Departments of 1 Cellular and Molecular Physiology and 2 Pediatrics, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In humans, two functional genes of
surfactant protein (SP) A, SP-A1 and
SP-A2, and several alleles of each functional gene have been characterized. SP-A is a multimeric molecule consisting of
six trimers. Each trimer contains two SP-A1 molecules and one SP-A2
molecule. Until now, it has been unclear whether a single SP-A
gene product is functional or whether there are functional differences
either among alleles or between single-gene SP-A products and SP-A
products derived from both genes. We tested the ability of in vitro
expressed SP-A variants to stimulate tumor necrosis factor (TNF)-
production by THP-1 cells. We observed that 1) single-gene
products and products derived from both genes stimulate TNF-
production, 2) there are differences among SP-A1 and
SP-A2 alleles in their ability to stimulate TNF-
production,
and 3) the increases in TNF-
production are lower after
treatment with the SP-A1 alleles than after treatment with the
SP-A2 alleles. Furthermore, coexpressed SP-As from
SP-A1 and SP-A2 genes have a higher activity compared
with SP-As from individual alleles or mixed SP-As from SP-A1
and SP-A2 genes. These data suggest that the SP-A-induced
increases in TNF-
levels differ among SP-A variants and appear to be
affected by SP-A genotype and whether SP-A is derived from one or both genes.
allele; macrophage; surfactant protein A; tumor necrosis factor-
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
PULMONARY SURFACTANT IS ESSENTIAL for normal lung
function. Human surfactant protein (SP) A, in addition to
surfactant-related functions (8), plays a role in innate host defense
and regulation of inflammatory processes in the lung (reviewed in Refs.
1, 4, 17, 26). The human SP-A locus is located on chromosome 10 q22-23 and consists of two functional genes, SP-A1 and
SP-A2, and one pseudogene (15). Several alleles
have been characterized for each functional gene (Fig.
1) (6, 19; reviewed in Ref. 7). These alleles are
classified based on nucleotide differences within the coding region,
which result in amino acid changes, and have the potential for
functional differences. The two SP-A genes are differentially
regulated during development and by agents such as glucocorticoids,
cAMP, and insulin (18, 23, 31, 40). The SP-A alleles appear to
be differentially regulated by dexamethasone, a synthetic
glucocorticoid analog (16, 18). Moreover, the two SP-A genes
may be differentially expressed in different human tissues and cells.
Both genes are expressed in lung alveolar type II cells, but only the
SP-A2 gene is expressed in tracheal and bronchial submucosal
gland cells (12, 38).
|
Human SP-A from bronchoalveolar lavage (BAL) fluid is a complex multimeric molecule. It consists of six trimers, and each trimer contains two SP-A1 molecules and one SP-A2 molecule (42, 43). SP-A is a collagenous C-type lectin or collectin and has four structural regions: a cysteine-containing amino terminus, a collagen-like domain, a neck region, and a carbohydrate recognition domain (29). These domains of the SP-A molecule may be involved in different aspects of SP-A function (reviewed in Ref. 28). It is unclear whether the functions of SP-A depend on a single domain or multiple domains. SP-A1 and SP-A2 alleles have differences in 10 amino acid residues involving three of the four SP-A domains (excluding the neck region) (reviewed in Ref. 7).
Human SP-A from BAL fluid regulates cytokine production in the macrophage-like cell line THP-1, and this effect is inhibited by surfactant lipids (20, 22). Human SP-A also stimulates the expression of cell surface markers that are associated with inflammation (21). These actions may play a role in the development and maintenance of the inflammatory response during normal lung host defense and also raise the possibility that SP-A is involved in the pathogenesis of inflammatory lung disease.
Because a number of SP-A variants have been characterized, several questions have been raised. 1) Are single-gene products functional? 2) Are there functional differences among single-gene products? 3) Are there functional differences between single-gene SP-A products and SP-A variants containing products from both genes?
In the present study, our goal was to assess the functional differences
among SP-A variants derived from either single or both SP-A
genes by studying the effect of SP-A alleles on tumor necrosis
factor (TNF)- production by THP-1 cells. We found that 1)
single-gene products stimulate THP-1 cells to produce TNF-
, 2) there are differences among SP-A1 and SP-A2
alleles in the level of TNF-
stimulation, and
3) coexpressed SP-As containing both SP-A1 and
SP-A2 gene products have a higher activity compared with SP-As
from individual alleles or mixed SP-As from SP-A1 and SP-A2 gene products.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell lines and cell culture conditions. The insect cell line Sf9 (GIBCO BRL, Life Technologies) from Spodoptera frugiperda was used for protein expression in this study. The cells were cultured in Sf-900 II SFM medium (GIBCO BRL) in an incubator at 28°C. For initial culture and transfection with recombinant bacmid (baculovirus shuttle vector) DNA, the cells were grown in a monolayer culture. For protein expression, the cells were cultured in suspension in a 250-ml flask with shaking at 100 rpm.
The THP-1 cell line was obtained from the American Type Culture
Collection (Manassas, VA). The cells were cultured in RPMI 1640 medium
(Sigma, St. Louis, MO) with 0.05 mM 2-mercaptoethanol containing 10%
(vol/vol) heat-inactivated fetal calf serum (FCS; Summit Biotechnology,
Ft. Collins, CO). The cells were grown in suspension in
75-cm2 T-flasks at 37°C in an atmosphere of 5%
CO2. The cells were split periodically to maintain the
cellular density between 1 × 105 and 1 × 106 cells/ml. THP-1 cells were differentiated with
108 M vitamin D3 (BIOMOL, Plymouth
Meeting, PA) for 72 h before treatment with SP-A.
