S.N. Bhattacharyya

Structural studies on a glycoprotein isolated from alveoli of patients with alveolar proteinosis

A major glycoprotein 36 000 molecular weight) has been isolated from lung lavage of patients with alveolar proteinosis and found to contain five residues of hydroxyproline, fifty residues of glycine, three residues of methionine, 3 mol of sialic acid, 4.4 mol of mannose, 4.0 mol of galactose, 6.0 mol of glucosamine, and 1 mol of fucose. Cyanogen bromide (CNBr) treatment of the glycoprotein resulted, as expected, in four peptides of apparent molecular weights of 18 000, 12 000, 5000 and 1000, respectively. The chemical compositions of the CNBr peptides indicate the presence of hydroxyproline and high amounts of glycine in all but one of the peptides; two of the four CNBr peptides contain carbohydrate. Gel filtration, acrylamide gel electrophoresis and end-group analyses of the native glycoprotein and its CNBr peptides indicate that the peptides are homogeneous. End-group analyses of the CNBr cleavage products assign the 18 000 molecular weight peptide to the NH2-terminal portion and the 1000 molecular weight peptide to the COOH-terminal portion of the native glycoprotein molecule. Pronase digestion of the 36 000 molecular weight glycoprotein, followed by gel filtration and cation exchange chromatography, resulted in two fractions. One fraction was acidic and contained all the carbohydrate, a high content of aspartic acid and no hydroxyproline. The other fraction was basic and contained 8.4% hydroxyproline, 14% proline, 28% glycine and no carbohydrate, suggesting the presence of collagen-like sequence in the peptide chain. Paper electrophoresis of the basic fraction demonstrated two components, the amino acid compositions of which are identical to those of collagen. Partial amino-terminal sequence analysis of one of the CNBr peptides (18 000 molecular weight) indicated the presence of -Fly-Pro-HyP-Gly-sequence in the peptide chain, which confirms our suggestion that collagen-like regions are present in the native glycoprotein molecule. Limited acid hydrolysis of the acidic fraction and subsequent fractionation of the acid hydrolysate using Dowex column yielded a fraction which produced brown colour with ninhydrin reagent. Paper chromatography of this fraction demonstrated a large component which also stained brown with ninhydrin reagent. After acid hydrolysis, this component was found to consist of equal amounts of asparitic acid and glucosamine, indicating that the N-acetylglucosamine of the oligosaccharides is linked to the asparagine residue of the peptide. No serine or threonine linkages are present.

Structure elucidation by one- and two-dimensional 360- and 500-MHz 1H NMR of the oligosaccharide units of two glycoproteins isolated from alveoli of patients with alveolar proteinosis

The structure of the oligosaccharide units of the glycoproteins of Mr 36,000 and 62,000 isolated from alveoli of patients with alveolar proteinosis have been determined by one- and two-dimensional 1H NMR spectroscopy at 500 and 360 MHz. Bi-, tri-, and tetraantennary glycans of N-acetyllactosaminic type have been found in high percentage. They are 1----6 monofucosylated and fully sialylated, the ratio NeuAc(2----3)/NeuAc X (2----6) increasing with increasing degree of branching.

Studies on structural relationship between two glyco-proteins isolated from alveoli of patients with alveolar proteinosis

Abstract A glycoprotein of molecular weight 62 000 has been isolated from lung lavage of patients with alveolar proteinosis and found to contain five residues of hydroxy-proline, 72 residues of glycine, three residues of methionine, 3 mol of sialic acid, 4 mol of mannose, 4.2 mol of galactose, 1 mol of fucose and 5.8 mol of N- acetylglucosamine . Cyanogen bromide (CNBr) treatment of the glycoprotein resulted in four peptides of molecular weights 18 000, 12 000, 5000 and 27 000, three of the peptides containing hydroxyproline and two of the peptides containing carbohydrates. The presence of hydroxyproline in the native glycoprotein and the -Gly-Pro-Hyp-Gly- sequence in the CNBr peptide of molecular weight 18 000 suggest that collagen-like regions may be present in the peptide chain. Limited trypsin digestion of the glycoprotein of molecular weight 62 000 resulted in two peptides of molecular weights 36 000 and 26 000, the former being a glycoprotein. The compositional and amino acid sequence analyses of these peptides indicate that the peptides of molecular weights 36 000 and 26 000 are proteolytic products of the glycoprotein of molecular weight 62 000 and that the peptide of molecular weight 26 000 is the COOH-terminal extension peptide of the native glyco-protein (molecular weight 62 000).

