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Lysosomal glycosidases and glycosphingolipids: New avenues for research

Rosa Alcalde Marques, A.

Publication date 2016 Document Version Final published version

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Citation for published version (APA): Rosa Alcalde Marques, A. (2016). Lysosomal glycosidases and glycosphingolipids: New avenues for research.

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UvA-DARE is a service provided by the library of the University of Amsterdam (https://dare.uva.nl) Download date:26 Sep 2021 Chapter 7

CHAPTER 5

Activity-based probes for retaining human β-D-galactosidases

Manuscript pending submission Chapter 7

5 Activity-based probes for retaining human β-D-galactosidases

André R. A. Marques1,*, Thomas J. M. Beenakker2,*, Chi-Lin Kuo3,*, Vrushali H. Jadhav2, Maria J. Ferraz3, Bogdan I. Florea2, Herman S. Overkleeft2, Johannes M. F. G. Aerts1,3

The work will be submitted in modified form as manuscript. T.J.M.B. designed and synthesized the activity- based probes; A.R.A.M. performed the biochemical analyses and C-L.K. performed competition experiments.

Introduction

The human body contains a great variety of glycoconjugates, including numerous compounds with a β-glycosidic linked galactose moiety. Among this plethora of structures are glycosphingolipids, N- and O-linked glycans in glycoproteins, glycosaminoglycans in proteoglycans and the disaccharide lactose, β-D-galactopyranosyl-(1→4)-D-glucose. Food is equally rich in such compounds including β- galactosyl containing plant structural polysaccharides. For the ongoing turnover of endogenous β- galactosides mammalian cells are equipped with β-galactosidases, enzymes able to hydrolyze terminal non-reducing β-D-galactosyl residues. Two specific intracellular β-galactosidases have been identified in man, both located in lysosomes and showing an acid pH optimum: acid β-galactosidase encoded by the GLB1 gene (locus 3p22.3) (1–3) and galactocerebrosidase encoded by the GALC gene (locus 14q31.3) (4–6). Furthermore, for the digestion and assimilation of β-D-galactosides the human body possesses an intestinal β-D-galactosidase activity. Enterocytes of the small intestine express at the surface of microvilli the enzyme lactase-phlorizin hydrolase (LPH), encoded by the LCT gene (locus 2q21.3) (7–10). This large enzyme contains two catalytic pockets with β-D-galactosidase and β-D- glucosidase activity respectively (11–14). The physiological relevance of all three enzymes is reflected by the pathologies observed during inherited deficiencies. Defects in GLB1 can result in GM1 gangliosidosis or in Morquio syndrome (Morquio B disease; MPS IVB), characterized by lysosomal accumulation of specific glycosphingolipids and glycosaminoglycans respectively (15–18). GM1 gangliosidosis is a neurodegenerative disorder, whereas Morquio B disease (MPS IVB) presents with dysostosis multiplex and normal central nervous system function (19). Secondary deficiency of lysosomal GLB1 is caused by inherited defects in cathepsin A, also named protective protein, which forms in lysosomes a complex with neuraminidase and GLB1. The corresponding inherited disease of the combined neuraminidase and GLB1 deficiency is named galactosialidosis (20). Deficiency of GALC results in another devastating neurological disorder, Krabbe disease (21). Finally, lactase deficiency causes lactose intolerance (22). All β-galactosidases employ the double displacement mechanism for catalysis (Figure 1A), based on the concerted action of a nucleophile and acid/base residue in the catalytic pocket. Characteristically this retaining reaction mechanism implies transient formation of a glycosylated enzyme through covalent linkage of the removed glycoside with the catalytic nucleophile, a glutamate

* The first three authors contributed equally to the experimental work described in this chapter.

neurodegenerative disorder, glycosp syndrome causes lactose intolerancecauses Characteristically this retaining reaction based on the concerted action of a nucleophileand a during i gluco results in a in results enzyme through covalent linkage removed the of glyco combinedneuraminidase and GLB1 deficiency isnamed lysosomes a complex with neuraminidase and caused byis inheriteddefects in cathepsin A, also namedprotective protein, multiplex and normal central nervous system function 2q21.3) in manin non- galactosides osse a itsia β intestinal an possesses surface ofsurface microvilli th galactosyl containing 14q31.3 and O lactose, based probes; A.R.A.M. performed the biochemical analyses and C and analyses thebiochemical A.R.A.M. performed probes; based Bogdan I. Florea I. Bogdan * β The humanbody contains grea Introduction work The A. Marques R. André ABPs for retaining β-galactosidases

The first three authors contributed equally to the experimental work in thischapter. described theto experimental equally first contributed authors The three - e glycosidic GLB1 reducing β sidase activityrespectively All T Activity 5 - , both located, both in lysosomess and linked glycans

hi he physiological relevance of ) β nherited deficiencies. Defects in GLB1 (7

- ngolipidsand glycosamin (4

will be submitted in modified form as manuscript. T.J.M.B. designed and synthesized the activity the synthesized designed and T.J.M.B. manuscript. form as modified in submitted will be

D –10) β (Morquio B(Morquio disease; MPS gene (locus nother –6) - -

g mammalian galactopyranosyl linkedgalactose moiety. Amongplethora structuresof this areglycosphingolipids, N alact . - . 2 D , Furthermo This largeThis enzyme contains two Herman S.

