Identification of Differentially Expressed Proteins in the Cervical
Mucosa of HIV-1-Resistant Sex Workers
Adam Burgener,*,†,⊥Julie Boutilier,†,⊥Charles Wachihi,‡Joshua Kimani,‡Michael Carpenter,†,‡
Garrett Westmacott,‡Keding Cheng,‡Terry B. Ball,†and Francis Plummer†,‡
Department of Medical Microbiology, University of Manitoba, Winnipeg, Manitoba, Canada R3T 2N2,
Department of Medical Microbiology, University of Nairobi, Nairobi, Kenya, and National Microbiology
Laboratory, Public Health Agency of Canada, Winnipeg, Manitoba, Canada R3G 1C4
Received June 4, 2008
Novel tools are necessary to understand mechanisms of altered susceptibility to HIV-1 infection in
women of the Pumwani Sex Worker cohort, Kenya. In this cohort, more than 140 of the 2000 participants
have been characterized to be relatively resistant to HIV-1 infection. Given that sexual transmission of
HIV-1 occurs through mucosal surfaces such as that in the cervicovaginal environment, our hypothesis
is that innate immune factors in the genital tract may play a role in HIV-1 infection resistance.
Understanding this mechanism may help develop microbicides and/or vaccines against HIV-1. A
quantitative proteomics technique (2D-DIGE: two-dimensional difference in-gel electrophoresis) was
used to examine cervical mucosa of HIV-1 resistant women (n ) 10) for biomarkers of HIV-1 resistance.
Over 15 proteins were found to be differentially expressed between HIV-1-resistant women and control
groups (n ) 29), some which show a greater than 8-fold change. HIV-1-resistant women overexpressed
several antiproteases, including those from the serpin B family, and also cystatin A, a known anti-
HIV-1 factor. Immunoblotting for a selection of the identified proteins confirmed the DIGE volume
differences. Validation of these results on a larger sample of individuals will provide further evidence
these biomarkers are associated with HIV-1 resistance and could help aid in the development of effective
microbicides against HIV-1.
Keywords: HIV-1 • innate immunity • HIV-1-resistance • mass spectrometry • two-dimensional gel
electrophoresis • biomarkers • antiproteases
The Human Immunodeficiency Virus type 1 (HIV-1) pan-
demic has continued unabated for 25 years. Currently, HIV-1
infects an estimated 40 million individuals worldwide.1Glo-
bally, the HIV-1 pandemic disproportionately affects women
due to biological and social factors. This is particularly evident
in sub-Saharan Africa and other developing areas of the world.2
As a result of the inexorable spread of HIV-1, slow HIV-1
vaccine development and the need for female-controlled HIV-
1-prevention technology, interest has grown in the develop-
ment of mucosal HIV-1 microbicides. Thus, the identification
of natural factors involved in protection against HIV-1 in the
genital mucosa is important.
A subset of 140 women out of a total of over 2000 participants
from the Pumwani Sex Worker cohort have been identified to
be relatively resistant to HIV-1 infection.3,4Previously described
resistance mechanisms, such as ∆-32-CCR5 polymorphisms,
have been discounted in this population as their cells are
readily infected in vitro and this genotype has not been
detected in this group.5-9Evidence suggests that the mucosal
layer in the cervicovaginal compartment plays a role in mediat-
ing resistance to HIV-1 infection, which is the first site of
contact for the HIV-1 virus. Studies have shown that protective,
innate and adaptive factors exist in this environment against
HIV-1. These include HIV-1-specific IgA, cellular immune
responses,10,11and a plethora of HIV-1 inhibitory factors such
as RANTES, SLPI, alpha/beta-defensins, lysozyme, lactoferrin,
calprotectin, cystatin, and histone H2A.12-22This suggests that
these, and potentially other undiscovered factors, may also be
playing a role in HIV-1 resistance, and understanding this
mechanism could aid in the development of microbicides and/
or vaccines against HIV-1.
To more thoroughly characterize differences in genital
secretions between HIV-1-resistant, -infected, and -uninfected
individuals, this study employed a gel-based proteomics tech-
nique, two-dimensional difference in-gel electrophoresis (2D-
DIGE). This technique allows the quantitation of differentially
abundant proteins in complex biological samples and has been
used previously to examine cervicovaginal fluid for biomarkers
of pre-term birth.23Here, we report the results of this study
which includes the identification of proteins present at dif-
ferential levels in cervical lavage fluid between HIV-1-resistant,
* To whom correspondence should be addressed. Adam Burgener, E-mail:
email@example.com. Telephone: (204) 789-5001. Fax: (204) 789-2018.
