THE JOURNAL OF BIOLOGICAL CHEMI8TRY Vol. 268, No. 9, Issue of March 25, pp. 6809-6815, 1993 Printed in U. S. A.

Isolation and Characterization of C-reactive Protein (CRP) cDNA and Genomic DNA from Xenopus Zueuis A SPECIES REPRESENTING AN INTERMEDIATE STAGE IN CRP EVOLUTION*

(Received for publication, July 23, 1992, and in revised form, December 16, 1992)

Leewen Lin and Teh-Yung Liu From the Division of Biochemistry and Biophysics, Center for Biologics, Evaluation and Research, Food and Drug Administration, Bethesda, Ma&& 20892

C-reactive protein (CRP) is a prototypic acute phase protein in human and rabbit. Although it is structurally and functionally conserved from invertebrate to hu- man, there are species-specific differences in patterns of expression and putative function. To further inves- tigate the biological significance, regulation, and evo- lution of CRP, we isolated Xenopus CRP and subse- quently derived and sequenced corresponding cDNA and the genomic clones. The structure and expression of Xenopus CRP were also compared to those of the other CRPs. Analyses of the amino acid sequence and the nucleotide sequence reveal that the mature Xeno- pus CRP is a 222-amino acid protein preceded by a 16- residue signal peptide. During development, Xenopus CRP is expressed, only when the liver appears, and therefore is not likely to play a role in early embryonic development. Compared to other species, Xenopus CRP is present at an intermediate low level of <1 bg/ml in the normal serum. Unlike human and rabbit CRP, Xen- opus CRP is not induced by turpentine or heatshock treatment. The heatshock consensus sequence (Woo, P., Korenberg, J. R., and Whitehead, A. S . (1985) J. Biol. Chem. 265, 4136-4142) are not present in the Xenopus CRP gene. It is suggested that Xenopus CRP represents a transitional period in CRP evolution when host defenses switched from primitive innate immunity to a much more complex immune system. The consti- tutive functions of CRP gradually became less essential as the result of the development of a complex immune system.

C-reactive protein (CRP),’ the prototypic acute phase pro- tein in humans, was first discovered in patients with pneu- monia and identified as a precipitin for the C-polysaccharide of the pneumococcal cell wall (Tillet and Frances, 1930). Calcium-dependent precipitation of this protein with the phosphocholine ligand of the C-polysaccharide provides the functional definition of CRPs. The role CRP plays in helping to survive inflammation and to repair tissue damage is still a matter of conjecture. I n vitro, CRP has been found to bind phosphocholine and chromatin. Robey et al., (1984) suggested

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

to the GenBankTM/EMBL Data Bank with accession numberfs) u)866. The nucleotide sequence(s) reported in this paper has been submitted

The abbreviations used are: CRP, C-reactive protein; PC, phos- phocholine; kb, kilobase(s); bp, base pair(s); KLH, keyhole lymphet hemocyanin; PAGE, polyacrylamide gel electrophoresis; IL, interleu- kin.

that CRP may act as a scavenger for chromatin released from damaged cells. In vitro, it, like the immunoglobulins, possesses the ability to promote agglutination and phagocytosis of bac- teria, and complement fixation (Gal and Miltenyi, 1955; Hok- ama et al., 1962; Kaplan and Volanakis, 1974; James et al., 1983; Robey et al., 1984, 1985). In rodents, in conjunction with platelets, CRP is able to mediate protection against immature schistosomes or directly inhibit schizont develop- ment of malaria sporozoites (Bout et al., 1986; Pied et al., 1989). The various biological functions attributed to CRP have been related to the host nonspecific defense mechanism against infectious agents and to wound healing (Pepys and Baltz, 1983). It is possible that CRP is multifunctional and there is truth in all of these proposals.

CRP is evolutionarily conserved and has been found in mammals and in the invertebrate Limulus polyphemus. Mem- bers of the CRP family reveal remarkable stable conservation of certain structural and functional properties, however, there are also species-specific differences in other characteristics even between relatively closely related species (Baltz et al., 1982). In some species of mammals, CRPs behave as acute phase proteins. Human and rabbit CRPs are present at low levels (less that 0.1 pg/ml), and the extent of their elevation can be as high as 3000-fold in response to acute phase stimuli; mouse CRP is a trace protein in normal serum, and rises to only about 2 pg/ml during acute phase response; rat CRP is a major normal serum protein present at a concentration between 300 and 600 pg/ml in sera from healthy adults, and it rises only moderately (900 pg/ml) in response to injury or inflammation. Furthermore, CRP is absent in Syrian hamster. On the other hand, in cold-blooded animals, CRPs seem to be constitutively expressed. Limulus CRP represents a major component of the hemolymph at a concentration of 1-5 mg/ ml (Robey and Liu, 1981). Although many bony fishes have appreciable amounts of CRP in their serum even in the absence of any overt acute phase stimulus, flounder (Plati- chthys flesus L.) lacks CRP (Baltz et al., 1982). The possible significance of these species-specific differences in the pattern of expression is not known nor is how these differences occurred during evolution.

