From the Division of Pulmonary/Critical Care Medicine, Department of Internal Medicine, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267
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ABSTRACT |
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The objective of the current study
was to examine the functional importance of the N-terminal domains of
surfactant protein A (SP-A) including the N-terminal segment from
Asn1 to Ala7 (denoted domain 1), the N-terminal
portion of the collagen domain from Gly8 to
Gly44 (domain 2), and the C-terminal portion of the
collagen-like domain from Gly45 to Pro80
(domain 3). Wild type recombinant SP-A (SP-Ahyp; where hyp
indicates hydroxyproline-deficient) and truncated mutant (TM) SP-As
containing deletions of domain(s) 1 (TM1), 2 (TM2), 1 and 2 (TM1-2),
and 1, 2, and 3 (TM1-2-3) were synthesized in insect cells and
purified by mannose-Sepharose affinity chromatography. N-terminal
disulfide-dependent dimerization was preserved at near wild type levels
in the TM1-2 (at Cys Pulmonary surfactant lines the distal airspaces and stabilizes the
gas exchanging alveolar units of the lung by reducing surface tension
(1). Surfactant is composed of phospholipids and proteins that are
organized as lattice-like arrays (e.g. tubular myelin) and
vesicular aggregates within the alveolar lining fluid, and as a film at
the alveolar air-liquid interface. Surfactant protein A
(SP-A)1 is a hydrophilic,
Ca2+-dependent phospholipid-binding protein
that is secreted into the airspace by alveolar type II cells (2). The
function of SP-A is not fully understood, but reports that SP-A binds
to a high affinity receptor on the surface of alveolar type II cells (3, 4), is required for the formation and/or stability of tubular
myelin (5, 6) and large surfactant aggregates (7), and protects
surfactant surface activity in the presence of foreign protein
contamination (8) suggest one or more roles in surfactant function. The
notion that SP-A is also a pulmonary host defense protein is supported
by membership in the structurally homologous collectin family of
antibody-independent opsonins (including mannose-binding protein A) (9,
10), reports that SP-A binds to, aggregates, and opsonizes multiple
microorganisms (for review see Ref. 11), and the finding that the SP-A
gene-targeted mouse is susceptible to infection with group B
streptococcus (12).
Rat SP-A is a large oligomer composed of 18 very similar subunits that
are distinguished by variable glycosylation at one or both
N-linked carbohydrate attachment sites (13, 14) and microheterogeneity in the N-terminal amino acid sequences. The latter
most probably arises by alternative site cleavage by signal peptidase,
which results in a slightly elongated variant containing an Ile-Lys-Cys
(IKC) sequence upstream of the predicted N terminus of Asn1
(15). The primary structure of the SP-A subunits are otherwise identical, composed of several discrete domains including the following
(16, 17): 1) a short N-terminal segment, 2) a collagen-like sequence of
Gly-X-Y repeats (where X is any amino
acid and Y is often Pro or hydroxyproline) containing a
midpoint interruption (Gly44), 3) a hydrophobic neck
domain, and 4) a carbohydrate recognition domain (CRD). Trimeric
association of monomeric subunits occurs by the folding of the
collagen-like domains into triple helices (18). The role of the neck
domain in the oligomeric assembly of SP-A has not been reported, but in
related collectins, the formation of The domains of SP-A that interact with type II cells and surfactant
phospholipids have been mapped by site-directed mutagenesis. The CRD of
the molecule contains binding sites for carbohydrate, the major surface
glycoprotein of the pulmonary pathogen Pneumocystis carinii,
the high affinity SP-A receptor on type II cells, and surfactant
phospholipids, especially dipalmitoylphosphatidylcholine (DPPC)
(21-24). We have recently reported that the N-terminal domains that
contribute to oligomeric assembly, including the interchain disulfide
bond at Cys6 and the collagen-like region, are essential
for SP-A function (25). The collagen-like region was required for
competitive binding to the SP-A receptor and the specific inhibition of
surfactant secretion from type II cells. Disruption of the
Cys6 interchain disulfide bond, by substitution of serine
for cysteine, blocked the aggregation of liposomes by SP-A and reduced
the inhibitory effect of SP-A on surfactant secretion from isolated
alveolar type II cells. Recent analyses indicate that the N-terminal
IKC sequence, and specifically the interchain disulfide-forming
cysteine at Cys In the current study, we used truncated mutant forms of SP-A to examine
the role of the N-terminal segment and defined regions of the
collagen-like domain in oligomeric assembly, phospholipid binding and
aggregation, and SP-A receptor-mediated functions.
Production of Mutant Recombinant Proteins--
The synthesis of
the mutant recombinant proteins TM1, TM2-3, and TM1-2-3 has been
previously reported (15). The TM2 protein was generated by similar
methods. Briefly, the TM2 mutant cDNA encoding the deletion of
amino acids Gly8-Gly44 of rat SP-A was produced
using mutagenic oligonucleotides by overlapping extension polymerase
chain reaction amplification of a 1.6-kilobase pair rat SP-A cDNA
(27, 28). The TM2 cDNA was ligated into the EcoRI site
of PVL 1392 (Invitrogen), and the correct orientation was confirmed
using KpnI. Nucleotide sequencing of the
Gly8-Gly44 coding region of the TM2 cDNA
confirmed the intended deletion and the absence of spurious mutations
(29). A recombinant baculovirus containing the TM2 cDNA for SP-A
was produced by homologous recombination in Spodoptera
frugiperda (Sf-9) cells following contransfection with linear
viral DNA and the PVL 1392/TM2 construct (BaculoGold, PharMingen).
