Isolation and Characterization of Two Glycoproteins from Patients with Alveolar Proteinosis

Abstract

The saline-insoluble particulate material obtained by pulmonary lavage from four patients with alveolar proteinosis was analyzed for total lipid (52%), protein (44%), and carbohydrate (4%). Sodium dodecyl sulfate-gel electrophoresis revealed only three major peptides in these patients. Molecular weights (from the gels) were 69,000, 62,000, and 36,000, and the latter two peptides were periodic acid-Schiff stain-positive. Amino-acid and sugar analyses were performed on the peptides cut from the gels and on peptides purified by Sephadex G-200 chromatography. The 69,000-molecular-weight peptide contained nor carbohydrate. Antibody studies and aminoacid analysis indicated that it was albumin. The 62,000-molecular-weight peptide contained 1% hydroxyproline, 1% hydroxylysine, 10% glycine, and 9% carbohydrate.The 36,000-molecular-weight peptide contained 1.2% hydroxyproline, 1% hydroxylysine, 13% glycine, 1.4% sialic acid, 2.6% glucose, 2.4% galactose, 2% mannose, 0.8% fueose, 0.4% glycosamine, and 0.6% galactosamine. Proteins extracted from kidney glomeruli with similar amino-acid and carbohydrate composition have been observed previously. Alveolar proteinosis, first described by Rosen, Castleman, and Liebow in 1958 (1), is a chronic pulmonary disease of unknown pathogenesis in which the alveoli and terminal bronchioles of the lung are filled with a periodic acid-Schiff-positive amorphous material. This material, which is insoluble in isotonic saline, can be removed in particulate form by pulmonary lavage (2, 3). The extractable lipid from the lavage material contains large amounts of phosphatidyl choline and cholesterol (4). In the present study, the major peptides from the saline-insoluble lavage material have been isolated and characterized. METHODSFour patients with alveolar proteinosis, diagnosed by a typical clinical course, radiographic and physiologic findings, and lung biopsy, underwent therapeutic pulmonary lavage with 15-20 liters of isotonic saline under the direction of either Dr. J. Ramirez-R. (1 patient) or Dr. J. Kylstra (3 patients) (2, 3). The lavage material was centrifuged at 5000 X g for 20 min to obtain a thick, yellow precipitate and a clear supernatant. Both fractions were stored at -20°.Solubilization and Sodium Dodecyl Sulfate (SDS) Gel Electrophoresis of the Lavage Precipitate. The lavage precipitate was homogenized four times in cold isotonic saline and collected by centrifugation (15,000 X g for 15 min). Total protein was determined by the Lowry et al. method (5), and total carbohydrate was determined by the phenol-sulfuricacid method (6), and the thiobarbituric-acid assay for sialic acid (7). Total lipid was estimated gravimetrically after extraction with chloroform-methanol (2: 1 by volume).The lavage precipitate was dissolved in 0.2% SDS (Fisher)-0.1% 2-mercaptoethanol (Eastman)-0.04 M Tris-HCl (Schwarz-Mann) (pH 7.5). Solubilization of this material could be achieved only in the presence of thiol reagents. A black substance, representing less than 5% of the material, was precip...

The pulmonary extracellular lining.

“…The particulate material from PAP lavage also contains large amounts of two glycoproteins, molecular weight 62,000 and 32,000 (117,(144)(145)(146)(147)(148), although these proteins are proteolytic fragments of a larger 80,000 molecular weight protein (148). The 80,000 protein is, in turn, a portion of a 250,000 molecular weight protein (149).…”

Isolation and characterization of two hydroxyproline-containing glycoproteins from normal animal lung lavage and lamellar bodies.

“…sulfate (SDS) -polyacrylamide gel electrophoresis revealed the presence of three major peptides, the mol wt of which were 69,000, 62,000, and 36,000 (4). The latter two peptides were PAS-positive.…”

“…In this study, we present compositional data on two glycopeptides isolated from lung lavage material of normal rabbit lung and lamellar bodies that are very similar to the unique glycopeptides isolated from lungs of patients with alveolar proteinosis (4 Preparation of lavage material. Normal rabbits (7-8lbs) were killed by air embolus, and the lungs were lavaged through the trachea, first with 150 ml (50 ml three times) of ice-cold isotonic saline and then with 150 ml (50 ml three times) of cold hypotonic EDTA (1 mM EDTA in 5 mM Tris-HCl buffer, pH 7.5).…”

“…Neutral sugars were analyzed by the method of Lee, Johnson, White, and Scocca (27) after the peptides were hydrolyzed with 2 N trifluoroacetic acid for 3 h at 110'C. In addition, glucose was measured with glucose oxidase method as described before (4). Amino sugars were also estimated on the single column of amino acid analyzer after the peptide was hydrolyzed in 4 N HCl for 6 h at 100'C, as described by Spiro (28).…”

Particles Causing Lung Disease

“…This lipid-glycoprotein mixture can be removed by bronchopulmonary lavage (114). The predominant lipid is dipalmitol phosphatidylcholine (DPL).…”