The physiological roles played by CRP are not evident, although the stability observed in their ligand binding speci- ficities implies that these proteins might have important functions which merit preservation. To examine the biological significance of CRP and its species-specific differences, stud- ies with Xenopus luevis were conducted. X. luevis was chosen as the experimental animal for investigation, because it oc- cupies an evolutionary place somewhere between L. polyphe- mus and mammals. In addition, X. laevis has been used to study the expression of many proteins during early develop-

6809

6810 Xenopus C-reactive Protein

ment and provides a system for study of developmental biol- ogy. Therefore, the expression of CRP in various embryonic stages could be examined.

EXPERIMENTAL PROCEDURES

Materials-Live frogs (X. loeuis) purchased from Nasco, Fort At- kinson, Wisconsin, were kept in tanks and fed with frog chow until use. Genescreen Plus membranes and [ C Y - ~ ~ P I ~ C T P were purchased from Du Pont-New England Nuclear. Restriction enzymes, T4 DNA ligase, and Klenow fragment were obtained from Promega. Reagents from DNA sequencing were purchased from United States Biochem- ical. Reagents for cDNA synthesis, X g t l O cloning vector, and pack- aging extract were obtained from Invitrogen. Reagents for polymerase chain reaction (PCR) were from Perkin-Elmer/Cetus. AH-Sepharose 4B-phosphocholine (AH-Sepharose 4B-PC) affinity resin was pre- pared as previously described (Oliveria et al., 1980).

Turpentine and Heatshock Treatment-Fifty p1 of turpentine was injected into the thigh muscle of each frog. Eighteen h after injection, the frogs were sacrificed, and the blood and livers were collected. The livers were stored in liquid nitrogen. The blood was centrifuged twice in a Beckman JB6 centrifuge at 3000 rpm for 10 min at 4 "C. The serum used for Western Blot analysis was heat-treated at 56 'C for 30 min to inactivate proteolytic enzymes, and stored at -20 "C. For heatshock treatment, the frogs were kept a t 30 "C for 18 h, and then the blood and livers were collected and processed as described above.

Purification and Amino Acid Sequencing of Xenopus CRP-The blood of turpentine-treated frogs was diluted with 2 volumes of PC column buffer (0.15 M NaC1, 0.01 M CaC12, 0.05 M Tris-C1, pH 7.4) and centrifuged in Beckman JB6 at 3000 rpm for 10 min at 4 "C, and the supernatant was withdrawn and recentrifuged. The resulting supernatant was passed through a column of AH-Sepharose 4B-PC column 1 X 5 cm) equilibrated with PC column buffer, then the column was washed in the same buffer followed by 2 M NaCl, 0.01 M CaClz, 0.05 M Tris-C1, pH 7.4, until the effluent had a constant Am value. The column was washed with the PC column buffer again until Am = 0. The CRP was eluted by including 0.1 M PC in the buffer, desalted by passing through a PD-10 column (Pharmacia LKB Bio- technology Inc.) and concentrated by ammonium sulfate precipitation (0.5 saturation). The protein was then subjected to SDS-PAGE and protein sequence analysis.

SDS-PAGE was done in the Laemmli system under reducing or nonreducing conditions (Laemmli, 1970) using the Pharmacia Phast- gel system. The amount of protein used for sequence analysis was determined by amino acid analysis on a Beckman 6300 amino acid analyzer after the protein was hydrolyzed by the methane sulfonic acid method (Simpson et al., 1976).

Both the intact protein (about 2 pg) and tryptic peptides generated from the treatment of the protein with trypsin were subjected to automated Edman degradation on an Applied Biosystem 470A protein sequencer on-line with an Applied Biosystem 120 PTH analyzer. To generate tryptic peptides, Xenopus CRP (about 2 pg) was precipitated with methanol/chloroform, redissolved in 50 pl of 100 mM Tris, pH 8.0, 1 mM CaC12, 4 M urea, and incubated with ~-1-tosylamido-2- phenylethyl chloromethyl ketone-treated trypsin (Promega) for 20 h at room temperature. After digestion, 100 pl of 8 M guanidine-HC1, 0.5 M Tris, 0.35 mM EDTA was added to the reaction mixture, and then the tryptic peptides were reduced by the addition of 2 pl of 100 mg/ml dithiothreitol and incubated at 37 "C for 60 min. In order to alkylate the cysteines, 2 pl of 4-vinylpyridine (Sigma) was added, and the mixture was incubated at 37 'C for 30 min. The sample was then chromatographed by reversed-phase HPLC on a Vydac C4 column loading with 0.1% (v/v) trifluoroacetic acid in water and applying a 0-35% gradient of 0.09% (v/v) trifluoroacetic acid in 80% (v/v) acetonitrile over 60 min, then 35-75% (v/v) over 30 min, and 75- 100% (v/v) over 15 min. A control was also run and compared to the sample to eliminate peptides derived from the trypsin.

