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Correction of Pulmonary Abnormalities in Sftpd
ⴚ/ⴚ
Mice
Requires the Collagenous Domain of Surfactant Protein D
*
Received for publication, January 23, 2006, and in revised form, June 19, 2006 Published, JBC Papers in Press, June 20, 2006, DOI 10.1074/jbc.M600651200
Paul S. Kingma
‡
, Liqian Zhang
‡
, Machiko Ikegami
‡
, Kevan Hartshorn
§
, Francis X. McCormack
¶
,
and Jeffrey A. Whitsett
‡1
From the
‡
Division of Pulmonary Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio 45229-3039,
§
Departments of Medicine and Pathology, Boston University School of Medicine, Boston, Massachusetts 02118-3393,
and
¶
Pulmonary/Critical Care Division, University of Cincinnati Medical Center, Cincinnati, Ohio 45267-0564
Surfactant protein D (SP-D) is a member of the collectin
family of innate defense proteins. Members of this family
share four distinct structural domains: an N-terminal cross-
linking domain, a collagenous domain, a neck region, and a car-
bohydrate recognition domain. In this study, the function of the
collagenous domain was evaluated by expressing a SP-D colla-
gen deletion mutant protein (rSftpdCDM) in wild type and SP-D
null mice (Sftpd
ⴚ/ⴚ
). rSftpdCDM formed disulfide-linked trim
-
ers that further oligomerized into higher order structures. The
mutant protein effectively bound carbohydrate and aggregated
bacteria in vitro. Whereas rSftpdCDM did not disrupt pulmo-
nary morphology or surfactant phospholipid levels in wild type
mice, the mutant protein failed to rescue the emphysema or
enlarged foamy macrophages that are characteristic of Sftpd
ⴚ/ⴚ
mice. Moreover, rSftpdCDM partitioned with small aggregate
surfactant in a manner similar to SP-D, but rSftpdCDM did not
correct the abnormal surfactant ultrastructure or phospholipid
levels observed in Sftpd
ⴚ/ⴚ
mice. In contrast, rSftpdCDM com
-
pletely corrected viral clearance and the abnormal inflamma-
tory response that occurs following pulmonary influenza A chal-
lenge in Sftpd
ⴚ/ⴚ
mice. Our findings indicate that the collagen
domain of SP-D is not required for assembly of disulfide-stabi-
lized oligomers or the innate immune response to viral patho-
gens. The collagen domain of SP-D is required for the regulation
of pulmonary macrophage activation, airspace remodeling, and
surfactant lipid homeostasis.
Surfactant protein D (SP-D)
2
is a member of the collectin
family of C-type lectins. Members of this family include surfac-
tant protein A (SP-A), SP-D, mannose-binding protein, conglu-
tinin, and CL-43. SP-A and SP-D contribute to the innate
immune system of the lung by binding and enhancing the clear-
ance of a variety of viral, bacterial, and fungal pathogens (1–3).
The collectins are defined by four structural domains shared by
all family members: a short amino-terminal cross-linking
domain, a triple helical collagenous domain, a neck domain,
and a carbohydrate recognition domain (CRD) (4–8). Three
neck domains associate to form a triple coiled-coil structure
that facilitates the assembly of the remaining domains into a
trimer (9). Final assembly of the trimer, thought to be the
minimal functional unit of collectins, occurs through disul-
fide bonds between the cysteine-rich amino-terminal
domains (10, 11). Trimers further combine into larger mul-
timeric complexes through disulfide-stabilized, noncovalent
interactions. Although larger structures are commonly
observed, SP-D exists predominantly as a tetramer of tri-
meric subunits (dodecamer) assembled into a cruciform
structure (10, 12).
Animal models of SP-D deficiency have revealed a complex
role for SP-D in pulmonary immunity and alveolar homeosta-
sis. Mice with a targeted deletion of the Sftpd gene (Sftpd
⫺/⫺
)
survived normally but developed gradually worsening pulmo-
nary inflammation, emphysema, and surfactant phospholipid
accumulations (13, 14). Sftpd
⫺/⫺
mice accumulate apoptotic
alveolar macrophages as well as enlarged, lipid-laden, macro-
phages that release metalloproteinases and reactive oxygen
species (15–19). Uptake and clearance of viral pathogens,
including influenza A and respiratory syncitial virus, were defi-
cient in Sftpd
⫺/⫺
mice (20, 21). In contrast, clearance of group
B Streptococcus and Hemophilus influenza was unaltered in the
absence of SP-D (19). However, oxygen radical release and pro-
duction of the proinflammatory mediators, tumor necrosis fac-
tor-
␣
, IL-1, and IL-6, were increased in Sftpd
⫺/⫺
mice when
exposed to either viral or bacterial pathogens (19 –21).
The roles of the various domains of SP-D in its complex
functions have been studied by expressing SP-D point
mutants, deletion mutants, or chimeric proteins of SP-D and
other collectins. For example, whereas expression of the full-
length rat Sftpd gene (rSftpd ) fully rescues the Sftpd
⫺/⫺
mouse phenotype, expression of a fusion protein that
included the N-terminal and collagen domains of SP-A fused
to the neck and CRD of SP-D (rSftpa/d) was not sufficient to
correct the emphysema or lipid accumulations characteristic
of Sftpd
⫺/⫺
mice, indicating that the collagenous and N-ter
-
minal domains of SP-D are essential for these functions (22).
* This work was supported by National Institutes of Health (NIH) Grants
HL56387 (to J. A. W.), HL63329 (to M. I.), and HL68861 (to F. X. M.) and
NHLBI, NIH, Grant HL60931 (to K. H.). The costs of publication of this article
were defrayed in part by the payment of page charges. This article must
therefore be hereby marked “advertisement” in accordance with 18 U.S.C.
Section 1734 solely to indicate this fact.
1
To whom correspondence should be addressed: Cincinnati Children’s
Hospital Medical Center, Division of Pulmonary Biology, 3333 Burnet
Ave., Cincinnati, OH 45229-3039. Tel.: 513-636-4830; Fax: 513-636-7868;
E-mail: jeff.whitsett@cchmc.org.
2
The abbreviations used are: SP-D, surfactant protein D; SP-A, surfactant pro
-
tein A; CRD, carbohydrate recognition domain; IL, interleukin; BALF, bron-
choalveolar lavage fluid; PBS, phosphate-buffered saline; IAV, influenza A
virus; ELISA, enzyme linked immunosorbent assay; MMP, matrix metallo-
proteinase; Sat PC, saturated phosphatidylcholine; SIRP, signal-inhibitory
protein.
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 34, pp. 24496 –24505, August 25, 2006
© 2006 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
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Substitution of serine for cysteine at positions 15 and 20 of
the N-terminal domain (rSftpd
Ser15,20
) results in a protein
that corresponds to a single trimeric arm of the SP-D
dodecamer but fails to correct the abnormal macrophages,
emphysema, or lipid accumulations in Sftpd
⫺/⫺
mice, sug
-
gesting that oligomers induced by the SP-D collagenous and
N-terminal domains are required for complete function (23).
