Biosynthesis of surfactant protein D - Contributions of conserved NH2-terminal cysteine residues and collagen helix formation to assembly and secretion

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-KI 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 Asn(70), Although mutants with serine substituted for Cys(15) and Cys(20) (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 crosslinked 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.