Secretory leukocyte protease inhibitor: Inhibition of human immunodeficiency virus-1 infection of monocytic THP-1 cells by a newly cloned protein

Article abstract of DOI:10.1016/S0045-2068(02)00008-1

The ability of the salivary protein, secretory leukocyte protease inhibitor (SLPI), to inhibit human immunodeficiency virus-1 (HIV-1) infection in vitro has been reported previously and has led to the suggestion that SLPI may be partially responsible for

Full text of DOI:10.1016/S0045-2068(02)00008-1

BIOORGANIC
CHEMISTRY
Bioorganic Chemistry 30 (2002) 249–263
www.academicpress.com
Secretory leukocyte protease inhibitor:
inhibition of human immunodeficiency
virus-1 infection of monocytic THP-1 cells by
a newly cloned protein
a
b
Nancy R.Shine, Susan C.Wang, Krystyna Konopka,a,*
b
a
€ €
Elizabeth A.Burks, Nejat Duzguneßs,
and Christian P.Whitman
b,
*
a
Department of Microbiology, School of Dentistry, University of the Pacific, San Francisco,
CA 94115, USA
b
Medicinal Chemistry Division, College of Pharmacy, The University of Texas, Austin,
TX 78712-1074, USA
Received 5 February 2002
Abstract
The ability of the salivary protein, secretory leukocyte protease inhibitor (SLPI),
to inhibit human immunodeficiency virus-1 (HIV-1) infection in vitro has been
reported previously and has led to the suggestion that SLPI may be partially
responsible for the low oral transmission rate of HIV-1.However, results contra-
dictory to these findings have also been published.These discrepancies can be at-
tributed to a number of factors ranging from the variability of macrophage
susceptibility to HIV infection to the quality of commercially available preparations
of SLPI.To resolve these differences and to study further the potential anti-HIV-1
activity of SLPI, the purified and re-folded protein, expressed from a synthetic gene,
was examined using human monocytic THP-1 cells.This newly cloned SLPI reduced
HIV-1Ba-L infection in differentiated THP-1 cells, in contrast to the results observed
* Corresponding authors.Fax: 1-415-929-6564 (KK) and 1-512-232-2606 (CPW).
E-mail addresses: kkonopka@sf.uop.edu (K. Konopka), whitman@mail.utexas.edu (C.P.
Whitman).
0045-2068/02/$ - see front matter Ó 2002 Elsevier Science (USA).All rights reserved.
PII: S0045-2068(02)00008-1

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N.R. Shine et al. / Bioorganic Chemistry 30 (2002) 249–263
when using commercially available preparations of SLPI.Interestingly, while the two
proteins displayed different anti-HIV effects they had comparable anti-protease ac-
tivity.The identification of the THP-1 cell line as a system that supports HIV rep-
lication, which can be inhibited by a preparation of SLPI now available in large
quantities, sets the stage for a thorough investigation of the molecular and structural
basis for the anti-HIV activity of SLPI. Ó 2002 Elsevier Science (USA).All rights
reserved.
Keywords: Secretory leukocyte protease inhibitor; Human immunodeficiency virus-1; THP-1
cell line; Synthetic gene
Secretory leukocyte protease inhibitor (SLPI)1 is a potent 11.7 kD serine protease
inhibitor found in mucosal secretions, including bronchial and cervical mucus [1],
seminal plasma [2], and secretions of the parotid and submandibular salivary glands
[3,4].SLPI inhibits two neutrophil serine proteases (neutrophil elastase and cath-
epsin G), the pancreatic proteases (a-chymotrypsin and trypsin) [5], as well as human
mast cell chymase [6] and tryptase Clara [7].Its presence in various mucosal secre-
tions suggests that its primary function is to protect mucosal tissues from proteo-
lytic degradation by neutrophil proteases released in response to inflammatory
stimuli [8,9].SLPI may also play a role in the host defense against certain bacterial
and fungal infections [10,11], as well as the pneumotropic Sendai and influenza A
viruses [7].
The presence of SLPI in salivary secretions has raised the possibility that it might
be one of the components responsible for the anti-human immunodeficiency virus
type-1 (HIV-1) activity of whole saliva and the low oral transmission rate of acquired
immune deficiency syndrome (AIDS) [12–15].However, the ability of SLPI to block
HIV infection remains controversial.Two laboratories have reported that recom-
binant SLPI (rSLPI) inhibits HIV-1 infection of macrophages and primary T-cells
[13–16,18,19], while a third group found that SLPI had no anti-HIV effect in human
T-cell lines, peripheral blood lymphocytes, and primary macrophages [17].2 We have
also observed significant variability in the ability of rSLPI to inhibit HIV-1 infection
1
Abbreviations used: BSA, bovine serum albumin; Cat G, human neutrophil cathepsin G;
Chy, chymotrypsin; CCR5, CC chemokine receptor 5; ELISA, Enzyme-linked immunosor-
bent assay; ESI-MS, electrospray ionization mass spectrometry; HIV-1, human immunode-
ficiency virus-1; HPLC, high-pressure liquid chromatography; HS, heparan sulfate; IPTG,
isopropyl-b-D-thiogalactoside; Kn, kanamycin; LB, Luria–Bertani medium; MIP, macro-
phage inflammatory protein; NE, human sputum elastase; PCR, polymerase chain reaction;
PES, polyether sulfone; RANTES, regulated upon activation, normal T-cell-expressed, and
presumably secreted; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis;
SLPI, secretory leukocyte protease inhibitor; SP, sulfopropyl; TCID50, tissue culture
infectious dose.
2
All of these studies have been performed using the same, commercially available,
preparation of SLPI obtained from either Synergen and/or R&D Systems.