Construct of plasmids and bacmids. cDNAs of human SP-A1 alleles 6A2 and 6A4 (18) and human SP-A2 alleles 1A, 1A0, 1A1, and 1A2 (10, 19) were separately amplified with the primer pair 861/862. The PCR products included the entire coding region of SP-A, ~100 bp of the 5'-untranslated region, and 400 bp of the 3'-untranslated region. The PCR products were cloned into T vector, and recombinant sequences were verified by sequencing. The cDNAs of the SP-A1 and SP-A2 alleles were isolated from the recombinant vector by digestion with Xho I and Sph I. The cDNAs were then cloned into donor plasmid pFastBac DUAL (GIBCO BRL) that had been previously digested with the same restriction enzymes. In addition, cDNA of allele 6A (10) was taken from plasmid pSP65-6A by using the restriction enzyme EcoR I and then inserted into the EcoR I site of donor plasmid pFastBac DUAL with standard methods (39). The expression of the SP-A gene in the bacmid was driven by the p10 promoter.
Recombinant plasmid pFastBac DUAL containing cDNA of the SP-A allele was transformed into Escherichia coli DH10Bac. The plasmid pFastBac DUAL contained site-specific transposition sequences (Tn7R and Tn7L) by which the foreign gene was transferred into the baculovirus genome (bacmid). Recombinant bacmid DNA was isolated and purified from the selected clones. To verify the transposition region in recombinant bacmid, the isolated bacmid DNA was used as a template for PCR with the pUC/M13 amplification primers. The PCR products confirmed that the insert sizes were correct for alleles 6A2, 6A4, 1A, 1A0, 1A1, 1A2 (3.9 kb), and 6A (3.5 kb).
Transfection, SP-A expression, and purification from baculovirus-mediated insect cell system. The recombinant bacmid DNA was then transfected into insect cell line Sf9 by means of CellFECTIN reagent (GIBCO BRL) according to the manufacturer's instructions. For each allele, at least three independent recombinant bacmid clones were used for transfection. Three days after bacmid DNA transfection, SP-A expression in both the medium and the insect cells was examined by Western blotting. The clones that produced a strong signal for SP-A on Western blots were chosen for further experimentation and were amplified twice.
Insect cell Sf9 cultures of 50 ml were grown in 250-ml flasks at 28°C with shaking. When the cell density reached 1 × 106 cells/ml, the culture was inoculated with 1 ml of a virus solution containing ~1 × 109 titer/ml of virus particles. The inoculated cells were cultured continuously with the same conditions. For initial experiments, the culture was sampled every 12 h to detect SP-A expression and secretion.
SP-A was recovered from the culture medium and purified by mannose-affinity chromatography according to the method of Fornstedt and Porath (11). Each column contained a 10-ml matrix of mannose-Sepharose. About 200 ml of medium from an infected culture were harvested 84 h after inoculation. Insect cells and fragments of cells were removed from the culture medium by centrifugation at 500 g for 10 min at 4°C. The supernatant was transferred to other tubes and diluted with an equal volume of sterile double-distilled H2O. Ca2+ in the diluted supernatant solution was adjusted to 10 mM with 1 M CaCl2. The SP-A-containing solution was passed through a mannose-Sepharose 6B column at ~30 ml/h. The column was washed with 80-100 ml of 5 mM Tris and 1 mM CaCl2 (pH 7.5). SP-A was eluted with an elution buffer containing 5 mM Tris and 2 mM EDTA (pH 7.5), and the eluted solution was collected in 1-ml fractions. The tubes containing SP-A were detected by gel electrophoresis and Western blotting and pooled. SP-A was purified from these fractions by repeating the mannose-affinity procedure. The purified SP-A was then dialyzed against 5 mM Tris (pH 7.5) that was changed at least three times.
Preparation of native human SP-A. Native human SP-A was purified from BAL fluid obtained from patients with alveolar proteinosis. Partial purification of SP-A from the crude BAL fluid was accomplished by centrifuging the BAL fluid to obtain a crude SP-A pellet. The pellet was washed with distilled water that solubilized SP-A. Ca2+ (5 mM) was then added, causing SP-A to self-aggregate. SP-A was then pelleted by centrifugation, separating it from most of the contaminating serum proteins that remained soluble. This procedure was repeated several times. SP-A was then further purified by preparative isoelectric focusing with a Rotofor apparatus (Bio-Rad, Richmond, CA). Briefly, crude proteinaceous material from BAL fluid was solubilized in 3 M urea and 20% glycerol. The solubilized protein was then combined with ampholytes (pH 3.0-10.0; Bio-Rad) and placed in the chamber of the Rotofor apparatus. Focusing was conducted at a constant power (5 W) until the voltage stabilized. NaCl was added to the dialysis bag at a final concentration of 1 M and then dialyzed against distilled water at 4°C for 4-7 days.
SP-A was concentrated with Amicon filter Centriprep-10 (Amicon,
Beverly, MD). All procedures were performed at 4°C or on ice. Protein concentration was determined with the microbicinchoninic acid
method (Pierce, Rockford, IL) with RNase A as a standard. SP-A was
stored at 70°C.
SP-A antiserum. An antiserum against human SP-A was prepared with SP-A purified from the lavage fluid from an alveolar proteinosis patient by preparative isoelectric focusing. The purification procedure used has been described in detail elsewhere (22). SP-A obtained with this method was >98% pure. Briefly, the antiserum was raised in a New Zealand White rabbit by immunizing the rabbit with SP-A mixed with Freund's adjuvant following standard methods. IgG was isolated from the serum with a protein A-Superose column (Pharmacia Amersham Biotech, Piscataway, NJ) on a Pharmacia fast-performance liquid chromatography system. The resultant IgG fraction recognized SP-A on Western blots with high affinity.
