Mucus glycoproteins from cystic fibrotic sputum. Macromolecular properties and structural 'architecture'.
Mucus glycoproteins (mucins) were isolated from sputum of patients with cystic fibrosis (CF) after separation into sol and gel phases. The mucus gel was solubilized with gentle stirring in 6 M-guanidinium chloride supplemented with proteinase inhibitors, and purification of mucins was subsequently achieved by isopycnic density-gradient centrifugation in CsCl/guanidinium chloride. Density-gradient centrifugation also revealed a heterogeneity of the macromolecules, the pattern of which varied between individuals, and mucins from the gel phase was pooled as 'heavy' and 'light' fractions. Gel chromatography on Sepharose CL-2B showed that the heavy fraction contained a larger proportion of smaller species than the 'light' fraction and that the gel phase mucins were much larger than those from the sol. An apparently homogeneous high-Mr mucin population from one individual contained approx. 70% (w/w) carbohydrate, the major sugars being N-acetylglucosamine (17.8%), N-acetylgalactosamine (6.7%), galactose (20.7%), fucose (13.2%) and sialic acid (11.4%). These mucins had an S020.w of 47 S, and an Mr of 15 x 10(6) -20 x 10(6), and rate-zonal centrifugation revealed a polydisperse size distribution [range (5-30) x 10(6)] with a weight-average Mr of 17 x 10(6). The whole mucins were visualized with electron microscopy as linear and apparently flexible threads, disperse in size. Reduction produced subunits which were included on Sepharose CL-2B, and subsequent trypsin digestion yielded high-Mr glycopeptides which were further retarded. The size distributions and fragmentation patterns of mucin from two other CF patients were the same, as studied by gel chromatography, rate-zonal centrifugation and electron microscopy. We conclude that CF mucins are heterogeneous in both size and buoyant density and that the various populations, though differing in buoyant density, share the same architecture and macromolecular properties and are, in this respect, similar to mucins from normal respiratory secretions [Thornton, Davies, Kraayenbrink, Richardson, Sheehan & Carlstedt (1990) Biochem. J. 265, 179-186] and human cervical mucus [Carlstedt & Sheehan (1989) SEB Symp. XLIII 289-316].
Thornton
D J
DJ
Department of Biochemistry and Molecular Biology, University of Manchester, U.K.
Sheehan
J K
JK
Lindgren
H
H
Carlstedt
I
I
D000293:Adolescent; D000328:Adult; D002241:Carbohydrates / Q000737:chemistry; D002499:Centrifugation, Density Gradient; D002648:Child; D002850:Chromatography, Gel; D003550:Cystic Fibrosis / Q000378:metabolism* / Q000473:pathology; D004797:Enzyme-Linked Immunosorbent Assay; D005782:Gels / Q000737:chemistry; D006801:Humans; D008970:Molecular Weight; D009077:Mucins / Q000737:chemistry* / Q000302:isolation & purification / Q000648:ultrastructure; D011108:Polymers; D013183:Sputum / Q000737:chemistry*; D013329:Structure-Activity Relationship
D002241:Carbohydrates; D005782:Gels; D009077:Mucins; D011108:Polymers