From the
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
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
We have previously reported that a 48-amino-acid peptide
termed
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
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,
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
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
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
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
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.
We thank Drs. Kiichiro Nakajima, and Kumiko Y.
Kumagaye (Peptide Institute Inc.), and Fred S. Esch (Eisai London
Research Laboratories) for the helpful discussions.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
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.
-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.
-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.
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.
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.
-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.
-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.
-Cys
, Cys
-Cys
,
and Cys
-Cys
, and one interchain disulfide
bond, Cys
-Cys
, that connects the two chains,
as shown in Fig. 2B.
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.
, 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) .
-Aga-TK,
-agatoxin-TK; HPLC, high
performance liquid chromatography; MALD, matrix-assisted laser
desorption.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.