†University of Manitoba.
⊥These authors contributed equally to this manuscript.
‡University of Nairobi.
§National Microbiology Laboratory, Public Health Agency of Canada.
4446 Journal of Proteome Research 2008, 7, 4446–4454
Published on Web 08/16/2008
10.1021/pr800406r CCC: $40.75
2008 American Chemical Society
HIV-1-infected, and HIV-1-uninfected commercial sex workers
(CSW). These proteins represent potential biomarkers of HIV-1
resistance in the Punwani Sex Worker cohort.
Materials and Methods
Study Population. The study was performed with the
University of Manitoba and Nairobi human research ethics
board approval. Samples were collected from consenting
women enrolled in the Pumwani Sex Worker cohort in Kenya.
Thirty-nine women were included in the study, which were
assigned to the following study groups based on HIV-1 status:
HIV-1-resistant (HIV-R, n ) 10), HIV-1-negative (HIV-N, n )
10), HIV-1-positive (infected) sex workers (HIV-P, n ) 10), and
HIV-1-uninfected women from the Mother to Child Transmis-
sion cohort (MCH, n ) 9) which were included as an additional
low risk of infection, HIV-1-uninfected control group. All of the
women from the sex worker cohort were actively engaged in
sex work at the time of sample collection. Selection criteria for
HIV-1 resistance included enrollment in the cohort for at least
3 years and remained uninfected (PCR and HIV-1 antibody
negative) for more than 3 years while engaged in sex work.
Criteria for the HIV-1-negative group were women enrolled in
the cohort for less than 3 years and were HIV-1 uninfected.
The average age for the HIV-R group was 40.6 ( 5.2 years;
HIV-P group 36.5 ( 5.3 years; HIV-N group 30 ( 6.83 years;
and MCH 39 ( 4.5 years. Women included in the study did
not have any other detectable STIs or bacterial infections of
the genital tract at the time of sample collection.
Cervical Lavage (CVL) Sample Collection. The endocervix
was washed with 2 mL of sterile 1× phosphate buffered saline
(PBS) and the lavage was collected from the posterior fornix.
Samples were placed into a 15 mL conical tube and centrifuged
to remove cellular debris, and the supernatant was stored at
-70 °C until analysis. For DIGE analysis, the CVL fluid was
pooled (500 µL from each sample) to form HIV-R, HIV-N, HIV-
P, or MCH groups. The pooled samples were concentrated by
Amicon ultracel 5 kDa MW filter, desalted, and precipitated
using the 2D Clean-Up Kit (GE Healthcare).
Protein Labeling with CyDye Fluors. Cervical lavage samples
were labeled with Cy2, Cy3 or Cy5 following the protocols in
the Ettan DIGE System User Manual (18-1173-17 Edition AA,
GE Healthcare). Briefly, 50 µg of protein (∼5 mg/mL) was
labeled with 400 pmol of dye. Labeling reactions were carried
out for 30 min followed by a 10 min lysine quench (1 µL of 10
mM lysine). Samples from each cohort (Gel one consisted of a
pooled sample, HIV-R and HIV-N sample, while gel two
consisted of a pooled sample, HIV-P and MCH sample) were
mixed and incubated in an equal volume of 2× 2D sample
buffer (7 M Urea, 2 M Thiourea, 4% CHAPS, 130 M DTT, 1%
(v/v) IPG buffer, GE Healthcare) for at least 10 min. After
reduction, samples were brought to a volume of 450 µL with
1× 2D sample buffer (7 M Urea, 2 M Thiourea, 2% CHAPS, 65
mM DTT, 0.5% (v/v) IPG buffer). All reactions were carried out
in the dark on ice. The labeled samples were then used for 2D-
DIGE analysis. The Cy2-labeled pooled sample consisted of an
equal amount of protein from all four populations and served
as an internal control against which all protein volumes were
Two-Dimensional Gel Electrophoresis. Labeled proteins
were separated on an IPGphor isoelectric focusing unit using
24 cm pH 4-7 IPG strips (GE Healthcare) in the dark using
the following profile: 10 h passive rehydration, 4 h at 30 V, 0.5
kVh stepped to 500 V, 1.0 kVh gradient to 1000 V, 13.5 kVh
gradient to 8000 V, 45 kVh stepped to 8000 V. Prior to the
second-dimension run, proteins in the IPG strips were reduced
and alkylated in SDS equilibration buffer (50 mM Tris-HCl, pH
8.8, containing 6 M Urea, 30% (v/v) glycerol, 2% (w/v) SDS and
0.002% (w/v) bromophenol blue) for 15 min in 1% (w/v) DTT
followed by 15 min in equilibration buffer containing 2.5%
(w/v) IAA. Samples were then processed simultaneously on 26
cm × 20 cm × 1 mm precast 10-20% gradient Optigels
(Nextgensciences) using the Ettan Dalt 6 Electrophoresis
System (GE Healthcare). Gels were run in the dark at 10 °C at
constant power (2 W/gel) overnight. The power was increased
to 100 W, and gels were run for 20 min past the dye front
migrating from the gel.
