Abstract
Of 14 identified epitopes on human GCase (acid beta-glucosidase), monoclonal antibodies (MCABs) recognizing 3 produced inhibition and 1 resulted in activation of GCase. MCABs F1 and F2 completely, and MCAB 61 partially (approximately 70%), inhibited GCase activity. Substrates and active site-directed inhibitors (specific sphingolipid and 5-amino-5-deoxyglucose derivatives) protected the enzyme from inhibition by MCAB F1 and F2, but not that by MCAB 61. Conduritol B epoxide did not protect GCase from the inhibition by these MCABs when covalently bound to the active site. These results indicated highly specific binding requirements of MCABs F1 and F2 for residues in a complex active site. In comparison, kinetic analyses using GCase transition state analogues, N-alkyl-glucosylamines, and MCAB 61 demonstrated that this MCAB "freezes" the conformation of the enzyme and inhibits GCase by preventing formation of a conformer needed for maximal catalytic rates. The activating MCAB 122 mimicked the effects of saposin C and competed with this natural activator for residues on the enzyme. Interaction of saposin A and saposin C or MCAB 122 with GCase produced a synergistic effect leading to a marked sensitization of the enzyme to these activators. No such synergism or additivity was found for the maximal catalytic rate since it could be achieved by saturating amounts of any one or combinations of these activators. In the presence of MCAB 61, only 15 to 25% of the maximal activation of GCase was obtained by saposin C or MCAB 122, indicating that the major activation effects of these effectors derived from an induction of a GCase conformational change. These results demonstrate that saposins A and C mediate their activating effects by binding to distinct sites on GCase. Furthermore, major components of the mechanisms for catalysis and saposin C activation are due to conformational changes during the transition state. These findings have implications for understanding the perturbations of GCase function due to the missense mutations which cause Gaucher disease.
Highlights
From the Division of Medical and Molecular Genetics, Departmentof Pediatrics, Mount Sinai Schoolof Medicine, New York, New Y o f k 10029-6574
We developed monoclonal antibodr ies (MCABs) to human GCase and used them to investigate the active site and the binding sites for saposins C and A
Effects of MCAB 61 on GCase Activity-Due to the partial inhibition induced by MCAB 61 and thevery rapid rate with which this MCAB binds to and inhibits GCase, studies with this antibody were conducted after maximal inhibition was obtained
Summary
From the Division of Medical and Molecular Genetics, Departmentof Pediatrics, Mount Sinai Schoolof Medicine, New York, New Y o f k 10029-6574. When phosphatidylserine was added to GCase-MCAB 122 complexes, the same degree of activation (about 12- to 15-fold) was obtained as in the presence of this negatively charged lipid alone.
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