Characterization of expressed SP-A and native human SP-A. Purity was determined by two-dimensional (2D) gel electrophoresis with the Immobiline DryStrip kit and Multiphor II horizontal SDS electrophoresis unit (Pharmacia). Immobiline DryStrips (pH 3.0-10.5) were hydrated with 8 M urea, 0.5% Triton X-100, 10 mM dithiothreitol, and 0.2% (vol/vol) acetic acid. Protein samples containing SP-A were dissolved in 9 M urea, 2% (vol/vol) Triton X-100, 2% (vol/vol) 2-mercaptoethanol, and 2% (vol/vol) Pharmalyte, pH 3-10 (Pharmacia). Three parts of the protein sample were then mixed with one part of the buffer containing 8 M urea, 2% (vol/vol) 2-mercaptoethanol, 2% (vol/vol) Pharmalyte (pH 3-10), 0.5% (vol/vol) Triton X-100, and 0.0025% (wt/vol) bromphenol blue and loaded on the DryStrips. Electrofocusing was done for 31,000 V · h. The strips were then removed and equilibrated for 10 min in 0.5 M Tris base, 0.5 N HCl, and 16 mM dithiothreitol (pH 6.8) and for an additional 10 min in 6 M urea, 0.05 M Tris base, 0.05 N HCl, 24 mM iodoacetamide, 30% (vol/vol) glycerol, and 1% SDS (pH 6.8). The equilibrated strips were transferred to 12.5% horizontal SDS gels for electrophoresis. The SDS gels consisted of a 33-mm stacking gel containing 5% polyacrylamide (29.2% acrylamide and 0.8% bis-acrylamide), 0.125 M Tris, and 0.1% SDS (pH 6.8) and a 77-mm separating gel containing 12.5% polyacrylamide, 0.375 M Tris, and 0.1% SDS (pH 8.8). Silver staining of gels was done with a method described by Rabilloud (36).
Lipopolysaccharide removal. As a part of the SP-A purification, we removed lipopolysaccharide (LPS) from the SP-A preparations by incubating the purified SP-A with polymyxin B-agarose (1 ml/mg SP-A) according to the manufacturer's instructions (Sigma). LPS content of the SP-A preparations was assessed with QCL-1000, a test employing Limulus amebocyte lysate (BioWhittaker, Walkersville, MD). Purified SP-A contained <0.1 pg LPS/µg SP-A. The LPS content of three independent preparations is expressed as mean ± SE.
Nonreducing and reducing SDS-PAGE and Western blotting. SDS-PAGE analysis was done following the procedure described by Laemmli (24). Samples of protein in reducing SDS-PAGE were mixed 3:1 by volume with loading buffer containing 200 mM Tris · HCl (pH 6.8), 400 mM dithiothreitol, 8% (wt/vol) SDS, 0.4% (wt/vol) bromphenol blue, and 40% (vol/vol) glycerol. In nonreducing SDS-PAGE, each protein sample was mixed 3:1 with loading buffer containing 200 mM Tris · HCl (pH 6.8), 8% (wt/vol) SDS, 0.4% (wt/vol) bromphenol blue, and 40% (vol/vol) glycerol. Samples were heated to 100°C for 5 min and subjected to electrophoresis on SDS-10% (wt/vol) polyacrylamide gels at 100 V at room temperature. Protein bands were visualized by silver staining and sized by comparison with broad-range molecular-weight standards (Bio-Rad).
After SDS-PAGE, the proteins were transferred to polyvinylidene difluoride membrane (0.2-µm pore size; Bio-Rad) with Towbin's buffering system and a Bio-Rad Mini Trans-Blot apparatus. The membrane was blocked by incubation in 3% skim milk and incubated in buffer containing a rabbit antiserum to human SP-A (1:1,000) and then in a solution containing a goat antiserum to rabbit IgG conjugated to alkaline phosphatase. The color was developed in 5-bromo-4-chloro-3-indoylphosphate p-toluidine salt/p-nitro blue tetrazolium chloride buffer (pH 9.5) according to the manufacturer's instructions (Bio-Rad).
Stimulation of THP-1 cells with SP-A and ELISA assay of
TNF-. After differentiation in
10
8 M vitamin D3 for 72 h, THP-1 cells
were washed and exposed to SP-A in concentrations ranging from 0 to 100 µg/ml for dose-response experiments or 50 µg/ml for other
experiments. Incubations were performed with 2 × 106
cells/ml in 24-well culture plates for periods extending from 0 to 24 h
in time-course experiments or 4 h in other experiments. FCS (10%) was
present in all incubations.
The TNF- ELISA assays were performed as recommended by the
manufacturer (PerSeptive Diagnostics, Cambridge, MA). The human TNF-
ELISA measured concentrations of TNF-
ranging from 12.5 to 1,000 pg/ml. A reference curve was obtained by plotting the TNF-
concentration of several dilutions of standard protein versus absorbance.
Statistical methods. TNF- data from three independent
experiments are expressed as means ± SE. The different levels of
TNF-
among various SP-As were compared and analyzed by
multiple-comparison ANOVA test. Differences were considered significant
when the P value was <0.05.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Baculovirus-mediated SP-A expression in insect cells. cDNAs of
three SP-A1 alleles (i.e., 6A, 6A2, and
6A4), and four SP-A2 alleles (i.e., 1A,
1A0, 1A1, and
1A2) were cloned into expression bacmids. These
cDNAs were driven by the baculovirus p10 promoter. After 3 days of
transfection, SP-A could be detected in the medium and the insect
cells. However, the viral titer from the initial transfection was low,
and thus before proceeding with protein expression, the virus was
amplified twice. The final viral titer after amplification reached at
least 1 × 109. SP-A was expressed by
inoculating 50 ml of insect cell culture at 1-2 × 106 cells/ml with 1 ml of a high viral titer solution.
