Tài liệu Báo cáo khóa học: The lysozyme of the starfishAsterias rubens A paradigmatic typei lysozyme docx

The lysozyme of the starfish Asterias rubens A paradigmatic type i lysozyme Sana Bachali 1 , Xavier Bailly 2 , Jacqueline Jolle ` s 3 , Pierre Jolle ` s 3, * and Jean S. Deutsch 1 1 E ´ quipe De ´ veloppement et E ´ volution, UMR 7622 ‘Biologie du de ´ veloppement’, CNRS et Universite ´ P & M Curie, Paris, France; 2 Station Biologique, Roscoff, France; 3 Laboratoire des Prote ´ ines, Universite ´ Paris V, France On the basis of a partial N-terminal sequence, Jolle ` s and Jolle ` s [Jolle ` s, J., & Jolle ` s, P. (1975) Eur. J. Biochem. 54, 19–23] previously proposed that the lysozyme from the starfish Asterias rubens represents a new form of lysozyme, called type i (invertebrate) lysozyme. Indeed, it differed from both the types c (chicken) and g (goose) known in other animals, as well as from plant and phage lysozymes. Recently, several proteins belonging to the same family have been isolated from protostomes. Here we report the com- plete mature protein sequence and cDNA sequence of the lysozyme from Asterias. These sequences vindicate the previously proposed homology between the starfish, a deuterostome, and protostome lysozymes. In addition, we present a structural analysis that allows us to postulate upon the function of several conserved residues. Keywords: cDNA; invertebrates; lysozyme; starfish; struc- ture. During recent years, interest in a new type of lysozyme, the invertebrate-type (i-type), has been growing. In 1996 Jolle ` s et al. [1] published the N-terminal sequences of lysozymes from two coastal bivalves belonging to the genus Mytilus and of four deep-sea bivalves belonging to the genera Bathymodiolus and Calyptogena. This lysozyme represented a model for the digestion of bacteria by the deep-sea bivalves [2]. A similar lysozyme was then described in other bivalves, Tapes japonica [3], and Chlamys islandica [4,5]. These authors noticed the striking similarity between the bivalve lysozyme and another protein, the so-called desta- bilase identified in the medicinal leech Hirudo medicinalis [6,7]. It was then determined that the leech destabilase also has lysozyme activity [8,9]. In a previous work [10], we reported the cDNA sequence of several bivalve lysozymes. We showed that, in addition to bivalve lysozymes, homologous sequences can be found in the genome of the nematode Caenorhab- ditis elegans and that of the fly Drosophila melanogaster,as well as expressed sequences tags from penaeid shrimps, indicating that these species possess putative proteins akin to the lysozyme i type. We performed a phylogenetic analysis of all of these sequences together with those of the more conventional lysozyme c type; the results suggested that these two lysozymes originate from a common gene ancestor, at least in the central exon coding for the active lysozyme domain [10]. In fact, the existence of a new type of lysozyme, lysozyme i, was proposed as early as 1975, on the basis of the N- terminal sequence of a lysozyme extracted from the starfish Asterias rubens [11]. All recently described type-i lysozymes, including putative proteins derived from nucleic acid sequences, belong to protostome species. Thus, it seems worthwhile to revisit the lysozyme i from the deuterostome invertebrate in which it has been described for the first time. In the present work, we present the protein sequence and the complete cDNA sequence of the lysozyme i from A. rubens. In addition, we present putative models of its secondary and tertiary structure. Material and methods Biological material The starfish A. rubens was collected near Roscoff (Brittany, France). For RNA extraction, samples were preserved in RNAlater TM solution (Ambion) to inactivate RNAases. Protein sequencing The A. rubens lysozyme was prepared according to Jolle ` s and Jolle ` s [11]. The lysozyme was reduced according to Jolle ` s et al. [12], using iodoacetamide for alkylation. Diges- tion by trypsin or carboxypeptidase (Worthington, Lake- wood, NJ, USA) 1 or by Staphylococcus aureus V8 proteinase (Miles) was performed for 18 h at 37 °Cin0.1 