Effect of cysteine 85 on biochemical properties and biological function of human surfactant protein A variants

Four "core" amino acid differences within the collagen-like domain distinguish the human surfactant protein A1 (SP-A1) variants from the SP-A2 variants. One of these, cysteine 85 that could form intermolecular disulfide bonds, is present in SP-A1 (Cys⁸⁵) and absent in SP-A2 (Arg⁸⁵). We hypothesized that residue 85 affects both the structure and function of SP-A1 and SP-A2 variants. To test this, wild-type (WT) variants, 6A² of SP-A1 and 1A⁰ of SP-A2, and their mutants (6A ^2(C85R) and 1A^0(R85C)) were generated and studied. We found the following: (1) Residue 85 affected the binding ability to mannose and the oligomerization pattern of SP-As. The 1A^0(R85C) and 6A ^2(C85R) patterns were similar and/or resembled those of WT 6A ² and 1A⁰, respectively. (2) Both SP-A WT and mutants differentially induced rough LPS and Pseudomonas aeruginosa aggregation in the following order: 1A⁰ > 6A² > 6A^2(C85R) > 1A^0(R85C) for Re-LPS aggregation and 1A⁰ > 6A ² = 6A^2(C85R) = 1A^0(R85C) for bacterial aggregation. (3) SP-A WT and mutants enhanced phagocytosis of P. aeruginosa by rat alveolar macrophages. Their phagocytic index order was 6A^2(C85R) > 1A⁰ > 6A² = 1A^0(R85C). The activity of mutant 1A^0(C85R) was significantly lower than WT 1A⁰ but similar to 6A². Compared to WT 6A², the 6A ^2(C85R) mutant exhibited a significantly higher activity. These results indicate that the SP-A variant/mutant with Arg⁸⁵ exhibits a higher ability to enhance bacterial phagocytosis than that with Cys ⁸⁵. Residue 85 plays an important role in the structure and function of SP-A and is a major factor for the differences between SP-A1 and SP-A2 variants.