Fresh monolayers of 107 Trichoplusia ni
(T. ni) cells were infected with plaque-purified recombinant
virus at a multiplicity of infection of 10 and then incubated with
serum-free media (IPL-41) supplemented with 0.4 mM ascorbic
acid and antibiotics for 72 h. Recombinant SP-A was purified from
the culture media by adsorption to mannose-Sepharose 6B columns in the
presence of 1 mM Ca2+ and elution with 2 mM EDTA (30). The purified recombinant SP-A was dialyzed
against 5 mM Tris (pH 7.4) and stored at Purification/Modification of Native SP-A--
Surfactant was
isolated by bronchoalveolar lavage of silica-pretreated Sprague-Dawley
rats (31) and floated on NaBr gradients (32, 33). SP-A was isolated and
purified from the surfactant pellicle by delipidation,
mannose-Sepharose affinity chromatography (30), and gel permeation
chromatography with Bio-Gel A-5m.
Protein Assays--
The SP-A content of tissue culture media
containing recombinant SP-A was determined with a rabbit polyclonal IgG
against rat SP-A using a sandwich enzyme-linked immunosorbent assay
(34). The lower limit of sensitivity of the assay was 0.20 ng/ml, and the linear range extended from 0.16 to 10.0 ng/ml. Routine protein concentrations were determined with the bicinchoninic protein assay kit
(BCA) (Pierce) using bovine serum albumin as a standard.
Analysis of Recombinant SP-A--
The effects of mutations on
the oligomeric structure of the recombinant proteins were assessed by
chemical cross-linking. Variant SP-As (10 µg) were incubated with the
5 mM disuccinimidyl glutarate (DSG, Pierce) in 20 mM HEPES containing 0.1 mM EGTA, 0.15 M NaCl, and 100 mM KCl at room temperature for
30 min. The reaction was stopped by addition of reducing sample buffer,
and the proteins were size-fractionated on 8-16% gradient SDS-PAGE gels and stained with Coomassie Blue.
Primary Culture of Alveolar Type II Cells and Secretion of
Phosphatidylcholine--
Experiments to assess the effect of
N-terminal deletions on the SP-A-mediated inhibition of surfactant
secretion were performed as described previously (14). Briefly,
alveolar type II cells were isolated from male Sprague-Dawley rats by
tissue dissociation with elastase and purification on metrizamide
gradients (35). The type II cells were seeded into tissue culture
flasks and incubated overnight in [3H]choline (0.5 µCi/ml) containing Dulbecco's modified Eagle's medium and 10%
fetal calf serum (D10) at 37 °C in a 10%
CO2 atmosphere. After washing, SP-A variants were tested
for their ability to antagonize
12-O-tetradecanoylphorbol-13-acetate (10 Receptor Binding--
A whole cell competitive binding assay was
performed to determine the ability of various recombinant forms of SP-A
to compete with 125I-rat SP-A for receptor occupancy on the
surface of isolated type II cells (36). Following isolation, 2 × 106 type II cells/35-mm dish were incubated overnight in
D10 at 37 °C in a 10% CO2 atmosphere. The
following morning, the nonadherent cells were removed by washing the
monolayers three times at 4 °C with 10 ml of Dulbecco's modified
Eagle's medium containing 1 mg/ml BSA. The monolayers were then
incubated with 1 µg/ml 125I-rat SP-A and various
recombinant SP-As in D10 for 3 h at 37 °C in a 10%
CO2 atmosphere. After washing three times on ice with buffer containing 50 mM Tris (pH 7.4), 100 mM
NaCl, 2 mM CaCl2, and 1 mg/ml BSA, the cells
were solubilized in 0.1 N NaOH, and radioactivity was
quantified in a gamma radiation counter.
Liposome Aggregation and Lipid Binding--
Liposome binding and
aggregation experiments were performed using lipids purchased from
Avanti Polar Lipids, as described previously (14). Unilamellar vesicles
were produced by probe sonication of lipid mixtures composed of
DPPC:egg PC:phosphatidylglycerol (PG), 9:3:2, and equilibrated with
SP-As (lipid:protein ratio 20:1 by weight) in 50 mM Tris,
150 mM NaCl buffer (buffer A) at 20 °C. Aggregation was
determined by measuring light scattering (A400 nm) at 1-min intervals after the addition
of 5 mM Ca2+ (final). For lipid binding,
multilamellar liposomes produced by vigorous vortexing of the same
lipid mixture were incubated with 10 µg/ml SP-A (lipid:protein ratio
50:1) in buffer A containing 2.0% BSA and 5 mM
Ca2+. Following incubation for 1 h at room
temperature, the reaction mixture was centrifuged at 14,000 × gav for 10 min and washed once, and the SP-A
content of the pellet and pooled supernatant fractions were determined
by enzyme-linked immunosorbent assay. Percent binding was defined as
SP-Apellet/(SP-Apellet + supernatant) × 100. Control experiments in which liposomes or Ca2+ were
individually deleted were also performed.
