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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 4 (August 15), 1999:
pp. 1418-1428
A Novel Serpin Expressed by Blood-Borne Microfilariae of the Parasitic
Nematode Brugia malayi Inhibits Human Neutrophil Serine
Proteinases
By
Xingxing Zang,
Maria Yazdanbakhsh,
Haobo Jiang,
Michael R. Kanost, and
Rick M. Maizels
From the Institute of Cell, Animal and Population Biology, University
of Edinburgh, Edinburgh, UK; the Department of Parasitology, Leiden
University Medical Center, Leiden, The Netherlands; and the Department
of Biochemistry, Kansas State University, Manhattan, KS.
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ABSTRACT |
Serine proteinase inhibitors (serpins) play a vital regulatory role
in a wide range of biological processes, and serpins from viruses have
been implicated in pathogen evasion of the host defence system. For the
first time, we report a functional serpin gene from nematodes that may
function in this manner. This gene, named Bm-spn-2, has been
isolated from the filarial nematode Brugia malayi, a causative
agent of human lymphatic filariasis. Polymerase chain reaction (PCR)
and Western blot experiments indicate that Bm-spn-2 is
expressed only by microfilariae (Mf), which are the long-lived
blood-dwelling larval stage. A survey of the greater than 14,000 expressed sequence tags (ESTs) from B malayi deposited in dbEST
shows that greater than 2% of the ESTs sequenced from Mf cDNA
libraries correspond to Bm-spn-2. Despite its abundance in the
microfilarial stage, Bm-spn-2 has not been found in any other
point in the life cycle. The predicted protein encoded by Bm-spn-2 contains 428 amino acids with a putative signal
peptide. Antibodies to recombinant Bm-SPN-2 protein react specifically with a 47.5-kD native protein in Mf extract. Bm-SPN-2 is one of the
largest of the 93 known serpins, due to a 22 amino acid
carboxy-terminal extension, and contains the conserved serpin signature
sequence. Outside these regions, levels of homology are low, and only a distant relationship can been seen to a Caenorhabditis elegans serpin. The Bm-spn-2 gene contains 6 introns, 2 of which appear to be shared by both nematode species. The B malayi introns
have an extended and conserved 3' splice site and are relatively
large compared with C elegans. A panel of mammalian serine
proteinases were screened and Bm-SPN-2 protein was found to
specifically inhibit enzymatic activity of human neutrophil cathepsin G
and human neutrophil elastase, but not a range of other serine
proteinases. It is possible that Bm-SPN-2 could function as a
stage-specific serpin in the blood environment of the microfilarial
parasite in protection from human immunity and thus may be a good
candidate for protective vaccine.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
SERINE PROTEINASE inhibitors (serpin)
genes comprise a large gene family,1 and their protein
products regulate a wide variety of proteinase-dependent physiological
functions, such as blood coagulation,2
fibrinolysis,3 activation of complement, and the
inflammatory response.4 In addition to these fluid-phase
reactions, serpins have critical roles in cell interactions and
mobility, eg, maspin suppresses invasion and motility of mammary tumor
cells5; plasminogen activator inhibitor 1 (PAI-1) blocks
migration of smooth muscle cells,6 whereas PAI-2 can
prevent apoptosis in cells exposed to tumor necrosis factor-
(TNF-).7
Infectious organisms have evolved an array of specific adaptations to
evade the host defence system.8.9 In view of the potency of
serpin regulation of biological processes, it seems likely that
pathogens may themselves encode serpins and use them to block host
defense functions. For example, virus-encoded serpins prevent the
proteolytic activation of interleukin-1 (IL-1),10-12 block granzyme activity,13 and protect infected cells from
Fas- and TNF-induced apoptosis.12,14 Less is known of
serpins from parasitic agents, but protease inhibitors have been
described that inhibit a diverse range of host functions, such as
neutrophil chemotaxis,15 blood
coagulation,16-18 and antigen processing.19
Lymphatic filariasis is one of the most important human tropical
diseases, with an estimated 120 million people infected and a further
900 million at risk of infection.20,21 It is caused by a
mosquito-transmitted nematode, Brugia malayi, which
migrates to the lymphatic vessels, where sexually reproducing adult
worms produce millions of microfilariae (Mf) that migrate into the
bloodstream, where they can reside for long periods ( 1 year). The
mechanisms by which circulating Mf avoid host immune defenses are
poorly understood.
Most serpin family members so-far characterized are from mammals,
insects, and viruses. Although a few serpin cDNA sequences have been
reported from helminth parasites,22,23 only in the free-living nematode Caenorhabditis elegans has the genomic
sequence of a serpin been determined.24 As part of our
ongoing research on identification of vaccine candidates from the human
filarial nematode B malayi, we have found a novel serpin gene
from the Mf stage of B malayi. We report here the isolation,
sequencing, and expression of this novel serpin gene, Bm-spn-2.
Moreover, we show that recombinant Bm-SPN-2 inhibits the enzymatic
activity of 2 serine proteinases from human neutrophils, cathepsin G
and elastase, suggesting a functional interaction between parasites and
host cells in the same blood environment.
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MATERIALS AND METHODS |
Antigens and cDNA expression libraries.
Mf were obtained from B malayi-infected jirds (Meriones
unguiculatus) purchased from TRS Laboratories (Athens, GA). Mf
obtained by extensive lavage of the peritoneal cavities of infected
jirds were separated from host cells over lymphocyte separation media and then passed through a Sephadex G-25 column.25 Fractions containing Mf without host cells were pooled. Mf proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the protein fraction of molecular weight 33-55 kD was
recovered by electro-elution. Protein concentrations were measured
using the Bradford assay (Pierce, Rockford, IL).
Six B malayi stage-specific cDNA expression libraries from L3,
adult males, adult females, and Mf were supplied by the Filarial Genome
Project (Williams et al, unpublished data; and Scott et al, unpublished
data).26,27 Further details on construction of
these libraries and their availability are posted at
http://helios.bto.ed.ac.uk/mbx/fgn/net/fgpresource.html.
Mice and immunization.
Eight-week-old female BALB/c mice were injected with 50 µg of either
Mf proteins with molecular weight 33-55 kD or Bm-SPN-2 recombinant
protein (see below) in complete Freund's adjuvant and boosted 4 weeks
later. Mice were killed at 6 weeks and antisera were collected.
Isolation and sequencing of Bm-spn-2 cDNA clone.
A B malayi Mf cDNA expression library was screened using immune
sera, preadsorbed against Escherichia coli lysate, obtained from BALB/c mice immunized with Mf proteins of molecular weight 33-55 kD. The immunoreactive plaques, detected using peroxidase-conjugated rabbit antimouse IgG (DAKO, Glostrup, Denmark) and the TMB membrane peroxidase substrate system (KPL, Gaithersburg, MD), were purified to
homogeneity by 2 subsequent rounds of screening. Bluescript phagemids
were then excised from positive clones using the manufacturer's protocols and sequenced.
Isolation and characterization of Bm-spn-2 gene.
