THE JOURNAL OF BIOLOGICAL CHEMISTRY
20802–20809, 1998
© 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
Isolation and Characterization of the Gene Encoding a Novel
Factor Xa-directed Anticoagulant from the Yellow Fever
Mosquito, Aedes aegypti*
(Received for publication, March 23, 1998, and in revised form, May 5, 1998)
Kenneth R. Stark‡ and Anthony A. James§
From the Department of Molecular Biology and Biochemistry, University of California, Irvine, California 92697-3900
Mosquito salivary glands secrete a number of proteins
that inhibit mammalian hemostasis and facilitate blood
feeding. We have isolated the protein product and cor-
responding cDNA of a gene designated Anticoagulant-
factor Xa (AFXa), that encodes the factor Xa (FXa)-di-
rected anticoagulant of the yellow fever mosquito, Aedes
aegypti. The protein was purified partially by cation
exchange chromatography and shown by enzyme activ-
ity profiles and SDS-polyacrylamide gel electrophoresis
analysis to have an Mr 5 54,000. The protein was purified
further by preparative SDS-polyacrylamide gel electro-
phoresis and subjected to internal protein sequencing,
and the sequence of five peptides was determined. De-
generate oligonucleotides were designed based on three
of the peptide sequences, and these were used to screen
an adult female salivary gland cDNA library from A.
aegypti. A 1.8-kilobase pair cDNA was isolated and
shown to encode a 415-amino acid conceptual transla-
tion product with a predicted molecular mass of 47.8
kDa that contains the five sequenced peptides. Hydro-
phobicity analysis predicts a 19-amino acid signal pep-
tide typical for secreted proteins. Northern analysis
demonstrated that AFXa is expressed only in female
salivary glands. Baculovirus-expressed AFXa protein
has the appropriate size and expected FXa-directed an-
ticoagulant activity. Analysis of the primary amino acid
sequence shows that the AFXa gene product has simi-
larities to the serpin superfamily of serine protease in-
hibitors and may represent a novel, highly diverged
member of this family.
Mosquitoes contribute significantly to worldwide morbidity
and mortality rates by transmitting pathogens responsible for
malaria and other diseases. The transmission of pathogens
occurs via salivation as the female mosquito takes a blood meal.
Mosquito saliva contains biochemically active molecules that
counteract vertebrate hemostasis and allow successful feeding
(1). In addition, studies with sandfly saliva and the parasites
that cause leishmaniasis have shown that the saliva of vector
insects can play a role beyond acting as a transfer medium and
can be involved in the enhancement of parasite infectivity (2).
* This work was supported by National Institutes of Health Grant
AI29746 and grants from the John D. and Catherine T. MacArthur
Foundation and the Burroughs-Wellcome Fund. The costs of publica-
tion of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
‡ Student in the Medical Scientist Program at the College of Medi-
cine, University of California, Irvine.
§ To whom correspondence should be addressed: Dept. of Molecular
Biology and Biochemistry, University of California, Bio. Sci. II, Rm.
3205, Irvine, CA 92697-3900. Tel.: 949-824-5930; Fax: 949-824-2814;
E-mail: aajames@uci.edu.
20802
Vol. 273, No. 33, Issue of August 14, pp.
Printed in U.S.A.
By understanding the biochemical actions of saliva and its role
in blood feeding, we hope to gain a better understanding of how
hematophagy contributes to disease transmission and identify
important targets for genetic manipulation toward the devel-
opment of parasite-refractory mosquitoes (3).
Mosquito saliva has been shown to contain platelet antiag-
gregating factors (4 – 6) and vasodilators (7, 8). Recently, we
used coagulation assays and factor-specific assays to demon-
strate that a number of mosquito species contain anticoagu-
lants (9). In a remarkable finding, we determined that al-
though all of the mosquito species we examined inhibited
Downloaded from www.jbc.org by on May 26, 2007
coagulation at the common pathway, the specific coagulation
factor that was inhibited differed between the hematophagous
subfamilies of mosquitoes. The culicine mosquitoes contained
anticoagulants that inhibited coagulation factor Xa (FXa)1, while
anopheline mosquitoes had thrombin-directed anticoagulants.
A biochemical analysis of the anticoagulant in the yellow
fever mosquito, Aedes aegypti, a culicine, showed that it was a
specific, reversible, noncompetitive, proteinaceous inhibitor of
FXa (10). The anticoagulant was shown to function at physio-
logical levels and had calcium and pH optima that corre-
sponded to those of FXa. Because anticoagulants do not form a
specific class of molecules, the A. aegypti anticoagulant was
isolated and analyzed to define further its mechanism of inhi-
bition and to understand its molecular origins. Oligonucleotide
primers based on partial peptide sequences of the purified
anticoagulant led to the isolation of a 1.8-kb cDNA. The corre-
sponding gene has been designated Anticoagulant-factor Xa
(AFXa). The cDNA encodes a conceptual translational product
of 415 amino acids with a predicted molecular mass of 47.8 kDa
and shares primary structural similarities with the serpin su-
perfamily of serine protease inhibitors. However, the AFXa
gene product has a unique inhibitory mechanism in comparison
with typical serpins (10), and therefore appears to be highly
diverged biochemically from the serpin family of proteins.
EXPERIMENTAL PROCEDURES
Materials—HEPES, rehydrogenated Triton X-100, CAPS, and
AEBSF (Calbiochem); bovine FXa and thrombin (Enzyme Research
Laboratories); and Chromozym X and TH, leupeptin, pepstatin, and