Image Analysis and Poststaining. Gels were imaged directly
between low-fluorescence glass plates on the Typhoon 9400
variable mode imager (GE Healthcare). The DIGE images were
previewed with ImageQuant software version 5.2 to ensure the
absence of detector saturation. After image acquisition, gels
were fixed overnight in a solution containing 40% methanol
and 10% acetic acid. Gels were then stored at 4 °C until further
processing. Finally, gels were subjected to silver staining as
Spot Volume Determination. DeCyder v.5.02 (GE Health-
care) was used to analyze the DIGE images. Scanned images
were cropped in ImageQuant software version 5.2 to remove
gel artifacts and then each image underwent spot detection in
the differential in-gel analysis (DIA) module without any
restrictions set. Protein maps were then exported to the
biological variance analysis module (BVA), where all gels were
land marked with at least 20 protein spots by hand, followed
by software alignment of the remaining proteins. Protein spots
were deemed of interest and identified based on the following
parameters: a volume ratio of at least g1.50 when dividing the
protein spot volume of resistant women to the average of all
others (i.e.: VHIV-R/VAVG:HIV-P,HIV-N,MCH) and clear separation
from other protein spots on the gel to ensure accurate spot
Protein Digest Preparation. Silver-stained gel spots were
destained with 1:1 ratio of 30 mM potassium ferracyanide to
100 mM sodium thiosulfate as described previously.25Gel spots
were washed and dried in 40% acetonitrile/100 mM ammonium
bicarbonate, followed by 100% acetonitrile wash, and dried in
a vacuum centrifuge. Gel spots were rehydrated with 200 µL
of trypsin (Trypsin Gold, Promega) at a concentration of 5 ng/
µL and incubated overnight at 37 °C.
LC-MS/MS Configuration. Nanoflow LC of tryptic peptide
samples was preformed with an Agilent 1100 nanoflow LC
system equipped with a C18precolumn (Zorbax 300SB-C18, 5
µm, 5 mm × 0.3 mm, Agilent) and a C18analytical column
(Zorbax 300SB-C18, 3.5 µm, 15 cm × 75 µm, Agilent). The
aqueous mobile phase (solution A) contained 5% acetonitrile
and 0.1% formic acid, and the organic mobile phase (solution
B) contained 95% acetonitrile and 0.1% formic acid. Samples
(5-µL injected) were loaded and washed on the precolumn for
5 min with solution A at 50 µL/min. Peptides were then eluted
off the precolumn and through the analytical column with a
125-min gradient from 1% to 40% solution B, 5-min gradient
from 40% to 95% solution B, and a 5-min rinse with 95%
solution B at a flow rate of 250 nL/min. The columns were
immediately re-equilibrated for 10 min at initial conditions
(100% solution A for the precolumn and 1% solution B for the
analytical column). Eluting peptides were injected via nano-
spray source into a QStar XL Qq-TOF (Applied Biosystems). The
Differentially Expressed Proteins in HIV-1-Resistant Sex Workers
Journal of Proteome Research • Vol. 7, No. 10, 2008
ion source was equipped with a 50-µm inner-diameter, fuse-
silica needle with a 15-µm tip (PicoTip Emitter, New Objective).
