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Activity-based probes for retaining β-: Novel tools for research and diagnostics

Kallemeijn, W.W.

Publication date 2014

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Citation for published version (APA): Kallemeijn, W. W. (2014). Activity-based probes for retaining β-glucosidases: Novel tools for research and diagnostics.

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Download date:01 Oct 2021 Chapter 4

Unambiguous identification of the acid/base and nucleophile in all human retaining β-glucosidases by activity-based probes

Wouter W. Kallemeijna, Martin D. Witteb,c, Tineke M. Voorn-Brouwera, Marthe T. C. Walvoortb,d, Jeroen D. C. Codéeb, Gijsbert A. van der Marelb, Rolf G. Boota, Herman S. Overkleeftb and Johannes M. F. G. Aertsa

Under review

Graphical abstract – Identification of the key acid/base and nucleophile residues in retaining β-glucosidases via site-directed mutagenesis of putative catalytic residues, then azide-mediated rescue of the enzymatic activity and by activity-based probe-labeling.

155 Chapter 4

Abstract Mammalian glycoconjugate catabolism is executed by an array of glycosidases, each tailored to hydrolyze specific endogenous or natural exogenous substrates. Retaining β- glucosidases operate by a mechanism in which the key amino acids driving glycosidic bond hydrolysis act as catalytic acid/base and nucleophile. Recently we designed two distinct classes of fluorescent cyclophellitol-type activity-based probes (ABPs) that exploit this mechanism to covalently modify the nucleophile in retaining β-glucosidases. Where cyclophellitol β-epoxide ABPs require a protonated acid/base for irreversible inhibition, i.e. labeling, β-aziridine ABPs do not. Here we illustrate the use of ABPs to unambiguously identify the key acid/base and nucleophile residues in the retaining β-glucosidases expressed by man. After site-directed mutagenesis of residues putatively involved in the reaction mechanism, rescue of enzymatic activity with sodium azide enables the identification of catalytic residues. Subsequently, the acid/base is identified by ABPs. The method was validated on GBA (CAZy glycosylhydrolase family GH30) and then applied to non-homologous (putative) β-glucosidases from GH1: GBA3, LPH, klotho, βklotho and KLPH. Finally, we identified the acid/base and nucleophile in non-lysosomal β-glucosidase GBA2 (GH116), recently implicated in apoptosis, oncogenesis and neurological disorders.

Author affiliations aDepartment of Medical Biochemistry, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands. bDepartment of Bio-organic Synthesis, Leiden Institute of Chemistry, Leiden University, Leiden, The Netherlands. cDepartment of Bio- Organic Chemistry, Stratingh Institute for Chemistry, University of Groningen, Groningen, The Netherlands. dDepartment of Biology and Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America.