Phospholipid Uptake by Type II Cells--
Uptake of phospholipid
liposomes by type II cells was performed according to the method of
Wright et al. (37) with minor modifications (38). Freshly
isolated alveolar type II cells (1 × 106/tube) were
incubated with unilamellar liposomes (100 µg/ml) composed of
[3H]DPPC (1600 cpm/nmol):egg PC:PG, 7:2:1, and SP-A
variants in 0.5 ml of Dulbecco's modified Eagle's medium, 10 mM HEPES (pH 7.4) for 1 h at 37 °C. The media and
cells were separated by centrifugation at 160 × g for
5 min at 4 °C, and the cells were washed three times in ice-cold
phosphate-buffered saline containing 1 mg/ml BSA. An additional volume
of 0.5 ml of phosphate-buffered saline was added to each tube, and the
cells and media were transferred to separate liquid scintillation vials
and counted. Percent uptake was calculated according to the equation:
[3H]DPPCcells/([3H]DPPCcells + 3[H]DPPCmedia) × 100.
Characterization of the Recombinant Proteins--
The structures
of the mutant recombinant proteins used in this study are shown in Fig.
1. Recombinant SP-A that is overproduced in insect cells has functional characteristics that are comparable to
the natural protein, despite incomplete hydroxylation of prolines in
the collagen-like region (designated "hyp" for
hydroxyproline-deficient) (14). For the purposes of this study we have
designated the domains of SP-A from N to C terminus as the follows:
I-K-C domain, domain 1 (N-terminal segment), domain 2 (N-terminal half
of the collagen-like domain), domain 3 (C-terminal half of the
collagen-like domain), neck, and CRD. The synthesis and physical
characterization of the mutant recombinant SP-As containing telescoping
deletions from Asn1 through the end of the N-terminal
segment (TM1 or Direct Binding of TM SP-As to Multilamellar Liposomes--
The
effects of N-terminal deletions on the direct binding of TM SP-As to
multilamellar liposomes was assessed by determining the percent of
added protein that cosedimented with the phospholipid pellet upon
centrifugation, as described previously (14). Specific binding was
defined by subtracting Ca2+-independent sedimentation of
SP-A (i.e. in the presence of EDTA) from total
Ca2+-dependent binding. As shown in Fig.
4, 53.8 ± 2.2% of
SP-Ahyp specifically bound to the liposomes, compared with
32.2 ± 2.5% of TM1 and 43.8 ± 1.6% of TM1-2. The TM2
protein exhibited wild type levels of phospholipid binding activity
(54.5 ± 2.6%), but binding by TM1-2-3 was negligible (2.1 ± 1.3%). We have previously reported that the binding of TM2-3 to
lipid is about half that of SP-Ahyp (25). These data
indicate that domain 2 of SP-A does not contribute to lipid binding and
that domain 3 is required for wild type levels of lipid binding
activity. The finding that TM1-2 bound to lipid approximately 80% as
effectively as SP-Ahyp demonstrates that N-terminal
disulfide linkage can functionally replace the N-terminal segment with
respect to lipid binding.
Aggregation of Phospholipid Liposomes by TM SP-As--
We have
previously determined that the Cys6 interchain disulfide
bond but not the collagen-like domain of rat SP-A is required for
aggregation of phospholipid liposomes (25). The role of the N-terminal
segment in aggregation was examined by incubation of truncated mutant
forms of SP-A with unilamellar liposomes in the presence of
Ca2+ and measurement of light scattering. As shown in Fig.
5, the maximal end point for light
scattering induced by SP-Ahyp was 0.0965 ± 0.0003 A400 units. The TM1 retained only minimal aggregation activity, approaching an end point for light scattering of
0.0233 ± 0.0007 A400 units. The TM1-2 and
TM1-2-3 were essentially nonfunctional in the assay with end points of
0.0062 ± 0.003 and 0.0030 ± 0.0001 A400 units, respectively. Comparison of the
complete loss of function in TM1-2-3 and the wild type aggregation
activity of the previously reported TM2-3 (also known as Competition of TM SP-As with 125I-Rat SP-A for Binding
to the SP-A Receptor on Type II Cells--
The binding of deletion
mutant SP-As to the receptor on the surface of isolated type II cells
was assessed in a competitive binding experiment, and the results are
shown in Fig. 6. SP-Ahyp
competed with 125I-rat SP-A for receptor occupancy in a
dose-dependent manner with an IC50 of 9.25 µg/ml, similar to what has been reported previously (14, 21, 38). The
TM1 and TM1-2-3, which have limited interchain linkage at the N
terminus, did not compete for receptor occupancy. The TM1-2 and the
TM2 both competed with 125I-rat SP-A for receptor binding
with IC50 values that were approximately 76 µg/ml
(estimated) and 35.5 µg/ml. These data suggest that N-terminal interchain disulfide bond formation is critical for the activity of
SP-A to compete for receptor occupancy and that domain 2 and the
N-terminal segment are not absolutely required. In addition, comparison
of the well preserved activity of TM2 and the loss of competitive
binding by the previously characterized TM2-3 protein (25) suggests
that domain 3 may play an important role in the binding of SP-A to the
receptor.