Data-dependent acquisition was used with a 10 s cycle: 1-s
interval for acquiring intact peptide signal (MS), and three 3-s
intervals for collision-induced dissociation of the 3 most intense
peptides signals in the initial 1-s interval (MS/MS). The MS m/z
range was 350-1500, and the MS/MS m/z range was 70-2000.
Collision energy was automatically determined by the data
acquisition software (Analyst QS 1.1). MS/MS data was acquired
for the entire LC run.
Data Analysis. The Mascot search engine (Matrix Science,
London, U.K.; version 2.1.03) was used to search the NCBInr
(20070922) database with the MS/MS data. The search param-
eters were as follows: taxonomy was restricted to Homo sapiens,
protein molecular weight was unrestricted, fixed modification
was Carbamidomethyl (C), variable modification was Oxidation
(M), peptide and fragment mass tolerance was ( 0.4 Da, and
up to one missed cleavage was allowed.
Criteria for Protein Identification. Scaffold (version Scaf-
fold-01_06_19, Proteome Software Inc., Portland, OR) was used
to validate MS/MS based peptide and protein identifications.
Individual Peptide identifications were accepted if they could
be established at greater than 95.0% probability as specified
by the Peptide Prophet algorithm,26while protein identifica-
tions were accepted if they could be established at greater than
80% probability and had at least 2 identified peptides. Protein
probabilities were assigned by the Protein Prophet algorithm.27
Proteins that contained similar peptides and could not be
differentiated based on MS/MS analysis alone were grouped.
Western Immunoblotting. Protein (1 µg) from each sample
was resolved by SDS-PAGE gels (Invitrogen) and transferred
to nitrocellulose membranes using the iBlot transfer system
(Invitrogen). The blots were blocked with 5% nonfat milk for
1 h at room temperature and incubated overnight at 4 °C with
the appropriate dilution of antibody. The blots were then
washed three times with TBST, and incubated with the ap-
propriate horseradish peroxidase (HRP)-conjugated secondary
antibody. The bands were developed with Immobilon detection
reagent (Millipore). The primary antibodies used and catalogue
numbers were as follows: mouse monoclonal anti-Cystatin A
(Cat. no. ab10442, Abcam), rabbit polyclonal anti-SerpinB13
(Cat. no. ab47743, Abcam), mouse monoclonal anti-SerpinB3
(Cat. no. ab55733, Abcam), and Heat-shock protein 70 mouse
monoclonal (Cat. no. ab6535, Abcam).
2D-DIGE Analysis of HIV-R, HIV-P, HIV-N, and MCH
CVL Fluid. To define differentially expressed proteins in CVL
fluid between HIV-1-resistant women and control groups, this
study used the 2D-DIGE multiplex proteomic approach. CVL
protein samples were differentially labeled with a fluorescent
cyanine dye and co-resolved in the same gel to obtain relative
quantitative information. Two independent 2D-SDS PAGE gels
were run: gel one included CVL protein from HIV-R and HIV-P
labeled with Cy5 and Cy3 fluorescent dyes, respectively; gel two
included MCH and HIV-N labeled with Cy5 and Cy3, respec-
tively. A Cy2-labeled internal standard control (pool of all four
groups) was multiplexed with the samples and co-resolved in
each of the gels. Gel images were imported into the DeCyder
BVA package software where protein volumes were normalized
to the internal control. Protein maps were matched and protein
volume ratios were determined for the resistant group versus
all three control groups both independently (e.g., HIV-R versus
HIV-P) or together (HIV-R vs average of the 3 others). Figure 1
shows the 2D-PAGE gels images of the co-electrophoresed
HIV-R sample (Figure 1A) and the HIV-P (Figure 1B) sample.
Analysis of the protein maps revealed that 72 protein spots
demonstrated a clear change in volume (at least g1.50) between
the HIV-R and HIV-P, and HIV-R versus the average of the 3
other groups. These spots were excised and analyzed by tandem
mass spectrometry. From these 72 picked protein spots, 26 of
them (representing 17 unique proteins) were identified confi-
dently by mass spectrometry and are numbered in Figure 1.
Table 1 shows the identity of these protein spots along with
the volume ratio change with respect to the HIV-R group.
