From the
Pulmonary surfactant protein D (SP-D) is a member of the
collectin subgroup of the C-type lectin superfamily that binds
glycosylated lipids such as phosphatidylinositol (PI) and
glucosylceramide (GlcCer). We have previously reported that the
carbohydrate recognition domain of SP-D plays an essential role in
lipid binding. However, it is unclear how the carbohydrate binding
property of SP-D contributes to the lipid binding. To clarify the
relationship between the lectin property and the lipid binding activity
of rat SP-D, we expressed wild-type recombinant rat SP-D (rSP-D) and a
mutant form of the protein with substitutions Glu-321
Pulmonary surfactant protein D (SP-D)
We have previously reported that the domains responsible for the
lipid binding properties of SP-A and SP-D are located in the CRDs of
the molecules(18, 19, 20) . The lipid binding
specificity of SP-D is quite different from that of SP-A, which binds
to dipalmitoylphosphatidylcholine (DPPC)(21) ,
galactosylceramide (GalCer), lactosylceramide, and
asialo-G
Comparison of the sequence at the carboxyl-terminal portions of
C-type lectin CRDs has demonstrated that SP-D can be categorized as a
mannose- and glucose-type lectin similar to serum mannose-binding
proteins (MBPs) and SP-A(24) . These studies also revealed that
Glu-187 and Asn-189 in mannose-binding protein A (MBP-A) are essential
for the sugar binding specificity of the
molecule(24, 25) . Mutations of Glu-187
In an effort to understand the
contribution of the lectin property of SP-D to glycolipid binding
activity, we introduced mutations into rat SP-D (Glu-321
The purpose of this study was to examine the relationship
between the carbohydrate binding property and the lipid binding
activity of SP-D. We have previously reported that the region of SP-D
responsible for the lipid binding property is located in the
CRD(18) . Both lipid ligands for SP-D (PI and GlcCer) are
glycolipids, an observation that raises the question of whether the
lipid binding property of SP-D is completely due to its sugar binding
specificity. To address this problem, we expressed wild-type
recombinant SP-D and the mutant protein with substitutions Glu-321
The substitutions Glu-321
rSP-D expressed in CHO-K1 cells appeared
essentially identical to native rat SP-D. This result is in agreement
with the recent observations by Crouch et al.(33) . The
difference of molecular size on SDS-PAGE between native and recombinant
molecules was <0.5 kDa (which was due to the heterogeneity of N-linked glycosylation of recombinant molecules), and rSP-D
eluted at exactly the same positions as native rat SP-D on gel
filtration and showed equal reactivity to the antibody raised against
native protein. In addition, the rSP-D used in this study interacted
with lipids in essentially the same manner as native rat SP-D. In
direct binding assays using either TLC plates or multilamellar
liposomes, there were no differences between the lipid binding
specificity of rSP-D and that of native SP-D: both the molecules bound
to PI and GlcCer, but not to DPPC, PG, or GalCer. The sedimentation of
rSP-D with PI liposomes was also identical to the value observed with
native rat SP-D(18) . Furthermore, rSP-D and native SP-D were
indistinguishable in competition with
Unlike its wild-type
counterparts, SP-D
The results obtained in experiments
examining the binding to GlcCer were more striking. rSP-D retained the
GlcCer binding property of the native molecule, while
SP-D
We also studied the effects of
various carbohydrates on the PI vesicle binding of the recombinant
molecules. The affinity of SP-D for PI is much higher than that for
inositol(14) . Inositol was the most effective competitor of PI
binding by rSP-D and SP-D
The studies presented also
demonstrate that the binding mechanisms of SP-D to PI and GlcCer are
not identical. Glu-321 and Asn-323 are essential for the GlcCer
binding, but are not critical for the PI binding. Furthermore, the
relative affinity of the protein for inositol was not affected by the
alteration of carbohydrate binding specificity (data not shown). These
observations suggest that the recognition domains of SP-D for PI and
GlcCer may overlap, but are not identical. While the binding of rSP-D
and the mutant protein to the diacylglycerol-containing lipids
(Gal
In addition to carbohydrate, the hydrophobic components of
glycolipids appear to play an important role in SP-D binding reactions.
Although SP-D binds PI and GlcCer, it fails to bind lyso-PI (14) and glucosylsphingosine on TLC plates (data not shown).
