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-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.
Brown-Augsburger
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P
Department of Pathology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Chang
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D
Rust
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Crouch
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EC
D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D003094:Collagen / Q000737:chemistry; D017124:Conserved Sequence; D006224:Cricetinae; D003432:Cross-Linking Reagents; D003545:Cysteine / Q000737:chemistry; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004220:Disulfides / Q000737:chemistry; D006023:Glycoproteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D066298:In Vitro Techniques; D008168:Lung / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D037663:Pulmonary Surfactant-Associated Protein D; D011663:Pulmonary Surfactants / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D014162:Transfection
D003432:Cross-Linking Reagents; D017931:DNA Primers; D018076:DNA, Complementary; D004220:Disulfides; D006023:Glycoproteins; D037663:Pulmonary Surfactant-Associated Protein D; D011663:Pulmonary Surfactants; D011994:Recombinant Proteins; D003094:Collagen; D003545:Cysteine