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

Patricia Brown-Augsburger, Kevan Hartshorn, Donald Chang, Kevin Rust, Catherine Fliszar, Howard G. Welgus, Edmond C. Crouch

Research output: Contribution to journalArticle

116 Scopus citations

Abstract

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 to 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 (RrSP-D), and have expressed the mutant (RrSP-Dser 15/20) in Chinese hamster ovary (CHO-K1) 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 Tm, and acquired susceptibility to proteolytic degradation. Like RrSP-D, RrSP-Dser 15/20 bound 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 antimicrobial properties of SP-D.

Original languageEnglish
Pages (from-to)13724-13730
Number of pages7
JournalJournal of Biological Chemistry
Volume271
Issue number23
DOIs
StatePublished - 1996

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