However, intranasal administration of a truncated SP-D tri-
mer (consisting of only the CRD and neck domains) into
Sftpd
⫺/⫺
mice partially corrects the apoptotic macrophages,
lipid accumulations, and elevated inflammatory mediators,
suggesting that the collagen domain is not essential for these
activities (15, 24). Since most collectins form sulfhydryl-sta-
bilized oligomers, the relatively large collagen domain of
SP-D suggests that this domain serves a purpose that is
beyond facilitating oligomerization of the SP-D CRD. Com-
parisons of SP-D, mannose-binding protein, and conglutinin
fusion proteins suggest the large collagen domain promotes
proper spacing of trimeric subunits in order to facilitate
cross-linking of separate microorganisms and microbial
aggregation (25). Therefore, to further investigate the function
of the SP-D structural domains, we genetically introduced into
wild type and Sftpd
⫺/⫺
mice lungs an SP-D collagen deletion
mutant protein (rSftpdCDM) that formed multimers via an
intact N terminus. Whereas expression of rSftpdCDM elicited a
protective response to influenza virus, the protein failed to cor-
rect the abnormal macrophage activation, emphysema, or lipid
abnormalities in Sftpd
⫺/⫺
mice, indicating that the SP-D col
-
lagenous domain is critical for normal regulation of lipid home-
ostasis, macrophage activity, and the structural integrity of
peripheral airspaces.
EXPERIMENTAL PROCEDURES
Animal Husbandry—Mice were handled in accordance with
approved protocols through the Institutional Animal Care and
use Committee at Cincinnati Children’s Hospital Medical Cen-
ter. All mice had been maintained in the vivarium in barrier
containment facilities. Sentinel mice in the colony were sero-
logically negative for common murine pathogens.
Generation of Transgenic Mice—
rSftpdCDM cDNA was generated
using recombinant PCR to delete a
177-amino acid sequence (Gly
26
–
Pro
202
) corresponding to the com
-
plete collagen domain from rat
SP-D (Fig. 1). The rSftpdCDM
cDNA was inserted into the EcoRI
site of 3.7hSPC/SV40 expression
vector and sequenced (26). The
transgene was microinjected into
FVB/N oocytes fertilized with
Sftpd
⫺/⫺
sperm by the Children’s
Hospital Transgenic Core facility,
and founders were identified by
transgene-specific PCR using
upstream primer 5⬘-GGAGACAA-
AATCTTCAGGGCG-3⬘ and down-
stream primer 5⬘-TTCGGATGGT-
GGCAGCATAG-3⬘. Transgenic animals were crossed with either
Sftpd
⫺/⫺
mice to generate rSftpdCDM
Tg⫹
/Sftptd
⫺/⫺
mice or wild
type mice to generate rSftpdCDM
Tg⫹
/Sftpd
⫹/⫹
mice (16).
Western Blot Analysis—Animals were weighed, anesthetized
by intraperitoneal injection of pentobarbital, and exsangui-
nated. Bronchoalveolar lavage was performed five times with 1
ml of normal saline. Typically, ⬎90% of the instilled volume was
recovered. For each lane, 40
l of the bronchoalveolar lavage
fluid (BALF) was dried, reconstituted in 15
l of Laemmli buffer
with or without sulfhydryl reduction with

-mercaptoethanol,
and resolved on 10 –20% SDS/Tris/glycine/polyacrylamide gel
(Novex, San Diego, CA). After separation and transfer to a
nitrocellulose membrane, protein was detected with rabbit
anti-mouse SP-D or guinea pig anti-rat SP-A antiserum (Seven
Hills Bioreagents, Cincinnati, OH) diluted 1:5000 in Tris-buff-
ered saline as previously described (27).
SP-D Purification—Previous studies demonstrated that inac-
tivation of the granulocyte-macrophage colony-stimulating
factor gene (Gmcsf
⫺/⫺
) in mice impaired SP-D clearance and
increased SP-D levels in BALF severalfold (28). Therefore, to
increase the amount of starting material for protein purifica-
tion, rSftpdCDM was purified from rSftpdCDM
Tg⫹
/Sftptd
⫺/⫺
/
Gmcsf
⫺/⫺
mice. Wild type mouse SP-D was purified from
Sftpd
⫹/⫹
/Gmcsf
⫺/⫺
mice that also carried a deletion of the two
expressed Sftpa genes in order to minimize the potential of
SP-A contamination. A similar Sftpa deletion was not possible
in the rSftpdCDM
Tg⫹
/Sftptd
⫺/⫺
/Gmcsf
⫺/⫺
mice; however,
contaminating SP-A was not detectable by silver stain gels in
rSftpdCDM preparations.
SP-D- or rSftpdCDM-containing BALF was applied to a mal-
tosyl-Sepharose (Sigma) column and selectively eluted with
manganese as previously described (29). The pooled fractions
were diluted 10-fold in 20 m
M Tris-HCl, pH 7.4, and 30 mM
CaCl
2
and applied to a 1-ml bed volume maltosyl-Sepharose
column. The column was stripped of lipopolysaccharide with
20 m
M Tris-HCl, pH 7.4, 20 mM n-octyl-

-D-glucopyranoside,
200 m
M NaCl, 2 mM CaCl
2
, 100
g/ml polymyxin and washed
with 20 m
M Tris-HCl, pH 7.4, 0.5 mM CaCl
2
, 200 mM NaCl. The
protein was eluted with 20 m
M Tris-HCl, pH 7.4, 200 mM NaCl,
FIGURE 1. Schematic representation of the rSftpdCDM transgene. The rSftpdCDM cDNA was generated
using recombinant PCR to delete the 177-amino acid sequence of the collagen domain from rat SP-D. The
rSftpdCDM cDNA was inserted into the EcoRI site of 3.7hSPC/SV40 expression vector and sequenced. The
transgene was identified by transgene-specific PCR primers used to generate the transgenic mice.
SP-D Collagen Domain
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1mM EDTA. Under the conditions employed, lipopolysacha-
ride concentration was typically ⱕ0.1 endotoxin units/
gof
protein.
SP-D Sizing and Detection—The size of rSftpdCDM multim-
ers was determined by gel filtration chromatography using
Sepharose CL-6B equilibrated in 20 m
M Tris-HCl, pH 7.4, 200
m
M NaCl, 5 mM EDTA, and 0.02% sodium azide. Purified rSft-
pdCDM was diluted in the same buffer and applied to the col-
umn (1.5 ⫻ 90 cm). rSftpdCDM protein concentration was
determined in each fraction using an enzyme-linked immu-
nosorbent assay (ELISA). Plates were washed five times
between incubations, and all washes and dilutions were carried
out with Tris-buffered saline, 0.1% Tween 20. A mouse anti-
SP-D monoclonal antibody was developed by exposing
Sftpd
⫺/⫺
mice to purified, full-length, human SP-D and select
-
ing cell lines that demonstrated a high affinity for SP-D and
minimal cross-reactivity with SP-A (Seven Hills Bioreagents,
Cincinnati, OH). ELISA plates were coated with this mono-
clonal antibody (1
g/ml, 100
l/well) in 0.1 M carbonate buffer,
pH 9.6, overnight at 4 °C and blocked with 1% bovine serum
albumin for 1 h at room temperature. Plates were washed, and
appropriate dilutions of standards and protein samples were
added and incubated for1hatroom temperature. Plates were
washed and incubated with rabbit anti-mouse SP-D antiserum
(100
l/well diluted 1:750) for 1 h. This was followed by wash-
ing and incubation with donkey horseradish peroxidase-conju-
gated anti-rabbit IgG (100
l/well diluted 1:10,000) (Jackson
Immunoresearch, West Grove, PA) for 1 h. After washing
plates again, TMB substrate (100
l/well) (BioFx Laboratories,
Owings Mills, MD) was added. The color reaction was stopped
after 10 min with 2
M H
2
SO
4
, and plates were read at 450 nm.
