©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Isolation and Characterization of a Peptide Isomerase from Funnel Web Spider Venom (*)

Yasushi Shikata , Tomohiro Watanabe , Tetsuyuki Teramoto , Atsushi Inoue , Yoshiyuki Kawakami , Yukio Nishizawa , Kouichi Katayama , Manabu Kuwada (§)

From the (1)Eisai Tsukuba Research Laboratories, Ibaraki 300-26, Japan

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

A novel peptide isomerase was purified from the venom of funnel web spider, Agelenopsis aperta. The complete primary structure of the isomerase has been established by sequence analyses of polypeptide chains, assignments of disulfide bridges, carbohydrate analyses, and mass spectrometry of sugar chains. The isomerase was found to be a 29-kDa polypeptide that consists of an 18-residue light chain and a 243-residue heavy chain connected by a single disulfide bridge. The heavy chain contains three intramolecular disulfide bridges and one N-linked oligosaccharide chain with a simple trimannosyl core structure. A sequence homology search showed a significant similarity of the enzyme with serine proteases, particularly around a putative catalytic triad of the isomerase. The isomerase specifically interconverts the configuration of Ser of a 48-amino-acid peptide, -agatoxin-TK, and the conversion rate from L-Ser to D-Ser was approximately two times faster than the reverse reaction.


INTRODUCTION

Biological systems depend on specific molecular recognition involving strict differentiation between the chiral forms of polypeptides. Translation machinery in protein synthesis has evolved to utilize only one of the chiral forms of amino acids, the L-form(1) . However, various peptides containing D-amino acids have been isolated from multicellular organisms, including frog skin dermorphins, deltorphins, a peptide that interacts with an adenosine receptor, and peptides isolated from an African giant snail(2) . Furthermore, proteins containing D-amino acids have also been found in aged human tissues, such as eye lens crystallins, myelin basic protein, erythrocyte proteins, and -amyloid peptides from Alzheimer disease brains(3) . It is therefore very important to elucidate the mechanism underlying the presence of D-amino acid residues in these peptides and proteins.

We have previously reported that a 48-amino-acid peptide termed -agatoxin-TK (-Aga-TK),()which was isolated from the venom of the funnel web spider, Agelenopsis aperta, contains a D-serine residue at position 46(4, 5) . -Aga-TK is a selective and potent blocker of P-type calcium channels in cerebellar Purkinje neurons and inhibits the release of amino acid neurotransmitters and monoamines from hippocampal and striatal slices (5, 6). Adams et al.(7) and Heck et al.(8, 9) also found this peptide and designated it -agatoxin-IVB. NMR analysis of -agatoxin-IVB has indicated that the peptide has a three-stranded antiparallel -sheet formed by four disulfide bonds (residues 4-38) and a flexible random coil tail consisting of the 10 carboxyl-terminal amino acids (residues 39-48)(7, 10) . Structure-function relationship studies of -Aga-TK indicated that a specific conformation of the carboxyl-terminal region, particularly the D-configuration of Ser, together with the -sheet structure may be essential for blockade of P-type calcium channels(5) .

-Aga-TK contains two serine residues at positions 28 and 46, of which only Ser is in the D-form. Recently, we have also found in the spider venom a related peptide with the same amino acid sequence and disulfide pairings as those of -Aga-TK except for the L-configuration of Ser, though this peptide is about six times less abundant than -Aga-TK in the venom(11) . These results raise the questions of why only Ser of the two serine residues is in the D-form and why the two -Aga-TKs containing opposite configuration at Ser are both present in the A. aperta venom. It seems likely that D-Ser may be formed from L-Ser in the nascent molecule of -Aga-TK as a post-translational modification, since it would be difficult to explain why only one serine residue of the two is translated in the D-configuration. Presumably the putative isomerase that converts L-Ser to D-Ser cannot convert L-Ser to D-Ser because of steric hindrance exerted by amino acids surrounding L-Ser. Heck et al.(8) have recently reported the presence in A. aperta venom of an isomerase that converts L-Ser to D-Ser residue in the -agatoxin-IVB molecule.