Figure 2 shows the expression of allele
6A2 in insect cells. SP-A protein could not be
detected in the medium during the first 24 h after inoculation. After
24 h, secreted SP-A was detectable in the medium. After 48 h, the
secreted SP-A protein in the medium accumulated significantly and
peaked in 72-84 h at 1.5 mg SP-A/100 ml medium. As a control, we
did mock transfections with the bacmid that did not contain SP-A cDNA. No SP-A protein was detected in the medium or cells from the mock transfection. Cells transfected with each of the seven SP-A
alleles (6A, 6A2, 6A4, 1A,
1A0, 1A1, and
1A2) successfully expressed proteins. In addition,
five coexpressed combinations
(1A0/6A2,
1A1/6A2,
1A/6A4,
1A0/6A4, and
1A2/6A4) in which the viral
titers of SP-A1 and SP-A2 were mixed at a 1:1 ratio
were also successfully expressed in this study. The patterns of protein
expression and secretion in all single-gene product SP-As and
coexpressed SP-As were similar to those shown with allele
6A2 results (Fig. 2).
|
Characteristics of single-allele SP-As and coexpressed SP-As.
SP-A recovered from the medium was purified by mannose-affinity chromatography as described in MATERIALS AND METHODS. Seven
single-gene SP-As and five coexpressed SP-As exhibited similar
properties during this chromatographic procedure (data not shown).
These results demonstrate that the mannose-binding properties in the presence of Ca2+ were maintained for the SP-A variants
derived from one or both SP-A genes. The results of
one-dimensional (1D) and 2D gel electrophoresis indicate 99% purity
for each of the seven single-gene products from alleles 6A,
6A2, 6A4, 1A,
1A0, 1A1, and
1A2 and for the five coexpressed SP-As from alleles
1A0/6A2,
1A1/6A2,
1A/6A4,
1A0/6A4, and
1A2/6A4. Figure
3 depicts the SP-A patterns of silver
staining and Western blotting of 1D and 2D gels for allele
6A2. No protein was detected by silver staining
after purification from mock-infected cultures.
|
Two representative SP-A alleles, 1A0 from
SP-A2 and 6A4 from SP-A1 as well as
coexpressed SP-A 1A0/6A4 were
analyzed for their disulfide-dependent oligomeric assembly by
nonreducing SDS-PAGE. The results in Fig. 4
demonstrate that single-gene products form dimers and monomers but with
a lower apparent molecular mass than native SP-A. The size
of SP-A monomers ranges between 25 and 29 kDa, and the size of SP-A
dimers ranges between 48 and 60 kDa. Similarly, coexpressed SP-A
1A0/6A4 has monomers, dimers,
trimers, and higher molecular forms as determined with nonreducing
SDS-PAGE. Human SP-A from BAL fluid used as a control has dimers and
higher molecular forms. These data indicate that 1)
disulfide-dependent oligomeric assembly does occur in both single-gene
products and coexpressed products of both genes in the insect cell
expression system and 2) coexpressed SP-A of both genes has a
different oligomeric assembly compared with that observed with
single-gene products.
|
Dose-response and time-course studies of SP-A effects on the
stimulation of TNF- production by THP-1 cells.
Vitamin D3-differentiated THP-1 cells were exposed to
single-gene SP-As, 6A2 of SP-A1 or
1A0 of SP-A2, at different concentrations
ranging from 0 to 100 µg/ml (Fig.
5A). TNF-
content in the medium
ranged from 16.9 pg TNF-
/ml in the negative control to 486 pg
TNF-
/ml with 100 µg SP-A/ml. The two positive
controls consisted of 50 µg/ml of native human SP-A from an alveolar
proteinosis patient and 100 pg/ml of LPS from E. coli serotype
055:B5. Both positive controls stimulated TNF-
production to
approximately the same degree as 50 µg/ml of single-gene SP-A. Figure
5B shows the LPS dose response. The data indicate that the
TNF-
levels were similar to the control level when LPS was
5
pg/ml. All of our protein preparations have LPS < 5 pg/ml (i.e., 50 µg of SP-A contains <5 pg of LPS). In subsequent experiments, we
used 50 µg SP-A/ml. Another group of controls included coexpressed
SP-A (1A0/6A4; 50 µg/ml) in
the presence and absence of polymyxin B (1 µg/ml) or polymyxin B
alone. LPS (100 pg/ml) in the presence and absence of polymyxin B was
also used. SP-A plus polymyxin B stimulated TNF-
production at a
level (609 ± 26 pg/ml) similar to that seen with SP-A alone (624 ± 32 pg/ml). Although LPS alone stimulated TNF-
production (397 ± 25 pg/ml), the LPS effect was almost completely inhibited by polymyxin B
(43 ± 24 pg/ml). The untreated control level was 28 ± 9 pg/ml of
TNF-
, and the level with polymyxin B alone was 28 ± 7.9 pg/ml of
TNF-
. Moreover, medium from mock-infected cells was subjected to
mannose-affinity purification and concentration similar to medium from
SP-A-expressing cells. Two additional controls of 50 µl of medium
with and without 50 µg/ml of SP-A from mock-infected cells were used.
The levels of TNF-
in both controls were 21 ± 7 and 412 ± 35 pg/ml, respectively. The former is similar to the background
TNF-
level (17 ± 4.6 pg/ml) in THP-1 cells that were not treated,
and the latter is not significantly different from that obtained with
only SP-A (394 ± 26.9 pg/ml).
|
Based on the SP-A dose-response experiments, SP-As from alleles
6A2 or 1A0 (50 µg/ml) were
used in time-course experiments. Figure 6
shows the time course of SP-A stimulation of TNF- production by
THP-1 cells. After 1 h of incubation with SP-A, TNF-
could be
detected in the culture medium of THP-1 cells at levels above
background. The content of TNF-
in the medium dramatically increased
from 1 to 4 h and reached a peak at 4 h. The content of TNF-
subsequently decreased, although it was still above background at
13 h. The levels of TNF-
in the medium varied slightly in
cells tested with 6A2 and 1A0.
