Isolation and partial characterization of a low molecular weight immunoglobin from snake (Psammophis sibilans) serum

Comp. Biochem.Physiol.Vol. 104B,No. 2, pp. 281-286, 1993 Printed in Great Britain

0305-0491/93 $6.00+ 0.00 © 1993Pergamon Press Ltd

ISOLATION A N D PARTIAL CHARACTERIZATION OF A LOW MOLECULAR WEIGHT I M M U N O G L O B U L I N FROM SNAKE (PSAMMOPHIS SIBILANS) SERUM HODA I. NEGM, AHMED M. Aao EISHA,* MOHAMED H. MANSOUR,t ABDEL HAKIM M. SAAD~ and FATHIM. ABDEL GALIL* Zoology Department, Faculty of Science, Monofia University; *Chemistry Department and #Zoology Department, Faculty of Science, Cairo University, Cairo 12613, Egypt

(Received 17 July 1992; accepted 21 August 1992) Abstract--l. A low molecular weight immunoglobulin fraction has been isolated by protein A affinitychromatography from the serum of non-immune snakes (Psammophis sibilans). 2. Analysis of the purified molecule by SDS-PAGE under non-reducing conditions indicated that the molecular weight of the whole Ig was 182 kDa, homologous to the IgY of lower vertebrates and distinct from rabbit IgG that was co-purified under identical conditions. 3. As determined by SDS-PAGE and gel filtration, in a dissociating buffer of the reduced and alkylated molecule, snake IgY was composed of a H-chain of molecular weight about 70 kDa and an L-chain of about 22 kDa. The H:L molar ratio was roughly 1:1 and the subunit chain composition of the purified Ig assumed to be H 2 L 2. 4. The amino acid composition of purified H- and L-chains of snake IgY was compared by the SAQ index differences in composition to rabbit IgG ),-chain as well as to/z-like and non-/z-polypeptides of primitive vertebrates. The snake polypeptide was, interestingly, more closely related in structure to the primitive Ig subunits than to the functionally-specific ),-chain of mammals.

INTRODUCTION Evidence has been reviewed which establishes that fish, as well as tetrapod vertebrates, possess low molecular weight immunoglobulins (LMW Ig) as distinct from the IgM class, which is ubiquitous in distribution among vertebrate species (Marchalonis, 1977). Although the final assessment of a structural homology among these immunoglobulins, and those of higher mammals, must entail comparison of amino acid sequences of heavy chains from the diverse species, sufficient data exist at present to allow some general conclusions to be drawn regarding the non-/z heavy chains in phylogeny. Based on variations among the various species tested, current views favour the possibility that IgG-like L M W polypeptides of reptiles and birds might not be homologous to those of amphibians. Subsequently, genes encoding the various ~-like heavy chains could have arisen independently during amphibian and reptilian evolution (Litman, 1976). Nevertheless, supporting evidence for this notion must await information on Ig structure and analyses of Ig genes among representatives occupying key position in vertebrate evolution which, as yet, is unavailable. Reptiles represent a pivotal position in the phylogeny of the immune system since they are considered the common ancester of birds and mammals. Available data suggest that reptiles exhibit a diversity of immune responses equivalent to the humoral and ~To whom correspondence should be addressed. 281

cell-mediated reactions of higher vertebrates (El Ridi et al., 1988). A precise analysis of immunoglobulin structure should facilitate further studies on humoral immunity in this group of primitive vertebrates. In the present study, the L M W IgY has been purified from the normal serum of the snake, Psammophis sibilans, by a single-step purification that involves protein A afffinity-chromatography. Unlike previous investigations, where purification of reptilian immunoglobulins required a combination of diverse preparative techniques (Ambrosius, 1976), the documented capacity of protein A to bind IgG and IgG-like molecules has been used in this study (Hsu and Du Pasquaier, 1984). Based on amino acid composition analyses, our data may have implications in the understanding of the structural interrelationships of L M W Ig classes in phylogeny.

MATERIALS AND METHODS

Animals Adult males and females of the snake, Psammophis sibilans (non-hibernator, diurnal, oviparous) were collected from Moudureit El Tahrir Area. Snakes were maintained in an animnal room at a temperature range of 18-25°C and provided with live lizards ad libitum. A total of 100 animals were used in the present study. Adult New Zealand rabbits weighing 3-5 kg were used and maintained in an animal room at room temperature and given chow ad libitum.

