Pulmonary surfactant apoprotein A structure and modulation of surfactant secretion by rat alveolar type II cells.
The pulmonary surfactant apoprotein with a reduced denatured molecular mass of 26-38 kDa (PSP-A) has recently been identified as an inhibitor of surfactant phospholipid secretion by isolated rat alveolar type II cells. We have investigated some of the structural determinants of PSP-A that are relevant to the inhibitory process. The PSP-A was isolated from rats given an intratracheal instillation of silica. The yield of PSP-A from silica-treated animals was 20-40-fold higher than that obtained from untreated animals. Reduction of PSP-A with 2-mercaptoethanol caused a reversible loss of biological activity that was restored by mild oxidation. Alkylation of the protein with excess iodoacetamide also led to inactivation, although titration with 5,5'-dithiobis-(2-nitrobenzoic acid) indicated that the protein initially contained no free sulfhydryl moieties. Neither alkylation nor reduction plus alkylation completely prevented the formation of oligomers as determined by gel permeation analysis. The apparent molecular mass of PSP-A at 4 degrees C in low ionic strength buffers was 1.6 megadaltons, and at 37 degrees C in normal ionic strength buffers was greater than 1.5 megadaltons. Removal of the oligosaccharide moiety with endoglycosidase F also had no effect upon biological activity. Five distinct monoclonal antibodies recognizing peptides epitopes on PSP-A were produced. All monoclonal antibodies exhibited similar affinity for PSP-A and recognized the delipidated and deglycosylated form. Four monoclonal antibodies reacted with epitopes on PSP-A that altered its function as an inhibitor. One monoclonal antibody was clearly ineffective at altering the activity of PSP-A. These results demonstrate that: 1) disulfide bonds are required for the activity of PSP-A, 2) disruption of disulfides does not prevent the formation of oligomeric forms of PSP-A, 3) the oligosaccharide moiety is not essential for biological activity, and 4) monoclonal antibodies can be used to map the epitopes responsible for biological activity.
D000478:Alkylation; D000818:Animals; D000911:Antibodies, Monoclonal; D001059:Apoproteins / Q000276:immunology / Q000302:isolation & purification / Q000494:pharmacology / Q000502:physiology*; D055598:Chemical Phenomena; D002621:Chemistry; D004220:Disulfides; D000939:Epitopes / Q000276:immunology; D008168:Lung / Q000032:analysis / Q000187:drug effects; D046911:Macromolecular Substances; D008297:Male; D008623:Mercaptoethanol / Q000494:pharmacology; D009844:Oligosaccharides; D010084:Oxidation-Reduction; D011650:Pulmonary Alveoli / Q000187:drug effects / Q000378:metabolism*; D037661:Pulmonary Surfactant-Associated Proteins; D011663:Pulmonary Surfactants / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology / Q000502:physiology*; D051381:Rats; D011919:Rats, Inbred Strains; D012822:Silicon Dioxide / Q000494:pharmacology; D013329:Structure-Activity Relationship
D000911:Antibodies, Monoclonal; D001059:Apoproteins; D004220:Disulfides; D000939:Epitopes; D046911:Macromolecular Substances; D009844:Oligosaccharides; D037661:Pulmonary Surfactant-Associated Proteins; D011663:Pulmonary Surfactants; C046616:pulmonary surfactant apoprotein; D008623:Mercaptoethanol; D012822:Silicon Dioxide