蛇毒Serine Protease Inhibitor及其Paralogues功能性基因體之研究(III)Functional Genomic of Snake Venom Serine Protease Inhibitor and Its Paralogues (III)

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行政院國家科學委員會專題研究計畫成果報告

蛇毒 Serine protease inhibitor 及其 Paralogues 功能性

基因體之研究(3/3)

計畫類別：個別型計畫

計畫編號： NSC94-2320-B-110-002-

執行期間： 94 年 08 月 01 日至 95 年 07 月 31 日

執行單位：國立中山大學生物醫學科學研究所

計畫主持人：張榮賢

計畫參與人員：程韻靜、陳顧中、高培修、王梨冠

報告類型：完整報告

處理方式：本計畫可公開查詢

中華民國 95 年 8 月 7 日

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本計畫主要目的在於探討台灣眼鏡蛇及台灣雨傘節蛇毒 Protease inhibitor

及其 Paralogous proteins 功能性基因體，以及其認知之細胞作用標的。由台灣

眼鏡蛇毒腺我們選殖出 Chymotrypsin inhibitor cDNA，並以 E. coli 製備其重組蛋

白，以定點突變方法分析其結構與功能之關係顯示氮端區域雖非其活性所需，但

卻對其結構穩定性及其抑制 Chymotrysin 活性有效期間有顯著影響。利用酵母

菌雜交系統，以

β

-Bungarotoxin B1 鏈為釣餌，發現 Potassium channel-interacting

protein3 (KChIP3)可能為

β

-Bungarotoxin B1 鏈細胞內作用標的，Pull down assay

及 BIACORE 分析均證實 KChIP3 和

β

-Bungarotoxin 及

β

-Bungarotoxin B1 鏈均產

生分子間結合反應，並且鈣離子可增強此一分子間結合反應。進一步以 PCR

方式選殖台灣眼鏡蛇 Chymotrypsin inhibitor 之 Genomic DNA，發現其與台灣雨

傘節鍵前神經毒

β

-Bungarotoxin B1 鏈基因具有相同基因結構，其核酸序列相似性

達 83%，顯示 Protease inhibitor 和

β

-Bungarotoxin B1 鏈具有演化上的同源性。同

樣台灣眼鏡蛇 Chymotrypsin inhibitor 與

β

-Bungarotoxin B2 鏈、B4 鏈、B5 鏈及

B6 鏈基因也具有相同基因構造，分析 Chymotrypsin inhibitor 及 B 鏈基因 Exon

及 Intron 區域，發現台灣眼鏡蛇 Protease inhibitor 及台灣雨傘節

β

-Bungarotoxin B

鏈基因演化過程有 Intron insertions 或 deletions 的現象，同時其 Exon region 經由

加速演化產生序列變異性。

關鍵詞: 台灣眼鏡蛇、台灣雨傘節、蛋白水解酵素抑制劑、同源蛋白、結構與功

能之關係、作用標的。

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The goals of the present study are to study the functional genomics of Taiwan

cobra (Naja naja atra) and Taiwan banded krati (Bungarus multicinctus) related to

protease inhibitors and paralogues, and explore the cellular targets interacting with the

protease inhibitors and paralogues. A cDNA encoding chymotrypsin inhibitor is

constructed from the cellular RNA isolated from the venom glands of Naja naja atra.

Cloned protein is expressed as a function protein from E.coli. Ala-screening

mutagenesis studies on the N-terminus of chymotrpsin inhibitor reveal that the

N-terminus is not involved in functional manifestation of the protease inhibitor.

However, alteration in the globally structural rigidity of mutated chymotrpsin

inhibitor affects the sustainable period in inhibiting chymotrypsin activity. Using

B1 chain of

β

-bungarotoxin as bait in yeast two-hybrid screen, we find that KChIP3 is

a binding protein of B1 chain. Pull down assay and BIACORE analysis further

proved that KChIP3 is associated with

β

-bungarotoxin as well as B1 chain.

Moreover, their interaction could be enhanced by the addition of Ca

2+

. The

genomic DNA encoding chymotrypsin inhibitor is amplified by PCR. The gene

shares virtually an identical structural organization with Taiwan banded krait

β

-bungarotoxin B1 chain gene. Moreover, the overall sequence identity of

chymotypsin inhibitor and

β

-bungarotoxin B1 chain genes is up to 83%. These

findings strongly suggest that chymotypsin inhibitor and

β

-bungarotoxin B1 chain

may have originated from a common ancestor. Likewise,

β

-bungarotoxin B chain

(B2, B4, B5 and B6 chains) genes also share the same genetic structure with

chymotrypsin inhibitor. Comparative studies on B chain and chymotrypsin

inhibitor genes show that intron insertions or deletions occur with the evolution of B

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protein-coding sequence of the genes.

Key words: Taiwan cobra; Taiwan banded krait; Protease inhibitor; Paralogous

proteins; Structure-function relationship; Functional targets.

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1. Cheng, Y.C., Yan, F.C. and Chang L.S. (2005) Taiwan cobra chymotrypsin

inhibitor: cloning, functional expression and gene organization. Biochem. Biophys.

Acta 1747, 213-220.

2. Lin, Y.L., Wu, P.F., Wu, T.T. and Chang, L.S. (2006) KChIP3, a binding protein

for Taiwan banded krait

β

-bungarotoxin. Toxicon 47, 265-270.

3. Cheng, Y.C., Chen. K.C., Lin, S.K. and Chang, L.S. (2006) Divergence of genes

encoding B chains of

β

-bungarotoxins. Toxicon 47, 322-329

4. Yan, F.J., Chen, C.P. Cheng, Y.C. and Chang, L.S. (2006) Mutagenesis studies on

the N-terminus and Thr-54 of Naja naja atra (Taiwan cobra) chymotrypsin

inhibitor. Protein J. in press.

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Taiwan cobra chymotrypsin inhibitor: cloning, functional

expression and gene organization

Yun-Ching Cheng, Fang-Jiun Yan, Long-Sen Chang*

Institute of Biomedical Sciences, National Sun Yat-Sen University, Kaohsiung 804, Taiwan Received 26 May 2004; received in revised form 16 November 2004; accepted 16 November 2004

Available online 8 December 2004

Abstract

A cDNA encoding chymotrypsin inhibitor was constructed from the cellular RNA isolated from the venom glands of Naja atra (Taiwan cobra). The resultant amino acid sequence showed that the mature protein is comprised of 57 amino acid residues with six cysteine residues. Cloned protein was expressed and isolated from the inclusion bodies of E. coli and refolded into a functional protein in vitro. Deleting the first three residues at its N-terminus caused a moderate increase in the inhibitory constant (Ki) against chymotrypsin. The genomic DNA

encoding the chymotrypsin inhibitor was amplified by PCR. The gene shares virtually an identical structural organization with the h-bungarotoxin B1 chain (a snake Kunitz/BPTI neurotoxic homolog) gene. Moreover, the overall sequence identity of the N. atra chymotrypsin inhibitor and h-bungarotoxin B1 chain genes was up to 83%. These findings strongly suggest that snake Kunitz/BPTI protease inhibitors and neurotoxic homologs may have originated from a common ancestor.