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Blood was obtained from normal snakes by decapitation and from normal rabbits by bleeding from the ear penna. Clots were allowed to stand for 2 hr at room temperature, then overnight at 4°C, and sera were separated by centrifugation at 3000 rpm for 30 min at 4°C. Sera were decomplemented at 56°C for 30 min, dialyzed against three exchanges of a dialysis buffer (0.05 M phosphate, pH 8.4 containing 0.15 M NaCI and 0.02% NAN3), and stored at - 2 0 ° C until used.

Gel filtration A column (2.5 × 100 cm) was packed with Sephadex G-200 (Pharmacia Fine Chemicals, Uppsala, Sweden) in dialysis buffer, pH 8.4, at room temperature. Samples of post-protein A column fractions were introduced to the column by an upward flow at a rate of 15ml/hr using an LKB peristaltic pump (Pharmacia). Samples were eluted using a dialysis buffer, pH 8.4, continuously monitored for protein at 280 nm, using a dual-channel LKB UV cord (Pharmacia) and collected as 10 ml aliquots using an LKB automated fraction collector. Samples were concentrated for analysis by dialysis against three exchanges of double-distilled water followed by freeze-drying.

Protein A-Sepharose 4B affinity chromatography A 5-ml column of immobilized protein A was prepared by coupling protein A to CNBr-activated Sepharose 4B (Pharmacia) at a ratio of 2 mg protein A to 1 ml swollen beads. The protein A column was equilibrated with 0.58% acetic acid containing 0.15 M NaC1, and then with phosphate-buffered saline (PBS), pH 7.4, containing 0.15 M NaC1. Sera were applied to protein A column at a flow-rate of 3 ml/hr and the protein A column washed with PBS, pH 7.4, containing 0.15 M NaC1 and 0.02% N a N 3. Material bound to the protein A column was eluted with 0.58% acetic acid containing 0.15 M NaC1 and 0.02% NaN 3. The eluted fractions were immediately brought to pH 8.0 with solid NaHCO3, dialyzed against two exchanges of PBS, pH 7.4, and then against several exchanges of distilled water, and freeze-dried.

Reductive cleavage of immunoglobulins Reduction and alkylation of purified snake Ig was performed by the method of Fleischman et al. (1961). Briefly, a 2% (w/v) solution of post-protein A column fractions in 0.55 M Tris-HC1, pH 8.2, was made up to 0.75 M with 2-mercaptoethanol and the mixture was kept at room temperature at 1 hr. The mixture was then cooled and an equal volume of 0.75 M cold iodoacetamide was added to alkylate the liberated sulfhydryl groups. The mixture was allowed to stand for 1 hr in the dark and the pH maintained at about 8.0 by the step-wise addition of trimethylamine. Reduced proteins were dialyzed against 1 N cold

acetic acid, centrifuged at 6000 rpm to remove insolubles and then fractionated by gel filtration on a Sephadex G-200 column, equilibrated and eluted with 1 N acetic acid.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) Proteins in whole sera or column fractions were separated on slab gels (5% and/or 10% acrylamide and 0.8% bisacrylamide) (Sigma Chemical Co., St Louis, MO) in the presence of SDS (BDH, Poole, U.K.) under reducing and non-reducing conditions, using the discontinuous buffer system of Laemmli (1970). Gels were electrophoresed on an LKB multiphore 2103 horizontal slab gel apparatus, at 20 mA constant current until the tracking dye (Bromophenol Blue, Sigma) was within 0.5 cm of the gel edge. Gels were stained with 0.12% (M/v) Coomassie Blue R-250 (Sigma) in 8% (v/v) acetic acid, 25% (v/v) ethanol, and photographed wet using a Kodacolor 100 ASA film. Molecular weight standards were phosphorylase B (94 kDa), BSA (67 kDa) ovalbumin (43 kDa), carbonic anhydrase (30kDa), soybean trypsin inhibitor (20 kDa) and c~-lactalbumin (14.9 kDa) and were all electrophoresed in sample buffer as described above.

Amino acid composition analysis Amino acid analysis was performed using a modified Beckman 121 automatic amino acid analyzer. Purified heavy (H) and light (L) chains of snake immunoglobulin were hydrolyzed under vacuum in 6 M HC1 for 24 and 66 hr at 110°C before analysis and the values of amino acids averaged over the two hydrolysis times. Half-cystine values were determined as cystic acid residues resulting from acid oxidation.