- devastating neurological disor galactosyl residues - osidasesemploy double the displacement mechanism based probes for retaining human β human forretaining probes based 1, the enzyme * , plant polysaccharides structural

in glycoproteins,in Thomas 3p22.3) cells are equipped with -

D (22) re, re, Overkleeft - galactosidaseactivity. E - whereas for the digestion and assimilation of β of assimilation and digestion the for . (1→4)

J. M. J. at variety of glycoconjugates, including

(1 (11 lactase –3) –14)

2 - . Two specific . Two , Beenakker ogly D

Johannes M. F. G. AertsG. F. M. Johannes and galactocerebrosidase encoded by the howing a Morquio B - IVB) all all - glucose . phlori

glycosaminoglycans proteoglycansin cans respectivelycans mechanism three enzymesreflected is by the pathologies observed , characterizedby zin hydrolase 2, . F * β der, Krabbe diseaseder, Krabbe n GLB1 , Chi ,

- catalytic pocket can galactosidases acid pH optimum: ood is intracellular disease (M nterocytes of the smallintestine nterocytes the of

- (19) Lin Kuo Lin implies transient forma . side withside the catalytic nucleo res . The . The corresponding inheriteddisease of the o te non troe o edgnu β endogenous of turnover ongoing the For equally g ult . c alactosialidosis

id/base residue in the catalyticresidueid/base thein Secondary deficiency lysosomalof GLB1

1,

- (LPH) L.K. performed competition experiments. competition L.K. performed 3 in GM1in gangliosidosis 3, (15 *

, β rich in such in rich lysosomal accumulation specificof

PS IVB PS , enzymes Vrushali H. Jadhav Vrushali H. - –18) s galactosidases have identified been

, encodedby the with with

- acid β acid (21) D . - galactosideshuman body the numerous GM1gangliosidosis is a ) β . Finally, deficiency lactase

-

- (20) for ablehydrolyze to terminal - D presents D- galactosidaseencoded by compounds -

galactosidase and β and galactosidase ti . Deficiency GALC of catalysis

galactosidases on of a and the disacchand the GALC compounds 2 phile, a

, L which

with dysostosis Maria J. Ferraz J. Maria CT or in in or exp

(Figure 1A) including glycosylated

gene (locus gene ress at theressat

glutamate forms in Morquio pocket.

with (locus aride aride - D

87 β 3 a - - - - - , ,

Chapter 5 88 Chapter 5

or aspartate, of the enzyme. Earlier, we have designed and synthesized activity-based probes (ABPs) for retaining glycosidases based on the use of differently configured cyclophellitol scaffolds (23–30). Here we report the synthesis of β-galactoside-configured cyclophellitol-aziridine tagged with an Two classes of ABPs were generated: cyclophellitol-epoxides with a grafted fluorophore of choice at alkyl azide (compound 1), and the generation of a fluorescent analogues by click chemistry C6 and cyclophellitol-aziridines with a grafted fluorophore at the aziridine-nitrogen. The first (compound 2) (Figure 1B-C). The inhibitor specificity of non-tagged compound 1 is compared to that generated ABP was a (β-glucoside-configured) cyclophellitol compound with engrafted fluorophore of β-galactoside-configured cyclophellitol epoxide (compound 3) (Figure 1C). The ability of at the C6 (compound 5), specifically labeling GBA (lysosomal glucocerebrosidase) with high affinity compound 2 to visualize β-galactosidases in cell and tissue extracts is demonstrated. Our findings (24). The second developed ABP was β-glucoside-configured cyclophellitol-aziridine (compound 4), demonstrate once more the generic applicability of cyclophellitol-aziridine scaffolds to generate labeling with high affinity β-glucosidases in class (GBA, GBA2, GBA3 and LPH β-glucosidase fluorescent ABPs for retaining glycosidases. Such ABPs have great potential in allowing future pocket) (25). At much higher concentration this type of ABP also labels out of class glycosidases, studies on properties of enzymes in their real life cellular and tissue context, in health and disease. such as lysosomal acid α–glucosidase (GAA), β-glucuronidase (GUS) (C.L. Kuo, J. Jianbing; unpublished findings) and the β-galactosidases GLB1 and GALC (chapter 4). Next, we generated higher affinity cyclophellitol-aziridine type ABPs for α-fucosidase (FUCA), β-glucuronidase and α- Materials and Methods glucosidases using correspondingly configured cyclophellitol scaffolds (30) (Jiang et al. unpublished). Again a more specific ABP for GALC was obtained using a β-galactoside-configured cyclophellitol- Probes epoxide grafted with fluorophore at C6 (chapter 1). Unfortunately, fluorophore engrafted β- The activity-based probes used in this study are depicted in Figure 1. The synthesis of the novel β-galactose galactoside-configured cyclophellitol-aziridine ABPs were not yet obtained due to synthetic configured probes 1 and 2 will be described in detail in the final manuscript. The identity of the β-galactose challenges (29). configured probes was checked with 1H NMR and 13C NMR spectroscopy. Azide-aziridine cyclophellitol 1: 1H

NMR (400 MHz, D2O): δ (ppm) = 3.93 (d, J = 8.0 Hz, 1H, H-2), 3.81 – 3.75 (m, 3H, H-4 and H-7), 3.34 (dd, J

= 0.2 Hz, 2.0 Hz, 1H, H-3), 3.27 (t, J = 6.8 Hz, 2H, CH2N3), 2.53 – 2.48 (m, 1H, CHHN), 2.13 – 2.04 (m, 2H,

