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Identification characterization and cloning of an immunoglobulin degrading enzyme in the cat flea Ctenocephalides felis.

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136
Silver et al.
Archives of Insect Biochemistry and Physiology 51:136–150 (2002)
Identification, Characterization, and Cloning of an
Immunoglobulin Degrading Enzyme in the Cat Flea,
Ctenocephalides felis
Gary M. Silver, Patrick J. Gaines,* Shirley W. Hunter, Joely D. Maddux, Rex E. Thomas,
and Nancy Wisnewski
The degradation of cat immunoglobulin G (IgG) in blood-fed adult C. felis midguts was examined. SDS-PAGE analysis of
dissected midgut extracts obtained from C. felis that had been blood fed for various times between 0 to 44 h revealed that by
24 h most of the high molecular weight proteins, including the heavy chain of IgG, were digested. A 31-kDa serine protease
with IgG degrading activity was purified from fed C. felis midguts by benzamidine affinity chromatography, hydrophobic
interaction chromatography, and cation exchange chromatography. Three primary cleavage products between 30- and 40-kDa
were observed when the purified protease was incubated with protein A purified cat IgG. N-terminal amino acid sequence
analysis of the products revealed that the IgG degrading protease cleaves after specific cysteine and lysine residues within the
hinge region of IgG. The enzyme is also capable of degrading other immunoglobulins, serum albumin, and hemoglobin,
suggesting that it may have roles in both combating the host’s immune system and providing nutrients for the flea. A cDNA
clone encoding the 265 amino acid IgG degrading protease proenzyme was isolated. When expressed in a baculovirus/insect
cell expression system, the recombinant protein had the same N-terminus as the processed 237 amino acid mature native
protein and possessed IgG degrading activity indistinguishable from the native protein. Arch. Insect Biochem. Physiol. 51:136–
150, 2002. © 2002 Wiley-Liss, Inc.
KEYWORDS: cat flea; immunoglobulin; IgG; serine protease; bloodmeal
INTRODUCTION
The cat flea (Ctenocephalides felis) is the most
common ectoparasite of dogs and cats worldwide.
Like other hematophagous arthropods, C. felis must
consume host blood to survive and reproduce. During this process, they consume large quantities of
host immunoglobulins, exposing the fleas to possible attack by the host immune system. Thus, it is
advantageous to the flea to be able to eliminate
immunoglobulins from the bloodmeal as rapidly
as possible. Previously, we reported that the majority of ingested cat immunoglobulin G (IgG)
passes through a feeding flea into its feces, but a
small amount of IgG persists in the flea’s hemolymph as long as it continues to feed (Vaughan
et al., 1998). In contrast, the amount of active IgG
in the midgut decreased as the flea continued to
feed, implying that IgG is either transported out
of the midgut or proteolytically degraded. In this
work, we examine the role that a specific serine
protease may play in the degradation of IgG.
In the midgut of mosquitoes and other hematophagous insects, serine proteases are produced
shortly after the initiation of bloodfeeding (Borovsky and Schlein, 1988). Most of the serine proteases
are bulk digestive enzymes related to mammalian
trypsins and chymotrypsins, while others have spe-
Heska Corporation, Fort Collins, Colorado
*Correspondence to: Patrick J. Gaines, Heska Corporation, 1613 Prospect Parkway, Fort Collins, CO, 80525. E-mail: gainesp@heska.com
Received 1 May 2002; Accepted 12 August 2002
© 2002 Wiley-Liss, Inc.
DOI: 10.1002/arch.10059
Published online in Wiley InterScience (www.interscience.wiley.com)
Archives of Insect Biochemistry and Physiology
Degradation of IgG in C. felis
cific functions such as processing hormones and
enzymes to active forms. Despite the fact that serine
proteases have been studied in detail in other insects, very little has been published on serine proteases in fleas. Prasad has shown that the rat fleas
Xenopsylla cheopis and Xenopsylla astia have detectable levels of protease activity in their midguts after feeding on rat blood for 5 days, but do not
distinguish serine protease activity in the midgut
from other types of protease activity (Prasad,
1979). Borovsky and colleagues measured the
amount of trypsin-like activity in the midguts of
blood-fed C. felis injected with a peptide hormone
that effects serine protease expression in mosquitoes and found that serine protease activity was
inhibited upon introduction of the mosquito hormone TMOF (Borovsky et al., 1990). Recently,
Gaines et al. (1999) described the cloning of 38
distinct serine protease genes from C. felis cDNA
libraries, including the gene encoding the serine
protease discussed in this article. Several of the
cloned genes encode proteins that are similar to
known insect and arachnid serine proteases, but
the specific functions of these proteases are unknown.
Immunoglobulin-degrading proteases have
been found in pathogenic bacteria (Plaut and
Bachovchin, 1994), parasites (Tamashiro et al.,
1987; Kong et al., 1994), and insects (Eisemann
et al., 1995; Pruett, 1993). These enzymes, which
belong to both the cysteine and serine protease
families, are thought to impart some protection to
the invading organism from the host’s immune system through the inactivation of host immunoglobulins, including both IgG and IgA. Importantly,
however, the other organisms known to contain
immunoglobulin-degrading enzymes are endoparasites, while C. felis is an ectoparasite.
In this report, data are presented for the first
time that show that C. felis expresses a serine protease that is capable of degrading host immunoglobulins. We fed C. felis on cat blood using an in
vitro feeding system. After 24 h of continuous feeding, most of the high molecular weight blood proteins in the C. felis midgut were no longer visible
by electrophoresis, implying that they were meNovember 2002
137
tabolized by midgut digestive proteases. Close examination of the protein profiles showed that the
heavy chain of cat IgG was nearly completely digested, while the light chain was relatively intact.
An investigation of the enzymes most responsible
for IgG degradation in C. felis midguts revealed that
one of these enzymes was a serine protease of approximately 31-kDa. Our results indicate that this
enzyme preferentially cleaves cat IgG in the hinge
region, but that it is also capable of cleaving other
blood proteins such as hemoglobin and serum albumin. We have cloned this enzyme from a C. felis
cDNA library, and have expressed active recombinant protein in a baculovirus/insect cell expression
system.