Genomic DNA was isolated from B malayi parasites. Mf were lysed
and digested in lysis buffer (100 mmol/L NaCl, 50 mmol/L EDTA, 1% SDS,
1% 2-ME, 10 µg/mL RNase, 100 µg/mL proteinase K, 100 mmol/L Tris-Cl, pH 8.5) at 65°C for 30 minutes and then extracted with phenol, phenol/chloroform, and chloroform. The DNA was precipitated and
washed with ethanol, and then resuspended in TE buffer. Genomic DNA
(100 µg/mL) was heated at 100°C for 10 minutes, put on ice for 10 minutes, and then used as template for polymerase chain reaction (PCR)
amplification using the following primers: SF1 (nt 1-26 of
Bm-spn-2 cDNA), 5'-AATATTGGCAATTCGCAATTATCCTC-3'; and SR4 (nt 780-761), 5'-ACGGTAGCGACACCGCTTGC-3'; or SF4
(nt 564-584), 5'-GGAGCCCGTAATATCGCTAGC-3'; and SR1 (nt
1418-1391), 5'-AAATTAATGCATTTTTTATTCAACATCA-3'. The PCR
reactions were performed 94°C for 5 minutes and 30 cycles of
94°C for 40 seconds, 55°C for 40 seconds, and 65°C for 2 minutes, with a final 10-minute extension period at 65°C. The
resulting products (SF1/SR4, 1,524 bp; SF4/SR1, 1,456 bp) were purified (GENECLEAN II; BIO 101, La Jolla, CA) and subcloned into
pMOSBlue T-vector (Amersham, Buckinghamshire, UK). At
least 4 independent clones of each target fragment were picked and
sequenced. Introns and exons were identified by comparing genomic
sequence and cDNA sequence.
Library PCR.
PCRs were performed on 1-µL aliquots of each cDNA library from B
malayi L3, adult males, adult females, and Mf, with the following gene-specific primers: SF3 (nt 261-280),
5'-GCCAGAGGTGAAACTGAGCG-3'; and SR4 (as above). Primers for
the B malayi tumor protein homolog-1 (tph-1)
corresponded to the published sequence28:
5'-AATGTTGATCTTCAAGGGATGCATTCAC-3' and
5'-TTTGTTTTTCTTCAATGAGTGCCTCCTT-3'. Forty cycles of
amplification were performed under the following cycling conditions:
94°C for 1 minute, 50°C for 1 minute, and 72°C for 50 seconds.
Reverse transcriptase-PCR (RT-PCR).
Total RNA from L3, adult males, adult females, and Mf was isolated
using RNAzol B Isolation of RNA kits (AMS, Witney, Oxfordshire, UK).
Reverse transcription was performed using oligo(dT) as the first primer
and 1 µg of total RNA according to the GeneAmp RT-PCR kit protocol
(Perkin-Elmer, Branchburg, NJ). Amplification of Bm-spn-2 cDNA
was performed using 1 µL of first-strand cDNA, with the following
gene-specific primers: SF5 (nt 886-906),
5'-GTGCACTTGACTCATCTCACG-3'; and SR2 (nt 1310-1283),
5'-CTAACCTTTGTCTTTTTTTCGGTGTTTCC-3'. Primers for the
tph-1 were used as described above. Thirty-five cycles of
amplification were performed under the following cycling conditions: 94°C for 1 minute, 55°C for 1 minute, and 72°C for 2 minutes.
5' rapid amplification of cDNA ends (5' RACE).
Total RNA from Mf was isolated using RNAzol B. Reverse transcription
was performed using SR4 (as described above) as the first primer and 2 µg of total RNA according to the 5'/3' RACE kit protocol (Boehringer Mannheim, Mannheim, Germany). Amplification
of the 5' end of Bm-spn-2 cDNA was performed using 5 µL
of dA-tailed first-strand cDNA, with oligo dT-anchor primer and a
gene-specific primer SR5 (nt 571-553)
5'-CGGGCTCCATAATTCATAC-3'. The resulting products were
purified (Microcon, Beverly, MA), subcloned into pGEM T-vector
(Promega, Madison, WI), and then sequenced.
Expression and purification of Bm-SPN-2 protein.
A cDNA fragment encoding Bm-SPN-2 without the putative N-terminal
signal peptide (amino acid residues 1-20) was amplified by PCR from the
Mf cDNA library using the following primers: SF2 5'-CAACAGTACTTTAAACCATTGTTCTG-3' and SR2 (as described
above). These primers were designed with an additional 5'
nucleotide for insertion and in-frame expression with the pET-29
T-Vector (Novagen, Madison, WI). PCR conditions were 35 cycles of
94°C for 1 minute, 55°C for 1 minute, and 72°C
for 2 minutes. The resulting 1,226-bp product was purified (GENECLEAN
II; BIO 101), ligated into pET-29 T-Vector, sequenced completely, and
then transformed into BL21(DE3) strain of E coli.
Transformed BL21(DE3) cells were incubated in LB containing kanamycin
(50 µg/mL) at 37°C for 4 hours and then 1 mmol/L
isopropyl-1--D-galactopyranoside (IPTG; Stratagene, La Jolla, CA)
was added. The cells were incubated for an additional 4 hours at 37°C and then at 4°C overnight. Cells were harvested
and sonicated. The recombinant Bm-SPN-2 containing 6 C-terminal
histidine residues (His-Tag) was purified by affinity column
chromatography over His-Bind resin (Novagen) under native conditions.
Genomic Southern blot.
Amplification of a B malayi genomic fragment was performed by
PCR using genomic DNA and gene-specific primers SF4 (as described above) and SR4 (as described above). A 408-bp PCR product, spanning exon 4, intron 4, and exon 5, was 32P-labeled by random
hexanucleotide priming and then used as probe. Five micrograms of B
malayi genomic DNA was digested with BamHI, EcoRI,
Xba I, and Xho I. Digested DNA was fractionated on a
0.8% agarose gel, transferred to nylon membranes (Hybond-N), and
hybridized with 32P-labeled probe. Blots were hybridized
for 24 hours at 65°C in a rotary hybridization oven (Hybaid) in
3× standard sodium citrate (SSC), 1% SDS, and 0.2% skimmed milk
containing 100 µg/mL sonicated herring sperm DNA. Washes
were performed at 65°C in 2× SSC, 1% SDS for 30 minutes and
then at room temperature in 0.1× SSC for 30 minutes, before
autoradiography at  70°C.
Western blot.
Lysates of L3, adult males, adult females, Mf, and BL21(DE3) cells
containing the pET-29/Bm-spn-2 plasmid after IPTG induction were prepared by resuspending pellets in denaturing SDS-PAGE sample buffer and boiling for 10 minutes. Insoluble cellular debris was removed by centrifugation at 13,000 rpm for 30 minutes, and SDS-soluble proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes. Blot strips were incubated with mouse antisera to
recombinant Bm-SPN-2 protein, diluted 1/4,000, and then with
peroxidase-conjugated rabbit antimouse IgG (DAKO). The bound antibodies
were detected by chemiluminescence on the addition of the luminol-based
ECL substrate (Amersham).
Sequence analysis.
All sequencing was performed on a Applied Biosystems 377 automated
sequencer by the ABI PRISM Dye Terminator cycle sequencing method
(Perkin-Elmer). Sequence analysis was performed using MacVector program
version 6.0 (Oxford Molecular Group, Oxford, UK). Sequence searching was performed with the BLAST algorithm29 using
the NCBI BLAST server. The GCG program PILEUP (Genetics Computer Group, University of Wisconsin, Madison, WI) and BOXSHADE were
used to perform multiple alignments. The tree reflecting evolutionary relationships was constructed by PAUP 3.1.130 using maximum parsimony, excluding constant, and uninformative sites.
Inhibitory activity assay.
