(Received for publication, April 20, 1995; and in revised form, May 19, 1995)
From the
Surfactant protein D (SP-D) is a member of the C-type lectin superfamily with four distinct structural domains: an amino terminus involved in forming intermolecular disulfides, a collagen-like domain, a neck region, and a carbohydrate recognition domain. A collagen domain deletion mutant (CDM) of SP-D was created by site-directed mutagenesis. A second variant lacking both the amino-terminal region and the collagen-like domain was generated by collagenase treatment and purification of the collagenase-resistant fragment (CRF). The CDM expressed in CHO-K1 cells formed the covalent trimers, but not the noncovalent dodecamers, typical of native SP-D. The CRF derived from recombinant SP-D formed only monomers. The CDM bound mannose-Sepharose and phosphatidylinositol (PI) as well as SP-D, but the binding to mannosyl bovine serum albumin and glucosylceramide was diminished by approximately 60%. The CRF displayed weak binding to mannose-Sepharose and PI and essentially no binding to mannosyl bovine serum albumin and glucosylceramide. Both SP-D and CDM altered the self-aggregation of PI-containing liposomes. SP-D reduced the density and the light scattering properties of PI aggregates.
These results demonstrate that the collagen-like domain is required for dodecamer but not covalent trimer formation of SP-D and plays an important, but not essential, role in the interaction of SP-D with PI and GlcCer. Removal of the amino-terminal domain of SP-D along with the collagen-like domain diminishes PI binding and effectively eliminates GlcCer binding.
Pulmonary surfactant is an extracellular complex of lipids and
proteins that stabilize the alveoli at the air-liquid
interface(1) . Surfactant protein D (SP-D) ()is a
hydrophilic glycoprotein that is synthesized and secreted by alveolar
type II cells (2, 3) and is loosely associated with
pulmonary surfactant. The protein can also be produced by non-pulmonary
cells (4) . SP-D is a member of the collectin subgroup of the
C-type lectin superfamily(5) , and its structure is
characterized by four distinct domains comprised of: 1) an
intermolecular disulfide containing amino-terminal region, 2) a
collagen-like domain, 3) a neck region, and 4) a carbohydrate
recognition domain (CRD)(6) . Electron microscopy reveals that
the protein exists as a dodecamer which is assembled into a cruciform
structure composed of four identical trimers(5, 7) .
In addition to cruciform-shaped oligomers, large radial aggregates
composed of 10 or more trimeric subunits have also been reported. The
basic cruciform structure shares similarity with bovine conglutinin and
CL-43(5, 8, 9) .
SP-D is known to bind the glycolipids phosphatidylinositol (PI) and glucosylceramide (GlcCer)(10, 11, 12) , and previous studies revealed that the lipid binding properties map to the carbohydrate recognition domain (13, 14) . SP-D also binds to several microorganisms and alveolar macrophages (15, 16, 17) via carbohydrate recognition. The interaction of SP-D and other collectins with microorganisms may constitute an important component of immunoglobulin-independent host defense. In contrast to the CRD, the role of other SP-D domains in measurable protein function is largely uncharacterized.
The role of the collagenous region of surfactant protein A (SP-A), another hydrophilic surfactant-associated glycoprotein that is structurally related to SP-D, has been studied using collagenase-resistant protein fragments (denoted CRF)(18, 19) . These studies revealed that the collagenous domain is not essential for lipid binding(19) , inhibition of lipid secretion from alveolar type II cells (18) , and binding to the high affinity receptor on type II cells(18) . Although the SP-A CRF retained several activities of its intact counterpart, its affinity for different ligands was reduced compared with its native form(18, 19) . Extrapolating from these observations, it is probable that the domains other than the CRD of SP-D can contribute to its interactions toward multiple ligands.