Keywords: Chymotrypsin inhibitor; Mutagenesis of N-terminus; Exon–intron organization; Naja atra

1. Introduction

In addition to enzymes and toxins, snake venom contains serine protease inhibitors Several Kunitz/BPTI inhibitors from the venom of Viperidae and Elapidae snakes have been isolated and sequenced [1–8]. Their physiological roles in the regulatory mechanisms that influence the proteases in coagulation, fibrinolysis and inflammation have been considered. Snake neurotoxic Kunitz/BPTI homologs, such as dendrotoxins, calciclu-dine and the B chain of h-bungarotoxin, are also well known to act as Ca2+ or K+ channel blockers [9–12]. Comparative analysis of the Kunitz/BPTI inhibitors and neurotoxic homologs suggested that the changes in active site residues resulted in both conformational adjustment

and functional divergence [13,14]. The specificity of Kunitz/BPTI inhibitors towards serine proteases is closely associated with P1 amino acid. However, besides surrounding the reactive site, the residues present in the weak contact loop are also important for the different interactions with various serine proteases [15]. Muta-genesis studies on BPTI have elucidated the structural elements for the activity of protease inhibitor [16–19]. Unlike BPTI, regional variation analysis suggested that the N-terminal residues of snake Kunitz/BPTI inhibitors are involved in intermolecular recognition with proteases

[20]. In the present study, the cDNA encoding the chymotrypsin inhibitor (designated as NACI) is con-structed from the cellular RNA of Naja atra (Taiwan cobra) venom glands. Then mutagenesis studies on its N-terminus are carried out. Moreover, the evolutionary relationship between snake venom protease inhibitor and neurotoxic Kunitz/BPTI homologs is discussed based on the organization of NACI and h-bungarotoxin (h-Bgt) B chain genes.

* Corresponding author. Fax: +886 7 5250197.

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2. Materials and methods

2.1. Preparation of mRNA from venom glands

Cellular RNA was isolated from N. atra venom glands that had been stored in liquid nitrogen immediately after the killing of the snake. Two deep-frozen glands from one snake were homogenized to extract RNA with a guanidinium isothiocyanate/phenol/chloroform isolation kit (Stratagene, USA).

2.2. PCR amplification and cloning of NACI

A previous study showed that the h-Bgt B chain is structurally homologous with Kunitz/BPTI inhibitors [21]. Thus, the sense and antisense primers with sequences ATGTCTTCTCGAGGTCTTCTTCTCC-3V and 5V-GGTCATCCAACACAGGTGCGGTT-3V were synthesized based on the signal peptide and 3V-noncoding regions of h-Bgt B1 chain cDNA [22], respectively. RT-PCR was performed in 100 Al of reaction buffer containing 100 mM Tris–HCl, pH 8.3, 1 mM dNTP, 25 AM antisense primer and 200 ng of RNA template. In the reverse transcription, cDNA synthesis was conducted with rTth reverse transcriptase (5 units) and 2 Al of 10 mM MnCl2at 70 8C For 15 min. Then

8 Al of chelating buffer containing 50% (v/v) glycerol, 100 mM Tris–HCl (pH 8.3), 1 M KCl, 7.5 mM EGTA and 0.5% (v/v) Tween-20 was added to the reaction. After adding 8 Al of 25 mM MgCl2 containing 25 AM sense primer, the

amplification was performed in a thermocycler for 35 cycles of 1 min at 94 8C, 1 min at 50 8C and 1 min at 72 8C. The PCR products were cloned into pGEM-T easy vector using the TA-cloning procedure (Promega), and the cDNA encoding NACI was confirmed by DNA sequencing. 2.3. Expression of NACI

Synthetic oligonucleotides were designed to produce an amplified DNA fragment spanning the open reading frame of NACI. The forward primer introduced a 5V-EcoRV site preceding Arg-1 of NACI: GATATCCGTCCAAGGTT-CTGTGAACTGGCT, with the reverse primer being 5V-GGTCATCCAACACAGGTGCGGTT-3V (PI-down). The PCR product was cloned into pGEM-T easy vector. The inserted DNA fragment was cut with EcoRV and EcoRI, then ligated into the large fragment of EcoRV/EcoRI-cut pET-29a(+).

The resulting plasmid was transformed into E. coli strain BL21(DE3). Transformants were selected on LB-agar plates supplemented with 50 Ag/ml ampicillin. For the purpose of gene expression, E. coli BL21(DE3) cells harboring the plasmid were grown at 37 8C in LB medium containing 50 Ag/ml ampicillin. After OD550reached 1.0,

isopropyl-h-d-thiogalactoside (IPTG) was added to a final concentration of 0.2 mM. The culture was induced for periods of up to 4 h. The cells were harvested and lysed using ultrasonication.

The recombinant NACI was found to appear exclusively in the inclusion bodies of E. coli. The inclusion bodies recovered from 1 l of bacterial culture were thoroughly washed with 10 mM Tris–1 mM EDTA (pH 8.0) containing 0.5% Triton X-100, and sulfonated with disodium 2-nitro-5-(sulfothio)-benzoate (NTSB) in 8 M urea, 0.3 M Na2SO3,

pH 8.5 [23]. The protein was precipitated with 1% acetic acid and dissolved in 10 ml of 50 mM sodium borate (pH 8.5) containing 5 mM EDTA, 8 M urea, and 4 mM reduced and 2 mM oxidized glutathione. Refolding was performed by a fourfold dilution with the same buffer without urea. After standing for 1 day at room temperature, the refolded protease inhibitor was further purified by HPLC on a SynChropak RP-P column (4.6 mm25 cm) and eluted with a linear gradient of 7.5%–75% acetonitrile for 50 min. The flow rate was 0.8 ml/min, and the effluent was monitored at 280 nm. The pET29a(+) possesses a thrombin site for cleavage of the fusion protein, thus the purified protein was hydrolyzed with thrombin at 37 8C for 20 h. The hydro-lysates were separated on a SynChropak RP-P column. Efforts were also made to express NACI using pET20b(+) vector. A 5V-NdeI site was introduced preceding with Arg-1 of NACI by PCR, and the resulting DNA fragment was ligated into the large fragment of Nde I/Eco RI-cut pET20b(+). As expected, the recombinant protein contained an extra Met but not a fusion peptide at its N-terminus. Following induction with IPTG, there was no appreciable amount of the recombinant protein in the cell extracts of E. coli harboring the NACI-pET20b(+) plasmid that could be detected by SDS-PAGE analysis. Thus, the recombinant NACI fusion protein was prepared using the expression vector pET29a(+).

To prepare mutated NACI, three up primers, G-up (5V-GATATCGGCCGTCCAAGGTTCTGTGAACTGGCT-3V), DN1-up (5V-GATATCCCAAGGTTCTGTGAACTGG-CTCCT-3V) and DN3-up (5V-GATATCTTCTGTGAACT-GGCTCCTTCAGC-3V), were synthesized. An EcoRV site was introduced into these up primers. The mutated DNA fragments were generated by PCR amplification using primer combinations G-up and down, DN1-up and PI-down and DN3-up and PI-PI-down, respectively. Conditions of PCR, subcloning and gene expression were the same as above. Mutated proteins were also found to appear exclusively in the inclusion bodies of E. coli. Refolding of the mutated proteins was carried out according to the procedure described above.