156 Catalytic residue identification with ABPs

etaining β-glucosidases hydrolyze β-D-glucose from various endogenous and natural exogenous substrates. In man, the documented retaining β-glucosidases R fulfill important physiological functions while belonging to different CAZy glycosylhydrolase families (CAZy GH1, GH30 and GH116, see Fig 1a); a classification based on their amino acid sequence, catalytic mechanism, structure and active-site residues1. Lysosomal (GBA, in CAZy GH30) degrades glucosylceramide and its deficiency causes Gaucher disease, a relatively common lysosomal storage disorder2−4. In addition, carriers of GBA mutations have recently been shown to be at increased risk for developing Parkinsonism5, 6. Non-lysosomal β-glucosidase (GBA2, in GH116) converts glucosylceramide to ceramide in the cytosol7−9 and has been implicated in ceramide-driven apoptosis10. GBA2 deficiency was recently also linked to a neurological disorder11, 12. Cytosolic β-glucosidase (GBA3, in GH1) hydrolyzes xenobiotic β-glucosides13, 14. GBA3 deficiency is not associated with a health risk15. Deficiency of -phlorizin (LPH, in GH1), an intestinal containing a functional β-glucosidase pocket next to a β-galactosidase pocket, causes lactose intolerance16–19. Finally, klotho, βklotho and KLPH (γklotho) are homologous to LPH and GBA3 and also categorized in CAZy family GH1, but all lack functional β-glucosidase activity20–25. Klotho has been reported to have β-glucuronidase activity towards artificial substrates when fused with an extracellular domain24, 25, while sialidase activity has also been reported26. The klotho- proteins interact with FGFs and FGF-receptors, modulating endocrine axes that regulate various metabolic processes27. Klotho is involved in processes20, whereas βklotho is essential in bile acid homeostasis and activity of brown adipose tissue21. A disease condition has not been associated with defects in KLPH. Despite absence of structural homology between β-glucosidases from GH1, 30 and 116, all hydrolyze glucosidic substrates through a double-displacement mechanism, driven by a discriminate nucleophile and acid/base residue (Fig 1a, b, next page)18. The nucleophile attacks the anomeric C1 carbon of the β-D-glucose moiety while the acid/base residue protonates the inter-glycosidic oxygen, resulting in aglycone release and concomitant nucleophile glycosylation28. Next, enzyme deglycosylation is promoted by the activation of an incoming water nucleophile by the deprotonated acid/base, thereby completing the hydrolysis event. Recently we reported the development of two different classes of activity-based probes (ABPs) for retaining β-glucosidases29, 30. The first class concerns cyclophellitol β-epoxides functionalized with a fluorescent BODIPY moiety grafted to the C6 position (MDW933, or ABP 1, see Fig 1c)29. They bind irreversibly to the nucleophile E340 of GBA in an acid/base- dependent manner (Fig 1d)29, 30. Cyclophellitol β-aziridines functionalized with a BODIPY group grafted to the aziridine nitrogen comprise the other class (MDW1044, or ABP 2, see Fig 1c)30, and bind nucleophiles of various β-glucosidases, independently of the acid/base status (Fig 1e)30. An elegant method has been developed in the past to identify these key residues, by using sodium azide as an external acid/base and/or nucleophile, allowing

157 Chapter 4

Figure 1 | Background. (a) Protein of all retaining β-glucosidases expressed in man, categorized in glycosylhydrolase families GH1, 30 and 116. (b) Double displacement mechanism of retaining β-glucosidases, with distinct acid/base (A/B, top) and nucleophile (N, bottom). (c) Structure formulas of cyclophellitol β-epoxide ABP 1 (MDW93329) and β- aziridine ABP 2 (MDW104430) and their nucleophile-binding mechanisms (d, e, respectively). Azide-mediated rescue of hydrolysis in case of absent acid/base (f) or nucleophile (g). catalytic activity rescue of enzyme molecules with a mutant catalytic residue (Fig 1f, g)31. Moreover, identification of the acid/base and nucleophile is achievable by determining the product’s stereochemistry. In retaining β-glucosidases, sodium azide-mediated rescue results retention of stereochemistry in case of acid/base absence (β-bond, Fig 1f) and inversion of stereochemistry when the nucleophile is lacking (α-bond, Fig 1g)31. In a series of related studies, Withers and co-workers have employed 2-deoxy-2- fluoroglycosides to identify the nucleophile of various retaining β-glycosidases, by (temporarily) trapping the nucleophile in a glycosyl-enzyme intermediate31−36. Trapping occurs due to the substitution of the C2-hydroxyl by fluorine, which markedly destabilizes the positive charge developed during the oxocarbenium ion-like transition states flanking the covalent glycosyl-enzyme intermediate, thereby effectively reducing the catalytic rates involved in the double-displacement mechanism by up to 107-fold. Introduction of ‘good’ aglycones, i.e. leaving groups with low pKa, such as 2,4- dinitrophenyl (pKa ~4.7), or fluorine (pKa ~3.2), result in faster formation of the glycosyl-

158 Catalytic residue identification with ABPs enzyme intermediate. However, the activated 2-deoxy-2-fluoroglucoside substrate can be hydrolyzed poorly but independently in absence of the acid/base, and the glycosyl-enzyme adduct hydrolyzes slowly over time31−36. We here report a convenient and sensitive method for identification of the acid/base and nucleophile residues in all known human retaining β-glucosidases. Our approach entails a combination of site-directed mutagenesis, subsequent identification of putative catalytic residues by sodium azide-mediated rescue of catalysis, and finally discrimination between the acid/base and nucleophile by ultra-sensitive detection of ABP-labeled β- glucosidases. The method was validated using GBA (GH30), and then applied to identify the catalytic residues of β-glucosidase(-like), non-homologous retaining β-glucosidases expressed in man (GBA3, LPH, klotho, βklotho, KLPH from GH1, and GBA2 from GH116).