The Inhibition of Surfactant Secretion from Type II Cells by the TM
SP-As--
We have previously reported that disruption of the
Cys6 interchain disulfide bridge markedly impairs all
inhibition of surfactant secretion by SP-A, but deletion of the
collagen-like domain blocks only specific (monosaccharide-reversible)
inhibition (25). In this study, we examined the role the N-terminal
segment and subdivisions of the collagen domain in inhibition of
secretion by SP-A (Fig. 7). The
concentration of SP-Ahyp required for half-maximal
inhibition of secretion (IC50) was approximately 5.5 µg/ml, which was somewhat higher than previously reported values of
0.1-1.0 µg/ml (14, 21, 25, 38). Inhibition by TM1, TM1-2, and TM2
was comparable with that seen with SP-Ahyp, with
IC50 values of 1.10, 0.18, and 0.63 µg/ml, respectively. In contrast to the potent inhibition by these proteins and the previously characterized TM2-3 protein (25), the TM1-2-3 was a poor
inhibitor of surfactant secretion (IC50 >65 µg/ml). This result indicates that interchain disulfide linkage is critical for
inhibition of surfactant secretion. To assess the specificity of
inhibition by the TM proteins, the experiment was also carried out in
the presence of Enhanced Uptake of Surfactant Liposomes by TM SP-As--
We have
previously reported that deletion of the collagen-like domain and
disruption of the Cys6 disulfide bond blocked the
SP-A-mediated uptake of lipid vesicles by type II cells (25). In the
absence of added SP-A, approximately 0.43 ± 0.09% of the
[3H]DPPC label was taken up by the cells (Fig.
9). In the presence of 50 µg/ml
SP-Ahyp, there was a more than a 4-fold increase in lipid
uptake, to 1.88 ± 0.28%. The two truncated proteins with the
greatest degree of N-terminal disulfide linkage, TM1-2 and TM2,
enhanced liposome uptake by 3-fold to 1.36 ± 0.22% and 3.5-fold
to 1.55 ± 0.20%, respectively. The TM1, which is only 17%
dimeric, had a weak effect on lipid uptake (1.7 fold enhancement to
0.710 ± 0.07%) and the TM1-2-3, which is monomeric with respect
to disulfide bond formation, was even less active (1.4-fold enhancement
to 0.61 ± 0.09%). We conclude that interchain disulfide linkage
but not domain 1 or domain 2 is essential for SP-A-mediated lipid
uptake by type II cells. The finding that TM2 is nearly as active as
SP-Ahyp and that TM2-3 is inactive in the assay (25) may
indicate that the sequences within domain 3 may play a role in lipid
uptake by SP-A.
The purpose of this study was to examine the individual functional
contributions of the major N-terminal domains of SP-A including the
interchain disulfide bonds, the N-terminal segment, and the N-terminal
and C-terminal portions of the collagen-like domain. The strategy
employed was to introduce telescoping N-terminal deletions through the
beginning, middle, and end of the collagen-like region of SP-A and
characterize the effects of the truncations on the interactions of SP-A
with type II cells and surfactant lipids. Pairwise comparisons of TM
proteins with complementary mutants that contained the N-terminal
segment (TM1 and SP-Ahyp, TM1-2 and TM2, and TM1-2-3 and
TM2-3) permitted dissection of the function of each of the N-terminal
domains. Partial preservation of N-terminal disulfide cross-linking at
Cys1) and TM2 proteins (at
Cys
1 and Cys6), and to a lesser extent in TM1
(at Cys
1), but not in TM1-2-3. Cross-linking analyses
demonstrated that the neck + CRD was sufficient for assembly of
monomers into noncovalent trimers and that the N-terminal segment was
required for the association of trimers to form higher oligomers. All
TM proteins except TM1-2-3 bound to phospholipid, but only the
N-terminal segment containing TM proteins aggregated phospholipid
vesicles. The TM1, TM1-2, and TM2 but not the TM1-2-3 inhibited the
secretion of surfactant from type II cells as effectively as
SP-Ahyp, but the inhibitory activity of each mutant was
blocked by excess
-methylmannoside and therefore nonspecific.
TM1 and TM1-2-3 did not enhance the uptake of phospholipids by
isolated type II cells, but the TM1-2 and TM2 had activities that were
72 and 83% of SP-Ahyp, respectively. We conclude the
following for SP-A: 1) trimerization does not require the collagen-like
region or interchain disulfide linkage; 2) the N-terminal portion of
the collagen-like domain is required for specific inhibition of
surfactant secretion but not for binding to liposomes or for enhanced
uptake of phospholipids into type II cells; 3) N-terminal interchain
disulfide linkage can functionally replace the N-terminal segment for
lipid binding, receptor binding, and enhancement of lipid uptake; 4)
the N-terminal segment is required for the association of trimeric
subunits into higher oligomers, for phospholipid aggregation, and for
specific inhibition of surfactant secretion and cannot be functionally replaced by disulfide linkage alone for these activities.
INTRODUCTION
Top
Abstract
Introduction
References
-helical coiled coil bundles in
this region is critical for trimerization (19). Fully assembled SP-A is
a hexamer of trimers that are laterally associated through the
N-terminal segment and the first half of the collagen-like domain and
stabilized by inter- and intratrimeric interchain disulfide bonds at
Cys
1 and Cys6 (18, 20).
1, is not essential for the interactions
of SP-A with type II cell or surfactant phospholipids (26).
EXPERIMENTAL PROCEDURES
0 °C.
7
M)-stimulated surfactant secretion by coincubation with the
monolayer for 3 h. In some experiments, 0.125 M
-methylmannoside was added simultaneously with the
12-O-tetradecanoylphorbol-13-acetate and SP-A to determine
if the SP-A effect was reversible with excess monosaccharides.
Secretion was measured using [3H]phosphatidylcholine (PC)
as a marker for surfactant and expressed as percent secretion
(radioactivity in the media/radioactivity in the cells + media).
