(Received for publication, December 16, 1995; and in revised form, January 29, 1996)
From the
Jararaca GPIb-BP, a snake venom protein composed of and
subunits purified from Bothrops jararaca, binds to
platelet glycoprotein (GP)Ib and functions as a receptor blocker for
von Willebrand factor binding to GPIb (Fujimura, Y., Ikeda, Y., Miura,
S., Yoshida, E., Shima, H., Nishida, S., Suzuki, M., Titani, K.,
Taniuchi, Y., and Kawasaki, T.(1995) Thromb. Haemostasis 74,
743-750). We present here the entire 142- and 123-residue amino
acid sequences of the respective
and
subunits and also
demonstrate that the platelet GPIb-binding site resides on the
and not on the
subunit based on an enzyme-linked immunosorbent
assay using biotin-labeled jararaca GPIb-BP and competing ligands.
Sequences of the
and
subunits were determined by analysis
of the intact S-pyridylethylated proteins and their peptides
generated by digestion with Achromobacter protease I, Staphyloccocus aureus V8 protease, pepsin, endoproteinase
Asp-N, or L-1-tosylamino-2-phenylethyl chloromethyl
ketone-trypsin. A 38-39% identity of amino acid sequence between
the
and
subunits of jararaca GPIb-BP was observed, as well
as a high degree of sequence identities (38-64%) with the
respective subunits of botrocetin (Usami, Y., Fujimura, Y., Suzuki, M.,
Ozeki, Y., Nishio, K., Fukui, H., and Titani, K(1993) Proc. Natl.
Acad. Sci. U. S. A. 90, 928-932) and the
-chain of
echicetin (Peng, M., Holt, J. C., and Niewiarowski, S.(1994) Biochem. Biophys. Res. Commun. 205, 68-72).
The first step in primary hemostasis is thought to be platelet
adhesion to exposed subendothelium at the site of vascular injury. This
process appears to involve the initial binding of plasma vWF ()to subendothelial tissue, followed by vWF binding to
platelet GPIb(1) .
To date, purification and
characterization of several GPIb-binding snake venom proteins have been
reported, including alboaggregin-B from Trimeresurus
albolabris(2) , echicetin from Echis
carinatus(3) , and agkicetin from Agkistrodon acutus(4) . These proteins bind to GPIb and function as receptor
blockers of vWF binding. More recently, Peng et al. (5) demonstrated that the reduced and S-alkylated
subunit of echicetin retains its inhibitory activity on vWF
binding to GPIb, although with less potency than that of the intact
protein, and reported the amino acid sequence of the
subunit.
However, the structural features and entire amino acid sequence of
GPIb-binding snake venom proteins have not yet been reported.
Recently, we purified the GPIb-binding snake venom proteins jararaca GPIb-BP(6) , flavocetin-A and -B(7) , and tokaracetin (8) from the venom of Bothrops jararaca, Trimeresurus flavoviridis, and Trimeresurus tokarensis, respectively. The characteristic features of these proteins, in particular their inhibitory effect on vWF binding to GPIb and on vWF-dependent platelet aggregation, appear to be closely similar to those of echicetin. However, which subunit of these proteins binds platelet GPIb remained unknown.
In the present study, as a first step in elucidating the
structure-function relationships of these GPIb-binding proteins, we
determined the complete amino acid sequences of both the and
subunits of jararaca GPIb-BP. Furthermore, we also demonstrated
that the GPIb-binding site of jararaca GPIb-BP resides on its
and
not its
subunit. This is the first report of the primary
structure of a snake venom GPIb-binding protein.
Figure 1:
Separation of and
subunits
of jararaca GPIb-BP. After reduction and S-pyridylethylation,
the jararaca GPIb-BP was subjected to RP-HPLC on a SynChropak RP-8
column using a trifluoroacetic acid/acetonitrile system at a flow rate
of 1.0 ml/min. Peptides were monitored at 280
nm.
Figure 3:
Summary of the sequence proof of jararaca
GPIb-BP. The proven sequences of specific peptides (underlined) are given in the one-letter code below the
summary sequence (bold type). The prefixes K, E, P, D, and T denote peptides generated by cleavage of PE-
and PE-
subunits of jararaca GPIb-BP with API, V8 protease,
pepsin, endoproteinase Asp-N, and TPCK-trypsin, respectively. Products
of API digest are numbered from the amino terminus to the carboxyl
terminus of the protein. Sequences written in uppercase letters were proven by Edman degradation; sequences in lowercase
letters indicate tentative identifications. Unidentified residues
are shown by a
or dashes.