Thus, the nature of the hydrophobic moiety influences the reactivity
and affinity of SP-D for the ligand. Consistent with this idea is the
observation that rSP-D and SP-D
The
interaction of SP-D with unilamellar PI liposomes reveals yet another
aspect of lipid interaction by the protein. SP-D diminishes the
Ca
In
summary, we expressed wild-type rSP-D and the mutant with substitutions
Glu-321
We thank Peggy Hammond for excellent secretarial
assistance.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
Gln and
Asn-323
Asp (SP-D
) in CHO-K1 cells. The
indicated mutations have previously been shown to change the
carbohydrate binding specificity of surfactant protein A and
mannose-binding protein from mannose > galactose to the converse.
rSP-D expressed in mammalian cells was essentially identical to native
rat SP-D in its lipid and carbohydrate binding properties. In contrast,
SP-D
was unable to bind GlcCer, but retained
binding activity toward PI liposomes and solid-phase PI. The efficiency
of SP-D
binding to PI liposome was
50% of
that of rSP-D in the presence of 5 mM Ca
,
but equivalent at 20 mM Ca
. Carbohydrates
competed for SP-D binding to PI such that maltose > galactose for
rSP-D, and the order was reversed for SP-D
.
Furthermore, SP-D
could bind to
digalactosyldiacylglycerol more effectively than rSP-D. These results
suggest the following. 1) The carbohydrate binding specificity of
SP-D
was changed from a mannose-glucose type to a
galactose type; 2) the GlcCer binding property of SP-D is closely
related to its sugar binding activity; and 3) the PI binding activity
is not completely dependent on its carbohydrate binding specificity.
(
)is
a hydrophilic glycoprotein synthesized and secreted by alveolar type II
cells(1, 2) . The primary structure of rat SP-D is
characterized by four distinct domains: 1) an N-terminal region
involved in intermolecular disulfide bonding, 2) a collagenous domain
composed of 59 Gly-X-Y repeats, 3) a neck domain, and
4) a carbohydrate recognition domain (CRD)(3) . In solution,
SP-D forms a 12-mer composed of four identical trimers(4) . The
protein can also form higher order oligomers(4) . SP-D is a
member of the C-type lectin superfamily and shares significant
structural homology with conglutinin, CL43, and surfactant protein A
(3, 5). While SP-A has been implicated as an important molecule in
surfactant lipid metabolism(6, 7, 8) , the
function of SP-D is not well understood. Several studies suggest that
SP-D may play a role in immunoglobulin-independent host defense
mechanisms in the
lung(9, 10, 11, 12, 13) . SP-D
also binds several glycolipids, including phosphatidylinositol (PI) (14, 15) and glucosylceramide (GlcCer)(16) .
Under some conditions, SP-D can antagonize the inhibitory effect of
SP-A upon lipid secretion by alveolar type II cells(17) .
Collectively, these observations suggest that interactions of SP-D with
lipid are likely to be important to its functions within the lung.
(22, 23) . However, the mechanism
of lipid binding by the proteins and the relationship between the
lectin property and the glycolipid binding activity are unclear.
Gln and
Asn-189
Asp in MBP-A altered the carbohydrate binding
specificity of the molecule from a mannose type to a galactose
type(24) . Similar results were also reported for analogous
mutations in SP-A(20) .
Gln and
Asn-323
Asp) to alter the sugar binding specificity of the
molecule. Using this mutant form of SP-D (SP-D
)
expressed in CHO-K1 cells, we show that the carbohydrate binding
specificity is essential for GlcCer binding by SP-D, but is not
critical for the PI binding property of the molecule.
Purification of Rat SP-D
Rat SP-D was isolated
from bronchoalveolar lavage fluids of Sprague-Dawley rats by the method
previously described(14) . Briefly, bronchoalveolar lavage
fluids of silica-treated rats were centrifuged at 33,000 g
at 4 °C for 16 h to sediment surfactant,
and the resultant supernatant was applied to a mannose-Sepharose
affinity matrix in the presence of 5 mM calcium. The
SP-D-containing fraction was then eluted in the presence of 2 mM EDTA, followed by gel filtration in the presence of 10 mM EDTA using a Bio-Gel A-15m column (Bio-Rad). The protein was
dialyzed against TBS (5 mM Tris-HCl, 150 mM NaCl, pH
7.4) at 4 °C and stored at -20 °C.
DNA Construction of SP-D and
SP-D
The 1.2-kilobase pair cDNA for rat
SP-D was isolated as reported previously (3) The SP-D cDNA,
ligated into pGem-7Zf(+) at the EcoRI site, was digested
completely with HindIII and partially with XbaI, and
the resultant full-length insert was subsequently ligated into a pEE14
plasmid vector (26) using the same restriction sites. The mutant
cDNA of SP-D with substitutions Glu-321 Gln and Asn-323
Asp (SP-D
) was produced using the polymerase
chain reaction and the overlapping extension method (27) and the
cDNA of SP-D as the template. This construct was also ligated into a
pEE14 vector using a unique XbaI site, and the orientation of
the insert was confirmed by restriction enzyme digestions using EcoRI and NotI. Mutations were confirmed by DNA
sequencing using the method of Sanger et al.(28) and
Sequenase.