apoprotonin (Boehringer Mannheim) were purchased from the indi-
cated manufacturers, and all other chemicals were from Sigma.
Mosquito Maintenance—A. aegypti (Rockefeller strain) were reared
at 22 °C, 80% relative humidity, 18:6 light/dark cycle, and fed on anes-
thetized guinea pigs and/or sugar cubes. The mosquitoes used in the
experiments were 3–5-day-old adults that had never fed on blood prior
to salivary gland dissection.
1
The abbreviations used are: FXa, factor Xa; AEBSF, 4-(2-aminoeth-
yl)benzenesulfonyl fluoride; bp, base pair(s); kb, kilobase pair(s); CAPS,
3-(cyclohexylamino)propanesulfonic acid; EDTA, ethylenediamine tet-
raacetic acid; PVDF, polyvinylidene difluoride; PAGE, polyacrylamide
gel electrophoresis; SGE, salivary gland extract; PAI-2, plasminogen
activator inhibitor-2.
This paper is available on line at http://www.jbc.org
 Mosquito Anticoagulant Is a Serpin
Salivary Gland Extract—Salivary glands were dissected in Aedes
saline (20 mM HEPES, 150 mM NaCl, 3.3 mM KCl, 2.2 mM MgCl2, 2.2
mM CaCl2, 1.8 mM NaHCO3, pH 7.0), transferred to a vial containing
homogenization buffer (50 mM HEPES, 150 mM NaCl, 10 mM EDTA,
0.05% rehydrogenated Triton X-100, pH 8.0), and stored at 270 °C until
needed. The salivary glands were thawed and disrupted by sonication
(Heat Systems Ultrasonics) in an ice-water bath at 40% output power
for 5 min. The homogenate was centrifuged (20,000 3 g) for 10 min at
4 °C, and the supernatant, salivary gland extract (SGE), was recovered
for purification of the anticoagulant or use in biochemical assays.
FXa Chromogenic Assay and Protein Assay—All biochemical assays
were performed in duplicate at 37 °C unless otherwise specified. Bovine
FXa (0.22 ng) was incubated with and without SGE in coagulation
assay buffer (50 mM Tris, 250 mM NaCl, 0.1% polyethylene glycol 8000,
0.1% bovine serum albumin, pH 8.3) with 5 mM CaCl2 for 15 min in a
final volume of 0.2 ml. Chromozym X (500 mM) was added, and FXa
activity was measured spectrophotometrically by following the release
of free p-nitroaniline with a continuous change in absorbency at 405 nm
(molar extinction coefficient 9.75 mM21 cm21) using a Thermo-max
kinetic microplate reader (Molecular Devices, Inc.). The reaction veloc-
ities were measured with and without the addition of SGE, and the
percentage of inhibition was expressed as follows: ((uninhibited veloc-
ity 2 inhibited velocity)/uninhibited velocity) 3 100%. Units of antico-
agulant activity are defined as percentage of inhibition of 0.22 ng of
FXa/1.0 ml of AFXa. Protein concentrations were determined using the
Bio-Rad protein assay with bovine serum albumin as a standard.
SDS-PAGE Analysis and Electrophoretic Transfer—SDS-PAGE was
performed in 10 or 12.5% polyacrylamide slab gels according to Lae-
mmli (11) and stained with Coomassie Brilliant Blue R-250 or silver-
stained (12). Gels were transferred to PVDF (Bio-Rad) membranes
using a tank electroblotter (Bio-Rad) in CAPS buffer containing 10%
methanol (13). For preparative SDS-PAGE, 0.1 mM thioglycolate was
added to the upper buffer reservoir as a scavenger of unpolymerized
acrylamide (14).
AFXa Purification and Peptide Sequencing—Salivary gland extract
with added protease inhibitor mixture (10 mM E-64, 0.1 mM leupeptin,
10 mg/ml pepstatin A, 2 mg/ml apoprotonin, and 0.2 mM AEBSF) was
used as the starting material for purification of the FXa-directed anti-
coagulant. Salivary gland extract was filtered (0.22 mm, ProX filter,
Phenix), total protein content was quantified, the sample was diluted
10-fold in buffer A (50 mM HEPES, pH 7.5), and the diluted sample was
loaded onto a 1.0-ml Mono S cation exchange column (Amersham Phar-
macia Biotech) at 0.5 ml/min. The column was washed with buffer A
until the base line returned to 0, and bound proteins were eluted at 0.5
ml/min with 10 ml of buffer A with 0.3 M NaCl, followed by a 20-ml
linear gradient from 0.3 to 0.8 M NaCl in buffer A. Relative protein
concentration was monitored by absorbance at 280 nm. 0.5-ml fractions
were collected with an LKB Frac-100 fraction collector and assayed for
FXa-inhibitory activity. Fractions with anticoagulant activity were an-
alyzed with SDS-PAGE to determine purity, and the peak active frac-
tions were pooled and purified further by 12.5% preparative SDS-
PAGE. Two gel-purified samples were submitted for internal protein
sequencing. One sample was electrophoretically transferred to a PVDF
membrane and submitted to the Harvard Microchemistry Laboratories
(under the direction of W. Lane) for cleavage with trypsin, peptide
separation, mass spectroscopy, and peptide sequencing. The second
sample was submitted as a gel slice to the W. M. Keck Foundation
Biotechnology Resource Laboratory at Yale University (under the di-
rection of K. Williams) for Lys-C digestion, peptide separation, mass
spectroscopy, and peptide sequencing. Amino-terminal protein sequenc-
ing was performed at the University of California, Irvine Sequencing
Facility (under the direction of R. Niece).
AFXa cDNA Isolation, Sequencing, and Characterization—Degener-
ate oligonucleotides were designed based on the amino acid sequence of
three peptides and synthesized using inosine where possible. Separate
oligonucleotides were used for degeneracies in the first position,
whereas nucleotide mixtures were used for third position degeneracies
(Table I). Nested gene amplification was used to amplify a specific
sequence corresponding to AFXa from a lgt11 female salivary gland
cDNA library (15). An aliquot of phage DNA was purified by treatment
with RNase and DNase (37 °C for 30 min) followed by proteinase K
treatment (37 °C for 5 min). The sample was extracted with one volume
