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Canine tracheal mucin glycoprotein was isolated from beagle dogs fitted with tracheal pouches. Following exclusion chromatography on Sepharose CL-4B, noncovalently associated proteins were further resolved by dissociative density gradient centrifugation in CsBr-guanidinium chloride, and the mucin was then extracted with chloroform-methanol. The delipidated high-density product obtained had a nominal molecular weight of about 10(6) and an overall composition characteristic for a mucin glycoprotein, viz., a high content of serine and threonine, about 80% carbohydrate by weight, the absence of mannose or uronic acid, measurable ester sulfate, and a Pronase-resistant domain of molecular weight (1.75-3.0) X 10(5) which contains essentially all of the saccharide residues. Noncovalently bound lipid amounted to 6-10% by weight and was primarily cholesterol and cholesteryl esters. Cleavage of disulfide bonds by performic acid oxidation resulted in the release of a protein (Mr 65,000) not otherwise resolved by sodium dodecyl sulfate gel electrophoresis or the purification scheme.

Following several model experiments, conditions were developed for optimal deglycosylation of tracheal mucin glycoproteins. Exposure of rigorously dried material to trifluoromethanesulfonic acid at 0 degree C for up to 8 h results in cleavage of essentially all fucose, galactose, and N-acetylglucosamine, about 80% of the N-acetylneuraminic acid (NeuNAc), and a variable amount of N-acetylgalactosamine (GalNAc), the sugar involved in linkage to protein. Residual N-acetylneuraminic acid is sialidase susceptible and apparently in disaccharide units, presumably NeuNAc2----GalNAc. The remaining N-acetylgalactosamine is mostly present as monosaccharides, and a few Gal beta 1----3GalNAc alpha units are also present; both are cleaved by appropriate enzymatic treatment. The saccharide-free proteins obtained from either human or canine mucin glycoproteins have molecular weights of about 100,000 and require chaotropic agents or detergents for effective solubilization.

A high-density mucin glycoprotein was isolated from human tracheobronchial secretions substantially free of contaminating protein, low-density glycoprotein, proteolytic enzymes, and lipid. A closely associated 65-kDa protein was discovered while investigating the effect of 2-mercaptoethanol treatment on the purified mucin glycoprotein. It has been established that the 65-kDa protein is neither alpha 1-antichymotrypsin nor human serum albumin, two proteins of similar molecular weight which are found in crude tracheobronchial secretions. This protein lacks cross-reactivity with antibodies directed against serum components and is presumably comparable to the 65-kDa protein similarly isolated from canine tracheal pouch secretions [Ringler et al. (1987) Biochemistry 26, 5322-5328]. Although both the presence of sulfhydryl groups and the ability to be reassociated with the mucin molecule have been established, it is not clear whether its association is due to direct disulfide bonding, hydrophobicity, or entrapment. It was found that 14C-methylated methemoglobin was an inappropriate substrate for measurement of proteolytic activity in mucin preparations due to inherent entrapment and clearance capabilities of mucin molecules.

The major glycoprotein from human tracheobronchial secretions and from primary explant cultures of human tracheal epithelium has been purified to apparent homogeneity. Mucin was solubilized in buffer and fractionated on Sepharose CL-4B, followed by CsBr density gradient centrifugation of the void volume fraction. High- and low-density fractions were obtained in ratios ranging from 2:1 to 5:1. The high-density (1.46) fraction appeared homogeneous by exclusion chromatography and recentrifugation in CsBr and had an amino acid composition characteristic of a mucin-type structure (threonine, serine, proline, glycine, and alanine comprise two-thirds of the amino acid residues). The carbohydrate, which is nearly 80% by weight, was O-glycosidically linked via GalNAc, sulfated (5.4% by weight), and contained fucose, galactose, glucosamine, galactosamine, and sialic acid. The low-density fraction had an amino acid composition distinct from that of the high-density fraction (threonine, serine, proline, glycine, and alanine comprise 51% of the amino acid residues) and a lower sulfate content. The size distribution of the saccharides in the low-density fraction was similar to that of the high-density fraction; the same sugars were present although the ratios were different. The low-density fraction contained 3 times more noncovalently associated lipid than did the high-density fraction. Several distinct classes of lipids were identified. Neutral lipids (mono-, di-, and triglycerides, cholesterol, and cholesteryl esters) comprised 56% of weight of the total lipid. Glycolipids and phospholipids were also identified. Palmitate (16:0), stearate (18:0), and oleate (18:1) were the major fatty acids in all classes of lipids.