Purification of canine surfactant-associated glycoproteins A. Identification of a collagenase-resistant domain.
A procedure for purification of surfactant-associated glycoproteins A from canine surfactant was established utilizing preparative isoelectric focusing as a major purification step in absence of detergents. The proteins migrated as charge trains, isoelectric points 4.2-5.0. Unglycosylated forms of surfactant-associated protein A1 (26 kDa) and glycoproteins A2 and A3 (32-36 kDa) were identified by silver-staining and immunoblot analysis. These forms were demonstrated to be identical polypeptides by fingerprint analysis of 125I-labeled peptides generated by tryptic-chymotryptic digests of the iodinated proteins. Size and charge heterogeneity were related to varying amounts of N-linked complex carbohydrates, including sialic acid, which were sensitive to endoglycosidase F and neuraminidase but resistant to endoglycosidase H. A collagenase-sensitive region was demonstrated which was required for sulfhydryl-dependent oligomerization of the molecule. Collagenase treatment resulted in removal of approx. 10 kDa from the glycoprotein molecule. Collagenase-resistant fragments of 21-23 kDa migrated with carbohydrate-dependent size and charge heterogeneity and were reduced to 16 kDa by endoglycosidase F. Amino acid composition of the surfactant glycoproteins demonstrated high glycine content which was diminished after digestion with collagenase. Several glycine-rich tryptic peptides were isolated by reverse-phase chromatography. Partial sequence information shows Gly-X-Y repeat sequences containing hydroxyproline residues. The major canine surfactant-associated proteins, glycoproteins A contain complex-type N-linked carbohydrate. In addition, a separate collagen-like peptide domain is present and is required for sulfhydryl-dependent oligomerization.
D000118:Acetylglucosaminidase; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D007525:Isoelectric Focusing; D008168:Lung / Q000032:analysis*; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D003012:Microbial Collagenase; D009439:Neuraminidase; D010446:Peptide Fragments / Q000032:analysis; D011510:Proteolipids / Q000302:isolation & purification*; D037661:Pulmonary Surfactant-Associated Proteins; D011663:Pulmonary Surfactants / Q000302:isolation & purification*
D000596:Amino Acids; D010446:Peptide Fragments; D011510:Proteolipids; D037661:Pulmonary Surfactant-Associated Proteins; D011663:Pulmonary Surfactants; D009439:Neuraminidase; D000118:Acetylglucosaminidase; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D003012:Microbial Collagenase