SITE-DIRECTED MUTAGENESIS OF CYS-15 AND CYS-20 OF PULMONARY SURFACTANT PROTEIN-D - EXPRESSION OF A TRIMERIC PROTEIN WITH ALTERED ANTIVIRAL PROPERTIES

Surfactant protein D (SP-D) molecules are preferentially assembled as dodecamers consisting of trimeric subunits associated at their amino t ermini, The NH2-terminal sequence of each monomer contains two conserv ed cysteine residues, which participate In interchain disulfide bonds, In order tea study the roles of these residues in SP-D assembly and f unction, we employed site-directed mutagenesis to substitute serine fo r cysteine 15 and 20 in recombinant rat SP-D (RsSP-D), lad have expres sed the mutant (RrSP-Dser15/20) in Chinese hamster ovary (CRO-RP) cell s, The mutant, which was efficiently secreted, bound to maltosyl-agaro se, but unlike RrSP-D, was assembled exclusively as trimers. The const ituent 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 co ntained a pepsin-resistant triple helical domain, it showed a decrease d T-m, and acquired susceptibility to proteolytic degradation, Like Rr SP-D, RrSP-Dser15/20 hound to the hemagglutinin of influenza A. Howeve r, it showed no viral aggregation and did not enhance the binding of i nfluenza A to neutrophils (PMN), augment PMN respiratory burst, or pro tect PMNs from deactivation, These studies indicate that amino-termina l disulfides are required to stabilize dodecamers, and support our hyp othesis that the oligomerization of trimeric subunits contributes to t he anti-microbial properties of SP-D.