Results

Proof of principle – The nucleophile (E340) and acid/base (E235) of GBA have been identified unequivocally (Fig 2a)29, 37. To validate our envisioned method, GBA isoforms with substituted nucleophile and/or acid/base residues were generated. For this purpose, COS-7 cells were transfected with constructs expressing myc/His-tagged human GBA or

Figure 2 | Proof-of-principle − Rescue of substrate hydrolysis. (a) Schematic depicting GBA with active-site pocket presenting acid/base E235 (left, grey) and nucleophile E340 (right, black). (b) Structure formulas of 4MU−, 4NP− and 2,4DNP- β-glucopyranoside (Glc). (c) Catalytic activity of GBA variants towards 4MU−, 4NP− and 2,4DNP-β-D-Glc relative to mock (black, hatched, white columns, respectively). Sodium azide-mediated rescue of hydrolysis of 4MU− (d), 4NP− (e) and 2,4DNP-β-D-Glc (f) by mock (empty plasmid; ¢–¢), wild-type GBA (●–●), E235G (p–p), E235Q (●–●), E340G (n–n), E340Q (u–u) and E235G/E340G (n–n). Data are average of triplicates, ± SD, with one-way ANOVA significance p < 0.05*, p < 0.001***.

159 Chapter 4 variants with E235G, E235Q, E340G, E340Q or E235G/E340G substitutions. All mutagenized GBA proteins were deficient in enzymatic activity towards artificial 4MU−, 4NP− and 2,4DNP-β-D-glucopyranoside (Glc) substrates (Fig 2b). Next, sodium azide was used to rescue the activity of GBA lacking either key residue, as sodium azide has been reported to act as external acid/base or nucleophile (Fig 1f, g)31, 38. Titration with sodium azide did not result in enhanced hydrolysis rate (rescue) of the acid/base and nucleophile mutants in combination with fluorogenic 4MU-β-D-Glc (Fig 2c). Partial, though significant rescue was observed with 4NP-β-D-Glc, exempting the acid/base and nucleophile-lacking E235G/E340G GBA (Fig 2b). The most pronounced rescue was achieved with 2,4DNP-β-D- Glc (Fig 2e). This can readily be explained by considering that 4MU and 4NP are inferior leaving groups to 2,4DNP, as its pKa is markedly lower (~4.08) compared to 4MU and 4NP (~7.79 and ~7.15, respectively)39−41; the lower the pKa, the weaker the base, the better the leaving group. At high concentrations, sodium azide severely impaired activity of GBA (see for instance Fig 2d), through a mixed type of competitive reversible− and linear-mixed inhibition (data not shown). To discriminate the acid/base from the nucleophile residue, cell lysates were labeled with β-epoxide ABP 1 or β-aziridine ABP 2 and visualized by SDS-PAGE and scanning (Fig 3a). β-Epoxide ABP 1 requires a priori a protonated acid/base and thus did not label E235G or E235Q GBA at their nucleophilic residues. Lack of a nucleophile in E340G and E340Q GBA inherently does not allow labeling. Addition of sodium azide restored ABP 1 labeling of E235G and E235Q GBA variants. β-Aziridine ABP 2 bound to the nucleophile E340 regardless of the acid/base status in GBA molecules (Fig 3a). Substitutions of E340 in GBA to G340 or Q340 inherently prevented labeling by ABPs (Fig 3a). Finally, lysates were ABP-

Figure 3 | Proof-of-principle − ABP labeling. (a) Labeling of GBA variants over-expressed in COS-7 cells, with excess β- epoxide ABP 1 (top half) or β-aziridine ABP 2 (bottom half); labeling in total lysates (first gel); ABP-labeling of GBA variants in the absence and presence of sodium azide (second and third gels) as detected after His-tag pull-down. Post pull-down α-myc loading control (bottom gel). (b) β-Epoxide ABP 1 and β-aziridine ABP 2 labeling at different sodium azide concentrations. Inter- gel comparisons facilitated by imiglucerase (asterisk; ❉) as positive control.