Expression and Isolation of Recombinant
Proteins
The recombinant proteins were expressed using the
glutamine synthetase amplification system(26) . The pEE14
plasmid vectors containing the cDNAs for wild-type SP-D and
SP-D were transfected into CHO-K1 cells using
Lipofectamine (Life Technologies, Inc.). Transfected cells were
incubated in glutamine-free Glasgow minimum essential medium (GMEM)
supplemented with 10% dialyzed fetal bovine serum (complete
GMEM-10(26) ) in the presence of 25 µM methionine
sulfoxamine for 7-10 days. Colonies were isolated using cloning
cylinders, transferred to complete GMEM-10 containing higher
concentrations of methionine sulfoxamine (100-500
µM), and incubated for another 10-15 days. The
colonies isolated from the highest methionine sulfoxamine concentration
were then cloned by limiting dilution. The clones that produced the
highest amount of recombinant proteins were identified using an SP-D
enzyme-linked immunosorbent assay. To isolate rSP-D, cloned cells (4
10
/150-mm dish) were incubated in complete GMEM-10
for 4-5 days until reaching confluence (40
10
/150-mm dish) and then changed to serum-free medium. 48 h
later, the medium was harvested and centrifuged to remove cell debris,
followed by dialysis (3
10
volume) against TBS at 4
°C. The dialyzed SP-D solution was then applied to a
mannose-Sepharose affinity matrix (bed volume of 5 ml) in the presence
of 5 mM calcium, and rSP-D was eluted using 2 mM EDTA. The eluted protein was dialyzed against TBS again and stored
at -20 °C. For SP-D
, which failed to
bind to mannose-Sepharose, the medium containing expressed protein was
directly applied to a protein A-Sepharose affinity column covalently
coupled with polyclonal anti-rat SP-D IgG (29). Immunoreactive SP-D was
eluted from the affinity matrix with 0.1 M glycine, pH 3.0;
immediately neutralized with 1 M Tris base; and then dialyzed
against TBS and stored at -20 °C.
Metabolic Labeling of Recombinant Proteins
The
recombinant proteins were metabolically labeled with
TranS-label (ICN). Confluent cells were washed twice with
phosphate-buffered saline and incubated in methionine- and
cysteine-free GMEM for 2 h. Next, the cells were incubated in the same
medium containing 20 µCi/ml Tran
S-label for 48 h. The
labeled proteins were isolated and stored as described above for
nonlabeled proteins. The specific activity of
S-labeled
rSP-D and SP-D
used ranged between 1500 and 4500
cpm/ng.
Lipids
DPPC, PI, phosphatidylglycerol (PG),
phosphatidylserine (PS), and cholesterol were purchased from Avanti
Polar Lipids, Inc. GlcCer, GalCer, galactosyldiacylglycerol (GalDAG),
and digalactosyldiacylglycerol (GalDAG) were purchased from
Sigma. Multilamellar and unilamellar liposomes were prepared as
described previously (18) and used for the lipid binding and
self-association assay described below.
Direct Binding of Recombinant Proteins to Multilamellar
Liposomes
100 µg of multilamellar liposomes were incubated
with 0.1 µg of S-labeled recombinant proteins in TBS
containing 5 mM calcium and 2% bovine serum albumin (binding
buffer) for 1 h at room temperature, followed by incubation at 0 °C
for 10 min. The incubation mixtures were then centrifuged at 10,000
g
at 4 °C for 10 min. The resultant
pellet was washed once with ice-cold binding buffer, and the amount of
protein sedimenting with liposomes was quantified using liquid
scintillation spectrometry. In some experiments, all the procedures
described above were performed at room temperature. In carbohydrate
competition experiments, the binding was performed in the presence of
0-200 mM carbohydrate as indicated.
Direct Binding of Recombinant Proteins to Lipids on
Thin-layer Chromatograms
Binding of S-labeled
recombinant proteins to solid-phase lipids on TLC plates was performed
as described previously (14) Briefly, 5-50 µg of
lipids were separated on TLC plates with organic solvent. Subsequently,
the plates were air-dried and soaked in blocking buffer (TBS containing
5 mM CaCl
, 2% bovine serum albumin, 1%
polyvinylpyrrolidone) for 1 h. Then the plates were overlaid with
S-labeled proteins in blocking buffer for 90 min at room
temperature. Finally, the plates were washed with ice-cold blocking
buffer, air-dried, and exposed to x-ray film at -80 °C for
1-5 days.