Typically, this assay results in absorbance changes that are
equal, parallel, and linear for mouse SP-D, rSftpdCDM, and
human SP-D concentrations between 10 and 150 ng/ml.
Carbohydrate Binding—Direct binding of SP-D to carbohy-
drate was detected by mixing BALF from 6 – 8-week-old mice
with maltosyl-Sepharose-linked beads at 4 °C for2hin4m
M
Tris-HCl, pH 7.4, and 5 mM CaCl
2
(23). Binding specificity and
calcium dependence were confirmed by the addition of 100 m
M
maltose and 10 mM EDTA to the binding reaction, respectively.
The samples were centrifuged at 10,000 ⫻ g for 1 min, and the
supernatants were removed. The amount of unbound SP-D in
the supernatant was determined by Western blot analysis.
Selective carbohydrate binding was performed as described
previously (30). Briefly, microtiter plates were coated with 10
g/ml mannan at 4 °C overnight, washed, and blocked with 1%
bovine serum albumin. BALF was incubated with increasing
concentrations of maltose, glucose, galactose, or GlcNAc. Sam-
ples were subsequently added to the coated plate and incubated
with rabbit anti-SP-D antibody followed by peroxidase-conju-
gated goat anti-rabbit IgG antibody. After washing, o-phe-
nylenediamine was added to each well, and the A
490 nm
was
measured. The concentration of sugar that inhibited 50% of
SP-D binding to the mannan-coated plate was defined as the
IC
50
.
Bacterial Aggregation—Bacterial aggregation was assessed by
measuring light transmission through a bacterial suspension
after the addition of SP-D. Purified rSftpdCDM, mouse SP-D
(150 nm based on the monomer molecular weight), or a control
reaction that contained protein buffer without SP-D was mixed
with 600
lofEscherichia coli Y1088 (OD
700 nm
⬃ 1) grown the
previous night and resuspended in Tris-buffered saline plus 5
m
M CaCl
2
(31). The OD
700 nm
was measured every 2.5 min, and
all values were reported as relative to the absorbance at t ⫽ 0.
The extent of aggregation was determined by the decrease in
the optical density of the bacterial suspension. Calcium
dependence was confirmed by the inhibition of aggregation in
the presence of 10 m
M EDTA.
Lung Morphology—Lungs from 12-week old mice were fixed
at 25 cm of water pressure with 4% paraformaldehyde in phos-
phate-buffered saline (PBS) and processed into paraffin blocks.
Sections (5
m) from each lobe were stained with hematoxylin
and eosin. Immunohistochemistry for SP-D was performed at
dilutions of 1:200 by using a rabbit polyclonal antibody gener-
ated to murine SP-D. Immune complexes were detected using
an avidin-biotin-peroxidase technique (Vectastain Elite ABC
kit, Vector Laboratories, Burlingame, CA).
Metalloproteinase Activity—Alveolar macrophages (5 ⫻ 10
5
)
were isolated by centrifuging BALF and cultured for 24 h in
AIM-V medium (Invitrogen). Proteinases in the conditioned
media were assayed by zymography as described previously
(32).
Phospholipid Analysis—Saturated phosphatidylcholine (Sat
PC) was measured in homogenized lung tissue and BALF from
6– 8-week-old mice (n ⫽ 6 – 8 mice for each genotype) as pre-
viously described (33).
Isolation of Large and Small Aggregate Sufactant—Large
and small aggregate surfactant lipids were isolated from
rSftpdCDM
Tg⫹
/Sftpd
⫹/⫹
mice (n ⫽ 6) as previously described
(14). Briefly, bronchoalveolar lavage was performed five times
with 1 ml of normal saline. BALF was centrifuged at 40,000 ⫻ g
over a 0.8
M sucrose cushion for 15 min. Small aggregate sur-
factant was collected from the supernatant. Large aggregate
surfactant was collected from the interface, diluted with normal
saline, and centrifuged again at 40,000 ⫻ g for 15 min. The large
aggregate pellet was dissolved in normal saline. An equal vol-
ume of each material was dried, diluted in Laemmli buffer, and
analyzed by Western blot.
Ultrastructure of lipid aggregates was determined in pooled
BALF samples (n ⫽ 5/pool) by electron microscopy from 6 – 8-
week-old mice as previously described (14). Briefly, large and
small aggregate surfactant was isolated from BALF; fixed with
glutaraldehyde, paraformaldehyde, and CaCl
2
in 0.1 M sodium
cacodylate buffer at 4 °C; and stained with osmium tetroxide,
potassium ferrocyanide, and uranyl acetate. After dehydration,
it was embedded in Embed812 resin (Electron Microscopy Sci-
ences, Fort Washington, PA), and ultrathin sections (90 nm)
were obtained. Random electron micrographs were taken, and
ultrastructure was evaluated.
Influenza A Virus—Experiments utilized influenza A virus
strain H
3
N
2
A/Phillipines/82 (IAV) and were performed as pre
-
viously described (34). Briefly, IAV was grown in chorioallan-
toic fluid of 10-day-old embryonated hen eggs. Virus was puri-
fied and stored frozen in PBS until use. Six-week-old (n ⫽ 6–8)
mice were anesthetized with inhaled isoflurane and inoculated
intratracheally with 5 ⫻ 10
5
fluorescent foci in 80
l of PBS.
SP-D Collagen Domain
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Quantitative IAV cultures were performed 3 days after inocu-
lation. Lungs were removed and homogenized in 2 ml of PBS,
and aliquots were frozen. IL-6, tumor necrosis factor-
␣
, and
interferon-
␥
concentrations were determined by ELISA kits
(R&D Systems, Minneapolis, MN). IAV titers were determined
by incubating lung homogenates with Madin-Darby canine
kidney monolayers for7hat37°C.Monolayers were washed,
fixed, and incubated with monoclonal antibody against IAV
nucleoprotein followed by a rhodamine-labeled goat anti-
mouse IgG. Fluorescent foci were counted, and the resulting
viral titer was expressed as fluorescent foci/g lung weight.
SP-D binding to IAV was monitored by hemagglutination
inhibition assays (n ⫽ 3). Purified mouse SP-D and rSftpdCDM
were compared. Sample protein was serially diluted in 96-well
round bottom plates with IAV in PBS with 0.5 m
M CaCl
2
and
0.5 m
M MgCl
2
. After incubating the protein/IAV mixture at
room temperature for 10 min, fertilized chicken egg erythro-
cytes were added, and the samples were incubated for an
additional 2 h. The minimal amount of SP-D or rSftpdCDM
needed to fully inhibit agglutination was observed, and the
number of hemagglutination units inhibited per pmol of
SP-D or rSftpdCDM was reported.