In the present study, we have purified from the venom of A. aperta and characterized a novel peptide isomerase, which specifically inverts the chirality at the Ser residue of -Aga-TK in a reversible manner. This is the first report to assign the structure of a peptide isomerase from an eukaryotic organism. We present here the complete primary structure of this isomerase including the polypeptide sequences of both chains, disulfide pairings, and glycosylation form. Sequence homologies to known serine proteases and enzymatic properties of the isomerase are also described and discussed.


MATERIALS AND METHODS

Isomerase Purification

Freeze-dried crude venom from the spider A. aperta was purchased from Spider Pharm (Feasterville, PA), and the venom was reconstituted in 20 mM EDTA to avoid proteolysis by metalloproteinases in the venom(8) . The crude venom was fractionated on a Pharmacia Superdex 75HR column (10 300 mm) equilibrated with phosphate-buffered saline, pH 7.0, at a flow rate of 0.4 ml/min.

Structure Determination

The light chain and heavy chain of the purified isomerase were separated by reversed-phase HPLC after reduction with dithiothreitol and S-carboxyamide methylation with iodoacetamide. The heavy chain was digested with lysyl endopeptidase (Wako Pure Chemical Industries) in 50 mM Tris-HCl buffer, pH 9.0, in the presence of 2 M urea at 37 °C. The heavy chain was also digested with Staphylococcus aureus V8 protease (Miles Scientific) and arginyl endopeptidase (Takara Shuzo) in 50 mM Tris-HCl buffer, pH 9.0, containing 2 M urea at 37 °C. The peptides generated with V8 protease were further digested with asparaginyl endopeptidase (Takara Shuzo) in 50 mM sodium acetate buffer, pH 5.0, containing 2 M urea at 37 °C. Chemical cleavages of Asn-Gly bonds and tryptophanyl bonds of the heavy chain were carried out as described by Steinman et al.(12) and Huang et al.(13) , respectively. For the analysis of disulfide pairings, intact isomerase was digested with modified trypsin (Promega) in 50 mM Tris-HCl buffer, pH 6.5, containing 2 M urea at 37 °C. All the peptides generated by these enzymatic digestions and chemical cleavages were purified by reversed-phase HPLC on a Vydac C18 column (4.6 150 mm) with a linear gradient of acetonitrile in 0.1% trifluoroacetic acid. Amino acid analysis was performed with a Beckman 6300 amino acid analyzer after hydrolysis with 6 N HCl according to the conventional method. Automated Edman degradation of the peptides was performed by Shimadzu PSQ-1 and PSQ-2 gas-phase protein sequencers equipped with on-line HPLC systems. Molecular mass was determined by a Kratos MALDI-II or a PerSeptive Voyager matrix-assisted laser desorption (MALD) mass spectrometer, and the accuracy of the mass spectrometric measurement was within 0.2%. Carbohydrate analysis of the intact isomerase and peptide fragments was performed by a Dionex DX-300 ion chromatograph system. Neutral and amino sugars were analyzed after hydrolysis of the glycopeptides with 2.5 N trifluoroacetic acid at 100 °C for 6 h, and neuraminic acid was determined after hydrolysis in 0.01 N HCl at 80 °C for 1 h. Carbohydrate chains of glycopeptides were removed with N-glycanase (Boehringer Mannheim) according to the manufacturer's protocol. Protein data base search was performed using the Gene Bright program (Hitachi Software Engineering) and NBRF, PIR, and SWISS-PROT data bases.

Isomerase Activities

Synthetic -[d-Ser]Aga-TK and -[L-Ser]Aga-TK were prepared as described previously(5) . These -Aga-TKs were incubated with the purified isomerase at the substrate-to-enzyme molar ratio of 3:1 in phosphate-buffered saline, pH 7.0, containing 2 mM EDTA, at 37 °C. The stereochemical conversion of the Ser residue in -Aga-TKs was assessed by determination of the peptide containing either L- or D-Ser using reversed-phase HPLC, as described in detail in the legend to Fig. 4. Enantiomer analysis of D- and L-amino acids in the converted peptides was performed as described before(5) .