M ammo- nium bicarbonate with an enzyme/substrate ratio of 1 : 50. Cyanogen bromide (Merck) cleavage was performed in Correspondence to J. S. Deutsch, E ´ quipe De ´ veloppement et E ´ volution, UMR 7622 ‘Biologie du de ´ veloppement’, CNRS et Universite ´ P&M Curie, 9 quai St-Bernard, case 241, 75252 Paris cedex 05, France. Fax: +33 14427 3253, Tel.: +33 14427 2576, E-mail: jean.deutsch@snv.jussieu.fr Note: The nucleotide sequence of the Asterias lysozyme i cDNA is available in the GenBank database under accession number AY390770. *Present address: MNHN, Paris and Mine ´ ralogie Cristallographie (LMCP) UMR 7590, Universite ´ P & M Curie, Paris (France), pl. Jussieu, case 115, 75252 Paris cedex 05, France. (Received 22 September 2003, revised 4 November 2003, accepted 11 November 2003) Eur. J. Biochem. 271, 237–242 (2004) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03915.x 70%formicacidfor48hat20°C. HPLC of the peptides was performed with a Waters chromatograph (model ALC/ GPC-204) using a Brownlee RP 300 column (22 · 4.6 cm) and absorbance at 220 and 280 nm was followed. Each peptide (0.2 nmol) was submitted to automated Edman degradation in an Applied Biosystems 470A protein sequencer. The phenylthiohydantoins of amino acids were identified by an on-line Applied Biosystems 120A PTH analyser. Synthesis of cDNA Total RNA was extracted using the RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. It was treated with RNAase-free DNAase I (Pharmacia) for 30 min at 37 °C. cDNA was synthesized by reverse transcription from DNAase-treated RNA using Moloney murine leukaemia virus reverse transcriptase (Stratagene) and an oligo-dT primer. RACE/PCR, cloning and sequencing A3¢ RACE/PCR was performed using this cDNA as a template, an oligo-dT primer and a degenerate primer (AS1, Table 1) designed from the N-terminal sequence of the A. rubens lysozyme determined by Jolle ` s and Jolle ` s[11]. This yielded too short a fragment to represent the complete 3¢ end of the lysozyme cDNA. Yet, its sequence showed a clear homology with other lysozyme i sequences [10]. We thought that this was due to inappropriate priming of the oligo-dT primer. The specific (nondegenerated) primer AS3 (Table 1) was determined from this first sequence fragment. Then a second PCR step was performed using AS3 and the oligo-dT. PCR were performed on cDNA in 20-lL reaction mix containing 5¢- and oligo-dT primers at 20 and 4 pmol, respectively, dNTP 10 m M and 5 U Qbiotaq DNA poly- merase (Q-Biogene). PCR cycles were as follows: 3 min at 94 °C followed by 30–40 cycles of 1 min at 94 °C, 1 min at 56–59 °C (depending on the primers), 1 min at 72 °Cand finally 10 min at 72 °C. After amplification, the PCR products were analysed by electrophoresis through 1% agarose gels and purified using the Jetsorb Kit (Genomed). They were cloned in a T-overhang vector derived from pBlueScript KS+ (Strata- gene), prepared according to Holton [13]. Sequencing was performed on both strands with the thermosequenase fluorescent-labelled primer cycle sequencing kit and 7-deaza-dGTP (Amersham Pharmacia). To expand the cDNA on its 5¢ side, the specific antisense primers AS3R and AS4R (Table 1) were used for reverse transcription. The cDNA was extended by terminal trans- ferase A (Biolabs). Two amplification steps were performed using AS3R/oligo-dT and AS4/oligo-dT at the annealing temperature of 57 °Cand58°C, respectively. The 5¢ RACE fragment was purified, cloned and sequenced as described above. Computer structural analysis The signal peptide and cleavage site of the putative Asterias lysozyme i protein were determined using the following software: SIGNAL P http://www.cbs.dtu.dk/services/SignalP/ and PSORT II http://psort.nibb.ac.jp/. Hydrophobic Cluster Analysis (HCA plots) [14] was performed using the DRAW- HCA software available online at http://smi.snv.jussieu.fr/ hca/. To get three-dimensional representations of catalytic centres of lysozyme i and compare it with that of lysozymes c, the primary sequences of Asterias and of Mytilus lysozymes i were submitted to the automated Protein Fold Recognition server 3 D-PSSM at http://www.sbg.bio.ic.ac.uk/ 