To investigate the inhibitory activities of Bm-SPN-2, 5 µL of each
individual serine proteinase was incubated with 10 µL of Bm-SPN-2
(0.4 µg/µL) in TBS buffer (150 mmol/L NaCl, 20 mmol/L Tris-HCl, pH
7.8) or bovine serum albumin (0.4 µg/µL) in TBS buffer as negative
control. After 10 minutes of incubation at room temperature, 0.7 mL of
an appropriate peptidyl-p-nitroanilide substrate solution (100 µmol/L in 50 mmol/L Tris-HCl, pH 7.8, 50 mmol/L NaCl) was added to
the mixture, and the residual enzyme activity was measured by
monitoring the absorbance change at 405 with time.31 The enzymes and their substrates used in the tests were bovine pancreatic trypsin (50 ng/µL; Sigma, St Louis, MO) and
D-Phe-L-pipecolyl-Arg-p-nitroanilide (Sigma); human plasmin
(0.1 µg/µL; Athens Research & Technology, Athens, GA) and
Z-D-Phe-Pro-Arg-p-nitroanilide (where Z stands for
benzyloxycarbonyl; a gift from Dr J. Tomich, Microchemical Core
Laboratory, Kansas State University); bovine pancreatic
-chymotrypsin (0.1 µg/µL; Sigma) and
N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (Sigma);
human neutrophil cathepsin G (1 µg/µL; Athens Research & Techology,
Inc) and N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (Sigma); porcine pancreatic elastase (0.1 µg/µL; Worthington, Lakewood, NJ) and
N-succinyl-Ala-Ala-Pro-Leu-p-nitroanilide (Sigma); and
human neutrophil elastase (1 µg/µL; Athens Research & Techology, Inc) and N-succinyl-Ala-Ala-Pro-Leu-p-nitroanilide
(Sigma). Where the proteinase activities in the presence of the
Bm-SPN-2 were lower than the control, a possible serpin-enzyme
interaction was further examined. Aliquots of the proteinase (1.0 µg/µL, 4.0 µL) were mixed with 0, 5, 10, 15, and 20 µL of the
concentrated Bm-SPN-2 (5.0 µg/µL). Different amounts of bovine
serum albumin (0.4 µg/µL) were also included to stabilize the
enzyme and to adjust the final volume up to 25 µL. After incubation,
residual enzyme activities were determined as described above.
Clotting assays were performed with an activated partial thromboplastin
time (APTT) kit (Sigma) to test whether Bm-SPN-2 inhibits the activity
of serine proteinases in the human coagulation pathway. Pooled normal
plasma (0.1 mL) and 0.1 mL of various concentrations of Bm-SPN-2 or TBS
buffer alone were incubated at 37°C for 5 minutes, and then 0.1 mL
of prewarmed APTT reagent was added. After 3 minutes, 0.1 mL of
prewarmed 0.020 mol/L calcium chloride solution was added and the
clotting time was recorded.
Assays of macrophage antigen processing and presentation were performed
to investigate whether Bm-SPN-2 inhibits the activity of serine
proteinases involved in the major histocompatibility complex (MHC)
class II pathway within macrophages. The murine macrophage cell line J774A.1 was treated with various concentrations of
Bm-SPN-2 for 30 minutes, and then viable Streptococcus pyogenes or synthetic peptides aa 17-31 or aa 308-319 of the S.pyogenes M5 protein were added and incubated for 3 hours. The macrophages were
fixed and incubated with the T-cell hybridoma, HX17 recognizing peptides aa 17-31, and HY2 recognizing peptides aa 308-319 for 24 hours. The culture supernatants were collected for IL-2
assay.32
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RESULTS |
Isolation and characterization of a cDNA for Bm-spn-2.
Studies of B malayi Mf proteins were undertaken to identify
prominent antigens recognized by the host immune system's T
lymphocytes. One fraction from Mf, containing proteins of 35-55 kD,
proved highly potent at inducing antigen-specific T-cell proliferation and cytokine production (Zang et al, unpublished data).
Antibodies were raised against this fraction by
immunizing BALB/c mice and were used to screen an Mf cDNA expression
library to identify the principal antigenic proteins. Three positive
clones were isolated, one of which was found to be a novel cDNA insert
and was analyzed as described below.
The sequence of the full-length cDNA was determined and is shown in
Fig 1A. The cDNA insert
contained 1,438 bp and a single open reading frame (ORF) starting at nt
27 and ending at nt 1311, which was followed by two more stop codons
and a potential polyadenylation signal sequence (AATAAA, nt 1398-1404)
15 to 20 nt upstream from a poly A tail. Like most B malayi
cDNAs, the GC content was low (35.1%). The ORF encoded a 428 amino
acid protein with 2 potential N-glycosylation sites at
asparagines 21 and 360 of the deduced amino acid sequence. Residues 1 through 20 formed a very strong hydrophobic region consistent with a
signal peptide function. The predicted mature protein, without this
signal sequence, would have a molecular mass of 47.5 kD and an
isoelectric point of 6.9.
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| Fig 1.
(A) Nucleotide and deduced amino acid sequences of the
Bm-spn-2 cDNA. Inferred amino acid numbering is in boldface.
The putative hydrophobic signal peptide (amino acids 1-20) is
underlined and 2 potential N-glycosylation sites (amino acids
21 and 360) are circled. The serpin motif (amino acids 353-357) and
serpin signature (amino acids 377-387) are boxed. The position of a
potential polyadenylation signal (nucleotide 1398-1404) is in boldface.
Primers used in PCRs are indicated with solid arrow. The nucleotide
sequence has been deposited in GenBank with the accession no. AF009825.
(B) Schematic of the gene structure of Bm-spn-2. The gene spans
2.7 kb and is split into 7 exons and 6 introns, as depicted. The
nucleotide sequence has been deposited in GenBank with the accession
no. AF009826.
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In GenBank searches for similar amino acid sequences, 85 significant
matches were found by the BLAST algorithm,29 all of which
belonged to the serine proteinase inhibitor, or serpin, superfamily.
One member of this superfamily from B malayi, previously described by Yenbutr and Scott,23 encodes a distantly
related gene we now refer to as Bm-spn-1. In light of this
similarity, the novel protein was designated Bm-SPN-2, and its gene was
designated Bm-spn-2.
Isolation and characterization of the Bm-spn-2 gene.
The Bm-spn-2 cDNA clone contained a 26-bp 5'-untranslated
region (UTR) and a 105-bp 3'-UTR, which allowed us to isolate the genomic copy of Bm-spn-2 by PCR. The Bm-spn-2
gene spanned 2572 bp (Fig 1B) and, on comparison to the cDNA sequence,
was found to consist of 7 exons and 6 introns with the intron/exon
boundaries conforming to the normal pattern (exon/GT...AG/exon) of
splice acceptor and splice donor sites. Intron lengths were remarkably
uniform, varying from 182 to 210 bp. At position 5 at the
3' end of the introns, T was present in all 6 introns. Digests of
B malayi genomic DNA with BamH I, EcoRI,
Xba I, and Xho I all yielded single bands that
hybridized with the 32P-labeled probe (data not shown),
indicating that Bm-spn-2 is either a single copy gene or is
represented by copies in identical genomic environments.
A comparison was made between Bm-spn-2 and the 12 genes from
B malayi for which full genomic sequence information is
available in GenBank. These contained a total of 99 exons and 86 introns, with each gene containing between 2 and 27 exons. The majority of both exons and introns range from 60 to 240 bp, with only 3/86 introns being smaller than 60 bp (Fig 2).
In this respect, Bm-spn-2 appears to be a typical B
malayi gene, in marked contrast to C elegans in which more
than half of the introns are shorter than 60 bp.33 Although
other lower eukaryotes show a marked shift towards AT abundance in
intronic sequences,34 the AT-richness of exons in the 13 known B malayi genes attenuates this effect. B malayi
exons and introns are 62% and 69% AT, respectively, whereas the
corresponding figures for C elegans are 54% and
70%.33 Although B malayi introns conform to the
GT-AG rule, in rare instances, GC or TT were used as 5' splice
sites and AC used as 3' splice site
(Table 1). B malayi introns have an
extended and conserved 3' splice site consensus sequence, TTTCAG,
in which the 5 position is T in 93% of the cases (Table 1).