Data Analysis—Where appropriate, either a representative
experiment from one mouse line was shown, or results from
each line were averaged, and data were analyzed by unpaired
Student’s t tests.
RESULTS
Transgenic Mouse Lines—rSftpdCDM
Tg⫹
/Sftpd
⫺/⫺
trans
-
genic mice were produced by nuclear injection of the
rSftpdCDM gene into FVB/N mice and backcrossed to
Sftpd
⫺/⫺
mice. The rat Sftpd gene is 92% identical (based on
nucleotide and mature protein amino acid sequence) to the
mouse Sftpd gene, and multiple studies indicate that mouse and
rat SP-D have nearly identical properties. Four founder mice
were identified using transgene-specific PCR on tail clip DNA.
Germ line transmission was demonstrated in all four lines, and
the transgene was inherited as an autosomal gene following
Mendelian inheritance. Survival and breeding were not influ-
enced by the rSftpdCDM
Tg⫹
transgene. Expression and secre
-
tion of transgenic protein rSftpdCDM was confirmed in all four
mouse lines by Western blot analysis of mouse BALF using a
rabbit anti-mouse SP-D antibody (data not shown). Two mouse
lines with similar levels of rSftpdCDM protein expression were
selected for further breeding and analysis. Experiments were
done in parallel with both lines, and results were similar.
Expression of rSftpdCDM Protein—As determined by ELISA
assays, levels of SP-D in BALF from wild type mice were 1.2
g/ml compared with rSftpdCDM levels of 11
g/ml in
rSftpdCDM
Tg⫹
/Sftpd
⫺/⫺
mice. Rabbit anti-mouse SP-D anti
-
body was used to detect SP-D and rSftpdCDM in BALF (Fig. 2).
The monomeric form of the mutant protein migrated under
reducing conditions at the predicted molecular mass of 22 kDa.
rSftpdCDM migrated slightly slower than the 60-kDa standard
under nonreducing conditions, indicating that the disulfide
linkages normally observed between monomeric chains in the
wild type N-terminal domain also formed in the mutant pro-
tein. To determine the ability of the rSftpdCDM to form high
order complexes, the protein was analyzed by Sepharose col-
umn chromotography. Under these conditions, the majority of
rSftpdCDM migrated between apoferritin and

-amylase
(molecular masses of 443 and 200 kDa, respectively) at a posi-
tion equaling 270 kDa, which is the molecular mass expected if
adding the N-terminal domain to the CRD trimer promoted
noncovalent interactions between four trimeric subunits
(dodecamer). Smaller peaks were observed at the expected
positions of molecules composed of 1, 2, 30, and more trimers,
suggesting that the purified mutant protein consists of a heter-
ogeneous population of multimers. Whereas the mutant pro-
tein effectively self-assembled to higher order structures, it did
not form intermediate molecular weight heteropolymers of
mutant and wild type protein when expressed in a wild type
SP-D background (data not shown).
RSftpdCDM Binds Carbohydrate—To compare the carbohy-
drate binding properties of wild type SP-D and rSftpdCDM,
binding to maltosyl-Sepharose was evaluated (Fig. 3). Protein
FIGURE 2. Analysis of bronchoalveolar lavage fluid and purified protein
from mice expressing rSftpdCDM. A, BALF from wild type (lanes 1 and 3) and
rSftpdCDM
Tg⫹
/ Sftpd
⫺/⫺
(lanes 2 and 4) mice were resolved on SDS-PAGE
under reducing (Reduced) conditions. Disulfide-linked trimers were resolved
under nonreducing (Non Reduced) conditions. Protein was detected with rab-
bit anti-mouse SP-D antibody. B, purified rSftpdCDM was resolved by column
chromatography in Sepharose CL-6B. Protein levels were determined by
ELISA and expressed as relative to the peak fraction. The arrows indicate the
peak position of eluted standards: blue dextran (a), thyroglobulin (b), apofer-
ritin (c),

-amylase (d ), alcohol dehydrogenase (e), albumin (f ), and carbonic
anhydrase (g).
SP-D Collagen Domain
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from both wild type and mutant BALF bound maltose. Binding
was inhibited by the addition of EDTA, indicating that rSftpd-
CDM maintained calcium-dependent lectin activity. In addi-
tion, binding to maltosyl-Sepharose was reversed by the addi-
tion of free maltose, confirming the specificity of maltose
interactions.
The saccharide binding preference of SP-D and rSftpdCDM
was assessed using BALF and inhibition of binding to yeast
mannan by competing saccharides (Table 1). Under the condi-
tions utilized, a higher binding affinity for the competing sac-
charide was indicated by a lower concentration of saccharide
needed to disrupt protein-mannan interactions. Maltose was
the preferred binding substrate of the carbohydrates tested
with an IC
50
of1mM with BALF from wild type mice, consistent
with results previously reported (27, 30). The order of binding
preference was maltose ⬎ glucose ⬎ galactose ⬎ GlcNAc for
both SP-D and rSftpdCDM. Whereas the mutant and wild type
proteins had similar relative saccharide binding preferences,
the IC
50
values observed with rSftpdCDM were lower than
SP-D. Assuming there are no compositional differences in the
mutant and wild type BALF that might influence binding activ-
ity, these results suggest that the mutant protein may have a
higher carbohydrate binding affinity.
rSftpdCDM Aggregates Bacteria—Previous studies dem-
onstrated that whereas mutant trimeric forms of SP-D
(rSftpd
Ser15,20
and trimeric CRDs) bind carbohydrates, they do
not effectively aggregate infectious particles (11, 31). Similar
results were observed in earlier work with a SP-D collagen dele-
tion mutant (35). However, unlike the current rSftpdCDM, the
earlier collagen deletion mutant did not form multimers. In
addition, studies with chimeric collectins containing domains
of SP-D, mannose-binding protein, and conglutinin suggest
that the relatively large collagen domain of SP-D increases bac-
terial aggregation activity (25). To determine if rSftpdCDM
induced bacterial aggregation, light transmission through a
suspension of E. coli was monitored after the addition of
purified rSftpdCDM (Fig. 4). The drop in absorbance in the
rSftpdCDM reaction indicates that the multimeric mutant
protein effectively aggregates bacteria. Aggregation by
rSftpdCDM was completely inhibited by the addition of
EDTA, confirming the calcium dependence for protein func-
tion. When compared with a reaction that contained an
equal molar concentration of wild type SP-D based on the
molecular weight of the monomer chain, the aggregation
activity of rSftpdCDM was slightly better than the wild type
protein. Thus, bacterial aggregation is not dependent on
appropriate spacing of the CRD by the collagenous domain of
SP-D.
Lung Morphology—Deletion of the mouse Sftpd gene caused
several distinct alterations in lung morphology, including
emphysema and accumulations of enlarged foamy macro-
phages (13, 16–18). To determine if the mutant protein influ-
enced these findings, pulmonary structure was assessed in wild
type and Sftpd
⫺/⫺
mice expressing rSftpdCDM
Tg⫹
(Fig. 5
). Im-
munostaining revealed marked expression of rSftpdCDM
Tg⫹
in
alveolar type II cells and bronchiolar epithelial cells. However,
rSftpdCDM did not alter the lung morphology in 12-week-old
wild type mice. Moreover, the mutant protein did not correct
the abnormal morphology typically observed in Sftpd
⫺/⫺
mice.