MATERIALS AND METHODS
Flea Tissues and Reagents
Ctenocephalides felis (C. felis) were contained in
screened chambers and allowed to feed either on
cats or on heparinized cat blood in a commercially
available in vitro feeding system (Flea Data, Inc.
Freeville, NY) based on a model described by Wade
and Georgi (1988). Unless indicated otherwise, all
fleas were fed for 24 h. The midguts from fed fleas
were dissected and immediately placed into ice
cold buffer. Cat IgG, rabbit IgG, bovine IgG, cat
serum albumin, and cat hemoglobin were purchased from Sigma-Aldrich Co. (St. Louis, MO).
Purified cat IgA and IgM were purchased from
Bethyl Laboratories, Inc. (Montgomery, TX). Cat
IgG was also purified in our laboratory via Protein
A Sepharose chromatography (Amersham Pharmacia Biotech, Piscataway, NJ). (1,3-3H)- diisopropylfluorophosphate (3H-DFP) was purchased from
DuPont-NEN (Wilmington, DE).
Electrophoresis
Unless specified otherwise, all SDS-PAGE was
performed using Novex® precast 14% Tris-Glycine
mini-gels (Invitrogen, Carlsbad, CA) under reducing conditions. Molecular weights were estimated
using either Mark12™ Wide Range Protein Standards (Invitrogen) or Low range SDS-PAGE stan-
138
Silver et al.
dards (Bio-Rad Laboratories, Hercules, CA). Gels
were silver stained using either the Silver Express®
staining system (Invitrogen) or the procedure of
Blum et al. (1987), except that the concentrations
of sodium thiosulfate and silver nitrate used were
half of the published values.
Flea Feeding Timecourse
C. felis were allowed to feed on cats for various
times between 1 to 44 h and the midguts from 20
fed fleas were dissected in gut dissection buffer (50
mM Tris-HCl, 100 mM CaCl2, pH 8.0) at designated timepoints. The midgut tissues were homogenized by freeze-fracture and bath sonication, and
clarified by centrifugation at 14,000g for 20 min.
The soluble material was then diluted to a final
concentration of approximately 1 midgut equivalent per microliter of gut dissection buffer. The
soluble midgut serine proteases were covalently labeled with 3H-DFP using the method generally described by Borovsky (1988). Specifically, the
extracts were labeled with 1 mCi of 3H-DFP, incubated for 18 h at 4°C, spotted onto 1 cm2 filter
paper, precipitated onto the filter by soaking the
filters in 10% (v/v) trichloroacetic acid, and quantified using a LS1801 liquid scintillation counter
(Beckman Instruments, Fullerton, CA). In addition,
1 midgut equivalent from each timepoint was analyzed by silver-stained SDS-PAGE.
Purification of Native Flea Midgut IgG Degrading
Activity
The midguts from approximately 10,000 cat
blood-fed C. felis were homogenized in benzamidine column buffer (20 mM Tris-HCl, 500 mM
NaCl, pH 8.0). The soluble proteins were then incubated overnight at 4°C with 3 ml of p-aminobenzamidine linked-Sepharose beads (Sigma-Aldrich
Co.). After incubation, the column was drained and
the beads were washed with 20 ml of benzamidine
column buffer. The beads were then incubated
overnight at 4°C with 100 mM Tris-HCl, pH 8.0,
containing 100 mM p-aminobenzamidine to elute
bound serine proteases. After the incubation, the
column was drained and the beads were washed
twice with 10 ml of benzamidine column buffer.
The eluted protein fractions were pooled and concentrated to 4.5 ml using an Amicon® Centriprep®
3 centrifugal concentrator (Millipore, Beverly, MA).
The sample was supplemented with 2 M (NH4)2
SO4 and applied to a polypropylaspartamide HIC
column (PolyLC, Columbia, MD). The column was
washed with 0.1 M KPO4, 2 M (NH4)2SO4, pH 6.5,
and eluted with a linear gradient to 0.1 M KPO4,
pH 6.5, and the resulting fractions were assayed
for IgG degrading activity. Active fractions were
pooled, dialyzed 2 h against 120 ml of 50 mM
NaOAc, pH 6.0, and applied to a PolyCAT A cation exchange column (PolyLC). The column was
washed with 50 mM NaOAc, pH 6.0, and eluted
with a linear gradient to 50 mM NaOAc, 1 M NaCl,
pH 6.0. Fractions containing the highest levels of
IgG degrading activity included a protein band that
migrated at about 31-kDa on the SDS-PAGE gel.
The protein concentration of these fractions was
determined by the Bio-Rad Protein Assay (Bio-Rad)
using bovine serum albumin as a standard. For
N-terminal sequencing, the 31-kDa protease was
resolved by SDS-PAGE and blotted onto a polyvinylidene difluoride (PVDF) membrane using
CAPS buffer (10 mM CAPS, pH 11, 0.5 mM DTT,
10% (v/v) methanol). The membrane was stained
with Coomassie Brilliant Blue and destained with
50% (v/v) methanol. The N-terminal amino acid
sequence of the 31-kDa protease was determined
using a 473A Protein Sequencer (Applied Biosystems, Foster City, CA) using standard techniques.
Preparation of Peptides With Cyanogen Bromide
Serine proteases from cat blood fed flea midguts were homogenized in gut dissection buffer and
purified using p-aminobenzamidine linked-Sepharose beads as described above. Approximately 150
mg of the purified protease pool was resolved by
SDS-PAGE on a preparative-well 14% Tris-Glycine
electrophoresis gel under reducing conditions. After electrophoresis, the proteins in the gel were visualized by staining for 30 min in Coomassie
Brilliant Blue stain [0.1% (w/v) Coomassie Blue
Archives of Insect Biochemistry and Physiology
Degradation of IgG in C. felis
R, 40% (v/v) methanol, 10% (v/V) acetic acid] and
destaining for 2.5 h in 50% (v/v) methanol. The
band corresponding to the 31-kDa protease was
excised with a razor blade. The protein was electroeluted, concentrated, and partially digested for
24 h with cyanogen bromide (CNBr) as described
by Silver and Fall (1995), except that a small
amount of acetic acid was added to the sample
after electroelution and concentration to lower the
sample pH and therefore reduce autodigestion by
the 31-kDa protease. After CNBr digestion, the peptides in the sample were resolved by SDS-PAGE on
an 18% Tris-Glycine electrophoresis gel under reducing conditions, blotted onto a PVDF membrane
and N-terminally sequenced.