N.R. Shine et al. / Bioorganic Chemistry 30 (2002) 249–263
251
in macrophages [20].The observed discrepancies may be due to the fact that the
susceptibility of macrophages to HIV-1 infection is influenced by isolation, culture
and infection conditions, donor variations, and maturation of macrophages at the
time of infection [21].
Since SLPI is thought to inhibit a step beyond virus binding, but before reverse
transcription [16,18], it is likely that it interacts with a cell surface molecule(s) in-
volved in virus entry and whose expression is variable depending on the experimental
conditions.It has been suggested that the anti-HIV effect of rSLPI does not depend
on its anti-protease activity, but rather the interaction of SLPI with cell surface
molecules other than CD4 [13,16,18,19].Although CD4 serves as the primary re-
ceptor for HIV-1, other cofactor(s) are essential for virus-cell membrane fusion re-
quired for virus entry [22,23].We found no correlation between the binding of rSLPI
to human T-cells and the expression of the cell surface proteins CD4, CD26, and
CCR5 [24].It has been reported recently that the cell surface glycosaminoglycan,
heparan sulfate (HS), may participate in HIV-cell attachment and virus entry
[25–27].The cationic SLPI forms a tight complex with the negatively charged
polysulfated glycosaminoglycan, heparin [28].Thus, binding of SLPI to HS ex-
pressed on the cell surface may affect HIV interactions with its receptors and inhibit
infection.However, treatment of differentiated THP-1 cells with heparinases does
not reduce SLPI binding [29].
It is possible that differences in the commercially available batches of rSLPI may
also contribute to the variability.It has been suggested that both instability and
susceptibility to oxidation may result in a loss of antiviral activity of rSLPI [18,19].
Studies in our laboratory, however, have eliminated degradation or oxidation of the
rSLPI molecule as the cause of the variable anti-HIV effect of rSLPI [20].
To address these difficulties, we have constructed an expression system to provide
large quantities of the SLPI protein, and used the THP-1 cell line as a model system
for the evaluation of the anti-HIV effect of SLPI.THP-1 cells [30,31] differentiate
into macrophage-like cells after treatment with phorbol 12-myristate 13-acetate
(PMA) [32] and are frequently used to study HIV macrophage/monocyte interac-
tions, as well as the relationship between cellular differentiation and virus production
[33–38].For the construction of the expression system, the human SLPI gene was
synthesized by a PCR-based strategy using long oligonucleotides and cloned into an
expression vector [39,40].Subsequently, the protein was overexpressed in Escherichia
coli, and the purified protein successfully folded as assessed by its ability to inhibit
chymotrypsin, elastase, and cathepsin G [41].This SLPI (designated sSLPI) 3 reduced
the infection of differentiated THP-1 cells by HIV-1Ba-L.The antiviral effect observed
with sSLPI was not observed with a commercially available preparation (i.e., rSLPI),
although both exhibited comparable anti-protease activity.The identification of a
system that supports HIV replication which can be inhibited by a preparation of
SLPI, coupled with the availability of an expression system for SLPI and its mutants
will now enable us to investigate the biochemical and structural basis for the anti-
3
For simplicity of the discussion, the SLPI produced by the synthetic gene will be designated
sSLPI while the commercially available preparation of SLPI will be designated rSLPI.

