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 crosslinking domain, a collagenous domain, a neck region, and a carbohydrate recognition domain. In this study, the function of the collagenous domain was evaluated by expressing a SP-D collagen deletion mutant protein (rSftpdCDM) in wild type and SP-D null mice (Sftpd/). rSftpdCDM formed disulfide-linked trimers that further oligomerized into higher order structures. The mutant protein effectively bound carbohydrate and aggregated bacteria in vitro. Whereas rSftpdCDM did not disrupt pulmonary 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/ 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/ mice. In contrast, rSftpdCDM completely corrected viral clearance and the abnormal inflammatory response that occurs following pulmonary influenza A challenge in Sftpd/ mice. Our findings indicate that the collagen domain of SP-D is not required for assembly of disulfide-stabilized oligomers or the innate immune response to viral pathogens. 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 surfactant protein A (SP-A), SP-D, mannose-binding protein, conglutinin, and CL-43. SP-A and SP-D contribute to the innate immune system of the lung by binding and enhancing the clearance 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 disulfide bonds between the cysteine-rich amino-terminal domains (10, 11). Trimers further combine into larger multimeric complexes through disulfide-stabilized, noncovalent interactions. Although larger structures are commonly observed, SP-D exists predominantly as a tetramer of trimeric 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 homeostasis. Mice with a targeted deletion of the Sftpd gene (Sftpd/) survived normally but developed gradually worsening pulmonary inflammation, emphysema, and surfactant phospholipid accumulations (13, 14). Sftpd/ mice accumulate apoptotic alveolar macrophages as well as enlarged, lipid-laden, macrophages that release metalloproteinases and reactive oxygen species (15–19). Uptake and clearance of viral pathogens, including influenza A and respiratory syncitial virus, were deficient 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 production of the proinflammatory mediators, tumor necrosis factor-, 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-terminal domains of SP-D are essential for these functions (22).
Kingma, PS, et al.
The Journal of Biological Chemistry 2006