Eight proteins were overexpressed and nine were underex-
pressed in the HIV-R group. The proteins that demonstrated
the greatest overexpression included serpin B3 (4.65-fold, spot
363) and serpin B4 (3.17-fold, spot 500), followed by alpha-2
macroglobulin-like 1 protein (2.72-fold, spot 502), cystatin A
(2.15-fold, spot 768), thioredoxin (2.23-fold, spot 751), S100A7
protein (1.88-fold, spot 791), serpin B13 (1.71-fold, spot 390),
serpin B1 (1.53-fold, spot 370). Rho-dissociation inhibitor was
Figure 1. Two dimensional DIGE analysis of proteins in cervical lavage fluid. 2D-E was performed on 50 µg of fluor-labeled CVL protein
using 24 cm isoelectric focusing strips with a pH range of 4-7 in the first dimension and SDS-PAGE (10-20% gradient) in the second.
The images represent protein spots that are more abundant (A) or less abundant (B) in the HIV-1-resistant group compared to the
control group. Protein spots that showed a >1.50-fold change in abundance are numbered.
Burgener et al.
4448 Journal of Proteome Research • Vol. 7, No. 10, 2008
the most underexpressed protein in the HIV-R group (8.43-
fold, spot 674), followed by haptoglobin (5.74-fold, spot 662),
IGHA1 protein (4.71-fold, spot 221), beta-actin (3.94-fold, spot
342), transglutaminase-3 (3.26-fold, spot 569), complement
component 3 (2.93-fold, spot 417), chloride intracellular chan-
nel 1 (1.65-fold, spot 513), and apolipoprotein A1 (1.50-fold,
To more directly assess the trend of protein differences, the
standardized log abundance of each protein spot was calculated
and compared across all groups. This was done by dividing
the individual protein spot volume by the internal control.
Thus, a 50% difference in spot volume would have a standard-
ized log value of ( 1.5, while no changes would have a value
of (1.0. When these values were plotted, different trends of
abundance were observed, and a visual representation of this
is shown in Figure 2. For simplicity, each protein spot was
assigned a trend letter (A, B, or C) depending on which it was
most closely matched. Proteins which were clearly segregated
in the HIV-R group from the others, either overexpressed or
underexpressed with at least 50% (standardized log ratio of >
(1.5) difference to the nearest group, such that all 3 controls
clustered together, fell into the “A” trend. An example of this
is serpin B3 (spot 363) where the HIV-R group was clearly
overexpressed (2.6) compared to the HIV-P (-4.0), HIV-N (-1.3)
and MCH groups (-1.5). Protein spots that were differentially
expressed in the HIV-R group and had one other group within
50% abundance, but separated from the other groups by at least
50%, were assigned the “B” trend. S100A7 protein (spot 791)
fell into this category, clustering the HIV-R group (1.6) with
MCH (1.4), but overexpressed compared to HIV-P (-1.5) and
HIV-N (-1.8). The “C” trend represents those protein spots
which showed differential expression in the HIV-R group to at
least one other group by 50%, but not in a clear pattern from
the other two. This was observed with serpin B1 (spot 370)
where HIV-R was overexpressed (1.3) compared to HIV-P
(-2.0), but not overly so to HIV-N (-1.1) and MCH (1.1).
Visual inspection of the gel images shows that there is a clear
cluster of overexpressed proteins in the right-hand portion of
the gel (Figure 1A). The majority of these spots were identified
as serpin B3. The separation of serpin B3 into distinct locations
highlights the resolution capacity of 2DE and ability to distin-
guish different isoforms from one another. Shifts in the
horizontal plane represents different pIs (i.e., protein spots 377
and 363) due to different post-translational modifications that
alter the charge state. Alternatively, shifts in the vertical plane
(i.e., protein spots 354 and 468) are most likely different
cleavage products although other large post-translational modi-
fications such as glycosylation and even phosphorylation have
been shown to dramatically effect protein migration in certain
cases. Serpin B3 is known to have multiple isoforms: a native
form of 44.5 kDa and pI of 6.36, with shorter versions at 38.5
kDa and pI of 6.29, and 35.5 kDa and pI of 7.98, which would
account for the presence of this protein in multiple spots at
different molecular weights and/or isoelectric points.