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HIV activity of SLPI.Some of these results have been presented earlier in prelimi-
nary form [42].
Experimental procedures
Materials.All reagents, buffers, and solvents were obtained from either Aldrich
Chemical or Sigma Chemical, unless noted otherwise.Tryptone and yeast extract
were obtained from Difco (Detroit, MI).The YM-3 ultrafiltration membranes were
obtained from Millipore (Bedford, MA).Isopropyl- b-D-thiogalactoside (IPTG),
thin-walled PCR tubes, and Genie Prep spin columns were obtained from Ambion
(Austin, TX).Syringe filters were obtained from Nalgene (Naperville, IL).The ex-
pression vector pET24a(+) was obtained from Novagen (Madison, WI).Restriction
enzymes, T4 DNA ligase, and low melting point agarose were obtained from Pro-
mega (Madison, WI).Reagents for the PCR were obtained from either Stratagene
(La Jolla, CA) or F.Hoffmann–La Roche (Basel, Switzerland).Oligonucleotides
were synthesized by Oligos Etc.(Wilsonville, OR).Human sputum elastase (NE) was
purchased from Elastin Products (Owensville, MO) and human neutrophil cathepsin
G (Cat G) was purchased from Calbiochem-Novabiochem (San Diego, CA).Se-
quencing grade chymotrypsin (Chy) was obtained from F.Hoffmann-La Roche.
Recombinant SLPI (rSLPI) was acquired from R&D Systems (Minneapolis, MN).
Bacterial strains. Escherichia coli strain DH5a was used for transformation of
ligated plasmids and E. coli strain BL21(DE3)pLysS was used for expression of the
recombinant proteins.Both strains were obtained from Novagen.Cells for general
cloning and expression were grown in LB media supplemented with kanamycin
(50–100lg=mL).The compositions of LB and SOC medium are described else-
where [43].
Cells and viruses.Monocytic THP-1 cells (American Type Culture Collection;
ATCC, TIB-202) were maintained at 37 °C, under 5% CO2 in RPMI 1640 medium
(Irvine Scientific, Santa Clara, CA) supplemented with 10% (v/v) heat-inactivated
fetal bovine serum (FBS) (Irvine Scientific), penicillin (100 U/mL), streptomycin
(100lg=mL), and
L-glutamine (2 mM) (RPMI medium/10) [30,31].The cells,
cultured as a single-cell suspension, were split 1:4 once a week to give a density
of 1 ꢀ 106 cells/mL.The supernatant of chronically infected H9 HTLV-IIIB cells
was used as a source of the HIV-1IIIB strain.The p24 concentration of the stock
was 150 ng/mL as determined by ELISA.A monocytotropic strain, HIV-1 Ba-L, was
obtained from Advanced Biotechnologies.(Columbia, MD) and propagated in
macrophages [44].The p24 concentration of the stock solution was 364 ng/mL,
and the TCID50 was ꢁ1 ꢀ 106/mL.
Protease substrates.The activities of chymotrypsin and cathepsin G were assayed
using the protease substrate N-succinyl–Ala–Ala–Pro–Phe–p-nitroanilide.The ac-
tivity of elastase was assayed using N-methoxysuccinyl–Ala–Ala–Pro–Val–p-nitro-
anilide.The synthetic chromogenic substrates were obtained from Sigma Chemical.
General methods.Techniques for restriction enzyme digestions, ligation, trans-
formation, and other standard molecular biology manipulations were based on
methods described elsewhere [43].Plasmid DNA was introduced into cells by

N.R. Shine et al. / Bioorganic Chemistry 30 (2002) 249–263
253
electroporation using a Cell-Porator Electroporation System (GibcoBRL, Gai-
thersburg, MD).DNA sequencing was done at the University of Texas (Austin)
Sequencing Facility.HPLC was performed on a Waters system using either a
TSKgel phenyl 5-PW hydrophobic column or a TSKgel SP-5-PW (cation exchange)
column (TosoHaas, Montgomeryville, PA).Protein was analyzed by sodium dodecyl
sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) under denaturing condi-
tions on 17% gels on a vertical gel electrophoresis apparatus obtained from Bio-Rad
[45].Protein concentrations were determined using the method of Waddell [46].
Kinetic data were obtained on either a Hewlett Packard 8452A Diode Array spec-
trophotometer or on a Vmax microplate reader (Molecular Devices, Sunnyvale, CA),
as indicated in the text.
Synthesis of the human SLPI gene.The synthesis of the human SLPI gene was
carried out by the PCR in three stages in a Perkin–Elmer DNA Thermocycler using
six long oligonucleotides, two oligonucleotide primers, and the PCR reagents [39,40].
The six long oligonucleotides ðꢁ75 bases each), corresponding to segments of the
full-length human SLPI gene [47], are shown in Table 1.Two oligonucleotide
primers, 50-GAGATATACATATGTCTGGTAAAAGC-30 (primer A) and 50-
GTCGACTTATGCTTTTACCGG-30 (primer B), were also synthesized in order to
amplify the PCR-synthesized gene.Primer A consists of a NdeI restriction site
(underlined) followed by 12 bases corresponding to the coding sequence for the SLPI
gene.Primer B consists of a SalI restriction site (underlined), followed by a stop
codon and 12 bases corresponding to the complementary sequence of the SLPI gene.
Table 1
The sequences of the six oligonucleotides used to synthesize the SLPI gene
Segment Aa
50-GA GAT ATA CAT ATG TCT GGT AAA AGC TTC AAA GCT GGC GTA TGC
CCG CCG AAA AAA TCC GCG CAG TGT CTG CGG-30
Segment B
50-ACA ACG TTT TTT ACC CGG GCA CTG CCA GTC GGA CTG GCA TTC CGG
TTT TTT GTA CCG CAG ACA CTG CGC GGA-30
Segment C
50-CCG GGT AAA AAA CGT TGT TGC CCG GAC ACC TGC GGC ATC AAA
TGC
CTG GAT CCG GTT GAT ACC CCG AAC CCG-30
Segment D
50-CAG CAT CAG ACA CTG GCC ATA GGT TAC CGG GCA TTT ACC CGG TTT
TCG ACG AGT CGG GTT CGG GGT ATC AAC-30
Segment E
50-GGC CAG TGT CTG ATG CTG AAC CCG CCG AAC TTC TGC GAA ATG GAC
GGC CAG TGT AAA CGA GAT CTG AAA TGC TGT-30
Segment Fa
50-G CTT GTC GAC TTA TGC TTT TAC CGG GGA AAC ACA AGA TTT GCC
GCA
CAT ACC CAT ACA GCA TTT CAG ATC TCG-30
a The NdeI restriction site in Segment A and the SalI restriction site in Segment F are
underlined.