A) B) H-5 and CHHN), 1.88 (d, J = 6.0 Hz, 1H, H-1), 1.85 (d, J = 6.0 Hz, 1H, H-6), 1.62 – 1.42 (m, 4H, 2 x CH2), O O O O O 13 1.40 – 1.22 (m, 8H, 4 x CH2). C NMR (101 MHz, D2O): δ (ppm) = 76.4 (C-3), 70.7 (C-2), 70.3 (C-4), 61.0 (C- O O OH O O OH H OH OH OH H OH 7), 59.2 (CH2N), 51.2 (CH2N3), 43.0 (C-6), 41.0 (C-1), 39.5 (C-5), 29.1 (CH2) 29.0 (CH2), 28.9 (CH2), 28.5 HO HO HO HO HO H HN + N 2 O O O R2 R (CH2), 27.0 (CH2), 26.4 (CH2). HRMS: calcd. for [C15H29N4O4] 329.21818; found 329.21833. BCN-Cy5 used OR1 HO HO O HO OH HO HO 1 HO HO H HO HO HO for conjugation to generate compound 2: H NMR (400 MHz, MeOD): δ (ppm) 8.26 (t, J = 13.1 Hz, 2H), 7.49 O O O O O (d, J = 7.4 Hz, 2H), 7.41 (t, J = 7.7 Hz, 2H), 7.33 – 7.22 (m, 4H), 6.66 (t, J = 12.4 Hz, 1H), 6.30 (dd, J = 13.7, O O O O O 7.0 Hz, 2H), 4.11 (t, J = 7.4 Hz, 2H), 3.92 (d, J = 6.8 Hz, 2H), 3.63 (s, 3H), 3.61 – 3.55 (m, 4H), 3.52 (t, J = 5.5 Hz, 4H), 3.34 (t, J = 5.5 Hz, 2H), 3.26 (t, J = 5.7 Hz, 2H), 2.39 – 2.30 (m, 2H), 2.28 – 2.15 (m, 4H), 2.12 – 2.03 C) (m, 3H), 1.87 – 1.79 (m, J = 7.7 Hz, 2H), 1.74 – 1.65 (m, 16H), 1.51 – 1.47 (m, J = 15.3, 8.6, 7.2 Hz, 2H), 1.34 O O – 1.26 (m, 2H), 0.80 – 0.60 (m, 3H). 13C NMR (101 MHz, MeOD): δ (ppm) 175.9, 175.3, 174.6, 155.5, 144.2, O N HO H O N N 143.5, 142.7, 142.5, 129.8, 129.7, 126.7, 126.2, 126.2, 123.4, 123.3, 112.1, 111.8, 104.4, 104.3, 99.5, 71.3, 71.3, HO H 2 H H HO 71.0, 70.6, 70.0, 50.5, 50.5, 49.6, 49.4, 49.2, 49.0, 48.8, 48.6, 48.4, 44.8, 41.6, 40.3, 36.6, 34.4, 31.6, 28.2, 28.0, HO + N 27.8, 27.3, 26.5, 26.3, 25.0, 24.2, 21.9. HRMS: calcd. for [C49H66N4O5] 790.50277; found 790.49779. N HO N3 HO N 7 N OH 7 N OH Enzymes N 1 2 Recombinant murine galactocerebrosidase (GALC) was expressed in human embryonic kidney 293 (HEK293) MeO cells as previously described (31). The mouse enzyme is 83% homologous to human GALC (31). The culture medium containing the secreted recombinant protein was used directly for fluorogenic substrate assays.

F N F B Fluorogenic substrate assay of recombinant GALC MeO N N N Culture medium containing recombinant GALC was diluted 2/1 (v/v) with McIlvaine (citrate/phosphate) buffer HO HO N F N pH 4.3 (10 µL total volume) and exposed to the indicated concentrations of compounds 1 or 3 (10 μL 2x HO HO B F HO O solution in H2O) for 30 min at 37°C, before addition of 100 µL substrate mix (0.23 mg/mL 4- O N N O N HO HO 5 HO methylumbelliferyl-β-D-galactopyranoside in McIlvaine pH 4.3/H2O 1/1 (v/v) with 0.2 M NaCl and 0.1% 4 N OH OH N OH BSA). After incubation at 37°C for 30 min, the reaction was quenched with 2.5 mL 0.3 M NaOH-glycine, pH 3 4 5 10.6 and fluorescence was measured with a fluorimeter LS55 (Perkin Elmer) using λex 366 nm and λem 445 nm. All samples were corrected for background fluorescence (sample without enzyme) and residual enzyme activity was calculated as compared to a control sample incubated in the same manner but without inhibitors. Figure 1 | ABPs for β-galactosidases. A) General mechanism for β-galactosidases. B) Covalent and Displayed values represent mean values from duplicate experiments. Graphpad Prism 5 software was used to irreversible inhibition of β-galactosidases by N-alkyl β-galactoside-configured cyclophellitol-aziridine. C) determine apparent IC values. Structures of compounds used in this study 1-5. 50 unpublished findings) labeling C6 the at generated in thisstudy used of compounds Structures higher affinity (24) challenges g epoxide grafted with fluorophore at C6 A glucosidases using correspondingly configured cyclophellitol scaffolds α acid lysosomal as such pocket) C6cyclophellitol and classesTwo ABPs of were generated: cyclophellitol for retainingglycosidasesfor reesbe niiin f β of inhibition irreversible Fig asp or alactoside gain a moregain a ure 1 ure 1 . Thesecond developed ABP was artate, theof enzyme. Earlier

(25) with high affinity

(compound C) A) |

ABP was a was ABP HO HO

HO HO (29) - HO config Bs o β for ABPs . Atmuchhigher. concentration this HO HO HO

specificABP GALC for was . cyclophe OH

1 OH 3 OH HO O O ured cyclophellitolured O N O O OR 5) O H

1 ( - and the HO HO β aziridines withgrafted a fluorophore at the , llitol - 7 - galactosidases. specifically labeling GBA N HO glucoside - based on 3 galactosidases by galactosidases –glucosidase OH HO β - HO aziridine type ABPs for α for ABPs type aziridine - g HO HO β lucosidases in class in (GBA,lucosidases GBA2, GBA3 LPH and N HO OH - HO O g O alactosidases GLB1 and GALC

- , O O the us OH configured) cyclophellit O we we 4 O H - O aziridine ABP β

H 1 4 N N - - have A)

g 5 e of of e N (GA N . lucoside

eea mcaim o β for mechanism General obtained using N 7 ( N designed and - chapter HO A), A), differently configuredcyclophellitol scaffolds alkyl N HO 5 H N typeof ABP also labels out N OH -

β HO O configured cyclophellitolconfigured (lysosomal glucocerebrosidase) B N O - - β g epoxideswith a grafted fluorophore OH O F H - s were nots lucuronidase O F galactoside OH 1) O - 2 O Me . fucosidase H N O Unfortunately a synthesized ol ol B) β - compound HO HO g O - alactoside Me

configured cyclophellitol configured 2 O OH F HO

O (FUCA) (GUS yet yet (chapter 4) (chapter F N - B O N H N galactosidases. B) Covalent and and Covalent B) galactosidases. (30) O N activity O aziridine- , obtained due to syntheticto obtaineddue O H

R ) with engrafted fluorophore - 2 (Jiang al et fluorophore 5 configured cyclophellitol (C.L. Kuo, J. Jianbing; J. (C.L. Kuo, - , aziridine β - - of class gly . Next, HO glucuronidase and based probes (ABPs) based HO HO HO nitrogen. The first N N N OH with highwith affi N HO HN N O OH

. unpublished) (compound O O β we generated nrfe β engrafted - - R aziridine O O o g 2 f choicef at lucosidase cosidases,

(23 –30) . C) C) . nity 4) α - - - , . .