Gene Ontology of Differentially Expressed Proteins in
HIV-1-Resistant Women. Differentially expressed proteins in
the HIV-R sample were grouped based on their primary
biological function according to their gene ontology. Table 2
shows the list of overexpressed proteins (top) in HIV-R and
underexpressed proteins (bottom) with their major biological
function listed. A visual representation of these groups is shown
in Figure 3. The majority of overexpressed proteins tended to
be those with antiprotease activity. A small portion of these
are involved in immune response and cellular proliferation. The
underexpressed proteins in the HIV-R group did not show a
clear trend in biological function clustering. Many are involved
in the immune response, cell organization, with some having
antiapoptotic activity, or involvement with ion transport/
Western Blots. To confirm the trends observed by 2D-DIGE,
immunoblots using antibodies specific for cystatin A, serpin
B3, and serpin B13 were performed on the same protein pools.
The results confirm the trends seen in the DIGE analysis for
these proteins and are shown in Figure 4. The top blot shows
serpin B3 and there is clear overabundance of all isoforms in
the HIV-R groups, at 45 kDa (native MW), 39, 36, and 32 kDa.
This matches the literature values of 44.5, 39.5, and 35.5 kDa.
The smaller 32 kDa isoform may represent a further cleavage
product. Serpin B13 also showed a clear overabundance in the
HIV-R sample, for all MWs of 46, 40, 38, and 29 kDa. Cystatin
A also followed the trend observed in the 2D-DIGE analysis
and is overabundant in the HIV-R group, with the other three
groups at roughly the same levels. To exclude the possibility
that the HIV-R sample was overloaded in the immunoblots,
another protein known to be present in cervicovaginal fluid,
HSP70, which was not found to be differentially abundant by
2D-DIGE, was used as a control. As of yet there is no defined
protein in cervical lavage fluid that is known to be present in
equal amounts across individuals, so the selection of HSP70
was simply to demonstrate a immunoblotting trend that
Figure 2. A representative diagram of protein abundance trends
observed in cervical lavage fluid between study groups. Each of
the proteins identified showed either an “A”, “B”, or “C” trend
of abundance. “A” trend proteins were those that were clearly
differentially abundant in the HIV-1-resistant group; the “B” trend
proteins were similar in abundance between the HIV-1-resistant
and one other group; and the “C” trend proteins were those that
did not clearly segregate the HIV-1-resistant group from the other
Differentially Expressed Proteins in HIV-1-Resistant Sex Workers
Journal of Proteome Research • Vol. 7, No. 10, 2008
differed from cystatin A, serpin B3, and serpin B13. The bottom
immunoblot shows that the HIV-R sample had relatively the
same amount of HSP70 as HIV-N, where the MCH and HIV-P
samples had slightly higher amounts.
To address the possibility that the overexpression of these
markers was due to an overabundance in a few samples,
immunoblots of cystatin A were performed on each individual
unpooled sample. The trends closely matched that seen with
Table 1. Abundance Ratios of Protein Spots That Are Overexpressed (A) and Underexpressed (B) in Cervical Lavage Fluid between
HIV-1-Resistant Women and Control Groups
standardized log ratioc
(A) Overexpressed Proteins in HIV-1-Resistant Women
Alpha 2-macroglobulin like-1
Cystatin A 768
S100A7 protein 791
Serpin B1 370
Q86VF3Serpin B3 14
(B) Underexpressed Proteins in HIV-1-Resistant Women
Rho GDP dissociation inhibitor674
IGHA1 protein 221
Complement component 3417
aVolume ratio: the standardized ratio between the HIV-1-resistant group versus the standardized average of the other three groups determined by
DIGE.bTrend: according to Figure 2.cStandardized log ratio: the log value of the sample spot volume ratio over that of the internal control (pool of all
Table 2. Biological Function of Differentially Expressed Proteins in Cervical Lavage of HIV-1-Resistant Women
numberproteinprotein biological function
Overexpressed in HIV-1-Resistant Women
Cysteine protease inhibitor
Serine protease inhibitor
Elastase inhibitor, broad-spectrum endopeptidase inhibitor
Cysteine protease inhibitor
Innate immune response, epidermis development
Cysteine protease inhibitor
Elastase/Cathepsin G inhibitor
Alpha 2-macroglobulin like-1 protein
Underexpressed in HIV-1-Resistant Women
Antiapoptosis, cell motility, cell adhesion
Ion homeostasis, defense response
Complement component 3 Immune response
Transglutaminase 3Cell organization
Chloride intracellular channel 1 Chloride transport, signal transduction
Rho GDP dissociation inhibitor
Burgener et al.