of phenol for 20 min and one volume of chloroform for 1 min. The DNA
was precipitated with one volume of ethanol in the presence of 0.3 M
sodium acetate. The RNase treatment was repeated followed by stand-
ard phenol/chloroform extraction and ethanol precipitation, and a
100-ng aliquot was used as the target DNA.
The location of primer sets C and D (Table I) within the putative
20803
AFXa sequence was surmised because they are based on the peptide
that contains the carboxyl terminus. Primer sets AC, AD, BC, and BD
were utilized for the first round of gene amplification. The nested step
was amplified using 2 ml of DNA from the above reactions as follows.
From the AD reaction, the primer sets AC, BD, and BC were used; from
the AC reaction, BC primer sets were used; from the BC reaction, AC
primer sets were used; and from the BD reaction, primer sets BC, AD,
and AC were used. The nested gene amplification reaction conditions
were as follows: 1 cycle of 94 °C for 3 min; 20 cycles of 92 °C for 30 s,
60 °C for 1 min with a 0.5 °C decrease each cycle, and 72 °C for 1 min;
20 cycles of 92 °C for 30 s, 50 °C for 1 min, and 72 °C for 1 min; and 1
cycle of 72 °C for 5 min. A unique 225-bp product was isolated from the
nested gene amplification reactions, cloned into pGEM-T vector (Pro-
mega), and sequenced using the chain-terminating, dideoxy method
(16). Conceptual translation of the primary sequence verified that this
amplified product corresponded to AFXa. The 225-bp product was la-
beled with [32P]dATP and [32P]dCTP by random prime reactions (Am-
ersham Pharmacia Biotech) and used to screen the cDNA library.
Positive clones were plaque-purified, and the clone containing the larg-
est insert, 8 –2B, was chosen for further characterization. For conven-
ience, this clone will here be called the “AFXa cDNA.” Phage DNA was
purified using Lambda DNA kits (Qiagen). The AFXa cDNA insert was
released by digestion with EcoRI, cloned into pBlueScriptIISK(2)
(Stratagene), and sequenced by dideoxy chain termination with l for-
ward and reverse primers and overlapping primers designed from the
sequencing results. Protein data base searches were performed with the
National Center for Biotechnology BLAST Network services (17). To
obtain a full-length cDNA sequence, two nested oligonucleotide primers
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(AFXa 59 R1, 59-TTCTGCCTGATAGTCTATCGCC-39; AFXa 59 R2, 59-
TATCATCCTGTGTGAAACACACC-39) were designed to the 59-end of
the AFXa cDNA. Heminested gene amplification was performed using
100 ng of purified cDNA from the phage library with l forward or
reverse primers and the nested 59-end AFXa primers as follows: 1 cycle
of 94 °C for 3 min; 30 cycles of 92 °C for 30 s, 55 °C for 1 min, and 72 °C
for 1 min; and 1 cycle of 72 °C for 5 min.
Northern Blot Analysis of AFXa—Total RNA was prepared using
Triazole (Invitrogen) from 3–5-day-old adult male and female mosqui-
toes, adult female salivary glands, and adult female carcasses without
heads and salivary glands. Poly(A)1 RNA was isolated using the Oli-
gotex mRNA kit (Qiagen). The RNA was separated on 1% agarose-
formaldehyde gels, transferred to a nylon filter (Zeta probe), and probed
with 32P-labeled AFXa cDNA. A. aegypti genomic DNA was prepared
(18) and used to amplify a 450-bp actin fragment using the primers
59-GGTATCCACGAAACTGTCTAC-39 (forward) and 59-TTGGACGCT-
GACAAGTATCAC-39 (reverse), designed to the 39-end of the A. aegypti
muscle actin gene (19). Gene amplification procedures were as follows:
1 cycle of 94 °C for 3 min; 40 cycles of 92 °C for 30 s, 52 °C for 1 min, and
72 °C for 1 min; and 1 cycle of 72 °C for 8 min. This actin fragment was
radiolabeled and used to reprobe the RNA blot. Before probing, the
filter was stripped with 0.1 3 saline-sodium citrate and 0.5% SDS in
two washes incubated at 95 °C for 20 min. All hybridizations were
performed using aqueous hybridization solutions at 65 °C (20).
Expression and Purification of Recombinant AFXa—Full-length
AFXa cDNA was generated by gene amplification of 100 ng of plasmid
DNA from clone 8 –2B using the primers 59-CTCGGATCCATGTATCT-
GAAGATAGTAATATTAGTCACC-39 (forward) and 59-TCGCCGCTC-
GAGAGTGCTGTAGCAGTGTTGACC-39 (reverse), designed to start at
amino acid 1 and end with the polyadenylation site. The primers were
also designed to contain a BamHI site at the 59-end and an XhoI site at
the 39-end for directional cloning. The error-proof VentR DNA polymer-
ase (New England Biolabs) was used in gene amplification reactions as
follows: 1 cycle of 94 °C for 3 min, 52 °C for 2 min, and 72 °C for 1.5 min;
5 cycles of 92 °C for 30 s, 52 °C for 1 min, and 72 °C for 1.5 min; 35 cycles
of 92 °C for 30 s, 62 °C for 1 min, and 72 °C for 1.5 min; and 1 cycle of
72 °C for 8 min. The amplified product was cloned into pGem-T (Pro-
mega) and subsequently subcloned into the baculovirus transfer vector,
pVL1393 (Invitrogen) using the BamHI/NotI cloning sites (NotI was
obtained from the pGem-T vector). The recombinant virus was trans-
fected into SF-9 cells and plaque-purified, and high titer virus for
recombinant protein production was expanded in Hi-5 cells (Invitro-
gen). Recombinant protein (580 mg of total protein) was harvested from
the supernatant of infected Hi-5 cells, and protease inhibitor mixture
was added. The sample was centrifuged (40,000 3 g, 30 min), diluted
3-fold with buffer A, and applied to a 1.0-ml Mono S column at 1.0
ml/min. The column was washed with buffer A until the base line
returned to 0, and bound proteins were eluted with a 20-ml linear NaCl
gradient. One-ml fractions were collected in tubes containing 10% glyc-
erol, and fractions were assayed for FXa-inhibitory activity.
20804 Mosquito Anticoagulant Is a Serpin