Assay for IgG Cleavage Activity
IgG cleavage activity was assayed by incubating
purified native or recombinant 31-kDa IgG degrading protease with Protein A-Sepharose purified cat
IgG in 0.1M Tris-HCl, pH 8.0. The reaction mixtures were incubated for various times at 37°C and
then analyzed by silver stained SDS-PAGE. A decrease in the amount of visible IgG heavy chain
and the appearance of cleavage products between
30- and 45-kDa indicated IgG cleavage activity.
Assay for Degradation of Other Blood Proteins
Characterization of the proteolytic activity of the
native 31-kDa protease was performed by assessing the digestion of several immunoglobulin heavy
chains as well as other blood proteins as described
above. Specifically, 1 mg each of commercially available cat IgG, cat IgA, cat IgM, cat serum albumin,
cat hemoglobin, bovine IgG, and rabbit IgG was
incubated with 200 ng purified native 31-kDa IgG
degrading protease in 27 ml 0.1M Tris-HCl, pH 8.0,
at 37°C for 18 h. After incubation, the reaction products were analyzed by silver-stained SDS-PAGE.
Determination of IgG Cleavage Site
Two experiments were performed to determine
the cleavage site specificity of the purified 31-kDa
November 2002
139
protease. In the first experiment, 10 mg of Protein
A-Sepharose purified cat IgG were incubated with
2 mg purified 31-kDa protease in 100 ml of 0.2 M
Tris-HCl, pH 8.0 at 37°C for 18 h. The reaction
mixture was resolved by SDS-PAGE, blotted onto
a PVDF membrane, and stained with Coomassie
Brilliant Blue. Bands of about 33- and 37-kDa were
excised and subjected to N-terminal amino acid
sequencing. To further investigate cleavage site
specificity of the purified 31-kDa protease, 15 mg
of Protein A-Sepharose purified cat IgG were incubated with 2 mg purified 31-kDa protease in a total volume of 300 ml of 50 mM Tris-HCl, pH 8.0,
at 37°C for 24 h. Aliquots of the incubation mixture were removed at 1, 2, 4, 6, 8, 12, 16, and 24
h after initiation of the incubation. Following removal, 20 ml of each aliquot were mixed with 1
ml of 20 mM p-aminobenzamidine and stored at
–80°C. The samples were resolved by SDS-PAGE,
and either silver stained or blotted onto a PVDF
membrane and stained with Coomassie Brilliant
Blue. A 34-kDa band observed in the 1-h timepoint
lane was excised and subjected to N-terminal
amino acid sequencing.
Library Construction
An adult C. felis cDNA library was prepared as
described by Gaines et al. (1999). Briefly, total RNA
was extracted from a pool of C. felis fed on cat
blood for 0.25, 2, 4, 8, 15, 24, and 48 h using Tri
Reagent™ (Molecular Research Center, Cincinnati,
OH) according to the manufacturer’s protocol.
Messenger RNA was subsequently extracted from
the total RNA using the FastTrack™ 2.0 kit (Invitrogen). The ZAP Express/Gigapack cloning kit
(Stratagene, La Jolla, CA) was used to generate a
lambda phage cDNA library from 2.0 mg of the
mRNA.
PCR Amplification
The N-terminal amino acid sequence “IVG
GEDVDISTCGXQ” was obtained following purification of the 31-kDa protease protein. A degenerate forward oligonucleotide PCR primer with the
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Silver et al.
sequence 5¢ GAA GAT GTW GAT ATT TCW ACA
TGT GG 3¢ was designed from this protein sequence, and was used with the M13 universal vector primer (5¢ TGT AAA ACG ACG GCC AGT 3¢)
in a PCR reaction. The conditions for the PCR reaction were as follows: 95°C for 40 sec, then 30
cycles of 95°C for 45 sec, 42°C for 45 sec, and 72°
for 60 sec. A final extension step of 72° for 5 min
completed the reaction. The reaction products were
separated on a 1% agarose gel, and the 960-bp fragment was cut from the gel and purified using a
QIAquick™ gel extraction kit (Qiagen, Chatsworth,
CA). The fragment was ligated into the pCR® 2.1
cloning vector (Invitrogen). The ligation reaction
was used to transform One Shot® INVaF¢ chemically competent E. coli cells (Invitrogen). A single
clone was isolated and sequenced using an ABI
Prism 377 DNA sequencer (Perkin Elmer, Foster
City, CA). The predicted translation of this DNA
fragment had 100% identity with an internal
amino acid sequence obtained from the CNBr digest of the purified 31-kDa protease confirming that
this was a fragment of the gene encoding the 31kDa protease. A homologous reverse PCR primer
was then designed that was complimentary to
nucleotides 691–718 of the DNA fragment described above. This primer, which has the sequence
5¢ GAA AAT GAA ATC CAC TTA AAC ATT ACG 3¢,
was used with the vector primer M13 reverse (5¢
CAG GAA ACA GCT ATG ACC 3¢) to amplify the
5¢ end of the gene encoding the 31-kDa protease.
When no obvious products were detected by agarose gel electrophoresis, the products from this PCR
reaction were diluted 1:25 and used as a template
for a second, nested PCR reaction. A second homologous reverse primer complementary to nucleotides 216–237 of the fragment described above
was designed with the sequence 5¢ CTC TTA TTG
TAC GAG GGA TGC 3¢. This primer was used with
the forward T3 vector primer (5¢ TAA TAC GAC
TCA CTA TAG GG 3¢) to amplify a DNA fragment
approximately 400 bp in length. This fragment
overlapped the 960 bp fragment by 216 bp, and
had 100% identity with this fragment in this region. In addition, the predicted coding region encoded a protein that had 100% identity with the
N-terminal amino acid sequence and the internal
amino acid sequence of the purified IgG degrading protease.