160 Catalytic residue identification with ABPs labeled in combination with a range of azide concentrations (Fig 3b). Again, no labeling of nucleophile mutants was detected, but binding of E235G, E235Q was complete from 0.01 M azide on (Fig 3b), sharply contrasting with the maximal rescue of 2,4DNP-β-D-Glc hydrolysis in combination with 0.5–1 M sodium azide (Fig 2d). Where ABP labeling of the β-glucosidase nucleophile requires a single reaction step (Fig 1d, e), substrate hydrolysis in the presence or absence of an acid/base involves two mechanistic steps (Fig 1a, f). Kinetic rates are strongly affected by protein-fold, active-site spaciousness and enzymatic inhibition by azide presence (at higher concentrations). Apparently, ABP labeling is less influenced by these factors even in the process of sodium azide-mediated rescue, precipitating it as a superior strategy to visualize acid/base mutants.

Application to GH1 family member β-glucosidase GBA3 – Next, we applied the above analysis strategy to human proteins categorized in GH family 1: GBA3, LPH, klotho, βklotho and KLPH (Fig 4a). Crystallography studies indicated that in GBA3 E165 and E373 function as acid/base and nucleophile, respectively42. Mutagenized GBA3 containing E165G and/or E373G eliminated activity towards 4MU−, 4NP− and 2,4DNP-β-D-Glc (Fig 4b). Titration with sodium azide resulted in a minor but significant rescue of E165G and E373G-containing GBA3 towards 4NP-β-D-Glc, indicating their involvement in substrate catalysis (Fig 4c). A more evident rescue of activity was observed by using 2,4DNP-β-D-Glc

Figure 4 | Cytosolic β-glucosidase GBA3. (a) Schematic depicting GBA3 with predicted acid/base (left, grey) and nucleophile (right, black). (b) Catalytic activity of GBA3 variants towards 4MU−, 4NP− and 2,4DNP-β-D-Glc relative to mock (black, hatched, white columns, respectively). Sodium azide-mediated rescue of 4NP− (c) and 2,4DNP-β-D-Glc (d) hydrolysis by mock (empty plasmid; ¢–¢), wild-type GBA3 (●–●), E165G (p–p), E373Q (●–●) and E165G/E373G (n–n). (e) Labeling of GBA3 variants over-expressed in COS-7 cells, with excess β-epoxide ABP 1 (top half) or β-aziridine ABP 2 (bottom half); labeling in total lysates (first gel); ABP-labeling of GBA3 variants in the absence and presence of sodium azide (second and third gels) as detected after His-tag pull-down. Post pull-down α-myc loading control (bottom gel). Data are average of triplicates, ± SD, with one-way ANOVA significance p < 0.001***.

161 Chapter 4 as substrate (Fig 4d). Substitution of E373 furthermore prevented labeling by ABP 1 and 2, whilst substitution of E165 allowed labeling by ABP 2, and labeling by ABP 1 in the presence of sodium azide (Fig 4e). Our results indicate that indeed E373 is the nucleophile and E165 the acid/base residue in GBA3.

Elucidation of β-glucosidase acid/base and nucleophile of LPH – Lactase/phlorizin hydrolase contains four domains with high homology with GBA3 and other GH 1 family members16−18. Only two domains of LPH, coded III and IV, have functional catalytic pockets with acid/bases E1065 and E1538, and nucleophiles E1273 and E1749, respectively (Fig 5a)16−18, 43. Pocket III possesses β-glucosidase activity, whereas pocket IV catalyzes hydrolysis of β-galactosides including lactose16−18, 43. We earlier noted that LPH can be labeled by ABPs 1 and 229, 30, but the interacting nucleophiles involved were not established. Substitution of acid/base E1065 or nucleophile E1273 to a glycine reduced LPH enzyme activity towards 4MU−, 4NP−, and 2,4DNP-β-D-Glc substrates by 89 to 93% (Fig 5b). Sodium azide restored activity of E1065− and E1273-substituted LPH towards 2,4DNP-β- D-Glc (Fig 5c) and to a lesser extent to 4NP-β-D-Glc. Release of D-glucose from lactose is 92−96% decreased by the E1538G and E1749G substitutions in pocket IV (Fig 5d).