7), 59.2 (CH studies studies on properties enzymesof their in real cellularlife and tissue context, healthin and di fluorescent ABPs for retaining glycosidases.Such ABPshave great potential in allowing future demonstrate once more the generic for compound β of (compound alkyl azide(compound configured probes was checked with was checked probes configured methylumbelliferyl pH 4.3 (10 µL total volume) and exposed to the indicated concentrations of compounds compounds of concentrations indicated the to exposed and volume) µL total (10 4.3 pH Culture medium containing recombinant GALC was diluted 2/1 with 2/1 was(v/v) GALC diluted McIlvaine medium recombinant Culture containing substr Fluorogenic assays. for fluorogenicsubstrate directlywasused recombinant protein secreted the medium containing cells described as previously (HEK293) kidney 293 in was human embryonic (GALC) expressed galactocerebrosidase murine Recombinant Enzymes (d, (d, 27.8, 27.3,26.5, 26.3,25.0, 24.2,21.9. 71.0, 70.6, 70.0, 50.5, 50.5, 49.6, 49.4, 49.2, 49.0, 48.8, 48.6, 48.4, 44.8, 41.6, 40.3, 36.6, 34.4, 31.6, 28.2, 28.0, 143.5, 142.7, 142.5, 129.8, 129.7, 126.7, 126.2, 126.2, 123. = 0.2 Hz, 2.0 Hz, 1H, H Hz, Hz, 1H, 2.0 0.2 = NMR (400 MHz, D MHz, NMR (400 probes configured The Probes Materials andMethods (CH

determine apparent IC apparent determine frommean values represent values Displayed as c wasactivity calculated 445 λem and nm residual and enzyme enzyme) without (sample fluorescence forbackground werecorrected 366 nm.All samples λex using Elmer) (Perkin LS55 fluorimeter a with measured was fluorescence and 10.6 mLwith 0 min,was 2.5 quenched for 30 37°C the at Afterreaction incubation BSA). 1.40 solution insolution H 7.0 Hz, 2H), H (m, 1.87 3H), (t, 3.34 4H), Hz, – ABPs for retaining β-galactosidases

- 1.26 (m, 2H), 0.80 5 and CH and 5

J conjugation 2

activity – ), 27.0 (CH =7.4 Hz, 2H), 7.41 (t, -

galactoside 1.22 (m, 8H, 4xCH Here wereport synthesisthe β of

-

H 2 based probes probes based 4.11 (t, (t, 4.11 2 ) (Figure2) 1B N), 51.2 (CH N), 1.88N), (d, – o iulz β visualize to 2

O) O) to generate compound generatecompound to 1.79 (m, 2 J ), 26.4 (CH

- = 5.5 Hz, 2H), 3.26 (t, 2H),3.26 Hz, 5.5 = - 1 configured cyclophellitol epoxide (compound 2 ate assay of recombinant GALC recombinant of assay ate β for 30 min at 37°C, before addition of 100 µL substrate mix (0.23 mg/mLmix addition (0.23 µL of 100 substrate before for 30 4 min 37°C, at

O): O): J - and and –

D 50 =7.4 Hz, 2H), 3.92 (d,

0.60 (m, 3H).

- values. - δ galactopyranoside in McIlvaine pH 4.3/H in pH McIlvaine galactopyranoside J 3), 3.27 (t,

2

(ppm) =3.93 (d, J =7.7 Hz, 2H), 1.74

used in this study are depicted in1 Figure instudy thisused depicted are 2 2 ompared to a control sample incubated in the same manner but without inhibitors. withoutmanner but inhibitors. sample insame incubated control the a to ompared will inofinidentity thedescribed detail the The finalbe manuscript. ). ). - N J 1 = 6.0 Hz, 1H, H Hz,= 1H, 6.0 C 2

(31) ), and ), generation the offluorescent a analogues by clickchemistry ). HRMS: calcd. for [C calcd. HRMS: ). 13 3 =7.7 Hz, 2H), 7.33 - ) ), 43.0 (C galactosidases in cell and tissueextracts is demonstrated. Our findings

C NMR (1 C NMR . Theinhibitor specificity non of . The mouse enzyme is 83% homologous to human GALC human. GALC to homologous mouseis 83% enzyme The

J 1

13 H NMR and and NMR H

= 6.8 Hz, 2H, CH Hz, 6.8 = 2 HRMS: calcd. for[C HRMS: calcd. C NMR (101 MHz, MeOD): MeOD): MHz, (101 C NMR :

- J 1 6), 41.0 (C 01 MHz, D MHz, 01 applicability of cyclophellitol H NMR (400 MHz, MeOD): MHz, MeOD): NMRH (400

- =5.7 Hz, 2H), 2.39 J galactoside

- = 8.0 Hz, 1H, H 1H, Hz, 8.0 = J 1), 1.851), (d,

– duplicate =6.8 Hz, 2H), 3.63 (s, 3H), 3.61

1.65 (m, 16H), 1.51 – 13

7.22 (m, 4H), 6.66 (t, C NMR spectr C NMR - 15 2

1), 39.5 (C O): O): H -

2 29 configured cyclophellitol experiments N δ J N 4, 1 4, 3

49 ), 2.53), (ppm) =76.4 (C = 6.0 Hz, 1H, H Hz, 1H, 6.0 = 4 O H - 2), 3.81 23.3, 112.1, 111.8, 104.4, 104.3, 99.5, 71.3, 71.3, 4 66 – ] + N

