TY - JOUR
T1 - Site-directed mutagenesis of cys-15 and cys-20 of pulmonary surfactant protein D
T2 - Expression of a trimeric protein with altered anti-viral properties
AU - Brown-Augsburger, Patricia
AU - Hartshorn, Kevan
AU - Chang, Donald
AU - Rust, Kevin
AU - Fliszar, Catherine
AU - Welgus, Howard G.
AU - Crouch, Edmond C.
PY - 1996
Y1 - 1996
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=0029891544&partnerID=8YFLogxK
U2 - 10.1074/jbc.271.23.13724
DO - 10.1074/jbc.271.23.13724
M3 - Article
C2 - 8662732
AN - SCOPUS:0029891544
VL - 271
SP - 13724
EP - 13730
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 23
ER -