HUMAN GLUCOSAMINE-6-SULFATASE DEFICIENCY - DIAGNOSTIC ENZYMOLOGY TOWARDS HEPARIN-DERIVED TRISACCHARIDE SUBSTRATES

Glucosamine-6-sulphatase (6S) activity towards a series of radiolabelled heparin-derived trisaccharide substrates was determined in cultured human skin fibroblast and leucocyte homogenates, and in urine supernatants of normal individuals and patients affected with 6S deficiency [Sanfilippo D syndrome; mucopolysaccharidosis (MPS) type IIID]. The N-sulphated and N-acetylated derivatives of the trisaccharide substrate O-(alpha-glucosamine 6-sulphate)-(1 --> 4)-L-O-(alpha-iduronic acid 2-sulphate)-(1 --> 4)-D-O-2,5-anhydro[1-H-3]mannitol 6-sulphate (GlcNH6S-IdoA2S-anM6S) were prepared by enzymic digestion of a pentasulphated tetrasaccharide isolated following the HNO2 deamination of heparin. Purified lysosomal enzymes and MPS-patient skin fibroblasts were used along with chemical degradation to confirm the structure of each of the substrates that were utilized to study the interaction of the enzyme activities required to degrade the highly sulphated regions of heparan sulphate. Human liver, skin fibroblast and urine 6S activities were separated by chromatofocusing into at least four and possibly up to six individual activities. 6S activities present in each of the tissues generally had similar catalytic properties, including K(m) values, pH optima and inhibition with NaCl, Na2SO4 and NaH2PO4. Leucocyte and skin fibroblast 6S activities towards GlcNAc6S-IdoA2S-anM6S were maximal at pH 4.1 and 3.9 respectively, with K(m) values of 2.8-mu-M and 0.9-1.7-mu-M respectively. Urine 6S activity towards GlcNAc6S-IdoA2S-anM6S was stimulated 30-fold by BSA at pH 3.9, which shifted the pH optimum from 5.1 to 4.2 and decreased the K(m) value at pH 4.2 from 4.0-mu-M to 0.5-mu-M. Residual 6S activity present in the skin fibroblast homogenates from MPS IIID patients was characterized for activity towards GlcNAc6S-IdoA2S-anM6S and observed to have similar pH optima and K(m) values to normal skin fibroblast 6S activities, although the residual 6S activity was less than 1 % of the normal control range.