The purpose of this study was to 1) construct and purify SP-D lacking the collagen domain (collagen deletion mutants (CDM)), 2) prepare and purify SP-D lacking the collagen domain and the amino terminus (CRF), 3) characterize some of the physical properties of the SP-D variants, and 4) determine the effects of the structural interactions upon lipid binding. The results demonstrate that the collagen-like region is required for dodecamer formation but not for trimer formation. In addition, the CDM variant retains 75% of phosphatidylinositol binding and 35% of GlcCer binding, whereas the CRF retains about 35% of the phosphatidylinositol binding but loses GlcCer binding.
Figure 1:
Schematic representation of recombinant
surfactant proteins. The domain structures of the monomeric subunit of
recombinant wild type (rSP-D), the collagen domain deletion
mutant form (CDM), and the collagenase-resistant fragment of
recombinant SP-D (CRF) are shown. The four major domains of
rSP-D are denoted D1 to D4, and the correponding
amino acid sequences are: D1, Ala-Asn
;
D2, Gly
-Pro
; D3,
Asp
-Val
; and D4,
Gly
-Phe
.
Figure 2: Electrophoretic analysis of recombinant proteins. Proteins were subjected to 8-16% SDS-PAGE under reducing (lanes a, b, c, g, h, and i) and nonreducing (lanes d, e, f, j, k, and l) conditions and visualized by Coomassie staining (lanes a-f) or autoradiography (lanes g-l). rSP-D (lanes a, d, g, and j), CDM (lanes b, e, h, and k), and CRF (lanes c, f, i, and l) are shown.
Figure 3:
Gel
chromatography of recombinant proteins. S-rSP-D (
),
CDM (
), and CRF (
) were subjected to chromatography using
a Bio-Gel A-1.5m column (A) and a Sephacryl S-200 column (B). The radioactivity of each fraction was quantified using
liquid scintillation spectrometry. The arrows show the peak
position of eluted standards: a, blue dextran; b,
thyroglobulin; c, aldolase; d, bovine serum albumin; e, ribonuclease A.
Figure 4:
Direct binding of recombinant proteins to
solid phase mannosyl-BSA. Aliquots ranging from 0 to 5 µg/ml of S-rSP-D (
), CDM (
) and CRF (
) were
incubated with mannosyl-BSA that had been previously adsorbed to
microtiter wells. The amount of recombinant protein bound to each well
was measured by liquid scintillation spectrometry. Values are mean
± S.E. of three experiments.
Figure 5:
Direct binding of S
recombinant proteins to multilamellar liposomes. Aliquots of 0.1 µg
of
S-rSP-D, [
S]CDM and
[
S]CRF were incubated with 100 µg of
multilamellar liposomes and the sedimentation of each protein with PI
(PI:PS:cholesterol, 30:40:30) (white column) and GlcCer
(GlcCer:PS:cholesterol, 30:40:30) (gray column) liposomes was
measured. Results are expressed as percent sedimentation (sedimented
radioactivity/total activity added). Values are mean ± S.E. of
three experiments.
Figure 6:
Direct binding of S
recombinant proteins to lipids on TLC plates. 5 µg of PI and GlcCer
were subjected to thin layer chromatography in the solvent system
chloroform:methanol:water (70:30:5). After drying the plates and
blocking nonspecific binding sites,
S-rSP-D (lane
a),
S-CDM (lane b), or
S-CRF (lane c) were incubated with the TLC plates for 90 min.
Binding of the radiolabeled proteins was visualized by autoradiography
after washing and air drying.
Figure 7:
Calcium requirements for S
recombinant protein binding to PI and GlcCer liposomes. Direct binding
of
S-rSP-D (
), CDM (
), and CRF (
) to PI (A) or GlcCer (B) liposomes was performed at the
indicated concentrations of calcium, and the results are expressed as
percent sedimentation. Values are mean ± S.E. of three
experiments.
Figure 8: The effects of recombinant proteins on the calcium-induced self-association of unilamellar liposomes. Aliquots of 5 or 20 µg/ml of rSP-D (A and D), CDM (B and E), or CRF (C and F) were preincubated with 100 µg/ml of unilamellar liposomes containing PI:DPPC (50:50) (A-C) or PI:DPPC (30:70) (D-F) for 1 min at room temperature. Next, calcium was added to final concentration of 5 mM and the change in absorbance at 400 nm was measured. ``Controls'' are the values in the absence of proteins. Data are from a representative one of three or four experiments.
