We present the nucleotide sequence of a cDNA coding for cyclophilin homologue of the sea urchin Hemicentrotus pulcherrimus. The 1,755-nucleotide sequence contains a 492-bp open reading frame corresponding to a translation product of 164 amino acids. Comparison of the deduced amino acid sequence with the previous data shows a high degree of conservation (∼80% homology). Southern blot analysis of genomic DNA suggests the presence of a multi-gene for sea urchin cyclophilin. Northern blot analysis indicates a mRNA size of ∼3 kb and that message is accumulated at blastula stage.
INTRODUCTION
Cyclophilin is an abundant cytosolic protein that has the activity of peptidyl prolyl cis-trans isomerase in almost all organisms (Fischer et al., 1989; Handschumacher et al., 1984; Takahashi et al., 1989). It has been known that peptidyl prolyl cis-trans isomerase involves in protein folding (Fischer and Bang, 1985; Lang et al., 1987). In human immune system, cyclophilin has a role of the receptor of the immunosuppressant, cyclosporin A (Handschumacher et al., 1984). The cyclophilin-cyclosporin A complex interferes the production of cytokines in T-cell (Koletsky et al., 1986) by inhibiting calcineurin, a calcium- and calmodulin-dependent protein phosphatase (Liu et al., 1991). Complementary DNAs of cyclophilin have been isolated from various organisms and deduced amino acid sequences are highly conserved (de Martin and Philipson, 1990; Haendler et al., 1987; Hasel and Sutcliffe, 1990; Stamnes et al., 1991), indicating that cyclophilin is one of the essential protein for living cells. However specific function and intrinsic ligand of cyclophilin have not been determined yet. The analysis of cyclophilin in embryonic development would lead to determine physiological function of it in the cell and/or organ.
In the present study, we report cloning and analysis of a cDNA coding for cyclophilin homologue in the sea urchin Hemicentrotus pulcherrimus. We have also demonstrated the temporal expression of the message during early embryonic development. A remarkable accumulation of cyclophilin message was occurred during gastrulation in sea urchin development.
MATERIALS AND METHODS
Screening of cDNA library and DNA sequencing
A cDNA library was constructed in λgt11 from poly(A)+ RNA isolated from early pluteus larva of the sea urchin H. pulcherrimus. Filters containing 20,000 plaques of the library were screened with the mouse cyclophilin homologue (Ohta, K., unpublished data). Single positive clone was isolated and subcloned into pUC119 vector for further analysis.
Nucleotide sequences were determined by the dideoxy chain termination method (Sanger et al., 1977) with the Sequenase Kit (United States Biochemical Co.) using [α-35S] dATP.
Southern blot analysis
Restricted digests of genomic DNA isolated from testis of mature sea urchin were fractionated on a 0.8% agarose gel, transferred overnight to a Hybond-N+ membrane (Amersham International plc). The cDNA insert, HPCyp-1, was labeled with [α-32P] dCTP to specific activities of approximately 109 cpm/μg. The filter was prehybridized at 65°C in 5×Denhardt's solution, 0.5% SDS, 0.9 M NaCI, 0.05 M NaH2PO4, 0.005 M EDTA, pH 7.5 and 100 μg/ml denatured salmon sperm DNA. Then it was hybridized overnight in the same solution to radiolabeled probe. The filter was subsequently washed at 65°C in 0.1 ×SSC and 0.1%SDS.
Northern blot analysis
Total RNA was isolated from unfertilized eggs and embryos of the sea urchin (Chomczynski and Sacchi, 1987), separated by agarose gel electrophoresis containing formaldehyde and transferred to a Hybond-N+ membrane. HPCyp-1 was labeled with [α-32P] dCTP and used as a probe. Hybridization was performed with the same procedure as in southern blot analysis except for use of 1 mg/ml of yeast tRNA instead of denatured salmon sperm DNA. Final washing was done with 0.2×SSC and 0.1% SDS at 65°C.
RESULTS AND DISCUSSION
A cDNA of sea urchin H. pulcherrimus was isolated from λ phage cDNA library constructed from early pluteus larva poly(A)+ RNA using a mouse cyclophilin homologue as a probe. The nucleotide sequence of the cDNA designated HPCyp-1 was determined by the dideoxy chain termination method as shown in Figure 1. The cDNA is 1,775 bp in length. Fifty base pairs of 5∣-untranslated region are followed downstream of this polyadenylation signal in the clone. by a start codon ATG lying in a favorable context for a Therefore this polyadenylation signal in the HPCyp-1 translation initiation according to the Kozak's criteria (Kozak, sequence we cloned may be not functional for 1981). An open reading frame of 492 bp codes for 164 polyadenylation. amino acids to yield a protein of estimated molecular weight The deduced amino acid sequence shows a high of 17,677. A stop codon TAA occurs at position 493. The degree of homology to cyclophilin family except for the N-non-coding region of 3∣-end of the cDNA is composed 1,210 bp including polyadenylation site (AATAAA) (Proudfoot, 1991). But poly(A)+ sequence is not contained downstream of this polyadenylation signal in the clone. Therefore this polyadenylation signal in the HPCyp-1 sequence we cloned may be not functional for polyadenylation.
The deduced amino acid sequence shows a high degree of homology to cyclophilin family except for the N-and C-terminal 20 to 30 amino acids (Fig. 2). HPCyp-1 product has 78.7%, 77.4%, 80.1% and 75.3% homology to human T-cell cyclophilin (Haendler et al., 1987), mouse cyclophilin (Hasel and Sutcliffe, 1990), Drosophila cyclophilin-1 (Stamnes et al, 1991) and Schizo-saccharomyces pombe cyclophilin (de Martin and Philipson, 1990), respectively. Comparison with the sequence deduced from a H. pulcherrimus cyclophilin cDNA, HPCyp-1, shows that the three consensus sequences, NGTGGKSIYG, LSMANAGPNTNGSQFF and WLDGKHVVFG (Koser et al., 1990) are conserved in the protein except the discrepancy in the second sequence; Pro-125 is replaced with Val in H.pulcherrimus protein. The single conserved tryptophan and the four cysteine residues are found in the predicted amino acid sequence.
In order to determine the different sequences that code for cyclophilin of H. pulcherrimus, genomic southern analysis was done under the high-stringency condition using the insert of the clone as a probe. Several bands were revealed in the blot (Fig. 3a). This result suggests the presence of additional sequences homologous to HPCyp-1. Same result was obtained using the probe derived from the EcoRI-Pvull fragment of the cDNA insert (data not shown). Because this fragment contains part of the complete coding region and 50 bp of 5∣-untranslated region, the hybridization pattern actually represents cyclophilin sequence. In this study, it is unknown whether these sequences reflect functional genes or pseudogenes.
Northern blot analysis was performed under the high-stringency condition using HPCyp-1 as a probe. The embry-onic stages used for the assay were unfertilized egg, early blastula, mesenchyme blastula, mid-gastrula and prism larva. A band of ∼3,000 nucleotides is revealed (Fig. 3b). The mRNA is not identified in unfertilized eggs and cleavage stage embryos. The cyclophilin message is originally accu-mulated at early blastula stage and then the expression level of mRNA is slightly increased. Thereafter a remarkable accumulation of the message is identified during gastrula-tion in H. pulcherrimus embryos.