2.30 (m, 2H), 2.28 - 329.21818; found 5), 29.1 (CH 4 – O - oscopy. oscopy. 2 tagged

O 1/1 NaCl M0.1%and with1/1 O (v/v) 0.2 δ 5 2.48 (m, 1H, C . – ]

+ Graphpad Prism 5 software was used to wasused to software Prism 5 Graphpad . The synthesis of thesynthesis of The . – (ppm) 175.9, 175.3, 174.6, 155.5, 144.2, 1.47 (m, 790.50277

δ 3.75 (m,H 3H,

(ppm) 8.26 (t, 3 J ) (Figure 1C). The ability of A

- - compound =12.4 Hz, 1H), 6.30 (dd, - aziridine scaffoldsgenerate to 3), 70.7 (C 6), 1.62 zide 2 ) 29.0 (CH J –

- = 15.3,= Hz, 8.6, 7.22H), 1.34

aziridine cyclophellitol cyclophellitol aziridine ; 3.55 (m, 4H), 3.52 (t,

found found H 329.21833. – - aziridine tagged an with

HN), 2.13 HN), – (citrate/phosphate) (citrate/phosphate) 2.15 (m, 4H), 2.12 .3 M NaOH M .3

- - 1.42 (m, 4H, 2x CH 2), 70.3 (C- 2), 1 4 and H and 4 J

790.49779 is compared to thatcompared to is =13.1 Hz, 2H), 7.49 2 ), 28.9 (CH 1 novel novel

(31) or

BCN - – 7), 3.34 (dd,

3 . The culture The . 2.04 (m, 2H, -

glycine, pH glycine, pH 4), 61.0 (C β β sease. . 1 μ 2x μL (10

- - - Cy5 Cy5 galactose galactose

galactose galactose J

2 = 13.7, J ), 28.5 – buffer buffer

1 = 5.5 5.5 =

used used 2.03 2.03 :

89 1 2 H ), ), J - -

Chapter 5 90 Chapter 5

Fluorogenic substrate assay using mouse kidney homogenates red fluorescent ABP 5. As a loading control gels were stained with Coomassie Brilliant Blue (CBB) and de- For measurement of β-galactosidase and β-galactocerebrosidase activity in mouse kidney homogenates we stained with milliQ water. followed the protocol previously described by Martino et al. (32) with slight modifications. Briefly, a volume of tissue lysate equivalent to 7.5 µg of protein was completed with water (6.25 µL total volume) and exposed to the 2-step labeling with ABP 2 indicated concentration of compound 1 or 3 (6.25 µL 2x solution in McIlvaine buffer pH 4.3) for 30 min at 1 μM compound 1 and 0.5 μM BCN-based fluorophore (1-(1-((1R,8S,9R)-bicyclo[6.1.0]non-4-yn-9-yl)-3,14-

37°C. Afterwards 12.5 µL of 110 µM AgNO3 in water (final concentration 11 µM) and 100 µL of substrate mix dioxo-2,7,10-trioxa-4,13-diazanonadecan-19-yl)-3,3-dimethyl-2-((1E,3E)-5-((E)-1,3,3-trimethylindolin-2- (0.23 mg/mL 4-methylumbelliferyl-β-D-galactopyranoside in McIlvaine pH 4.3) were added and the mixture ylidene)penta-1,3-dien-1-yl)-3H-indol-1-ium) were combined in 10 μL milliQ water for 3 h at RT to generate was incubated for 15 min (for β-galactosidase) or 120 min (for GALC) at 37°C. The reaction was then quenched compound 2. Cell lysate, tissue homogenates, and culture medium of GALC-overexpressed HEK293T cells (50 with 2.5 mL 0.3 M NaOH-glycine pH 10.6 and the fluorescence was measured with a fluorimeter as described μg total protein per sample) were pre-incubated in 10 μL 150 mM McIlvaine buffer pH 4.5 for 5 min on ice, above. GLB1 activity was calculated by subtracting the contribution of GALC from the total β-galactosidase followed by the incubation with the mixture containing the coupled compound 2 (10 μL) for 1 h at 37°C. Protein activity. Displayed values represent mean values from triplicate experiments. Graphpad Prism 5 software was samples were denatured with the above described method, and then run on 10% SDS-PAGE gels. Wet slab gels used to determine apparent IC50 values. were scanned for fluorescence using Bio-rad ChemiDoc MP Imager, using Cy5 channel (λex 650 nm and λem 695 nM (band pass 55). Coomassie Brilliant Blue (CBB) was used on the same gels for loading control. Expression and labeling of human lactase phlorizin hydrolase (LPH) Primers were designed based on NCBI reference sequence NG_008104.2. Full-length cDNA sequence was cloned into pcDNA3.1 in frame with the myc/His vector (Invitrogen). Confluent HEK293 cells were transfected Results with empty pcDNA3.1 vector or the vector with the described insert, in conjunction with FuGENE (Roche), and harvested after 72 h by scraping in 25 mM potassium phosphate (KPi) buffer (pH 6.5, supplemented with 0.1% (v/v) Triton X-100 and protease inhibitor cocktail (Roche)). A volume equivalent to 50 µg of protein was pre- Characterization of inhibitory properties of β-galactoside configured cyclophellitol-aziridine incubated with inhibitor 1 or 3 (final concentration 1 and 0.5 µM) dissolved in water for 30 min at 37 °C, and cyclophellitol-epoxide for various human β-galactosidases followed by labelling with 0.5 µM (final concentration) of ABP 4 for 30 min at 37°C. We firstly determined the inhibitory action of compound 1 for GLB1, GALC and LPH β-D- galactosidase activities. For this purpose we used HEK293 cells overexpressing GALC and kidney Lactase activity assay lysates containing relatively high GLB1 activity. Enzyme activities were measured 4- The activity was measured by determination of released glucose from lactose, as previously described (14). methylumbelliferyl–β-D-galactoside as substrate at optimal conditions for each condition as early Briefly, 10 µL of lysate of HEK293 cells overexpressing LPH (see above) were pre-incubated for 30 min at described (chapter 1). Assay conditions for GALC and GLB1 activity were such that contribution of 37°C with 10 µL of the inhibitors dissolved in water. Afterwards 30 µL of KPi buffer, 125 µL of 60 mM sodium other enzymes to the generation of fluorescent 4-methylumbelliferone was minimal. For measurement citrate pH 6.0 and 125 µL of 100 mM lactose were added to each sample and incubated for 30 min at 37 °C. To stop the reaction the samples were boiled for 3 min at 100 °C. Finally, the samples were centrifuged for 10 min of LPH activity we used lysates of HEK293 cells over-expressing LPH. Enzymatic activity was at maximum speed and the supernatant was used to measure glucose with the Amplex Red glucose assay kit determined by quantification of release of glucose from lactose. Table 1 shows that compound 1 is a (Invitrogen, UK) according to the manufacturer’s instructions. very potent, irreversible, inhibitor of all three β-D-galactosidases tested. The most sensitive enzyme was GLB1 (IC50 = 8.1 nM), followed by LPH (IC50 = 11.2 nM) and GALC (IC50 = 21.2 nM). A In vitro labeling assay using mouse tissue homogenates comparison with β-galactoside-configured cyclophellitol (compound 3) reveals differences in