E. coli Expression of Recombinant 31-kDa Protease
A forward primer complimentary to the region
encoding the N-terminus of the purified protein
was designed with the sequence 5¢ GCG GGA TCC
TAT AAA TAT GAA ACT TTT GGT AGT TTT TGC G
3¢. This primer was used with a reverse primer complimentary to the region encoding the predicted
C-terminus of the protein with the sequence 5¢
GCT CTA GAC CAC TTA AAC ATT AGC ATA TTT
TTC 3¢ to amplify the gene encoding the 31-kDa
protease by overlap PCR. A Bam HI restriction endonuclease cleavage site was included at the 5¢ end
of the forward primer, and a Xba I cleavage site
was included at the 5¢ end of the reverse primer.
The DNA fragments described above containing the
5¢ and 3¢ ends of the gene encoding the 31-kDa
protease were used together as templates in the
overlap PCR reaction. The conditions for the PCR
were as follows: 95°C for 45 sec, then 25 cycles of
95°C for 45 sec, 45°C for 45 sec, and 72°C for
120 sec. A final extension step of 72°C for 5 min
completed the reaction. The products of the reaction were separated on a 1% agarose gel, and an
approximately 980 bp product was cut from the
gel and purified using a gel purification kit (Qiagen). The fragment was digested with Bam HI and
Xba I enzymes (New England Biolabs, Beverly, MA)
and purified using a nucleotide removal kit (Qiagen). The E. coli expression vector lambda PR/T2ori/
S10HIS-RSET-A9 (Tripp et al., 1995) was also digested with Bam HI and Xba I, then treated with
calf intestinal alkaline phosphatase (Gibco BRL),
and purified with the QIAquick™ nucleotide removal kit (Qiagen) prior to ligation. One milliliter of the ligation reaction was used to transform
One Shot® BL-21 E. coli cells (Novagen, Madison,
WI). A single clone was isolated, grown in Luria
broth minimal media, and induced with 0.5 mM
IPTG. Production of the recombinant 31-kDa protease was determined by Western blot using the
T7 tag antibody system (Novagen).
Archives of Insect Biochemistry and Physiology
Degradation of IgG in C. felis
141
Purification of E. coli Recombinant and Generation of
Cat Antiserum
nization. The serum used for subsequent Western
blots was obtained from the day 47 bleed.
One liter of E. coli cells expressing the recombinant 31-kDa protease was homogenized by probe
sonication and lysozyme treatment in 50 mM Tris
buffer, pH 8.0, containing 50 mM NaCl, 1 mM
PMSF. After several rounds of sonication and centrifugation, the protein was solubilized in column
buffer (PBS, pH 8.0, 8M urea, 1 mM PMSF, and 1
mM -mercaptoethanol) and the supernatants from
the solubilization protocol were assessed for the
presence of recombinant 31-kDa protease by Western blotting using antibodies against the T7 tag.
All subsequent steps were performed in column
buffer, with the pH adjusted and/or other components added as indicated. The protein suspension
was then applied to a nickel HiTrap™ Metal Chelating Column (Amersham Pharmacia) in column
buffer and the recombinant protein eluted from
the column by adjusting the pH of the column
buffer to 4.5. The sample was then loaded onto a
HiTrap SP-Sepharose cation exchange column
(Amersham Pharmacia) in column buffer, pH 4.5,
and eluted with a linear gradient of column buffer,
pH 4.5, containing 1 M NaCl. The partially purified recombinant 31-kDa protease was then reapplied to a Nickel HiTrap Metal Chelating Column
in column buffer, pH 8.0, and eluted with a linear
gradient of 0 to 1M imidazole in column buffer.
The purified proteins were concentrated and
diafiltered into PBS, pH 7.6, containing 8M urea
and 1 mM PMSF using a Centricon® Plus-20 centrifugal concentrator (Amicon Inc., Beverly, MA).
Protein concentration was determined by the BioRad protein assay using bovine serum albumin as
a standard. The recombinant 31-kDa protease was
diluted to 1 mg/ml in PBS and emulsified in an
equal volume of TiterMax® research adjuvant
(CytRx Corporation, Norcross, GA). To generated
cat anti-31-kDa protease antibodies, a domestic
shorthair cat was immunized with 50 mg of the
recombinant protein by a single site subcutaneous
injection. A second injection was administered 32
days later. Bleeds were obtained prior to immunization and 32 and 47 days after the initial immu-
Expression of the Recombinant 31-kDa Protease in
Baculovirus
November 2002
A forward primer complementary to the 5¢ end
of the 31-kDa protease coding region (5¢ GCG
GGA TCC TAT AAA TAT GAA ACT TTT GGT AGT
TTT TGC G 3¢) was used with a reverse primer
complementary to the 3¢ end of the coding region (5¢ GCT CTA GAC CAC TTA AAC ATT AGC
ATA TTT TTC 3¢) to amplify the gene encoding
the full length 31-kDa protease, including the signal sequence. A Bam HI cleavage site was included
in the forward primer, and a Xba I cleavage site
was included in the reverse primer. The DNA fragments containing the 5¢ and 3¢ ends of the 31kDa protease gene were used together as templates
for the reaction, as described above. Conditions
for the overlap PCR reaction were the same as described above. The products were separated on a
1% agarose gel, and an approximately 1-kB fragment was excised and purified using a QIAquick™
gel purification kit (Qiagen). The PCR fragment
was digested with Bam HI and Xba I, purified, and
ligated into the baculovirus expression plasmid
pVL1393, which had been previously digested, alkaline phosphatase treated, and purified as described above. One microliter of the ligation
reaction was used to transform E. coli INVaF¢ cells
(Invitrogen). The pVL1393-31-kDa protease plasmid DNA was isolated from a single clone and
co-transfected into S. frugiperda cells (Invitrogen)
with BaculoGold™ wild type linear baculovirus
DNA (PharMingen, San Diego, CA) by standard
calcium phosphate transfection procedures. The
supernatant containing virus was harvested 10
days post-transfection. Recombinant virus was
plaque purified two times prior to assessing protein expression following transfection of HighFive™ cells BTI-TN-5B1-4 (Invitrogen). Cells were
grown at 1 ´ 106/ml and infected at a MOI of 1.0
for 48 h at 27° C. Cell culture supernatants were
harvested by spinning at 3,600 rpm for 15 min.
Protein was analyzed by Western blot analysis us-
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Silver et al.
ing the cat antibody generated against the purified E. coli recombinant 31-kDa protease. Two
bands of approximately 31- and 35-kDa, which
were not present in the untransfected High-Five™
negative control supernatants, were detected using the anti-31-kDa protease antiserum.