Figure 9:
The distributions of SP-D variants and PI
containing unilamellar liposomes within self-forming Percoll gradients.
The sedimentation of lipid alone is shown in A. 20 µg/ml
of rSP-D (B), CDM (C), or CRF (D) were
preincubated with 100 µg/ml of unilamellar liposomes composed of
PI:DPPC (50:50) in the presence of 5 mM calcium for 5 min and
overlaid on TBS containing 5 mM calcium and 40% Percoll. The
gradients were formed by centrifugation at 30,000 gav
1 h, and the amount of protein and liposome in each fraction
was measured as described under ``Materials and Methods.''
The distributions of liposomes in the absence of protein (
),
liposomes in the absence of protein and calcium (
), liposome in
the presence of protein (
), protein in the absence of liposome
(
), and protein in the presence of liposome (
) are
shown.
The purpose of this study was to examine the roles of the
intermolecular disulfide bond containing NH terminus and
the collagenous region of rat SP-D in the structure and the function of
the protein. SP-D is a member of the C-type lectin superfamily that
forms oligomeric structures due to its NH
-terminal
disulfide bonds and its collagenous domains(5, 7) .
The macromolecular organization of SP-D is based upon a reiterated
trimerization of a single polypeptide. In electron micrographs, each
trimer forms a radial arm ending in a globular domain. Trimerization
within the arm is a consequence of interaction of collagen-like domains
that form triple helices. Four trimeric subunits are covalently coupled
to form cruciform structures. However, higher order oligomers
containing more than four trimeric subunits have also been
described(7, 9) . In order to examine the relationship
of SP-D structure to function, we expressed wild type recombinant rat
SP-D and a variant in which the collagenous domain was deleted using
CHO-K1 cells. The rat rSP-D was also treated with bacterial collagenase
to obtain a proteolytic fragment.
The wild type recombinant rat SP-D was identical in its size and interaction with lipids, to its native form as reported previously (14) . The macromolecular structure of rSP-D expressed in CHO cells has also been shown to be similar to that of native form by Crouch et al.(7) . For these reasons, the recombinant molecules expressed in mammalian cells are ideal to study the relationship between structure and function.
The
CRF obtained from the collagenase digestion of rSP-D in these studies
appeared as monomer in nonreducing SDS-PAGE and as an 18-kDa
polypeptide when examined by gel filtration chromatography. In
contrast, the CDM appeared primarily as trimers in nonreducing SDS-PAGE
and three times the size of the CRF by gel filtration chromatography.
Because both the CDM and the CRF possess no long arms (D2 domain), the
molecular sizes of both proteins obtained by gel filtration
chromatography using globular proteins as standards should be very
close to the actual values. Accordingly, the CRF should exist as a
monomer and CDM as a trimer in solution. These observations indicate
that the two cysteine residues in the NH terminus (D1
domain) are likely to be the only cysteine residues involved in the
intertrimeric disulfide bond formation of rSP-D, and these findings are
consistent with observations using enzymatically digested native rat
SP-D(25) . Furthermore, the fact that the CDM does not form
higher order oligomers than trimers suggests that the D2 domain
(collagenous domain) is important not only for the triple helical
structure but also necessary for the formation of the dodecameric
structure of SP-D.
The oligomeric state of the CRF of SP-D utilized
in these studies was different from that of other collectin family CRFs
reported previously. The CRF of recombinant human mannose-binding
protein appears as dimers and trimers as well as monomers(26) ,
and rat mannose-binding protein's CRF forms trimers(27) .
The CRF of native rat SP-D has also been reported to form trimers in
the presence of Ca(25) . However, we have
been unable to obtain reliable gel filtration results of CRF in the
presence of Ca
. These differences may occur as a
consequence of disulfide interchange among CRDs that convert
intramolecular pairings to intermolecular pairings. Such interchanges
may be stabilized by noncovalent interactions among CRDs.