An API
digest of the PE- subunit was separated by RP-HPLC as shown in Fig. 2. The peptides were designated with a prefix K as K1
through K15 according to their order in the final amino acid sequence.
Six of 11 major peptides thus isolated were subjected to ISMS analysis (Table 2) and sequence determination with a protein sequencer (Fig. 3), although peptide K15 did not provide an appreciable
signal by ISMS analysis. Peptide K3 extended the amino-terminal
sequence obtained with the intact
subunit to Lys-44. Among peptic
peptides of the PE-
subunit, separated by RP-HPLC (data not shown)
and designated with a prefix P, peptide P25 was found to extend the
sequence of peptide K4 and overlapped peptides K9, K10 (free Lys), and
K11. The overlap of peptides K9, K10, and K11 was established by
analysis of peptide D15 isolated from an Asp-N endoproteinase digest of
the PE-
subunit and designated with a prefix D (data not shown).
Among V8 peptides of the PE-
subunit with a prefix E (data not
shown), peptide E21 extended the sequence of peptides K11 and K12 (free
Lys) and overlapped the sequence of peptide K14. Finally, peptide E22
extended the sequence of peptide K14 to the carboxyl terminus of the
subunit. The carboxyl-terminal residue of peptide E22 was not
unambiguously identified by sequence analysis, but ISMS analysis
indicated the presence of Arg in addition to the sequence obtained.
Figure 2:
Separation of peptides generated by
digestion of the PE- subunit with API. The digest was separated by
RP-HPLC as described for Fig. 1on a SynChropak RP-18 at a flow
rate of 1.3 ml/min and monitoring at 206 nm. Purified peptides are
identified by a K prefix as described in Fig. 3.
Thus, the amino-terminal sequence of residues 1-44 and the
carboxyl-terminal sequence of residues 45-142 were unambiguously
determined by direct sequencing of the intact subunit and peptides
derived from several digests of the PE- subunit. However, although
direct evidence for the overlap of these two sequences, namely that of
peptides K3 and K4, was not obtained by analysis of any peptide, ISMS
analysis showed an MH
value of 17,458 ± 3.7 for
the PE-
subunit, which coincides within experimental error with
the value calculated from the sequence of residues 1-142,
indicating no amino acid residue between residues 44 and 45.
Figure 4:
Amino acid sequence homology between the
and
subunits of jararaca GPIb-BP, botrocetin(20) ,
and the
subunit of echicetin(5) . There is a high degree
of homology (38-39% identity) between the
and
subunits of jararaca GPIb-BP. Numbers indicate positions of
amino acid residues from the amino terminus of the
subunit of
jararaca GPIb-BP. Gaps have been inserted to maximize homology.
Identical residues to those of both the
and
subunit of
jararaca GPIb-BP are boxed.
An API digest of the PE- subunit
was separated by RP-HPLC in a similar manner to that in Fig. 3(data not shown). The peptides were designated with a
prefix K as K1 through K6 according to their order in the final amino
acid sequence, and all were subjected to ISMS analysis (Table 2)
and sequence determination with a protein sequencer (Fig. 3).
The sequences of all K peptides were completely determined until the
carboxyl terminus except for peptide K4, and their calculated molecular
masses showed good agreement with those observed by ISMS analysis (Table 2). The sequence of peptide K6 showed some
microheterogeneities at residues 115 (Gln/Glu) and 117 (Phe/Val), which
were also identified with tryptic peptides T10 and T11, and agreed with
the results by ISMS analysis (Table 2). In addition, the sequence
of peptide K4 [2] derived from an API digest of the protein
isolated from another batch of the snake venom showed Ser at residue 53
in place of Leu identified with peptide K4.
The sequence of peptide
K3 extended the amino-terminal sequence obtained with the intact
protein to Lys. The overlaps of peptides K3-K6 were
obtained by analysis of some of either the peptic (designated with a
prefix P) or V8 protease (designated with a prefix E) peptides. There
were some problems in analysis of the carboxyl-terminal region of
peptide K4. Sequencer analysis of peptides K4 and K4 [2]
provided a partial sequence of residues 36-60 but became
ambiguous after residue 61. Analysis of two peptic peptides P40 and
P43, particularly of the former, extended the sequence of peptide K4
but still with tentative identification of Asn at residue 73 and Glu at
residue 78 and ambiguous identification at residues 65 and 72. Residues
65 and 72 were both assumed to be Trp by sequence homology to other
snake venom proteins of the same family (Fig. 4). The molecular
masses calculated on this assumption showed good agreement with
MH
values of the intact PE-
subunit and peptide
K4 as observed by either ISMS or MALD/TOF mass spectrometry,
respectively (Table 2). These mass spectrometric analyses also
indicated the absence of an amino acid residue between Glu
and Trp
, which agreed with isolation of peptide E5
in terms of substrate specificity of V8 protease. However, Asn
should still be regarded as a tentative identification.
