Abstract
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.
Highlights
Lung surfactant is a mixture of phospholipids, neutral lipids, and surfactant protein A (SP-A)1 SP-B, SP-C, and SP-D, which are secreted into the air spaces by alveolar type II cells and Clara cells of the distal pulmonary epithelium [1]
Recombinant rat SP-A (rSP-A) was slightly smaller under reducing conditions and less extensively cross-linked by interchain disulfide bonds under nonreducing conditions
The atypical morphological appearance of Tubular Myelin (TM) in the SP-AϪ/Ϫ,D/A mouse supports a primary role for the N-terminal domain of SP-A in the organization of tubular myelin
Summary
Lung surfactant is a mixture of phospholipids, neutral lipids, and surfactant protein A (SP-A) SP-B, SP-C, and SP-D, which are secreted into the air spaces by alveolar type II cells and Clara cells of the distal pulmonary epithelium [1]. Disruption of interchain disulfide bond formation at the N terminus of SP-D by C15S and C20S substitutions limits oligomeric assembly to trimerization and blocks SP-Dmediated functions in vitro [23], and in vivo [24]. These data suggest that the N-terminal segments of SP-A and SP-D are critical for interactions with surfactant phospholipids and microbial ligands. Lungspecific overexpression of the C15S,C20S SP-D in SP-Dϩ/ϩ mice disrupted oligomeric assembly of the endogenous SP-D and produced air space 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
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