2.4. Inhibition of chymotrypsin activity

Assays were performed with 0.1 M Tris–HCl (pH 8.0) containing 10 mM CaCl2in a total volume of 500 Al. The

reactions were initiated through adding the enzyme or the mixtures of enzyme and inhibitor into the substrate (N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide) containing buffer solution, and the initial rate of p-nitroanilide ( pNA) was monitored continuously at 405 nm. An appropriate amount

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of chymotrypsin (16 nM) was incubated with varying concentrations of substrate ranging from 0.1 to 0.8 mM. In order to determine the inhibitory potency of the protease inhibitor, chymotrypsin (16 nM) was incubated with varying concentrations of inhibitor (240 to 480 nM) at 37 8C for 30 min prior to activity assay. The slope (Km/Vmax) of lines

obtained from the Lineweaver–Burk representation (1/V vs. 1/[S]) of saturation curves at different inhibitor concen-tration were plotted against the concenconcen-tration of inhibitor. The inhibitory constant (Ki) of the chymotrypsin/inhibitor

complex was determined from the intercept point of the x-axis[24].

2.5. PCR amplification and cloning of the NACI precursor N. atra liver was ground to a fine powder in liquid nitrogen. The genomic DNA was extracted from the powder according to the procedure previously described

[25]. Two oligonucleotide primers based on the promoter region and the 3V-noncoding region of the h-Bgt B1 chain gene[26]with the forward sequence AACCCCAATTG-CAGACAGTGAACAGG-3V and the reverse 5V-TGGTCCAGGGCAGAGAGCAGGGTC-3V were synthe-sized. PCR reaction was carried out using Advantage genomic PCR kit (Clontech Laboratories Inc.). The procedure was carried out essentially according to the manufacturer’s protocol. The resulting products were cloned into pGEM-T easy vector using the TA-cloning

procedure (Promega), and the gene encoding NACI was confirmed by DNA sequencing.

2.6. Comparison of nucleotide sequence and homology search

In the comparison and analysis of the determined nucleotide sequences, a software package (PC/GENE program, Stratagene) was used for sequence alignment according to percent sequence identity. BLAST searches against non-redundant databases were carried out via the Internet using the software at the Website (http://www.ncbi. nlm.nih.gov/).

2.7. Other tests

Determination of the protein sequence, reduction and S-carboxymethylation (RCM) of protein, mass analysis, circular dichroism measurement, SDS-PAGE and native gel analyses were performed in essentially the same manner as previously described[23–25].

3. Results and discussion

PCR amplification of the N. atra venom gland cDNA mixtures with the primers designed from h-Bgt B1 chain cDNA generated a PCR fragment estimated to be

approx-Fig. 1. Nucleotide and deduced protein sequences of the precursor of NACI. (A) The nucleotide sequence of 313 base pairs is shown above the amino acid sequences of 81 residues including a signal peptide of 24 amino acid residues. The boxes indicate the primers used to amplify NACI cDNA. The cDNA sequence of NACI appears in the EMBL, GeneBank and DDBJ nucleotide sequence databases under the accession number AJ586046. (B) Aligned signal peptides of the h-bungarotoxin B1 chain (h-Bgt B1 chain), Vipera ammodytes ammodytes trypsin inhibitor (VAATI), Pseudonaja textilis textilis textilinin1 (PTxln1) and NACI.

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imately 300 bp (data not shown). The DNA fragment was subcloned by a TA-cloning kit. More than 10 positive clones were selected for nucleotide sequencing. BLAST searches revealed that the deduced amino acid sequence of the selected clones (Fig. 1) was similar to snake Kunitz/BPTI inhibitors and neurotoxic homologs. In terms of the finding that the cDNAs of h-Bgt B1 chain, Viper ammodytes protease inhibitors and Pseudonaja textilis textilis plasmin inhibitors have a highly conserved signal peptide region with 24 amino acid residues [22,27,28], the N-terminus of the mature protein would presumably be Arg (Fig. 1). This protein is comprised of 57 amino acids with six cysteine residues, which are located at homologous positions like those of the snake Kunitz/BPTI family protein (Fig. 2). In protease inhibitors, the reactive site residue (P1) generally corresponds to the specificity of the cognate enzymes, i.e., inhibitors with P1 Lys and Arg tend to inhibit trypsin and those with P1 Leu, Met, Phe, Tyr and Trp tend to inhibit chymotrypsin[15]. The protein contains a P1 Phe and thus is designated as N. atra chymotrypsin inhibitor (NACI). The key amino acids such as Gly12, Tyr23, Phe33, Tyr35, Gly37, Asn43 and Phe45, which are possibly in contact with protease[29], are located essentially in the same positions in NACI as in other Kunitz/BPTI protease inhibitors (Fig. 2). To subclone the NACI into the expression vector of pET29a(+), a new primer was designed in order to create an EcoRV site preceding the Arg-1 of NACI. The antisense

Fig. 2. Amino acid sequence alignments of snake Kunitz/BPTI protease inhibitors and neurotoxic homologs. The amino acid residues are numbered using BPTI as reference. Oh11, Ophiophagus hannah chymotrypsin inhibitor; BFCI, Bungarus fasciatus chymotrypsin inhibitor; NNCI, Naja naja chymotrypsin inhibitor; VAACI, Vipera ammodytes ammodytes chymotrypsin inhibitor; NNTI, N. naja trypsin inhibitor; EMTI, Eristocophis macmahonii trypsin inhibitor; HHTI, Hemachatus haemachatus trypsin inhibitor; VAATI, V. ammodytes ammodytes trypsin inhibitor; PTxln1, Pseudonaja textilis textilis textilinin 1; PTxln3, P. textilis textilis textilinin 3; BcKIa, Bungarus candidus Kunitz inhibitor a; h-Bgt B1 chain, Bungarus multicinctus h-bungarotoxin B1 chain; Dendrotoxin B, Dendroaspis polylepis polylepis dendrotoxin B; Dendrotoxin I, D. polylepis polylepis dendrotoxin I; Alphdendrotoxin, Dendroaspis angusticeps a-dendrotoxin; Dendrotoxin k, D. polylepis polylepis dendrotoxin k; Dendrotoxin E, D. polylepis polylepis dendrotoxin E; BPTI, bovine pancreatic trypsin inhibitor. The arrow (A) indicates the P1 residue.

Fig. 3. Purification of the recombinant NACI fusion protein and G-NACI. The refolded proteins were applied on a SynChropak RP-P column equilibrated with 0.1% TFA, and eluted with a linear gradient of 7.5–75% acetonitrile for 50 min. The flow rate was 0.8 ml/min and the eluate was monitored at 280 nm. The arrows indicate the recombinant G-NACI fusion protein (line a) and G-NACI (line b), respectively. Inset: (A) SDS-PAGE analyses of G-NACI fusion protein and G-NACI. Lane 1, molecular markers (prestained SDS/PAGE standards from Bio-Rad); lane 2, G-NACI fusion protein; lane 3, G-NACI. (B) Native gel analyses of G-NACI fusion protein and G-NACI. Lane 1, G-NACI fusion protein; lane 2, G-NACI.