FIG. 1. Purification of AFXa from

crude salivary gland extract. A, sali-
vary gland extract prepared from 2000
pairs of dissected glands (4.9 mg of total
protein) was resolved on a 1.0-ml Mono S
cation exchange column. Bound proteins
were eluted (solid line (OD 280 nm)) with
a 10-ml step gradient of 0.3 M NaCl, fol-
lowed by a 20-ml gradient from 0.3 to 0.8
M NaCl (dotted line (NaCl gradient)).
0.5-ml fractions were collected and as-
sayed for FXa inhibitory activity. The
peak activity was contained in fractions
34 –38 (open circles (FXa inhibition)). B,
10-ml aliquots from samples 30 – 41 were

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subjected to 12.5% SDS-PAGE and silver-
stained. A 54-kDa protein band was de-
termined to correspond to the major activ-
ity peak (indicated by an arrow). The
fraction numbers are indicated for each
respective lane with a bar over the frac-
tions containing the peak activity. Molec-
ular mass markers in kDa are shown to
the right of the gel.

Multiple Sequence Alignment—Comparison and alignment of the
carboxyl terminus of the AFXa translation product were performed
with the similar regions of the following serpins identified from blast
analysis: plasminogen activator inhibitor-2 from Homo sapiens (P05120
(21)), maspin from Rattus norvegicus (U58857),2 neuroserpin from Gal-
lus gallus (Z71930 (23)), a-1 antitrypsin from H. sapiens (K02212 (24)),
BmYP44 from Brugia malayi (U04206 (25)), and antichymotrypsin II
from Bombyx mori (S19546 (26)). The sequences were aligned using the
Pileup multiple sequence alignment program with a gap weight of 12
and a gap extension penalty of 4 (Genetics Computer Group, University
of Wisconsin). Gap alignments were adjusted to visually emphasize
insertions and deletions except within the reactive site loop, where
maximum alignment of amino acid sequence flanking the reactive cen-
ter P1 was emphasized.
RESULTS
Purification and Peptide Sequencing of the Mosquito FXa-
directed Anticoagulant—We had shown previously that adult
female A. aegypti salivary glands contain a potent FXa-directed
anticoagulant activity and provided a preliminary biochemical
characterization (10). In order to characterize further the an-
ticoagulant, strategies were developed to isolate and purify the
2
Y. Y. Umekita, R. A. Richard, and S. Liao, unpublished GenBank
accession number.
protein using adult female A. aegypti SGE as the starting
material. Mono S cation ion exchange chromatography was
used to fractionate SGE prepared from 2000 dissected female
glands, and this produced a high yield of active protein in a
mixture of proteins (Fig. 1A). SDS-PAGE analysis of the active
fractions demonstrated that a protein with an Mr 5 54,000
coincided with the FXa-inhibitory activity peak (Fig. 1B). The
54-kDa species was confirmed to correspond to the majority of
the anticoagulant activity by densitometry analysis of the pro-
tein bands shown on the gel (data not shown). Fractions 34 –37
from the Mono S column containing the protein with the peak
anticoagulant activity were pooled, further purified by prepar-
ative SDS-PAGE, and transferred to PVDF membrane, and the
54-kDa band was excised and submitted for protein sequencing
analysis. Approximately 10 pmol of the putative anticoagulant
were subjected to tryptic digestion prior to internal protein
sequencing. This analysis yielded two sequenced peptide frag-
ments, H46 and H23 (Table I). A gene amplification screen of
an adult female A. aegypti salivary gland cDNA library using
degenerate oligonucleotides designed based on the sequences of
the H46 and H23 peptides failed to produce any true products,
nor did an oligonucleotide-based library screen reveal any pos-
itive phage (data not shown).
 Mosquito Anticoagulant Is a Serpin 20805
TABLE I
Amino acid sequence of internal peptides from the AFXa protein and sequence of degenerate oligonucleotides used in gene amplification
Peptidea Amino acid sequenceb Degenerate oligonucleotidesc