Figure 5 | Lactase/phlorizin hydrolase LPH. (a) Schematic depicting LPH with predicted acid/base (E1065, E1538; light grey sticks) and nucleophile (E1273, E1749; black sticks) in pockets III and IV. (b) Catalytic activity of LPH variants towards 4MU−, 4NP− and 2,4DNP-β-D-Glc relative to mock (black, hatched, white columns, respectively). (c) Sodium azide-mediated rescue of 2,4DNP-β-D-Glc hydrolysis by mock (empty plasmid; ¢–¢), wild-type LPH (●–●), E1065G (p–p), E1273G (●–●), E1538G (p– p), E1749G (♢–♢) and E1273G/E1749G (n–n). (d) β-D-Gal activity of LPH variants, relative to mock, measured as liberated β- D-Glc. (e) Labeling of LPH variants over-expressed in COS-7 cells, with excess β-epoxide ABP 1 (top half) or β-aziridine ABP 2 (bottom half); labeling in total lysates (first gel); ABP-labeling of LPH variants in the absence and presence of sodium azide (second and third gels) as detected after His-tag pull-down. Post pull-down α-myc loading control (bottom gel). Data are average of triplicates, ± SD, with one-way ANOVA significance p < 0.05*, p < 0.001***.

162 Catalytic residue identification with ABPs

Substitution of catalytic residues in pocket III, E1065G and E1273G, also affect the hydrolysis of lactose in pocket IV (reduction by 63−67%), similar to previously published data (Fig 5d)43. In contrast, LPH molecules with substitutions in pocket IV (E1538G and E1749G) show normal hydrolysis of 2,4DNP-β-D-Glc in pocket III (Fig 5b, c). Similarly, LPH molecules with substitutions in both pockets III and IV are completely restored by sodium azide to wild type β-glucosidase activity (Fig 5c). Labeling with β-epoxide ABP 1 of G1065 LPH only occurred in the presence of sodium azide, confirming E1065 as acid/base residue (Fig 5e). G1273 LPH was not bound by ABP 1 in the absence or presence of sodium azide (Fig 5e), while its enzymatic activity is rescued (2,4DNP-β-D-Glc, Fig 5c), indicating that E1273 is indeed the nucleophile. Pocket IV of LPH seems not targeted by ABPs 1 or 2 since mutagenesis of its nucleophile and acid/base residue (E1538 and E1749, respectively) did not influence ABP labeling (Fig 5e) nor resulted in a significantly reduced β-glucosidase activity (Fig 5b). It seems therefore that substitutions in pocket IV have little impact on pocket III, but

Figure 6 | Klotho, βklotho and KLPH. (a) Schematic depicting klotho, βklotho and KLPH with putative acid/bases (grey) and nucleophiles (black) in predicted pockets. (b) Labeling of klotho, βklotho, KLPH and positive controls (wild-type and E235G GBA) over-expressed in COS-7 cells, with excess β-epoxide ABP 1 (top half) or β-aziridine ABP 2 (bottom half); labeling in total COS-7 lysates (first gel); ABP-labeling in the absence and presence of sodium azide (second and third gels) as detected after His-tag pull-down. Post pull-down α-myc loading control (bottom gel).

163 Chapter 4 intriguingly not vice versa. Wild type LPH is irreversibly inhibited by ABPs 1 and 2 in its hydrolysis of 2,4DNP-β-D-Glc. Low IC50 values are observed (1.3−1.4 and 0.19−0.21 μM for ABP 1 and 2, respectively). In contrast, ABP 1 and ABP 2 are no potent irreversible inhibitors of lactose hydrolysis mediated by pocket IV, again indicating that the activity- based probes interact exclusively with pocket III of LPH.

CAZy GH1 members klotho, βklotho and KLPH – Sequence analysis indicates that klotho and βklotho possess two pockets and KLPH one. Moreover, each is predicted to lack either an acid/base or nucleophile (Fig 6a, previous page). Absence of enzymatic activity, towards 4MU−, 4NP− and 2,4DNP-β-D-Glc was confirmed (data not shown) by no demonstrable ABP labeling (Fig 6b). Even after incubations of 18 hours with excess of either ABP 1 or 2 (1 mM) and the presence of sodium azide, no labeling was observed, suggesting these proteins’ pockets have acquired conditions unsuitable for labeling with cyclophellitol β- epoxide– and β-aziridine type ABPs, and do not possess β-glucosidase activity.