Synthesis of Oligodeoxyribonuckotides-Oligodeoxyribonucleotides were synthesized by the phosphoramidite method on an Applied Biosystems 380B DNA synthesizer (Caruthers et al., 1982).

RNA Purification-Xenopus embryonic or adult liver total RNA was isolated by the guanidinium thiocyanate method as described by Chirgwin et al. (1979). Polyadenylated RNA was purified by oligo(dT)-cellulose chromatography (Aviv and Leder, 1972). Xenopus embryos were prepared as follows. Eggs were fertilized in uitro and incubated at 20 "C. Embryonic stages were determined by comparing the morphology of the embryos with the normal table described by Nieuwkoff and Faber (1956). The staged embryos were rinsed with

water and stored at -20 "C until use. Construction of cDNA Library-The cDNA was synthesized from

7 pg of poly(A)+-RNA isolated from turpentine-treated Xenopus liver by the method of Okayama and Berg (1982), as modified by Gubler and Hoffman (1983), except that 10 pCi of [ C X - ~ ~ P I ~ C T P was included in the reaction. After the ends of the cDNA were made blunt with T4 DNA polymerase, the cDNA was ligated to adaptors consisting of two oligonucleotides: 5'-AATTCGCGGCCGC-3' and 3"GCGCCGGCG- PO,-5'. This cDNA was size-fractionated on a CL-4B-Sepharose column equilibrated with 10 mM Tris-C1, pH 7.5, 100 mM NaCl, 1 mM EDTA (Ausbel et al., 1987). The size of the cDNAs in each fraction was determined on a 1% agarose gel. The fractions containing cDNAs larger than 0.5 kb were pooled, ligated to EcoRI cut, alkaline phosphatase-treated X g t l O arms and packaged in uitro. Escherichiu coli strains C600 and C600hfr were used to titer the recombinant phages.

otides, CRPX3 (5'-ACCACCAAACGAATCCTGCTCCTGCCC-3', Cloning of Partial Xenopus CRP cDNA by PCR-Two oligonucle-

which is complementary to the conserved Ca2+ binding region in CRPs (Nguyen et al., 1986; Dang et al., 1985), and CRPX4 (5'- GCYTTYGTRTTYCCCAARCC-3', where R = A or G, and Y = C or T), which is based on the N-terminal sequence of the frog CRP, were used as the primers in the PCR. The reaction mixture contained one-hundredth of the first strand cDNA synthesis reaction as tem- plate, 10 mM Tris-C1, pH 8.3 (at 25 "C), 50 mM KCl, 1.5 mM MgC12, 0.01% (w/v) gelatin, 0.2 mM dATP, 0.2 mM dCTP, 0.2 mM dGTP, 0.2 mM dTTP, 1.0 p~ CRPX3, 1.0 p~ CRPX4,2.5 units of Taq DNA polymerase. The reaction began with an initial melting step of 5 min at 94 "C, then the amplificaiton cycles followed. The cycle profile was: melting (94 "C, 1 min), annealing (50 'C, 1 min), and polymeri- zation (72 "C, 2 min). Thirty-five cycles were carried out, and at the last cycle the polymerization step was extended to 10 min. The reaction mixture was extracted with phenol/chloroform (l:l), and the PCR product was precipitated with ethanol. Then the PCR product was phosphorylated by T4 kinase, blunt-ended, and loaded on an agarose gel. There was one major band of 400 bp in size, which was excised, purified, and subsequently cloned into pUC13 (Pharmacia) at the SmaI site (pXCRP-p4) for sequence analysis.

Screening of Xenopus cDNA and Genomic Libraries-All cloning procedures were carried out according to the standard protocols (Maniatis et al, 1982). The cDNA library was plated on E. coli strain CGOOhfl-, and duplicate filters were made. Plasmid pXCRP-p4 was digested with EcoRI and PstI, the resulting fragment was gel-purified, labeled with 32P by random priming (Feinberg and Vogelstein, 1983), and used as a probe for screening. Two phages (XXCRP-cl and XXCRP-c4) were positive, and plaque-purified. The phage DNAs were isolated, and digested with EcoRI subcloned into Bluescript (pXCRP-cl and pXCRP-c4, respectively).