The rSP-D utilized in these studies behaved identically to its native form in its interaction with the carbohydrates. The CDM bound to mannose-Sepharose as efficiently as rSP-D; however, its affinity toward solid phase mannosyl-BSA was significantly reduced compared to wild type. Furthermore, only 20% of the CRF bound to a mannose-sepharose affinity matrix and it failed to bind to solid phase mannosyl-BSA. These findings indicate that the degree of oligomerization of the molecules is a critical determinant of the high affinity binding to immobilized carbohydrates.
The binding properties of recombinant molecules toward glycolipids were also studied. In direct binding to multilamellar liposomes, the activity of CDM toward PI liposomes was quite close to that of its wild type counterpart; however, the affinity for GlcCer liposomes was decreased to about 30% of that of rSP-D. The CRF bound to PI liposomes with approximately 30% of the efficiency of rSP-D, and it failed to show significant binding to GlcCer liposomes. The results obtained from the direct binding assay using solid phase glycolipids on TLC plates were essentially the same as those from the liposome binding studies. These findings indicate that the oligomerization of the molecule contributes to the high affinity binding to glycolipids, especially to GlcCer, despite the fact that the binding domain of SP-D for glycolipids has been mapped to its CRD (D4 domain)(13, 14) . We have previously identified important differences between PI binding and GlcCer binding based upon site-directed mutagenesis(14) . The finding that CRF coordinately lost solid phase carbohydrate and GlcCer binding activity, but retained significant PI binding, suggests that the structural determinants of SP-D required for GlcCer binding and PI binding are different. This latter conclusion is consistent with previous findings in which engineering of altered specificity of carbohydrate binding demonstrated different mechanisms for PI and GlcCer binding(14) .
The interactions of PI-containing unilamellar
liposomes and the recombinant proteins demonstrate that the cruciform
structure of SP-D can significantly alter lipid-lipid interaction.
rSP-D reduced the calcium-dependent self-association process, whereas
CDM enhanced it. The effect of CRF was much smaller than those of rSP-D
and CDM; however, it reduced the initial change of the calcium-induced
self-aggregation of liposomes. Our preferred interpretation of these
results is that liposomes containing 50% PI will form tightly packed
aggregates in which Ca bridges apposing liposomes. In
the presence of rSP-D the liposomes interact with the SP-D and are
effectively held apart by as much as the end to end distance between
the globular heads which is 114 nm, thereby effectively creating an
open lattice. This arrangement would account for the lower buoyant
density of rSP-D-liposome complexes compared with
Ca
-liposome complexes. In contrast to the wild type
protein, the CDM is likely to be composed of only trimeric globular
domains which effectively cross-link liposomes with a spacing of as
little as 9 nm. The increased A
occurring with
CDM may be due to recruitment of more liposomes into aggregates than
with Ca
alone. Evidence for this latter
interpretation is found in Fig.9C in which the amount
of lipid in high buoyant density aggregates significantly increases in
the presence of CDM. When liposomes contain 30% PI, their aggregation
in response to Ca
alone is barely detectable, but the
inclusion of CDM greatly enhances the process. Such enhancement by CDM
illustrates that it is a more effective aggregating agent than
Ca
alone and is entirely consistent with the above
interpretations. The apparent monomeric structure of CRF in conjunction
with its PI binding may function to weakly interfere with
Ca
bridging of liposomes and principally reduce the
rate of lipid aggregation.
In summary, we expressed, purified and characterized rSP-D and its CDM and CRF variants. The CDMs appeared as trimers and the CRF as monomers, indicating that the collagenous domain is essential for the assembly of the dodecameric structure of SP-D. Both CRF and CDM retained significant PI binding activity. The GlcCer binding activity was reduced in CDM and lost in CRF. These findings further substantiate important structural differences between GlcCer and PI binding sites in SP-D.