Twitcher mice (twi/twi) together with wild-type (wt) littermates were generated by crossing heterozygous inhibitor sensitivities among the examined human β-D-galactosidases (see Table 1). The lowest IC50 twi (+/twi) mice in-house. The heterozygous C57BL/6J B6.CE-Galc /J mice (stock number 000845) were obtained for compound 3 was observed for GLB1, followed by GALC and LPH. from The Jackson Laboratory (Bar Harbor, USA). Mouse pups were genotyped as previously described (33). Mice (± 3 weeks old) received the rodent AM-II diet (Arie Blok Diervoeders, Woerden, The Netherlands). Animals were housed, and animal experiments were conducted, according to approved protocols by the Table 1 | IC values of GALC, GLB1 and LPH towards compound 1 and 3. Institutional Animal Welfare Committee of the Academic Medical Centre Amsterdam in the Netherlands. 50 Animals were first anesthetized with a dose of Hypnorm (0.315 mg/mL phenyl citrate and 10 mg/mL fluanisone) and Dormicum (5 mg/mL midazolam). The given dose was 80 µL/10 g body weight. Animals were Enzyme Compound 1 Compound 3

sacrificed by cervical dislocation. Tissues were collected, snap frozen in liquid N2 and stored at -80 °C. Later, GALC 0.0212 μM 0.0391 μM homogenates from the frozen material were made in KPi buffer. A volume of tissue homogenate equivalent to GLB1 0.0081 μM 0.0217 μM 50 µg of protein was pre-incubated with inhibitor 1 or 3 (final concentration 0.5 and 0.25 µM) dissolved in LPH 0.0112 μM 5.657 μM water for 30 min at 37 °C, followed by labelling with 0.25 µM (final concentration) of ABP 5 for 30 min at 37°C and finally with 2.5 µM (final concentration) of ABP 4 for 30 min at 37°C. Ability of compound 1 to block the labelling of β-D-galactosidases with high concentration of SDS-PAGE analysis and fluorescence scanning fluorescent β-glucoside configured cyclophellitol-aziridine Protein samples (recombinant enzyme, cell and tissue homogenate) were denatured by adding 5x Laemmli sample buffer containing 2-mercaptoethanol (1/5th of sample volume) and boiling for 4 min at 100°C. The High concentrations of fluorescent β-glucoside-configured cyclophellitol-aziridine (ABP 4) also label samples were then run on a 7.5 or 10% SDS-PAGE gel and wet slab gels were scanned for fluorescence using retaining β-D-galactosidases (chapter 4). We exploited this for the present investigation. We the Typhoon Variable Mode Imager (Amersham Biosciences, Piscataway, NJ, USA), using λex 488 nm and λem examined whether pre-incubation with compound 1 blocks labeling of β-galactosidases with ABP 4. 520 nm (band pass 40) for green fluorescent ABPs 2 and 4 and λex 532 nm and λem 610 nM (band pass 30) for Indeed, labelling of GALC and GLB1 by ABP 4 was blocked by the β-galactoside-configured sample buffer containing 2 containing samplebuffer Laemmli by were 5x denatured adding homogenate) tissue and enzyme, cell samples (recombinant Protein SDS- 7Cadfnlywt . M(ia ocnrto)o ABP of (final µM withconcentration) 2.5 finally and 37°C a nuae o 5mn(o β (for min 15 for incubated was incubated ( mice ( Twitcher In vitro to according UK) (Invitrogen, 37°C. Afterwards 37°C. e 2 m(adps 0 o re loecn ABP fluorescent green for 40) pass (band nm 520 λem Imager (Amersham Mode Variable theTyphoon water for 30 min at 37 °C, followed by labelling with 0.25 µM (final ABP µM of concentration) with 0.25 min °C,water followed for30 by37 at labelling 50 µg of protein KPi buffer. weremade material in frozen from the homogenates (0.23 mg/mL 4 above. GLB1 activity was calculated by subtracting the contribution of GALC from the total β total the from GALC of contribution IC determineapparent used to the subtracting by was software Prism5 Graphpad experiments. values from triplicate calculated mean values represent activity. Displayed was activity GLB1 above. mLNaOH with M 2.5 0.3 Primers were designed based on NCBI reference sequence NG_008104.2. Full NG_008104.2. sequence reference NCBI on based weredesigned Primers (LPH) phlorizin hydrolase lactase human of labeling and Expression (v/v) Triton X with 0.1% supplemented buffer6.5, (pH (KPi) mM phosphate potassium scraping25 in h by after 72 harvested and (Roche), with FuGENE insert,in conjunction described with the thevector or vector with pcDNA3.1 empty w cells HEK293 Confluent (Invitrogen). vector myc/His the with frame in into pcDNA3.1 cloned at m was activity The activity assay Lactase byfollowed labelling indicated concentration of compound concentration indicated the to exposed and µL volume) total water (6.25 with was completed µg of protein 7.5 to equivalent tissue lysate by Martino previously described theprotocol followed β of measurement For kidney homogenates mouse using assay substrate Fluorogenic sacrificed by cervical dislocation. Tissues were collected, snap frozen in liquid N liquid frozen in snap werecollected, Tissues dislocation. by cervical sacrificed were Animals weight. g body was µL/10 80 given dose The midazolam). mg/mL Dormicum(5 and fluanisone) Institutiona by the protocols approved to according wereconducted, animalexperiments and