A Xenopus genomic library, made with homozygous (HD-I) DNA, was a generous gift from Dr. Igor Dawid of the National Institutes of Health. This library was constructed by partial Sau3AI digestion of Xenopus genomic DNA and cloning into X phage EMBL 4. Approxi- mately 5 X 10' phages were plated on E. coli strain LE392, and screened with 3ZP-labeled EcoRI fragment of XCRP-cl. One phage was positive (XXCRP-g2). It was plaque-purified, and the phage DNA was isolated.

Analysis of Recombinant X Phage DNA-The restriction map of the cloned gene was constructed by digestion of phage DNA with various restriction endonucleases (Maniatis et al., 1982). DNA restric- tion fragments were separated in an agarose gel, blotted onto Genescreen Plus membrane, and hybridized to 32P-labeled EcoRI fragment of XCRP-cl according to the manufacturer's recommenda- tions. Two overlapping restriction fragments, a 2.5-kb PstI fragment and a 4-kb BamHI fragment (XCRP-g2p2 and XCRP-g2bl, see Fig. 2) were subcloned into Bluescript plasmid and sequenced.

Cloning of 5' Xenopus CRP cDNA Using Rapid Amplification of cDNA Ends (RACE)-In order to obtain the 5' sequence of Xenopus CRP mRNA, 5'-end RACE was carried out as described by Frohman, (1990). Briefly, a set of three nested oligonucleotides were made: 1) primer 1, 5'-TTTCAGGATTGCATACGTGGT-3', complementary to nucleotides 97 to 117; 2) primer 2, 5"TGCCAAC- CAAATCTTGTG-3', complementary to nucleotides 47 to 67; 3) primer 3,5'-GGCAAGAAAGATAAACCACAG-3', complementary to nucleotides 16 to 36. First, cDNA was made by reverse transcriptase using 1 pg of total liver RNA and 10 ng of primer 1, and, after removing the excess primers by centrifugation through Centricon (Amicon), tailed with dATP by terminal transferase (Bethesda Re- search Laboratories). CRP-specific cDNA was amplified twice using

Xenopus C-reactive Protein

PCR. First amplificaiton reaction contained 1/10 of the polyadenyl- ated cDNA, dT17, and primer 2. Excess primers were removed after the reaction. The second amplification contained 1/50 of the first amplification product, dT17, and primer 3. PCR reactions were carried out as described previously. The final PCR product was phosphoryl- ated by T4 kinase, blunt-ended, and cloned into Bluescript a t the EcoRV site.

Northern Blot Analysis-Twenty pg of total RNA from each sample was loaded in formaldehyde/agarose gel, electrophoresed, transferred onto a Genescreen Plus membrane, and hybridized with 32P-labeled XCRP-cl EcoRI fragment according to the manufacturer's recom- mendations. The membrane was then exposed to x-ray film.

DNA Sequencing and Sequence Analysis-The cDNA fragments and the genomic fragments were subcloned into either pUC13 or Bluescript plasmid. The complete sequence was determined by San- ger's dideoxy chain termination method (Sanger and Coulson, 1975), modified for double-stranded sequencing (Guo et al., 1983), employing sequencing strategies of the exonuclease I11 deletion method (Hein- koff, 1984) and synthetic oligonucleotide primers. Nucleotide se- quence data were compiled and analyzed by the Genetics Computer Group sequence analysis software package (Devereux et al., 1984) and BLASTP program (Altschul et al., 1990).

Preparation of Anti-peptide Antibody Against Xenopus CRP- Based on Jameson/Wolf antigenic index (Jameson and Wold, 1988), an N-bromoacetyl-16-amino-acid peptide, Br-CH2-C- LRGQATTQPKROSKTL (corresponding to CRP amino acid 195- 210, except that Cyszo7 was substituted by Ser), was synthesized with an Applied Biosystems peptide synthesizer using T-Boc chemistry. After purificaiton by reversed-phase HPLC and characterization by amino acid analysis, this bromoacetylated peptide was coupled to keyhole lymphet hemocyanin (KLH) by the method of Robey and Fields (1989). Briefly, KLH was first derivatized with iminothiolane, then the mixture was passed through a Sephadex G-25 column. The modified KLH and the N-bromoacetylated peptide were mixed and stirred for 3 h at room temperature. The peptide-KLH conjugate was recovered following dialysis of the mixture against 0.1 M NH.HCO3 and injected into a rabbit to raise antibody.

Western Blot Analysis-Serum samples were loaded on 16% SDS- PAGE and run under either reducing or nonreducing conditions. Proteins were transferred from the gel to Immobilon-P membrane (Millipore) by electrophoresis. The protein blots were treated sequen- tially with TBST (100 mM Tris, 150 mM NaC1, 0.1% Tween 20, pH 7.4) for 5 min, blocking solution (5% non-fat dry milk in TBST) for 60 min, three times with TBST for 5 min each, and incubated with antiserum (1:200 dilution in TBST) for 16 h at 4 "C. Then the blots were washed three times with TBST for 5 min each, incubated with goat anti-rabbit IgG-alkaline phosphatase conjugate (Promega) for 60 min, and washed three times with TBST for 5 min each. Nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate were used for color development. The color reaction was stopped by rinsing filters in distilled water.

