The role of pulmonary collectin N-terminal domains in surfactant structure, function and homeostasis, in vivo .

The N-terminal domains of the lung collectins, surfactant proteins A (SP-A) and D (SP-D), are critical for surfactant phospholipid interactions and surfactant homeostasis, respectively. To further assess the importance of lung collectin N-terminal domains in surfactant structure and function, a chimeric SP-D/SP-A (D/A) gene was constructed by substituting nucleotides encoding amino acids Asn(1)-Ala(7) of rat SP-A with the corresponding N-terminal sequences from rat SP-D, Ala(1)-Asn(25). Recombinant D/A migrated as a 35-kDa band on reducing SDS-PAGE and as a ladder of disulfide-linked multimers under nonreducing conditions. The recombinant D/A bound and aggregated phosphatidylcholine containing vesicles as effectively as rat SP-A. Mice in which endogenous pulmonary collectins were replaced with D/A were developed by human SP-C promoter-driven overexpression of the D/A gene in SP-A(-/-) and SP-D(-/-) animals. Analysis of lavage fluid from SP-A(-/-,D/A) mice revealed that glycosylated, oligomeric D/A was secreted into the air spaces at levels that were comparable with the authentic collectins and that the N-terminal interchange converted SP-A from a "bouquet" to a cruciform configuration. Transmission electron microscopy of surfactant from the SP-A(-/-,D/A) mice revealed atypical tubular myelin containing central "target-like" electron density. Surfactant isolated from SP-A(-/-,D/A) mice exhibited elevated surface tension both in the presence and absence of plasma inhibitors, but whole lung compliance of the SP-A(-/-,D/A) animals was not different from the SP-A(-/-) littermates. Lung-specific overexpression of D/A in the SPD(-/-) mouse resulted in hetero-oligomer formation with mouse SP-A and did not correct the air space dilation or phospholipidosis that occurs in the absence of SP-D. These studies indicate that the N terminus of SP-D 1) can functionally replace the N terminus of SP-A for lipid aggregation and tubular myelin formation, but not for surface tension lowering properties of SP-A, and 2) is not sufficient to reverse the structural and metabolic pulmonary defects in the SP-D(-/-) mouse.


Introduction
Lung surfactant is a mixture of phospholipids, neutral lipids and surfactant proteins (SP-) 1 A, B, C, and D, which are secreted into the airspaces by alveolar type II cells and Clara cells of the distal pulmonary epithelium (1). Although the primary function of surfactant is to reduce surface tension, the contribution of each molecular component to surface activity is not completely understood. Surfactant phospholipids form a film at the air- However, SP-A -/and SP-D -/mice also exhibit abnormalities of surfactant structure, metabolism and function (8)(9)(10). Surfactant isolated from SP-A -/mice does not contain 6 metalloproteinases and oxidant species (12). All of these defects are corrected by lung specific expression of the cognate collectin in the SP-A -/and SP-D -/mice (13,14).
The structural basis of SP-A and SP-D surfactant functions have been explored by mutagenesis using in vitro and in vivo analyses. The primary structure of both proteins includes an N-terminal segment containing interchain linkages formed by Cys residues, a collagen-like region of gly-x-y repeats, a hydrophobic 'neck' domain and a carbohydrate recognition domain (CRD) (15,16). Trimeric association of subunits occurs by the folding of the collagen-like domains into triple helices (17) and coiled-coil bundling of alpha helices in the neck (18). In the fully assembled molecules, the N-terminal sequences and disulfide-bonds of the pulmonary collectins stabilize the parallel arrangement of six trimers that characterizes the "bouquet" structure of SP-A and the radial alignment of four trimers that imparts the cruciform organization to SP-D (19,20).
SP-A and SP-D bind to carbohydrate and lipid ligands by their CRDs, but high affinity interactions require oligomeric assembly mediated by N-terminal crosslinking of trimeric arms. This configuration facilitates simultaneous engagement of individual collectin molecules with multiple sites on membranes and microbial surfaces. Deletion of the collagen-like domain from SP-A, which limits oligomeric assembly to simple trimers and hexamers, reduces binding to liposomes but does not block liposome aggregation (21).
Deletion of the N-terminal segment of SP-A (22), or selective disruption of interchain disulfide bond formation by Cys6Ser substitution, limits assembly to simple trimers and blocks SP-A mediated liposome aggregation and binding (21). Disruption of interchain disulfide bond formation at the N-terminus of SP-D by Cys15Ser and Cys20Ser 7 substitutions limits oligomeric assembly to trimerization, and blocks SP-D mediated functions in vitro (23), and in vivo (24). Collectively, these data suggest that the Nterminal segments of SP-A and SP-D are critical for interactions with surfactant phospholipids and microbial ligands.
Recently Zhang et al. reported that genetic replacement of endogenous mouse SP-D (mSP-D) with a mutant SP-D containing disrupted interchain disulfide linkages (Cys15Ser, Cys20Ser) failed to correct the alveolar phospholipidosis and airspace dilation that occur in the SP-D -/mouse (24). In addition, lung specific overexpression of the Cys15Ser, Cys20Ser SP-D in SP-D +/+ mice disrupted oligomeric assembly of the endogenous SP-D and produced airspace dilation and foamy macrophage formation, without phospholipidosis (24). These data suggested that the in vivo activity of SP-D is dependent on its oligomeric structure. The purpose of this study was to examine the role of the N-terminal segment-dependent oligomeric structure of SP-A and SP-D in their functions, in vivo. were purified from the culture media by adsorption to mannose-Sepharose 6B columns in the presence of 1 mM Ca 2+ and elution with 2 mM EDTA. The purified recombinant D/A was dialyzed to remove EDTA, and then stored at -20°C. Rat SP-A and SP-D were purified from the bronchoalveolar wash of silica-treated animals using previously published methods (22,27). Small amounts of D/A required for immunoblot analysis were purified from SP-D -/-,D/A mice by sedimentation of surfactant at 15,000 x g, butanol extraction of the washed surfactant pellet, dialysis of the insoluble proteins, and mannose-Sepharose affinity chromatography. To obtain sufficient D/A for sizing by gel filtration chromatography, SP-A -/-,D/A mice were lavaged two weeks after intranasal instillation of silica (28). D/A was separated from other lavage proteins and surfactant phospholipids by cosedimentation with the surfactant pellet, repeated washing with 1 mM CaCl 2 in 150 mM NaCl, and elution from the pellet with isotonic saline containing 2 mM EDTA. The surfactant lipids were pelleted by high speed centrifugation and the supernatant containing D/A was stored at -20°C.