RESULTS
Asn1-Ala7), the midpoint of
the collagen-like region (TM1-2 or
Asn1-Gly44), and end of the collagen region
(TM1-2-3 or
Asn1-Pro80) have been
previously reported (15). For this study, an additional mutant SP-A
containing a nested deletion of the proximal collagen-like region (TM2
or
Gly8-Gly44) was produced using similar
methods. The TM2 migrated slightly more rapidly than the
SP-Ahyp on SDS-PAGE gels under reducing conditions,
consistent with the deletion of 37 amino acids (Fig.
2). Like the SP-Ahyp, the TM2
formed disulfide-linked multimers at least as large as tetramers under
non-reducing conditions. This migration profile is consistent with
interchain disulfide bond formation at both sulfhydryls which are
available for interchain linkage, at Cys
1 and
Cys6. We have previously reported that disulfide bond
formation at Cys
1 in TM1 and TM1-2 results in 17 and
51% dimers, respectively, based on densitometry of nonreducing
SDS-PAGE gels. SP-Ahyp, by comparison, is 60% dimers and
higher oligomers (15). The TM1-2-3 is monomeric due to the
near-absence of the Cys
1-containing tripeptide sequence
at the N terminus (<3% of polypeptide chains), presumably because the
mutations introduced lead to exclusive signal peptidase cleavage site
at the Cys
1-Ala81 bond rather than the
alternative site cleavage that occurs in SP-Ahyp, TM1, and
TM1-2 (15). The preservation of partial and near wild type levels of
Cys
1-dependent disulfide cross-linking at the
N terminus of TM1 and TM1-2, respectively, allows for evaluation of
the functional consequences of the peptide sequence deletions that are
distinct from those that are due to loss of covalent interchain
linkage. Noncovalent interactions that also contribute to the assembly
of the SP-A oligomer were analyzed by DSG cross-linking, and the
results are shown in Fig. 3. Treatment of
the SP-Ahyp with DSG followed by size fractionation on
reducing SDS-PAGE resulted in the appearance of a series of 8-9
distinct bands, indicating that the same number of polypeptide chains
were closely associated in the soluble protein, most likely as three
trimers. Previously reported gel filtration chromatography data are
also consistent with nonameric assembly of SP-Ahyp (25).
Cross-linking analyses further revealed that the TM1-2-3 was a
noncovalent trimer, demonstrating that collagen-like region and
interchain disulfide bond formation are not required for
triple-stranded folding of SP-A during synthesis or to maintain the
association of three chains in the fully processed protein. The TM1 and
TM1-2 did not form detectable oligomers greater than trimers, but the TM2 was at least hexameric. These results indicate that N-terminal interchain disulfide linkage is not sufficient for the association of
trimers and that either the N-terminal segment itself or two interchain
bridge-forming cysteines are required for the interaction. We have
recently reported that individual disruption of Cys6 or
Cys
1 by substitution with Ser did not prevent the
assembly of large SP-A oligomers, based on cross-linking
analyses2 and gel exclusion
chromatography (25, 26). Taken together, these data suggest that the
neck + CRD are sufficient for trimerization of SP-A, and that the
N-terminal segment plays an important role in the association of
trimeric subunits in solution that is not solely attributable to
disulfide bond formation.
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Fig. 1.
Schematic of TM SP-As. Wild type
recombinant SP-A, SP-Ahyp, contains an N-terminal extension
composed of amino acids Ile-Lys-Cys (IKC), an N-terminal segment
(NTS) denoted domain 1, a collagen-like region divided by a
midpoint interruption at Gly44 into domains 2 and 3, a neck
region, and a carbohydrate recognition domain (A). Truncated
mutant (TM) recombinant SP-As TM1, TM1-2, TM1-2-3, and TM2
are shown (B). Branched structures represent
N-linked carbohydrate.
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Fig. 2.
Electrophoretic analysis of TM2 SP-A.
Rat SP-A, wild type recombinant SP-A (SPAhyp), and TM2
SP-As were subjected to 8-16% SDS-PAGE under reducing and nonreducing
conditions and stained with Coomassie Brilliant Blue. hyp,
hydroxyproline-deficient.
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Fig. 3.
Cross-linking analysis of oligomeric
association of polypeptide chains of TM SP-As. The wild type
recombinant SP-A (SP-Ahyp), TM1, TM1-2, TM1-2-3, or TM2
were incubated with the cross-linking reagent DSG for 30 min at room
temperature and then size-fractionated on 8-16% reducing SDS-PAGE
gels and stained with Coomassie Brilliant Blue. hyp,
hydroxyproline-deficient.
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Fig. 4.
Direct binding of TM SP-As to
phospholipid liposomes. Multilamellar liposomes were incubated
with SP-Ahyp, TM1, TM1-2, TM1-2-3, or TM2 at room
temperature for 1 h. Following centrifugation, SP-A in the pellet
and supernatant were quantified by enzyme-linked immunosorbent assay.
Specific binding was determined by subtraction of SP-A sedimentation in
the presence of EDTA from total binding. The data shown are mean ± S.E., n = 3. hyp,
hydroxyproline-deficient.
G8-P80)
(25) indicates that covalent interchain linkage, or the N-terminal segment peptide sequence, or both, are required for aggregation. At
least one (25), but not both N-terminal interchain bonds are required,
since disruption or deletion of Cys
1 that forms
interchain bonds does not block aggregation (26). The aggregation
activity of TM2 was nearly identical to SP-Ahyp,
approaching a maximal end point of 0.0965 ± 0.0008 A400 units. Comparison of the TM1-2 and TM2
results indicate that the N-terminal segment, and not simply N-terminal
interchain disulfide-linkage (present in near wild type levels in
TM1-2), is required for lipid aggregation by SP-A. Collectively, these
data are consistent with a role for SP-A self-association via the
N-terminal segment polypeptide domain in the aggregation of
phospholipid vesicles.