RESULTS

Purification and Characterization of the Xenopus CRP- Xenopus CRP was purified using the phosphocholine affinity matrix. Isolation of Xenopus CRP proved to be much more difficult than from other species due to its low serum content (<I pg/ml), nonresponsiveness to induction, and tendency to form insoluble aggregates during purification.

The purity of the Xenopus CRP prepared by the PC-affinity column was assessed by SDS-PAGE and by protein sequence analysis. Under nonreducing conditions, multiple protein bands with apparent molecular weights of > 100,000 were observed. Under reducing conditions two protein bands of M, 24,000 f 2,000 and 75,000 & 2,000 appeared. Amino acid sequence analysis of the protein preparation (containing both the 75- and the 24-kDa proteins) revealed a single seqeunce. Western Blot analysis of this protein preparation under re- ducing conditions showed only the 24-kDa protein band, whereas, under nonreducing conditions, multiple high molec- ular weight bands were detected (Fig. 1). On the other hand, Western Blot analysis of Xenopus serum showed a single band of MI 24,000 f 2,000 under reducing conditions and a band of 50,000 f 2,000 under nonreducing conditions (Fig. 2).

681 1

1 2

c

FIG. 1. SDS-PAGE of Xenopus CRP purified by the PC column. The gel is 8-25% acrylamide using the Laemmli system. Lane I, reduced Xenopus CRP; lane 2, nonreduced Xenopus CRP. A, Coomassie Blue staining. B, Western blot analysis using antipeptide antibody.

A 1 2

?@O.OOO - ,>8.000 - 43.000 - ?9,000-

18,400-

1.1.300- "

B 1 2

200 000 - n7:.100- CR.DO0-

43.00Q - ?o.@oo-

FIG. 2. SDS-PAGE analysis of Xenopus CRP in serum. The gel is 16% acrylamide using the Laemmli system. Lane 1, under reducing condition; lane 2, under nonreducing condition. A, Coo- massie Blue staining. B, Western blot analysis using antipeptide antibody.

These results suggest that the subunit structure of the Xeno- pus CRP exists as a dimer of M, 50,000 f 2,000 in serum but, upon purification, forms aggregates that are not completely dissociable in SDS under nonreducing conditions. In the presence of reducing agent and SDS, the aggregates partially dissociate to the monomeric form of MI 24,000 & 2,000 and an aggregate of MI 75,000 f 2,000.

Amino-terminal sequence was obtained in low yield (about lo%), most likely due to the amino-terminal Gln residue (see Fig. 4), which tends to cyclize to pyrrolidone carboxylic acid. Part of this sequence, VFLFPK (residues 8-13, see Fig. 4) was confirmed by sequence analysis of an isolated trypic

6812 Xenopus C-reactive Protein

peptide, and because of its low degeneracy in codon selection, the sequence VFLFPKP was chosen to design an oligonucle- otide probe.

Cloning of Partial CRP cDNA by PCR-Based on the partial amino-terminal sequence determined for the Xenopus CRP, and the sequence of the conserved Ca2+-binding region (underlined in Fig. 4), two oligonucleotides were designed as primers for use in the PCR. The major PCR product, which had the expected size (about 400 bp), was cloned into pUC13 (XCRP-p4, Fig. 3) and sequenced. Excluding the primers, the sequence extends from nucleotides 90-453 (corresponding to amino acid 15-135, Fig. 4). The deduced amino acid sequence revealed a Ser at position 15 and Ile-Ile-Leu at position 133- 135 as expected.

Screening of cDNA Library and Sequence Analysis of the cDNA Clones-Two clones, XXCRP-cl and XXCRP-c4, that hybridized with the PCR fragment, were isolated from ap- proximately 5 x 10‘ recombinant phages. Both recombinant phages were plaque-purified, and phage DNA was isolated and digested with either EcoRI or NotI. The digestion revealed that the XXCRP-cl and XXCRP-c4 cDNA inserts (Fig. 3) lacked internal EcoRI and NotI sites and were about 950 and 650 bp in size, respectively. Both cDNA inserts were sub- cloned into Bluescript at EcoRI sites, and the complete nu- cleotide sequences were determined using the double-stranded dideoxy chain termination method. XCRP-cl cDNA is 943- bp long and is 294 nucleotide longer than the XCRP-c4 at the 5’-end.

