The Cys6 Intermolecular Disulfide Bond and the Collagen-like Region of Rat SP-A Play Critical Roles in Interactions with Alveolar Type II Cells and Surfactant Lipids

Abstract

Pulmonary surfactant forms a phospholipid-rich monomolecular film at the air-liquid interface of the lung which is essential for the mechanical stability of the alveolar gas exchanging unit (1, 2). Surfactant protein A (SP-A) 1 is a 650-kDa oligomeric glycoprotein secreted by the pulmonary epithelium which has been reported to play important roles in surfactant homeostasis (3-5), the formation of tubular myelin (6, 7), the maintenance of monolayer integrity (8, 9), and in host defense (10, 11). SP-A shares a number of conserved structural features with other "collectins," a subset of the C-type lectin superfamily (12), including an NH 2 -terminal region containing interchain disulfide bonds, a collagen-like domain, a triple helical neck region and COOH-terminal carbohydrate recognition domain (CRD). Monomeric SP-A subunits fold into trimers through the formation of triple helices within the collagen-like region and neck region of the protein. Electron microscopic (13) and biochemical data (14) reveal that trimers further assemble into hexagonal arrays which are laterally associated through the first half of the collagen regions and stabilized by disulfide bonds near the NH 2 terminus (15). Studies with chimeric (16) or mutated (18 -20) 2 forms of SP-A have demonstrated the involvement of the COOH-terminal domains of the protein in ligand and receptor binding, the regulation of surfactant secretion and lipid uptake by type II cells.Several laboratories have explored the functional role of the collagen-like domain of SP-A. Treatment of SP-A with collagenase produces a peptide composed of the neck and CRD of the protein, termed the collagenase-resistant fragment (CRF), which has been used to assess functional properties of the collagen-like region. The CRF binds dipalmitoylphosphatidylcholine (DPPC) (21), mixed phospholipid liposomes (22), and carbohydrate (23) with lower affinity than the native protein and does not aggregate DPPC-containing phospholipid vesicles

The Role of Pulmonary Collectin N-terminal Domains in Surfactant Structure, Function, and Homeostasis in Vivo

“…Nucleotide sequencing of the entire D/A coding region was performed to confirm correct splicing and the absence of spurious mutations (26). The D/A gene was ligated into the unique EcoRI site of the baculovirus transfer vector, PVL 1392 (22), or the 3.7-kb hSP-C plasmid (21), which contains a 3.7-kb human surfactant protein C promoter. Orientation was confirmed by restriction digestion with KpnI and BamHI for the PVL 1392/D/A and hSP-C/D/A constructs, respectively.…”

“…This configuration facilitates simultaneous engagement of individual collectin molecules with multiple sites on membranes and microbial surfaces. Deletion of the collagen-like domain from SP-A, which limits oligomeric assembly to simple trimers and hexamers, reduces binding to liposomes but does not block liposome aggregation (21). Deletion of the N-terminal segment of SP-A (22) or selective disruption of interchain disulfide bond formation by C6S substitution limits assembly to simple trimers and blocks SP-A-mediated liposome aggregation and binding (21).…”

“…Deletion of the collagen-like domain from SP-A, which limits oligomeric assembly to simple trimers and hexamers, reduces binding to liposomes but does not block liposome aggregation (21). Deletion of the N-terminal segment of SP-A (22) or selective disruption of interchain disulfide bond formation by C6S substitution limits assembly to simple trimers and blocks SP-A-mediated liposome aggregation and binding (21). Disruption of interchain disulfide bond formation at the N terminus of SP-D by C15S and C20S substitutions limits oligomeric assembly to trimerization and blocks SP-Dmediated functions in vitro (23), and in vivo (24).…”

Pulmonary Surfactant Protein-A (SP-A) Restores the Surface Properties of Surfactant after Oxidation by a Mechanism That Requires the Cys6 Interchain Disulfide Bond and the Phospholipid Binding Domain

“…The recombinant SP-A ⌬N1-P80 (⌬N1-P80) lacking the NH 2 -terminal and collagenlike regions, is trimeric (31). The rSP-A ⌬G8-P80 (⌬G8-P80) is a collagen deletion mutant containing the NH 2 terminus, the neck region, and the CRD (29). The mutant rSP-A hyp-C6S (C6S), which has Cys 6 replaced by a serine, forms trimers but not larger oligomeric forms (29).…”

“…The rSP-A ⌬G8-P80 (⌬G8-P80) is a collagen deletion mutant containing the NH 2 terminus, the neck region, and the CRD (29). The mutant rSP-A hyp-C6S (C6S), which has Cys 6 replaced by a serine, forms trimers but not larger oligomeric forms (29). The two CRD mutant proteins, rSP-A hyp-E195A (E195A) and rSP-A hyp-D215A (D215A), bear point mutations in amino acids predicted to contribute to coordination of a calcium ion involved in PL and carbohydrate binding (28).…”