Identification of catalytic residues in GBA2 from GH116 – The non-lysosomal β- glucosidase GBA2 has recently received considerable attention. A key function of the enzyme in apoptosis has been proposed, but its catalytic residues currently remained elusive7−12. Recently, the acid/base and nucleophile of a GBA2-homologue in S. solfataricus have been identified38. Protein alignment of GBA2 homologues present in various species and S. solfataricus revealed several conserved aspartate and glutamate residues, which could harbor its acid/base and nucleophile residues (Fig 7a, b). We substituted these aspartic– and glutamatic acid residues individually to glycines (E527G, D659G, D663G, E667G, D673G and D677G, see schematic in Fig 7a). Cells were transfected with C-terminally myc/His-tagged human wild-type GBA2, or the before mentioned isoforms. Each GBA2 isoform exhibited enzymatic activity towards artificial 4MU−, 4NP−, and 2,4DNP-β-D-Glc substrates, except GBA2 with E527, D677 or

Figure 7 | Mapping of catalytic residues in GBA2 – In silico prediction. (a) Schematic depicting GBA2 putative catalytic residues (thin sticks), with possible acid/base D667 (grey sticks) and nucleophile E527 (black sticks). (b) Amino-acid sequence alignment of GBA2 homologues from various species, with conserved aspartic (D) and glutamic acids (E) highlighted from a.

164 Catalytic residue identification with ABPs

Figure 8 | Non-lysosomal β-glucosidase GBA2 – Functional analysis. (a) Catalytic activity of LPH variants towards 4MU−, 4NP− and 2,4DNP-β-D-Glc relative to mock (black, hatched, white columns, respectively). (b) Sodium azide-mediated rescue of 2,4DNP-β-D-Glc hydrolysis by mock (empty plasmid; ¢–¢), wild-type GBA2 (●–●), E527G (p–p), D677G (●–●), D659G (¢– ¢), D663G (r–r), E667G (o–o), D673G (♢–♢) and E527G/D677G (o–o). (c) Labeling of GBA2 variants over-expressed in COS-7 cells, with excess β-epoxide ABP 1 (top gel) or β-aziridine ABP 2 (second gel); ABP-labeling of GBA2 variants in the absence and presence of sodium azide (fourth and fifth gels) as detected after His-tag pull-down. Post pull-down α-myc loading control (bottom). Data are average of triplicates, ± SD, with one-way ANOVA significance p < 0.05*, p < 0.001***. both substituted (Fig 8a). Titration with sodium azide subsequently revealed activity- rescue towards 2,4DNP-β-D-Glc only in the case of E527G and D677G GBA2, consistent with a catalytic role (Fig 8b). Use of 4NP-β-D-Glc gave similar results. The acid/base and nucleophile were thereafter identified by labeling cell lysates containing E527G and D677G GBA2 with ABP 1 or 2, and subsequent scanning of SDS-PAGE (Fig 8c). β-Epoxide ABP 1 did not bind covalently to GBA2, as previously reported29, 30. β-Aziridine ABP 2 labeled all GBA2 isoforms except E527G GBA2, indicating E527 is the putative nucleophile of GBA2. Consistent with this, the enzymatic activity of E527 GBA2 was rescued by sodium azide (Fig 8b, c). Labeling of D677G GBA2 by ABP 2 and its susceptibility for activity-rescue by sodium azide in combination with 4NP− and 2,4DNP-β-D-Glc indicates D677 is the acid/base in GBA2.

Discussion Retaining β-glucosidases fulfill important functions in the human body as is perhaps illustrated best by the deleterious consequences of GBA deficiency causing Gaucher disease, and the emerging implication of GBA2 in cancer and neuronal pathologies. Better understanding of human β-glucosidases requires identification of their crucial catalytic amino acids for catalysis, the nucleophile and acid/base residues. An elegant method has been developed in the past to identify these key residues, by using sodium azide as an external acid/base and/or nucleophile, allowing catalytic activity rescue of enzyme

