The envelope protein (gp120) of human immunodeficiency virus (HIV) contains highly conserved mannosylated oligosaccharides. These glycoconjugates contribute to resistance to antibody neutralization, and binding to cell surface lectins on macrophages and dendritic cells. Mannose-binding lectin (MBL) binds to gp120 and plays a role in defence against the virus. In this study it is demonstrated that surfactant protein D (SP-D) binds to gp120 and inhibits HIV infectivity at significantly lower concentrations than MBL. The binding of SP-D was mediated by its calcium-dependent carbohydrate-binding activity and was dependent on glycosylation of gp120. Native dodecameric SP-D bound to HIV gp120 more strongly than native trimeric SP-D. Since one common polymorphic form of SP-D is predominantly expressed as trimers and associated with lower blood levels, these individuals may have less effective innate defence against HIV. A chimeric protein containing the N-terminal and collagen domains of SP-D linked to the neck and carbohydrate-recognition domains of MBL (called SP-D/MBLneck+CRD) had greater ability to bind to gp120 and inhibit virus replication than either SP-D or MBL. The enhanced binding of SP-D/MBLneck+CRD was dependent on assembly into higher molecular mass multimers (i.e. a trimeric form of the chimera did not bind to a greater extent than MBL). Hence, the enhanced binding of SP-D compared with MBL results from distinctive properties of its N-terminal and/or collagen domains. SP-D is present in lung and airway fluids, as well as in blood and various mucosal locations, and could, like MBL, play a role in restricting HIV transmission or replication in vivo.

Surfactant protein D (SP-D) plays important roles in the host defense against infectious microorganisms and in regulating the innate immune response to a variety of pathogen-associated molecular pattern. SP-D is mainly expressed by type II cells of the lung, but SP-D is generally found on epithelial surfaces and in serum. Genotyping for three single-nucleotide variations altering amino acids in the mature protein in codon 11 (Met(11)Thr), 160 (Ala(160)Thr), and 270 (Ser(270)Thr) of the SP-D gene was performed and related to the SP-D levels in serum. Individuals with the Thr/Thr(11)-encoding genotype had significantly lower SP-D serum levels than individuals with the Met/Met(11) genotype. Gel filtration chromatography revealed two distinct m.w. peaks with SP-D immunoreactivity in serum from Met/Met(11)-encoding genotypes. In contrast, Thr/Thr(11) genotypes lacked the highest m.w. form. A similar SP-D size distribution was found for recombinant Met(11) and Thr(11) expressed in human embryonic kidney cells. Atomic force microscopy of purified SP-D showed that components eluting in the position of the high m.w. peak consist of multimers, dodecamers, and monomers of subunits, whereas the second peak exclusively contains monomers. SP-D from both peaks bound to mannan-coated ELISA plates. SP-D from the high m.w. peak bound preferentially to intact influenza A virus and Gram-positive and Gram-negative bacteria, whereas the monomeric species preferentially bound to isolated LPS. Our data strongly suggest that polymorphic variation in the N-terminal domain of the SP-D molecule influences oligomerization, function, and the concentration of the molecule in serum.

Collectins play important roles in innate defence against viral, fungal and bacterial pathogens. CL-43, a bovine serum collectin, which appears to have evolutionarily evolved from surfactant protein D (SP-D), shows unique structural and functional properties. In the present study, we describe the initial characterization of a recombinant CL-43 expressed in mammalian cells. Like natural CL-43, the recombinant is secreted as trimeric forms that show a preference for mannose and N-acetyl mannosamine. The natural and recombinant proteins have significantly higher haemagglutination-inhibiting activity against influenza A virus (IAV) than recombinant trimeric forms of SP-D. In contrast with the more highly multimerized forms of SP-D, namely conglutinin or mannose-binding lectin, CL-43 did not induce viral or bacterial aggregation and did not enhance IAV-induced neutrophil H(2)O(2) generation. Like SP-D, CL-43 also strongly enhanced neutrophil uptake of IAV. However, the mechanism of this enhanced internalization is different from that of SP-D in that it did not require viral aggregation. These studies establish that the trimeric structure of CL-43 is specified by its primary sequence and indicate that this naturally occurring trimeric collectin has unique antiviral activities. These findings could facilitate the development of recombinant collectins with novel antimicrobial properties.

Surfactant protein D (SP-D) belongs to a subgroup of mammalian collagenous Ca(2+)-dependent lectins known as the collectins. It is thought to play a significant role in the innate immune response against microorganisms within the lungs and at other mucosal surfaces. This report documents the isolation and characterization of SP-D purified from porcine lung lavage using mannan affinity chromatography and gel filtration. Ultrastructural analysis shows both dodecameric and higher order oligomeric complexes of SP-D. The molecular mass of monomeric porcine SP-D (50 kD) is larger than that of SP-D from humans (43 kD). The difference in mass is due to the presence of an Asparagine-linked glycosylation in the carbohydrate recognition domain of porcine SP-D, which is absent in SP-D of other species investigated so far. Analysis of this carbohydrate moiety indicates that it is a highly heterogeneous, complex type oligosaccharide which is sialylated. The heterogeneity of oligosaccharide sialylation results in the existence of many differently charged porcine SP-D isoforms. The removal of the carbohydrate moiety reduces the inhibitory effect of porcine SP-D on influenza A virus haemagglutination. Therefore, the carbohydrate moiety may influence interactions with pathogens.