Mucus glycoconjugate complexes released from feline trachea by a bacterial toxin.
This paper describes low-density mucus glycoconjugates released from feline trachea by dirhamnolipid (DRL), a toxin from Pseudomonas aeruginosa. Mucus glycoconjugates in feline tracheas were radiolabeled in vivo with 3H-proline and 14C-glucose. Control mucus and that released by 200 micrograms/ml DRL were dissolved in guanidine hydrochloride buffer (GuHCl) and chromatographed on Sepharose CL-2B. Molecules eluting in the void volume (V0) of the column were isolated by isopycnic density gradient centrifugation in CsCl/GuHCl. All samples gave peaks of radiolabeled and periodic acid/Schiff (PAS)-reactive material at rho = approximately 1.50 and approximately 1.60 g/ml, but DRL-stimulated samples contained low-density material (rho < 1.32 g/ml), also PAS-reactive and radiolabeled. Control secretions incubated with DRL in vitro did not form low-density material. In Triton X-100 (1% vol/vol), a nonionic detergent, low-density material behaved as smaller molecules, running in the partially included volume (Vi) of the column of Sepharose CL-2B, but still in the V0 of Sephacryl S-300. Incubation with chondroitinase ABC, heparinase II and III, and keratanase failed to change its elution profile on S-300, evidence against glycosaminoglycans; but proteolysis with trypsin or proteinase K gave two peaks, peptide fragments near the totally included volume of the column and glycopeptides in V0. The V0 glycopeptides banded between 1.50 and 1.55 g/ml in a CsCl gradient and eluted as a single peak in the Vi of Sephacryl S-400, suggesting a distinct homogeneous glycopeptide, smaller than those from normal mucins. The main 14C-labeled sugars in this glycopeptide were fucose, glucosamine, galactosamine, and galactose, consistent with a mucin. Thus, DRL releases stable but noncovalent complexes containing one or more distinct mucinlike glycoconjugates, probably combined with lipids and peptides. We discuss their possible relevance to airway diseases, including cystic fibrosis.
Fung
D C
DC
Department of Physiology, St. George's Hospital Medical School, London, United Kingdom.
Somerville
M
M
Richardson
P S
PS
Sheehan
J K
JK
D000818:Animals; D001427:Bacterial Toxins / Q000494:pharmacology*; D002415:Cats; D005260:Female; D006001:Glycoconjugates / Q000378:metabolism*; D006017:Glycolipids / Q000494:pharmacology*; D008297:Male; D008970:Molecular Weight; D009093:Mucus / Q000737:chemistry / Q000378:metabolism*; D017830:Octoxynol / Q000494:pharmacology; D011550:Pseudomonas aeruginosa; D013501:Surface-Active Agents; D014132:Trachea / Q000737:chemistry* / Q000187:drug effects*
D001427:Bacterial Toxins; D006001:Glycoconjugates; D006017:Glycolipids; D013501:Surface-Active Agents; C051310:rhamnolipid hemolysin, Pseudomonas aeruginosa; D017830:Octoxynol