Measurement of Calcium-induced Phospholipid
Self-agglutination
100 µg/ml unilamellar liposomes
containing PI and DPPC (50:50, w/w) or PS and DPPC (50:50, w/w) was
preincubated with native and recombinant proteins (5-20
µg/ml) for 1 min in TBS. Subsequently, calcium was added to a final
concentration of 5 mM, and the change in absorbance at 400 nm
was measured for 5 min using a Beckman DU-64 spectrophotometer at 20
°C. In the absence of lipid, SP-D does not exhibit any light
scattering in the presence of calcium.
Other Procedures
Protein concentrations were
measured by the bicinchoninic acid assay (Pierce) using bovine albumin
as a standard. SDS-polyacrylamide gel electrophoresis (PAGE) was
performed according to the method of Laemmli(30) .
Immunoblotting of native and recombinant proteins was performed by the
method of Towbin et al.(31) using polyclonal anti-rat
SP-D IgG as a probe. Size fractionation of native and recombinant
proteins was performed by gel filtration chromatography using Bio-Gel
A-15m (1.5 100-cm column) in the presence of 10 mM EDTA, 0.15 M NaCl at room temperature.
Characterization of Wild-type Recombinant SP-D and
SP-D
Wild-type rSP-D and SP-DExpressed in CHO-K1
Cells
were
expressed using glutamine synthetase amplification under methionine
sulfoxamine selection. Production of these recombinant proteins ranged
from 3 to 6 mg/48 h/liter of culture medium when confluent cells in
150-mm dishes were incubated with 25 ml of medium. rSP-D was purified
using mannose-Sepharose 6B affinity chromatography, and >95% of the
protein was recovered. In contrast, SP-D
failed
to bind the mannose-Sepharose affinity matrix, requiring that the
protein be isolated using an anti-rat SP-D IgG affinity column. The
recovery of the mutant protein was >90% after affinity column
purification. Fig. 1shows the SDS-PAGE analysis of native rat
SP-D, rSP-D, and SP-D
(lanes a-f).
Native rat SP-D appeared at 43 kDa under reducing and denaturing
conditions and formed disulfide-bonded trimers (
120 kDa) under
nonreducing conditions. rSP-D and SP-D
appeared
identical to each other and formed disulfide-bonded trimers under
nonreducing conditions. The molecular size of both recombinant proteins
was slightly smaller than that of native rat SP-D (<0.5 kDa under
reducing conditions). The difference in molecular size between native
and recombinant molecules was not observed when the proteins were
treated with N-glycanase (data not shown). This latter result
suggests that a minor difference in oligosaccharide structure may exist
between native SP-D and its recombinant form expressed in CHO-K1 cells. Fig. 1also shows the autoradiography of
S-labeled
rSP-D and SP-D
(lanes g-j). The
radioactive proteins appeared essentially identical to their unlabeled
counterparts, and little degradation of the labeled proteins was
observed. Fig. 2shows the immunoblotting of the native and
recombinant proteins. Each protein (100 ng) was subjected to SDS-PAGE
and transferred to a nitrocellulose sheet, and the reactive antigens
were detected using anti-rat SP-D IgG as a probe. In spite of the
mutations, SP-D
reacted to the antibody as well
as native and wild-type recombinant SP-D. Under reducing conditions,
all three proteins yield identical immunoreactive forms. Examination of
the proteins under nonreducing conditions indicates that the major
forms are nearly identical, although several minor bands appear in the
native SP-D lane, suggesting that a small fraction of the preparation
has been proteolyzed.
Figure 1:
Electrophoretic analysis of recombinant
proteins. Proteins were subjected to 8-16% SDS-PAGE under
reducing (lanes a-c, g, and h) and
nonreducing (lanes d-f, i, and j)
conditions and visualized by Coomassie Blue staining (lanes
a-f) or autoradiography (lanes g-j). Native
rat SP-D (lanesa and d), rSP-D (lanesb, e, g, and i),
SP-D (lanesc, f, h, and j) are shown.
Figure 2:
Immunoblotting analysis of recombinant
proteins. 100 ng of native rat SP-D, rSP-D, and SP-D were subjected to SDS-PAGE under reducing (lanes
a-c) and nonreducing (lanes d-f) conditions
and transferred to nitrocellulose sheets. The immunoreactive materials
were detected using polyclonal antibody against rat SP-D. Native rat
SP-D (lanesa and d), rSP-D (lanesb and e), and SP-D
(lanesc and f) are
compared.