However, the time-course profile of TNF-
production was identical for the two variants. Based on the dose-response and time-course results, in subsequent experiments we used 50 µg SP-A/ml and a time
point of 4 h to compare the effects of different SP-A variants.
|
Comparison of single-gene SP-A variants on the stimulation of
TNF- production. A total of seven single-gene SP-A
variants were produced by in vitro expression. Three of them were
SP-A1 alleles, i.e., 6A, 6A2, and
6A4, and the other four were SP-A2 alleles,
i.e., 1A, 1A0, 1A1, and
1A2. Three independent transfections and protein
isolations were performed for each SP-A variant. The LPS content of the
allele preparations used in the study, i.e., 6A,
6A2, 6A4, 1A,
1A0, 1A1, and
1A2, was 0.055 ± 0.013, 0.053 ± 0.016, 0.056 ± 0.018, 0.055 ± 0.011, 0.071 ± 0.009, 0.061 ± 0.008, and
0.044 ± 0.009 pg/µg SP-A, respectively. Three independent
experiments were performed involving treatment of THP-1 cells with
SP-A. After stimulation with SP-A at a concentration of 50 µg/ml for
4 h, the content of TNF-
measured by ELISA in THP-1 cell culture
supernatants is shown in Fig. 7, A
and B. All seven SP-A alleles stimulated TNF-
production by THP-1 cells, and the levels of TNF-
in all treatments
were substantially higher than that of the negative control. The levels
of TNF-
for SP-A1 alleles 6A, 6A2, and
6A4 were 256 ± 46.9, 236 ± 26.9, and 201 ± 11.5 pg TNF-
/ml, respectively, with no significant differences among
these three alleles. The levels of TNF-
for SP-A2 alleles
1A, 1A0, 1A1, and
1A2 were 413 ± 15.7, 358 ± 14.0, 298 ± 20.9, and 391 ± 12.5 pg TNF-
/ml, respectively. Among these alleles, the
levels of TNF-
for 1A, 1A0, and
1A2 were significantly higher than that for
1A1 (P < 0.05).
|
In addition, when we compared the effects between SP-A1 (three
alleles as a group) and SP-A2 (four alleles as a group), the level of TNF- resulting from treatment with SP-A2 alleles
was significantly higher than that with the SP-A1 alleles
(P < 0.01).
Comparison of various SP-A types on the stimulation of
TNF- production. In addition to studying the
effects of single-gene products, we also examined several SP-A variants
derived from both SP-A1 and SP-A2 genes, coexpressed
SP-A variants, and mixed SP-As that were mixed at 1:1 after being
individually expressed with native SP-A from BAL fluid as a positive
control. All five coexpressed SP-As
(1A0/6A2,
1A1/6A2,
1A/6A4,
1A0/6A4, and
1A2/6A4; LPS content was 0.037 ± 0.011, 0.042 ± 0.006, 0.041 ± 0.007, 0.060 ± 0.013, and 0.052 ± 0.006 pg/µg SP-A, respectively), the three mixed
SP-As (1A/6A2,
1A1/6A2, and
1A2/6A2), and the native human
SP-A stimulated TNF-
production by THP-1 cells. The levels of
TNF-
in the medium of THP-1 cells were 421 ± 12.1, 489 ± 43.3, 632 ± 21.5, 602 ± 37.5, and 591 ± 31.6 pg TNF-
/ml after treatment with the coexpressed SP-As
1A0/6A2,
1A1/6A2,
1A/6A4,
1A0/6A4, and
1A2/6A4, respectively. Among
them, the three coexpressed SP-As from
1A/6A4,
1A0/6A4, and
1A2/6A4 had significantly
higher levels of TNF-
compared with the other two coexpressed SP-As
from 1A0/6A2 and
1A1/6A2 (Fig. 7C).
There were no marked differences among the three mixed SP-As (Fig.
7D). When we compared the levels of TNF-
in response to
various SP-As, we found that treatment with the coexpressed SP-As
resulted in much higher levels of TNF-
compared with the mixed SP-As
or the SP-A1 and SP-A2 single-gene products.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Human SP-A is a complex multimeric molecule coded by two very similar
genes, SP-A1 and SP-A2. Although most of the SP-A in BAL fluid is multimeric, recent evidence suggests that some monomeric SP-A exists (14). It is not known whether this monomeric SP-A is
functional, and because of the very high degree of similarity between
the two SP-A genes, it is not yet possible to isolate a
single-gene product from BAL fluid. Our goal in this study was to
explore whether single-gene products are functional and whether differences exist among SP-A variants derived from one gene and/or whether the activity of SP-A variants derived from a single gene differs from that observed for coexpressed SP-As (derived from both
genes). In the present study, we observed that the single-gene products
are functional and differences among single-gene products and between
single-gene products and coexpressed SP-As are detected as assessed by
their ability to stimulate TNF- by THP-1 cells.
