THE CYS(6) INTERMOLECULAR DISULFIDE BOND AND THE COLLAGEN-LIKE REGIONOF RAT SP-A PLAY CRITICAL ROLES IN INTERACTIONS WITH ALVEOLAR TYPE-IICELLS AND SURFACTANT LIPIDS

Rat pulmonary surfactant protein A is an oligomer of 18 polypeptide ch ains which are associated by triple helix formation in the collagen-li ke domain and interchain disulfide bridges at the NH2 terminus. The ro les of the intermolecular bond at Cys(6) and the collagen-like domain (Gly(8)-Pro(80)) in the interactions of SP-A with phospholipids and al veolar type II cells were investigated using mutant forms of the prote in. Wild type SP-A (SP-A(hyp)), SP-A with the substitution Cys(6) --> Ser to prevent disulfide formation (SP-A(hyp,C6S)), and SP-A with the collagen-domain deleted (SP-A(Delta G8-P80)) were synthesized sized in insect cells using recombinant baculoviruses. The SP-As were glycosyl ated and secreted from the invertebrate cells and the binding affiniti es of the wild type and mutant proteins for the mannose-Sepharose matr ix used for purification were nearly identical. The SP-A(hyp) and SP-A (Delta G8-P80) were at least nonameric in solution based on gel exclus ion chromatography, and demonstrated extensive sulfhydryl-dependent ol igomerization under nonreducing conditions. The SP-A(hyp,C6S) was also oligomeric in solution and formed disulfide-dependent dimers, indicat ing the presence of at least one additional interchain disulfide bond. The SP-A(Delta G8-P80) but not the SP-A(hyp,C6S) aggregated lipid ves icles at 20 degrees C and augmented the surface tension lowering effec t of extracts of natural surfactant. The SP-A(Delta G8-P80) competed p oorly with native SP-A for receptor occupancy on isolated alveolar typ e II cells and was a potent but nonspecific (concanavalin A-like) inhi bitor of surfactant secretion. In contrast, the SP-A(hyp,C6S) partiall y competed for receptor occupancy and weakly inhibited surfactant secr etion in a specific manner, Neither the SP-A(Delta G8-P80) nor the SP- A(hyp,C6S) supported the association of phospholipid liposomes with ty pe II cells. We conclude that: 1) the Cys(6) interchain disulfide bond of SP-A is required for aggregation of liposomes and for potent inhib ition of surfactant secretion, 2) The collagen-like region is required for competition with I-125-SP-A for receptor occupancy and specific i nhibition of surfactant secretion in the presence of competing sugars. 3) Both the NH2-terminal disulfide and the collagen-like region are r equired to enhance the association of phospholipid vesicles with type II cells.