Am. J. Respir. Cell Mol. Biol., Vol 12, No. 3, 03 1995, 296-306.
Mucus glycoconjugate complexes released from feline trachea by a bacterial toxin
DC Fung, M Somerville, PS Richardson and JK Sheehan
Department of Physiology, St. George's Hospital Medical School, London, United Kingdom.
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.
