Site-directed mutagenesis of Cys-15 and Cys-20 of pulmonary surfactant protein D - Expression of a trimeric protein with altered anti-viral properties

Surfactant protein D (SP-D) molecules are preferentially assembled as dodecamers consisting of trimeric subunits associated at their amino termini, The NH2-terminal sequence of each monomer contains two conserved cysteine residues, which participate In interchain disulfide bonds, In order tea study the roles of these residues in SP-D assembly and function, we employed site-directed mutagenesis to substitute serine for cysteine 15 and 20 in recombinant rat SP-D (RsSP-D), lad have expressed the mutant (RrSP-Dser15/20) in Chinese hamster ovary (CRO-RP) cells, The mutant, which was efficiently secreted, bound to maltosyl-agarose, but unlike RrSP-D, was assembled exclusively as trimers. The constituent monomers showed a decreased mobility on SDS-polyacrylamide gel electrophoresis resulting from an increase in the size and sialylation of the N-linked oligosaccharide at Asn-70. Although RrSP-Dser15/20 contained a pepsin-resistant triple helical domain, it showed a decreased T-m, and acquired susceptibility to proteolytic degradation, Like RrSP-D, RrSP-Dser15/20 hound to the hemagglutinin of influenza A. However, it showed no viral aggregation and did not enhance the binding of influenza A to neutrophils (PMN), augment PMN respiratory burst, or protect PMNs from deactivation, These studies indicate that amino-terminal disulfides are required to stabilize dodecamers, and support our hypothesis that the oligomerization of trimeric subunits contributes to the anti-microbial properties of SP-D.