Deletion Mapping of N-terminal Domains of Surfactant Protein A

Crystal Structure of Trimeric Carbohydrate Recognition and Neck Domains of Surfactant Protein A

“…The N187S substitution eliminates the only site of N-linked glycosylation in the CRD. The fragment forms a stable non-covalent trimer in solution (34). In the crystal, the monomers of the trimer are related by crystallographic symmetry.…”

“…A recombinant rat SP-A containing only the amphipathic linking domain and the CRD and lacking the consensus for asparagine-linked glycosylation (⌬N1-P80,N187S) was synthesized in insect cells using baculovirus vectors, as described previously (34). The ⌬N1-P80,N187S was purified by mannose-Sepharose affinity chromatography, dialyzed against 5 mM Tris and 50 mM NaCl and concentrated to at least 3 mg/ml.…”

Processing of Pulmonary Surfactant Protein B by Napsin and Cathepsin H

“…Recombinant baculovirus was produced by homologous recombination in Spodoptera frugiperda cells as described by McCormack et al (30). Fresh monolayers of Trichoplusia ni cells were infected with plaque-purified recombinant virus at a multiplicity of infection of 2 and cultured in serum-free media for 72 h. Recombinant SP-B ⌬C was purified from the culture medium of infected insect cells by NTA affinity chromatography, under nondenaturing conditions, as described previously (28).…”

Pulmonary Surfactant Protein A Up-Regulates Activity of the Mannose Receptor, a Pattern Recognition Receptor Expressed on Human Macrophages

“…Carbohydrate-deficient recombinant SP-A proteins retain structural and biologic functions, including oligomerization, aggregation of phospholipid liposomes, binding to immobilized carbohydrate, inhibition of lipid secretion from type II cells, and competition for receptor occupancy on type II cells (23). The synthesis of the mutant recombinant rat SP-A protein containing a nested deletion of the proximal collagen-like region (Gly 8 -Gly 44 ) (TM2), truncation of the protein at the neck region resulting in a protein lacking the collagen and the NH 2 -terminal regions (TM1-2-3), and an amino acid substitution at the Asn 187 site (TM1-2-3 ser187 ) were made as previously described (27,28). Purity of the SP-A preparation was assessed by SDS-PAGE.…”