In the present study, we used recombinant DNA technology and the baculovirus-mediated insect cell expression system to express in vitro SP-A from either one or both genes. In vitro expressed SP-As from this system can be purified to >99% purity by mannose-affinity chromatography. The success of this purification demonstrates that SP-As expressed in vitro in insect cells from either a single gene or both SP-A genes have the capacity to bind mannose. Indeed, some SP-A functions, such as the ability to bind to bacteria as part of its role in innate host defense, depend on this property (4, 13)
It has been previously reported (20, 22) that native human SP-A can
stimulate TNF- production in macrophage-like THP-1 cells. In the
present study, we demonstrate that purified SP-A products from a single
gene or from both genes also have this capability. SP-A is a secreted
protein that is expressed in type II alveolar epithelial cells in the
lung and then secreted into the lung alveolar space (33). As the SP-A
precursor is processed to form mature SP-A, it undergoes several
posttranslational modifications including N-linked glycosylation,
hydroxylation of proline residues, sialic acid addition to the
oligosaccharide, and signal peptide cleavage (8, 9, 35). These
posttranslational modifications may be involved in some SP-A functions
(29, 30). The SP-As expressed in insect cells do not undergo all of the
posttranslational modifications found in native SP-A. This is evident
in the patterns produced by the in vitro expressed protein on 1D and 2D
gels compared with those of native human SP-A. Rat SP-A expressed by
insect cells is deficient in hydroxyproline, suggesting that this
posttranslational modification does not occur in this system (29). In
addition, an altered pattern of glycosylation was found in rat SP-A
protein produced by this system (30). However, rat SP-A appears to be an effective inhibitor of the secretion of surfactant lipids from isolated type II cells (29). Our results and other published reports (29, 30) confirm that SP-As expressed in vitro by insect cells provide a model for the study of some normal SP-A functions such as the stimulation of immune cells, binding of carbohydrates, and the inhibition of surfactant lipid secretion from
isolated type II cells. In addition, the baculovirus-mediated insect
cell expression system provides a relatively simple, safe, and easy way
to obtain adequate amounts of protein, especially when many alleles and
combinations of alleles need to be produced.
The pattern of reducing and nonreducing SDS-PAGE indicated that either single-gene products or coexpressed SP-A from both genes from insect cells have a lower apparent molecular mass than native SP-A. The difference in the apparent molecular mass of the in vitro expressed alleles is likely to be due to the lack of proline hydroxylation by the insect cell system (29). It has been previously shown that hydroxylation of prolines retards SP-A migration (34). However, other differences in glycosylation between in vitro expressed and native SP-A that may account for the differences in molecular mass could not be eliminated. Single-gene products in nonreducing SDS-PAGE are predominantly in the form of dimers and monomers, and coexpressed SP-A (1A0/6A4) appears to consist of monomers, dimers, trimers, and higher molecular forms. This suggests that there is a different pattern of oligomeric assembly in coexpressed SP-As than in SP-A from single genes. This may also reflect the nature of heterodimers versus homodimers due to the presence of an extra cysteine residue at amino acid 85 of SP-A1. This observation also indicates that heterooligomers can form between SP-A proteins derived from separate viruses produced in the insect cell expression system. However, we cannot currently assess the exact ratio of SP-A1 to SP-A2.
To our knowledge, this is the first time that a single human SP-A gene product has been tested for its function. The results support the notion that SP-A in tracheal and bronchial submucosal gland cells where only a single-gene product is made (12, 38) is functional and can probably modulate cytokine expression. The presence of only SP-A2 mRNA in these cells suggests that SP-A2 may have its own functional significance. However, it is unclear what the exact function of the single-gene product is in specific human tissues and cells. In addition to its functions related to surfactant biology, SP-A is also involved in innate host defense (2) and regulation of inflammation (20, 22). Perhaps as a reflection of these host defense properties, SP-A has been found to be expressed in other tissues or cells such as gastric and intestinal epithelial cells (37) and the Eustachian tube (32). Until now, it was not clear what the effects of the SP-A genetic makeup and/or its oligomeric structure are on its function. Our study provides some information about this, although further investigation is required.
All of the allelic products of SP-A1 and SP-A2 that
were tested effectively stimulate TNF- production by THP-1 cells,
but the resulting levels of TNF-
vary among them. The SP-A2
products have a higher level of activity than the SP-A1
products. Among the alleles of SP-A2, alleles 1A,
1A0, and 1A2 all have higher
activity than 1A1. These variations may be due to
the presence of different amino acid residues in the SP-A peptide.
Between SP-A1 and SP-A2, there are four amino acid
residues at positions 66, 73, 81, and 85 of the collagen-like domain
that are different. Alleles 1A, 1A0, and
1A2 contain glutamine at position 223, and allele
1A1 contains lysine (7). Amino acid substitutions
within a protein can lead to quantitative or qualitative changes in the
activity of the particular protein (3, 27, 41). Whether the differences observed between the in vitro expressed SP-A alleles correspond to functional differences in vivo is not known.
We coexpressed SP-A1 and SP-A2 gene products by inoculating baculoviruses containing both genes at a ratio of 1:1 (1 × 109 titer in a 50-ml culture). This means that each cell received an average of 20 viral particles from SP-A1 and SP-A2. However, the ratio of SP-A1 to SP-A2 in each cell may vary. Therefore, it is possible that coexpressed SP-As are the result of a variable ratio and not a definite ratio of 1:1. We originally designed an expression vector for both genes by using a donor plasmid, pFastBac DUAL, in this study. The plasmid can express two genes such as SP-A1 and SP-A2, but many attempts failed to produce recombinant bacmid. The highly homologous SP-A1 and SP-A2 genes are at reverse orientation and separated by only ~200 bp. We believe that either this structure may not be transferred intact into bacmid by site-specific transposition processes or such a structure is not stable during the bacmid genome replication in E. coli.
One interesting result obtained in this study is that coexpressed SP-As have a much higher activity than the other SP-A types such as single-gene products or mixed SP-A. SP-A1 alleles have a cysteine at position 85, but SP-A2 alleles do not. This cysteine may form an intermolecular disulfide bond that helps to stabilize the SP-A multimeric structure. Several models of the human SP-A molecule suggest that this cysteine may play a role in linking the different peptides with an intermolecular disulfide bond (5, 25, 43). If this is the case, this may help explain, in part, the observed results. For example, the additional disulfide bond in coexpressed SP-As may increase stability of the molecules or change their molecular structure. The pattern on nonreducing SDS-PAGE gels reveals that coexpressed SP-A consisting of both gene products forms higher molecules than single-gene products. This further supports our conclusion. This disulfide bond probably is not present when these alleles are mixed after individual expression of each allele and purification.