3dpssm/ [15,16] and successfully yielded putative three- dimensional structures. The figures were drawn using SWISS - PDBVIEWER [17]. Results and discussion Determination of the primary structure of the protein The primary structure of the lysozyme from A. rubens was determined by amino acid sequence analysis of the intact carboxy-methylated protein and of constituent peptides obtained through digests by trypsin, S. aureus V8 protein- ase, carboxypeptidase and cyanogen bromide treatment (see Material and methods). The results are summarized by the sequence shown on Fig. 1. cDNA cloning and sequencing cDNA was prepared from soft tissues of a single A. rubens specimen. As a starting point, we used degenerate primers designed from the N-terminal sequence determined by Jolle ` s and Jolle ` s [11] (Table 1). The complete cDNA sequence was determined on PCR products after several rounds of 3¢ and 5¢ RACE/PCR (see Material and methods and Table 1). Thus, the cDNA sequence was determined independently of the biochemically determined protein sequence described in the above paragraph. The cDNA sequence agreed with the protein sequence without ambiguity (Fig. 2) and allowed confirmation of the data from the biochemical analysis in two cases when an overlapping peptide was missing. The predicted ORF from the cDNA is slightly longer than the biochemically deter- mined protein sequence. Computer analysis (see Material and methods) permitted us to postulate a signal peptide of 16 amino acids and its cleavage site. Ten more amino acids are found in the predicted translated protein upstream of the serine that is found to be the N-terminal residue of the extracted protein. This could be due to an artefactual cleavage at the fragile S–S peptide bound (Fig. 2) during the purification of the protein. Alternatively, this could be the physiological form of the protein, taking into account that the bivalve type i lysozymes have approximately the same Table 1. Primer sequences. Primer (5¢fi3¢) Corresponding peptide AS1 GGTTGCCTGAGRTGYATHTG a GCLRCIC AS3 GGGCTATTGGTCAGACGCTACACTC GYWSDATL AS3R GAGTGTAGCGTCTGACCAATAGCC GYWSDATL AS4R GATCTGATACGGTCCACACGACAG LSCGPYQI a H ¼ A or C or T; R ¼ AorG;Y¼ CorT. 238 S. Bachali et al. (Eur. J. Biochem. 271) Ó FEBS 2003 length at their N-terminal side [3,4,10]. The coding sequence is followed by a 3¢ noncoding tail of 172 nucleotides and is preceded by a 5¢ noncoding leader of 101 nucleotides. Up to now, complete protein sequences of i-type lysozymes were only available from protostome species. We aligned the Asterias lysozyme sequence with type i proteins for which a lysozyme activity has been demonstra- ted (Fig. 3). This alignment supports the homology of the starfish lysozyme with protostome proteins as proposed in our previous work [10]. Comparison between the lysozyme of the starfish, a deuterostome species, with the previously known lysozymes i provides the opportunity to reveal conserved residues over about 600 million years. Of about 120 amino acids, as many as 35 are identical, and 13 are similar (Fig. 3). The starfish lysozyme is less rich in cysteines than the protostome lysozymes i (10 vs. 13). Relative to the other known i lysozymes, it presents a four-residue insertion (residues 55–58 on Fig. 3). Comparison with the second exon of the human lysozyme c that comprises the active site reveals both similarities between the two types of lysozymes and residues specific to the i-type (Fig. 3). A BLAST search with the lysozyme sequence of A. rubens in the sequence database of the National Center for Biotechnology Information (NCBI) shows significant sequence similarities with the destabilase of the medicinal leech H. medicinalis [7], with the bivalve lysozyme sequences determined in our previous work [10], with the lysozyme of the bivalve Tapes japonica [3], with the so-called chlamysin of Chlamys islandica [4,5], and also with a hypothetical secreted protein of the nematode Caenorhabditis elegans and with putative gene products retrieved from the genomes of the fly Drosophila melanogaster and of the mosquito Fig. 1. Chemically determined primary struc- ture of the A. rubens