Pair-wise comparisons of amino acid compositions The amino acid compositions of purified H- and L-chains of snake Ig were compared to each other and to those of other vertebrates by the SAQ index, devised by Marchalonis and Weltman (1971), which is calculated for J amino acid as:

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where x represents the mole percentage of amino acid i for two proteins a and b. The degree of relatedness was reciprocally related to the value of the SAQ units (i.e. the smaller the value, the greater the relatedness). R E S U L T S AND D I S C U S S I O N

Dialyzed P. sibilans serum was mixed with an equal volume of PBS, pH 7.4, and subjected to protein A affinity chromatography. Samples of normal rabbit serum were similarly processed and applied in parallel to the same affinity column under identical conditions. The results of a typical elution profile for both serum samples are shown in Fig. l(a), where

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Elution volume (ml) Fig. 1. Protein A-affinity chromatography (A) and SDS-PAGE profiles (B) of purified Ig of snake (S) and rabbit (R). Normal snake serum (NSS; O) and/or normal rabbit serum (NRS; ©) were separately loaded onto the protein-A column at 3 ml/hr and washed with PBS, pH 7.4. Bound materials (B) were eluted with 0.58% acetic acid, neutralized, dialyzed and concentrated prior to electrophoresis on a 5% SDS-PAGE (non-reducing) along with similarly processed unbound fractions (U). Protein was estimated by A2s0 nm reading and the position of molecular weight markers indicated. approximately 7 and 13% of the total protein in snake and rabbit sera, respectively, were retained and eluted by the addition of 0.58% acetic acid. Analyzed by S D S - P A G E under non-reducing conditions (Fig. lb), the bound and eluted fractions in rabbit

serum consisted of a predominant component of 150 k D a which, in accordance with published observations (Goudswaard et al., 1978), represented a purified fraction of IgG. The snake bound and eluted fractions, on the other hand, also consisted of a single

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macromolecule, but with a molecular weight larger than that o f rabbit IgG, and estimated at 182 k D a (Fig. lb). It is n o t e w o r t h y that the estimated molecular weight o f the protein A fraction o f the snake serum was significantly lower than the 1200 kDa IgM o f the anuran X e n o p u s laevis, which is reported to bind protein A under similar conditions (Hadji-Azimi and Schwager, 1982; Hsu and Du Pasquier, 1984). To examine the molecular form o f the native portein A - b o u n d snake Ig in serum, a sample o f whole snake serum was c o m p a r e d to a sample o f the fraction not b o u n d to protein A by gel filtration through a Sephadex G-200 column in a non-dissociating buffer. The elution profile in b o t h cases was Table 1. Amino acid composition* of snake and hagfish IgH- and L-chains and the rabbit y-chain Snake Snake Hagfish Hagfish Rabbit H-chain L-chain H-chain L-chain v-chain Asx 9.71 8.80 9.01 10.73 11.27 Thr 8.07 7.75 8.89 7.97 8.05 Ser 8.76 11.90 9.30 9.28 14.49 Glx 13.05 12.35 12.14 10.11 8.05 Pro 6.17 5.15 6.47 5.83 4.83 Gly 3.44 3.90 2.88 4.07 6.44 Ala 4.26 3.95 4.76 4.98 4.83 Cys 2.78 2.35 3.12 2.01 1.61 Val 7.37 6.95 6.81 7.55 6.44 Met 1.20 0.71 0.89 1.60 1.61 Ile 6.36 3.70 6.10 5.01 1.61 Leu 6.91 7.75 7.45 8.82 6.44 Tyr 3.47 6.55 6.75 7.33 4.83 Phe 3.85 4.30 4.17 4.33 3.22 His 2.00 1.85 t.94 1.60 4.83 Lys 5.23 5.70 6.23 5.30 8.05 Arg 4.85 6.05 5.76 4.86 3.22 *All analyses were calculated as being equivalent to the mean number of each amino acid residue per 100 residue. +Data recalculated from Hill et al., 1966.