4450Journal of Proteome Research • Vol. 7, No. 10, 2008
the pooled samples with the HIV-1-resistant women having
medium to high levels in almost all individuals (9 of the 10)
(data not shown).
In humans, heterosexual transmission for HIV-1 occurs
across the genital mucosa. The risk of transmission is mediated
by the ability of the virus to gain access to susceptible target
cells. This is dependent on many factors, including the number
of cells which bind HIV-1 and allow it to migrate through this
barrier to infect CD4+ T-cells,28,29damage to the integrity of
the epithelial barrier due to mucosal trauma, inflammation,
and ulceration gives HIV-1 direct access to activated macroph-
ages and T-cells30and the presence of mucosal antiretroviral
factors, including defensins, RANTES, elafin, SLPI, histatins,
statherin, mucins, lysozyme, lactoferrin, and cystatins, affects
this process.13-22,31Therefore, factors that may influence the
integrity of the epithelial barrier, reduce inflammation, aid in
wound repair, or affect abundance and/or effectiveness of
antiviral factors could contribute to altered susceptibility to
This study used a gel-based approach to examine the
cervicovaginal proteome of HIV-1-resistant sex-workers. Steps
were taken to minimize the possibility that these results were
due to natural biological variation between study groups. This
included having a high cutoff threshold of protein selection to
those with only >1.5-fold change in volume despite the ability
of DIGE to reliably detect changes as little as 20%.32In addition,
the protein expression profiles of three control groups were
compared to HIV-1-resistant women to increase the stringency
of biomarker selection. The possibility exists that differential
expression of these proteins could be the result of significantly
over/under abundance in a few individuals which could have
skewed the pooled sample. Although not all biomarkers were
validated on an individual basis, immunoblots of cystatin A
confirmed the overexpression trend observed in the pooled
samples. Ongoing studies on the entire commercial sex worker
cohort, including over 100 HIV-1-resistant women, will be used
to confirm these results.
Comparison of protein profiles revealed that a group of
antiproteases were upregulated in HIV-1-resistant women.
These included those in the serpin B family (B1, 3, 4, and 13),
alpha-2 macroglobulin-like 1, and cystatin A. Of these, cystatin
A is of particular interest given its known anti-HIV-1 proper-
ties.33The exact mechanism of how cystatin A acts against
HIV-1 is not known, but it has been implicated in the interfer-
ence of HIV-1 viral processing, especially during the late stages
of the viral life cycle.34It also blocks epithelial-lymphocyte
interactions which can contribute to HIV-1 infection.34Cysta-
tin’s cysteine antiprotease activity may directly affect the
function of Vif, a viral protein essential for HIV-1 replication
and, therefore, reduce HIV-1 infection of CD4+ T cells.35
Although we found cystatin A to be overexpressed, we have
not yet determined the physiological concentration in these
women. Cystatin A has moderate antiviral activity at physi-
ological concentrations in human saliva (30% inhibition at 2
µg/mL) but increasing in effectiveness at higher concentrations
(90% inhibition at >20 µg/mL).33Therefore, determining the
actual level of overexpression in HIV-1-resistant women will
further support a role in defense against HIV-1 infection.
Serpins B1, B4, and B13 are all inhibitors of cathepsin G, an
inflammatory protease.36-38Cathepsin G is found in high
amounts in cervicovaginal fluid and it is a chemoattractant for
monocytes/neutrophils and also stimulates T-cells which are
targets for HIV-1 infection.39Cathepsin G is known to enhance
HIV infectivity in vitro,40and cleave anti-HIV-1 factors such
as RANTES to render them less effective against HIV-1.41
Therefore, an overabundance of serpins to antagonize cathe-
psin G action may be protective by lowering its inflammatory
capability and potentially lowering targets for HIV-1 infection
in the genital tract. Indeed, specific studies have shown that
serpin B1 is crucial to the innate mucosal immune system by
Figure 3. Biological functional clustering of overexpressed (A)
and underexpressed (B) proteins in cervical lavage fluid of HIV-
1-resistant women. Identified proteins were classified according
to their major biological function according to their gene ontology.