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primer was the same as that used for amplification of NACI cDNA. The amplified DNA was inserted into a TA-cloning vector and finally subcloned into the expression vector pET29a(+) using the EcoRV/EcoRI restriction sites. Recombinant clones were initially grown in 10-ml cultures and induced with IPTG. Aliquots of cell extracts were analyzed by SDS-PAGE. It was found that the expressed protein appeared exclusively in the inclusion bodies (data not shown). Thus, the recombinant proteins were subjected to refolding with NTSB essentially according to the procedure described previously[23]. The NACI fusion protein in which NACI fused with a peptide with the sequence MKETAAAK-FERQHMDSPDLGTLVPRGSMADI was purified by HPLC, and was shown to have an inhibitory effect on

chymotrypsin activity with an inhibitory constant Kiof 279

nM. NACI fusion protein (up to 0.2 mg/ml) was soluble in Tris buffer for activity assay. However, the poor solubility of NACI fusion protein in PBS buffer was noted, thus only a marginal amount of NACI could be obtained after thrombin digestion. As shown in Fig. 2, the number of N-terminal residues in snake venom protease inhibitors varied mostly from four to six residues using the first Cys residue as a reference point. Thus, a new primer was designed to include an additional Gly residue before Arg-1 of NACI with the resulting protein (G-NACI) having an N-terminal sequence similar to that noted in the Ophiophagus hannah chymo-trypsin inhibitor (Oh11) (Fig. 2). The recombinant G-NACI fusion protein was refolded with NTSB and then purified by Fig. 4. Inhibition of chymotrypsin activity in the presence of G-NACI. Lineweaver–Burk plot for the determination of Km/Vmaxvalues of chymotrypsin activity

on N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide in the absence (

.

) or presence of 240 nM (o), 320 nM (z), 400 nM (5) and 480 nM (n) G-NACI, respectively. Inset, Km/Vmaxreplot of calculating Ki.

Fig. 5. Schematic drawing showing the NACI mutants. The fused peptide comprises 25 amino acid residues (white box) and the peptide GSMADI (white circles). The segment of 25 amino acid residues is removed by thrombin digestion. The N-terminal segment RPRFC (gray circles) and the gray boxes of 52 amino acid residues represent the mature NACI protein. An extra Gly residue was inserted before Arg-1 of NACI, and the resulting protein is designated as G-NACI. The inhibitory constant (Ki) of the chymotrypsin/inhibitor complex was determined as shown inFig. 4.

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HPLC (Fig. 3). Meanwhile, its N-terminal fused peptide was removed by thrombin cleavage, and the resulting G-NACI was purified on a SynChropak RP-P column. The homoge-neity of the G-NACI fusion protein and G-NACI was verified by SDS-PAGE and native gel (Inset ofFig. 3). The results of protein sequence determination and mass analysis (data not shown) also supported that the fused peptide was removed by

thrombin digestion from G-NACI. Both G-NACI fusion protein and G-NACI were capable of inhibiting chymotryp-sin activity. The Ki value was determined as the x-axis

intercept of the line in the plot of Km/Vmaxvs. [I] values (inset

ofFig. 4). It was found to be 180 and 133 nM for G-NACI fusion protein and G-NACI, respectively. Two N-terminal mutants, NACI(DN1) and NACI(DN3), were constructed

Fig. 6. Organization and nucleotide sequence of NACI gene. (A) Identification and size determination of PCR-amplified genomic DNAs. (B) The deduced amino acid sequence is presented below the coding parts of the exons, and the signal peptide is indicated by gray boxes. The putative TATA box is indicated by bold letters and gray boxes. The exon regions are underlined. The genetic structure of NACI appears in the EMBL, GeneBank and DDBJ nucleotide sequence databases under the accession number AJ586047. (C) Schematic drawing showing the genetic organization of NACI and h-Bgt B1 chain genes. The gaps in the NACI gene represent the missing part relative to the h-Bgt B1 chain gene.

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with deleting Arg and Arg-Pro-Arg, respectively (Fig. 5). After thrombin cleavage, NACI(DN1) and NACI(DN3) showed Ki values of 148 and 355 nM, respectively. While

NACI(DN3) had a slightly reduced inhibitory activity compared to G-NACI, its gross conformation did not differ greatly from that of G-NACI fusion protein and G-NACI as evidenced by CD spectra (data not shown).

Comparative analyses of NACI and h-Bgt B1 chain cDNAs show a high degree of sequence identity in their signal peptide and 3V-noncoding regions. Thus, one pair of primers designed from the promoter and 3V-noncoding regions of the B1 chain gene[26]was employed to amplify genomic DNA encoding NACI. As shown in Fig. 6A, a genomic DNA with a size of approximately 3000 bp was amplified by one pair of primers as described in Materials and methods. Alignment of the genomic DNA with cDNA sequence allows us to unambiguously assign the exon– intron organization. As shown inFigs. 6B and C, the NACI gene is organized with three exons and two introns, which are virtually identical to those reported for the h-Bgt B1 chain gene [26]. Moreover, protein-coding regions for NACI and h-Bgt B1 chain were interrupted by introns in similar positions. The sequences of all exon–intron junc-tions agree with the GT/AG rule. Intron 1 is smaller in size than intron 2. The overall sequence identity of NACI and h-Bgt B1 chain genes was up to 83.1%. The exon 2 region of NACI and h-Bgt B1 chain genes encoded most of the mature proteins, and the sequence of exon 2 was more diversified than the flanking introns. The sequence identity for exon 1, intron 1, exon 2, intron 2 and exon 3 of the two genes are 88.0%, 90.9%, 60.5%, 90.7% and 87.9%, respectively. The signal peptide of NACI and h-Bgt B1 chain genes is encoded by the first exon.

Cardle and Dufton [20] proposed that both the N-terminal region and the antiprotease site of snake Kunitz/ BPTI inhibitors are involved in interactions with proteases. The first four residues (Tyr1, Asn2, Arg3 and Leu4) of tick anticoagulant protein that adopts the BPTI fold have been demonstrated to be important in exerting its inhibitory action [30]. In contrast, the slight change in Ki value

observed on the N-terminal NACI mutants does not support the mechanism in which the N-terminal residues of NACI form part of the active chymotrypsin inhibitory region. Regarding these details, however, it must be noted that the physiological target protein of NACI still remains to be identified.

The findings that the NACI gene and h-Bgt B1 chain genes share the same exon–intron organization and a high degree of nucleotide sequence identity support the idea that the two genes might have originated from the same ancestor. Analysis of the nucleotide sequences from the snake Kunitz/ BPTI protein family suggested that this family evolved by gene duplication followed by diversification[28]. Previous studies revealed that an accelerated evolution occurred within the exon regions of the venom gland phospholipase A2and three-finger toxin genes resulting in the functional

diversities observed in these proteins. Nevertheless, the introns of these genes had evolved at a similar rate and were highly conserved[25,31–36]. Taken together, an accelerated evolution may occur with the evolution of the snake Kunitz/ BPTI protein family too.

Acknowledgements

This work was supported by Grant NSC 92-2320-B110-013 from the National Science Council, ROC (to L.S. Chang). The authors express their sincere gratitude to Prof. C.C. Yang, Department of Life Sciences, National Tsing Hua University for his constant encouragement during this study.

