecular weight, giving a result similar to that from human, which had been used as a test for the program. The sedimentation velocity experiments gave an approximate indication of molecular weight. In both EDTA-containing Tris buffer and the dissociating guanidinium, the major component appeared to be of Mr 15 0 0 0 - 2 0 0 0 0 , which would be in the range of subunit weight obtained by SDS-polyacrylamide :gel electrophoresis. The non-covalently linked subunits [2] readily dissociate and only a small proportion were still present in the EDTA-containing buffer as the 7.4 S molecule. The approximate Mr of 150 000 corresponds very closely with the value obtained from gel filtration. It was not clear which factors controlled the association-dissociation reactions of the lumpsucker C-reactive protein and, because of the presence of small amounts of low molecular weight material in some preparations, sedimentation equilibrium studies were not carried out at this stage. Volanakis et al. [25] calculated a weight average molecular weight of 118000 for human C-reactive protein using sedimentation equilibrium and a sedimentation coefficient, S2o,w, of 6.5 S. Electron microscopy has revealed human C-reactive protein to be formed of five subunits arranged in a single disc [26], while the subunits of plaice C-

48 TABLE I AMINO ACID COMPOSITION OF LUMPSUCKER,MAMMALIANAND AN INVERTEBRATE C-REACTIVEPROTEIN Amino acid

Lys His Arg Asp Asn Thr Ser Glu Gin Pro Gly Ala Val Met lie Leu Tyr Phe Trp Cys

mol/100 mol Lumpsucker a

Human b

Rabbit c

Mouse c

Limulus d

5.61 ± 0.10 2.56 ± 0.12 5.41 ± 0.08 10.54 ± 0.04 n.a. 4.96 ± 0.03 7.89 ± 0.12 10.78 ± 0.23 n.a. 3.20 ± 0.08 8.59 -+0.13 5.29 ± 0.05 7.13 ± 0.13 3.11 ± 0.34 3.35 ± 0.05 8.85 ± 0.07 2.18 ± 0.08 6.13 ± 0.07 4.39 ± 0.16 n.d.

6.42 1.07 3.21 4.28 3.74 6.42 9.63 7.49 3.21 5.88 7.49 4.81 9.09 1.07 4.81 7.49 3.74 6.42 2.67 1.07

6.12 1.33 2.75 9.95 n.a. 4.64 9.13 9.95 n.a. 5.41 8.77 5.25 7.75 1.94 4.79 8.88 5.25 6.07 1.02 1.02

6.82 1.21 3.12 9.71 n.a. 4.62 9.42 10.00 n.a. 5.89 8.38 5.37 6.87 1.62 4.51 8.21 4.97 5.43 2.72 1.16

6.2 5.6 2.0 10.8 n.a. 6.2 6.8 13.0 n.a. 3.4 9.2 4.7 5.9 0.9 5.0 9.8 1.9 4.0 2.0 2.0

a Prepared by electrofocusing [2]. Values represent the mean ± S.E. of seven preparations. b Calcualted from the data of Oliveira et al. [14]. c Calculated from the data of Oliveira et al. [21 ]. d Calculated from the data of Robey and Liu [31]. n.a., not assayed. n.d., not detectable.

reactive protein appear to be arranged as two pentameric discs [22]. Lumpsucker C-reactive protein has not been examined in the electron microscope, but if one assumes a similarity to plaice then the molecule would be composed of 10 subunits. If an Mr of 150 000 represents the intact molecule, then subunits of Mr 15 000 would be necessary. Volanakis et al. [25] found some discrepancies between the subunit M r of 2 4 0 0 0 for human C-reactive protein, calculated from SDS-polyacrylamide gel electrophoresis and that of 21 000 from the primary sequence [14]. We found that human C-reactive protein gave a molecular weight of 23 400 on SDS-polyacrylamide gel electrophoresis and assume that C-reactive proteins exhibit some irregularity in their mobility in SDSpolyacrylamide gels [22]. When human C-reactive protein was run in our system and corrected to its

k n o w n molecular weight, the lumpsucker Mr was reduced b y approximately 1000. Mammalian C-reactive protein has been reported to be associated with lipid [27,28]. Pontet et al. [29] found the C-reactive protein in rabbit serum to be exclusively bound to low density lipoproteins. These factors might influence electrophoretic mobility, together with the ready binding of C-reactive protein in the presence of Ca 2÷, to many electrophoretic media [30]. The C-reactive protein isolated from lumpsucker eggs did not appear to contain carbohydrate and any associated lipid was removed b y the Hokama method of preparation. Although the number of subunits contributing to the intact C-reactive protein molecule of the lumpsucker has still to be clarified, the molecular weight studies and amino acid composition are consistent

49 with the evidence in lower vertebrates of molecules homologous with mammalian C-reactive protein.

Acknowledgements We thank Mr. S.N. Forrest and Mr. B. Cran for computer analyses and Mr. J.C. Stewart for ultracentrifugation studies. Dr. M.B. Pepys provided the plaice C-reactive protein and the rabbit antiserum to it, together with the h u m a n C-reactive protein.

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