165 Chapter 4 molecules with a mutant catalytic residue31. However, large sodium azide concentrations are required in conjunction with substrates augmented with optimized leaving groups, whilst only resulting in partial activity rescue. Moreover, at higher sodium concentrations the enzyme is markedly inactivated/inhibited. We have described here a complementary method that capitalizes on the unique features of our recent design of cyclophellitol β-epoxide– and β-aziridine type ABPs for β- glucosidases. The β-epoxide ABP 1 and β-aziridine ABP 2 probes fundamentally differ in ability to label β-glucosidases: Both ABPs bind covalently to the nucleophile, but β-epoxide ABPs require a functional acid/base and β-aziridine ABPs do not. As we demonstrated, β-aziridine ABP 2 covalently links with the nucleophile of GBA, GBA2, GBA3 and the β-glucosidase pocket III of LPH, independently of the presence of a functional acid/base residue. In contrast, labeling by β-epoxide ABP 1 of GBA, GBA3, and the β-glucosidase pocket III of LPH is prohibited by substitution of the acid/base glutamate and similarly this eliminates enzymatic activity. These phenomena can be rescued through addition of sodium azide as external acid/base. Binding of β-epoxide ABP 1 to the nucleophile in absence of the acid/base is completely rescued by employing several orders of magnitude less azide as required for substrate assays. Combining the outcome of labeling with ABPs 1 and 2 of wild-type and site-directed mutagenized β-glucosidases with the sodium azide-mediated rescue of their enzymatic activity and labeling, allowed us to identify the acid/base and nucleophile in all human β- glucosidases studied: GBA (E235/E340), GBA3 (E165/E373), LPH (pocket III: E1065/E1273) and GBA2 (D677/E527). The catalytic residues designated in GBA2 overlap with the recently postulated acid/base and nucleophile of GBA2-homologue SSO1353 from S. solfataricus38. Of interest, the proteins klotho, βklotho and KLPH, although evolved from β-glucosidases given the close homology to members of GH1, are not labeled by either ABP. Apparently these proteins no longer support suitable conditions required for binding ABPs of described architecture in some rudimentary β-glucosidase-like pocket24. It therefore seems very unlikely these proteins are still able to act as β-glucosidases, although klotho has been described to have β-glucuronidase activity24. We therefore deem it unlikely that these proteins possess β-glucosidase activity. Nevertheless, the described approach is generic and can also be employed to non-human retaining β-glucosidases30. In conclusion, our study presents a new application for activity-based retaining β- glucosidase profiling, that is, sensitive identification of the acid/base and nucleophile residues without requiring extensive knowledge of the ’ three-dimensional structure. Broadening of this approach beyond β-glucosidases to other classes of retaining glycosidases such as β- or should be feasible through variation of the cyclophellitol structure30.