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KChIP3: A binding protein for Taiwan banded krait

b-bungarotoxin

Ya-Ling Lin

a,1

, Pei-Fung Wu

b,1

, Tony T. Wu

c

, Long-Sen Chang

a,

*

a

Institute of Biomedical Sciences, National Sun Yat-Sen University, Number 70, Lien-Hai Road, Kaohsiung 80424, Taiwan, ROC

b

Department of Kinesiology and Health, National University of Kaohsiung, Kaohsiung 700, Taiwan, ROC

c

Division of Urology, Kaohsiung Veterans General Hospital, Kaohsiung 813, Taiwan, ROC Received 22 August 2005; accepted 27 October 2005

Available online 13 December 2005

Abstract

Using B1 chain of b-bungarotoxin (b-Bgt) as bait in yeast two-hybrid screen, we found that KChIP3 was a binding protein of B1 chain. Thus, protein–protein interaction between b-Bgt and KChIP3 is investigated in the present study. Pull-down assay showed that recombinant KChIP3 proteins were associated with b-Bgt as well as B1 chain, whereas the inability of KChIPs 1, 2 and 4 to bind with b-Bgt was observed. Although Ca2C_{was not a crucial factor essential for the binding of KChIP3 with b-Bgt}

and B1 chain, their interaction could be enhanced by the addition of Ca2C_{. Alternatively, the association of A1 chain of b-Bgt}

with KChIP3 was marginally detected. The dissociation constant of b-Bgt with KChIP3 were 12.2 and 6.08 mM in the absence and presence of 2 mM Ca2C, respectively. Moreover, native KChIP3 from rat brain was to be isolated by b-Bgt-Sepharose. These observations indicate that KChIP3 is a binding protein of b-Bgt. In view of the multiple functions of KChIP3 in neuronal cells, the interaction of KChIP3 with b-Bgt may represent an event for the manifestation of the biological activities of b-Bgt. q2005 Elsevier Ltd. All rights reserved.

Keywords: b-Bungarotoxin; KChIP3; Recombinant A and B chains; Protein–protein interaction

1. Introduction

b-Bungarotoxin (b-Bgt), the main presynaptic phospho-lipase A2 (PLA2) neurotoxin purified from the venom of

Bungarus multicinctus (Taiwan banded krait), consists of two dissimilar polypeptide chains, the A chain (w14 kDa) and B chain (w7 kDa), cross-linked by an interchain disulfide bond (Kondo et al., 1982a,b). Their A chains are structurally homologous to PLA2, while B chains share

similarity with trypsin inhibitor, toxin I and dendrotoxin (Kwong et al., 1995). Although chemical modification studies revealed that the A chain is an active subunit

responsible for the phospholipase activity and neurotoxic effect of b-Bgt (Yang and Lee, 1986; Chang and Yang, 1988), the B chain showed activity in blocking voltage-gated KC

channel (Benishin, 1990; Wu et al., 1998) Herkert et al. (2001)have recently reported that b-Bgt induced apoptosis of cultured neuronal cells through a KC

-channel-mediated internalization. It may be possible that b-Bgt may interact with intracellular proteins as those reported for snake presynaptic PLA2 neurotoxins such as taipoxin, crotoxin

and ammodytoxins (Dodds et al., 1997; Hseu et al., 1999; Sribar et al., 2001; 2003). In order to address this suggestion, probing the b-Bgt-binding proteins in rat brain was carried out using B1 chain as bait in yeast two-hybrid screen. The positive clones identified thus were subjected to nucleotide sequence analysis. By homology searches of established nucleotide databases, our results revealed that rat KChIP3 was a putative binding protein of B1 chain. The yeast cells

* Corresponding author. Tel.: C886 7 525 5813; fax: C886 7 525 0197.

E-mail address: [email protected] (L.-S. Chang).

1

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co-transformed with pAS2-1-B1 chain and pACT2-rat KChIP3 grew on plate lacking Leu, Trp, and His (Fig. 1), suggesting that B chain interacted with rat KChIP3.

KChIP3, also known as calsenilin and DREAM appears to possess multiple functions in different cell compartments of cell by regulating completely different target molecules, such as potassium channel on cell membrane, presenilin in endoplasmic reticulum and DRE sequence of DNA in the cell nucleus (An et al., 2000; Carrion et al., 1999; Buxbaum et al., 1998; Ledo et al., 2000; 2002). Moreover, KChIP3 exhibits a pro-apoptotic function, and causes the cells to be more susceptible to apoptotic triggers (Jo et al., 2001; 2003; 2004; Lilliehook et al., 2002). To date, four KChIP families including KChIP1, KChIP2, KChIP3 and KChIP4 have been identified (An et al., 2000; Ohya et al., 2001; Morohashi et al., 2002), but their physiological functions are not exclusively identical (Holmqvist et al., 2002; Shibata et al., 2003). All KChIPs share a high degree of sequence homology at their C-terminal region, which is made up of four EF-hands, but their N-terminus sequence is notably diversified. The present study is conducted to further demonstrate that b-Bgt interacts specifically with KChIP3.

2.1. Yeast two-hybrid screening

Yeast two-hybrid screens were carried out according to a standard protocol (Clontech). Briefly, B1 chain was cloned in frame with Gal4 DNA binding domain in the pAS2-1 vector (MATCHMAKER Two-Hybrid system, Clontech) to yield pAS2-1-B1 chain bait plasmid. A rat brain cDNA library was screened by co-transforming yeast YRG-2 (sratagene) with pAS2-1-B1 chain bait plasmid and rat brain library plasmid DNA (Clontech). The positive clones have the ability to grown on Trp, Leu and His dropout media

supplemented with 3-aminotriazole (3-AT, an inhibitor of HIS3) and turn blue in b-galactosidase filter assay. 2.2. Preparation of b-Bgt, recombinant A1 chain and recombinant B1 chain

b-Bgt was isolated from Bungarus multicinctus (Taiwan banded krait) venom according to the method described in

Liou et al. (2004). Recombinant A1 and B1 chains of b-Bgt were prepared essentially according to the procedure described byWu and Chang (2001).

2.3. Cloning of human and rat KChIP3

The coding sequences of human KChIP3 (NM_013434) were amplified by PCR from human brain Marathon ready cDNA library (Clontech) as described inLin et al. (2004a). Rat KChIP3 (AF297118) was amplified by PCR from rat brain Marathon ready cDNA library (Clontech). The primer rK3-down (50-CTAGATGACGTTCTCAAACAGCTGC-30) was designed from the C-terminus of rat KChIP3 (rKChIP3). The primer AP-1 was provided by the same kit. The conditions for the PCR reactions were essentially identical to those described in the manufacturer’s protocol. The resulting products were cloned into pGEM-T easy vector using the TA-cloning procedure (Promega), and the cDNA encoding rKChIP3 was confirmed by DNA sequencing.

2.4. Preparation of recombinant KChIP proteins

The restriction enzyme-cutting sites NcoI and EcoRI were introduced into 50- and 30-ends immediate with the first amino acid and termination codon of rKChIP3, respectively. The restriction enzyme-cutting sites NdeI and XhoI were introduced into 50- and 30-ends immediate with the first amino acid and the C-terminus of human KChIP3, respectively. The resulting human KChIP3 and rKChIP3 cDNAs were ligated into the large fragments of NdeI/XhoI-cut pET22b(C) and NcoI/EcoRI-NdeI/XhoI-cut pET30a(C), respect-ively. The recombinant human KChIP3 (designated as KChIP3) has an extra six-His residue at its C-terminus. Recombinant KChIP3 proteins were expressed and purified according to the method described inLin et al. (2004a,b).

Recombinant human KChIPs 1, 2 and 4 were prepared essentially according to the procedure previously described (Chang et al., 2003; Lin et al., 2004a,b).