RNA blot analysis indicated that Xenopus CRP is encoded by a 1.0-kb mRNA. In order to obtain the 5‘-end of the mRNA, 5’ RACE was performed as described under “Exper- imental Procedures.” The products were blunt-ended, phos- phorylated, and subcloned into Bluescript at EcoRV site. Plasmid DNA from 16 clones were isolated, and the sizes of their insert were compared on a 3% agarose gel. The four clones with longest inserts were sequenced and compared with the genomic sequence.

The complete nucleotide sequence of the Xenopus CRP cDNAs is shown in Fig. 4 (nucleotides -45 to 929). For simplicity of discussion, a numbering system is used in which nucleotide position +1 denotes the translational initiation site. The location of the initiator methionine was assigned by comparing with other CRPs and by the fact that it is the first methionine residue encountered in the open reading frame.

Isolation and Sequencing of the Xenopus CRP Gene-After screening of 500,000 recombinant genomic phages in the Xenopus XEMBL4 genomic library, one phage was detected

XCRP-g2b 1

P

with random primed, 32P-labeled XCRP-cl fragment. The insert of the genomic clone, designated XXCRP-g2, was ana- lyzed by restriction enzyme digestions and DNA blot analyses. Two overlapping fragments, a 4-kb BamHI fragment and a 2.5-kb PstI fragment (XCRP-g2bl and XCRP-g2p2, Fig. 3), were subcloned into Bluescript for sequence analysis. The nucleotide sequence of the Xenopus CRP gene is shown in Fig. 4. There are seven discrepancies between the genomic sequence and the cDNA sequence, which cause four amino acid changes, ie. Pro-24-Ala, Leu-EiO-Phe, Gln-135-Leu, and Val-165-Ile. Since the genomic library and the cDNA library were constructed from different frogs, these discrepancies are probably due to individual variation or genetic polymorphism of Xenopus.

The Xenopus CRP gene is composed of three exons and two introns. The sequences around the splice sites are con- sistent with the consensus sequences for exon-intron bound- aries of eukaryotic genes. The first intron, which is not present in other CRP genes, interrupts the 5”untranslated sequence. The position of the second intron is similar to that in mam- malian CRP genes, however, it is 1.8 kb in length which is much longer than the mammals.

Regulation of Xenopus CRP Gene Expression-In an at- tempt to understand the regulation of Xenopus CRP gene expression, we measured the levels of CRP protein in the serum in untreated and turpentine-treated frogs by Western Blot using antipeptide antibody (Fig. 5). Although the anti- body cross-reacted with some other proteins present in the frog serum, a 24-kDa protein, which has the correct molecular mass, was the major protein band recognized by the antipep- tide antibody. Although there was some variation in individual serum CRP levels, insignificant induction of CRP production was observed upon turpentine treatment. Sera from heatsh- ocked frogs and two frogs infected with unidentified agents were also examined. The level of serum CRP in these animals was not significantly different from the normal serum (data not shown).

CRP mRNA levels in total liver RNA isolated from the same frogs used for Western Blot analyses were measured by RNA blot analysis. No significant differences were observed in the CRP mRNA level of the normal and the turpentine- treated frogs either (data not shown).

The possibility that CRP is involved in embryonic devel- opment was examined. Western Blot analyses indicated that CRP was not present in Xenopus eggs. RNA blot analyses of total RNAs prepared from Xenopus embryos at various de- velopmental stages indicated that CRP mRNA was not de-

B /M

B

kc-! XCRP-g2p2

FIG. 3. Cloning of the cDNA and B P P E H the gene encoding Xenopus CRP. I I I I I Restriction map and the subclones of . M L\\\\\\\\\\I XCRP-92 Xenopus CRP cDNA and genomic frag- menta. B, BamHI; E, EcoRI; H, HindIII; P , PstI. Exon 1 is the black box; Exon 2 and 3 are hatched boxes. -

500 bp U

XCRP-c 1 XCRP-c4 XCRP-p4

Xenopus C-reactive Protein 6813

FIG. 4. Sequence of Xenopus CRP. Nucleotide and deduced amino acid sequence of Xenopus CRP gene. For nucleotide sequence, the translational initiation site is designated as nucleotide 1. cDNA sequence identical to genomic sequence is expressed as dash lines. Pol- yadenylation signal is underlined. Intron sequences are in brackets. For amino acid, negative numbers correspond to the signal peptide, while positive numbers represent residues of the mature protein. Amino acid residues in parentheses are derived from cDNA sequence. Regions to which oligonucleotides were synthe- sized as primers for the polymerase chain reaction are underlined. Also underlined are the amino-terminal sequences deter- mined by Edman degradation; upper line, from intact protein sequencing; lower line, regions to which oligonucleo- tides were synthesized.

