Isolation, purification, and properties of respiratory mucus glycoproteins

Abstract

The major glycoprotein from human tracheobronchial secretions and from primary explant cultures of human tracheal epithelium was 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 2/3 of the amino acid residues). The carbohydrate, which is nearly 80% by weight, was O-glycosicially linked via GalNAc [N-acetyl galactosamine], 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% by 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.