Y91 (K)TDVRLPQFTLRIK [91F-1] 59-AARACIGAYGTICGNCTNCC-39

[91F-2] 59-AARACIGAYGTIAGRCTNCC-39
[91F-3] 59-AARACIGAYGTICGNTTRCC-39
[91F-4] 59-AARACIGAYGTIAGRTTRCC-39
Y68 (K)NAVPLGDVIQK [68F-1] 59-AARAAYGCIGTICCICTNGG-39
[68F-2] 59-AARAAYGCIGTICCITTRGG-39
Y58 (K)SIGPESDEISXDRPPRYQ-COO2 [58R-1] 59-YTGRTANCGIGGIGGNCGRTC-39
[58R-2] 59-YTGRTAYCTIGGIGGNCGRTC-39
[58R-3] 59-YTGRTANCGIGGIGGRCTRTC-39
[58R-4] 59-YTGRTAYCTIGGIGGRCTRTC-39
[58CR-1] 59-ATYTCRTCNGAYTCIGGICC-39
[58CR-2] 59-ATYTCRTCRCTYTCIGGICC-39
H46 (R/K)SGMQNFLSTSDIDR
H23 (R/K)RFAAMR
a
Peptide designations are based on the fraction number of the reverse phase high performance liquid chromatography purification of the
peptides. The letter “Y” refers to the Keck Biotechnology Protein Sequencing Facility at Yale, and the letter “H” refers to the Harvard
Microchemistry Laboratory.
b
Amino acid sequences are listed in the standard single-letter code. Italicized amino acids in Y68 and Y58 were ambiguous. X was undeter-
minable at the time of sequencing. The end of peptide Y58 is shown as the carboxyl terminus in italics because of the lack of a lysine residue
(confirmed by mass spectroscopy). Amino acids shown in parentheses are deduced based on the specificity of the protease digestion properties.
c
Nucleotides are denoted with standard single letter designation and written in 59 to 39 format. The designations in brackets indicate the
oligonucleotide name, where the number refers to the peptide, “F” refers to forward primers, and “R” refers to reverse direction primers.
Degeneracies in the sequence are denoted as “Y” for G or C, “R” for A or G, and “N” for A, G, T, or C. “I” is for inosine. Oligonucleotides were mixed
as a single set for the gene amplification-based library screen as follows: set A (91F-1, 91F-2, 91F-3, and 91F-4), set B (68F-1 and 68F-2), set C

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(58CR-1 and 58CR-2), and set D (58R-1, 58R-2, 58R-3, and 58R-4).

Salivary gland extract from 10,000 dissected female glands
was subjected to Mono S chromatography in two separate
batches of 12.5 mg of total protein each. 1.0-ml fractions were
then collected and assayed for FXa-inhibitory activity. Peak
activity was found in fractions 18 –20 in both batches to give a
77.5% yield recovery of FXa-inhibitory activity. Fractions
18 –20 from both batches were combined and concentrated on a
Centricon-30 filter, and half of the sample was applied to 12.5%
preparative SDS-PAGE followed by transfer to PVDF mem-
brane, and the other half was reserved for preparative SDS-
PAGE without transfer to a membrane. The PVDF sample was
utilized for amino-terminal protein sequencing, and the gel
slice was submitted for Lys-C digestion and internal protein
sequencing. Amino-terminal protein sequencing revealed a
blocked N terminus. Analysis of Lys-C digestion products re-
sulted in the determination of the primary sequence of three
peptides: Y91, Y68, and Y58 (Table I). Peptide Y58 was identified
as the carboxyl terminus of the protein, because it lacked a lysine
residue at its carboxyl end, a characteristic of Lys-C digestion
reactions. This conclusion was confirmed by mass spectroscopy of
the peptide (data not shown). The amino acid X in peptide Y58
was undeterminable by spectroscopy or mass spectroscopy, and
we concluded that it most likely represents an unusual modifi-
cation of cysteine. This conclusion was confirmed when the pri-
mary nucleotide sequence of this region was determined.
Anticoagulant cDNA Isolation and Cloning—Degenerate ol-
igonucleotides were designed based on the standard triplicate
code using the amino acid sequences of the peptides Y91, Y68,
and Y58 (Table I). These oligonucleotides were used for a gene
amplification screen of an adult female A. aegypti salivary
gland cDNA library. Using primers designed to peptide Y58 as
the carboxyl terminus of AFXa, a strategy of nested gene am-
plification was developed to generate a DNA fragment to be
used as a probe for the cDNA library screen (Fig. 2A). Ampli-
fication of the cDNA library with primer sets A and D followed
by reamplification with primer sets B and C resulted in a
unique 225-bp DNA fragment. When this fragment was se-
quenced and the conceptual translation product was examined,
the amino acids from peptides Y68 and Y58 were revealed,
including amino acids that were not utilized in the design of the
oligonucleotides (Fig. 2B). These results confirm that this frag-
ment corresponded to a cDNA with sequence homology to the
AFXa gene. The 225-bp DNA fragment was labeled and used as
a probe to screen the salivary gland cDNA library. Approxi-
mately 750,000 plaques were screened in duplicate, and 100
positive colonies were identified. Twenty plaques were sub-
jected to secondary screening, and 13 of these were purified by
tertiary screening. The phage were analyzed for DNA insert
size by gene amplification using oligonucleotide primers based
on the sequence of the l-phage arms. Phage DNA was prepared
from four clones containing the largest insert sizes, and clone
8 –2B (AFXa cDNA) was selected and subcloned for further
analysis. The cDNA insert was sequenced completely in both
directions and contained a 1873-bp fragment corresponding to
nucleotides 17–1885 (Fig. 3).3 The 59-end of this phage clone
lacked an ATG initiation codon and therefore represented an
incomplete cDNA. Nested primers were designed to the 59-end
of the clone, and gene amplification using DNA prepared from
the cDNA library was utilized to recover a fragment containing
more of the 59-end. This procedure yielded nucleotides 1–16 in
the composite sequence and included the ATG start codon.
The 1245-bp open reading frame, nucleotides 6 –1250, en-
codes a conceptual translation product of 415 amino acids with
a calculated molecular mass of 47,800 Da. The sequences of all
five peptides determined by protein sequencing were identified
in the conceptual translation product, as were four potential
N-linked glycosylation sites. A hydrophobicity plot identified
the first 19 amino acids as a potential secretion signal peptide
(data not shown). The cDNA also contains an unusually long
39-untranslated region, nucleotides 1251–1870, with inverted
repeats at nucleotides 1473–1501 and 1815–1843, flanked by
8-bp duplications at nucleotides 1465–1472 and 1844 –1851.
Anticoagulant mRNA Expression Profile—The AFXa cDNA
was used as a probe to determine if its corresponding gene
showed a tissue-specific expression pattern matching that pre-
viously determined for the anticoagulant activity (10). Two
hybridizing species of RNA of approximately 1.9 and 1.6 kb
were detected in preparations from total females and female
salivary glands (Fig. 4). The 1.6-kb signal is sufficiently broad
3
This sequence has GenBankTM accession number AF050133.
20806 Mosquito Anticoagulant Is a Serpin