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Fig. 5.
Calcium-dependent aggregation of
phospholipid liposomes by TM SP-As. Unilamellar liposomes were
incubated with SP-Ahyp, TM1, TM1-2, TM1-2-3, or TM2 in
Tris buffer (pH 7.4) in a quartz cuvette. After equilibration for 2 min
at 20 °C, 5 mM Ca2+ was added and light
scattering (A400 nm) was measured in a
spectrophotometer. The data shown are mean ± S.E.,
n = 3.
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Fig. 6.
Competition of TM SP-As with
125I-rat SP-A for receptor occupancy on isolated alveolar
type II cells. Primary cultures of alveolar type II cells were
incubated with 0.5-1 µg/ml 125I-rat SP-A and various
concentrations of rat SP-A, SP-Ahyp, TM1, TM1-2, TM1-2-3,
or TM2 for 3 h at 37 °C. The monolayers were washed, dissolved
in 0.1 N NaOH, and counted in a gamma radiation counter as
described under "Experimental Procedures." hyp,
hydroxyproline-deficient.
-methylmannoside, a monosaccharide that has been
shown to block the nonspecific inhibition of surfactant secretion by
lectins such as concanavalin A (36). The inhibition by all of the TM
proteins was at least partially blocked by excess
-methylmannoside,
which is never seen for rat SP-A or SP-Ahyp (Fig.
8). Collectively, these results indicate
that interchain disulfide bond formation at Cys
1 can
functionally replace the N-terminal segment and/or the Cys6
interchain disulfide bond with respect to nonspecific inhibition of
surfactant secretion. However, specific inhibition of surfactant secretion requires the N-terminal segment and both the N-terminal and
C-terminal portions of the collagen-like domain.
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Fig. 7.
Inhibition of phospholipid secretion from
alveolar type II cells by TM SP-As. Isolated alveolar type II
cells in primary culture were incubated overnight with
[3H]choline to label cellular phosphatidylcholine. The
secretagogue 12-O-tetradecanoylphorbol-13-acetate
(TPA) and SP-Ahyp, TM1, TM1-2, TM1-2-3, or TM2
were added and incubated for 3 h at 37 °C. Media and cells were
harvested and counted in a scintillation counter. Percent secretion was
defined as count in the media/count in the media + cells. The data
shown are mean ± S.E., n = 3. hyp,
hydroxyproline-deficient.
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Fig. 8.
Assessment of specificity of inhibition of
secretion by TM SP-As. Inhibition of secretion by
SP-Ahyp, TM1, TM1-2, TM1-2-3, TM2, or TM2-3 was
performed as outlined in Fig. 7 except that 0.125 M
-methylmannoside was added to the media. hyp,
hydroxyproline-deficient.
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Fig. 9.
Enhanced association of phospholipid
liposomes with alveolar type II cells by TM SP-As. Unilamellar
liposomes labeled with trace [3H]DPPC were incubated with
monolayers of isolated alveolar type II cells in the presence of 50 µg/ml SP-Ahyp, TM1, TM1-2, TM1-2-3, or TM2. Following
incubation for 1 h, the cells were washed, dissolved in 0.1 N NaOH, and counted in a scintillation counter. The data
shown are mean ± S.E., n = 3.
DISCUSSION
1 in TM1 and TM1-2 facilitated examination of the
functional consequences of loss of the N-terminal segment and the
N-terminal half of the collagen-like domain without the confounding
influences of complete loss of covalent interchain linkage. Data
from this study and previous reports that indicate the domains of SP-A
that are essential or not essential for several SP-A functions are
outlined in Table I.
Domains of SP-A that are individually essential (+) or nonessential
() for individual functions
The assembly of SP-A can be conceptualized in two parts, the folding of
monomeric subunits into trimers and the association of trimers into an
octadecamer. Three domains of SP-A have potential roles in the
trimerization of SP-A as follows: the neck domain, the collagen-like
region, and interchain disulfide bonds. The observation that
collagenase-digested SP-A remains associated as a noncovalent trimer
indicates that intermolecular forces in the neck + CRD are sufficient
to support the oligomeric association of three strands (39).
Crystallographic analyses of related collectins demonstrate that the
CRDs fold independently and that the neck regions form tightly
associated triple-stranded bundles of -helical coiled coils (19, 40,
41). Data from this study are consistent with that model. Cross-linking
analyses indicated that the TM1-2-3 was composed of three strands,
indicating that the neck domain is sufficient for trimeric assembly.
This result further indicates that folding of the neck + CRD into a
functional conformation (as judged by competence to bind
mannose-Sepharose) does not require the collagen-like region, the
N-terminal segment, or interchain disulfide bonds and that the folding
of SP-A likely proceeds from the C terminus to the N terminus, as has
reported in other soluble collagens and collectins (42).