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In stage 1 of the PCR, a 25-lL reaction mixture consisted of 10ꢀ PCR buffer
(200 mM Tris–HCl, pH 8.8, 20 mM MgSO4, 100 mM KCl, 100 mM ðNH4Þ SO4, 1%
2
Triton X-100, 1 mg/mL nuclease-free BSA, 2:5lL), dATP, dTTP, dGTP, and dCTP
(1lL each from 5 mM stock solutions), segments A–F (1lL each from 1lM stock
solutions), primers A and B (2lL each from 10lM stock solutions), Pfu DNA
polymerase (0:25lL of a 2.5 U/lL solution), and de-ionized water (11:25lL).The
reaction mixture was contained in a thin-walled reaction tube and layered with sterile
paraffin oil.The PCR protocol consisted of 25 cycles, a 2-min incubation period at
94 °C preceding the 25 cycles, and a 10-min incubation period at 72 °C following the
25 cycles.Each cycle consisted of three steps: denaturation at 94 °C for 45 s, an-
nealing at 55 °C for 45 s, and elongation at 72 °C for 2 min.The reaction tubes were
maintained at 4 °C upon completion.The amplified products were extracted from the
oil [48,49], electrophoresed on a 2% agarose gel, and visualized by UV transillumi-
nation.The band corresponding to a 344 bp product was excised and extracted from
the gel [49,50].
In the second stage of PCR, the gel extract (containing the 344 bp product) was
used as the template for amplification by the PCR.Three reaction mixtures were
made up (25lL each) as described above, with two modifications.The gel extract
(2lL) was used in place of the six long oligonucleotides and two mixtures contained
additional quantities of MgCl2 (0.5 and 1:0lL from a 25 mM solution).The PCR
protocol consisted of 25 cycles, a 3-min incubation period at 94 °C preceding the 25
cycles, and a 5-min incubation period at 72 °C following the 25 cycles.Each cycle
consisted of three steps: denaturation at 94 °C for 1 min, annealing at 55 °C for 75 s,
and elongation at 72 °C for 75 s.Upon completion, the reaction mixtures were
treated as described above.UV transillumination of the gel showed that all three
reaction mixtures generated a product consisting of ꢁ344 bp.
In the last stage of PCR, the SLPI gene was further amplified using the gel extract
containing the 344 bp product.A 200 lL-reaction mixture consisted of 10ꢀ PCR
buffer (20lL), MgCl2 (4:0lL from a 25 mM stock solution), dATP, dTTP, dGTP,
and dCTP (1:6lL each from 25 mM stock solutions), gel extract (16lL), primers A
and B (16lL each from 10lM stock solutions), Pfu DNA polymerase (2lL of a
2.5 U/lL solution), and de-ionized water (124:4lL).The mixture was divided into
four equal portions and subjected to the PCR protocol used in stage two.The PCR
product was isolated and stored [49].
The resulting PCR product and the pET24a(+) were digested with NdeI and SalI
restriction enzymes [43], purified on a 1% agarose gel, and extracted from the gel [49].
The linearized vector and the SLPI gene were ligated using T4 ligase (3 U) at 16 °C
overnight [43].The DNA was precipitated from the mixture [43] and re-suspended in
sterile water (10lL).Aliquots (2 lL) were used to transform E. coli strain DH5a by
electroporation following the manufacturerÕs directions.Subsequently, an aliquot
(10lL) of the transformed cells was incubated in SOC media (1 mL) for 1 h, cen-
trifuged for 3 min, and a portion of the supernatant (900lL) removed.The cell pellet
was re-suspended in the remaining media and plated onto an LB/Kn (100lg=mL)
agar plate and grown overnight at 37 °C.Eleven single colonies were chosen
at random and screened for the presence of insert by the PCR using two primers:
one that is specific for the T7 promoter region of the pET-24a(+) vector