FIG. 2. Gene amplification strategy

for screening a salivary gland-spe-
cific cDNA library. A, degenerate oligo-
nucleotides (arrows) were designed to the
peptide fragments Y91, Y68, and Y58
(checkered bars). Nested gene amplifica-
tion was used to produce a fragment from
the A. aegypti female salivary gland
cDNA library as described under “Exper-
imental Procedures.” A unique 225-bp
fragment was generated from the nested
amplification using “B-C” primer sets,
and the sequence is shown below. B, con-
ceptual translation product of the 225-bp
DNA fragment is shown using standard
single-letter amino acid code. The amino
acid sequences in italics represent por-
tions of the peptide fragments Y68 and
Y58 and include amino acids that were
not used in the design of the degenerate
oligonucleotides (underlined).

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FIG. 3. Composite nucleotide sequence of the AFXa cDNA. The cDNA phage clone, 8 –2B, was sequenced with overlapping primers
(nucleotides 17–1885). Gene amplification was used to obtain additional sequences beyond the initiation codon (nucleotides 1–16) by amplification
of the A. aegypti female salivary gland cDNA library using nested primers to the 59-end of clone 8 –2B and l forward or reverse primers. Numbers
refer to the nucleotide sequence. The conceptual amino acid sequence is shown below the nucleotide sequence using the standard single-letter
amino acid code. The first 19 amino acids are double underlined to indicate the presumed signal peptide based on hydrophobicity plots.
Single-underlined amino acids in boldface type indicate the five sequenced peptides. Potential N-linked glycosylation sites are indicated with a
filled triangle. An asterisk indicates the carboxyl terminus of the protein. The initiation codon, ATG, the stop codon, TAA, and the presumed
polyadenylation signal sequence, AAATAAA, are shown in boldface type and underlined.

such that it could represent two mRNAs with similar molecular
weights or a highly expressed single mRNA. This pattern of
hybridization indicates that AFXa gene expression is specific to
the salivary glands as had been shown previously by analysis of
anticoagulant activity in dissected tissues. A muscle actin
probe was used as a control to confirm the presence of RNA in
negative lanes; the absence of a signal in the salivary gland
RNA preparations is expected based on its previously described
expression profile (19) and the lack of muscles in the glands.
Expression and Purification of Recombinant AFXa in Bacu-
lovirus—To confirm that the isolated cDNA corresponded to
that of the FXa-directed anticoagulant, the full-length AFXa
cDNA was expressed in baculovirus, and infected cell superna-
tants were analyzed for the production of FXa-inhibitory activ-
 Mosquito Anticoagulant Is a Serpin
ity. Analysis of duplicate samples for the presence of FXa-
inhibitory activity demonstrated that infected cells produced a
peak of activity at 72 h postinfection, followed by a decrease in
activity that results most likely from degradation of the recom-
FIG. 4. Northern analysis of AFXa expression in A. aegypti.
Poly(A)1 RNA was isolated from males (M), females (F), and female
carcasses without heads and salivary glands (C), and 3 mg of each was
loaded onto a 1% agarose-formaldehyde denaturing gel. Total RNA was
isolated from female salivary glands (SG), and 2 mg was loaded onto the
agarose gel. The top panel (AFXa) was probed with the 32P- labeled
EcoRI fragment from the AFXa cDNA. Approximate molecular sizes of
the AFXa RNA species are shown in kb and indicated with an arrow.
The blot was then stripped and reprobed with a 32P-labeled muscle
actin DNA fragment (bottom panel). The two gels are depicted, one
20807
binant protein (Table II). The extract (30 ml) was applied to a
1.0-ml Mono S cation exchange column using similar conditions
developed for the native protein except that bound proteins
were eluted with a salt gradient from 0 to 1.0 M NaCl (Fig. 5A).
An analysis of the peak activity showed two major proteins at
Mr 5 54,000 and Mr 5 40,000 (Fig. 5B). The smaller species is
most likely a degradation product, because it was not observed in
the initial analytical gel. Therefore, the recombinant AFXa prod-
uct shows FXa-directed anticoagulant activity, elutes off the
Mono S column with approximately the same salt concentration
as the native protein, and has the same molecular weight.
Anticoagulant Amino Acid Sequence Analysis—Having
shown that the isolated AFXa cDNA does indeed correspond to
the anticoagulant, the predicted amino acid sequence was com-
pared with those of other proteins in the data banks. Blast
analysis of the AFXa product revealed similarity within three
domains with several members of the serpin superfamily of
proteins. For descriptive purposes, we have defined the do-
mains as follows: domain I, 131–249; domain II, 262–358; and
domain III, 371– 413, with the numbers referring to the amino
acid positions in the AFXa gene product. The highest overall
score was with plasminogen activator inhibitor-2 (PAI-2) from
human placenta (blast score of 150). The overall amino acid