The results in Fig. 3show the size
fractionation of the proteins by gel chromatography. S-Labeled rSP-D (Fig. 3A) and
SP-D
(Fig. 3B) were analyzed using
a Bio-Gel A-15m column, and the radioactivity of each fraction was
quantified by liquid scintillation spectrometry. Most of the
S-labeled rSP-D eluted at the position of 1200 kDa (peakb), which corresponds to the main peak of
native rat SP-D(18) . There also appeared small peaks at the
positions of 2100 kDa (peaka) and 600 kDa (peakc), and both peaks were also observed in the elution of
native rat SP-D. The radioactivity present in each peak fraction was 4%
at 2100 kDa, 73% at 1200 kDa, and 13% at 600 kDa. The size distribution
of the recombinant proteins is similar to that of native SP-D, although
up to 10% of the native protein may be present in the 2100-kDa fraction
in some preparations. The elution pattern of
S-labeled
SP-D
was identical to that of rSP-D (Fig. 3B). While the results in Fig. 1indicate
some minor heterogeneity in N-linked glycosylation between
native and recombinant proteins, the oligomerization of rSP-D as well
as SP-D
expressed in CHO-K1 cells is essentially
the same as that of native rat SP-D.
Figure 3:
Gel
chromatography of rSP-D and SP-D.
S-Labeled rSP-D (A) and SP-D
(B) were subjected to chromatography using a Bio-Gel
A-15m column. The radioactivity of each fraction was quantified using a
liquid scintillation spectrometer as described under ``Materials
and Methods.'' The arrows show the peak positions
corresponding to 2100 kDa (arrowa), 1200 kDa (arrowb), and 600 kDa (arrowc).
Direct Binding of Recombinant Proteins to Multilamellar
Liposomes
Direct binding of S-labeled recombinant
proteins to multilamellar liposomes composed of PI, DPPC, GlcCer, or
GalCer was performed, and the results are shown in Fig. 4. In
these experiments, 0.1 µg of
S-labeled rSP-D or
SP-D
was incubated with 100 µg of the
different liposomes in the presence of 5 mM calcium, and
sedimentable radioactivity was measured. In Fig. 4A,
>90% of the rSP-D sedimented with PI liposomes, while none appeared
with DPPC liposomes. In addition, nearly 90% of the rSP-D
coprecipitated with GlcCer liposomes, and none was found with GalCer.
These observations are essentially the same as the result obtained
using native rat SP-D reported previously(18) . However,
SP-D
behaved quite differently from rSP-D. Only
50% of the SP-D
bound to PI liposomes and was
sedimentable. Strikingly, there was no binding of SP-D
to GlcCer liposomes. SP-D
also failed to
sediment with GalCer and DPPC liposomes, similar to its wild-type
counterpart. This latter result with GalCer is important since the
mutations introduced were expected to switch carbohydrate specificity
to favor galactose binding. The experiments described in Fig. 4A were repeated using populations of SP-D that
were purified by gel filtration to correspond only to the dodecamer
population, and the results are shown in Fig. 4B. The
data clearly indicate that there are no significant differences between
the selected dodecamer population of SP-D and the total population in
the interaction of the proteins with liposomes.
Figure 4:
Direct binding of S-labeled
recombinant proteins to multilamellar liposomes. Aliquots of 0.1 µg
of
S-labeled rSP-D (blackcolumns) or
SP-D
(graycolumns) were
incubated with 100 µg of multilamellar liposomes, and the
sedimentation of each protein with PI (PI:PS:cholesterol, 30:40:30),
DPPC (DPPC:PS:cholesterol, 30:40:30), GlcCer (GlcCer:PS:cholesterol,
30:40:30), and GalCer (GalCer:PS:cholesterol, 30:40:30) liposomes was
measured. Results are expressed as percent sedimentation (sedimented
radioactivity/total activity added). Values are means ± S.E. of
three experiments. A contains data for all size oligomers of
recombinant and mutant SP-D, and B contains data for purified
dodecameric oligomers of recombinant and mutant
SP-D.
Direct Binding of Recombinant Proteins to Lipid on TLC
Plates
Direct binding of S-labeled recombinant
proteins (2 µg/ml) to lipids (5 µg/lane) on TLC plates was also
performed as described under ``Materials and Methods,'' and
the results are shown in Fig. 5.