m o n i t o r e d using pure rabbit IgG and monomeric BSA as internal markers and the peak composition assessed by non-reducing S D S - P A G E . As shown in Fig. 2, whole snake serum resolved into six main peaks, o f which the second peak constituted p r e d o m inantly a 182 kDa c o m p o n e n t . This c o m p o n e n t was selectively depleted in the elution profile o f the u n b o u n d fraction to protein A which, otherwise, showed a profile similar to whole serum (data not shown). This, in turn, was indicative o f the occurrence o f the protein A - b o u n d snake Ig in serum as a stable m o n o m e r o f 182 kDa. The subunit composition of the protein A - b o u n d snake Ig was revealed by gel filtration (Fig. 3a) and by S D S - P A G E (Fig. 3b) in a dissociating buffer following reduction and alkylation of the intact molecule along with purified fractions o f rabbit IgG and hagfish IgM, which were similarly treated in parallel. It was evident that the 182 kDa snake Ig resolved into 70 and 22 kDa polypeptides that correlated, in terms o f structural organization, to rabbit IgG and hagfish IgM H- and L-chains, respectively. The molar ratio, calculated from the molecular weights o f snake Ig Hand L-chains, was similar to those o f rabbit IgG and Table 2. SAQ values for comparisonsamong snake and hagfish lg H-chain and L-chains and the rabbit 7-chain Snake Snake Hagfish Hagfish Rabbit H-chain L-chain H-chain L-chain ~'-chain Snake H-chain 0 Snake L-chain 32.4 0 Hagfish H-chain 16.5 18.5 0 Hagfish L-chain 32.6 23.6 16.9 0 Rabbit ~-chain 117.45 ND* 117.27 ND 0 *ND: Not determined.

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Table 3. SAQ values for comparisonsamong H- and L-chainsof snake and lower vertebrate Igs Snake Snake Lungfish Lungfish Dogfish7S Dogfish Shark H-chain L-chain H-chain* L-chain* H-chain* L-chain* L-chaint Snake H-chain 0 . . . . . . Snake L-chain 32.4 0 . . . . . Lungfish H-chain 30.2 29.7 0 . . . . Lungfish L-chain 33.9 22.3 5.1 0 ---Dogfish 7S H-chain 6.1 23.0 25.6 27.3 0 --Dogfish L-chain 28.9 5.8 22.5 13.3 18.0 0 -Dogfish 17S H-chain 19.3 12.8 14.1 13.7 12.4 12.8 77.2 Shark L-chain 82.7 50.8 91.6 70.0 70.6 60.0 0 Amino acid compositionswere taken from *Marchalonisand Edelman (1966); tLobb and Clem (1981).

hagfish IgM which, in all cases, was roughly l : l . Given that the intact molecule is 182 kDa, it can be assumed that snake Ig is present mainly in an HEL 2 composition in serum, a fundamental structure of immunoglobulins in all vertebrates (Marchalonis, 1977). Although the occurrence of snake Ig in a monomeric form, with no evidence of the presence of multiple forms of the molecule, or the presence of J chain-like components, rules out its relatedness to the IgM class, the isolated H-chain appeared to be larger than those reported for non-# polypeptide chains. On one hand, the snake Ig H-chain was obviously distinct from the 38 kDa H-chain of the IgN reported to exist in reptiles and aves (Marchalonis, 1977). On the other hand, the 7 0 k D a snake H-chain seemed to be homologous to the v-chain of the IgY molecule which, however, was reported to vary in molecular weight in various vertebrates. Thus, the polypeptide was identified as 59.SkDa in lizards (Natrajan and Muthukkaruppan, 1984), 60-63 kDa in chelonians (Kubo et al., 1973; H/idge and Ambrosius, 1984; Andreas and Ambrosius, 1989) and 65 kDa in birds (Leslie and Clem, 1969; Zimmerm a n et al., 1971). In a n u r a n amphibians, differential glycosylation accounted for the presence of two molecular weight variants of the v-polypeptide (Hsu and D u Pasquier, 1984) and this may be extended to interpret the observed variations in different animal models. To allow further structural characterization of purified snake IgY, separated H- and L-chains were analyzed for their amino acid composition and compared to their structural counterparts in hagfish IgM and rabbit IgG. As shown in Table 1, the composition showed a notably high percentage of the more hydrophilic residues (glutamic acid, aspartic acid and serin residues) than of the more hydrophobic residues (methionine, phenylalanine and isoleucine residues), indicating an overall hydrophilicity for purified snake Ig subunits comparable to that noted for other Ig polypeptides. Using the SAQ index of differences in each residue, the amino acid composition of the snake IgY subunits was compared to that of primitive Ig molecules as well as to the rabbit ~,-chain. As shown in Tables 2 and 3, the snake v-chain revealed a closer relatedness to /t-chains of the most primitive cyclostomes, and to the non-p H-chain of the dogfish CBPB 10~2--F