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| Fig 2.
Length distribution of exons and introns of all 13 B
malayi genes so far characterized. The survey included 99 exons and
86 introns. Each bar represents the number of exons and introns in each
size class.
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Mf-specific abundant transcription of Bm-spn-2 gene.
Two Bm-spn-2 gene-specific primers, SF3 in exon 2 and SR4 in
exon 5, were designed to amplify a 520-bp region of cDNA. Using 4 B
malayi stage-specific cDNA expression libraries from L3, Mf, adult
males, and adult females as templates, transcription of Bm-spn-2 was shown to be restricted to the Mf stage (data not shown). The identity of a 520-bp PCR product from the Mf cDNA library
was confirmed by sequencing. First-strand cDNA were then prepared from
these 4 stages and RT-PCR was performed with 2 gene-specific primers,
SF5 in exon 5 and SR2 in exon 7, and generated a product of the
expected length (425 bp) only in Mf-stage
(Fig 3). The absence of a Bm-spn-2
product in the adult female implies that embryonic Mf within the uterus
do not transcribe this gene. Parallel PCRs were performed with primers
specific for another B malayi gene, tph-1, which is
known to be expressed in all stages.28 A
tph-1-specific 575-bp band was found in all 4 stages from cDNA expression libraries or RNAs. In this respect, Bm-spn-2 differs quite markedly from Bm-spn-1, which is expressed in all
life-cycle stages.23
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| Fig 3.
Transcription of Bm-spn-2 at different
development stages of B malayi. RNAs isolated from L3, Mf,
adult males (lane M), and adult females (lane F) were processed to
obtain first-strand cDNA. The cDNAs were then used as templates for PCR
amplification of a 425-bp fragment from Bm-spn-2 or a 575-bp
fragment from B malayi tph-1.
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To date, the Filarial Genome Project has sequenced a total of
approximately 14,351 expressed sequence tags (ESTs), including 2,334 ESTs from Mf. Of these, 49 ESTs corresponded to Bm-spn-2, all
of which were isolated from the Mf cDNA library. This suggests an
abundance level of 2.1% of Mf mRNAs. These data are consistent with
our PCR results and confirm that Bm-spn-2 is an unusually highly expressed transcript in the Mf stage.
The trans-spliced leader (SL) primer and a reverse primer in exon 5 were designed for PCR using the Mf cDNA library or first-strand cDNA
from Mf as templates. Even after 40 rounds of amplification, no band
was detected in ethidium-stained gels (data not shown). Moreover, 2 reverse primers in exon 5 and 4 were designed for 5' RACE and no
SL was present at the 5' end of Bm-spn-2 cDNA, suggesting
that Bm-spn-2 mRNA is not trans-spliced. This forms another contrast to Bm-spn-1, which is known to contain the
spliced leader sequence in its mRNA.23
Expression of Bm-SPN-2 in E coli and purification of the
recombinant protein.
The Bm-spn-2 cDNA ORF without the signal peptide was subcloned
into the pET-29 E coli expression vector, and recombinant
Bm-SPN-2 was expressed as a fusion protein containing a C-terminal
hexahistidine-tag for affinity purification. The recombinant fusion
protein was expressed at very high levels on induction with IPTG, and
soluble material was purified by affinity chromatography. The purified recombinant Bm-SPN-2 had an apparent molecular mass of 52 kD on SDS-PAGE (Fig 4).
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| Fig 4.
Recombinant and native Bm-SPN-2 protein. Western
blot of lysates of BL21(DE3) cells containing the pET-29/Bm-spn-2
plasmid after IPTG induction (lane 1) and extracts of Mf (lane 2),
L3 (lane 3), adult males (lane 4), and adult females (lane 5) probed
with mouse antirecombinant Bm-SPN-2. Recognition of a 52-kD band in
lane 1 and of a 47.5-kD band in lane 2 is arrowed.
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Native protein.
Specific antibodies obtained from mice immunized with recombinant
Bm-SPN-2 bound the recombinant Bm-SPN-2 protein and also recognized a
single endogenous Bm-SPN-2 protein in Western blots of Mf proteins as
antigens (Fig 4, lane 2); no reactivity was seen with normal mouse
sera. The native Bm-SPN-2 had an apparent molecular mass of 47.5 kD.
This result was in good agreement with the predicted molecular weight
of the mature protein encoded by Bm-spn-2 ORF. Western blot
analyses were also performed with proteins from L3, adult males, and
adult females, but no reactivity was found (Fig 4, lanes 3 through 5).
These data confirm that Bm-spn-2 is expressed only in the Mf stage.
Homology with other serpins.
The amino acid sequence of Bm-SPN-2 shows a low level of overall
similarity with the other identified serpins at the level of 20% to
26%. However, certain characteristic features of serpins are evident,
and of the 51 residues identified as highly conserved within the serpin
superfamily,35 33 are identical in Bm-SPN-2 and several
more show conservative substitutions (Fig
5A). Figure 5A compares Bm-SPN-2 with 5 known serpins, namely human
1-antitrypsin (1-AT),35 human squamous cell carcinoma
antigen 1 (SCCA1),36 C elegans serpin
(Ce-spn),24 chicken ovalbumin-like gene Y protein (gene
Y),37 and mouse proteinase inhibitor 3 (SPI3).38
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| Fig 5.
Comparison of Bm-SPN-2 with other serpins: human
 1-antitrypsin (1-AT), C elegans serpin (Ce-spn), chicken
gene Y protein (gene Y), mouse proteinase inhibitor 3 (SPI3), and human
squamous cell carcinoma antigen 1 (SCCA1). (A) Amino acid sequences:
the alignment of amino acid sequences using PILEUP and BOXSHADE
programs was based on the 1-AT crystal structure35,40 in
which the conserved helices and strands are defined and was manually
adjusted to give the best fit. Gaps were introduced in sequences for
optimal alignment. The 1-AT sequence is given without the signal
sequence (MPSSVSWGILLLAGLCCLVPVSLA). Identical residues are highlighted
in black and similar residues in gray shading. The 51 starred residues
are those categorized as conserved in the larger serpin superfamily.
The scissile bond is marked with an arrow. (B) Intron/exon positions in
serpins. Thick lines indicate coding regions, thin lines indicate
untranslated regions, and gaps have been introduced to optimize
alignment; arrows marked by uppercase letters indicate positions of
introns. Sequences were aligned on the basis of amino acid similarity
with the scale showing amino acid positions beginning with the first
methionine of Bm-SPN-2. Introns are shaded according to their phase:
open (phase 0), shaded (phase 1), and solid (phase 2). Vertical lines
connect introns considered to be in identical positions.
|
|
The serpin signature of FRANHPFLYAI (aa 377-387, Fig 1A) corresponds
well to that, for example, of chicken gene Y, FRADHPFLFFI. The serpin
motif or hinge is less well conserved, with only E353 and G355
conforming to the consensus. Between these two motifs is the reactive
site loop of serpins, a stretch of approximately 14 amino acids
containing the cleavable bait attacked by proteases. Serpin sequences
here show extensive variation,31,39 and Bm-SPN-2 is
dissimilar to any known serpin in this region. Other novel structural
features of Bm-SPN-2 include the absence of a highly conserved
dipeptide Ile-Asn from within helix F and the proximity of a potential
N-glycosylation site between the serpin motif and the reactive
loop. Most interestingly, of all 93 serpin sequences deposited in
public databases, Bm-SPN-2 is one of the largest and has the longest
C-terminus other than antiplasmin.35 All of these features
would be more clearly understood in the light of a structure for the
protein. The 3-dimensional structures of serpins such as
1-AT,40 PAI-1,41 and antithrombin
III,42 have been solved, and these provide a useful
framework for analyzing other members of the superfamily.