Figure 4. Immunoblots of potential biomarkers of HIV-1-resis-
tance in cervical lavage fluid. Protein (1 µg) from each sample
group was resolved on SDS-PAGE gels and blotted with specific
antibodies. Both Serpin B3 and Serpin B13 show multiple
isoforms with molecular weights ranging from ∼45 to ∼30 kDa.
Differentially Expressed Proteins in HIV-1-Resistant Sex Workers
Journal of Proteome Research • Vol. 7, No. 10, 2008
protecting against cathepsin G and elastase-mediated inflam-
matory damage during host infection by Pseudomonas aerugi-
nosa.42In addition, serpin B4 has been proposed to protect
the epithelial barrier against chymase-induced inflammation
and tissue degradation.37It is interesting to note that other
serpins have anti-HIV-1-specific properties. A recent study
showed that the C-terminal region of Serpin A1 has strong
antiviral activity,43and that its receptor, CD91, is upregulated
and overexpressed in individuals infected with HIV-1 who are
true long-term nonprogressors.44
Serpin B1 and A2ML1 have specific elastase inhibitory
properties.36,45Although not identified in this study, human
neutrophil elastase (HNE), a serine protease, is known to be
present in the cervicovaginal compartment.46It has been
suggested that it can increase the risk of HIV-1 infection by
enhancing myeloid-related protein (MRP-8) expression,47an
inflammatory protein in cervicovaginal fluid known to stimu-
late HIV-1 production. HNE is also associated with increased
risk of intrauterine transmission of HIV-148and can impair
wound healing.49-51It is possible that overexpression of serpin
B1 and A2ML1 may be protective by antagonizing these actions
of HNE and/or maintaining the integrity of the epithelial
barrier. However, whether the concentration differences have
biological significance is unknown and it may be possible that
a small increase has little effect on total anti-elastase activity
Some of the downregulated proteins in HIV-1-resistant sex
workers identified in this study have known roles in HIV-1
infection. Complement component 3 serves as a chemoattrac-
tant that contributes to the generation of a specific immune
response. It is activated upon HIV-1 infection and can enhance
infection by allowing opsonized virus to bind to complement
receptor positive cells and infect them more efficiently.52It also
allows the fusion to nonpermissive cells, such as follicular
dendritic cells. Rho dissociation inhibitor has been implicated
in mediating HIV-1-infected cell migration though tight junc-
tions.53Beta-actin, a normal cytoskeletal component in cells,
is known to bind to HIV-1 reverse transcriptase and may be
involved in HIV-1 secretion.54Apolipoprotein A1, surprisingly,
is known to bind gp41 of HIV-1 and act as a competitive
inhibitor for binding to the cell surface,55-57although apoli-
poprotein levels are known to be affected upon HIV-1 infection,
so this may be a result of HIV-1 infection rather than being
related to HIV-1-resistance.
The identification of novel protein biomarkers that are
associated with HIV-1 resistance is important to understanding
what mediates protection against infection. This study found
that HIV-1-resistant sex workers differentially express proteins
in cervical mucosal fluid compared to HIV-1-infected and HIV-
1-uninfected counterparts. The most biologically interesting of
these include those from the serpin B family, alpha-1 macro-
globulin-like 1, and cystatin A, which have antiprotease and/
or anti-inflammatory properties. Cystatin A is known to have
anti-HIV-1 activity in vitro and it may be contributing, by itself
or in concert with other factors, to a protective environment
against HIV-1 infection. The hope is to use this information to
aid in the design or improvement of microbicides against HIV-
1. This technique will be carried out on a larger sample of
individuals to confirm their association with resistance to HIV-1
infection. The potential role these proteins play in HIV-1
infection is currently under investigation.
Acknowledgment. We thank the women from both
the Punwami Sex Worker and Mother to Child Transmission
cohorts for their dedicated and consistent participation in
this study. This work was funded by a grant from the Bill
and MelindaGates Foundation
Challenges in Global Health Initiative, the National Institute
Canadian Institutes of Health Research (CIHR). Francis A.
Plummer is a Tier I Canadian research chair in Resistance
and Susceptibility to Infections.
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