Figure 4: Stereochemical conversion of -[L-Ser]Aga-TK (A) and -[D-Ser]Aga-TK (B) by the peptide isomerase and time course of the reaction (C). A and B, L-TK and D-TK represent -[L-Ser]Aga-TK and -[D-Ser]Aga-TK, respectively. The -Aga-TKs were incubated with the isomerase at 37 °C for 4 days under the conditions described under ``Materials and Methods,'' and the reaction products were analyzed by reversed-phase HPLC. A Vydac C18 column (4.6 150 mm) was equilibrated with 25% acetonitrile in 0.1% trifluoroacetic acid, and the peptides were eluted with a linear gradient of 25-35% acetonitrile in 40 min at a flow rate of 1 ml/min. C, -[L-Ser]Aga-TK was incubated with the isomerase for 1, 4, 14, and 29 days under the same conditions as described above, and the conversion percentages of -[D-Ser]Aga-TK to total -Aga-TKs are plotted.




RESULTS AND DISCUSSION

The peptide isomerase was purified to homogeneity from the crude venom of A. aperta by a simple size-exclusion HPLC procedure as shown in Fig. 1. The isomerase activity was found in fraction 4, and the homogeneity of the enzyme was confirmed by the observation of a single band upon SDS-polyacrylamide gel electrophoresis and a single molecular ion of 29,418 Da upon MALD mass spectrometry. Purification of the enzyme from 500 µl of the crude venom yielded 50 nmol of isomerase. Under the purification conditions used, the metalloprotease activity was detected in fraction 2, and P-type calcium channel blockers, -Aga-TK and -Aga-IVA, were co-eluted in fraction 7.


Figure 1: Purification of the peptide isomerase from A. aperta venom. Fifty microliters of the crude venom was fractionated by Superdex 75HR size-exclusion chromatography as described under ``Materials and Methods.'' The isomerase activity was found in peak 4, shown in black. The molecular masses indicated were determined by using a Pharmacia molecular weight calibration kit.



An amino-terminal sequence analysis of the purified peptide isomerase yielded two amino acid sequences with approximately equal recovery, suggesting that the isomerase consisted of two polypeptide chains connected by a disulfide bond(s). This was confirmed by the finding that reduction of the isomerase with 2-mercaptoethanol resulted in a decrease of molecular mass determined by SDS-polyacrylamide gel electrophoresis. The two polypeptide chains were separated by reversed-phase HPLC after reduction and carboxyamide methylation of the enzyme. A direct Edman degradation of the light chain demonstrated that the chain consisted of 18 amino acid residues including one cysteine, as depicted in Fig. 2A, and the sequence was in good agreement with its molecular mass of 1982 Da (S-alkylated form) determined by MALD mass spectrometry.


Figure 2: The peptide alignment used for the determination of the sequence of the peptide isomerase (A) and a diagram of the primary structure of the isomerase (B). A, thick black lines indicate the residues identified by Edman degradation, and dashed lines indicate the sequence that was not identified. The designations K, E, R, N, and NG represent peptides generated with: K, lysyl endopeptidase; E, S. aureus V8 protease; R, arginyl endopeptidase; N, asparaginyl endopeptidase; NG, Asn-Gly bond cleavage; W, tryptophanyl bond cleavage. Peptides are numbered in order of elution from the HPLC column. B, residue numbers of the light chain are denoted as numbers with apostrophes to differentiate them from those of the heavy chain. Disulfide bonds are shown by broken lines.



The amino acid sequence of the heavy chain was determined by a combination of amino-terminal sequence analysis of heavy chain and Edman degradation and amino acid composition analysis of the peptide fragments generated with lysyl endopeptidase (K-peptides), S. aureus V8 protease (E-peptides), arginyl endopeptidase (R-peptides), and asparaginyl endopeptidase (N-peptides), and by chemical cleavages of Asn-Gly bonds (NG-peptides) and tryptophanyl bonds (W-peptides), as summarized in Fig. 2A. These peptides are numbered in order of elution from the HPLC column. The amino-terminal sequence analysis allowed the assignment of residues 1-39. Most of the sequences of the heavy chain were obtained by analysis of 19 non-overlapping K-peptides generated with lysyl endopeptidase. Automated Edman degradation of each K-peptide provided complete amino acid sequence through the carboxyl-terminal residue, and the sequence was confirmed by amino acid composition analysis of the peptide. The remainder of the sequence of the heavy chain and overlaps of the K-peptides were obtained by Edman degradation of six E-peptides, two EN-peptides, two NG-peptides, one R-peptide, and one W-peptide. Sequence analysis of peptide E6 and its subpeptide, E6N4, provided overlaps of amino-terminal K-peptides, K2, K12, K18, K19, and K13. Analysis of peptide R2 provided the overlap of K13 and K7, and analysis of peptide E4 and its subpeptide, E4N3, provided additional sequences and overlaps of K7, K6, K8, K10, K11, K9, K1, K14, and K16. Analysis of peptides E3, E2, E5, and W4 provided additional sequences and overlaps of K14, K16, K15, and K17. Analysis of peptide NG2 provided overlaps of the carboxyl-terminal three K-peptides, K4, K5, and K3. Finally, the entire sequence of the heavy chain was verified by molecular mass determination of the six E-peptides by MALD mass spectrometry. These data revealed that the heavy chain consists of 243 amino acid residues and contains seven cysteines aligned as shown in Fig. 2A.