Enlarged, foamy macrophage accumulations and emphysema
were detected readily in both transgenic mouse lines expressing
rSftpdCDM
Tg⫹
in a Sftpd
⫺/⫺
background. Therefore, whereas
FIGURE 3. SP-D and rSftpdCDM proteins bind maltosyl-Sepharose. BALF
from rSftpdCDM
Tg⫹
/Sftpd
⫹/⫹
mice was incubated with maltosyl-Sepharose
beads followed by centrifugation to pellet the beads. The resulting superna-
tant was evaluated by SDS-PAGE and Western analysis. Binding of endoge-
nous SP-D (SP-D) or mutant rSftpdCDM (CDM) was indicated by the absence
of protein in the supernatant. BALF was incubated with (⫹) or without (⫺)
beads or with beads and EDTA (EDTA) or excess free maltose (Maltose).
FIGURE 4. rSftpdCDM aggregates E. coli. Bacterial suspensions (n ⫽ 3) were
incubated with 150 nM purified rSftpdCDM (CDM, open triangles) or wild type
SP-D (SP-D, closed circles). Absorbance values were recorded at the indicated
times before (t ⫽ 0) and after the addition of the indicated protein and
reported as relative to the absorbance value at t ⫽ 0. Aggregation was indi-
cated by the clustering of bacterial particles and a subsequent drop in absorb-
ance. Aggregation with rSftpdCDM was completely reversed by the addition
of EDTA to the reaction mixture (EDTA, open circles). Control (closed circles)
was protein buffer alone. S.E. values were ⬍10% for all time points.
TABLE 1
Binding of SP-D and rSftpdCDM to carbohydrates
BALF from wild type and rSftpdCDM
Tg⫹
/Sftpd
⫺/⫺
mice was incubated in mannan-
coated ELISA plates with increasing concentrations of competing carbohydrate.
The saccharide concentration required to inhibit 50% of the binding (IC
50
)toman
-
nan is reported (n ⫽ 2).
Saccharide (IC
50
)
Maltose Glucose Galactose GlcNAc
mM
SP-D 1 8 20 ⬎100
rSftpdCDM 0.5 1 5 20
SP-D Collagen Domain
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targeted expression of the full-length rat Sftpd gene in Sftpd
⫺/⫺
mice fully rescues the Sftpd
⫺/⫺
phenotype (36), expression of
rSftpdCDM
Tg⫹
does not correct the abnormal lung morphol
-
ogy in Sftpd
⫺/⫺
mice.
MMP-9 and MMP-2 Activity—Proteinase activity gels were
used to assess MMP-9 and MMP-2 activity in media containing
alveolar macrophages from wild type, Sftpd
⫺/⫺
, and
rSftpdCDM
Tg⫹
/Sftpd
⫺/⫺
mice (Fig. 6
). Whereas minimal
MMP-9 and MMP-2 activity were observed in wild type sam-
ples, metalloproteinase activity was markedly elevated in
both Sftpd
⫺/⫺
and rSftpdCDM
Tg⫹
/
Sftpd
⫺/⫺
mice, indicating that rSft
-
pdCDM does not correct the
increased metalloproteinase pro-
duction by alveolar macrophages
from Sftpd
⫺/⫺
mice (32).
Lung Phospholipids and Surfac-
tant Structure—SP-D selectively
interacts with small aggregate sur-
factant in adult mice and regulates
the uptake and catabolism of surfac-
tant lipids by alveolar type II cells
(13, 14). Consequently, mice lacking
SP-D have 2–5-fold higher surfac-
tant pool sizes (13, 16). Moreover,
surfactant isolated from Sftpd
⫺/⫺
mice has abnormally large aggregate
lipid structures and small aggre-
gates consisting of atypical multila-
mellated forms (14). To evaluate if
rSftpdCDM corrected surfactant
phospholipid pool sizes, Sat PC
in BALF and lung homogenates
were assessed in wild type and
Sftpd
⫺/⫺
mice with and without
rSftpdCDM
Tg⫹
(Fig. 7
). Expression
of the mutant protein did not alter
alveolar, tissue, or total Sat PC levels
in wild type mice, and it did not cor-
rect the elevated Sat PC levels in
Sftpd
⫺/⫺
mice. In addition, to deter
-
mine if rSftpdCDM corrected the
abnormal surfactant ultrastructure
characteristic of Sftpd
⫺/⫺
mice,
large and small aggregate surfactant
was examined by electron micros-
copy (Fig. 8). The large aggregate fraction from Sftpd
⫺/⫺
and
rSftpdCDM
Tg⫹
/Sftpd
⫺/⫺
mice contained abnormal, large
lamellated lipid structures. The small aggregate fraction in
wild type mice consisted of single layer sheets or vesicles,
whereas atypical multilayered structures predominated in
the small aggregate fraction from Sftpd
⫺/⫺
and rSftpdCD
-
M
Tg⫹
/Sftpd
⫺/⫺
mice. Despite the failure of rSftpdCDM to
correct surfactant lipid structure, analysis of large and small
aggregate surfactant from rSftpdCDM
Tg⫹
/Sftpd
⫹/⫹
mice
revealed that rSftpdCDM partitioned with small aggregate
surfactant in a manner that was similar to SP-D (Fig. 9). This
is in contrast to SP-A, which segregated primarily with large
aggregate surfactant. Therefore, these results demonstrate
that the collagen domain of SP-D is not required for the
selective partitioning of SP-D with small aggregate surfac-
tant, but it is required for normal surfactant ultrastructure
that in turn influences uptake by alveolar type II cells (14).
Correction of Influenza A Infection by rSftpdCDM—Previous
in vitro studies demonstrated that SP-D binds IAV and
enhances IAV binding and uptake by neutrophils (31, 37, 38).
Decreased viral clearance and enhanced inflammation was
observed in Sftpd
⫺/⫺
mice exposed to intratracheal IAV (21). As
FIGURE 5. rSftpdCDM does not correct lung morphology in Sftpd
ⴚ/ⴚ
mice. Lungs were fixed and stained
with hematoxylin and eosin. A, lungs from wild type mice; B, rSftpdCDM
Tg⫹
in a wild type background; C,
immunostaining of SP-D in wild type mice; D, Sfpd
⫺/⫺
mice; E, rSftpdCDM
Tg⫹
in Sftpd
⫺/⫺
background; F, immu
-
nostaining of rSfpdCDM in a Sftpd
⫺/⫺
background. The arrowheads point to enlarged, foamy macrophages.
The arrows point to SP-D immunostaining in type II cells and bronchiolar epithelial cells. Bars, 100
m. The
insets show the same tissue under higher magnification.
FIGURE 6. rSftpdCDM does not correct increased metalloproteinase
activity in Sftpd
ⴚ/ⴚ
mice. Proteinase activity in conditioned media from
alveolar macrophages isolated from wild type (WT), rSftpdCDM
Tg⫹
/Sftpd
⫺/⫺
,
and Sftpd
⫺/⫺
mice was evaluated on zymogram gels. BALF was diluted as
shown, and MMP-2 and MMP-9 activity was indicated by a clear band at 72
and 92 kDa, respectively.