Purification of Baculovirus Recombinant 31-kDa
Protease
Recombinant 31-kDa protease was precipitated
from 6 liters of baculovirus supernatants by the
addition of 50–95% saturating (NH4)2SO4. The
presence of the recombinant 31-kDa protease in
the resulting pellet was confirmed by Western blot
analysis using cat anti-31-kDa protease antiserum.
The pellet was resuspended in 70 ml of modified
benzamidine column buffer (50 mM Tris-HCl,
400 mM NaCl, pH 8.0) and applied to a 3-ml
column containing p-aminobenzamidine-linked
Sepharose beads (Sigma). After an overnight incubation at 4°C, the column was washed with
modified benzamidine column buffer and active
proteases were eluted from the column by the addition of 14 ml modified benzamidine column
buffer supplemented with 100 mM benzamidine
(Sigma). The recovered proteases were diafiltered
into Tris buffered saline (TBS), pH 7.2, containing 1 mM benzamidine and applied in five
aliquots to a Bio-Silect™ SEC 125-5 size exclusion
chromatography column (Bio-Rad). The column
was eluted with TBS, pH 7.2. Column fractions
were analyzed by silver-stained SDS-PAGE and assayed for the presence of recombinant 31-kDa protease by Western blot. The identity of the purified
recombinant 31-kDa protease was verified by Nterminal sequencing.
RESULTS
Discovery of IgGase Activity in Flea Midguts
We initially observed the degradation of cat
IgG heavy chain while studying the increase in
active serine proteases in the C. felis midgut upon
initiation of blood feeding. Figure 1A shows that
3
H-DFP-labelable midgut serine proteases increase
nearly linearly with time as the fleas are allowed
to feed for 44 h on live cats, reaching a maximum level between 34 and 40 h. We have followed the serine protease levels for 2 days beyond
this, and have found no significant changes after
the 40-h timepoint (data not shown), suggesting
that once C. felis have fed for 2 days, the amount
of serine protease activity in their midguts remains
relatively constant. The fact that labelable serine
protease levels increased after the 8-h timepoint
is consistent with Northern blot data that shows
the mRNA encoding the 31-kDa protease (referred
to as CfSP-28) is not detected at 0 or 2 h following feeding, but is readily detected at 8 h following feeding (Gaines et al., 1999). The midgut
samples that were used in these labeling studies
were also examined by silver-stained SDS-PAGE
(Fig. 1B). After 15 h of feeding, most of the high
molecular weight blood proteins were either
eliminated or degraded. In particular, neither intact cat serum albumin, at 66-kDa, nor intact cat
immunoglobulin G (IgG) heavy chain, at 55-kDa,
was visible after the 15-h timepoint. In contrast,
analysis by Western blot shows that while intact
cat IgG heavy chain was eliminated from the guts,
the level of intact light chain in the guts remained
relatively unchanged over the 44-h timecourse
(data not shown).
Purification of Native Flea Midgut IgG Degrading
Activity
To investigate the degradation of cat IgG heavy
chain in flea guts, it was necessary to identify and
purify flea enzyme(s) having IgG degrading capabilities. The soluble proteins from approximately 10,000 cat blood fed flea midguts were
passed over a p-aminobenzamidine affinity column designed to specifically retain serine proteases. Using an in vitro IgG degradation assay,
we found that a significant portion of the IgG degrading activity was found in the protein fraction
that had bound to the p-aminobenzamidine column (data not shown), suggesting that some of
the enzyme(s) of interest were members of the
serine protease family. Following elution of the
Archives of Insect Biochemistry and Physiology
Degradation of IgG in C. felis
143
Fig. 1. Timecourse of feline fed flea midguts. An increase
in serine proteases corresponds to a decrease in high molecular weight proteins in fed flea midguts. A: Midgut extracts containing serine proteases were labeled with
3
H-DFP, precipitated with TCA onto filter paper, and quantified by scintillation counting. Ten midgut equivalents
were assayed at each timepoint. Error bars represent ± 1
standard deviation from the mean of at least 3 assays. B:
The soluble proteins in one midgut equivalent from each
timepoint were analyzed by silver-stained SDS-PAGE on a
14% Tris-Glycine gel. The arrow at 55-kDa indicates the
heavy chain of feline IgG. Cat IgG was identified by Western blot (data not shown), and the 66-kDa band was assumed to be serum albumin based on the predicted size
of 66-kDa for the mature protein (Hilger et al., 1996) and
the fact that serum albumin is predicted to be the dominant protein at this molecular weight in mammalian
blood.
bound serine proteases, hydrophobic interaction
and cation exchange chromatography were employed to identify serine proteases possessing IgG
degrading activity. Fractions from the cation exchange column were incubated overnight with purified cat IgG and analyzed by SDS-PAGE. Judging
by the visible decrease in IgG heavy chain, column fraction 23 (eluting at approximately 300
mM NaCl) had the greatest IgG heavy chain cleavage activity (Fig. 2A). SDS-PAGE analysis of the
fractions showed that fraction 23 contained a
dominant 31-kDa protein (Fig. 2B). To further
characterize the 31-kDa protein and to aid in molecular cloning of the gene, N-terminal and internal amino acid sequences were obtained. The
purified 31-kDa protein was subjected to SDSPAGE, electroblotted onto a PVDF membrane, and
N-terminally sequenced by Edman degradation.
To obtain internal sequence, the protein was iso-
lated by SDS-PAGE, electroeluted, and digested
with CNBr. The resultant peptide fragments were
blotted onto a PVDF membrane and N-terminally
sequenced. The N-terminal sequence of the intact protein, IVGGEDVDISTCGWQiSFQSENLH
FCGGSIIAPK, is similar to other insect serine proteases (52% identical over 34 amino acids with
hypodermin B from cattle grub, Hypoderma lineatum; Moire et al., 1994). A 22-kDa CNBr-generated peptide had the same N-terminal amino
acid sequence as the intact protein. The sequence
from a 21-kDa CNBr-generated peptide was more
ambiguous due to contamination by the 22-kDa
peptide. By subtracting the N-terminal sequence
of the 22-kDa peptide from the sequence obtained
from the 21-kDa peptide, the resulting sequence
for the 21-kDa peptide was determined to be (H/
R)P(A/S)YNKRADYDF(D/P)VA, where “/” represents an ambiguity between 2 residues.