In these experiments, two pairs of heterogeneous peptides were found among the K-peptides of the heavy chain. The first pair was K10 and K11, and these peptides were clearly separated by reversed-phase HPLC, though they had identical amino acid compositions corresponding to residues 105-119. The Edman degradation of the two peptides indicated that K10 has the sequence Asn-Gly at residues 113-114, while the sequencing of K11 was blocked at residue 113. These data clearly demonstrated that K11 was a product of -rearrangement of the Asn-Gly bond (14), and the structural alteration had probably occurred during preparation of the peptide fragment. The second pair of heterogeneous peptides was K14 and K16, which were found to have identical amino acid compositions and amino acid sequences corresponding to residues 142-157. These peptides involved the Ile-Pro-Ile-Ile-Pro-Trp sequence, in which bulky amino acids are adjacent to the two proline residues. These data strongly suggested that K14 and K16 might be stereoisomers arising from cis-trans isomerization of the two proline residues (15). Further investigation is needed to determine whether or not the stereoisomers are present in the native isomerase molecule.

The disulfide pairings of the eight cysteines in the peptide isomerase were determined by amino acid analysis and Edman degradation of the cystine-containing peptides generated by limited proteolysis with trypsin. The amino acid analysis showed four tryptic peptides, each of which contained only one cystine residue. Edman degradation of these peptides demonstrated that the isomerase contains three intramolecular disulfide bridges in the heavy chain, namely Cys-Cys, Cys-Cys, and Cys-Cys, and one interchain disulfide bond, Cys-Cys, that connects the two chains, as shown in Fig. 2B.

The carbohydrate analysis of the peptide isomerase demonstrated that the heavy chain contained approximately 4% carbohydrates by weight, comprising mannose, N-acetylglucosamine, and fucose. This result suggested the presence of an N-linked oligosaccharide chain(s) in the heavy chain. Glycosylated residues were identified by the carbohydrate analysis of all Lys peptides generated by lysyl endopeptidase digestion, described above. The analysis indicated that only one peptide, namely K9, was glycosylated. Edman degradation of K9 resulted in failure to detect a phenylthiohydantoin-amino acid only at residue 127. After the deglycosylation of K9 with N-glycanase, the peptide showed an increased retention time on reversed-phase HPLC, and phenylthiohydantoin-Asp was identified at residue 127 by Edman degradation. These data clearly indicated that an oligosaccharide chain is covalently attached to Asn of the heavy chain and that the amino acid sequence at this site (Asn-Ala-Thr; residues 127-129) matches the consensus sequence of potential N-glycosylation sites. The carbohydrate composition of K9 was determined to be 3.0 mol of mannose, 2.2 mol of N-acetylglucosamine, and 0.9 mol of fucose/mol of the peptide. The molecular mass of the carbohydrate moiety of K9 was estimated to be 1041 Da by MALD mass spectrometry. Therefore, it seems reasonable to conclude that the oligosaccharide chain attached to Asn consists of a simple trimannosyl core structure, Man3-GlcNAc2-Fuc (calculated, 1038 Da), as shown in Fig. 2B. On the basis of these data, we have established the complete primary structure of the peptide isomerase, including the sequences of the two chains, the locations of the four disulfide pairings, and the structure of the single N-linked carbohydrate chain.