2.5. Pull-down assay

Recombinant KChIPs in 0.05 M Tris–HCl (pH 7.5) containing 0.05 M NaCl were incubated with 100 ml of 50% b-Bgt-Sepharose beads or B1 chain-Sepharose beads at room temperature for 2 h in the absence or presence of 10 mM Ca2C. Alternatively, b-Bgt, A1 chain and B1 chain were incubated with 100 ml of 50% KChIP3-Sepharose beads in the presence of 10 mM Ca2C. The protein-bead

Fig. 1. The interaction of B1 chain with rat KChIP3 in yeast two-hybrid system. Media lacking Leu, Trp and His show the interaction between the bait (B1(C55S) chain) and the interaction protein (rat KChIP3). 1, pAS2-1-B1 chain and pACT2-rat KChIP3 was co-transformed into yeast YRG-2; 2, pAS2-1-B1 chain was transformed into yeast YRG-2; 3, pACT2-rat KChIP3 was transformed into yeast YRG-2; 4, pAS2-1-B1 chain and pACT2 was co-transformed into yeast YRG-2.

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complexes were washed three times with 1 ml of binding buffer (0.05 M Tris–HCl, 0.05 M NaCl, pH 7.5, with or without 10 mM Ca2C), and the bound proteins were eluted with 100 ml of elution buffer (0.05 M Tris–HCl, 1 M NaCl, 10 mM EGTA, pH 7.5). Then 10 ml of eluted samples were subjected to SDS-PAGE and immunoblotting analyses.

2.6. Isolation of b-Bgt-binding protein from rat brain protein extracts

Female Wistar rats (about 150 g) were decapitated and exsanguinated. The following procedures were carried out at 4 8C. The brain was homogenized in 0.05 M Tris–HCl (pH 7.5) containing 0.05 M NaCl and 10 mM CaCl2. The

homogenate was centrifuged at 13,000g for 30 min, and ammonium sulfate (34.3 g/100 ml) was added to the supernatant to precipitate the brain proteins. The precipitate was collected by centrifugation and dialyzed against 4 l of 0.05 M Tris–HCl (pH 7.5) containing 0.05 M NaCl and 10 mM CaCl2. The preparation was applied to a column of

b-Bgt-Sepharose (7 ml bed volume) equilibrated with the same buffer. The column was washed with 50 ml 0.05 M Tris–HCl (pH 7.5) containing 0.05 M NaCl and 10 mM CaCl2, and the bound proteins were eluted with elution

buffer (0.05 M Tris–HCl, 1M NaCl, 10 mM EGTA, pH 7.5). The eluted proteins were subjected to SDS-PAGE and immunoblotting analyses, and rKChIP3 was visualized with anti-KChIP3 antibodies (Santa Cruz Biotechnology).

2.7. Surface plasma resonance (SPR) experiment

Association between b-Bgt and KChIP3 were measured with a BIAcore X instrument (BIAcore AB, Uppsala, Sweden). KChIP3 was immobilized on an NTA sensor chip. The binding assay was performed with a constant flow rate of 5 ml/min at 25 8C with b-Bgt concentrations in the range of 25–2500 nM. b-Bgt was dissolved in 10 mM Hepes (pH 7.4) containing 150 mM NaCl and 0.005% surfactant P20. The experiments for assessing the effect of Ca2C on the interaction between KChIP3 and b-Bgt were carried out by adding 2 mM Ca2C in Hepes buffer. The chip was regenerated with the 10 mM Hepes (pH 8.3) containing 150 mM NaCl, 0.35 M EDTA and 0.005% surfactant P20. For fitting of the binding kinetics, BIAevaluation version 3.0 software (BIAcore AB) was applied, and the 1:1 Langmuir binding model was chosen.

2.8. Other tests

Fluorescence measurement, CD measurement, SDS-PAGE analyses, immunoblotting, covalent coupling of proteins to CNBr-activated Sepharose 4B were carried out essentially in the same manner as previously described (Chang et al., 2003; Lin et al., 2004a,b).

3. Results and discussion

As shown in Fig. 2, the secondary structure of recombinant rKChIP3 and KChIP3 indicates a typical pattern of a-helical EF-hands containing negative peaks at 222 and 208 nm, respectively. The negative peaks in the CD spectra of rKChIP3 and KChIP3 changed upon the addition of Ca2C, suggesting that the Ca2C-binding caused an alteration in the gross structure of KChIP proteins. To further determine the binding affinity of KChIP3 with Ca2C, titration of KChIP3-8-anilinonaphthalene-1-sulfonate (ANS) complex with Ca2C was carried out according to the procedure described byLin et al. (2004b). As illustrated inFig. 3, the emission intensity of KChIP3-ANS complex increased in parallel with increasing concentration of Ca2C until the saturation level was reached. The Ca2C-binding affinity of KChIP3 proteins was calculated from the change in intensity of ANS fluorescence. It revealed that KChIP3 possessed two types of Ca2C-binding sites, namely high-affinity (Kd1Z0.014 mM) and low-affinity (Kd2Z

0.762 mM) binding sites. Likewise, the Ca2C-binding constants Kd1 and Kd2 of rKChIP3 were 0.015 and

1.205 mM, respectively. These results indicated that the EF-hands of recombinant rKChIP3 and KChIP3 bound functionally with Ca2C.

As shown inFig. 4A and B, the binding of rKChIP3 and KChIP3 to b-Bgt-Sepharose increased with increasing KChIP3 concentration. Although KChIP3 proteins inter-acted with b-Bgt in the absence of Ca2C, their interaction was enhanced by the addition of Ca2C. Similar results were also noted with the binding of rKChIP3 and KChIP3 to B1 chain-Sepharose (Fig. 4A and B). In view of the fact that the B chain of b-Bgt itself did not exhibit a Ca2C-binding ability (Chang and Yang, 1993), the enhanced effect of Ca2Con the interaction of KChIP3 proteins with b-Bgt and B1 chain should arise from a subtle conformational change of KChIP3 proteins being induced by Ca2C. Alternatively, pull-down assay using KChIP3-Sepharose indicated that, in

Fig. 2. CD spectra of KChIP3 and rKChIP3. The CD spectra of proteins were measured in the absence or presence of 10 mM Ca2C.

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contrast to b-Bgt and B1 chain, A1 chain showed a marginally detectable affinity for binding with KChIP3-Sepharose in the presence of Ca2C(data not shown). These reflected that both subunits in b-Bgt molecule could bind with KChIP3, but the interaction of b-Bgt with KChIP3 may depend more heavily on B chain than on A chain. To demonstrate that b-Bgt was associated with native KChIP3 as well, the rat brain proteins partially purified by ammonium sulfate precipitation were applied through a column of b-Bgt-Sepharose. As shown inFig. 4C,

b-Bgt-binding protein of rat brain showed an immunoreactivity toward anti-KChIP3 antibodies, and had a molecular size similar to that of rKChIP3. Moreover, in sharp contrast to KChIP3, human KChIPs 1, 2 and 4 were not pulled down by b-Bgt-Sepharose at all (Fig. 5). These findings emphasized the notion of a specific interaction between b-Bgt and KChIP3.

To determine the dissociation constant of b-Bgt with KChIP3, surface plasma measurements were carried out. KChIP3 was coupled to an NTA sensor chip with the His-Tag at its C-terminus. Binding was observed upon injection of different concentrations of b-Bgt either in the absence or presence of 2 mM Ca2C. As shown in Fig. 6, Ca2C enhanced the interaction of KChIP3 with b-Bgt. The dissociation constant of 12.2 and 6.08 mM was obtained in the absence and presence of Ca2C, respectively.

