Isolation and Characterization of the Gene Encoding a Novel Factor Xa-directed Anticoagulant from the Yellow Fever Mosquito, Aedes aegypti*

Kenneth R. StarkDagger and Anthony A. James§

From the Department of Molecular Biology and Biochemistry, University of California, Irvine, California 92697-3900

    ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Mosquito salivary glands secrete a number of proteins that inhibit mammalian hemostasis and facilitate blood feeding. We have isolated the protein product and corresponding cDNA of a gene designated Anticoagulant-factor Xa (AFXa), that encodes the factor Xa (FXa)-directed anticoagulant of the yellow fever mosquito, Aedes aegypti. The protein was purified partially by cation exchange chromatography and shown by enzyme activity profiles and SDS-polyacrylamide gel electrophoresis analysis to have an Mr = 54,000. The protein was purified further by preparative SDS-polyacrylamide gel electrophoresis and subjected to internal protein sequencing, and the sequence of five peptides was determined. Degenerate 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 translation product with a predicted molecular mass of 47.8 kDa that contains the five sequenced peptides. Hydrophobicity analysis predicts a 19-amino acid signal peptide 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 anticoagulant activity. Analysis of the primary amino acid sequence shows that the AFXa gene product has similarities to the serpin superfamily of serine protease inhibitors and may represent a novel, highly diverged member of this family.

    INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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). 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 development of parasite-refractory mosquitoes (3).

Mosquito saliva has been shown to contain platelet antiaggregating factors (4-6) and vasodilators (7, 8). Recently, we used coagulation assays and factor-specific assays to demonstrate that a number of mosquito species contain anticoagulants (9). In a remarkable finding, we determined that although all of the mosquito species we examined inhibited 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 physiological levels and had calcium and pH optima that corresponded 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 inhibition 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 corresponding 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 superfamily 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
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Abstract
Introduction
Procedures
Results
Discussion
References

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 indicated 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 anesthetized 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.

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 -70 °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 × 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 µM) 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 mM-1 cm-1) using a Thermo-max kinetic microplate reader (Molecular Devices, Inc.). The reaction velocities were measured with and without the addition of SGE, and the percentage of inhibition was expressed as follows: ((uninhibited velocity - inhibited velocity)/uninhibited velocity) × 100%. Units of anticoagulant 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 Laemmli (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 µM E-64, 0.1 mM leupeptin, 10 µg/ml pepstatin A, 2 µg/ml apoprotonin, and 0.2 mM AEBSF) was used as the starting material for purification of the FXa-directed anticoagulant. Salivary gland extract was filtered (0.22 µm, 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 Pharmacia 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 analyzed with SDS-PAGE to determine purity, and the peak active fractions 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 direction of K. Williams) for Lys-C digestion, peptide separation, mass spectroscopy, and peptide sequencing. Amino-terminal protein sequencing was performed at the University of California, Irvine Sequencing Facility (under the direction of R. Niece).

AFXa cDNA Isolation, Sequencing, and Characterization-- Degenerate 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 lambda gt11 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 standard 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 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 µl 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 (Promega), 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 labeled with [32P]dATP and [32P]dCTP by random prime reactions (Amersham Pharmacia Biotech) and used to screen the cDNA library. Positive clones were plaque-purified, and the clone containing the largest insert, 8-2B, was chosen for further characterization. For convenience, 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(-) (Stratagene), and sequenced by dideoxy chain termination with lambda  forward 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 (AFXa 5' R1, 5'-TTCTGCCTGATAGTCTATCGCC-3'; AFXa 5' R2, 5'-TATCATCCTGTGTGAAACACACC-3') were designed to the 5'-end of the AFXa cDNA. Heminested gene amplification was performed using 100 ng of purified cDNA from the phage library with lambda  forward or reverse primers and the nested 5'-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 mosquitoes, adult female salivary glands, and adult female carcasses without heads and salivary glands. Poly(A)+ RNA was isolated using the Oligotex 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 5'-GGTATCCACGAAACTGTCTAC-3' (forward) and 5'-TTGGACGCTGACAAGTATCAC-3' (reverse), designed to the 3'-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 × 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 5'-CTCGGATCCATGTATCTGAAGATAGTAATATTAGTCACC-3' (forward) and 5'-TCGCCGCTCGAGAGTGCTGTAGCAGTGTTGACC-3' (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 5'-end and an XhoI site at the 3'-end for directional cloning. The error-proof VentR DNA polymerase (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 (Promega) 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 transfected into SF-9 cells and plaque-purified, and high titer virus for recombinant protein production was expanded in Hi-5 cells (Invitrogen). Recombinant protein (580 µg 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 × 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% glycerol, and fractions were assayed for FXa-inhibitory activity.

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 Gallus gallus (Z71930 (23)), alpha -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 center P1 was emphasized.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

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 anticoagulant, strategies were developed to isolate and purify the 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 = 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 protein 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 preparative 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 fragments, 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 positive phage (data not shown).


