Physicochemical characterization of a minor mucin component from cystic fibrosis tracheobronchial secretions

Abstract

A minor mucin glycoprotein component (HTM-2) was purified from the tracheobronchial secretions of two cystic fibrosis patients using a protocol established in our laboratory. The secretions were solubilized in 0.1 M Tris-HCl buffer (pH 7.5) containing 0.22 M potassium thiocyanate and fractionated on a Bio-Gel A-5m column, followed by digestion with DNAase, rechromatography on the same column and chromatography on hydroxyapatite which resolved the major mucin (HTM-1) from the minor mucin component (HTM-2). The mucin component HTM-2 was further purified using Superose 6 chromatography. SDS-composite gel (2% polyacrylamide + 0.5% agarose) and 6% polyacrylamide gel electrophoresis showed that the purified HTM-2 was totally free of low-molecular-weight contaminants. Equilibrium density sedimentation centrifugation of purified HTM-2 using CsCl gradients also showed the absence of proteoglycans and other low-molecular-weight proteins. Comparison of carbohydrate and amino acid compositions of the two mucin components indicated that HTM-2 was quite different from the major mucin, HTM-1, reported earlier from our laboratory (Biochemistry, 24, 7334, 1985). This suggested that HTM-2 has a different polypeptide core and is perhaps a different gene product. The effects of 6 M guanidine-HCl and different concentrations of NaCl on the molecular size of HTM-2 and its ability to form aggregates was also investigated using the technique of static light scattering. In buffer containing 6 M guanidine-HCl, HTM-2 had a weight-average molecular weight of ≈ 4.5·10 6. However, in the presence of buffer containing 0.03, 0.10 or 0.15 M NaCl, the molecular weight of HTM-2 was estimated to be ≈ 11·10 6. These data suggest aggregation of HTM-2 in the presence of a range of NaCl concentrations. In contrast to HTM-1, which is a more anionic glycoprotein, the apparent molecular size of HTM-2 did not decrease at the higher NaCl concentration.