November 2002
144
Silver et al.
Fig. 2. Purification of feline flea IgG degrading protease
by HPLC. Cation exchange chromatography fractions were
assayed for IgG degrading activity by overnight incubation with purified feline IgG and were analyzed by silverstained SDS-PAGE on a 14% Tris-Glycine gel under
reducing conditions. A: Twenty microliters of each cation
exchange column fraction were incubated with 250 ng feline IgG for 18 h at 37°C and analyzed by SDS-PAGE. The
decrease in IgG heavy chain (near 55-kDa) and the increase in cleavage products (30–45 kDa) indicated IgG degrading activity. B: Twenty microliters of each cation
exchange column fraction showing the proteins contained
in each fraction. The fraction with the highest IgG degrading activity, fraction 23, contains a dominant 31-kDa protein as well as some minor proteins at approximately
25-kDa.
Determination of IgG Cleavage Site
membranes and the 33-, 34-, and 37-kDa bands
N-terminally sequenced (Table 1). The 33- and 34kDa cleavage products have amino acid sequences
that are identical to each other with the exception
of an additional four amino acids at the N-terminal end of the 34-kDa polypeptide. This sequence
is also identical to the sequence in the hinge region of cat IgG heavy chain (Kanai et al., 2000).
The 37-kDa cleavage product had an N-terminal
amino acid sequence that is similar to the variable
N-terminal sequence of other mammalian IgG
molecules.
Upon closer inspection of the electrophoresis
gel in Figure 2A column fractions 22–25, three
bands of approximately 33-, 34-, and 37-kDa appeared to be IgG heavy chain cleavage products.
To determine whether or not these were cleavage
products, and to determine where the IgG heavy
chain was being cleaved, purified cat IgG was incubated with the purified 31-kDa protease for various times between 1 to 24 h and the reaction
products were analyzed by SDS-PAGE (Fig. 3). By
1 h, a reaction product was clearly visible at 34kDa (Fig. 3, band A). By 4 hours, bands at 33and 37-kDa (Fig. 3, bands B and C) were visible,
and by 24 h, bands B and C were dominant over
band A. Interestingly, these bands were somewhat
stable to further cleavage, as there is only a minor
increase in smaller cleavage products during the
incubation period. The reaction products from the
1- and 18-h incubations were blotted onto PVDF
Cleavage of Other Blood Proteins
To see if the flea 31-kDa IgG degrading protease
is specific for cat IgG or if it can cleave other proteins, purified flea 31-kDa protease was incubated
with several other immunoglobulins, cat serum albumin, and cat hemoglobin. Figure 4 shows that
all of the other potential substrates were cleaved
Archives of Insect Biochemistry and Physiology
Degradation of IgG in C. felis
145
these molecules are cleaved in a manner similar to
cat IgG. In contrast, cat serum albumin and cat hemoglobin were digested extensively. For example,
the albumin was cleaved into dozens of bands, none
of which were clearly dominant, and cat hemoglobin was cleaved so extensively that no cleavage products were visible. Based on these data, it appears
that while the flea 31-kDa protease cleaves immunoglobulins at a few specific sites, it is also capable
of thoroughly degrading other blood proteins.
Cloning of the Flea IgG Degrading 31-kDa Protease
Fig. 3. Timecourse of IgG digestion. Two micrograms of
purified 31-kDa IgG degrading protease were incubated
with 1.5 mg feline IgG for 24 h at 37°C. Ten microliters of
the reaction mixture were removed at each of the timepoints shown and analyzed by silver stained SDS-PAGE
in a 14% Tris-Glycine gel under reducing conditions. Bands
A, B, and C indicate the primary cleavage products in the
order that they appeared. These three bands were blotted
onto a PVDF and N-terminally sequenced by Edman degradation; the resulting sequences are shown in Table 1.
to some degree by the flea 31-kDa protease, but
some were much better substrates than others. Cat
IgG and cat IgA were the best immunoglobulin substrates, while cat IgM was degraded the least. Interestingly, all of the immunoglobulins were cleaved
and formed product bands that were similar in molecular weight to the cat IgG products, implying that
A PCR primer based on the N-terminal sequence obtained from the purified native 31-kDa
protease was used in conjunction with an antisense
vector primer to amplify the flea 31-kDa protease
gene from a cat blood fed whole flea cDNA library.
The resulting 960 base pair PCR product encoded
a predicted sequence that included a region identical to the deduced amino acid sequence obtained
from the 21-kDa peptide generated by CNBr digestion of the native 31-kDa protease. To obtain
the complete 5¢ end of the gene, antisense PCR
primers based on sequence from the initial 960
base pair PCR product were used in conjunction
with sense vector primers in a series of nested reactions. A 400 base pair PCR product that overlapped the 960 base pair fragment by 216 base
pairs contained the remaining coding regions at
the 5¢ end of the sequence, which encoded the fulllength flea 31-kDa protease. The Genbank database
accession numbers are AF053913 for the nucleotide
sequence and AAD21833 for the protein sequence.
TABLE 1. IgG Cleavage Site Determination*
Peptide
Amino acid sequence
Region of IgG
heavy chain
Feline IgG
Feline IgG 33 kDa
Feline IgG 34 kDa
Bovine IgG
Human IgG
Feline IgG 37 kDa
VRKTDHPPGPKPCDCPKCPPPEMLG
XPPPEMLGGPSIFIFPPKPKDDLLIKRK
DCPKCPPPEMLGGPSIFIFPPKPKDDLLIKRKSEV
QVQLRESGPSLVKPSQTL-SLTCTV
EVQLLEQSGAEVKKPGASVKVSCKA
DVQLVESGNVLVPPGNT--KVDKTV
Hinge
Hinge
Hinge
N-term variable
N-term variable
N-term variable
*Purified feline IgG was incubated for 18 h with purified 31-kDa protease. The dominant cleavage products were separated by electrophoresis and subjected to amino
acid sequencing. The resulting peptide sequences were aligned with published sequences for feline (accession no. BAA32230, Kanai et al. 2000), bovine (accession no.