N.R. Shine et al. / Bioorganic Chemistry 30 (2002) 249–263
255
(50-TAATACGACTCACTATAGG-30) and one that is specific for a sequence of the
T7 terminator region of the pET-24a(+) vector (50-TAGTTATTGCTCAGCGGT-
30).The presence of the insert is indicated by the observation of a PCR product of the
appropriate size (ꢁ500 bp).Accordingly, the 11 individual colonies were treated as
described elsewhere [49] and the resulting cell suspension was used as the template in
the described PCR protocol [49].The products were analyzed by agarose gel elec-
trophoresis.Positive colonies were grown in liquid LB/Kn medium (10 mL,
100lg=mL) overnight, and the newly constructed plasmid (designated pET24a–
SLPI) was isolated [51].The isolated plasmid was treated as described [49] and stored
at )20 °C.For protein expression, an aliquot (1 lL) was introduced into E. coli strain
BL21(DE3)pLysS by electroporation as described above.
For sequencing, the plasmid (pET24a–SLPI) was isolated from an overnight
growth of E. coli strain DH5a cells by the alkaline lysis method [43], treated with
2.5 M ammonium acetate, precipitated with ethanol, and suspended in TE buffer
(10 mM Tris–Cl, 1 mM EDTA, pH 8.0). Subsequently, a portion of the DNA
(ꢁ15lg) was exchanged into water using an Ambion Genie Prep spin column
according to the manufacturerÕs directions.
Expression of the SLPI gene.A single colony of the expression strain was used to
inoculate 50 mL of LB/Kn (50lg=mL).After overnight growth at 37 °C, 5 mL of the
culture was used to inoculate 750 mL of LB/Kn (50lg=mL) in a 2-L Erlenmeyer
flask, giving an initial OD600 of ꢁ0.05. Cultures were grown to an OD600 of 0.6–0.8 at
37 °C with vigorous shaking and then induced with isopropyl-b-
D-thiogalactoside
(IPTG) (0.5 mM final concentration). Incubation was continued for 3–4 h at 37 °C.
Cultures were harvested by centrifugation (7000g, 12 min) and stored at )80 °C.
Typically, 4.5 L of culture grown under these conditions yields ꢁ8.5 g of cells.
Purification and folding of SLPI.SLPI was purified by a modification of the
procedure of Kohno et al.[41].In a typical procedure, the cells ( ꢁ8.5 g) were thawed
and suspended in a volume of 50 mM sodium acetate buffer (20 mL, pH 6.0, Buffer
A) that was 2.5 times their weight. The cells were disrupted at 4 °C by sonication with
one pulse (1 min) from a Heat Systems W-385 sonicator equipped with a 0.5-in.
tapped horn delivering approximately 330 W/pulse.The solution was made 05.–
1.0 mM in phenylmethylsulfonyl fluoride and 6-aminocaproic acid, and 0.1 mg/mL in
DNase.Sonication was continued for three 5-min intervals, which were spaced 5 min
apart. Each interval consisted of 2.5-s pulses spaced at 2.5-s intervals. After cen-
trifugation (35,000g, 30 min), the supernatant was made 1 M in ammonium sulfate
and stirred for 1 h at 4 °C.Subsequently, the solution was centrifuged (27,000 g,
30 min), and the supernatant was injected in two portions (ꢁ10 mL each) into a
Phenyl 5-PW column (150 ꢀ 21:5 mm) attached to a Waters HPLC system.The
column had previously been equilibrated with 50 mM sodium acetate buffer, pH 6.0,
containing 1 M ammonium sulfate (Buffer B) at a flow rate of 5 mL/min.The column
was washed with Buffer B for 10 min followed by a decreasing ammonium sulfate
gradient (1.0–0.0 M) in 50 min. At the end of the gradient, the column was washed
with Buffer A for 10 min.The eluent was collected in 10-mL fractions.The protein
eluted from 0.3 to 0.1 M ammonium sulfate as determined by the distinctive orange/
brown color of the fractions and confirmed by SDS–PAGE.The appropriate frac-
tions were collected, pooled (final volume ꢁ125 mL), and diluted with an equal