In summary, in the present study, we have shown that single-gene
products of SP-A expressed in vitro are functional with respect to
their ability to stimulate THP-1 cells to produce TNF-. There are
differences among SP-A1 and SP-A2 alleles in the degree
of stimulation. Coexpressed SP-As of SP-A1 and SP-A2
have a higher activity compared with that from individual alleles or
SP-As from mixed SP-A1 and SP-A2.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Susan DiAngelo, Scott Phillips, and Jill Hayden for expert technical assistance.
![]() |
FOOTNOTES |
---|
This work was supported by National Heart, Lung, and Blood Institute Grants R37-HL-34788-S1 (to J. Floros) and HL-54683 (to D. S. Phelps) and National Institute of Environmental Health Sciences Grant R01-ES-09882-01 (to J. Floros).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: J. Floros, Dept. of Cellular and Molecular Physiology (H166), The Pennsylvania State Univ. College of Medicine, 500 University Dr., Hershey, PA 17033 (E-mail: jxf19{at}psu.edu).
Received 9 August 1999; accepted in final form 16 November 1999.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Batenburg, JJ,
and
Haagsman HP.
The lipids of pulmonary surfactant: dynamics and interactions with proteins.
Prog Lipid Res
37:
235-276,
1998[ISI][Medline].
2.
Benne, CA,
Kraaijeveld CA,
van Strijp JAG,
Brouwer E,
Harmsen M,
Verhoef J,
van Golde LMG,
and
van Iwwarden JF.
Interactions of surfactant protein A with influenza A viruses: binding and neutralization.
J Infect Dis
171:
335-341,
1995[ISI][Medline].
3.
Broun, P,
Shanklin J,
Whittle E,
and
Somerville C.
Catalytic plasticity of fatty acid modification enzymes underlying chemical diversity of plant lipids.
Nature
282:
1315-1317,
1998.
4.
Crouch, EC.
Collectins and pulmonary host defense.
Am J Respir Cell Mol Biol
19:
177-201,
1998
5.
Elhalwagi, BM,
Damodarasamy M,
and
McCormack FX.
Alternate amino terminal processing of surfactant protein A results in cysteinyl isoforms required for multimer formation.
Biochemistry
36:
7018-7025,
1997[ISI][Medline].
6.
Floros, J,
DiAngelo S,
Koptides M,
Karinch AM,
Rogan PK,
Nielsen H,
Spragg RG,
Watterberg K,
and
Dieter G.
Human SP-A locus: allele frequencies and linkage disequilibrium between the two surfactant protein A genes.
Am J Respir Cell Mol Biol
15:
489-498,
1996[Abstract].
7.
Floros, J,
and
Hoover RR.
Genetics of the hydrophilic surfactant proteins A and D.
Biochim Biophys Acta
1408:
312-322,
1998[ISI][Medline].
8.
Floros, J,
and
Phelps DS.
Pulmonary surfactant.
In: Anesthesia: Biologic Foundations, edited by Biebuyck J,
Lynch C, III,
Maze M,
Saidman LJ,
Yaksh TL,
and Zapol WM.. Philadelphia, PA: Lippincott-Raven, 1997, p. 1259-1279.
9.
Floros, J,
Phelps DS,
and
Taeusch HW.
Biosynthesis and in vitro translation of the major surfactant-associated protein from human lung.
J Biol Chem
260:
495-500,
1985
10.
Floros, J,
Steinbrink R,
Jacobs K,
Phelps D,
Kriz R,
Recny M,
Sultzman L,
Jones S,
Taeusch HW,
Frank HA,
and
Fritsch EF.
Isolation and characterization of cDNA clones for the 35-kDa pulmonary surfactant-associated protein.
J Biol Chem
261:
9029-9033,
1986
11.
Fornstedt, N,
and
Porath J.
Characterization studies on a new lectin found in seeds of Vicia ervilia.
FEBS Lett
57:
187-191,
1975[ISI][Medline].
12.
Gross, KL,
Kumar AR,
and
Snyder JM.
SP-A2 gene expression in human fetal lung airways.
Am J Respir Cell Mol Biol
19:
613-621,
1998
13.
Haagsman, HP,
Hawgood S,
Sargeant T,
Buckley D,
White RT,
Drickamer K,
and
Benson BJ.
The major lung surfactant protein, SP 28-36, is a calcium-dependent, carbohydrate-binding protein.
J Biol Chem
262:
13877-13880,
1987
14.
Hickling, TP,
Malhotra R,
and
Sim RB.
Human lung surfactant A exists in several different oligomeric states: oligomer size distribution varies between patient groups.
Mol Med
4:
266-275,
1998[ISI][Medline].
15.
Hoover, RR,
and
Floros J.
Organization of the human SP-A and SP-D loci at 10q22-23. Physical and radiation hybrid mapping reveals gene order and orientation.
Am J Respir Cell Mol Biol
18:
353-362,
1998
16.
Hoover, RR,
Thomas KH,
and
Floros J.
Glucocorticoid inhibition of human SP-A1 promoter activity in NCI-H441 cells.
Biochem J
340:
69-76,
1999[ISI][Medline].
17.
Johansson, J,
and
Curstedt T.
Molecular structure and interactions of pulmonary surfactant components.
Eur J Biochem
244:
675-693,
1997[Abstract].
18.
Karinch, AM,
Dieter G,
Ballard PL,
and
Floros J.
Regulation of expression of human SP-A1 and SP-A2 genes in fetal lung explant culture.