In vivo replacement of mSP-A and mSP-D by the chimeric D/A collectin-Swiss
Black/C129J SP-A -/and SP-D -/mice were developed from embryonic stem cells by targeted disruption of the endogenous mouse collectin genes, and maintained by breeding with Swiss Black mice, as previously described (8). Portions of the hSP-C/D/A construct by guest on March 23, 2020 http://www.jbc.org/ Downloaded from that were unnecessary for expression of the transgene were removed by digestion with Nde I and Not I. Lung specific overexpression of D/A in SP-A -/mice was accomplished by injection of the male pronucleus of fertilized SP-A -/mouse eggs with the hSP-C-D/A transgene, followed by uterine implantation in SP-A -/females (29). SP-A -/-,D/A progeny identified by PCR were expanded by breeding with SP-A -/mice. The D/A transgene was bred into the SP-D -/background by crossing the SP-A -/-,D/A mice with SP-D -/mice, using the genotyping strategies outlined below. Progeny which screened positively for the D/A transgene in the first round were bred in brother/sister matings. Progeny of the second generation which screened positively for the D/A transgene were screened for the gene-   Surfactant isolation and Sat PC measurement-Groups of mice from each genotype were anesthetized by intraperitoneal injection with pentobarbital sodium and exsanguinated by transection of the abdominal aorta. The chest was opened, and the proximal trachea was cannulated with a 20-gauge blunt needle. Alveolar lavage was performed by three cycles of instillation of saline to full lung expansion followed by gentle aspiration, repeated five times (total approximately 5 mls) for each animal (11). Large-aggregate surfactant was isolated from the pooled lavage fluid by centrifugation at 40,000 x g over a 0.8 M sucrose 13 cushion for 15 min (11). The large-aggregate surfactant then was collected from the interface, diluted with 0.15 M NaCl, and centrifuged again at 40,000 x g for 15 min. The pellet was suspended in normal saline and stored at -20°C. After lavage as above, lung tissue was homogenized in saline. Sat PC was measured by extracting the alveolar lavage sample or lung homogenates with chloroform/methanol (2:1), treatment with OsO 4 , separation by alumina column chromatography and phosphorus analysis as described (8). The trachea was cannulated with an 18 gauge metal needle, and mice were ventilated with a quasisinusoidal waveform at a frequency of 160 breaths/min and a tidal volume of measured with an oscillation technique that has been previously described (33). Regular ventilation was stopped and the mouse was allowed to passively expire to relaxation volume while PEEP was maintained. Low-amplitude flow oscillation was delivered to the lung over a 16 sec period of apnea. Tracheal pressure and tracheal volume measured during this maneuver were used to calculate respiratory compliance (33).

Specimen preparation for electron microscopy-Large
Histology-Mouse lungs (12 weeks old) were fixed at 25 cm of water pressure with 4% paraformaldehyde in PBS, and processed into paraffin blocks. Seven micrometer sections from each lobe were stained with hematoxylin and eosin and examined under light the microscope.

Statistics-Comparisons between the SP-A -/and SP-A -/-,D/A mouse lines, and the SP-D -/-
and SP-D -/-,D/A mouse lines were made using a two tailed t-test. The variables included SP-A levels, Sat PC levels, tubular myelin dimensions, lung compliance and surface tension. Data were expressed as mean ± S.E. unless otherwise noted, and p values of less than 0.05 were considered significant. (1.3± 0.3 x 10 -3 a.u./sec, p<0.01), but less than that of rSP-A (6.3 ± 0.5 x 10 -3 a.u./sec, p < 0.01) (Fig. 3 B, inset). As expected, the rSP-D did not aggregate the Sat PC containing vesicles. Collectively, these data indicate that rec. D/A was secreted from eucaryotic cells, assembled into disulfide-linked oligomers, and bound to carbohydrate and phospholipid ligands.

Development of SP-A -/-,D/A and SP-D -/-,D/A mouse lines-Mouse SP-A or SP-D were
replaced with D/A by human SP-C promoter directed expression of the D/A transgene in the distal lung epithelium of collectin deficient mice (Fig. 4). The SP-A -/and SP-D -/gene targeted mice were developed as previously described (8,9).  The recent availability of genetically engineered animal models has facilitated the study of pulmonary collectin structure and function, in vivo (14,24). Because the SP-A Nterminal segment is important for self-association and phospholipid interactions in vitro, we postulated that it plays a primary role in TM structure, in vivo (22). SP-A is known to be required for TM formation in vitro (39) and in vivo (8,14),        shown. Previously published data from SP-A -/-,rSP-A mice are included for comparison (14). Values are mean ± S.E., n = 3-4. * p < 0.05 for SP-A -/vs. SP-A -/-,D/A mice.