Degradation of keratan sulphate by β-N-acetylhexosaminidases A and B

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Abstract
Enzymic cleavage of .beta.-N-acetylglucosamine residues of keratan sulfate was studied in vitro by using as substrate a [bovine cornea] [3H]glucosamine-labeled desulfated keratan sulfate with N-acetylglucosamine residues at the non-reducing end. Both lysosomal .beta.-N-acetylhexosaminidase A and B are proposed to participate in the degradation of keratan sulfate on the basis of the following observations. Homogenates of fibroblasts from patients with Sandhoff disease, but not those from patients with Tay-Sachs disease, were unable to release significant amounts of N-acetyl[3H]glucosamine. On isoelectric focusing of .beta.-N-acetylhexosaminidase from human liver the peaks of keratan sulfate-degrading activity coincided with the activity towards p-nitrophenyl .beta.-N-acetylglucosaminide. A monospecific antibody against the human enzyme reacted with both enzyme forms and precipitated the keratan sulfate-degrading activity. Both isoenzymes had the same apparent Km of 4 mM but the B form was approximately twice as active as the A form when compared with the activity towards a chromogenic substrate. Differences were noted in the pH-activity profiles of both isoenzymes. Thermal inactivation of isoenzyme B was less pronounced towards the polymeric substrate than towards the .pi.-nitrophenyl derivative.