werehoused, Animals AM the weeksrodent received old) 3 (± Mice described as previously weregenotyped pups USA). Mouse Harbor, (Bar Laboratory Jackson from The sam stop the reaction the samples were boiled for 3 min at 100 min at for3 wereboiled samples the reaction the stop 125 and 6.0 pH citrate 37°C Briefly, 10 +/twi ples were on a then run ples aximum speed and the supernatant was used to measure glucose with the Amplex Red glucose glucose Red Amplex the with glucose measure to was used supernatant the and speed aximum PAGE analysis and fluorescence scanning fluorescence and analysis PAGE with 10 mice in ) Animals were first anesthetized with a dose of Hypnorm (0.315 mg/mL phenyl citrate and 10 mg/mL 10 and mg/mL with phenyl citrate (0.315 of Hypnorm dose a firstwere anesthetized Animals labeling assay using mouse tissue homogenates mousetissue using assay labeling with l Animal Welfare Committee of the Academic Medical Centre Amsterdam in the in Netherlands. Amsterdam Centre Medical Academic the Committeeof Animal Welfare l µ flst fHK9 el vrxrsigLH(e bv)were above) (see LPH cells overexpressing of HEK293 lysate L of µ - - 0 n rtaeihbtrccti Rce) oueeuvln o5 go rti was protein µg of 50 to volumeequivalent A (Roche)). cocktail inhibitor protease and 100 L of the inhibitors dissolved in water. Afterwards 30 Afterwards water. in dissolved Linhibitors of the -methylumbelliferyl- os.Teheterozygous C57BL/6J B6.CE house. The inhibitor twi/twi was measured by determination of released glucose from lactose, as previously described previously as lactose, from glucose released of by determination measured 25µ f110µM AgNO 12.5 µL of with pre µ - oehrwith together ) aatsds adβ and galactosidase L of 100 mM lactose were added to each sample and incubated for 30 min for to37 at each sample incubated and were mMadded L of 100 lactose 1 - - nuae with inhibitor incubated 0.5 lcn H1. n h loecnewsmaue with as fluorimeter described a measured was the and fluorescence 10.6 pH glycine or or -m 7.5 or 50 the M(ia ocnrto)o ABP of (finalµM concentration) ercaptoethanol (1/ ercaptoethanol 3 - alues. v aatsds)o 2 i frGL)a 7C h ecinwas thenquenched reaction The 37°C. at GALC) min (for 120 or galactosidase) (final concentration 1 and 0.5 µM) dissolved in water for 30 min water for30 °C, 37 at in dissolved (final µM) 0.5 and concentration 1 manufacturer’s instructions. manufacturer’s 10% SDS- 10% β -D- 1 wild- or or galactopyranoside in McIlvaine pH 4.3) were added and the mixture the and wereadded 4.3) pH in McIlvaine galactopyranoside 3 - 3 galactocerebrosidase activity in mouse in wekidney homogenates activity galactocerebrosidase nwtr(ia ocnrto 1µ)ad10µ fsbtaemix substrate µL of 100 and µM) 11 concentration water (final n AEgladwtsa eswr cne o loecneusing fluorescence for wetwere gelsscanned slab geland PAGE type (wt) littermates were generated by crossing heterozygous by crossing type were littermates generated (wt) 62 L2 ouini clan ufrp 4.3) for 30 min at buffer pH solution in McIlvaine (6.25 µL 2x i - 5 II diet (Arie Blok Diervoeders, Woerden, The Netherlands). Woerden, The Diervoeders, Blok (Arie diet II th isine, ictwy J S) sn e 8 mand nm 488 λex using USA), NJ, Piscataway, Biosciences, f sample volume) and boiling for 4 min The 4 f100°C. at for sample boiling volume) and 1 o or or et al. s 4 3 - n e 3 madλm60n bn as3)for 30) pass (band nM 610 λem and nm 532 λex and °C 4 Galc (final concentration 0.5 and 0.25 µM) in dissolved 0.25 and 0.5 (final concentration (32) o 0mna 37°C. for 30min at . ial,tesmlswr etiue o 0min for10 werecentrifuged samples the Finally, 4 twi with slight for A Jmc soknme 085 were obtained mice 000845) number /J (stock µ volume of tissue homogenate equivalent to equivalent homogenate tissue of volume 30 min 30 L of KPi L of modifications at 37°C. buffer, 125 - 2 length cDNA sequence was sequence cDNA length nd stored at nd stored pre a - incubated for 30 min for30 at incubated . µ Briefly, a volumeof Briefly, a f6 Msodium L ofmM 60 5 ere transfected ere -galactosidas - for 30 min a min 30 for 80 °C. Later, 80 assay °C (33) (14) . To To . pre kit kit e - t . . 695 nM(band pass 55) were scan samples were denatured with the above described method, with described above werethe denatured samples by thefollowed μg total protein total μg compound ylidene)penta- dioxo Indeed, label galactosidase 2- stained withwater. milliQ μM 1 examinedwhether pre was very determinedby quantification release of of glucosefrom Table lactose. 1 LPHof lysates H fluorescent A C Results ABP fluorescent red for compoundfor comparison with other enzymesgeneration the to fluorescent of 4 described methylumbelliferyl eann β retaining We firstly determined the and cyclophellitol-epoxide inhibitor sensitivities ABPs for retaining β-galactosidases igh concentrations fluorescentof bility ofbility compound 1 to block haracterization of inhibitory properties of step labeling with ABP 2 ABP with step labeling GLB1 - potent, irreversible,potent, inhibitor all three of 2,7,10 compound activity ned containing relatively high GLB1 activity.

