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.
Rat pulmonary surfactant protein A is an oligomer of 18 polypeptide chains which are associated by triple helix formation in the collagen-like domain and interchain disulfide bridges at the NH2 terminus. The roles of the intermolecular bond at Cys6 and the collagen-like domain (Gly8-Pro80) in the interactions of SP-A with phospholipids and alveolar type II cells were investigated using mutant forms of the protein. Wild type SP-A (SP-Ahyp), SP-A with the substitution Cys6 --> Ser to prevent disulfide formation (SP-Ahyp, C6S), and SP-A with the collagen-domain deleted (SP-ADeltaG8-P80) were synthesized in insect cells using recombinant baculoviruses. The SP-As were glycosylated and secreted from the invertebrate cells and the binding affinities of the wild type and mutant proteins for the mannose-Sepharose matrix used for purification were nearly identical. The SP-Ahyp and SP-ADeltaG8-P80 were at least nonameric in solution based on gel exclusion chromatography, and demonstrated extensive sulfhydryl-dependent oligomerization under nonreducing conditions. The SP-Ahyp,C6S was also oligomeric in solution and formed disulfide-dependent dimers, indicating the presence of at least one additional interchain disulfide bond. The SPADeltaG8-P80 but not the SP-Ahyp,C6S aggregated lipid vesicles at 20 degrees C and augmented the surface tension lowering effect of extracts of natural surfactant. The SP-ADeltaG8-P80 competed poorly with native SP-A for receptor occupancy on isolated alveolar type II cells and was a potent but nonspecific (concanavalin A-like) inhibitor of surfactant secretion. In contrast, the SP-Ahyp,C6S partially competed for receptor occupancy and weakly inhibited surfactant secretion in a specific manner. Neither the SP-ADeltaG8-P80 nor the SP-Ahyp,C6S supported the association of phospholipid liposomes with type II cells. We conclude that: 1) the Cys6 interchain disulfide bond of SP-A is required for aggregation of liposomes and for potent inhibition of surfactant secretion. 2) The collagen-like region is required for competition with 125I-SP-A for receptor occupancy and specific inhibition of surfactant secretion in the presence of competing sugars. 3) Both the NH2-terminal disulfide and the collagen-like region are required to enhance the association of phospholipid vesicles with type II cells.
McCormack
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Lord and Taylor Laboratory for Lung Biochemistry, Anna Perahia Adatto Clinical Laboratories, Denver, Colorado 80206, USA.
Pattanajitvilai
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Stewart
J
J
Possmayer
F
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Inchley
K
K
Voelker
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DR
D000818:Animals; D003094:Collagen / Q000378:metabolism*; D003545:Cysteine / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism*; D008081:Liposomes; D011485:Protein Binding; D011510:Proteolipids / Q000737:chemistry / Q000378:metabolism*; D011650:Pulmonary Alveoli / Q000378:metabolism*; D037662:Pulmonary Surfactant-Associated Protein A; D037661:Pulmonary Surfactant-Associated Proteins; D011663:Pulmonary Surfactants / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D004220:Disulfides; D008081:Liposomes; D011510:Proteolipids; D037662:Pulmonary Surfactant-Associated Protein A; D037661:Pulmonary Surfactant-Associated Proteins; D011663:Pulmonary Surfactants; D011994:Recombinant Proteins; D003094:Collagen; D003545:Cysteine