A 0 7 10 16 23 31 40

FIG. 5. Western blot analysis of Xenopus CRP gene expres- sion. Sera from control and turpentine-treated frogs were subjected to Western blot analysis. Frogs 1-3, untreated; and frogs 4-6, turpen- tine-injected. Three pg of serum protein was electrophoresed on a 16% polyacrylamide gel, transferred onto Immobilon membrane and CRP was detected using CRP-specific anti-peptide antibody as de- scribed under "Experimental Procedures."

tected in the oocytes, nor at stages 7 (blastula stage), 10 (gastrula stage), 16 (mid-neural fold stage), 23 (initial tail- bud stage), and 31 (tail-bud stage). The earliest stage that CRP mRNA was clearly detected on the RNA blot was at stage 40 (late tail bud stage) (Fig. 6). The timing of CRP expression corresponds to liver formation. Therefore, CRP is not likely to play a role in early embryonic development, but could be used as a marker for liver maturation.

DISCUSSION

Comparison of Xenopus CRP Gene and Protein Structures with CRPs from Other Species-Xenopus CRP gene encodes a 1.0-kb mRNA with an open reading frame of 714 bases. The size of the Xenopus CRP message is smaller than the mam- malian ones by about 1.0 kb at the 3"untranslated region. The deduced amino acid sequence consists of 238 amino acids with a signal peptide of 16 amino acids. The M, calculated from the amino acid composition of the mature protein se-

- 28s

- 18s

0 0 7 10 16 23 31 40

.&.L "&%id

FIG. 6. Northern blot analysis of RNAs from various devel- opmental stages. Total RNAs were isolated from Xenopus eggs (0) or from embryos at stages 7,10,16, 23,31, and 40. Twenty pg of each was electrophoresed onto a 1.2% agarose/formaldehyde gel. The RNA was transferred onto Gene Screen Plus membrane and hybridized with 32P-radiolabeled XCRP-cl fragment. A , autoradiogram; B, ethid- ium bromide staining.

6814 Xenopus C-reactive Protein

quence is 25,230, which agrees with the apparent M, of about 24,000 f 2,000 determined by SDS-PAGE. Based on this finding, the Xenopus CRP is most likely not glycosylated, although there is a potential N-glycosylation site at Asn-32. Human, rabbit, and mouse CRPs are ot glycosylated although the Limulus CRP does contain glycosyl groups. The subunit size of CRP is remarkably conserved in evolution, being -24 kDa in all species examined.

Xenopus CRP shows 45.1, 41.9, 38.5, and 23.8% overall identity in amino acid sequence to human, rabbit, mouse, and Limulus CRPs, respectively. There are several conserved re- gions with identity ? 60% (Fig. 7). The putative calcium- binding region (region 4 in Fig. 7; amino acid 132-146 of Xenopus CRP) is highly conserved (87% identity), from horse- shoe crab through Xenopus to mammals. To a lesser extent, the amino acid sequence preceding and around the proposed PC-binding site (region 1 in Fig. 7; amino acids 28-66 of Xenopus CRP) is also conserved (60% identity), however, the basic amino acid residues assumed to play an important role in PC-binding (Liu et al., 1982), are not present in the Xen- opus CRP. The binding loci for PC are most likely the result of the folding of the molecule rather than the consequence of a linear arrangement of certain amino acid residues (Segal et al., 1974; Liu et al., 1991).

Other conserved regions span amino acid residues 95-114 and 150-171 of the Xenopus CRP (regions 2 and 4, Fig. 7). These two regions are also similar to stretches of amino acid sequences in serum amyloid P-components, which belong to the same pentaxin family as the CRPs. The sequence of HXCXS/TWXS found in region 2 has been found in all Pentaxin family proteins (Bairoch, 1991), and in the Limulus CRP (Nguyen et al, 1986). In the case of the Xenopus CRP, although it contains this sequence, it is not known whether the disulfide-linked monomeric subunits are assembled into a pentaxin structure. Limulus CRP, which has a hexagonal structure (Fernandez-Mayan et al., 1968), also has this “sig- nature” sequence.

The mammalian CRPs contain a pair of conserved Cys residues that are involved in disulfide bond formation. The corresponding Cys residues in the Xenopus CRP are Cys-36 and Cys-97. Three additional Cys residues are present in the Xenopus CRP. One or more of these additional Cys residues are probably involved in interchain disulfide linkage, since Xenopus CRP exists as a dimer under denatured, nonreducing condition in the serum (Fig. 2). Dogfish CRP, which contains 3 Cys residues, also has this dimeric structure (Robey et al., 1983). This characteristic of a disulfide-linked dimer also belongs to mammalian immunoglobulins, and aside from fi- bronectin, the rarity of disulfide-linked homopolymers

throughout nature is indicative of their specialized and con- strained biosynthetic production. This structure is important for the divalent nature of the immunoglobulins which is essential for their cross-linking of antigens. The protein se- quence deduced from the genomic and cDNA sequence deter- mined for the Xenopus CRP would predict that the dimeric form of the Xenopus CRP is formed post-translationally by the interaction of monomers. Thus, although the covalent structure of CRPs bears no resemblance to immunoglobulins (Osmond et al., 1977) at the macromolecular level, the disul- fide-linked dimeric structure of the dogfish and the Xenopus CRPs may be an indication of common evolutionary origin with immunoglobulins.