lysozyme. Phenylalanine 112 is drawn in low case (f) because it was ambiguous. >, Amino acid determined by automated Edman degradation; <, amino acid determined by carboxypeptidase degradation; +, trypsin cut; +, trypsin peptide sequenced by the Edman technique; ¼¼ ¼ ¼, S. aureus V8 protease peptide, sequenced by the Edman technique; ––––, cyanogen bromide (BrCN) peptide, sequenced by the Edman technique. Fig. 2. cDNA sequence of the A. rubens lysozyme. Noncoding nucleotides (nt.) are shown in small case letters and the coding sequence is shown in upper case letters. Putative polyadenylation signals are boxed 3 . The N-terminal amino acid of the mature protein is boxed. The predicted signal peptide is in grey. Ó FEBS 2003 Lysozyme of Asterias rubens (Eur. J. Biochem. 271) 239 Anopheles gambiae. On the other hand, no significant similarity was found when BLAST searches were performed on the complete or near-complete genome sequences of deuterostome species, such as the mamalians Homo sapiens and Mus musculus, the teleost fishes Takifugu rubripes and Danio rerio and the urochordate Ciona intestinalis. The presence of type i genes in the three branches of the metazoan tree [18], the protostome Ecdysozoa (including arthropods and nematodes) and Lophotrochozoa (inclu- ding molluscs and annelids) and deuterostomes (starfish) brings evidence that a lysozyme i gene was present in the bilaterian ancestor. Given the present genomic data, it must Fig. 3. Alignment of the A. rubens lysozyme sequence with other lysozymes. The complete mature starfish protein was aligned with all protostome i-type proteins for which a lysozyme activity has been demonstrated. Aru, A. rubens; Med, Mytilus edulis; Cis, Chlamys islandica; Tja, Tapes japonica; Hme, H. medicinalis. Numbering is that of the starfish lysozyme. For comparison, the second exon of the human lysozyme-c (Has, Homo sapiens) is also aligned. Below are noted secondary structure elements: h marks a residue involved in an a-helix, b a residue involved in a beta-turn. Residues conserved in all c-type lysozymes [21] are underlined. The two active acidic residues of the c-type lysozymes are boxed. Conserved residues in all i-type lysozymes are in grey. Conserved cysteines are noted by s above the Asterias sequence. Fig. 4. Hydrophobic cluster analysis. The primary sequence is represented on a roll mimicking a-helices. The primary sequence is drawn twice. A dashed line follows one of these primary sequences. Prolines (P) and glycines (G) that break a-helices are represented as w and r, respectively. The hydrophilic residues serines (S) and threonines (T) are represented by h. Residues that are distant on the primary sequence may appear close to each other on this type of diagram, thus revealing hydrophobic clusters (boxed). (A) HCA plot of the Asterias lysozyme; hydrophobic residues conserved in all lysozymes i are in grey. (B) Second exon of the human lysozyme c. Conserved hydrophobic residues between lysozymes i and c are in grey. 240 S. Bachali et al. (Eur. J. Biochem. 271) Ó FEBS 2003 have been lost in several deuterostome lineages. Compar- ative genomics is developing rapidly. Complete genomes of a greater panel of species will be soon available. This will allow us to assess whether or not the lysozyme i gene has been lost from the origin of the whole vertebrate or even the whole chordate lineages. If this is true it this would fully justify the name given of ‘invertebrate’ lysozyme. Hydrophobic cluster analysis Primary structure comparison between such distantly related proteins as i-type and c-type lysozymes provides significant yet insufficient data on functionally important residues. To understand this issue further, we performed a hydrophobic cluster analysis [14]. This analysis permits one to relate residues that are not close to each other along the linear sequence, but may come close under secondary structure, forming hydrophobic clusters or pouches (Fig. 4A). The N-terminal half of i lysozymes is homologous to the second exon of vertebrate c-type lysozymes (Fig. 3 and [10]). Fig. 4B shows the HCA plot of this part of the human lysozyme. A number of hydrophobic