Recombinant pulmonary surfactant protein D. Post-translational modification and molecular assembly.
Pulmonary surfactant protein D (SP-D) is a member of a family of collagenous C-type lectins that includes the serum mannose binding proteins and surfactant protein A. Recent studies have shown that rat SP-D (rSP-D) molecules are assembled as tetramers of trimeric subunits (12 mers) and that dodecamers can participate in higher orders of molecular assembly involving interactions of the amino-terminal peptide domains. In order to further study the assembly of SP-D in vitro, Chinese hamster ovary K1 cells were transfected with a full-length rat SP-D cDNA, and stable transfectants with high levels of SP-D production (approximately 6 x 10(6) dodecamers/cell/24 h) were obtained using a glutamine synthetase selection system. The secreted molecules (RrSP-D), which were purified by affinity chromatography on maltosyl-agarose, comigrated with rSP-D on SDS-polyacrylamide gel electrophoresis in the presence and absence of reduction, and coeluted with rSP-D dodecamers from 4% agarose. The major bacterial collagenase-resistant peptide showed a decreased mobility on reduction consistent with the formation of intrachain disulfide bonds. A 17-kDa pepsin-resistant fragment was isolated following overnight digestion with pepsin at 27 degrees C, confirming the formation of a triple helical domain comparable in size and thermal stability to that of natural SP-D. The expressed protein contained sialylated endoglycosidase F-sensitive carbohydrate; amino acid analysis of acid and alkaline hydrolysates demonstrated essentially normal levels of hydroxyproline, hydroxylysine, and hydroxylysine-glycosides. Electron microscopic studies showed a molecular structure indistinguishable from lung SP-D, with a similar small subpopulation of molecules showing higher orders of multimerization. Solid-phase neoglycoprotein binding assays gave the same saccharide inhibition profile as natural rat SP-D, and both proteins showed efficient saccharide-dependent agglutination of Escherichia coli. These studies demonstrate that a single genetically distinct chain type can account for the various and complex molecular assemblies of SP-D, and further verify the potential physiologic significance of the disulfide-bonded multimers and higher aggregates isolated from rat, bovine, and human lung lavage.
Crouch
E
E
Department of Pathology, Washington University School of Medicine, St. Louis, Missouri 63110.
Chang
D
D
Rust
K
K
Persson
A
A
Heuser
J
J
D000371:Agglutination; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D016466:CHO Cells; D002850:Chromatography, Gel; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D005822:Genetic Vectors; D006023:Glycoproteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000648:ultrastructure; D008854:Microscopy, Electron; D008970:Molecular Weight; D011499:Protein Processing, Post-Translational; D037663:Pulmonary Surfactant-Associated Protein D; D011663:Pulmonary Surfactants / Q000096:biosynthesis* / Q000302:isolation & purification / Q000648:ultrastructure; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000648:ultrastructure; D015183:Restriction Mapping; D014162:Transfection
D000596:Amino Acids; D006023:Glycoproteins; D037663:Pulmonary Surfactant-Associated Protein D; D011663:Pulmonary Surfactants; D011994:Recombinant Proteins