AAA98647, Berens et al., 2001), and human (accession no. CAB75705, Hoffman et al., 2000) IgG. The 33- and 34-kDa peptides align with the hinge region of feline
IgG, while the 37-kDa peptide has similarity with the N-termini of the heavy chain variable region of bovine and human IgG.
November 2002
146
Silver et al.
Fig. 4. Cleavage of various proteins by 31-kDa protease.
Two hundred nanograms of purified 31-kDa protease were
incubated with 1 mg of various potential substrate proteins
for 18 h at 37°C and analyzed by SDS-PAGE. A decrease in
the amount of an intact protein and the appearance of
smaller peptides indicates that the protease was able to
cleave that protein. Lanes 1, feline IgG; 2, feline IgG +
31-kDa protease; 3, bovine IgG; 4, bovine IgG + 31-kDa
protease; 5, rabbit IgG; 6, rabbit IgG + 31-kDa protease;
7, feline IgA; 8, feline IgA + 31-kDa protease; 9, feline
IgM; 10, feline IgM + 31-kDa protease; 11, 31-kDa protease; 12, feline serum albumin; 13, feline serum albumin + 31-kDa protease; 14, feline hemoglobin; 15, feline
hemoglobin + 31-kDa protease.
The complete cDNA clone encodes a 265 amino
acid proenzyme that is processed to a 237 amino
acid mature enzyme with an estimated molecular
weight of 25,250 and an isoelectric point of 7.82
(Gaines et al., 1999). The predicted sequence is
highly similar to other insect serine proteases, including a vitellin-degrading protease precursor
from the silkworm, Bombyx mori (Ikeda et al.,
1991). The cDNA cloning, sequence analysis, and
mRNA expression patterns of the gene encoding
the 31-kDa protease are described more completely
in Gaines et al. (1999; referred to as CfSP-28).
ture 31-kDa protease were used to amplify a complete clone of the flea 31-kDa protease gene using the DNA fragments described above. The 980
base pair product was then subcloned into the E.
coli expression vector lambda PR/T2ori/S10HISRSET-A9, which adds 27 amino acids to the Nterminus of the recombinant protein, including
a his6 sequence for affinity purification and a sequence recognized by a T7 monoclonal antibody
(Novagen). The recombinant 31-kDa protease was
produced in One Shot® BL-21 E. coli cells, purified by a combination of affinity chromatography on a Nickel HiTrap™ Metal Chelating Column
and cation exchange chromatography on a HiTrap™
SP-Sepharose column, and used to immunize a
cat to generate anti-31-kDa protease polyclonal
antibodies.
Expression and Purification of Recombinant 31-kDa
Protease Expressed in E. coli
PCR primers complimentary to the regions encoding the predicted N- and C- termini of the ma-
Archives of Insect Biochemistry and Physiology
Degradation of IgG in C. felis
147
Expression and Purification of Recombinant 31-kDa
Protease Expressed in Insect Cells
PCR primers complimentary to the 5¢ and 3¢
ends of the coding region of the 31-kDa protease
were used to amplify a full-length clone of the flea
31-kDa protease gene using the DNA fragments described above. The 1-kB pair product was ligated
into the expression plasmid pVL1393, amplified
in INVaF¢ E. coli cells and transfected into S.
frugiperda cells along with baculovirus DNA. Recombinant virus was collected from the supernatant of the transfected cells, plaque purified, and
used to transfect High-Five™ cells for recombinant
protein expression. Two bands of approximately
31- and 35-kDa were detected in supernatant extracts by Western blot using the cat antiserum generated against the purified E. coli recombinant
31-kDa protease. Active recombinant 31-kDa protease was purified from 6 liters of High-Five™ cell
supernatants by a combination of ammonium sulfate precipitation, affinity chromatography on paminobenzamidine-linked Sepharose beads, and
size exclusion chromatography. The final preparation yielded approximately 300 mg of pure 31-kDa
protease of that ran at approximately 31-kDa on
SDS-PAGE. Interestingly, the 35-kDa band did not
bind to the p-aminobenzamidine affinity column,
implying that the 35-kDa band is an inactive variant of the protein, possibly the unprocessed proenzyme.
Comparison Between Native 31-kDa Protease and
Baculovirus-Expressed Recombinant 31-kDa Protease
Analysis of the purified baculovirus-expressed
recombinant 31-kDa protease indicated that the recombinant enzyme was properly processed by the
S. frugiperda cells. It had the same apparent mass
by SDS-PAGE as the native 31-kDa protease (approximately 31-kDa), and N-terminal sequence
analysis of the recombinant 31-kDa protease
yielded the same amino acid sequence as native
31-kDa protease, IVGGEDVDIST. A comparison of
the IgG degrading activities of the native and recombinant 31-kDa proteases was achieved by inNovember 2002
Fig. 5. Recombinant IgG degrading activity. Active 31-kDa
protease was expressed in baculovirus-infected insect cells
and purified by affinity and size exclusion chromatography. Two hundred nanograms of purified recombinant or
native 31-kDa protease were incubated with 1 mg of feline
IgG for 18 h at 37°C and analyzed by SDS-PAGE under
reducing conditions. The dominant cleavage products are
the same molecular weight and of similar intensity as those
produced by native 31-kDa protease indicating that the
recombinant and native enzymes have the same specificity and activity. Lanes 1: molecular weight standards; 2:
recombinant 31-kDa protease; 3: native 31-kDa protease;
4: recombinant 31-kDa protease with feline IgG; 5: native
31-kDa protease with feline IgG; 6: feline IgG.
cubation with purified IgG. Figure 5 shows that
the dominant cleavage products produced by coincubation of IgG with the recombinant 31-kDa
protease (Fig. 5, lane 4) have the same molecular
weight as those produced by the native enzyme
(Fig. 5, lane 5), indicating that the native and recombinant enzymes have the same specificity for
the IgG hinge region. Thus, it appears that the
baculovirus expressed recombinant 31-kDa protease is processed as expected and is a fully functional IgG degrading protease with the same
activity and specificity as the native enzyme.
148
Silver et al.
DISCUSSION
We first observed the digestion of cat IgG heavy
chain while studying the activity of serine proteases in C. felis midguts. The data in this article
show that C. felis digests host IgG quickly and
precisely. Within a few hours after the initiation
of feeding on cat blood, the heavy chain of cat
IgG is no longer visible by gel electrophoresis.