At the level of gene structure, Bm-spn-2, which contains 6 introns, also made an interesting comparison with 5 other serpins (Fig
5B). Introns E and F of Bm-spn-2 are positioned exactly as introns C and D in Ce-spn, supporting a close relationship
between the nematode genes, but no vertebrate serpins contained introns at the same point. The genes for chicken gene Y protein, murine SPI3
and human SCCA1, had 5 identical intron positions, but these were not
found either in the mammalian 1-AT gene or in either of the nematode sequences.
Phylogenetic analysis.
Of the 93 members of serpin superfamily currently in accessible
databases, 62 are vertebrate (mainly mammalian) and 11 are from
invertebrates, including insects, crayfish, horseshoe crabs, and
nematodes, whereas 5 are from plants and 15 are from viruses. A
phylogenetic comparison of all invertebrate serpins was performed using
PAUP to indicate the likely evolutionary relationship of Bm-spn-2 to known genes (Fig 6).
This analysis divides invertebrate serpins into 2 groups (91%
bootstrap confidence): group I comprising serpins from Tachypleus
tridentatus (horseshoe crab), and group II containing serpins from
B malayi, C elegans, Bombyx mori (Silkworm), Manduca sexta (tobacco hornworm), and Pacifastacus
leniusculus (signal crayfish). Within the latter group,
Bm-spn-1 and Ce-spn form a subgroup that is quite
distant from Bm-spn-2.
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| Fig 6.
Phylogenetic trees showing the relationship between the
predicted protein encoded by Bm-spn-2 and all other recorded
invertebrate serpins. Numbers above branches show the percentage of
bootstrap support for each clade. GenBank, Protein Identification
Resource, and SWISS-PROT accession numbers of the sequences used were
as follows: T tridentatus intracellular coagulation inhibitor
2, A55533; T tridentatus intracellular coagulation inhibitor,
D14483; Bm-spn-1 (Bm-SERPIN), U04206; Ce-spn (C elegans
serpin), U50301; B mori antitrypsin, D00738; B mori
antichymotrypsin II, P80034; M sexta serpin-1, U58361; M
sexta serpin-2, U79184; B mori antichymotrypsin, D13895;
P leniusculus proteinase inhibitor II, X82642;
Bm-spn-2, AF009825.
|
|
Inhibitory activity of recombinant Bm-SPN-2.
To screen for potential inhibitory function of Bm-SPN-2, we tested its
ability to inhibit a panel of mammalian serine proteinases with
different substrate specificities. Bm-SPN-2 inhibited the enzymatic
activity of the human neutrophil cathepsin G and human neutrophil
elastase in a dose-dependent manner (Fig
7), but showed no inhibitory activity against bovine pancreatic
trypsin, human plasmin, bovine pancreatic -chymotrypsin, and porcine
pancreatic elastase. The inhibition of the neutrophil proteinases by
Bm-SPN-2 is strikingly specific, considering that human neutrophil
cathepsin G and bovine pancreatic -chymotrypsin hydrolyze the same
substrates as do human neutrophil elastase and porcine pancreatic
elastase.
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| Fig 7.
Concentration-dependent inhibition of human neutrophil
cathepsin G and human neutrophil elastase activation by Bm-SPN-2.
Recombinant Bm-SPN-2 at different concentrations was mixed with human
neutrophil cathepsin G or human neutrophil elastase before the addition
of specific substrates. Changes in the rate of substrate cleavage were
used to calculate the percentage of inhibition at each Bm-SPN-2
concentration.
|
|
To test whether Bm-SPN-2 inhibits the activity of serine proteinases in
human coagulation pathway, the APTT was measured in the presence of
Bm-SPN-2, but no prolongation of the APTT was observed (data not
shown). This suggests Bm-SPN-2 dose not inhibit the serine proteinases
of the clotting pathway such as factor X and Hageman factor.
Serine proteinases are involved in processing and presentation of 2 epitopes of the S pyogenes M5 protein to CD4+ T
lymphocytes.32 To investigate whether Bm-SPN-2 inhibits
this antigen-processing pathway, the ability of macrophages to present these epitopes from S pyogenes to T cells was measured in the presence of Bm-SPN-2. No change in stimulation was observed (Delvig et
al, unpublished observation). This suggests that Bm-SPN-2
does not inhibit the activity of serine proteinases involved in antigen processing within macrophages.
| |
DISCUSSION |
The wide distribution of serpins and their ability to regulate a
variety of divergent proteinases show that they play a major regulatory
role in a host of biological processes. We describe here the isolation,
characterization, expression, and inhibitory function of a new serpin
gene, Bm-spn-2, from the human lymphatic filarial nematode
B malayi. The Bm-spn-2 gene, which appears to be a
single copy in the genome and consists of 7 exons, was found to have Mf
stage-specific expression. The 2 serpins that have been found in B
malayi, Bm-spn-1 and Bm-spn-2, are quite different. They share low homology and are not closely related, as shown by
phylogenetic analysis. Bm-spn-1 is expressed in most life
stages and its mRNA contains the 22 nt nematode 5' SL, whereas
Bm-spn-2 is expressed only in the Mf stage and its mRNA does
not contain SL. At present, the gene structure of Bm-spn-1 is
not available. It will be of interest to compare the gene structure,
chromosome position, and physiological roles of these two serpins.
We analyzed 13 B malayi genes for which complete sequence is
available and found that Bm-spn-2 was typical of this set.
B malayi genes have relatively large introns compared with the
nematode model C elegans33 and, like C
elegans, the introns are markedly AT-rich. This is a property
shared with introns of other invertebrates and plants34,43
that is possibly related to effective recognition and splicing of
introns.43 It is interesting to note that B malayi
introns have an extended and conserved 3' splice site in which T
at 5 position is very highly conserved, a feature noted previously only in C elegans.33 In
contrast, introns from most other organisms have only a combination of
an upstream polypyrimidine tract and a YAG consensus at their 3'
boundaries. This suggests that the 3' intron boundary may be a
more important element in B malayi intron recognition than in
most other organisms. Unusually, the exon sequences in these 13 B
malayi genes are also AT-rich, much more so than C elegans
exons, and no explanation is yet available for this finding.
The serpin gene family predates the common ancestor of nematodes,
arthropods, and chordates and may consequently be more than 1,200 million years old.44 Based on vertebrate serpin gene
organization, it has been proposed that the ancestral gene would have
had a large number of introns and that modern serpin genes derived from this by duplication have undergone varied intron
deletion.45 This model, consistent with the general
introns-early hypothesis,46 contrasts with a gain-and-loss
explanation, which would envisage an ancestral serpin gene with between
0 and 3 introns. A comparison of the intron/exon structure of
Bm-spn-2 with 5 other serpins show sets of introns found only
in nematode genes and others present only in vertebrate genes. Several
serpin genes also possessed unique introns. Under the introns early
mechanism, the ancestral serpin gene would have needed nearly 20 introns to account for the pattern observed in just 7 extant genes.
Therefore, it seems that intron gain-and-loss is a more likely
explanation of serpin gene evolution, as postulated for other gene
families.47 We hope that, as more genes encoding serpin
become available, the serpin family can be divided into subgroup on the
basis of gene organization and position, and this may provide an
alternate way to advance our fundamental knowledge of evolutionary relatedness.
