Recombinant pulmonary surfactant protein D. Post-translational modification and molecular assembly.

E Crouch,D Chang,K Rust,A Persson,J Heuser

doi:10.1016/s0021-9258(17)40752-6

E Crouch, D Chang + Show 3 more

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https://doi.org/10.1016/s0021-9258(17)40752-6

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Abstract

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.

Highlights

Pulmonary surfactant protein D (SP-D) ias member of serum mannose-binding proteina,nd bovine conglutinin a family of collagenous C-typelectins that includes the (Crouch et al, 1993a; Sastry and Ezekowitz,1993).SP-D is serum mannose bindingproteins and surfactant protein secreted into the air spacesby alveolar type I1 cells, and into
Protein and cDNA sequencing studies have revealed that eachSP-Dchainconsists of a short noncollagenousaminoterminal domain containingtwo cysteine residues,an uninterrupted collagen-like sequence (59 Gly-X-Y triplets in rat and selectionsystem.The secreted molecules(RrSP-D), human SP-D), a short connecting sequence, and a noncollagwhich werepurified by affinity chromatography on male-nous carboxyl-terminal domain that shows high sequence hotosyl-agarose, comigrated with rSPo-Dn SDS-polyacryl- mology with other memberosf the C-type lectin family (Crouch amide gel electrophoresis in the presence and absence et al, 1993a; Limet al., 1993; Lu et al, 1992; Rust et al, 1991; of reduction, and coeluted with rSP-D dodecamers fromShimizu et al, 1992)
The expression of SP-D in CHO cells with the assembly of dodecamers apparently identical to natural rSP-D establishes that theinformation required for the assembly of these structures is specified intheprimarystructure of SP-D

Summary

Towhom correspondence should be addressed

Dept.of Pathology, fied Eaele’s medium: MSX. methionine sulfoximine; PAGE, .pol.yacry~ l-. The abbreviations used are: SP-D,surfactant protein D; rSP-D, rat Chem., in press. Tionand assembly in a mammalian transfection system These studies establish that a single chain type is sufiicient for the formation of SP-D dodecamers and the higherorder molecular assemblies identified in silicotic rat and human proteinosis pulmonary lavage. 0.5% (w/v) deoxycholate, 1% (v/v) Triton X-100, 0.1%(w/v) SDS, 2.5mM EDTA, 0.1 mM phenylmethanesulfonyl fluoride2, .5 mM N-ethylmaleimide, pH 7.5) and examinedby SDS-PAGE (Persson etal., 1988). Degradatiue Analysis-Peptic digestions were performedfor 12-16 h at 27 “C in 0.5 M acetic acid in the presencoef 100 pg/ml porcine pepsin A. Amino acid analysis of acid and alkaline hydrolysates, and digestions

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