This suggests that there is a high level of IgG cleaving activity in the fed flea midgut and that the
enzyme(s) responsible are induced by blood feeding. To study this phenomenon, midguts from
10,000 fleas fed on cat blood for 24 h were homogenized and the extracts were applied to an
affinity column that specifically retained serine
proteases. While there was clearly IgG degrading
activity in the material that was not retained by
the column, a significant portion of the IgG degrading material was bound to the column and
was, therefore, presumed to be comprised of one
or more serine proteases. These enzymes were purified by HPLC and the majority of IgG degrading activity was attributed to a specific 31-kDa
protein. N-terminal and internal amino acid sequences were obtained from the purified 31-kDa
protease and the gene encoding the 31-kDa enzyme was cloned from blood-fed flea cDNA libraries. The full-length clone was expressed using
both E. coli and baculovirus expression systems
and expressed recombinant protein was purified.
The baculovirus expressed recombinant showed
IgG degrading activity comparable to the native
enzyme, and N-terminal amino acid sequencing
showed that the baculovirus recombinant was endogenously cleaved at the same position as the
native proenzyme. Time course assays with the
purified native flea 31-kDa protease showed that
it rapidly cleaved cat IgG heavy chain at two specific sites within the hinge region and that the
remainder of the IgG molecule, including the light
chains, was relatively resistant to degradation by
this enzyme. In fact, even after a 24-h incubation
period, little evidence of additional cleavage products was apparent. Given that host blood passes
rapidly through the flea’s digestive tract while the
flea is feeding (Dryden and Rust, 1994), it is likely
that most of the IgG heavy chain is cleaved only
in the hinge region leading to intact F(ab¢)2 and
Fc fragments. This is significant because an intact
F(ab¢)2 fragment may still be able to bind its target antigen. This hypothesis was corroborated by
a study that assessed the specific binding activity
of antibody found in the midguts, gutless bodies,
and feces obtained from fleas after feeding on an
immunized cat. C. felis midguts, gutless bodies,
and feces were all found to contain active antibody or F(ab¢)2 fragments capable of complexing
with a specific antigenic target (Vaughan et al.,
1998). Thus, it appears that ingested antibodies
cleaved by the flea midgut 31-kDa IgG degrading
protease are still capable of binding C. felis antigens, although the combined F(ab¢)2 and Fc fragment functionality may be lost.
There have been several reports of enzymes
that cleave immunoglobulins in the hinge region
leading either to intact Fab or F(ab¢)2 fragments.
Several medically important bacteria, including
Neisseria gonorrhoeae, Neisseria meningitidis, and
Haemophilus influenzae have been shown to contain serine proteases that cleave human IgA, presumably as a way of evading the host’s immune
system (Plaut and Bachovchin, 1994). Although
the cleavage activity appears similar to the flea
31-kDa protease, these enzymes cleave only after
proline residues in the hinge region. In contrast,
the flea 31-kDa protease specifically cleaves after
cysteine and lysine residues, but not after proline
residues found in the IgG hinge region. In addition, no other natural substrates have been found
for the bacterial IgA proteases, but the flea 31kDa protease also cleaves cat IgA, serum albumin,
hemoglobin, rabbit IgG, and bovine IgG. Immunoglobulin degrading enzymes have also been described in parasites (Tamashiro et al., 1987; Kong
et al., 1994) and insects (Eisemann et al., 1995;
Pruett, 1993) that evade the host’s immune system, but this activity has not been described in
ectoparasitic arthropods that remain hidden from
the host immune system, such as fleas, mosquitoes, and ticks. For example, endoparasitic cattle
grub larvae (Hypoderma lineatum), which survive
Archives of Insect Biochemistry and Physiology
Degradation of IgG in C. felis
longer than 8 months within the connective tissues of their bovine host, produce an immunoglobulin degrading enzyme called hypodermin A,
which may serve a role in protecting the larvae
from the host’s immunoglobulins. In contrast, the
ectoparasitic soft tick, Ornithodoros moubata, does
not digest host IgG, and passes intact, fully active
antibody into its hemolymph through the gut wall
(Minoura et al., 1985). It is, therefore, surprising
that C. felis, which live their entire lives outside
of the host and for the most part are hidden from
the host’s immune system, have apparently developed a mechanism to fight the host’s immune
system. In terms of function, the C. felis 31-kDa
protease is quite similar to hypodermin A. Both
are serine proteases capable of cleaving host IgG
in the hinge region forming intact F(ab¢)2 and Fc
fragments, and both can cleave a variety of other
blood proteins. Hypodermin A has been shown
to cleave bovine IgG, sheep IgG, goat IgG, horse
IgG, bovine serum albumin, and bovine hemoglobin (Pruett, 1993). Similarly, the flea 31-kDa
protease can cleave other blood proteins in addition to cat IgG. The broad substrate specificity of
the flea 31-kDa protease suggests that the enzyme
may serve dual roles: to protect the flea against
non-specific immunoglobulins and to help digest
the bloodmeal. Similar roles have also been ascribed to hypodermin A.
Many studies discuss the prospects of creating
vaccines against arthropod parasites (Kay and
Kemp, 1994; Jacobs-Lorena and Lemos, 1995;
Willadsen et al., 1993), but few successful vaccines have been developed. The most successful
arthropod vaccine developed to date has been
against the cattle tick, Boophilus microplus, using a
protective antigen, Bm86, isolated from the tick’s
brushborder membrane. Attempts at developing
vaccines against other arthropods, such as the
sheep blowfly (Lucilia cuprina) and the cattle grub
(Hypoderma lineatum) , have been less successful,
but are still under development. Perhaps vaccines
against the later organisms have been difficult to
develop because these insects have evolved sophisticated mechanisms, including IgG degrading enzymes, for evading the host immune system.
November 2002
149
ACKNOWLEDGMENTS
The authors thank the staff of the Heska flea
insectary for their efforts in providing flea tissues,
Jean Escudero for her work with baculovirus,
Deanna Scott for purifying the E. coli recombinant,
Sean Lupien and Cindy Bozic for their work in protein and DNA sequencing, and Drs. Dan T. Stinchcomb and Carol Talkington Verser for a critical
review of this manuscript.
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Archives of Insect Biochemistry and Physiology
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