The serpin superfamily has been constructed on the basis of sequence
similarities at the protein level.48 All members of the
serpin superfamily are thought to share a common highly ordered tertiary structure, defined by x-ray diffraction for the prototype molecule 1-AT35,40 which consists of 9 -helices and
3 -sheets. Although the predicted amino acid sequence of Bm-SPN-2
has a low overall homology to the sequences of serpins from other
species, its sequence is identical or conserved at most of the
characteristic amino acid positions. The serpin signature, which
includes the hinge distal to the reactive loop, is, for example, well
conserved. However, analysis of the serpin motif (in the proximal
hinge) is ambiguous. Two residues in Bm-SPN-2 (E353 and G355) are those found in 100% of functional serpins.49 Few of the other
residues in Bm-SPN-2 follow the consensus for small nonpolar
sidechains, which are thought to be necessary to allow the
conformational change associated with inhibitory
function.49,50 Further work will establish whether the
consensus sequence requires redefinition in the light of
phylogenetically removed genes such as those from nematodes and
trematodes.22
Genes that are expressed in a stage-specific manner have important
functional roles in the parasite life cycle and thus may provide
targets for the development for novel immunoprophylactic or
chemoprophylactic agents. It is significant that Bm-spn-2 is an
abundant transcript restricted to the Mf stage, and the presence of a
putative signal peptide (amino acids 1-20) indicates that that the
mature protein could be released as a secretory product to perform some
function in parasite survival. In several systems, serpins from
infectious organisms have been identified as factors that help
infectious organisms to evade the host defence
system.10,12,15,18 The ability of Bm-SPN-2 to inhibit 2 human neutrophil-derived serine proteinases, cathepsin G and elastase,
may be important in this context. Genes encoding cathepsin G, cathepsin
G-like lymphocyte granzymes B and H, and / chains of the T-cell
receptor are closely linked on human chromosomal band
14q11.2.51 Stimulated polymorphonuclear neutrophils release
cathepsin G concomitantly with neutrophil elastase, and these
proteinases mediate antimicrobial activity,52 degradation
of extracellular matrix, vasoregulation, and IL-8
processing.53 Furthermore, cathepsin G binds to T
lymphocytes and NK cells54,55 and exerts a mitogenic effect
on T lymphocytes, which are dependent on retaining enzymatic
activity.56 Cathepsin G is also known to be a chemokinetic
stimulant for T lymphocytes and chemoattractant for
monocytes.55 We suggest that neutrophils, representing 55%
of human blood leukocytes, would be the primary cell type to interact
with B malayi Mf and that release of Bm-SPN-2 neutralizes the
stimulating properties of cathepsin G. It is interesting to note a
study that identified a human squamous cell carcinoma antigen, SCCA2,
as a novel serpin that also inhibits cathepsin G.57 These
data suggest that a possible common strategy between parasites and
tumors is the production of a cathepsin G inhibitor to counteract
immune activation.
One of the remarkable features of the Mf stage of B malayi is
its longevity (>1 year in the bloodstream), which is testimony to the
effectiveness of immune evasion by this parasite. The survival of Mf
provides not only a reservoir of infection in an endemic community, but
also a clear target for intervention. Bm-SPN-2 seems an important
component of parasite survival strategies, so it may prove to be an
equally appropriate target for vaccination or pharmaceutical attack to
clear infection both from afflicted individuals and endemic
communities. Our preliminary results show that specific immune
responses against Bm-SPN-2 do indeed mediate the clearance of B
malayi Mf, supporting the contention that this serpin may be a
critical component of the host-parasite relationship.
| |
ACKNOWLEDGMENT |
The authors thank the Filarial Genome Project for cDNA libraries; Drs
Alexei Delvig and John Robinson (University of Newcastle) for testing
Bm-SPN-2 in an antigen processing assay; and Alex Loukas, Bill Gregory,
Andrew MacDonald, Franco Falcone, and Mark Dorris for invaluable
assistance. We also thank Drs Judith Allen and Mark Blaxter for advice
and critical review of the manuscript.
| |
FOOTNOTES |
Submitted November 11, 1998; accepted April 19, 1999.
Supported by grants from the European Union (INCO-DC IC18-CT95-0014)
and from the National Institutes of Health (GM41247).
The nucleotide sequence data reported in this paper are available in
GenBank database under the accession nos. AF009825 and AF009826.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Rick M. Maizels, PhD,
Institute of Cell, Animal and Population Biology,
University of Edinburgh, Edinburgh EH9 3JT, UK; e-mail:
r.maizels{at}ed.ac.uk.
| |
REFERENCES |
1.
Potempa J, Korzus E, Travis J:
The serpin superfamily of proteinase inhibitors: Structure, function, and regulation.
J Biol Chem
269:15957, 1994[Free Full Text]
2.
Carrell R, Evans DL, Stein PE:
Mobile reactive centre of serpins and the control of thrombosis.
Nature
353:576, 1991[Medline]
[Order article via Infotrieve]
3.
Collen D, Lijnen HR:
Basic and clinical aspects of fibrinolysis and thrombolysis.
Blood
78:3114, 1991[Free Full Text]
4.
Travis J, Salvesen GS:
Human plasma proteinase inhibitors.
Annu Rev Biochem
52:655, 1983[Medline]
[Order article via Infotrieve]
5.
Zou Z, Anisowicz A, Hendrix MJ, Thor A, Neveu M, Sheng S, Rafidi K, Seftor E, Sager R:
Maspin, a serpin with tumor-suppressing activity in human mammary epithelial cells.
Science
263:526, 1994[Abstract/Free Full Text]
6.
Stefansson S, Lawrence DA:
The serpin PAI-1 inhibits cell migration by blocking integrin alpha V beta 3 binding to vitronectin.
Nature
383:441, 1996[Medline]
[Order article via Infotrieve]
7.
Dickinson JL, Bates EJ, Ferrante A, Antalis TM:
Plasminogen activator inhibitor type 2 inhibits tumor necrosis factor -induced apoptosis. Evidence for an alternate biological function.
J Biol Chem
270:27894, 1995[Abstract/Free Full Text]
8.
Gooding LR:
Virus proteins that counteract host immune defenses.
Cell
71:5, 1992[Medline]
[Order article via Infotrieve]
9.
Maizels RM, Bundy DAP, Selkirk ME, Smith DF, Anderson RM:
Immunological modulation and evasion by helminth parasites in human populations.
Nature
365:797, 1993[Medline]
[Order article via Infotrieve]
10.
Ray CA, Black RA, Kronheim SR, Greenstreet TA, Sleath PR, Salvesen GS, Pickup DJ:
Viral inhibition of inflammation: Cowpox virus encodes an inhibitor of the interleukin-1 converting enzyme.
Cell
69:597, 1992[Medline]
[Order article via Infotrieve]
11.
Petit F, Bertagnoli S, Gelfi J, Fassy F, Boucraut-Baralon C, Milon A:
Characterization of a myxoma virus-encoded serpin-like protein with activity against interleukin-1 -converting enzyme.
J Virol
70:5860, 1996[Abstract]
12.
Kettle S, Alcami A, Khanna A, Ehret R, Jassoy C, Smith GL:
Vaccinia virus serpin B13R (SPI-2) inhibits interleukin-1-converting enzyme and protects virus-infected cells from TNF- and Fas-mediated apoptosis, but does not prevent IL-1-induced fever.
J Gen Virol
78:677, 1997[Abstract]
13.
Quan LT, Caputo A, Bleackley RC, Pickup DJ, Salvesen GS:
Granzyme B is inhibited by the cowpox virus serpin cytokine response modifier A.