In the second step of oligomeric assembly, six trimeric subunits become
associated through the N-terminal domains including the collagen-like
domain, the N-terminal segment, and the N-terminal interchain disulfide
bonds. We previously characterized mutant proteins containing
disruption of the Cys6 interchain disulfide bond, the
Cys1 interchain disulfide bond, or deletion of the
collagen-like domain (25, 26). Neither interchain disulfide bridges or
the collagen-like region were found to be individually required for
assembly of SP-A into a nonameric oligomer, the predicted structure of
the wild type recombinant protein SP-Ahyp (25). In the
current study, the oligomeric assembly of SP-A was investigated by
chemical cross-linking experiments. The largest oligomer of TM1-2 that
was detected by the cross-linking analyses was a trimer, indicating
that the C-terminal half of the collagen-like domain and extensive
disulfide cross-linking at the N terminus were not sufficient to
support the association of trimeric subunits into higher oligomers. The
complementary mutant protein TM2 was a hexamer, however, which strongly
implicates the N-terminal segment in the interaction between trimeric
subunits of SP-A. This property of the N-terminal segment does not
appear to be attributable to the availability of two (rather than one)
interchain disulfide bonds flanking this domain, since cross-linking
analyses2 and gel filtration chromatography indicated that
the SP-Ahyp, C-1S and SP-Ahyp, C6S were also
composed of multiple trimeric subunits (25, 26). Collectively, the
findings that reduced and alkylated SP-A (33), SP-Ahyp, C6S (25), and SP-Ahyp, C-1S (26)
remain associated as high molecular weight oligomers are consistent
with the notion that strong noncovalent intermolecular forces in the
neck, collagen-like region, and the N-terminal segment, and not
interchain disulfide bonds, are the major determinants of SP-A assembly
into trimeric and supratrimeric oligomers.
SP-A binds to surfactant lipids and to phospholipid liposomes that
contain DPPC. Lipid binding can be blocked by point mutations in the
CRD, providing strong evidence that the major lipid-binding site
resides in that domain (21). However, mutations in the N-terminal
region also reduce binding to lipid vesicles. We have previously
reported that the disruption of the Cys6 interchain
disulfide bond reduced binding to liposomes by 66% compared with
SP-Ahyp (25). In this study, deletion of the N-terminal
segment which includes Cys6 reduced lipid binding by only
40%. Greater retention of lipid binding by TM1 may have been related
to more extensive dimerization by disulfide bridging at
Cys1 in TM1 (17% dimeric) compared with the
SP-Ahyp, C6S (<5% dimeric). Lipid binding by the TM1-2
mutant, which is extensively dimerized, was about 81% of that of
SP-Ahyp, and wild type levels of lipid binding were
exhibited by TM2. These data eliminate the N-terminal half of the
collagen-like domain as an essential element for lipid binding and
demonstrate that N-terminal interchain disulfide linkage can
functionally replace the N-terminal segment with respect to lipid
binding. Two interchain disulfide bonds are not required for lipid
binding since deletion of Cys
1 was silent (26), but the
greater lipid binding activity of TM2 compared with TM1-2 suggests
that other subdomains of the N-terminal segment (e.g.
hydrophobic residues, carbohydrate moiety) may contribute to lipid
interactions. The characterization of the TM proteins also provides
some information about the role of the C-terminal half of the
collagen-like region in lipid binding. The TM1-2-3, which contains
only the neck + CRD of SP-A and is not disulfide-bridged, did not
exhibit detectable lipid binding in our assay, but the N-terminal
segment containing TM2-3 had lipid binding activity that was about 1/2
that of SP-Ahyp (25). Comparison of lipid binding
activities of TM2 and TM2-3 indicates that the C-terminal half of the
collagen-like domain may contribute to lipid binding (25).
Lipid aggregation is blocked by disruption of the Cys6
interchain disulfide bond (25) and by point mutations in the CRD (21). In this study, deletion of the N-terminal segment which contains Cys6 also greatly reduced lipid aggregation, and the larger
N-terminal deletions contained in TM1-2 and TM1-2-3 completely
eliminated the activity. Addition of the N-terminal segment to the the
TM1-2-3 protein (to produce the previously characterized TM2-3 (25)) restores aggregation activity, suggesting that covalent interchain disulfide bridging, the N-terminal segment polypeptide sequence, and/or
the N-terminal oligosaccharide moiety are required for the function.
Functional comparison of the TM1-2 and TM2 proteins elucidated the
role of these three N-terminal domains in lipid aggregation. Unlike the
lipid binding function of SP-A, extensive disulfide bond formation at
Cys1 in the TM1-2 mutant could not functionally replace
the N-terminal segment in the aggregation assay. However, the
N-terminal segment containing TM2 aggregated liposomes as well as
SP-Ahyp. The availability of a second interchain
disulfide-forming cysteine (at Cys6 within the N-terminal
segment) is not a likely explanation for difference in function between
TM1-2 and TM2, since we have recently shown that only one interchain
disulfide bridge is sufficient (26). Another model for SP-A-mediated
liposome aggregation that has been proposed is self-association by a
lectin interaction between the CRD and SP-A-associated carbohydrate
(43), which is attached to Asn1 of the
aggregation-competent TM2 but is absent from the N terminus of the
nonfunctional TM1-2. We believe that this mechanism is unlikely, based
on our previously reported finding that recombinant SP-A which contains
no carbohydrate, produced by synthesis in the presence of tunicamycin
or genetic ablation of both the Asn1 and Asn187
consensus sequences for glycosylation, aggregates lipid vesicles (14).
Collectively, these data indicate that the N-terminal segment is
required for liposome aggregation and that the N-terminal interchain
disulfide cross-linking is required but is not sufficient for the
activity. We propose a model for liposome aggregation in which SP-A
binds to phospholipid vesicles via the CRD and links adjacent liposomes
by interactions between N-terminal segments.