S-Labeled rSP-D
bound to PI and GlcCer, but not to PG or GalCer (Fig. 5, laneb). In contrast,
S-labeled
SP-D
bound to PI, but not to PG, GlcCer, or
GalCer (Fig. 5, lanec). Increasing the amount
of either SP-D
(10 µg/ml) or GlcCer (50
µg) had no effect upon binding. These results are essentially the
same as those derived from the direct binding assay using multilamellar
liposomes (described above) and indicate that Glu-321 and Asn-323 of
SP-D are important for the GlcCer binding property and partially affect
the PI binding activity of SP-D.
Figure 5:
Direct
binding of S-labeled recombinant proteins to lipids on TLC
plates. 5 µg of PI, PG, GlcCer, and GalCer were developed in the
solvent system chloroform:methanol:water (70:30:5). Lipids were
visualized with iodine vapor (lanea). The same
amount of lipids was also developed as described above, and
S-labeled rSP-D (laneb) or
SP-D
(lanec) binding was
performed on TLC plates and visualized by autoradiography as described
under ``Materials and Methods.''
Characterization of PI Binding Property of Recombinant
Proteins
The direct binding of recombinant proteins to
multilamellar liposomes was also examined at different concentrations
of calcium, and the results are shown in Fig. 6. rSP-D bound to
PI liposomes in a Ca-dependent manner, and the
maximum binding was observed at 2.5 mM calcium. The binding
was reduced slightly as the calcium concentration was increased above
2.5 mM. The binding of rSP-D to PI liposomes at 2.5 and 20
mM calcium was 96.4 and 83.3% of the maximum attainable
binding, respectively. The SP-D
binding to PI
liposomes was also Ca
-dependent; however, the binding
below 2.5 mM Ca
was significantly lower than
that of rSP-D. The binding of the mutant protein to PI liposomes
increased to the level of the wild-type protein at 20 mM Ca
. The binding at 2.5 and 20 mM calcium was 40.3 and 80.3% of the theoretical maximum,
respectively. Next, we studied the effect of temperature upon binding (Fig. 7). The PI liposome binding of recombinant proteins was
performed at 20 °C for 1 h, and the incubation mixtures were
transferred to 0 °C or left at 20 °C for an additional 10 min
as described under ``Materials and Methods.'' There was no
difference in the binding of rSP-D to PI liposomes at 20 and 0 °C;
however, the PI binding of SP-D
at 20 °C was
significantly reduced compared with that at 0 °C (52% at 0 °C
and 21% at 20 °C). These results suggest that Glu-321 and Asn-323
affect the stability of SP-D/PI interactions at low Ca
concentrations and elevated temperatures.
Figure 6:
Calcium requirement of S-labeled recombinant protein binding to PI liposomes.
Direct binding of
S-labeled rSP-D (
) and
SP-D
(
) to PI liposomes (100 µg of PI)
was performed with varying concentrations of calcium, and the results
are expressed as percent sedimentation as described under
``Materials and Methods.'' Values are means ± S.E. of
three experiments.
Figure 7:
Temperature dependence of S-labeled recombinant protein binding to PI liposomes.
S-Labeled rSP-D (blackcolumns) or
SP-D
(graycolumns) was
incubated with PI liposomes (100 µg of PI) for 1 h at 20 °C,
and the incubation mixtures were transferred to 0 °C or left at 20
°C for another 10 min. The results are expressed as percent
sedimentation as described under ``Materials and Methods.''
Values are means ± S.E. of three
experiments.
Light Scattering Measurement of Calcium-induced
Self-association of Unilamellar Liposomes
Self-association of
unilamellar liposomes composed of either PI and DPPC (50:50) or PS and
DPPC (50:50) was measured in the presence of native and recombinant
proteins as described under ``Materials and Methods.''
Liposomes that contain a high percentage of negatively charged
phospholipids such as PI and PS self-associate in the presence of
calcium. Fig. 8A shows the effect of native rat SP-D on
the self-association of PS containing unilamellar liposomes. SP-D has
no significant effect upon either the rate or extent of PS vesicle
aggregation as monitored by an increase in A.
Mutation-bearing SP-D gave the same result as rSP-D in its interaction
with PS vesicles. Next, we studied the effects of the proteins on the
self-association of PI liposomes. rSP-D reduced the A
change effected by Ca
in a
concentration-dependent manner (Fig. 8B). The effect of
20 µg/ml rSP-D was almost the same as that of 10 µg/ml rSP-D.
The absorbance at 6 min in the presence of 0, 5, 10, and 20 µg/ml
rSP-D was 0.063, 0.046, 0.033, and 0.031, respectively, and these
values correspond well to those found with native SP-D (data not
shown). SP-D
also reduced the change in A
of PI liposomes (Fig. 8C);
however, the effect was significantly less than that of rSP-D. The
absorbance at 6 min in the presence of 20 µg/ml mutant protein was
0.041. These results show that rSP-D and SP-D
specifically interact with unilamellar PI liposomes and that the
efficiency of SP-D
binding to PI and capacity to
alter liposome aggregation are lower than those of rSP-D.