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Fig. 1.   Purification of AFXa from crude salivary gland extract. A, salivary 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, followed by a 20-ml gradient from 0.3 to 0.8 M NaCl (dotted line (NaCl gradient)). 0.5-ml fractions were collected and assayed for FXa inhibitory activity. The peak activity was contained in fractions 34-38 (open circles (FXa inhibition)). B, 10-µl aliquots from samples 30-41 were subjected to 12.5% SDS-PAGE and silver-stained. A 54-kDa protein band was determined to correspond to the major activity peak (indicated by an arrow). The fraction numbers are indicated for each respective lane with a bar over the fractions containing the peak activity. Molecular mass markers in kDa are shown to the right of the gel.

                              
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Table I
Amino acid sequence of internal peptides from the AFXalpha protein and sequence of degenerate oligonucleotides used in gene amplification

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 membrane, 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 resulted 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 modification of cysteine. This conclusion was confirmed when the primary nucleotide sequence of this region was determined.

Anticoagulant cDNA Isolation and Cloning-- Degenerate oligonucleotides 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 amplification was developed to generate a DNA fragment to be used as a probe for the cDNA library screen (Fig. 2A). Amplification 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 sequenced 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 fragment 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. Approximately 750,000 plaques were screened in duplicate, and 100 positive colonies were identified. Twenty plaques were subjected 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 lambda -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 5'-end of this phage clone lacked an ATG initiation codon and therefore represented an incomplete cDNA. Nested primers were designed to the 5'-end of the clone, and gene amplification using DNA prepared from the cDNA library was utilized to recover a fragment containing more of the 5'-end. This procedure yielded nucleotides 1-16 in the composite sequence and included the ATG start codon.


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Fig. 2.   Gene amplification strategy for screening a salivary gland-specific cDNA library. A, degenerate oligonucleotides (arrows) were designed to the peptide fragments Y91, Y68, and Y58 (checkered bars). Nested gene amplification was used to produce a fragment from the A. aegypti female salivary gland cDNA library as described under "Experimental Procedures." A unique 225-bp fragment was generated from the nested amplification using "B-C" primer sets, and the sequence is shown below. B, conceptual translation product of the 225-bp DNA fragment is shown using standard single-letter amino acid code. The amino acid sequences in italics represent portions 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 5'-end of clone 8-2B and lambda  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.

The 1245-bp open reading frame, nucleotides 6-1250, encodes 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 3'-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 previously 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 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.


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Fig. 4.   Northern analysis of AFXa expression in A. aegypti. Poly(A)+ RNA was isolated from males (M), females (F), and female carcasses without heads and salivary glands (C), and 3 µg of each was loaded onto a 1% agarose-formaldehyde denaturing gel. Total RNA was isolated from female salivary glands (SG), and 2 µg 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 above the other, such that their appropriate lanes align.

Expression and Purification of Recombinant AFXa in Baculovirus-- 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 supernatants were analyzed for the production of FXa-inhibitory activity. 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 recombinant 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 = 54,000 and Mr = 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 product 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.

                              
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Table II
Factor Xa inhibitory activity in supernatants of baculovirus-infected cells expressing recombinant AFXalpha protein


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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 µg 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.

Anticoagulant Amino Acid Sequence Analysis-- Having shown that the isolated AFXa cDNA does indeed correspond to the anticoagulant, the predicted amino acid sequence was compared 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 domains 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 identity between the AFXa product and PAI-2 is 20%; however, similarities in amino acids as defined by Altschul et al. (17) are as high as 50-60% within the three domains. A multiple sequence alignment shows that the AFXa product has similarities with proteins that originate in a number of different species ranging from filarial worms to humans (Fig. 6). The three domains listed above are not only conserved in sequence, but also in their approximate location and spacing when compared with members of the serpin gene family. A comparison of the reactive site loop with AFXa and known serpin proteins illustrates that while there is remarkable conservation in the flanking amino acid sequences (amino acids 333-347 in AFXa and domain III), AFXa shows a greater degree of divergence than is typical for serpin proteins. Specifically, the reactive site loop in AFXa appears to be truncated in comparison with the serpins. Furthermore, P17 within the hinge region is typically a glutamic 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 significant divergence is evident and makes AFXa a unique protein.


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Fig. 6.   Multiple sequence alignment of the AFXa gene product with members of the serpin superfamily. A sequence alignment of several known serpins 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 domains near the carboxyl termini. Gaps were adjusted to allow maximum alignment to emphasize insertions and deletions except within the reactive site loop, where maximum alignment of the reactive center P1 was emphasized. The sequences have been shaded to emphasize identities (black) and similarities (gray) when conserved among 50% of the sequences utilized. Arrows and roman numerals I, II, and III delineate the three domains identified from the blast analysis. 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 human PAI-2, rat maspin (Mas), chicken neuroserpin (Nspn), human alpha -1-antitrypsin (A1AT), B. malayi BmYP44 (BmY), and silk moth antichymotrypsin II (ACT2).

