Keratanase-sensitive cell surface glycans are synthesized by cultured tracheal epithelial cells

Canine tracheal epithelial cells grown in monolayer culture appear undifferentiated and are uniformly devoid of secretory granules. Cells at confluency were radiolabeled with [35S]SO4 and washed extensively with fresh unlabeled medium (2 h). Brief trypsin treatment of the cells caused release of 35S-labeled products which were harvested by immediate transfer at 40C into isolation buffer containing protease inhibitors and 0.4 mM saccharo-1,4-lactone and 2-acetamido-galactonolactone to minimize endogenous protease and glycosidase activities. Gel filtration on Sepharose C14B in associating conditions yielded two 35S fractions, one with apparent MW≥106 da and another at 3x105 da (Kavg=0.39). β-elimination in 0.05 M NaOH/1.0 M NaBH4 yielded 35S products of greatly reduced MW (10,000 da) which were excluded from Bio-Gel P-10, characteristics of glycosaminoglycan chains. This sulfated fraction was resistant to chondroitinase ABC, heparinase and heparan sulfate lyase. However, it was completely degraded to oligosaccharides (2000 to ≥500 da) by endo-β-galactosidase or keratanase. This suggests that sulfate is carried on glycosaminoglycan chains which have a polylactosamine repeating unit typical of keratans and which are attached to protein moiety through alkali-labile linkage. Immunofluorescence studies using a monoclonal antibody directed to a lactosamine sequence show intense localized staining of the tracheal epithelial surface