Human glucocerebrosidase mediates formation of xylosyl-cholesterol by ß-xylosidase and transxylosidase reactions.

Deficiency of glucocerebrosidase (GBA), a lysosomal ß-glucosidase, causes Gaucher disease. The enzyme hydrolyzes ß-glucosidic substrates and transglucosylates cholesterol to cholesterol-ß-glucoside. Here we show that recombinant human GBA also cleaves ß-xylosides and transxylosylates cholesterol. The xylosyl-cholesterol formed acts as an acceptor for the subsequent formation of di-xylosyl-cholesterol. Common mutant forms of GBA from patients with Gaucher disease with reduced ß-glucosidase activity were similarly impaired in ß-xylosidase, transglucosidase, and transxylosidase activities, except for a slightly reduced xylosidase/glucosidase activity ratio of N370S GBA and a slightly reduced transglucosylation/glucosidase activity ratio of D409H GBA. XylChol was found to be reduced in spleen from patients with Gaucher disease. The origin of newly identified XylChol in mouse and human tissues was investigated. Cultured human cells exposed to exogenous ß-xylosides generated XylChol in a manner dependent on active lysosomal GBA but not the cytosol-facing ß-glucosidase GBA2. We later sought an endogenous ß-xyloside acting as donor in transxylosylation reactions, identifying xylosylated ceramide (XylCer) in cells and tissues that serve as donor in the formation of XylChol. UDP-glucosylceramide synthase (GCS) was unable to synthesize XylChol but could catalyze the formation of XylCer. Thus, food-derived ß-D-xyloside and XylCer are potential donors for the GBA-mediated formation of XylChol in cells. The enzyme GCS produces XylCer at a low rate. Our findings point to further catalytic versatility of GBA and prompt a systematic exploration of the distribution and role of xylosylated lipids.