Characteristics of Human Surfactant-Associated Glyoproteins A

Abstract

Surfactant-associated glycoprotein A [molecular weight (Mr) = 34,000, isoelectric point (pi) 4.6-5.0] and its sulfhydryl dependent oligomers were purified and partially characterized from surfactant obtained from human alveolar lavage. Two major forms of the protein were identified by silver stain and immunoblot analysis of surfactant using human surfactant-associated glycoprotein A antisera: glycoprotein A2, Mr = 34,000 and glycoprotein Ai, Mr = 28,000. The larger form was reduced to Mr = 28,000 by treatment with endogylcosidase F, indicating the presence of complex N-linked oligosaccharide on the molecule. Charge heterogeneity was decreased and the isoelectric point increased by treatment with neuroaminidase, supporting the presence of sialic acid. Homology between the proteins Mr = 34,000 and 28,000 was confirmed by analysis of two-dimensional tryptic and chymotryptic peptides of 125I-iodo-glycoproteins Ai and A2 which were identical. The protein was very rich in glycine and its amino acid composition was similar to that of glycoprotein A previously reported for the dog and rat. Treatment of glycoproteins A with bacterial collagenase resulted in the generation of highly glycosylated peptides Mr = 20,000- 22,000, pi 4.6-5.0, which no longer formed sulfhydryldependent oligomers, supporting the presence of significant collagen-like region in the molecule. In the absence of reducing agents, glycoprotein A from surfactant was present as sulfhydryl-dependent dimers and larger oligomers. Higher molecular weight aggregates of glycoproteins A were also present in lavage material even after sulfhydryl reduction. Glycoproteins A were identified in surfactant from amniotic fluid, normal adult lung lavage, human cadaver lung lavage, and material obtained from lung lavage from a patient with alveolar proteinosis. Alveolar proteinosis proteins contained larger amounts of the higher molecular weight aggregates and smaller molecular weight proteolytic fragments of glycoproteins A than material obtained from other sources. Peptide mapping of the I25Iiodinated aggregates, approximately Mr = 50,000, 70,000, 100,000, and greater were identical to glycoproteins A (Mr = 34,000 and 28,000) from normal human lung lavage. A smaller immunoreactive form (Mr = 20,000) shared several peptides but lacked others, supporting its origin as a proteolytic fragment of glycoproteins Ai or A2. Human glycoprotein A2 is a complex N-linked glycoprotein likely representing the glycosylated form of a polypeptide precursor Mr = 28,000.