Early proteolytic cleavage with loss of a C-terminal fragment underlies altered processing of the beta-galactosidase precursor in galactosialidosis.

Processing of human beta-galactosidase (beta-GAL) was studied in permanently transfected Chinese hamster ovary (CHO) cells and compared with that in normal cells and in cells from subjects with GM1-gangliosidosis, galactosialidosis and I-cell disease. Biosynthesis of beta-GAL in CHO cells results in the synthesis of an 88 kDa glycosylated and phosphorylated monomer precursor which is enzymically active and is secreted into the medium. Post-translational processing begins at the C-terminal end of the protein and gives rise to structurally related 67 and 64 kDa mature forms. These are subsequently degraded to give several inactive products of which a 50 kDa and a 18 kDa species are prominent. In normal fibroblasts only the 84 kDa precursor is readily detected inside cells, while the 88 kDa precursor is the only form secreted from cells in the presence of ammonium chloride. Processing of the precursor in normal fibroblasts results in the appearance of both the 67 and 64 kDa mature forms, which are also degraded to give 50 and 18 kDa products, as in transfected CHO cells. As affected controls, GM1-gangliosidosis cells showed a general loss of all forms of the enzyme, while in I-cell fibroblasts only the 84 kDa precursor and an 18 kDa degradation form were prominent. In galactosialidosis fibroblasts, taken from two different subjects, processing of beta-GAL was characterized by the respective appearance of intermediate 80 and 72 kDa enzymically inactive polypeptides, at levels lower than the normal amounts of the 67 and 64 kDa mature forms and higher than the normal amounts of degradation products, one of which is of 45 kDa and arises by endoproteolytic cleavage of the 80 kDa polypeptide. Incubation for up to 72 h in medium containing leupeptin, a potent inhibitor of thiol-dependent proteases, resulted in a significantly increased level of beta-GAL activity to near normal levels in fibroblasts from one galactosialidosis subject. Concordant with this, the abundance of the 84 kDa precursor was increased and the levels of the 80 kDa, 45 kDa and 18 kDa digestion products were diminished. However, in fibroblasts from the second galactosialidosis subject, the amount of the abnormal 72 kDa polypeptide was not influenced by leupeptin treatment. Leupeptin treatment did not increase enzymic activity levels in normal, GM1-gangliosidosis or I-cell disease fibroblasts, despite the fact that the production of the 50 kDa and 18 kDa degradation products was blocked in the presence of leupeptin. We concluded that in galactosialidosis the leupeptin-inhibitable proteolytic cleavage of a small fragment causes a conformational change of the precursor that precludes its further normal processing and results in its enzymic deficiency. This early abnormal trimming of beta-GAL is ascribable to a deficiency in the functional protective protein, the function of which is absolutely essential to render beta-GAL cryptic from at least two distinct and separate proteolytic attacks that together remove at least 12 kDa from the C-terminal end of the enzyme.