SP-D Collagen Domain
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determined by hemagglutination inhibition assays, rSftpdCDM
effectively bound IAV and inhibited IAV-mediated hemagglu-
tination (Fig. 10). Moreover, hemagglutination inhibition activ-
ity of rSftpdCDM was ⬃2-fold greater than that observed with
an equal molar (based on the molecular weight of a SP-D or
rSftpdCDM monomer) amount of wild type SP-D. To further
evaluate the anti-IAV activity of rSftpdCDM, IAV was admin-
istered to mouse lungs intratracheally, and the viral titers and
cytokine response (Fig. 11) were measured 3 days later. In con-
trast to Sftpd
⫺/⫺
mice, no detectable IAV was recovered from
the wild type or rSftpdCDM
Tg⫹
/Sftpd
⫺/⫺
lung homogenates. In
addition, the increased IL-6, tumor necrosis factor-
␣
, and inter-
feron-
␥
levels observed in IAV-challenged Sftpd
⫺/⫺
mice were
restored to wild type levels in rSftpdCDM
Tg⫹
/Sftpd
⫺/⫺
mice.
Taken together, these results demonstrate that rSftpdCDM
completely corrects viral binding, clearance, and inflammatory
responses observed in Sftpd
⫺/⫺
mice.
DISCUSSION
SP-D plays multiple complex roles in pulmonary physiology,
including binding and clearing of infectious pathogens, regula-
tion of the innate host defense system, airspace remodeling, and
surfactant phospholipid metabolism (1–3). In the present
study, the function of the SP-D collagen domain was evaluated
by expressing a SP-D collagen deletion mutant in the lungs of
wild type and Sftpd
⫺/⫺
mice.
Deletion of the collagen domain resulted in the secretion of
an ⬃22-kDa protein that migrated as a trimer under nonreduc-
ing conditions. These findings are consistent with in vitro stud-
ies describing an SP-D collagen deletion mutant that was
expressed in Chinese hamster ovary cells by Ogasawara and
Voelker (35, 39). In contrast to these earlier studies, the major-
ity of the present rSftpdCDM formed higher order complexes
of four trimeric subunits (dodecamer) when expressed in the
mouse respiratory system. The reason for this disparity is unclear,
but given the close proximity of the regions deleted, it probably
reflects differences in the protein expression systems utilized. In
FIGURE 7. Increased lung saturated phosphatidylcholine in rSftpdCD-
M
Tgⴙ
/Sftpd
ⴚ/ⴚ
mice. Alveolar, tissue, and total Sat PC levels were deter
-
mined in wild type (Sftpd
⫹/⫹
) and SP-D null (Sftpd
⫺/⫺
) mice with (open bars)or
without (closed bars) the rSftpdCDM transgene (n ⫽ 6 – 8 mice for each gen-
otype). Values were normalized for body weight. Sat PC levels in Sftpd
⫺/⫺
mice were significantly (p ⱕ 0.05) higher than wild type controls. The addition
of rSftpdCDM did not significantly (p ⫽ 0.48, 0.47, and 0.40 for alveolar, tissue,
and total, respectively) correct this increase. The error bars indicate S.E.
FIGURE 8. rSftpdCDM does not correct surfactant ultrastructure in
Sftpd
ⴚ/ⴚ
mice. Ultrastructure of large aggregate (LA) and small aggregate
(SA) surfactant isolated from wild type (Sftpd
⫹/⫹
CDM⫺), Sftpd
⫺/⫺
(Sftpd
⫺/⫺
CDM⫺), and rSftpdCDM
Tg⫹
/Sftpd
⫺/⫺
(Sftpd
⫺/⫺
CDM⫹) mice. The
closed arrowheads point to normal large aggregate lamellar bodies and small
aggregate single layer sheets and vesicles. The open arrowheads point to
abnormal large lipid structures in large aggregate surfactant. The arrows
point to atypical multilayered structures in small aggregate surfactant. Pho-
tomicrographs are representative of samples pooled from three mice of each
genotype.
SP-D Collagen Domain
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addition, the finding that adding the N terminus to the trimeric
CRD facilitates dodecamer formation supports previous results
that indicate that the structural predilection for dodecamers is
contained within the N-terminal domain (40).
Ogasawara and Voelker (35) demonstrated that the collagen
deletion mutant bound mannose-Sepharose and phosphatidyl-
inositol with affinities comparable with wild type SP-D, but
binding to mannosyl bovine serum albumin and glucosylcera-
mide was somewhat diminished. Although specific binding
constants were not determined in the present work, when com-
paring equal molar quantities of SP-D and rSftpdCDM, the
mutant protein binding activity for virus, bacteria, and carbo-
hydrate was consistently equal to or greater than wild type
SP-D, demonstrating that deletion of the collagen domain does
not inhibit substrate binding.
Despite the considerable binding affinity of rSftpdCDM,
expression of the mutant protein failed to correct the aberrant
alveolar macrophage activity characteristic of Sftpd
⫺/⫺
mice
(13, 16 –18). Foamy macrophages, increased secretion of met-
alloproteinases, and emphysema persisted despite expression
of high levels of rSftpdCDM. In contrast, influenza virus bind-
ing, clearance, and the associated cytokine response medi-
ated by rSftpdCDM were similar to or better than observed
in wild type mice. Similar findings of abnormal base-line
FIGURE 9. rSftpdCDM partitions with small aggregate surfactant. BALF
was isolated from rSfptdCDM
Tg⫹
/Sftpd
⫹/⫹
mice, and surfactant lipids were
separated into small (SA) and large (LA) aggregate fractions by centrifugation.
A, SP-D (SP-D) and rSftpdCDM (CDM) were detected in the BALF prior to lipid
isolation (BAL) and in small aggregate lipids by Western blot with anti-SP-D
antibody. B, identical samples were analyzed with anti-SP-A antibody. SP-A
(SP-A) was detected in BALF and in large aggregate surfactant.
FIGURE 10. rSftpdCDM binds influenza A virus. Binding to influenza A virus
was determined by hemagglutination inhibition assays (n ⫽ 3). The number
of hemagglutination units inhibited (HAI) per pmol of rSftpdCDM (CDM)or
wild type SP-D (SP-D) is shown. Error bars, S.E.
FIGURE 11. rSftpdCDM corrects response to Influenza A virus in Sftpd
ⴚ/ⴚ
mice. IL-6, tumor necrosis factor-
␣
(TNF
␣
), and interferon-
␥
(IFN
␥
) levels as
well as influenza A viral titers were determined in lung homogenates 3 days
after intratracheal installation of virus into wild type (WT), rSftpdCDM
Tg
⫹/
Sftpd
⫺/⫺
(CDM), or Sftpd
⫺/⫺
(Sftpd
⫺/⫺
) mice (n ⫽ 6 – 8 mice for each geno
-
type). Viral titers are expressed as relative values with Sftpd
⫺/⫺
mice normal
-
ized to 100%. Inflammatory cytokine levels and viral titers were
significantly higher (p ⬍ 0.05) in Sftpd
⫺/⫺
mice when compared with wild
type or rSftpdCDM
Tg⫹
/Sftpd
⫺/⫺
mice. Error bars, S.E.