Figure 8:
SP-D variants alter calcium-induced
self-association of unilamellar liposomes. Proteins (5-20
µg/ml) were preincubated with 100 µg/ml unilamellar liposomes
for 1 min at room temperature. Next, calcium was added to a final
concentration of 5 mM, and the change in absorbance (ABS) at 400 nm was measured. Controls are the values
in the absence of proteins. A, PS liposomes (PS:DPPC, 50:50)
with native rat SP-D; B, PI liposomes (PI:DPPC, 50:50) with
rSP-D; C, PI liposomes (PI:DPPC, 50:50) with
SP-D. Data are from a representative one of three
to five experiments.
Carbohydrate Competition for SP-D Binding to PI
Liposomes
Direct binding of rSP-D or SP-D to PI vesicles was performed in the presence of 25-100
mM sugar (inositol, maltose, glucose, mannose, galactose, and N-acetylgalactosamine), and the results are presented in Fig. 9. The values are expressed as the percentage of the control
value obtained in the absence of the sugars. Inositol was the most
effective inhibitor of PI binding of rSP-D (Fig. 9A).
The order of the competition for rSP-D binding to PI vesicles was
inositol > maltose > glucose > mannose > galactose, and it
corresponded well with the order of the carbohydrate binding affinity
of native rat SP-D(32) . N-Acetylgalactosamine showed
no effect even at a concentration of 200 mM (data not shown).
By comparison, the binding of SP-D
to PI vesicles
was most effectively inhibited by inositol; however, galactose became
the second most effective competitor in the binding, consistent with
the hypothesis that the introduced mutations should increase the
affinity of the protein for galactose. The order of the competition for
mutant protein binding to PI vesicles was inositol > galactose >
maltose > mannose > glucose. The most dramatic changes in rank
order affinity for carbohydrate competition were found for maltose and
galactose. For rSP-D, the IC
values of maltose and
galactose were 18.5 and >200 mM, respectively. For mutant
SP-D, the IC
values of maltose and galactose were 38.5 and
27.5 mM, respectively. These results clearly indicate the
sugar binding specificity of the mutant protein,
SP-D
, changed from a mannose-glucose type to a
galactose type; however, inositol remained the most effective ligand
for SP-D.
Figure 9:
Carbohydrate competition for S-labeled rSP-D and SP-D
binding to
PI multilamellar liposomes. Direct binding of
S-labeled
rSP-D (A) or SP-D
(B) to PI
liposomes (100 µg of PI) was performed in the presence of
0-100 mM concentrations of the indicated sugars, and the
results are expressed as percent sedimentation as described under
``Materials and Methods.'' Inositol (
), maltose
(
), glucose (
), mannose (
), galactose (
), and N-acetylgalactosamine (GalNac) (
) were used as
competitors. Values are means ± S.E. of three
experiments.
Direct Binding of Recombinant Proteins to Other
Glycolipids
To examine further changes in the ligand binding
specificity of the mutant protein, we investigated the direct binding
of the recombinant proteins to GalDAG and GalDAG. Fig. 10shows the direct binding of the
S-labeled
recombinant proteins to GalDAG and Gal
DAG on a TLC plate.
Both rSP-D and SP-D
bound to Gal
DAG,
but not to GalDAG, and the binding of SP-D
to
Gal
DAG was significantly greater than that of rSP-D. Next,
we performed the direct binding of the proteins to GalDAG and
Gal
DAG liposomes in the presence of 5 mM calcium (Fig. 11). No significant binding was observed for either protein
toward GalDAG liposomes; however, Gal
DAG liposomes bound
significantly more SP-D
than rSP-D. The
difference in binding to Gal
DAG between rSP-D and
SP-D
became more significant when the reactions
were performed in the presence of 20 mM calcium (16% in rSP-D
and 38% in SP-D
; data not shown).
Figure 10:
Direct binding of S-labeled
recombinant proteins to GalDAG and Gal
DAG on TLC plates. 5
µg of GalDAG and Gal
DAG were developed on TLC plates in
chloroform:methanol:water (70:30:5) and visualized by iodine vapor (lanea). Identical amounts of lipids were developed
as described above, and direct binding of
S-labeled rSP-D (laneb) and SP-D
(lanec) was performed as described under ``Materials and
Methods.''