and the lungfish, than to the rabbit y-chain. This observation could be accounted for by assuming that, during vertebrate evolution, IgY may have been the earliest L M W Ig class to emerge. This should, however, be tested by sequence studies on snake v-polypeptide and its structural homologs in amphibians and birds. Such information may provide clues to the structural relationship of the v-polypeptide to the primitive IgG H-chains, and may ultimately establish a unitary class for all primitive L M W Ig molecules. Acknowledgements--We thank Prof. Susumo Tomonaga, Yamaguchi University, Japan, for a generous gift of purified hagfish immunoglobulins. The authors are deeply indebted to Deutsche Gesellsschaft fiir Technische Zusammenarbeit-Gitz (F.R.G.) for donating protein monitoring equipment.

REFERENCES

Ambrosius H. (1976) Immunoglobulins and antibody production in reptiles. In Comparative Immunology (Edited by Marchalonis J. J.), pp. 298-334. Blackwell, Oxford. Andreas E. M. and Ambrosius H. (1989) Surface immunoglobulinon lymphocytes of the tortoise, Agrionemys horsfieldii. Devl comp. ImmunoL 13, 167-175. El Ridi R., Zada S., Afifi A., E1 Deeb S., El Rouby S., Farag M. and Saad A. H. 0988) Cyclic changes in the differentiation of lymphoid cells in reptiles. Cell. Diff. 2,4, 1-8. Fleischman J. B., Pain R. and Porter R. R. (196 l) Reduction of T-globulins. Archs Biochem. Biophys. 1, 174-186. Goudswaard J., Van der Donk J. A. and Noordzij A. 0978) Protein A reactivity of various mammalian immunoglobulins. Scan. J. Immunol. 8, 21-28. H/idge D. and Ambrosius H. (1984) Evolution of low molecular weight immunoglobulins. IV. IgY-like immunoglobulins of birds, reptiles and amphibians, precursors of mammalian IgA. Mol. Immunol. 8, 699-707. Hadji-Azimi I. and Schwager J. (1982) Biochemical studies of Xenopus laevis lymophocyte surface immunoglobulins. Devl comp. Immunol. 6, 703-715. Hill R. L., DeLaney R., Fellows R. E. and Lebovitz H. E. (1966) The evolutionary origins of the immunogiobulins. Proc. natn. Acad. Sci. U.S.A. 56, 1762-1769. Hsu E. and Du Pasquaier L. (1984) Studies on Xenopus immunoglobulins using monoclonal antibodies. Mol. Immunol. 21, 257-270. Kubo R. T., Zimmerman B. and Grey H. M. (1973) Phylogeny of immunoglobulins. In The Antigens (Edited by Sela M.), Vol. l, pp. 417-477. Academic Press, New York.

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Laemmli U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685. Leslie G. A. and Clem L. W. (1969) Phylogeny of immunoglobulin structure and function. III. Immunoglobulins of chicken. J. exp. Med. 130, 1337. Litman G. W. (1976) Physical properties of immunoglobulins of lower species: a comparison with immunoglobulins of mammals. In Comparative Immunology (Edited by Marchalonis J. J.), pp. 239 275. Blackwell, Oxford. Lobb C. J. and Clem L. W. (1981) Phylogeny of immunoglobulin structure and function. XII. Secretory immunoglobulins in the bile of the marine teleost, Archosargus probatocephalus. Molec. Immunol. lg, 615--621.

Marchalonis J. J. (1977) Immunity In Evolution. Harvard University Press, Cambridge, MA. Marchalonis J. J. and Edelman G. M. (1966) Polypeptide chains of immunoglobulins from smooth dog-fish (Mustelus canis). Science 154, 1567 1568. Marchalonis J. J. and Weltman J. K. (1971) Relatedness among proteins: a new method of estimation and its application to immunoglobulins. Comp. Biochem. Physiol. 38, 609-625. Natrajan K. and Muthukkaruppan V. K. (1984) Immunoglobulin classes in the garden lizard, Calotes versicolor. Devl comp. Immunol. 8, 845-854. Zimmerman B., Shalatin N. and Grey H. M. (1971) Structural studies on the duck 5.7 S and 7.8 S immunoglobulins. Biochemistry 10, 482 488.