SP-D Collagen Domain
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macrophage activation in the setting of a normal response to
influenza virus were also described in studies utilizing an
SP-D conglutinin (SftpdCong) fusion protein consisting of
the N terminus and collagen domains of rat SP-D and the
neck and CRD of conglutinin (27). Several interesting consid-
erations regarding SP-D are raised from these observations.
First, although the rSftpdCDM
Tg⫹
/Sftpd
⫺/⫺
alveolar macro
-
phages have an abnormal histological appearance and base-line
level of activity, they still are able to mount an effective and
relatively normal inflammatory response to the infectious
pathogen, influenza A. Second, although limited by the fact that
only one infectious pathogen was tested, the normal cytokine
response observed following influenza A viral infection sug-
gests that the elevated base-line macrophage activity in
Sftpd
⫺/⫺
mice is not due to the inability to clear a persistent low
level infection. Finally, the divergence of macrophage base-line
activity and the inflammatory response to viral infection in
rSftpdCDM
Tg⫹
/Sftpd
⫺/⫺
mice suggests that SP-D regulates
these processes through two independent pathways.
Previous studies by Gardai et al. (41) described simultaneous
inhibitory and stimulatory roles for SP-D in macrophage regu-
lation that were proposed to be mediated through two compet-
ing signaling cascades. In the first, SP-D inhibited NF-
B and
subsequent immune cell activation through binding of the CRD
to the signal-inhibitory regulatory protein
␣
(SIRP
␣
). In the
second, binding to SIRP
␣
was inhibited by the presence of an
infectious particle within the CRD, thereby allowing interactions
between the collagenous domain or N terminus of SP-D and the
macrophage-activating receptor calreticulin/CD91. This model is
supported by evidence from the collagen deletion mutant
described by Ogasawara and Voelker (35) as well as data derived
from a recombinant fragment of SP-D consisting of only a
trimeric CRD that indicate that both proteins inhibited alve-
olar macrophage activation, presumably through interactions
with SIRP
␣
(41). However, this model also predicts that the
fully functional CRD of rSftpdCDM in the present study would
bind SIRP
␣
and inhibit macrophage activation. Moreover, rSft-
pdCDM-mediated stimulation of macrophage activation
through calreticulin/CD91 would be limited by the absence of
a collagen domain. Therefore, the model proposed by Gardai
et al. (41) might predict that the rSftpdCDM
Tg⫹
/Sftpd
⫺/⫺
mouse would display an alveolar macrophage phenotype that is
predominantly anti-inflammatory. The enlarged foamy macro-
phages, elevated metalloproteinases, and emphysema indicate
that rSftpdCDM
Tg⫹
/Sftpd
⫺/⫺
mice are in a proinflammatory
state at base line and seemingly contradict the model proposed
by Gardai et al. (41). As with any mutational study, the current
findings may be a result of an unanticipated change in the struc-
ture of the CRD or neck or N-terminal domains of rSftpdCDM
as a result of the collagenous domain deletion. This qualifica-
tion notwithstanding, an alternative explanation exists that
may resolve this discrepancy. The present study suggests that
SP-D regulates activation of the pulmonary immune system
through two independent pathways. The first would control
activation of alveolar macrophages in the presence of infectious
particles and might involve the competing activities of SIRP
␣
and calreticulin/CD91. Activation of these receptors would be
appropriately balanced by the CRD and oligomerized N termi-
nus of rSftpdCDM and would explain the wild type response to
influenza virus exhibited by the rSftpdCDM
Tg⫹
/Sftpd
⫺/⫺
mice.
A second pathway would control the base-line level of alveolar
macrophage activation in the lung and in the absence of appro-
priate regulation elicit the phenotype of enlarged foamy mac-
rophages, increased metalloproteinases, and emphysema. The
results of the present study suggest that rSftpdCDM does not
effectively activate this pathway. Similar aberrant macrophage
activation was reported with SftpdCong, rSftpd
Ser15,20
, and
rSftpa/d (22, 23, 27). Therefore, whereas the receptors and sig-
naling molecules that mediate this pathway are unknown, SP-
D-mediated regulation of base-line macrophage activity, alve-
olar remodeling, and surfactant homeostasis requires a
multimeric SP-D containing the collagen domain.
The failure of rSftpdCDM to correct the enlarged foamy mac-
rophages, emphysema, and pulmonary phospholipid accumu-
lations observed in Sftpd
⫺/⫺
mice is in contrast to earlier work
with a recombinant fragment of SP-D consisting of a trimeric
CRD and neck domain, which partially corrected the abnormal
macrophages and surfactant pool sizes in Sftpd
⫺/⫺
mice (15).
The reason why a single trimeric CRD would partially resolve
the abnormalities that an oligomerized trimeric CRD fails to
correct is uncertain, but it may reflect unanticipated changes
that sometimes occur in mutant proteins or differences in the
concentration of the mutant SP-Ds utilized in each study.
Alternatively, this inconsistency may suggest a complex inter-
play between the N-terminal domain and the CRD in macro-
phage regulation and control of phospholipid pool sizes.
Although alveolar macrophages and type II epithelial cells
equally contribute to surfactant phospholipid catabolism, pre-
vious studies demonstrated that SP-D regulates surfactant pool
size by enhancing surfactant uptake by alveolar type II cells (14).
Specifically, SP-D maintains normal surfactant ultrastructure
by selectively interacting with small aggregate surfactant,
thereby facilitating surfactant uptake by type II cells. The lipid
binding specificity of collectins is mediated by the CRD in vitro
(42–45) and intranasal administration of trimeric CRDs
decreased intra-alveolar lipid accumulations in Sftpd
⫺/⫺
mice
(15, 24). In contrast, the SP-D CRD and neck domain in the
rSftpa/d fusion protein failed to correct surfactant lipid ultra-
structure or lower the phospholipid levels when expressed in
Sftpd
⫺/⫺
mice (22). In the present study, the addition of the
SP-D N terminus to the CRD failed to correct surfactant ultra-
structure or increased phospholipid levels. Therefore, normal
surfactant ultrastructure and the uptake of surfactant by type II
cells depend on the collagen domain.
Although rSftpa/d included the neck and CRD of SP-D, anal-
ysis of large and small aggregate surfactant lipids demonstrated
that rSftpa/d partitioned with large aggregate surfactant in a
manner similar to SP-A (22). In contrast, rSftpdCDM, which
included the neck, CRD, and N-terminal domains of SP-D, par-
titioned with small aggregate surfactant like the full-length
SP-D protein. Taken together, these studies suggest that
whereas phospholipid binding in vitro may be mediated by
the CRD, the structural features of SP-D (and SP-A) that
influence partitioning between small versus large aggregate
surfactant are contained within the N-terminal domain.