residues overlap between this plot and the corresponding part of the HCA plot of the Asterias lysozyme. The same overlap is obtained when comparing the human lysozyme and/or the chicken lysozyme with the protostome lysozymes i listed in Fig. 3 (data not shown). In the C-terminal half other hydrophobic residues are conserved among the i-type lysozymes (Fig. 4A). The natural substrate of lysozymes is a polymer of N-acetyl-glucosamine and of N-acetyl-muramic acid. Up to six sugar rings get into the cleft of the lysozyme c at subsites called A–F. The D subsite is the active site where the sugar chain is cleaved. In the chicken and human lysozymes c the hydrophobic cluster IYW (IWW in chicken) (Fig. 4B) is involved in interactions with sugar rings [19]. This hydro- phobic cluster is very well conserved in lysozymes i (Fig. 3 and Fig. 4A,B). We postulate that this function is conserved in i-type lysozymes. It is likely that the other conserved hydrophobic clusters are involved in similar interactions. Three-dimensional modelling A partial three-dimensional structure of the starfish lyso- zyme i model was successfully generated by the 3 D - PSSM software program (see Material and methods). In this model, some links remain uncertain. They correspond to variable parts of the proteins among the various i-type lysozymes. They probably represent loosely structured loops between more structured helices and/or sheets. Despite these missing parts, this computer-based three- dimensional model is recognized as a lysozyme with a high probability score ( PSSM E-value: 4.67 e-5) 2 .Inparticular,a Fig. 5. Putative three-dimensional model of the Asterias lysozyme i. These figures were generated with the help of the SWISS - PDBVIEWER software. (A) ApartoftheputativestructureoftheAsterias lysozyme, from residues L9 to L50 (according to numbering in Fig. 3). The side chains of E16 and S34 that we postulate to be the active enzymatic residues (see text) are shown. (B) Homologous part of the human lysozyme from the model deposited in the SwissProt data bank under the accession number 1IY3 (residues W28–K69, according to the sequence of the human lysozyme). The side chains of E35 and D53 that are the known active residues in c-type lysozymes are shown. Ó FEBS 2003 Lysozyme of Asterias rubens (Eur. J. Biochem. 271) 241 part of the three-dimensional structure of the Asterias lysozyme (Fig. 5A) is very similar to the known structure of the active site of the human c lysozyme (Fig. 5B). The putative structure of the Mytilus lysozyme i is almost identical (data not shown). The critical glutamate (E) of lysozyme c active site is conserved in lysozymes i. In contrast, the active aspartate (D52 according to chicken numbering) is not conserved (Fig. 3). In a previous paper we postulated that its role could be played by another D residue [10]. The present putative three-dimensional structure does not support this hypothesis. On the other hand, the tertiary structure supports the homology between the active D of lysozymes c and a conserved serine (S), as proposed on the basis of primary structure (Fig. 3). We determined the atomic distances between the oxygen atom of this S and those of the active E. They fall (8.1–8.5 A ˚ ) within the range of the distances between active atoms in the lysozyme c active site (5.9–8.2 A ˚ ). We thus propose that the S34 (according to numbering in Fig. 3) is the active residue for sugar chain cleavage in lysozymes i. A similar situation is found in g-type lysozymes, where E73 is an analogue of the active glutamate of lysozymes c, but no aspartate analogue is found [20]. Acknowledgements We are grateful to Prof. A. Toulmond for providing us with the facilities of the Station Biologique de Roscoff and constant support. S. Bachali is recipient of a PhD fellowship of the Tunisian government. We thank three anonymous referees for their comments that helped improve the manuscript. References 1. Jolle ` s, J., Fiala-Medioni, A. & Jolle ` s, P. (1996) The ruminant digestion model using bacteria already employed early in evolu- tion by symbiotic molluscs. J. Mol. 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