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N.R. Shine et al. / Bioorganic Chemistry 30 (2002) 249–263
volume of 6 M guanidine hydrochloride at 4 °C.After stirring for 1 h, the mixture
was concentrated by ultrafiltration (Amicon YM-3 membrane) to about 34 mL.
Subsequently, SLPI was folded using a protocol described elsewhere, although
sodium acetate buffer was used in place of Tris buffer [41].The diluted mixture
containing the refolded SLPI was filtered through a 0:2lM syringe filter to remove
the cloudy white precipitate, and concentrated by ultrafiltration to a final volume
of 14 mL.
The concentrate was then injected in two portions (7 mL each) into the TSKgel SP 5-
PW column (150 ꢀ 21:5 mm) attached to a Waters HPLC system.The column had
previously been equilibrated with Buffer A, at a flow rate of 5 mL/min.The column was
washed with the equilibration buffer for 10 min followed by an increasing NaCl gra-
dient (0.0–1.0 M) in 60 min. The eluent was monitored at 254 nm and collected in 10-
mL fractions. SLPI eluted from 0.5 to 0.6 M NaCl as determined by SDS–PAGE. The
most pure fractions were collected, pooled, and concentrated by ultrafiltration.The
concentrate was loaded onto a SephadexG-75 column (100 ꢀ 2:5 cm) equilibrated with
20 mM sodium phosphate buffer (pH 7.4) at a flow rate of ꢁ0.5 mL/min. Fractions
(10 mL) were collected, analyzed by SDS–PAGE, and the purest fractions were com-
bined (>95% as assessed by SDS–PAGE) and concentrated.This procedure yielded
about 6 mg of the purified and folded SLPI.For the biological studies, the SLPI
preparation was exchanged into 10 mM Hepes (pH 7.4) containing 100 mM NaCl.
Mass spectrometry.The mass of the purified SLPI was determined by electrospray
ionization mass spectrometry (ESI-MS) using an LCQ Finnigan octapole electro-
spray mass spectrometer.The samples for ESI-MS were prepared and analyzed as
previously described [52].
Assays for the anti-protease activity of SLPI.The ability of SLPI to inhibit the
activities of three proteases (NE, Chy, and Cat G) was determined using a fixed
concentration of protease in the presence and absence of different concentrations of
SLPI.The assay mixture consisted of 50 mM Hepes buffer (pH 74.) containing
100 mM NaCl, protease (final concentration, NE ¼ 20 nM, Chy ¼ 10 nM, and Cat
G ¼ 60 nM), and SLPI.For the chymotrypsin assay, 10 mM CaCl 2 was included.For
NE, nine concentrations of sSLPI (8–40 nM) and seven concentrations (8–32 nM) of
rSLPI were used.For Chy, 11 concentrations (2–18 nM) of both preparations were
used.For Cat G, eight concentrations (10–100 nM) of both preparations were used.
After 30 min, the Chy substrate (33 lM), the NE substrate (66lM), or the Cat G
substrate (66lM) was added.Stock solutions of substrate were made up in dimethyl
sulfoxide.Activity was monitored by following the rate of the appearance of p-nit-
roaniline at 405 nm using a Vmax microplate reader.The chromophore is generated by
the protease-catalyzed hydrolysis of the appropriate substrate.Assays were carried
out in 96-well microplates at 25 °C.The ratio of the reaction rate in the presence of
inhibitor to that in the absence of inhibitor, a, was determined by fitting the inhi-
bition data to Eq.(1):
2
1=2
ð½EoŠ þ ½IoŠ þ KappÞ À fð½EoŠ þ ½IoŠ þ KappÞ À 4½EoŠ½IoŠg
i
i
a ¼ 1 À
;
ð1Þ
ð2½EoŠÞ
where [Eo] and [Io] are the total concentrations of protease and SLPI, respectively,
and Kiapp is the apparent inhibition constant [53,54].Subsequently, the inhibition

N.R. Shine et al. / Bioorganic Chemistry 30 (2002) 249–263
257
constant, Ki; was calculated by correcting the Kapp values for the effect of substrate
concentration using Eq.(2):
i
Kiapp ¼ Kið1 þ ½SoŠ=KmÞ;
ð2Þ
where [So] is the substrate concentration, and Km is the Michaelis constant.The Km
values were determined separately using the Michaelis–Menten equation and found
to be 90lM (Chy), 140lM (NE), and 120lM (Cat G).The data from all experi-
ments were fitted by non-linear least-squares fit using the Enzfitter software (Elsevier
Science Publishers, Amsterdam).
Infection of PMA-treated THP-1 cells with HIV-1Ba-L.THP-1 cells, growing as a
single-cell suspension, were differentiated by treatment with phorbol 12-myristate 13-
acetate (PMA).Cells were plated at 1 ꢀ 106 cells/mL in 48-well plates and treated
with a 160 nM (ꢁ100 ng/mL) solution of PMA for 24 h at 37 °C.After treatment with
PMA the cells adhered and became flat and amoeboid in shape.Seven days after
PMA treatment, THP-1 (THP-1/PMA) cells were pre-incubated with either rSLPI or
sSLPI for 2 h at 37 °C and infected with HIV-1Ba-L.SLPI remained present during
the 2-h infection period at 37 °C.Control cells were treated similarly but without the
SLPI.After infection, the viral supernatant was removed, the cells were washed three
times and cultured in RPMI medium/10 (1 mL).Infection was monitored by the viral
p24 level in harvested culture supernatants using ELISA plates obtained from the
AIDS Vaccine Program (NCI-Frederick Cancer Research and Development Center;
Frederick, MD).
Results
Synthesis of the human SLPI gene.SLPI consists of 107 amino acids and is en-
coded by a 321 bp gene.In the PCR-based gene synthesis strategy, long oligonu-
cleotides having overlapping bases are joined together using the short overlapping
bases as primers to extend the long nucleotides [39,40].In the same reaction vessel,
the PCR-generated DNA fragment can then be amplified using two short terminal
primers.Accordingly, the full-length SLPI gene was divided into six long oligonu-
cleotides of about equal length (ꢁ75 bases each) with overlaps of 18–21 bases be-
tween the oligonucleotides (Table 1).The oligonucleotides were designed on the basis
of the published DNA sequence for the human SLPI gene [47].Segment A contains
an NdeI restriction site (underlined) followed by 60 bases corresponding to the se-
quence of the SLPI gene.These bases encode amino acids Ser-2–Arg-21.Segment B
contains 72 bases corresponding to the complementary sequence of the SLPI gene,
which codes for the amino acids Ser-16–Cys-39.Segment C contains 72 bases cor-
responding to the sequence of the SLPI gene and encodes amino acids Pro-34–Pro-
57.Segment D contains 72 bases corresponding to the complementary sequence of
the SLPI gene, which codes for the amino acids Val-52–Lys-75.Segment E contains
75 bases corresponding to the sequence of the SLPI gene and encodes Gly-70–Cys-
94.Finally, segment F contains a SalI restriction site, a stop codon, and 60 bases
corresponding to the complementary sequence of the SLPI gene.This sequence