In summary, our study provides further support for the
SP-D Collagen Domain
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importance of the distinct molecular domains of SP-D in medi-
ating the complex functions of this protein. The finding that the
collagen deletion mutant fails to correct the abnormal lung
morphology or surfactant lipid homeostasis of Sftpd
⫺/⫺
mice
supports a role of the collagen domain that is beyond proper
spacing of trimeric subunits for bacterial aggregation. Assimi-
lating the results of this study with those of prior reports on
SP-D reveals that whereas our understanding of SP-D is
improving, inconsistencies still exist. The CRD is critical for
binding to lipopolysaccharide, viruses, bacteria, fungus, and lip-
ids. Nonetheless, SP-D interactions with small aggregate sur-
factant and uptake of surfactant by alveolar type II cells in vivo
do not depend on the CRD alone. The collagen domain is
required for surfactant lipid structure and metabolism, but it is
not needed to effectively aggregate bacteria or to suppress
inflammatory responses to influenza A virus. The N-terminal
domain is critical for partitioning with large versus small aggre-
gate surfactant lipids in adult mice, and interactions between
interchain N-terminal domains are essential for oligomeriza-
tion, which, in turn, influences CRD binding affinity, phospho-
lipid catabolism, and the inflammatory response to infectious
pathogens. Finally, at the time of this writing, all SP-D mutant
proteins that delete or substitute even a single domain of SP-D
fail to fully correct the enlarged, foamy macrophages that are
characteristic of Sftpd
⫺/⫺
mice, suggesting that this function of
SP-D is dependent on a full-length, multimeric protein.
Acknowledgments—We are grateful to Mitchell R. White for invalu-
able advice and technical assistance in influenza A viral assays and to
Sarah K. Yoshimura for critical reading of the manuscript.
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SP-D Collagen Domain
AUGUST 25, 2006 • VOLUME 281 • NUMBER 34 JOURNAL OF BIOLOGICAL CHEMISTRY 24505
at University of Cincinnati/Medical Center Libraries on September 6, 2015http://www.jbc.org/Downloaded from
VOLUME 280 (2005) PAGES 12799 –12809
Interaction of the mammalian endosomal sorting
complex required for transport (ESCRT) III protein
hSnf7-1 with itself, membranes, and the AAA
ⴙ
ATPase
SKD1.
Yuan Lin, Lisa A. Kimpler, Teresa V. Naismith, Joshua M. Lauer,
and Phyllis I. Hanson
The plasmid used to express FLAG-hSnf7N (residues 1–116) in this
paper had an unintended missense mutation changing serine at residue
2 to cysteine. We found that this cysteine was palmitoylated. Changing
it back to serine decreased the amount of hSnf7N associated with mem-
branes from all for the mutant fragment containing cysteine to approx-
imately half for the wild-type fragment containing serine. The images of
FLAG-hSnf7N in Figs. 7B and 8 represent average cells expressing
mutant (Cys-2) hSnf7N, whereas for wild-type (Ser-2) hSnf7N, these
images correspond to cells expressing high levels of protein. All other
plasmids are as indicated, and the conclusions of the paper remain
unchanged.
VOLUME 280 (2005) PAGES 28382–28387
Role of the N-terminal domain of the human DMC1
protein in octamer formation and DNA binding.
Takashi Kinebuchi, Wataru Kagawa, Hitoshi Kurumizaka,
and Shigeyuki Yokoyama
PAGE 28385:
Due to an inadvertent error, the wrong image was presented in Fig.
4C. Fig. 4C should appear as shown below. The figure legend and text
remain unchanged.
VOLUME 281 (2006) PAGES 8888 – 8897
Dynamic changes in histone H3 lysine 9 methylations.
IDENTIFICATION OF A MITOSIS-SPECIFIC FUNCTION FOR
DYNAMIC METHYLATION IN CHROMOSOME
CONGRESSION AND SEGREGATION.
Kirk J. McManus, Vincent L. Biron, Ryan Heit, D. Alan Underhill,
and Michael J. Hendzel
The concentration of a drug that was employed, adenosine dialde-
hyde, was erroneously reported as 25 m
M. The concentration that was
employed was actually 250
M.
VOLUME 281 (2006) PAGES 23436 –23444
Conditional deletion of hypothalamic Y2 receptors
reverts gonadectomy-induced bone loss in adult mice.
Susan J. Allison, Paul Baldock, Amanda Sainsbury, Ronaldo Enriquez,
Nicola J. Lee, En-Ju Deborah Lin, Matthias Klugmann, Matthew During,
John A. Eisman, Mei Li, Lydia C. Pan, Herbert Herzog, and Edith M. Gardiner
PAGE 23436:
Dr. Klugmann’s name was misspelled in the author line. The correct
spelling is shown above.
VOLUME 281 (2006) PAGES 31553–31561
The first structure from the SOUL/HBP family of heme-
binding proteins, murine P22HBP.
Jorge S. Dias, Anjos L. Macedo, Gloria C. Ferreira, Francis C. Peterson,
Brian F. Volkman, and Brian J. Goodfellow
PAGE 31554:
Column 2, first line: The concentrations should read “(4.0
M)or
hemin (3.5
M)...”
FIGURE 4C
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 50, pp. 38966 –38968, December 15, 2006
© 2006 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
38966 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281• NUMBER 50 • DECEMBER 15, 2006
ADDITIONS AND CORRECTIONS
This paper is available online at www.jbc.org
We suggest that subscribers photocopy these corrections and insert the photocopies in the original publication at the location of the original
article. Authors are urged to introduce these corrections into any reprints they distribute. Secondary (abstract) services are urged to carry
notice of these corrections as prominently as they carried the original abstracts.
VOLUME 281 (2006) PAGES 11627–11636
PTP-PEST couples membrane protrusion and tail retraction via VAV2 and p190RhoGAP.
Sarita K. Sastry, Zenon Rajfur, Betty P. Liu, Jean-Francois Cote, Michel L. Tremblay, and Keith Burridge
PAGE 11632:
In Fig. 5, panel D was inadvertently omitted and is shown below. The figure legend is correct as it appears.
FIGURE 5
Additions and Corrections
DECEMBER 15, 2006 • VOLUME 281 • NUMBER 50 JOURNAL OF BIOLOGICAL CHEMISTRY 38967
VOLUME 281 (2006) PAGES 24496 –24505
Correction of pulmonary abnormalities in Sftpd
ⴚ/ⴚ
mice requires the collagenous domain of surfactant protein D.
Paul S. Kingma, Liqian Zhang, Machiko Ikegami, Kevan Hartshorn, Francis X. McCormack, and Jeffrey A. Whitsett
PAGE 24501:
Fig. 5: An incorrect image was used for the panel A inset. The correct image is shown below.
FIGURE 5
Additions and Corrections
38968 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281• NUMBER 50 • DECEMBER 15, 2006
McCormack and Jeffrey A. Whitsett
Ikegami, Kevan Hartshorn, Francis X.
Paul S. Kingma, Liqian Zhang, Machiko
Domain of Surfactant Protein D
Mice Requires the Collagenous
-/-
Sftpd
Correction of Pulmonary Abnormalities in
Lipids and Lipoproteins:
doi: 10.1074/jbc.M600651200 originally published online June 20, 2006
2006, 281:24496-24505.J. Biol. Chem.
10.1074/jbc.M600651200Access the most updated version of this article at doi:
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