In mammals, the concentration of serum CRP may increase 100-1000-fold under inflammatory conditions (Kushner, 1982, Syin et al., 1986). In vitro, it has been demonstrated that the human CRP gene expression is induced by IL-6 (Li et al., 1990). Woo et al. (1985), noted that in the 5’-end of the human CRP gene, there are three regions whose sequences are similar to the heatshock consensus sequence, CTn- GARnnTTnAG (Simon et al., 1985). In the Xenopus CRP gene, neither the IL-6-responsive element, nor the heatshock consensus sequences are found at the 5‘ upstream region. By protein and mRNA analyses, we have also shown that the Xenopus CRP gene is not inducible by either heatshock or an inflammantory agent that induces the production of IL-6 in mammals. The absence of the IL-6-responsive elements in the 5’ upstream region of Xenopus CRP gene, therefore, re- enforces the proposal by Li et al. (1990), that these elements are intimately involved in the induction of CRPs in human and rabbit under acute-phase condition. It is not known whether there is IL-6 present in the Xenopus.

Evolution and Biological Significance of C-reactive Pro- teins-CRP is a primitive pattern recognition, lectin-like molecule. It is evolutionarily conserved and is found from the invertebrate, L. polyphemus, to mammals. A number of func- tional capabilities have been demonstrated for CRP, however, the importance of these functions may vary from species to species. The marked differences in the mode of expression of CRP in various species again suggests functional differences. On the other hand, the antiquity and remarkable conservation of structure and phosphocholine-binding property of CRP in evolution indicate importance of at least certain functions. It is possible that the biological significance of CRP switched along with its mode of existence.

It has been suggested that CRP might serve the role of immunoglobulins in Limulus (Robey and Liu, 1981). The absence of immunoglobulins, the presence of multiple CRP genes, the polymorphic nature of CRP, and the lectin-like

1

. I . I I : .:I ..Ill1 1 1 1 1 1 1 1 1 1 . ~ : .lll:ll

FIG. 7. Amino acid sequence comparison of mature human and Xenopus CRPs. Human CRP and Xenopus CRP sequences (derived

boxed and numbered. from genomic sequence) were aligned using the Genetics Computer Group sequence analysis software package. The conserved regions were

Xenopus C-reactiue Protein 6815

property of CRP in Limulus make this an attractive proposal. It is possible that with the development of a more specific and complex immune system, the anti-infective function of CRP became less important and eventually nonessential. In Xenopus, the immune system has already developed, although its B-cell repertoire is less diverse than mammals (Du Pas- quier et al., 1989). In addition, there are antimicrobial peptides secreted from frog skin guarding it from infection (Bevins and Zasloff, 1990). Corresponding to the presence of these sys- tems, CRP expression in Xenopus is decreased to an inter- mediate low level. In mammals, where advanced immune systems exist, the constitutively expressed CRP levels drop to a minute amount. Furthermore, other pattern recognition proteins or specialized defense agents in animals, e.g. lipo- polysaccharide binding protein (Tobias et al., 1988, Weiss et at., 19781, and mannose binding protein (Ezekowitz and Stahl, 1988). can also provide innate immunity. These proteins or agents could take over the biological role of CRP to mediate constitutive protection against infection and could replace CRP. Therefore, CRP may not be essential for that function.

In summary, we have isolated the CRP gene from Xenopus and demonstrated that it is expressed at an intermediate low level compared to other species, and is not induced by inflam- matory agents. During development, Xenopus CRP is not expressed until the liver is formed. Studies of CRP structure and function in different stages of evolution reveal that this protein has evolved in its mode of expression, and thus has served different roles. Xenopus CRP could represent a tran- sitional period in CRP evolution when host defenses switched from primitive, nonspecific pattern recognition molecules to a much more complex immune system. The biological func- tions of CRP gradually became less essential as the result of the development of complex immune system.

Acknowledgments-we thank Drs. Milan Jamrich and Neil D. Goldman for valuable advice, Dr. Igor B. Dawid for the Xenopus genomic library, Robert A. Boykins for amino acid analysis and John B. Ewell for protein sequence determination. The expert assistance of S. Unger in the preparation of this paper is deeply appreciated.

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Cell 40. R0.5-Rl7