Although we could not find a known sequence homologous with that of the light chain of the peptide isomerase, the amino acid sequence of the heavy chain shows similarity with those of serine proteases from snake venoms, batroxobin from Bothrops moojeni(16) and flavoxobin from Trimeresurus flavoviridis(17) , and with those of mammalian serine proteases, human kallikrein (18) and bovine thrombin (19), as shown in Fig. 3. The entire heavy chain sequence shows 24, 26, 26, and 35% identity with batroxobin, flavoxobin, thrombin, and kallikrein, respectively. Interestingly, the homologies with the serine proteases are particularly marked at the regions around the putative catalytic triad of the isomerase, His, Asp, and Ser. These results suggested that the putative catalytic sites and/or the surrounding region in the isomerase molecule may play an important role in biological activity of the peptide isomerase.


Figure 3: Sequence homologies between peptide isomerase and several serine proteases. Arrowheads indicate the putative catalytic triad of the isomerase, which is predicted by the sequence similarities around the active site residues of the serine proteases. Alignments are made to maximize homology, and spaces indicate residues that are absent. Identical residues in these sequences are boxed.



Isomerase activity was evaluated in terms of the stereochemical conversion of Ser determined by reversed-phase HPLC analysis of the reaction products using -[L-Ser]Aga-TK and -[D-Ser]Aga-TK as substrates. The specificity of isomerase inversion was confirmed by enantiomer analysis of amino acids of the reaction products. As shown in Fig. 4, A and B, the isomerase specifically inverted both the L- and D-configuration of Ser of -Aga-TKs in a reversible manner. Under these conditions, 59% of L-Ser residue of -Aga-TK were converted to D-Ser within 4 days, but only 14% of D-Ser residue was changed to the L-form during the same period. A time course study of the inversion of -[L-Ser]Aga-TK showed that the reversible reaction reached an equilibrium after 14 days and products were found to be 71% of L-Ser-form and 29% of D-Ser-form of -Aga-TK after 29 days, as shown in Fig. 4C. Based on these data, the conversion rate of the L-Ser-form to the D-Ser-form was estimated approximately two times faster than that of the reverse reaction. Although the interconversion activity of the purified isomerase appeared to be very low, the concentrations of isomerase and -Aga-TK in these experiments, 9 and 28 µM, respectively, were significantly lower than the predicted concentrations of the two molecules in the A. aperta vemon, 100 and 760 µM, respectively. These results indicate that the isomerase plays a role in the maturation of -Aga-TK in the venom, since -[D-Ser]Aga-TK is an 80-90-fold more potent blocker of P-type calcium channels than is -[L-Ser]Aga-TK as described previously(5) .

Since several kinds of amino acid racemases isolated from bacteria contain a covalently bound pyridoxal phosphate(20) , we have investigated cofactor dependence of the purified isomerase. Spectrophotometric analysis showed that the isomerase lacks any significant absorbance at wavelengths higher than 300 nm. Addition of pyridoxal phosphate showed no effect on the isomerase activity. These results indicated that the isomerase neither contains pyridoxal phosphate nor requires this cofactor for its activity. Additionally, the isomerase activity is unaffected by the inclusion of 5 mM EDTA in assay mixtures. This suggests that the enzymic isomerization does not require metal ions, though the possibility of the involvement of tightly bound metal ions cannot be eliminated.

In conclusion, we have established the complete primary structure and characterized some unique properties of a peptide isomerase from A. aperta venom. This is the first report to describe the structure of a peptide isomerase from an eukaryotic organism. The isomerase can be distinguished from amino acid racemase from bacteria (20), since these bacterial enzymes convert the configuration only of free amino acids. Further studies on the enzymatic properties and three-dimensional structure of the isomerase should provide more definitive data as to its function.


FOOTNOTES

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

§
To whom correspondence should be addressed: Dept. of Physical and Analytical Chemistry, Eisai Tsukuba Research Laboratories, 5-1-3 Tokodai, Tsukuba-shi, Ibaraki 300-26, Japan. Tel.: 81-298-47-5652; Fax: 81-298-47-2037.

The abbreviations used are: -Aga-TK, -agatoxin-TK; HPLC, high performance liquid chromatography; MALD, matrix-assisted laser desorption.


ACKNOWLEDGEMENTS

We thank Drs. Kiichiro Nakajima, and Kumiko Y. Kumagaye (Peptide Institute Inc.), and Fred S. Esch (Eisai London Research Laboratories) for the helpful discussions.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.