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above the other, such that their appropriate lanes align.
identity between the AFXa product and PAI-2 is 20%; however,
similarities in amino acids as defined by Altschul et al. (17) are
TABLE II as high as 50 – 60% within the three domains. A multiple se-
Factor Xa inhibitory activity in supernatants of baculovirus-infected quence alignment shows that the AFXa product has similari-
cells expressing recombinant AFXa protein
ties with proteins that originate in a number of different spe-
Hours postinfection Sample 1a Sample 2a cies ranging from filarial worms to humans (Fig. 6). The three
h total units domains listed above are not only conserved in sequence, but
24 3994 2461 also in their approximate location and spacing when compared
48 54,062 63,449 with members of the serpin gene family. A comparison of the
72 88,225 94,062 reactive site loop with AFXa and known serpin proteins illus-
96 75,309 NDb
trates that while there is remarkable conservation in the flank-
a
1 unit 5 amount of inhibition of Factor Xa by 1.0 ml of viral ing amino acid sequences (amino acids 333–347 in AFXa and
supernatant in the standard factor Xa inhibitory assay described under
domain III), AFXa shows a greater degree of divergence than is
“Experimental Procedures.”
b
Not determined. The sample was harvested for purification at 72 h typical for serpin proteins. Specifically, the reactive site loop in
postinfection. AFXa appears to be truncated in comparison with the serpins.

FIG. 5. Purification of recombinant AFXa. A, full-length AFXa cDNA generated by gene amplification was cloned into a baculovirus
expression vector. Cell supernatant (30 ml) from recombinant virus-infected Hi-5 cells was harvested at 72 h postinfection and applied to a 1.0-ml
Mono S column (580 mg of total protein). Bound proteins were then eluted (solid line (OD 280 nm)) with a 20-ml gradient from 0 to 1.0 M NaCl
(dotted line (NaCl gradient)), 1.0 ml-fractions were collected in 10% glycerol, and samples were assayed for FXa-inhibitory activity (open circles
(FXa inhibition)). B, molecular mass marker sizes in kDa are shown to the left of the gel (lane 1). Fractions 12 and 13 were pooled and analyzed
on 10% SDS-PAGE stained with Coomassie Blue (lane 2). An arrow indicates the presumed active species at 54 kDa. The lower molecular weight
species most likely represents a product of degradation.
20808 Mosquito Anticoagulant Is a Serpin
FIG. 6. Multiple sequence align-
ment of the AFXa gene product with
members of the serpin superfamily. A
sequence alignment of several known ser-
pins with the AFXa gene product was
compiled using the GCG Pileup program
with a gap weight of 12 and a gap length
weight of 4. The serpin sequences were
chosen based on their similarities after a
blast analysis of the AFXa cDNA that
identified conservation within three do-
mains near the carboxyl termini. Gaps
were adjusted to allow maximum align-
ment to emphasize insertions and dele-
tions except within the reactive site loop,
where maximum alignment of the reac-
tive center P1 was emphasized. The se-
quences have been shaded to emphasize
identities (black) and similarities (gray)
when conserved among 50% of the se-
quences utilized. Arrows and roman nu-
merals I, II, and III delineate the three
domains identified from the blast analy-
sis. The reactive site loop containing the
hinge region (P17–P9) are indicated and
labeled. The reactive center, P1, where
proteolytic cleavage has been identified or
predicted is indicated with a triangle. The
sequences used for the alignment are hu-

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man PAI-2, rat maspin (Mas), chicken neu-
roserpin (Nspn), human a-1-antitrypsin
(A1AT), B. malayi BmYP44 (BmY), and
silk moth antichymotrypsin II (ACT2).