Biochim Biophys Acta
1398:
192-202,
1998[ISI][Medline].
19.
Karinch, AM,
and
Floros J.
5' Splicing and allelic variants of the human pulmonary surfactant protein A genes.
Am J Respir Cell Mol Biol
12:
77-88,
1995[Abstract].
20.
Kremlev, SG,
and
Phelps DS.
Surfactant protein A stimulation of inflammatory cytokine and immunoglobulin production.
Am J Physiol Lung Cell Mol Physiol
267:
L382-L388,
1994.
21.
Kremlev, SG,
and
Phelps DS.
Effect of SP-A and surfactant lipids on expression of cell surface markers in the THP-1 monocytic cell line.
Am J Physiol Lung Cell Mol Physiol
272:
L1070-L1077,
1997
22.
Kremlev, SG,
Umstead TM,
and
Phelps DS.
Surfactant protein A regulates cytokine production in the monocytic cell line THP-1.
Am J Physiol Lung Cell Mol Physiol
272:
L996-L1004,
1997
23.
Kumar, AR,
and
Snyder JM.
Differential regulation of SP-A1 and SP-A2 genes by cAMP, glucocorticoids, and insulin.
Am J Physiol Lung Cell Mol Physiol
274:
L177-L185,
1998
24.
Laemmli, UK.
Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature
227:
680-685,
1970[ISI][Medline].
25.
Lu, J,
and
Sim RB.
Collectins and collectin receptors.
In: New Aspects of Complement Structure and Function, edited by Erdei A.. Austin, TX: Landes, 1994, p. 85-106.
26.
Mason, RJ,
Greene K,
and
Voelker DR.
Surfactant protein A and surfactant protein D.
Am J Physiol Lung Cell Mol Physiol
275:
L1-L13,
1998
27.
Matsushita, M,
Ezekowitz RAB,
and
Fujita T.
The Gly-54 Asp allelic form of human mannose-binding protein (MBP) fails to bind MBP-associated serine protease.
Biochem J
311:
1021-1023,
1995[ISI][Medline].
28.
McCormack, F.
The structure and function of surfactant protein A.
Chest
11:
114S-119S,
1997.
29.
McCormack, FX,
Calver HM,
Watson PA,
Smith DL,
Mason RJ,
and
Voelker DR.
The structure and function of surfactant protein A: hydroxyproline- and carbohydrate-deficient mutant protein.
J Biol Chem
269:
5833-5841,
1994
30.
McCormack, FX,
Pattanajitvilai S,
Stewart J,
Possmayer F,
Inchley K,
and
Voelker DR.
The Cys6 intermolecular disulfide bond and the collagen-like region of rat SP-A play critical roles in interaction with alveolar type II cells and surfactant lipids.
J Biol Chem
272:
27971-27979,
1997
31.
McCormick, SM,
and
Mendelson CR.
Human SP-A1 and SP-A2 genes are differentially regulated during development and by cAMP and glucocorticoids.
Am J Physiol Lung Cell Mol Physiol
266:
L367-L374,
1994
32.
Paananen, R,
Glumoff V,
and
Hallman M.
Surfactant protein A and D expression in the porcine Eustachian tube.
FEBS Lett
452:
141-144,
1999[ISI][Medline].
33.
Phelps, DS,
and
Floros J.
Localization of surfactant protein synthesis in human lung by in situ hybridization.
Am Rev Respir Dis
137:
939-942,
1988[ISI][Medline].
34.
Phelps, DS,
and
Floros J.
Proline hydroxylation alters the electrophoretic mobility of pulmonary surfactant-associated protein A.
Electrophoresis
9:
231-233,
1988[ISI][Medline].
35.
Phelps, DS,
Floros J,
and
Taeusch HW.
Post-translational modification of the major human surfactant-associated proteins.
Biochem J
237:
373-377,
1986[ISI][Medline].
36.
Rabilloud, TA.
Comparison between low background silver diammine and silver nitrate protein stains.
Electrophoresis
13:
429-439,
1992[ISI][Medline].
37.
Rubio, S,
Lacaze-Masmonteil T,
Chailley-Heu B,
Kahn A,
Bourbon JR,
and
Ducroc R.
Pulmonary surfactant protein A (SP-A) is expressed by epithelial cells of small and large intestine.
J Biol Chem
270:
12162-12169,
1995
38.
Saitoh, H,
Okayama H,
Shimura S,
Fushimi T,
Masuda T,
and
Shirato K.
Surfactant protein A2 gene expression by human airway submucosal gland cells.
Am J Respir Cell Mol Biol
19:
202-209,
1998
39.
Sambrook, J,
Fritsch EF,
and
Maniatis T.
Molecular Cloning: A Laboratory Manual (2nd ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory, 1989.
40.
Scavo, LM,
Ertsey R,
and
Guo BO.
Human surfactant proteins A1 and A2 are differentially regulated during development and by soluble factors.
Am J Physiol Lung Cell Mol Physiol
275:
L653-L669,
1998
41.
Summerfield, JA,
Sumiya M,
Levin M,
and
Turner MW.
Association of mutation in mannose binding protein gene with childhood infection in consecutive hospital series.
BMJ
314:
1229-1232,
1997
42.
Voss, T,
Eistetter H,
and
Schaefer K.
Macromolecular organization of natural and recombinant lung surfactant protein SP28-36 structure homology with the complement factor C1q.
J Biol Chem
201:
219-227,
1988.
43.
Voss, T,
Melchers K,
Scheirle G,
and
Schafer KP.
Structural comparison of recombinant pulmonary surfactant protein A derived from two human coding sequences: implication for the chain composition of natural human SP-A.
Am J Respir Cell Mol Biol
4:
88-94,
1991[ISI][Medline].