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N.R. Shine et al. / Bioorganic Chemistry 30 (2002) 249–263
Table 2
Inhibition of proteases by sSLPI and rSLPI
Inhibitor
Ki (nM)
Chy
Cat G
NE
sSLPI
rSLPI
0:21 ꢂ 0:03
0:10 ꢂ 0:02
5:3 ꢂ 0:6
6:7 ꢂ 0:7
0:13 ꢂ 0:02
0:55 ꢂ 0:05
encodes amino acids Arg-89–Ala-108.4 Using these six segments, the two short PCR
primers, and the PCR reagents, the SLPI gene was constructed and amplified in
sufficient quantities for subsequent manipulations after three separate rounds of the
PCR.The sequence of the synthetic gene was confirmed by DNA sequencing.
Cloning, expression, purification, and folding of the sSLPI.The PCR fragment
containing the synthetic gene was cloned into a pET24a(+) vector using standard
techniques and expressed in E. coli strain BL21 (DE3)pLysS.The soluble portion of
the expressed SLPI was partially purified by hydrophobic interaction chromato-
graphy and subjected to a previously reported folding protocol [41].The refolded
SLPI was then purified to homogeneity (as assessed by SDS–PAGE) by cation-
exchange and size exclusion chromatography.The molecular mass of sSLPI was
determined by ESI-MS and found to be 11,711 Da, which is within experimental
error of the calculated mass (11,726 Da).Typically, this procedure yielded about
1.3 mg of pure, folded SLPI per liter of cell culture.
Inhibition of proteases by SLPI.The inhibitory activities of the sSLPI and rSLPI
on chymotrypsin, human sputum elastase, and human neutrophil cathepsin G were
determined by assuming that SLPI acts as a tight binding inhibitor, as described
previously [53,54].The results are summarized in Table 2.The values are comparable
and consistent with those reported [47,55].The results indicate that sSLPI is a more
potent inhibitor of elastase and chymotrypsin than of cathepsin G.
Effect of SLPI on HIV-1 infection of PMA-treated THP-1 cells.The ability of
SLPI (10lg=mL) to inhibit infection of PMA-treated THP-1 cells by HIV-1Ba-L was
investigated.At the same concentration of rSLPI, McNeely et al.[16] observed
ꢁ80% inhibition of p24 production in primary macrophages infected with HIV-1Ba-L
;
this potent inhibition of virus production was sustained for 3 weeks of culture.PMA-
treated THP-1 cells were infected efficiently with the macrophage-tropic (M-tropic)
HIV-1Ba-L isolate (Fig.1), while a very low level or complete lack of viral replication
was observed with the T-cell tropic (T-tropic) HIV-1IIIB isolate (data not shown).
Under our experimental conditions, sSLPI reduced significantly HIV-1Ba-L infection,
as assessed by viral p24 production.Ninety percent inhibition of viral replication was
observed at 5 days post-infection and ꢁ80% inhibition at days 7 and 11 post-
infection (Fig.1).In contrast, commercially available rSLPI did not inhibit HIV-1
infection in these cells.
4
SLPI consists of 107 amino acids.The numbering system (1–108) includes the initiating
N-formylmethionine, which is removed in E. coli.Thus, the sequence of the purified protein
is identical to that reported for the human SLPI [47].

N.R. Shine et al. / Bioorganic Chemistry 30 (2002) 249–263
259
Fig.1.The effect of sSLPI and rSLPI on p24 production in PMA-treated THP-1 cells.Seven
days post-differentiation, THP-1 cells in 48-well plates, were exposed to sSLPI or rSLPI, at
10lg=mL, as described in the Experimental procedures.Viral p24 antigen production was
determined on days 5, 7, and 11.Data represent the mean and standard deviations obtained
from triplicate wells, and are representative of three independent experiments.
Discussion
In this study, we have addressed two issues hampering efforts to understand
SLPIÕs role in the prevention of HIV-1 infection.We have developed an expression
system from which active SLPI can be obtained in sufficient quantities for future
mechanistic and structural experiments.We have also determined that our sSLPI
inhibits HIV infection of PMA-treated THP-1 cells, whereas commercial prepara-
tions of SLPI (i.e., rSLPI) do not, although both have comparable anti-protease
activity.These results raise the question as to why these two preparations of SLPI
exhibit different anti-HIV effects.The availability of our sSLPI will enable us to
investigate the biochemical and structural basis for the anti-HIV activity of SLPI.
The expression system for SLPI was created by synthesizing the human gene and
cloning the synthetic gene into a T7 expression system.In addition to generating a
source for SLPI and a template for mutants, we pursued this strategy because the T7
expression system is amenable to isotope labeling in a MOPS-buffered minimal
medium for structural studies [56].The relatively small size of SLPI (107 amino
acids) and the fact that it does not have post-translational modifications (e.g., gly-
cosylation) make it feasible to perform a structural analysis of the parent protein and
mutants by NMR spectroscopy.These experiments require significant quantities of
isotopically labeled protein (15N and 13C), which are now achievable using this
expression system.

