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

Abstract

Deficiency of glucocerebrosidase (GBA), a lysosomal β-glucosidase, causes Gaucher disease. The enzyme hydrolyzes β-glucosidic substrates and transglucosylates cholesterol to cholesterol-β-glucoside. Here we show that recombinant human GBA also cleaves β-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 β-glucosidase activity were similarly impaired in β-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 β-xylosides generated XylChol in a manner dependent on active lysosomal GBA but not the cytosol-facing β-glucosidase GBA2. We later sought an endogenous β-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 β-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.

Xylose‐Configured Cyclophellitols as Selective Inhibitors for Glucocerebrosidase

“…Following the observation that GBA is capable to metabolize β‐ d ‐xylosides, [32] we were interested to determine whether xylose‐configured cyclophellitols can be exploited as GBA specific inhibitors. Our study revealed that xylo ‐configured cyclophellitol 1 is indeed a potent inhibitor of GBA and poorly reacts with GBA2 or GBA3 in vitro , in intact cells, and zebrafish larvae.…”

“…In fact, the labeling of GBA2 by a xylo ‐configured cyclophellitol aziridine 2 is somewhat surprising given the finding that GBA2 does not hydrolyze 4‐methylumbelliferyl‐β‐ d ‐xylose. [32] We therefore checked whether the xylo ‐cyclophellitol aziridine ABP 6 could label GBA2 at alternative sites other than the catalytic nucleophile, and found that it could label neither the catalytic nucleophile mutant (E527G substitution) nor a combined substitution of catalytic nucleophile and acid/base residue (E527G/D667G substitution), suggesting that the labeling still proceeds via the catalytic nucleophile, identical to that of the broad‐spectrum β‐ d ‐glucosidase cyclophellitol aziridine ABP 10 . Possibly, despite that xylose is not an ideal substrate sugar for GBA2, the aziridine is reactive enough to allow the covalent bonding of the xylo ‐configured cyclophellitol aziridine to the GBA2 nucleophile.…”

“… [31] GBA is also able to use β‐ d ‐xylosides as donors in transglycosylation reactions, generating xylosylcholesterol and di‐xylosylcholesterol. [32] In contrast to GBA, GBA2 is not active towards β‐ d ‐xylosides and the activity of GBA3 towards these substrates is very low. [32] It thus appears that the presence of the pendant CH 2 OH group that distinguishes β‐ d ‐glucosides from β‐ d ‐xylosides is a prerequisite for affinity for GBA2 and GBA3.…”

“… [32] In contrast to GBA, GBA2 is not active towards β‐ d ‐xylosides and the activity of GBA3 towards these substrates is very low. [32] It thus appears that the presence of the pendant CH 2 OH group that distinguishes β‐ d ‐glucosides from β‐ d ‐xylosides is a prerequisite for affinity for GBA2 and GBA3. The flexibility of GBA for substrates with a modification at the glucose‐C6 is also reflected by its selective reactivity with O8‐modified cyclophellitol‐based inhibitors and ABPs and with those of glucose‐C6 modified substrates.…”

Design, Synthesis and Structural Analysis of Glucocerebrosidase Imaging Agents

“…One issue surrounding ABPs is their occasional cross‐reactivity with related glycosidases. Of note, GBA can be by inhibited both gluco ‐ and xylo ‐configured substrates, with demonstrated activity against 4‐methylumbelliferyl‐β‐xyloside; [60] conversely, galacto ‐configured cyclophellitol epoxides have proved inactive against GBA. [61] Nevertheless, studies on other glycosidases have shown that galacto ‐configured inactivators occasionally bind to glucosidases.…”