Figure 5:
Competition binding assays. Inhibition of
reduced and
subunits of jararaca GPIb-BP on the binding of
biotin-labeled jararaca GPIb-BP to immobilized platelets was measured
as described under ``Experimental Procedures.'' Nonspecific
binding was determined in the presence of a 50-fold excess of unlabeled
jararaca GPIb-BP. Specific binding obtained by subtraction of
nonspecific binding from the total was plotted.
, intact jararaca
GPIb-BP;
, reduced
subunit;
, reduced
subunit.
We determined the complete amino acid sequence of jararaca
GPIb-BP isolated from the venom of Bothrops jararaca. Jararaca
GPIb-BP consists of two subunits, designated the and
subunits. The
subunit consists of 142 amino acid residues, and
the
subunit consists of 123 amino acid residues. Calculated M
values of the
and
subunits (17,456.8
and 15,035.0/14,987.9, respectively) were in good agreement with their
apparent M
values estimated by SDS-PAGE (6) and coincide with their MH
values observed
by ISMS. In the carboxyl terminus of the
subunit of jararaca
GPIb-BP, substitutions, at least at the residues of Leu
,
Gln
, and Phe
, were observed in the same or
other batches, suggesting the presence of polymorphism. When the
sequences of jararaca GPIb-BP and the other proteins are aligned as
shown in Fig. 4, considerable homology is seen (38-64%
identity) between jararaca GPIb-BP and both botrocetin and echicetin.
The identity of the amino acid sequence between the
subunits of
jararaca GPIb-BP and echicetin was 52-54%, whereas the identity
between the
subunit of jararaca GPIb-BP and the
subunit of
echicetin was 38%. As indicated in Fig. 5, the platelet
GPIb-binding site resides on the
and not on the
subunit.
Peng et al.(5) reported that the biological activity
of echicetin also resides in the
subunit. It is interesting that
the identity of the amino acid sequence between the
subunits of
jararaca GPIb-BP and echicetin was much higher than that between the
subunit of jararaca GPIb-BP and the
subunit of echicetin.
However, the
subunit of echicetin retains its activity even after
reduction and S-alkylation(5) , whereas the
subunit of jararaca GPIb-BP loses its activity on S-alkylation
for unknown reasons. Botrocetin, isolated from the venom of B.
jararaca, is also a heterodimeric protein that has almost the same M
as jararaca GPIb-BP (20) but that binds
to vWF and not GPIb(21) . Furthermore, Ozeki et al. (22) in our laboratory isolated a galactoside-binding lectin
from the same venom whose structure is similar to GPIb-BP and
botrocetin but that is clearly a homodimeric sugar-binding protein. It
is now clear that B. jararaca snake venom contains several
proteins with homologous structures but with clearly distinct binding
characteristics. The amino-terminal sequences of
alboaggregin-B(23) , flavocetin-A and -B(7) ,
tokaracetin(8) , and agkicetin (4) are highly
homologous; the primary structure of jararaca GPIb-BP may reveal a
structure typical of these GPIb-binding proteins.
In regard to the
location of inter- and intra-chain disulfide bridges, the and
subunits contain 7 half-cystines each (Fig. 3). The number
of half-cystine residues and their positions in the sequence of
jararaca GPIb-BP are almost identical with those in the
and
subunits of botrocetin and with the
subunit of echicetin,
indicating that the disulfide bond pairings should be similar in these
proteins. Thus, from the known disulfide pairings of
botrocetin(20) , those of jararaca GPIb-BP may occur as
follows:
, 6-17, 39-136, and 111-128;
,
2-13, 30-119, 96-111. It is also likely that one
interchain disulfide bond is formed between Cys
of the
subunit and Cys
of the
subunit.
Although
the present results provide information about the specific structural
elements associated with the GPIb-binding ability of jararaca GPIb-BP,
further studies on the structure-function relationship of the
subunit of jararaca GPIb-BP should help clarify the pathophysiological
functions of platelet adhesion to exposed subendothelium at the site of
vascular injury and may help in the discovery of a lead molecule for a
low molecular GPIb antagonist.