Isolation and characterization of a pulmonary glycoprotein from human amniotic fluid

A glycoprotein of the molecular weight of 36 000 has been isolated from human amniotic fluid. The glycoprotein was found to contain sialic acid, galactose, mannose, fucose, glucosamine, hydroxyproline and relatively high amounts of glycine. End-group analyses resulted in a single NH2-terminal residue indicating that the glycoprotein was homogeneous. The data indicate that this unique collagen-like glycoprotein, which is immunologically identical to a major alveolar glycoprotein found in alveoli of patients with alveolar proteinosis, is also a major protein in the human amniotic fluid. The idea that the pulmonary constituents enter the amniotic fluid cavity during fetal lung development is also confirmed by this report.

Characterization of the collagen- and non-collagen-like regions present in a glycoprotein isolated from alveoli of patients with alveolar proteinosis

A major glycoprotein of Mr 36 000 has been isolated from the lung lavage of patients with alveolar proteinosis and found to contain five residues of hydroxyproline, 50 residues of glycine, 2.6 mol of sialic acid, 4.8 mol of mannose, 4.1 mol of galactose, 0.9 mol of fucose, 7.0 mol of N-acetylglucosamine. Limited pepsin digestion of this glycoprotein resulted in six peptides, three of which contained hydroxyproline and nearly 30% glycine and two of which contained all the carbohydrate present in the native glycoprotein. All three peptides containing hydroxyproline and high content of glycine were found to contain -Gly-x-y-Gly- type sequences. Partial amino acid sequence analyses indicate that this glycoprotein is composed of alternating short collagen- and non-collagen-like peptide sequences.

Structural studies on a collagen-like glycoprotein isolated from lung lavage of normal animal.

A major glycoprotein of Mr = 36 000 has been isolated from lung lavage of normal rabbit and purified to homogeneity by gel chromatography. The glycoprotein was found to contain 4 residues hydroxyproline, 50 residues glycine, 3 residues methionine, 2.6 mol sialic acid, 4.0 mol galactose, 5.0 mol mannose, 0.9 mol fucose and 7.0 mol glucosamine. Cyanogen bromide treatment of the glycoprotein resulted in four peptides with molecular weights of 18 000, 12 000, 5000 and 1000, three of the peptides containing hydroxyproline and two of the peptides containing carbohydrates. Partial NH2-terminal amino acid sequence analysis on one of the cyanogen bromide peptides (Mr = 18 000) indicated the presence of -Gly-Pro-Hyp-Gly- sequence in the peptide chain, suggesting that collagen-like region(s) may be present in this glycoprotein.

Isolation and characterization of subunits of human Clq

Abstract Extensive reduction and alkylation of purified Clq from human serum by urea, dithiothreitol and iodoacetamide resulted in three subunits. The estimated molecular weight of these subunits by sodium dodecyl sulfate-acrylamide gel electrophoresis was found to be 29 000, 27 000 and 22 000, respectively. The complete amino acid analysis of these subunits indicates the presence of hydroxyproline and a high amount of glycine in all three subunits. Hydroxylysine was found to be present only in the 29 000 and 27 000 molecular weight peptide. The 22 000 molecular weight peptide was found to contain an additional unidentified basic amino acid. The carbohydrate compositions of the three subunits indicate the presence of amino sugars, sialic acid, galactose and glucose in the 29 000 molecular weight peptide. The 27 000 molecular weight peptide contained amino sugars, glucose and sialic acid, whereas the 22 000 molecular weight peptide contained no carbohydrate. The data indicate that Clq is an unique glycoprotein which has no similarity in composition to hydroxylated peptides found in human alveoli, basement membrane or collagen.

Characterization of a glycoprotein isolated from a continuous tumor-cell line of human lung carcinoma

A glycoprotein with a molecular weight of 62 000 has been isolated from a tumor-cell line, A549, and purified to homogeneity by gel chromatography. The glycoprotein contained sialic acid, galactose, mannose, N-acetylglucosamine and a relatively high amount of glutamic acid and proline. The data indicated that the overall composition of this glycoprotein was different from that of the glycoprotein of Mr 62 000 isolated from lung lavage of patients with alveolar proteinosis. The glycoprotein did not react with the antiserum raised against glycoprotein of Mr 62 000 isolated from lung lavage of patients with alveolar proteinosis.

Characterization of pepsin-resistant peptides present in a glycoprotein isolated from lung lavage of normal rabbit.

A glycoprotein of Mr 36 000 has been isolated from lung lavage of normal rabbit and purified to homogeneity by gel chromatography. Three peptides containing hydroxyproline and nearly 30% glycine have been isolated and purified from pepsin-digested native glycoprotein. Partial NH2-terminal amino acid sequence analysis on one of the peptides indicated the presence of -Gly-Pro-Hyp-Gly- sequence in the peptide chain, suggesting that collagen-like region(s) may be present in this glycoprotein.