Furthermore, P17 within the hinge region is typically a glu-
tamic acid residue (27), while in AFXa it is a histidine residue.
The predicted P1 for AFXa aligns as an arginine that would be
expected for a FXa-specific serine protease inhibitor; however,
cleavage at this site has not been verified. Thus, AFXa shares
primary amino acid sequence in common with typical serpin
proteins, including the appropriate spacing of domains, but sig-
nificant divergence is evident and makes AFXa a unique protein.
DISCUSSION
Anticoagulants of natural origin have been the subject of
intensive research since the discovery of the thrombin-inhibi-
tor, hirudin, from the hematophagous leech (28). Anticoagu-
lants characterized in a number of different organisms come
from unique families of molecules and generally share little in
common with one another except that they inhibit factors from
the coagulation cascade. For example, hirudin is a unique
thrombin inhibitor that is unlike any other serine protease
inhibitor. The tick anticoagulant peptide is a kunitz-type in-
hibitor of FXa (29), as is antistasin from the Mexican leech (30).
In contrast, two anticoagulants have been described and char-
acterized from the kissing bug, Rhodnius prolixus. One is a
kazal-type inhibitor of thrombin (31), and the other is a nitro-
phorin-containing protein that inhibits factor VIIIa-mediated
activation of FXa (32). Many other anticoagulants have been
described in terms of their activity, but few others have been
characterized at their primary structure ( for a review, see Ref.
33). Until recently, mosquitoes were largely ignored in these
efforts, especially amid early reports that mosquitoes did not
contain anticoagulants (34, 35).
Recently, we demonstrated the presence of anticoagulants in
a number of mosquito species and showed that culicine mos-
quitoes produced FXa-directed anticoagulants (9, 10). Here, we
have characterized further the FXa-directed anticoagulant
from the culicine mosquito, A. aegypti, and used information
from the amino acid sequence to isolate a cDNA that corre-
sponds to the anticoagulant gene. The evidence supporting the
conclusion that the AFXa cDNA corresponds to the correct gene
includes analyses of the primary sequence of the isolated pro-
tein and conceptual translation product, gene expression pro-
files, and expression of recombinant protein. The 415-amino
acid open reading frame of the AFXa cDNA has the sequences
of all five peptides that were determined in the analysis of the
isolated protein. These sequences include two peptides not
utilized for the library screening. Furthermore, the open read-
ing frame of the AFXa cDNA also has four N-linked glycosyla-
tion sites and a putative cleavable signal leader peptide, both
features characteristic of a secreted salivary protein (1). In fact,
glycosylation most likely accounts for the molecular weight
difference observed in the SDS-PAGE analyses (Mr 5 54,000)
when compared with that calculated from the conceptual trans-
lation product (Mr 5 47,800). The stage-, sex-, and tissue-
specific expression pattern of the AFXa gene as detected by
cDNA hybridization to mRNA is consistent with the expression
profile of the salivary gland anticoagulant established using
biochemical assays (10). Finally, AFXa cDNA expressed in
baculovirus produces a protein that has FXa-inhibitory activity
and the appropriate molecular weight.
Analysis of the primary nucleotide sequence of the AFXa
cDNA shows that it has an unusually long 39-untranslated
region, and based on Northern analysis, salivary glands may
contain as many as three mRNA species of differing size. We
have evidence that the 39-untranslated region of clone 8 –2B
includes the remnants of a transposable element, and we have
isolated and sequenced three alleles of the AFXa gene includ-
ing one without the extended 39-end region.4 The presence or
absence of this putative element in the transcribed region of
AFXa accounts for the size differences in the mRNA molecules.
Molecular blast analysis of the AFXa conceptual transla-
tional product revealed intriguing amino acid sequence simi-
larities with serpin-like serine protease inhibitors in at least
three domains. The highest degree of identities and similarities
was with the arginine-serpin, plasminogen activator inhibi-
tor-2, from human, mouse, and rat. The multiple sequence
alignment analysis performed with AFXa and serpins from a
4
K. Stark, P. Ho, T. Schaub, and A. A. James, unpublished results.
 Mosquito Anticoagulant Is a Serpin
wide variety of species demonstrated conservation of amino
acid sequences within these three domains as well as conser-
vation of the length of the domains and spacing between the
domains. Conservation of sequence was also observed for the
regions flanking the reactive center. Although remarkable sim-
ilarities and identities exist among AFXa and serpins, signifi-
cant differences are also evident indicating divergence from the
serpin gene family. Specifically, the reactive site loop of AFXa
appears to be truncated in comparison with the serpin super-
family. Also, the hinge region of AFXa shows differences from
the serpin family, particularly with a substitution of histidine
for the typical glutamic acid residue at P17. This P17 has been
identified as glutamic acid for 39 serpins analyzed (27). The
hinge region of serpins is proposed to be essential for protease
inhibition by promoting a conformational change that allows a
tight interaction with the active site of serine proteases fol-
lowed by cleavage of the P1–P19 peptide bond (36). Precedence
for considerable variability among serpins has been observed.
For example, ovalbumin has structural characteristics of the
serpin superfamily but is not known to inhibit any serine pro-
teases (27, 36). However, ovalbumin has been shown to form a
reactive site loop by crystallography data and by susceptibility of
this region to proteolysis. In another example, the reactive center
20809
There are three subfamilies of mosquitoes, two of which, the
Anophelinae and Culicinae, are hematophagous. The molecu-
lar characterization of proteins involved in blood feeding may
offer clues to the evolutionary relationship among these sub-
families (5, 6, 9). The ubiquitous presence of genes encoding
serpins in higher eukaryotic organisms makes them available
for recruitment to new functions during the evolutionary ad-
aptation to hematophagy. The molecular divergence of AFXa
from other members of the serpin superfamily argues that this
protein has undergone significant evolution while becoming an
effective salivary anticoagulant. In this perspective, it will be
interesting to determine the structural nature of other mos-
quito anticoagulants, particularly the thrombin inhibitors from
the Anophelinae.
Acknowledgments—We thank Dr. Dan Knauer and an anonymous
reviewer for suggestions on presentation of the data, Dr. David Fouts
for advice and help in the production of the baculovirus recombinant
AFXa proteins, Nimesh Ladawala and Vladimir Vasquez for assistance
with salivary gland dissections, Theresa Schaub for technical assist-
ance with DNA sequencing and plasmid construction, and Lynn Olson
for help preparing the manuscript.
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