In the present study, our data reveal that b-Bgt interacts specifically with KChIP3 and rKChIP3. Sequence alignment shows that rKChIP3 and KChIP3 share up to 95% of their sequence identity. However, amino acid substitutions noted with rKChIP3 and KChIP3 occur mostly in the N-terminus. It is evident that the N-terminus of rKChIP3 and KChIP3 should not be directly involved in the binding with b-Bgt. In fact, deleting the N-terminus of KChIP3 did not appreciably affect the binding of KChIP3 with b-Bgt (data not shown). According to the finding that A1 and B1 chains could bind with b-Bgt, the KChIP3 binding region within the b-Bgt molecule should consist of both A and B chains. EF-hand proteins including TCBP-49, crocalbin and calmodulin also have also been reported to be physiological targets of snake presynaptic PLA2neurotoxins, i.e. taipoxin,

crotoxin and ammodytoxins (Dodds et al., 1997; Hseu et al., 1999; Sribar et al., 2001). The findings that different PLA2

neurotoxins interact with different EF-hand proteins are in line with the proposition that different PLA2 neurotoxins

exert different pharmacological effects because they recognize different acceptors (Bon, 1997). Nevertheless,

Fig. 4. The binding of rKChIP3 and KChIP3 to b-Bgt-Sepharose and B1 chain-Sepharose in the absence or presence of Ca2C_.

Different concentrations (Lanes 1, 3, 5 and 7, 70 mM; Lanes 2, 4, 6 and 8, 140 mM) of (A) rKChIP3 and (B) KChIP3 were incubated with b-Bgt-Sepharose or B1 chain-Sepharose, respectively. (C) Rat brain protein that bound with b-Bgt-Sepharose was analyzed by immunoblotting. Recombinant human KChIP3 has a molecular size similar to native rKChIP3. Lane 1, recombinant human KChIP3; Lanes 2 and 3 were the proteins eluted from the b-Bgt-Sepharose column. The amount of protein loading in Lane 3 was two-fold that in Lane 2.

Fig. 3. Fluorescence emission spectra of KChIP3-ANS complex in the presence of various concentrations of Ca2C. The sample cuvettes contained 7.5 mM KChIP3 per milliliter of 0.025 M Tris– 0.1 M NaCl (pH 8.0) and 7.5 mM ANS in the presence of various concentrations of Ca2Cas indicated.

Fig. 5. Pull-down of human KChIP proteins using b-Bgt-Sepharose. The four lanes on the left show that the KChIP protein is able to bind with b-Bgt. M, Molecular markers (Amersham Biosciences, phosphorylase b, 90 kDa; albumin, 66 kDa; ovalbumin, 45 kDa; carbonic anhydrase, 30 kDa; trypsin inhibitor, 20.1 kDa; a-lactalbumin, 14 kDa). The four lanes on the right are recombinant KChIPs 1, 2, 3 and 4, respectively.

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the functional involvement of these EF-hand proteins in manifesting the biological activities of presynaptic PLA2

neurotoxins still remained to be resolved. Recent results (Sribar et al., 2003) showed that ammodytoxins could associate with 14-3-3 protein g and 3 isoforms in addition to calmodulin. This implies that the pharmacological activities noted with presynaptic PLA2 neurotoxins may not be

attributed merely to an interaction with EF-hand proteins. In addition to affect presynaptic nerve terminal function of the neuromuscular junction, b-Bgt had been reported to induce widespread neuronal cell death throughout the mammalian and avian CNS (Hanley and Emson, 1979; Ciutat et al., 1996). Moreover, electrophysiological analysis in the central nervous system revealed that b-Bgt had a action on reducing transmitter release and neuronal excitability in rat hippocampal cells (Halliwell and Dolly, 1982a,b). Recent studies (Herkert et al., 2001; Shakhman et al., 2003) suggested that b-Bgt acted as an extremely potent inducer of neuronal apoptosis when applied to rat hippocampal cultures. Noticeably, biochemical and immunochemical characterization of KChIP3 in mouse brain indicated that this protein was predominantly expressed in the cerebellum and hippocampus (Zaidi et al., 2002). Together with the findings that KChIP3 exhibits a pro-apoptotic function (Jo et al., 2001; 2003; 2004; Lilliehook et al., 2002) and b-Bgt induces apoptosis of cultured neuronal cells through a KC

-channel-mediated internalization (Herkert et al., 2001), the involvement of KChIP3 in b-Bgt-induced apoptotic cell death (Herkert et al., 2001) seems to be possible.

Acknowledgements

This work was supported by grant NSC94-2320-B110-002 from the National Science Council, ROC (to L.S. Chang) and grant VGHNSU94-004 from Kaohsiung Veterans General Hospital.

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Divergence of genes encoding B chains of b-bungarotoxins

Yun-Ching Cheng, Ku-Chung Chen, Shu-Kai Lin, Long-Sen Chang *

Institute of Biomedical Sciences, National Sun Yat-Sen University, Kaohsiung 804, Taiwan, ROC

Received 22 August 2005; accepted 8 November 2005 Available online 2 February 2006

Abstract

The structural organization of the genes encoding B2, B4, B5 and B6 chains of b-bungarotoxins are reported in this study. These genes shared virtually identical overall organization with three exons interrupted by two introns in similar positions. On the contrary, intron 1 of these genes had a similar size, a notable variation with the size of intron 2 was observed. It was found that two regions at the second intron of B1 and B2 chains were absent in that of B4, B5 and B6 chains. RT-PCR analyses indicated that Bungarus multicinctus venom gland, heart, liver and muscle expressed the RNA transcripts showing sequence similarity with the intronic segment being deleted in B4, B5 and B6 chain genes. This reflects that the ancestral gene of the intronic segment might insert in multiple loci of B. multicinctus genome. Comparative analyses of B chain genes showed that the protein-coding regions of the exons are more diverse than introns, except for in the signal peptide domain. These results suggest that intron insertions or deletions occur with the evolution of B chains, and that accelerated evolution may diversify the protein-coding sequence of B chain genes same as snake phospholipase A2, neurotoxin and cardiotoxin genes.

Keywords: b-Bungarotoxin; B chain; Genomic structure; Intron insertion or deletion; Intronic RNA transcripts. Evolutionary divergence

1. Introduction

b-Bungarotoxin (b-Bgt), the main presynaptic phospho-lipase A2 (PLA2) neurotoxin purified from the venom of

Bungarus multicinctus (Taiwan banded krait), consists of two dissimilar polypeptide chains, the A chain (w14 kDa) and B chain (w7 kDa), cross-linked by an interchain disulfide bond (Abe et al., 1977; Kondo et al., 1982a,b). Their A chains are structurally homologous to PLA2, and

the B chains share similarities with the trypsin inhibitor, toxin I and dendrotoxin (Kwong et al., 1995). Eight A chain cDNAs and three B chain cDNAs have been cloned from B. multicinctus venom glands (Wu et al., 1998; Wu and Chang, 2001). Random combination of the A and the B chains should produce a number of b-Bgt isotoxins. Modern chromatographic techniques and amino acid analysis have revealed that there are at least 16 isoforms

of b-Bgt (Chu et al., 1995). Previous studies indicate that A and B chains are encoded separately by different genes, and the A and B chain genes do not originate from a common ancestor (Wu and Chang, 2000). These findings suggest that the intact b-Bgt molecules should be derived from the pairing of A and B chains after their mRNAs are translated. Although A chains and snake PLA2 enzymes share a

common origin, genetic divergence including sequence variations in protein-coding regions, exon-intron organiz-ation and the size of intron is noted with A chain genes and PLA2 genes (Chu and Chang, 2002). Moreover, it was

found that the skipping of exon changes the signal peptide sequences of A chains (Chu and Chang, 2002). Alter-natively, limited studies on the evolutionary divergence of B chain gene and its homologous have been reported. Recent results (Cheng et al., 2005) have shown that Naja naja atra chymotrypsin inhibitor (NACI) gene shares virtually an identical structural organization and high degree of sequence identity with B1 chain gene, suggesting that Kunitz/BPTI protease inhibitors and B chains may

* Corresponding author. Fax: C886 7 5250197.