J Biol Chem
270:10377, 1995[Abstract/Free Full Text]
14.
Smith KG, Strasser A, Vaux DL:
CrmA expression in T lymphocytes of transgenic mice inhibits CD95 (Fas/APO-1)-transduced apoptosis, but does not cause lymphadenopathy or autoimmune disease.
EMBO J
15:5167, 1996[Medline]
[Order article via Infotrieve]
15.
Leid RW, Suquet CM, Bouwer HGA, Hinrichs DJ:
Interleukin inhibition by a parasite proteinase inhibitor, taeniaestatin.
J Immunol
137:2700, 1986[Abstract]
16.
Cappello M, Vlasuk GP, Bergum P, Hunag S, Hotez PJ:
Ancylostoma caninum anticoagulant peptide: A hookworm-derived inhibitor of human coagulation factor Xa.
Proc Natl Acad Sci USA
92:6152, 1995[Abstract/Free Full Text]
17.
Cappello M, Hawdon JM, Jones BF, Kennedy WP, Hotez PJ:
Ancylostoma caninum anticoagulant peptide: Cloning by PCR and expression of soluble, active protein in E. coli.
Mol Biochem Parasitol
80:113, 1996[Medline]
[Order article via Infotrieve]
18.
Stassens P, Bergum PW, Gansemans Y, Jespers L, Laroche Y, Huang S, Maki S, Messens J, Lauwereys M, Cappello M, Hotez PJ, Lasters I, Vlasuk GP:
Anticoagulant repertoire of the hookworm Ancylostoma caninum.
Proc Natl Acad Sci USA
93:2149, 1996[Abstract/Free Full Text]
19.
Bennett K, Levine T, Ellis JS, Peanasky RJ, Samlogg IM, Kay J, Chain BM:
Antigen processing for presentation by class II major histocompatibility complex requires cleavage by cathepsin E.
Eur J Immunol
22:1519, 1992[Medline]
[Order article via Infotrieve]
20.
World Health Organization:
Lymphatic Filariasis: The Disease and Its Control: WHO Technical Report Series 821. Geneva, Switzerland, World Health Organization, 1992.
21.
Michael E, Bundy DAP, Grenfell BT:
Re-assessing the global prevalence and distribution of lymphatic filariasis.
Parasitology
112:409, 1996
22.
Blanton RE, Licate LS, Aman RA:
Characterization of a native and recombinant Schistosoma haematobium serine protease inhibitor gene product.
Mol Biochem Parasitol
63:1, 1994[Medline]
[Order article via Infotrieve]
23.
Yenbutr P, Scott AL:
Molecular cloning of a serine proteinase inhibitor from Brugia malayi.
Infect Immunity
63:1745, 1995[Abstract]
24.
Wilson R, Ainscough R, Anderson K, Baynes C, Berks M, Bonfield J, Burton J, Connell M, Copsey T, Cooper J, Coulson A, Craxton M, Dear S, Du Z, Durbin R, Favello A, Fraser A, Fulton L, Gardner A, Green P, Hawkins T, Hillier L, Jier M, Johnstone L, Jones M, Kershaw J, Kirsten J, Laisster N, Latreille P, Lightning J, Lloyd C, Mortimore B, O'Callaghan M, Parsons J, Percy C, Rifken L, Roopra S, Saunders D, Shownkeen R, Sims M, Smaldon N, Smith A, Smith M, Sonnhammer E, Staden R, Sulston J, Thierry-Mieg J, Thomas K, Vaudin M, Vaughan K, Waterston R, Watson A, Weinstock L, Wilkinson-Sproat J, Wohldman P:
2.2 Mb of contiguous nucleotide sequence from chromosome III of C. elegans.
Nature
368:32, 1994[Medline]
[Order article via Infotrieve]
25.
Allen JE, Lawrence RA, Maizels RM:
APC from mice harbouring the filarial nematode, Brugia malayi, prevent cellular proliferation but not cytokine production.
Int Immunol
8:143, 1996[Abstract/Free Full Text]
26.
Blaxter ML:
The filarial genome network.
Parasitol Today
11:441, 1995
27.
Blaxter ML, Raghavan N, Ghosh I, Guiliano D, Lu W, Williams SA, Slatko B, Scott AL:
Genes expressed in Brugia malayi infective third stage larvae.
Mol Biochem Parasitol
77:77, 1996[Medline]
[Order article via Infotrieve]
28.
Gregory WF, Blaxter ML, Maizels RM:
Differentially expressed, abundant trans-spliced cDNAs from larval Brugia malayi.
Mol Biochem Parasitol
87:85, 1997[Medline]
[Order article via Infotrieve]
29.
Gish W, States DJ:
Identification of protein coding regions by database similarity search.
Nat Genet
3:266, 1993[Medline]
[Order article via Infotrieve]
30.
Swofford DL:
PAUP: Phylogenetic Analysis Using Parsimony.3.1.1 Ed. Champaign, IL, Illinois Natural History Survey, 1991.
31.
Jiang H, Kanost MR:
Characterization and functional analysis of 12 naturally occurring reactive site variants of serpin-1 from Manduca sexta.
J Biol Chem
272:1082, 1997[Abstract/Free Full Text]
32.
Delvig AA, Robinson JH:
Different endosomal proteolysis requirements for antigen processing of two T-cell epitopes of the M5 protein from viable Streptococcus pyogenes.
J Biol Chem
273:3291, 1998[Abstract/Free Full Text]
33.
Blumenthal T, Steward K:
RNA processing and gene structure, in
Riddle DL,
Blumenthal T,
Meyer BJ,
Priess JR
(eds):
C.elegans II. Cold Spring Harbor, NY, Cold Spring Harbor Laboratory, 1997, p 117.
34.
Csank C, Taylor FM, Matindale DW:
Nuclear pre-mRNA introns: Analysis and comparison of intron sequences from Tetrahymena thermophila and other eukaryotes.
Nucleic Acids Res
18:5133, 1990[Abstract/Free Full Text]
35.
Huber R, Carrell RW:
Implications of the three-dimensional structure of -1-antitrypsin for structure and function of serpins.
Biochemistry
28:8952, 1989
36.
Schneider SS, Schick C, Fish KE, Miller E, Pena JC, Treter SD, Hui SM, Silverman GA:
A serine proteinase inhibitor locus at 18q21.3 contains a tandem duplication of the human squamous cell carcinoma antigen gene.
Proc Natl Acad Sci USA
92:3147, 1995[Abstract/Free Full Text]
37.
Heilig R, Muraskowsky R, Kloepfer C, Mandel JL:
The ovalbumin gene family: Complete sequence and structure of the Y gene.
Nucleic Acids Res
10:4363, 1982[Abstract/Free Full Text]
38.
Sun J, Rose JB, Bird P:
Gene structure, chromosomal localization, and expression of the murine homologue of human proteinase inhibitor 6 (PI-6) suggests divergence of PI-6 from the ovalbumin serpins.
J Biol Chem
270:16089, 1995[Abstract/Free Full Text]
39.
Sun J, Ooms L, Bird CH, Sutton VR, Trapani JA, Bird PI:
A new family of 10 murine ovalbumin serpins includes two homologs of proteinase inhibitor 8 and two homologs of the granzyme B inhibitor (proteinase inhibitor 9).
J Biol Chem
272:15434, 1997[Abstract/Free Full Text]
40.
Loebermann H, Tokuoka R, Diesenhofer J, Huber R:
Human 1-proteinase inhibitor. Crystal structure analysis of two crystal modifications, molecular model and preliminary analysis of the implications for function.
