Alessandra d'Azzo

The gene encoding human protective protein (PPGB) is on chromosome 20

Normal lymphocyte prometaphase chromosome spreads were hybridized in situ using single- and double-color fluorescence techniques. The results obtained with either the 1.8-kb protective protein cDNA or a 12-kb genomic fragment of the human protective protein gene as probe demonstrate that the PPGB gene is localized on the long arm of chromosome 20. This assignment was confirmed by hybridization with whole chromosome DNA libraries.

Correction of combined β-galactosidase/neuraminidase deficiency in human fibroblasts

The combined deficiency of β-galactosidase and neuraminidase in human fibroblasts can be corrected to nearly normal values. This can be accomplished by addition of concentrated culture medium obtained after NH4Cl stimulation of different types of human fibroblasts, including those with an isolated β-galactosidase or neuraminidase deficiency. The corrective factor is a macromolecular glycoprotein, which is labile at 60°C. Its uptake by human fibroblasts is competitively inhibited by mannose-6-phosphate and its corrective action within β-gal−/neur− fibroblasts continues during a “chase” of 72 hours.

The presence of a reduced amount of 32-kd “protective” protein is a distinct biochemical finding in late infantile galactosialidosis

SummaryThe biochemical defect underlying the late infantile form of galactosialidosis has been investigated in fibroblasts from two patients presenting with this phenotype. Immunoprecipitation experiments demonstrated that a reduced amount of 32-kd “protective” protein and a normal amount of its precursor are present in late infantile galactosialidosis fibroblasts, while neither of the two polypeptides are detectable in early infantile and juvenile/adult fibroblasts. Leupeptin treatment led to a slight increase in the amount of 54-kd and 32-kd polypeptides in both late-infantile galactosialidosis cell lines. Uptake studies in one of the two cell lines confirmed the hypothesis that a block in the maturation of the protective protein is responsible for the late infantile type of galactosialidosis. This mutation seems to be a distinct finding in all patients affected by this form of the disease.

Evidence for the deficiency of β-glucosidase-activating factor in fibroblasts of patients with I-cell disease

SummaryReduced activity of β-glucosidase was shown in the cultured skin fibroblasts of four patients with I-cell disease when the enzyme was tested without the use of detergents. In the presence of taurocholate and triton X100 β-glucosidase activity was normal. This suggested a deficiency of a β-glucosidase-activating factor in I-cell fibroblasts rather than of the enzyme itself. The deficiency of β-glucosidase activity was corrected to some extent by mixing cell lysates, and more effectively by cocultivation and fusion of I-cell disease and Gaucher fibroblasts. These results present evidence for the presence of a β-glucosidase-activating factor in normal and Gaucher fibroblasts. In fibroblasts of patients with I-cell disease this activator is probably deficient, as is the case for most lysosomal enzymes.