Specific binding to the SP-A receptor requires Ca2+ and is not sensitive to the presence of excess monosaccharides (36). Mutagenesis experiments indicate that SP-A receptor binding requires cooperative interactions between the major receptor recognition site in the CRD and domains involved in oligomeric assembly, including the collagen-like domain and Cys6 interchain disulfide bonds. Specifically, we have shown point mutations in the CRD (21, 38) and deletion of the collagen-like domain (25) eliminates competitive binding to the SP-A receptor. Analysis of the TM1-2 and TM2 proteins was used to map domains within the collagen-like region that are required for receptor binding. Competition for receptor occupancy by the TM2 protein was less effective than SP-Ahyp at low SP-A concentrations but was comparable to SP-Ahyp at the highest concentrations tested. The TM1-2 protein, which is 51% dimeric, also competed for binding, albeit at a level that was less than SP-Ahyp or TM2. The TM1 did not compete for receptor occupancy, most probably because of the limited degree of interchain disulfide linkage at the N terminus (17% dimeric). Our interpretation of these data is that the N-terminal segment is not absolutely essential for receptor binding and can be functionally replaced by interchain disulfide linkage at the N terminus. The C-terminal half of the collagen-like domain likely contributes to competitive receptor binding, however, since the TM1-2 and TM2 proteins were effective competitors and the TM2-3 protein was not.
Binding to the SP-A receptor on type II cells is coupled to inhibition of secretion of surfactant. Although several lectins can inhibit surfactant secretion, their activities are blocked by excess carbohydrate and are therefore nonspecific (36). In contrast, inhibition of secretion by SP-A is insensitive to the presence of monosaccharide competitors. We recently reported that deletion of the collagen-like domain (TM2-3) of SP-A blocks specific inhibition of surfactant secretion (25). The TM1 data presented in Fig. 8 indicates that, in addition to the collagen-like domain, the N-terminal segment is also required for specific inhibition of surfactant secretion. Monosaccharide-sensitive inhibition by the TM2 protein demonstrates that at least one determinant of specific inhibition within the collagen-like region maps to the N-terminal half of the domain. The integrity of the CRD is also important for specific inhibition, since a mutant protein containing the substitutions Glu195 to Gln and Arg197 to Asp produces weak, monosaccharide-reversible inhibition (38). Collectively, these data indicate that the Ca2+ and carbohydrate binding region of the CRD, the N-terminal segment, and the collagen-like domain are critical for specific inhibition of surfactant secretion by SP-A (Table I). Further studies will be required to determine if sequences within the C-terminal half of the collagen-like domain and the neck region contribute to specific inhibition of surfactant secretion.
The finding that the TM1 and TM1-2 were potent nonspecific inhibitors of surfactant secretion (monosaccharide-sensitive) indicates that the N-terminal segment is not required for this activity. Some degree of disulfide cross-linking is required, however, since the TM2-3 is potent nonspecific inhibitor of surfactant secretion, whereas the TM1-2-3 is a very weak nonspecific inhibitor. The data suggest that the minimal structural requirements for potent nonspecific inhibition of secretion by SP-A include the neck + CRD and disulfide-linkage at the N terminus.
The results of this study and prior studies indicate that SP-A-enhanced uptake of [3H]DPPC into isolated type II cells requires the collagen-like domain, interchain disulfide linkage, and the CRD. In general, the activity of the TM mutants in the uptake assay paralleled the extent of disulfide linkage at the N terminus. The TM1-2 and TM2 proteins which have near wild type levels of interchain linkage at the N terminus enhanced lipid uptake to an extent that was comparable to SP-Ahyp. On the other hand, the TM1 protein which is less extensively disulfide-bridged, had little activity in the assay. The previously characterized interchain disulfide-deficient mutant SP-Ahyp, C6S was also only a weak promoter of lipid uptake (25). Comparison of the well preserved functional activity of TM2 and the minimal activity of the previously described TM2-3 suggests that the Gly45-Pro80 but not the Gly8-Gly44 portion of the collagen-like domain of SP-A contain sequences that are critical for the property of SP-As to enhance the association of klipids into type II cells.
In summary, deletion mutagenesis of the N-terminal domains of SP-A
reveals that lipid binding requires interchain disulfide linkage, lipid
aggregation requires the N-terminal segment and interchain bridging,
receptor competition and SP-A-modulated uptake of surfactant by type II
cells requires N-terminal interchain bonds and the C-terminal half of
the collagen-like region, and specific inhibition of surfactant
secretion requires all N-terminal domains except the IKC region.
Functional mapping of the molecule may ultimately reveal the minimum
SP-A structural requirements for the formation of tubular myelin, for
improving the resistance of surfactant to protein inhibition, and for
antimicrobial defense.
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FOOTNOTES |
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* This research was supported by National Institutes of Health Grant P30ES-0609606, a Career Investigator Award from the American Lung Association, and the Medical Research Service of the Department of Veteran Affairs.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. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 513-558-0480;
Fax: 513-558-4858; E-mail: frank.mccormack{at}uc.edu.
The abbreviations used are: SP-A, surfactant protein A; TM, truncated mutant; CRD, carbohydrate recognition domain; IKC, isoleucine-lysine-cysteine; BSA, bovine serum albumin; DSG, disuccinimidyl glutarate; DPPC, dipalmitoylphosphatidylcholine; PC, phosphatidylcholine; PG, phosphatidylglycerol; PAGE, polyacrylamide gel electrophoresis.
2 F. X. McCormack, unpublished data.
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