J Mol Biol
177:531, 1984[Medline]
[Order article via Infotrieve]
41.
Mottonen J, Strand A, Symersky J, Sweet RM, Danley DE, Geoghegan K F, Gerard RD, Goldsmith EJ:
Structural basis of latency in plasminogen activator inhibitor-1.
Nature
355:270, 1992[Medline]
[Order article via Infotrieve]
42.
Carrell R, Stein PE, Fermi G, Wardell MR:
Biological implications of a 3 A structure of dimeric antithrombin.
Structure
2:257, 1994[Abstract/Free Full Text]
43.
Goodall GJ, Filipowicz W:
The AU-rich sequences present in the introns of plant nuclear pre-mRNAs are required for splicing.
Cell
58:473, 1989[Medline]
[Order article via Infotrieve]
44.
Wray GA, Levinton JS, Shapiro LH:
Molecular evidence for deep precambrian divergences amony metazoan phyla.
Science
274:568, 1996[Abstract/Free Full Text]
45.
Strandberg L, Lawrence D, Ny T:
The organization of the human-plasminogen-activator-inhibitor-1 gene. Implications on the evolution of the serine-protease inhibitor family.
Eur J Biochem
176:609, 1988[Medline]
[Order article via Infotrieve]
46.
Gilbert W, de Souza SJ, Long M:
Origin of genes.
Proc Natl Acad Sci USA
94:7698, 1997[Abstract/Free Full Text]
47.
Stoltzfus A, Spencer DF, Zuker M, Logsdon JM Jr, Doolittle WF:
Testing the exon theory of genes: The evidence from protein structure.
Science
265:202, 1994[Abstract/Free Full Text]
48.
Marshall CJ:
Evolutionary relationships among the serpins.
Phil Trans R Soc Lond Series B
342:101, 1993[Medline]
[Order article via Infotrieve]
49.
Hopkins PCR, Whisstock J:
Function of maspin.
Science
265:1893, 1994[Free Full Text]
50.
Stein PE, Carrell RW:
What do dysfunctional serpins tell us about molecular mobility and disease?
Nat Struct Biol
2:96, 1995[Medline]
[Order article via Infotrieve]
51.
Caughey GH, Schaumberg TH, Zerweck EH, Butterfield JH, Hanson RD, Silverman GA, Ley TJ:
The human mast cell chymase gene (CMA1): Mapping to the cathepsin G/granzyme gene cluster and lineage-restricted expression.
Genomics
15:614, 1993[Medline]
[Order article via Infotrieve]
52.
Belaaouaj A, McCarthy R, Baumann M, Gao Z, Ley TJ, Abraham SN, Shapiro SD:
Mice lacking neutrophil elastase reveal impaired host defense against gram negative bacterial sepsis.
Nat Med
4:615, 1998[Medline]
[Order article via Infotrieve]
53.
Padrines M, Wolf M, Walz A, Baggiolini M:
Interleukin-8 processing by neutrophil elastase, cathepsin G and proteinase-3.
FEBS Lett
352:231, 1994[Medline]
[Order article via Infotrieve]
54.
Yamazaki T, Aoki Y:
Cathepsin G binds to human lymphocytes.
J Leukoc Biol
61:73, 1997[Abstract]
55.
Chertov O, Ueda H, Xu LL, Tani K, Murphy WJ, Wang JM, Howard OMZ, Sayers TJ, Oppenheim JJ:
Identification of human neutrophil-derived cathepsin G and azurocidin/CAP37 as chemoattractants for mononuclear cells and neutrophils.
J Exp Med
186:739, 1997[Abstract/Free Full Text]
56.
Hase-Yamazaki T, Aoki Y:
Stimulation of human lymphocytes by cathepsin G.
Cell Immunol
160:24, 1995[Medline]
[Order article via Infotrieve]
57.
Schick C, Kamachi Y, Bartuski AJ, Cataltepe S, Schechter NM, Pemberton PA, Silverman GA:
Squamous cell carcinoma antigen 2 is a novel serpin that inhibits the chymotrypsin-like proteinases cathepsin G and mast cell chymase.
J Biol Chem
272:1849, 1997[Abstract/Free Full Text]
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S. C. Pak, V. Kumar, C. Tsu, C. J. Luke, Y. S. Askew, D. J. Askew, D. R. Mills, D. Bromme, and G. A. Silverman
SRP-2 Is a Cross-class Inhibitor That Participates in Postembryonic Development of the Nematode Caenorhabditis elegans: INITIAL CHARACTERIZATION OF THE CLADE L SERPINS
J. Biol. Chem.,
April 9, 2004;
279(15):
15448 - 15459.
[Abstract]
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P. Schierack, R. Lucius, B. Sonnenburg, K. Schilling, and S. Hartmann
Parasite-Specific Immunomodulatory Functions of Filarial Cystatin
Infect. Immun.,
May 1, 2003;
71(5):
2422 - 2429.
[Abstract]
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X. Zang, P. Taylor, J. M. Wang, D. J. Meyer, A. L. Scott, M. D. Walkinshaw, and R. M. Maizels
Homologues of Human Macrophage Migration Inhibitory Factor from a Parasitic Nematode. GENE CLONING, PROTEIN ACTIVITY, AND CRYSTAL STRUCTURE
J. Biol. Chem.,
November 8, 2002;
277(46):
44261 - 44267.
[Abstract]
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A. S. MacDonald, M. I. Araujo, and E. J. Pearce
Immunology of Parasitic Helminth Infections
Infect. Immun.,
February 1, 2002;
70(2):
427 - 433.
[Full Text]
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A. Loukas and P. Prociv
Immune Responses in Hookworm Infections
Clin. Microbiol. Rev.,
October 1, 2001;
14(4):
689 - 703.
[Abstract]
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W. R. Atchley, T. Lokot, K. Wollenberg, A. Dress, and H. Ragg
Phylogenetic Analyses of Amino Acid Variation in the Serpin Proteins
Mol. Biol. Evol.,
August 1, 2001;
18(8):
1502 - 1511.
[Abstract]
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X. Zang, A. K. Atmadja, P. Gray, J. E. Allen, C. A. Gray, R. A. Lawrence, M. Yazdanbakhsh, and R. M. Maizels
The Serpin Secreted by Brugia malayi Microfilariae, Bm-SPN-2, Elicits Strong, but Short-Lived, Immune Responses in Mice and Humans
J. Immunol.,
November 1, 2000;
165(9):
5161 - 5169.
[Abstract]
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W. F. Gregory, A. K. Atmadja, J. E. Allen, and R. M. Maizels
The Abundant Larval Transcript-1 and -2 Genes of Brugia malayi Encode Stage-Specific Candidate Vaccine Antigens for Filariasis
Infect. Immun.,
July 1, 2000;
68(7):
4174 - 4179.
[Abstract]
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M. Lizotte-Waniewski, W. Tawe, D. B. Guiliano, W. Lu, J. Liu, S. A. Williams, and S. Lustigman
Identification of Potential Vaccine and Drug Target Candidates by Expressed Sequence Tag Analysis and Immunoscreening of Onchocerca volvulus Larval cDNA Libraries
Infect. Immun.,
June 1, 2000;
68(6):
3491 - 3501.
[Abstract]
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A. Loukas, M. Hintz, D. Linder, N. P. Mullin, J. Parkinson, K. K. A. Tetteh, and R. M. Maizels
A Family of Secreted Mucins from the Parasitic Nematode Toxocara canis Bears Diverse Mucin Domains but Shares Similar Flanking Six-cysteine Repeat Motifs
J. Biol. Chem.,
December 8, 2000;
275(50):
39600 - 39607.
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INTRODUCTION