Analysis of N-acetylgalactosamine-4-sulfatase protein and kinetics in mucopolysaccharidosis type VI patients.

A sensitive and specific, monoclonal antibody-based immunoquantification assay has facilitated determination of the N-acetylgalactosamine-4-sulfatase (4-sulfatase) protein content in cultured fibroblasts from normal controls and mucopolysaccharidosis type VI (MPS VI) patients. The assay enabled the quantification of 4-sulfatase protein by using a panel of seven monoclonal antibodies and has shown that fibroblasts from 16 MPS VI patients contained less than or equal to 5% of the level determined for normal controls. Fibroblasts from the most severely affected patients contained the lowest levels of 4-sulfatase protein, usually with few epitopes detected, while fibroblasts from mildly affected patients had higher levels of 4-sulfatase protein, with all seven epitopes detected. The pattern of epitope expression is proposed to reflect the conformational changes in the 4-sulfatase protein that arise from different mutations in the 4-sulfatase gene. Immunoquantification in combination with a specific and highly sensitive 4-sulfated trisaccharide-based assay of enzyme activity in these MPS VI patient fibroblasts enabled the determination of residual 4-sulfatase catalytic efficiency (kcat/Km). The capacity of fibroblasts to degrade substrate (catalytic capacity) was calculated as the product of 4-sulfatase catalytic efficiency and the content of 4-sulfatase in fibroblasts. One patient, 2357, with no clinical signs of MPS VI but with reduced 4-sulfatase activity and protein (both 5% of normal) and dermatansulfaturia, had 5% of normal catalytic capacity. The other 15 MPS VI patient fibroblasts had 0%-1.4% of the catalytic capacity of fibroblasts from normal controls and were representative of the spectrum of MPS VI clinical phenotypes, from severe to mild.(ABSTRACT TRUNCATED AT 250 WORDS)