Abstract
Surfactant protein D (SP-D) is preferentially secreted as dodecamers consisting of four collagenous trimers cross-linked by disulfide bonds. In these studies, we examined the biosynthesis of wild-type rat SP-D (RrSP-D) and selected mutants by stably transfected CHO-K1 cells to determine the roles of a conserved N-linked oligosaccharide, the collagen helix, and interchain disulfide bonds in SP-D assembly and secretion. The major intracellular form of RrSP-D accumulated in the RER as complexes containing up to four trimeric subunits. Disulfide cross-link formation and RrSP-D secretion were selectively inhibited by 2,2'-dipyridyl, an inhibitor of prolyl and lysyl hydroxylase, and by 2 mM dithiothreitol, but unaffected by tunicamycin or elimination of the consensus sequence for glycosylation at Asn70. Although mutants with serine substituted for Cys15 and Cys20 (RrSP-Dser15/20) are secreted as trimeric subunits, proteins with single cysteine substitutions were retained in the cell. Surprisingly, the secretion of RrSP-Dser15/20 was unaffected by 2,2'-dipyridyl. These studies strongly suggest that the most important and rate-limiting step for the secretion of SP-D involves the association of cross-linked trimeric subunits to form dodecamers stabilized by specific inter-subunit disulfide cross-links. Interference with collagen helix formation prevents secretion by interfering with efficient disulfide cross-linking of the NH2-terminal domain.
Highlights
Surfactant protein D (SP-D) is preferentially secreted as dodecamers consisting of four collagenous trimers cross-linked by disulfide bonds
We examined the biosynthesis of wild-type rat SP-D (RrSP-D) and selected mutants by stably transfected CHO-K1 cells to determine the roles of a conserved N-linked oligosaccharide, the collagen helix, and interchain disulfide bonds in SP-D assembly and secretion
We have used stably transfected CHO-K1 cells expressing wild-type and selected mutant recombinant wild-type rat SP-D (RrSP-D) as a model system for examining the biosynthesis of SP-D
Summary
Surfactant protein D (SP-D) is preferentially secreted as dodecamers consisting of four collagenous trimers cross-linked by disulfide bonds. The secretion of RrSP-Dser15/20 was unaffected by 2,2-dipyridyl These studies strongly suggest that the most important and rate-limiting step for the secretion of SP-D involves the association of crosslinked trimeric subunits to form dodecamers stabilized by specific inter-subunit disulfide cross-links. Previous biochemical and molecular studies have identified at least three structural features that could play critical roles in the assembly and secretion of SP-D These include the conserved asparagine-linked oligosaccharide at Asn, the triple helical conformation of the collagenous domain, and the conserved NH2-terminal cysteine residues
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