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Anticoagulants of natural origin have been the subject of intensive research since the discovery of the thrombin-inhibitor, hirudin, from the hematophagous leech (28). Anticoagulants 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 inhibitor of FXa (29), as is antistasin from the Mexican leech (30). In contrast, two anticoagulants have been described and characterized from the kissing bug, Rhodnius prolixus. One is a kazal-type inhibitor of thrombin (31), and the other is a nitrophorin-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 mosquitoes 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 corresponds 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 protein and conceptual translation product, gene expression profiles, 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 reading frame of the AFXa cDNA also has four N-linked glycosylation 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 = 54,000) when compared with that calculated from the conceptual translation product (Mr = 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 3'-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 3'-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 including one without the extended 3'-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 translational product revealed intriguing amino acid sequence similarities 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 inhibitor-2, from human, mouse, and rat. The multiple sequence alignment analysis performed with AFXa and serpins from a wide variety of species demonstrated conservation of amino acid sequences within these three domains as well as conservation 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 similarities and identities exist among AFXa and serpins, significant 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 superfamily. 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 followed by cleavage of the P1-P1' 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 proteases (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 and hinge region of maspin are different from typical inhibitory serpins (37), and our alignment suggests that the maspin reactive site loop may also be truncated, similar to AFXa.

Our previous biochemical characterization of the FXa-directed anticoagulant from A. aegypti established reversible, noncompetitive, and noncovalent inhibition kinetics (10). The physiological relevance of the anticoagulant activity was shown by the failure of clot formation in the volume of blood characteristic of a typical blood meal in the presence of the expected amount of salivary gland protein secreted during feeding (10). Furthermore, 50% inhibition of 0.22 ng of FXa was observed using 1 µg of total protein from salivary gland extract. From our AFXa protein purification efforts, we estimated that 1 µg of total protein of SGE corresponds to 0.2-2 ng of anticoagulant. A crude estimate based on the similarity in molecular masses of FXa (46 kDa) and AFXa (56 kDa) is roughly consistent with a 1:1 ratio of inhibitor to enzyme expected for a physiological inhibitor.

Our kinetic data suggest that AFXa interacts with an exosite on FXa and not within the substrate binding domain. Furthermore, we did not observe the formation of an SDS-stable complex between FXa and crude salivary gland extract, although recently, the significance of these SDS-stable complexes has been questioned, since efforts to isolate the acyl intermediates have been unsuccessful (38). Based on these data, we predicted that AFXa would not share homologies with the serpin superfamily (10). However, the primary sequence analysis shows clearly that AFXa has structural similarities with serpins. The divergence of AFXa within the hinge region may account for the novel inhibition kinetics we observed and our inability to match the molecular structure of AFXa with its biochemical characteristics. Interestingly, the analysis of tick anticoagulant peptide demonstrated amino acid sequence similarity with Kunitz-type inhibitors, but the reaction kinetics are different from typical Kunitz-type inhibitors (22). Because serine protease inhibitors are typically regulatory molecules, while the anticoagulant serine protease inhibitors from hematophagous arthropods are necessary to facilitate their unique feeding strategies, the distinct evolutionary pressures on these molecules may account for the divergences observed among the typical serine protease inhibitors and anticoagulants. We are currently undertaking a biochemical analysis of recombinant AFXa to better determine its mechanism of inhibition.

There are three subfamilies of mosquitoes, two of which, the Anophelinae and Culicinae, are hematophagous. The molecular characterization of proteins involved in blood feeding may offer clues to the evolutionary relationship among these subfamilies (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 adaptation 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 mosquito anticoagulants, particularly the thrombin inhibitors from the Anophelinae.

    ACKNOWLEDGEMENTS

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 assistance with DNA sequencing and plasmid construction, and Lynn Olson for help preparing the manuscript.

    FOOTNOTES

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

Dagger Student in the Medical Scientist Program at the College of Medicine, 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{at}uci.edu.

The abbreviations used are: FXa, factor Xa; AEBSF, 4-(2-aminoethyl)benzenesulfonyl fluoride; bp, base pair(s); kb, kilobase pair(s); CAPS, 3-(cyclohexylamino)propanesulfonic acid; EDTA, ethylenediamine tetraacetic acid; PVDF, polyvinylidene difluoride; PAGE, polyacrylamide gel electrophoresis; SGE, salivary gland extract; PAI-2, plasminogen activator inhibitor-2.

2 Y. Y. Umekita, R. A. Richard, and S. Liao, unpublished GenBank accession number.

3 This sequence has GenBankTM accession number AF050133.

4 K. Stark, P. Ho, T. Schaub, and A. A. James, unpublished results.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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