Pulmonary surfactant protein-D (SP-D) is a member of the collectin family of C-type lectins that is synthesized in many tissues including respiratory epithelial cells in the lung. SP-D is assembled predominantly as dodecamers consisting of four homotrimeric subunits each. Association of these subunits is stabilized by interchain disulfide bonds involving two conserved amino-terminal cysteine residues (Cys-15 and Cys-20). Mutant recombinant
rat SP-D lacking these residues (RrSPDser15/20) is secreted in cell culture as trimeric subunits rather than as dodecamers. In this study, transgenic mice that express this mutant were generated to elucidate the functional importance of SP-D oligomerization in vivo. Expression of RrSP-Dser15/20 failed to correct the pulmonary phospholipid accumulation and emphysema characteristic of SP-D null (mSPD2/2) mice. Expression of high concentrations of the mutant protein in wild-type mice reduced the abundance of disulfide cross-linked oligomers of endogenous SP-D in the bronchoalveolar lavage fluid and demonstrated a phenotype that partially overlapped with that of the SP-D2/2 mice; the animals developed emphysema and foamy macrophages without the associated abnormalities in alveolar phospholipids typical of SP-D2/2 mice. Development of foamy macrophages in SP-D-deficient mice is not secondary to the increased abundance of surfactant phospholipids. Disulfide cross-linked SP-D oligomers are required for the regulation of surfactant phospholipid homeostasis and the prevention of emphysema and foamy macrophages in vivo.
Surfactant protein-D (SP-D)1 is a member of a family of collagenous host defense lectins termed collectins (1, 2). SP-D is secreted into the distal airways and alveoli of the lung (3–5) but is expressed also in other tissues (6–8). Each 43-kDa SP-D monomer consists of an NH2-terminal domain containing two conserved cysteine residues (Cys-15 and Cys-20), a collagenous domain, a short neck sequence, and a COOH-terminal carbohydrate recognition domain (CRD) (9–12). Electron microscopy and proteinase digestion studies demonstrated that SP-D monomers are assembled into tetramers of trimeric subunits (dodecamers) (13, 14). The two NH2-terminal cysteine residues of SP-D are critical in the formation of interchain disulfide bonds that stabilize the dodecameric structure (13). Substitution of serine for cysteine at positions 15 and 20 of the mature protein results in the efficient secretion of trimeric subunits corresponding to a single arm of the SP-D dodecamer (15). Mutant recombinant rat protein migrates as a monomer on SDS-polyacrylamide gel electrophoresis in the absence of sulfhydryl reduction and elutes as a trimer rather than as a dodecamer from gel filtration columns under nondenaturing conditions (16). The collagen domain forms a triple helix as assessed by protease digestion (16). The trimeric protein is also a functional lectin with the same saccharide selectivity as the wild-type protein. RrSP-Dser15/20 binds to the hemagglutinin of the influenza virus in a CRD-dependent manner (15, 16). However, the mutant SP-D does not aggregate particulate ligands such as viral particles and competitively inhibits aggregation mediated by wild-type SP-D (15, 16).
High affinity binding of SP-D to various ligands in vitro requires a trimeric CRD (17–19). However, previous studies using trimeric CRDs, RrSP-Dser15/20, and variably multimerized fractions of recombinant human SP-D suggest that trimers are functionally univalent and have a restricted range of biological activities. For example, trimeric CRDs elicit the chemotaxis of neutrophils and mononuclear phagocytes in vitro (20) and neutralize the respiratory syncytial virus in vivo (21). In addition, RrSP-Dser15/20, similar to wild-type SP-D, inhibits stimulated proliferation of T-lymphocytes (22). However, other activities of SP-D involve ligand aggregation and require higher orders of covalently stabilized oligomerization. For example, trimeric CRDs inhibit the hemagglutinin of the influenza virus, but unlike SP-D dodecamers, they cannot mediate viral aggregation, enhance viral binding to neutrophils, or enhance the oxidative response to a bound virus (16, 19, 23).
Zhang, L, et al.
The Journal of Biological Chemistry 2001