Figure 11:
Direct binding of S-labeled
recombinant proteins to GalDAG and Gal
DAG liposomes.
S-Labeled rSP-D (blackcolumns) or
SP-D
(graycolumns) was
incubated with 100 µg of multilamellar liposomes, and the
sedimentation of each protein with GalDAG (GalDAG:PS:cholesterol,
30:40:30) or Gal
DAG (Gal
DAG:PS:cholesterol,
30:40:30) liposomes was measured. The results are expressed as percent
sedimentation as described under ``Materials and Methods.''
Values are means ± S.E. of three
experiments.
Gln and Asn-323
Asp in an effort to alter the sugar
binding specificity of the molecule.
Gln and Asn-323
Asp were chosen based upon the results of
a study conducted with serum MBP-A that demonstrated that analogous
mutations resulted in the alteration of the sugar binding specificity
of the molecule from a mannose-glucose type (24) to a galactose type.
The homology of the amino acid sequence at corresponding regions
between rat SP-D and MBP-A (3, 24) suggests the
possibility that identical substitutions in rat SP-D may also change
the carbohydrate binding specificity of the molecule. This same
approach has recently been used with the related protein
SP-A(20) .
S-labeled rSP-D for
binding to solid-phase PI (data not shown).
was unable to bind to a
mannose-Sepharose affinity matrix. Mutant SP-D was isolated using an
antibody affinity column, and it appeared to be identical to rSP-D when
analyzed by SDS-PAGE, gel filtration, and immunoblotting. However,
SP-D
behaved quite differently from rSP-D in its
interactions with lipids. The sedimentation of SP-D
with PI liposomes at 2.5 mM Ca
was
about half of that of rSP-D. In addition, SP-D
required higher concentrations of calcium (20 mM) as
well as lower temperature conditions than rSP-D to achieve equivalent
binding efficiency. Experiments examining the disruption of PI
self-aggregation also indicated that the affinity of
SP-D
for this lipid was lower than that of rSP-D.
These observations demonstrate that Glu-321 and Asn-323 play an
important role in the expression of the PI binding property of SP-D,
but are not absolutely required.
did not bind to GlcCer. These results
indicate that Glu-321 and Asn-323 of SP-D are essential for the GlcCer
binding activity of the molecule.
; however, the rank
order for competition by other carbohydrates was markedly altered.
Maltose was the second most effective competitor for rSP-D binding to
PI, and galactose was insignificant as a competitor. In contrast, for
SP-D
binding to PI, galactose was the second most
effective competitor. These results show that SP-D
still possessed the carbohydrate binding properties, but that its
binding specificity had been altered to a galactose >
mannose-glucose type. Furthermore, SP-D
bound to
Gal
DAG with higher affinity than rSP-D. These observations
demonstrate clear evidence of altered carbohydrate binding specificity
by SP-D
.
DAG and PI) is similar, the binding to
ceramide-containing lipids (i.e. GlcCer) is markedly
different. The results suggest that the carbohydrate specificity of
SP-D may play a more dominant role in interactions of the protein with
ceramide-containing lipids than with diacylglycerol-containing lipids.
bind
Gal
DAG, but not GalCer ( Fig. 4and Fig. 5),
ceramide dihexoside, and ceramide trihexoside (data not shown).
-dependent self-aggregation process by an unknown
mechanism. Our speculation is that SP-D may form an ordered lattice in
which the protein is interposed between liposomes and thus reduces the
absorption of light that occurs when liposomes are agglutinated by
Ca
. In the experiments described in Fig. 8, the
highest levels of SP-D give a liposome:SP-D ratio of
1. The
cruciform structure of the SP-D oligomer and the location of the CRDs
at the extreme ends of the molecules constitute a favorable structural
arrangement for preventing liposome/liposome interactions.
Gln and Asn-323
Asp (SP-D
)
using the glutamine synthetase system and CHO-K1 cells. Both of the
recombinants appeared to be identical to native rat SP-D in terms of
the size and extent of oligomerization. rSP-D behaved essentially the
same as native rat SP-D in the interactions with carbohydrates and
lipids. The affinity of SP-D
for PI was
significantly reduced compared with that of rSP-D, and the GlcCer
binding property was completely ablated in the mutant. The carbohydrate
binding specificity of the mutant was altered to a galactose type from
a mannose-glucose type. Glu-321 and Asn-323 are therefore essential for
the GlcCer binding property of SP-D, but are not absolutely required
for PI binding.
DAG,
digalactosyldiacylglycerol; PAGE, polyacrylamide gel electrophoresis;
G
, Il
NeuAc-GgOse
Cer.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.