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have originated from a common ancestor. However, comparative analyses of NACI gene and B chain gene reveal that an approximate 1.8 Kb long is absent from the intron 2 of NACI gene. Whether the intron insertions or deletions also occurred with the evolution of B chain genes or this represented an event for differentiating the evolutionary divergence between B chain genes and Kunitz/BPTI protease inhibitor genes still remained to be elucidated. To address this question, cloning of B chain genes is undertaken in the present study.

2.1. PCR amplification and cloning of B chain precursors Bungarus multicinctus liver was ground to a fine powder in liquid nitrogen. The genomic DNA was extracted from the powder according to the procedure previously described (Chu and Chang, 2002). Two oligonucleotide primers based on the promoter region and 30-noncoding region of the b-Bgt B1 chain gene (Wu and Chang, 2000) with the forward sequence 50 -AACCCCAATAGCAGACAGTGAACAGG-30 (P1) and the reverse one 50-TGGTCCAGGGCAAGAAGCAG GGTC-30 (P2) were synthesized. Moreover, a sense primer with the sequence 50-ATGTC TTCTGGAGGTCT TCTTCTCC-30 (P3) was designed from the signal peptide of B1 chain cDNA (Wu et al., 1998). The PCR products were amplified by primer combinations P1 and P2 and P3 and P2, respectively. PCR reaction was carried out using Advantage genomic PCR kit (Clontech Laboratories Inc.), and the procedure was essentially conducted according to the manufacturer’s protocol. The resulting products were cloned into pGEM-T easy vector using the TA-cloning procedure (Promega).

2.2. Analyses of RNA transcripts derived from intron 2 of B1 chain gene

Cellular RNA was isolated from the tissues of Bungarus multicinctus including venom glands, heart, liver and muscle by a guanidinium isothiocyanate/phenol/chloroform isolation method as previously described (Chang et al., 1998). The sense and antisense primers with sequences 50 -CCAGCCGAAATCCCATCAAGTACA-30 and 50-TTC CGGCGCGCAGTAAAGACCC-30 were synthesized from the second intronic sequence of B1 chain gene (Wu and Chang, 2000) at positions 4432–4455 and 4563–4584, respectively. RT-PCR reaction was carried out essentially according to the procedure described byChang et al. (1998). The products were cloned into pGEM-T easy vector (Promega) for DNA sequencing.

2.3. Comparative analysis of nucleotide sequence and homology search

A software package (PC/GENE program, Stratagene Ltd., USA) was used for sequence alignment according to percent sequence identity for comparing and analyzing the determined nucleotide sequences,. BLAST searches against non-redundant databases were carried out via the Internet using the software at the Website (http://www.ncbi.nlm.nih. gov/). The program used to search the putative regulatory elements was TFSEARCH, which is freely available at the website http://www.cbrc.jp/research/db/TFSEARCH.html. The number of nucleotide substitutions per sie (Kn) in the non-coding regions (introns), and the numbers of nucleotide substitutions per synonymous site (Ks) and per nonsynon-ymous (Ka) in the protein-coding regions were computed for pairs of B chain genes according to the method of

Comeron (1999).

2.4. Isolation and purification of b-Bgt isotoxins

Crude Bungarus multicinctus (Taiwan banded krait) venom was separated into 23 fractions on a SP-Sephadex C-25 column (2.5 cm!95 cm) as previously described (Chang et al., 2002). The fractions were further purified by reverse phase HPLC on a SynChropak RP-P column (4.6 mm!25 cm) equilibrated with 0.1% trifluoroacetic acid (TFA) and eluted with a linear gradient of 7.5–75% acetonitrile for 50 min. The flow rate was 0.8 ml/min and the effluent was monitored at 280 nm.

Fig. 1. Identification and size determination of PCR-amplified genomic DNAs. Electrophoresis was carried out in 1% agarose gel. Lane 1, DNA markers; Lane 2: PCR products amplified from Bungarus multicinctus genomic DNAs.

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2.5. Other tests

Mass analysis, reduction and S-carboxymethylation of proteins and SDS-PAGE were performed in essentially the same manner as previously described (Chang et al., 2005). 3. Results and discussion

As shown in Fig. 1, two PCR products with the size of approximately 3.2 and 4.6 kb were amplified from

B. multicinctus genome using primers P1 and P2. The DNA fragments were then subcloned with a TA cloning kit, and more than 20 clones were selected for nucleotide sequencing. Alignment with the genomic sequence with B chain cDNAs allowed us to identify unambiguously the exon-intron boundaries. In addition to B1 chain gene, two genomic DNAs with the length of 4699 and 3243 bp encoding B2 chain and B4 chain precursors, respectively, were identified. B1 chain, B2 chain and B4 chain precursors all composed of three exons and two introns. In the

Table 1

Comparison of structural organization of B chain and NACI genes Exon No. Exon length (bp) 50-Splice donor

sequence

Intron no. Intron length (bp)

30-Splice acceptor sequence

B1 chain 1 85 TCATCgtgag 1 570 cacagGGGAT

2 162 TCTTGgtaag 2 3128 tttagTGTAT

3 150

B2 chain 1 85 TCATCgtgag 1 578 cacagCGGAT

2 162 TCTTGgtaag 2 3096 tttagAGTAT

3 81

B4 chain 1 85 TCATCgtgag 1 580 cacagCGGAT

3 84

B5 chain 1 85 TCATCgtgag 1 578 cacagCATAT

2 162 TCTTGgtaag 2 1242 tacagGATGA

3 79

B6 chain 1 79 TCATCgtgag 1 571 cacagCATAT

3 82

NACI 1 79 TCATCgtgag 1 572 cacagGGTTC

2 162 TGTTGgtaag 2 1275 tacagGATGA

3 72

The first exon was temporarily assigned to start at the first arnino of signal peptide region.

Fig. 2. Amino acid sequence alignments of B chains. The arrows indicated the positions of protein-coding regions interrupted by introns. Amino acid sequence of B1 (accession no. Y12100), B 2 (accession no. Y12101) and B3 (accession no. AJ242991) chains was deduced from cDNAs, and that of B4, B5 and B6 chains was deduced from the protein-coding regions of genomic DNAs. The conserved cysteine residues are indicated by gray boxes.

蛇毒Serine Protease Inhibitor及其Paralogues功能性基因體之研究(III)Functional Genomic of Snake Venom Serine Protease Inhibitor and Its Paralogues (III)

行政院國家科學委員會專題研究計畫 成果報告