166 Catalytic residue identification with ABPs

Experimental procedures

General methods – Cyclophellitol β-epoxide ABP 1 and β-aziridine ABP 2 were synthesized as described earlier29, 30. Chemicals were obtained from Sigma-Aldrich if not otherwise indicated. Recombinant GBA (imiglucerase) was purchased from Genzyme. COS-7 cells were cultured in HAMF12-DMEM (Invitrogen) supplied with 10% (v/v) FCS. Amino-acid sequence alignments were performed in ClustalOmega 1.2.0. Molecular cloning and site-directed mutagenesis – Design of cloning primers and for mutation introduction were based on NCBI Reference sequences NM 000157.3 (GBA), NM 020944.2 (GBA2), NM 020973.3 (GBA3), NG 008104.2 (LPH), NM 004795.3 (klotho), NM 175737.3 (βklotho) and NM 207338.2 (KLPH). All full- length cDNA sequences were cloned in pcDNA3.1 in frame with the myc/His vector (Invitrogen). Site-directed mutagenesis was performed using the QuickChange II XL Site-Directed Mutagenesis Kit (Agilent Technologies) with primers shown in Table 1. Protein expression and isolation – Confluent COS-7 cells were transfected with pcDNA3.1 empty vector (Mock) or vector with described insert in conjunction with FuGENE (Roche), and harvested after 72 hours by scraping in 25 mM potassium phosphate buffer (pH 6.5, supplemented with 0.1% (v/v) Triton X-100, inhibitor cocktail (Roche)). After determination of the protein concentration (BCA kit, Pierce), lysate was aliquoted and frozen at –80 °C. Enzyme activity assays – Assays were performed at 37 °C. Cellular extracts were pre-incubated for 30 min 29, 30 with inhibitors of GBA (1 mM conduritol β-epoxide (CBE); IC50 9.49 mM) , and/or GBA2 (20 nM N-(5- 44, 45 adamantane-1-yl-methoxy)pentyl)-deoxynojirimycin (AMP-DNM), IC50 2 nM) to isolate enzyme of interest. GBA activity was measured in 150 mM McIlvaine buffer (pH 5.2), 0.2% (w/v) sodium taurocholate, 0.1% (v/v) Triton X-100, protease inhibitor cocktail (Roche) and 0.1% (w/v) BSA. GBA2, GBA3 and LPH activities were measured in the same buffer without detergents at pH 5.8, 6.5 and 6.0, respectively. Where specified, enzyme assays were performed with 3.75 mM fluorescent 4-methylumbelliferyl-β-D-glucopyranoside (4MU-β-D-Glc), chromogenic 20 mM 4-nitrophenyl β-D-glucopyranoside (4NP-β-D-Glc), or 20 mM 2,4-dinitrophenyl β-D- glucopyranoside (2,4DNP-β-D-Glc). Activity of lactase was quantified by measuring liberated D-glucose from 100 46 mM lactose . Measurements of ABP 1 and 2 IC50 for LPH were performed by pre-incubating cell lysates with an appropriate range of inhibitor dilutions for 30 min at 37 °C, whereafter residual β-galactosidase and β-glucosidase activity was measured with 100 mM lactose and 3.75 mM 4MU-β-D-Glc. Liberated 4MU was fluorimetrically 29, 30 determined (λEX 365 nm, λEM 425 nm) , whereas 4NP-β-D-Glc and 2,4DNP-β-D-Glc reactions were stopped with excess 1 M ice-cold Na2CO3 (pH 10.2) and extinction of 4NP and 2,4DNP were determined at λABS 405 nm −1 −1 −1 −1 (ε4NP 8.94 M cm , ε2,4DNP 9.27 M cm ). In vitro ABP labeling – Samples (10 μg total protein) were labeled with ABP 1 or 2 in appropriate McIlvaine buffer without BSA at 37 °C in 40 μL total volume. GBA, GBA2 were labeled with 1 μM ABP for 1 h; GBA3, LPH with 10 μM ABP for 1 h; klotho, βklotho and KLPH with 1 mM for 18 h. After labeling, and when applicable, His- tagged proteins were purified with TALON beads following manufacturer’s instructions (Clontech). Samples were then denatured with 5 μL Laemmli buffer (50% (v/v) 1M Tris-HCl, pH 6.8, 50% (v/v) 100% glycerol, 10% (w/v) DTT, 10% (w/v) SDS, 0.01% (w/v) bromophenol blue), boiled for 4 min at 100 °C, and separated by electrophoresis on 7.5% (w/v) SDS-PAGE gel running continuously at 90 V29. Wet slab gels were scanned on fluorescence using the Typhoon Variable Mode Imager (Amersham Biosciences) using λEX 488 nm and λEM 520 nm (band pass filter 40 nm) for green fluorescent ABP 1/3. Western blotting was accomplished by transfer of the protein for 1 h at 12 V, followed by blocking of the membrane with 2% (w/v) BSA in TBST buffer (50 mM Tris- HCl, pH 7.4, 150 mM NaCl, 0.1% (v/v) Tween-20), overnight treatment with 1:2,000 diluted primary mouse α- myc mAb (Cell Signalling, b118, 2% (w/v) BSA in TBST), washing with TBST for 20 min (repeated 6 times), followed by 1:10,000 diluted secondary rabbit α-mouse IRD680 (Cell Signalling, 2% (w/v) BSA in TBST), subsequent washing with TBST for 20 min (repeated 6 times), and read-out on Odyssey infrared scanner. Sodium azide-mediated rescue – Samples were incubated with ABP or artificial substrate in the presence of 0–5 M sodium azide (equilibrated to pH 5.2 with Tris-HCl). After substrate reactions, tubes were centrifuged (3,000 rpm for 3 min at room temperature) whereafter the supernatant was collected, vigorously mixed and

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liberated chromogenic/fluorogenic product measured. After ABP labeling reactions, proteins were precipitated with 10% (w/v) trichloroacetic acid for 10 min on ice, centrifuged at 10,000 rpm for 10 min, and resulting pellet was washed thrice with cold 50% (v/v) aceton in nanopure water prior to further use for His-tag pull-down or directly for SDS-PAGE.

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Supplementary Information

Supplementary Table 1 | Primers. Employed for site-directed mutagenesis of GBA, GBA2, GBA3 and LPH.

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