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N.R. Shine et al. / Bioorganic Chemistry 30 (2002) 249–263
Structural analysis of SLPI is a prerequisite to in-depth mechanistic experiments.
This protein is rich in cysteine residues that form a total of eight disulfide bonds
[57].One obvious concern for preparations of SLPI is whether the protein is
properly folded with the eight correctly paired disulfide linkages.Different con-
formations of SLPI, a result of incorrect formation of disulfide bonds, may be one
explanation for the variability in experiments examining the anti-HIV activity of
SLPI.
The anti-HIV activity of rSLPI was reported by two laboratories [13–15,18,19],
while this effect was not confirmed by others [17], and variable results have been
observed in our laboratory [20].All these studies have been performed using the
same, commercially available, preparation of rSLPI obtained from Synergen and/or
R&D Systems.Comparison of the anti-HIV effect of rSLPI obtained by different
laboratories is complicated by the fact that macrophages were cultured for different
time periods before infection.McNeely et al.[16,18] cultured monocytes on glass for
10–14 days before infection with HIV-1Ba-L.Shugars et al.[19] used 5-day old
macrophages cultured on plastic, while Turpin et al.[17] cultured monocytes on
plastic for 7–10 days prior to infection with HIV-1ADA
.
The susceptibility of macrophages to HIV-1 infection is greatly influenced by
isolation, culture and infection conditions, donor variations, and macrophage
maturation at the time of infection [21,58].Freshly isolated monocytes are resistant
to HIV-1 infection, and the susceptibility of macrophages to HIV-1 infection is
greatly influenced by their stage of differentiation at the time of infection [59].The
receptor for the b-chemokines RANTES, MIP-1a, and MIP-1b, CCR5, was iden-
tified as the major co-receptor for the entry of macrophage tropic (M-tropic) strains
of HIV-1 [58,60].The expression of CCR5 on the cell surface significantly increases
as monocytes mature to macrophages over 1–7 days of adherence, and then de-
creases to lower levels between days 10 and 14 [61].These changes in surface ex-
pression of CCR5 correlate with CCR5 mRNA and the susceptibility of
macrophages to productive infection [61–63].
The established monocytic THP cell line [30,31] has been used as an alternative
to primary macrophages, because, after stimulation with PMA, the cells differ-
entiate into adherent macrophage-like cells, mimicking the monocyte-to-macro-
phage transformation [32].More than 80% of PMA-treated THP-1 cells
change morphologically and adhere to the substratum.Undifferentiated THP-1
cells are susceptible to infection by T-cell tropic (T-tropic) HIV-1 isolates [34,37]
and become susceptible to M-tropic isolates after treatment with differentiat-
ing agents [38].We have characterized thoroughly the cell surface expression of
the HIV receptor CD4 and the chemokine receptors CXCR4 and CCR5,
both during propagation and following differentiation [64].Cell surface CD4
plays a primary role in determining how efficiently THP-1 cells can be infected
with X4 HIV-1IIIB and R5 HIV-1Ba-L isolates.PMA-induced downregulation of
CD4 significantly increases infection with the R5 virus, HIV-1Ba-L, and differen-
tiated THP-1 cells are infected efficiently with HIV-1Ba-L.Therefore, differentiated
THP-1 cells and the R5, HIV-1Ba-L virus were used in our study.In this study,
we have shown that sSLPI inhibits infection of PMA-treated THP-1 cells by
HIV-1Ba-L
.

N.R. Shine et al. / Bioorganic Chemistry 30 (2002) 249–263
261
Having established a system to study the anti-HIV effect of SLPI, experiments to
identify specific cell surface protein(s) to which SLPI binds can be pursued.Using
competitive binding studies, McNeely et al.[18] suggested that the anti-HIV activity
of SLPI does not involve inhibition of cell-surface serine proteases.A putative re-
ceptor for SLPI was also identified.However, these binding studies were performed
using freshly isolated monocytes while the infectivity studies were performed using
10-day-old adherent macrophages.Cell surface proteins are differentially expressed
during the transition from monocyte to macrophage and freshly isolated monocytes
are essentially refractory to HIV-1 infection [65].Hence, the results of the compet-
itive binding assays may have to be performed directly with PMA-treated THP-1
cells, the same cells that are used to show the anti-HIV activity of SLPI.
Acknowledgments
The research was partially supported by the Pacific Dental Research Foundation,
Grant No.516 (KK), and by funds from the University of the Pacific School of Den-
tistry (DRES03-001) (KK).SCW is a Fellow of the American Foundation for Phar-
maceutical Education.Electrospray ionization mass spectrometry was performed by
the analytical instrumentation service core supported by Center grant ES 07784.
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