The Longer Isoform and Cys-1 Disulfide Bridge of Rat Surfactant Protein A Are Not Essential for Phospholipid and Type II Cell Interactions

Rat SP-A is a heterooligomer of two closely related isoforms, that requires interchain disulfide linkage for several functions including SP-A-mediated phospholipid vesicle aggregation and modulation of surfactant secretion and uptake by isolated alveolar type II cells. While the Cys6 disulfide bond of rat SP-A is known to be critical for function, the importance of the second interchain disulfide linkage within the N-terminal Isoleucine-3-Lysine-Cysteine-1 (IKC) sequence of the alternatively processed isoform is not clear. To examine the role of the Cys-1-dependent multimerization in SP-A function, we disrupted the Cys-1 disulfide bond by deletion of the IKC sequence (SP-Ahyp, DeltaIKC) or the substitution Cys-1 to Ser (SP-Ahyp,C-1S) in mutant recombinant proteins produced in insect cells. N-terminal sequence analyses revealed that the mutations influenced signal peptidase cleavage specificity, resulting in an increase in the abundance of the longer isoform of SP-Ahyp,C-1S and in N-terminal truncation of a fraction of the SP-Ahyp,DeltaIKC polypeptides at Gly8. On nonreducing SDS-PAGE analysis, both mutant proteins migrated as monomers and dimers but not the higher multimers that are characteristic of the wild-type recombinant protein (SP-Ahyp). Cross-linking analyses demonstrated that the association between trimeric SP-A subunits remained intact despite disruption of the Cys-1 bond. The SP-Ahyp,C-1S eluted in the same volume as SP-Ahyp from the gel sizing column with an apparent mass of 440 kDa, indicative of association of at least 9-10 monomers. The phospholipid binding and aggregation activities of the SP-Ahyp,C-1S were approximately 75% and 55% of the SP-Ahyp, respectively, but the SP-Ahyp,DeltaIKC was functionally comparable to SP-Ahyp. Similarly, both mutant proteins regulated the secretion and uptake of surfactant from isolated type II cells, but the SP-Ahyp,DeltaIKC was somewhat more potent than the SP-Ahyp,C-1S. Competitive binding to the SP-A receptor on type II cells was reduced by both Cys-1 mutations. We conclude that neither Cys-1-dependent multimerization nor the longer SP-A isoform is absolutely required for oligomeric association of trimeric SP-A subunits, SP-A/phospholipid interactions, or the regulation of surfactant secretion or uptake from type II cells by rat SP-A.