2 (chapter 1) (chapter

. Cell. -trioxa- -D-galactosidases (IC

β 1,3 Table 1 for fluorescence using Bio for fluorescence l -glucoside configured cyclophellitol-aziridine

ing GALC of and activit

3 was observed for incubation with incubation per sample) werepre 50

-dien-1- 1 lysate,

we used

= 8.1 nM= 4,13 and and – β 5 ies β . -galactoside- . As a loading control gels were stained with Coomassie Brilliant Bl Brilliant Coomassie with stained were gels loading control As . a | among examined the human . Assay conditions for GALC and GLB1were activitysuch that contribution of -D- Coomassie Brilliant Blue (CBB) wasused (CBB) Blue CoomassieBrilliant IC -diazanonadecan- - . μM 0.5 yl) . For thispurpose we HEK293 used incubation withincubation compound tissue homogenates tissue

galactosideas substrate at optimal condition 50 -3

values of GALC, GLB1 andLPH towards compound GALC Enzyme LPH GLB1 s of HEK293 lysate of s H ), followedby), for variousfor human β

inhibitory inhibitory - the mixture containing the coupled themixture coupled containing the indol BCN (chapter 4).

GLB1

configuredcyclophellitol (compound GLB1 -1- -

the the - incubated in 10 incubated β based fluorophore( -gluco ium) - rad ChemiDoc MP Imager, using Cy5 channel Cy5 Imager, using MP ChemiDoc rad 19- labelling of action of compound , followed by , and , yl)

Compound were combined in 10 μL milliQ water for 3 h at RT at h 3 for water milliQ μL 10 in combined were by ABP 0.0112 μM 0.0081 0.0212 μM side

LPH - 3,3 We exploitedWe this for thepresent investigation. culture culture - β - configured cyclophellitolconfigured dimethyl -methylumbelliferone -D-g cells over (IC

β -galactosidases μM μL μL blocks labelin blocks 1 -g β 4 β 50 -D-g

medium of GALC alactosidases tested. mostThe sensitive enzyme alactosideconfigured cyclophellitol 1 and then run on 10% SDS -D-galactosidases highwith concentration of

150 mM and GALC

1- was

= 11.2 nM)= and -2- (1

alactosidases (see1 alactosidases Table -

- ((1 ((1 Enzyme activities were measured 4 expressing LPH.expressing Enzymaticactivity was lce b te β the by blocked Compound

on the same gels for for gels same the on E 0.0217 μM 0.0391 μM compound cellsoverexpressing GALC and R 5.657 μM McIlvaine buffer McIlvaine buffer ,3 ,8 1

E S for for LPH. )-5- ,9 g R )- ((E of β of - GLB1 bicyclo[6.1.0]non-4- overexpressed HEK293T cells HEK293T overexpressed was minimal. For

s for s 2

)- 3

GALC

- (10 μL) for 1 h at 37°C. Protein -

1,3,3 shows that compoundshows that aziridine ( galactosidases with 3 ) reveals) , GAL

- each condition -trimethylindolin-2- PAGE gels. Wet slab gels gels slab gels.Wet PAGE - pH 4.5 galactoside loading control.

(IC

1 and (λex 650 nm and λem and nm 650 (λex C and ). ). 50 ABP ue (CBB) and de and (CBB) ue

Thelowest IC for 5 mi 5 for = 21.2 nM = 3. diff measurement

yn ) 4)

-configured erences in in erences

P β LPH -9- to generateto - also labelalso azir

n on ice, n on ice,

as earlyas yl) kidney ABP i 1 - i 3,14 dine dine ). A A ). -D- We (50 (50 s a

91 4. 50 - - -

Chapter 5 92 Chapter 5

cyclophellitol-aziridine 1 (Figure 1). A similar phenomenon was observed with β-galactoside- configured cyclophellitol-epoxide 3 (Figure 1). For more convenient analysis of the gels, the interfering labeling of lysosomal β-glucosidase GBA with ABP 4 was prevented by its pre-labelling with specific cyclophellitol-epoxide ABP 5. Unlike compound 3, compound 1 partly blocked the labelling of GBA by ABP 4.

Figure 3 | Compound 2 labels β-galactosidases in cells and tissues. A) Pre-clicking compound 1 and BCN- based fluorophore resulted in compound 2, which labels GLB1 and GBA in fibroblast lysate (left lanes), and GALC precursor in cell culture medium of HEK293 cells with GALC overexpression (right lanes). The gel slabs were stained with Coomassie Brilliant Blue (CBB) as loading control. B) Compound 2 labels GALC in mouse kidney homogenate (left lanes), and in mouse brain homogenate both GALC and GLB1 (right lanes).

Discussion

The investigations in this chapter provide evidence that β-galactoside-configured cyclophellitol- aziridines hold great potential as scaffold for the development of fluorescent ABP allowing visualization of β-D-galactosidases. Follow-up of this investigation is needed and will have to Figure 2 | Compound 1 blocks the labelling of β-galactosidases by ABP 4. Pre-incubation of mouse kidney encompass the synthesis of pure compound 2 (work in progress) and subsequent investigations on its (A) and brain (B) homogenates (50 µg of protein) with inhibitors 1 or 3 prevents labelling of GALC and GLB1 by ABP 4. GBA was labelled with specific ABP 5. C) Pre-incubation of lysates of HEK293 cells overexpressing applicability for labelling of enzymes in intact cells. Earlier it has been noticed that β-D-glucoside, α- LPH (50 µg of protein) with inhibitor 1, but not with inhibitor 3, prevents labelling of LPH by ABP 4. The slab D-galactoside, α-L-fucoside-configured cyclophellitol-aziridines engrafted with fluorophores are cell gels were stained with Coomassie Brilliant Blue (CBB) as loading control. permeable and label their lysosomal target enzymes in situ. It seems therefore very likely that this will also hold for fluorescent β-galactoside-configured cyclophellitol aziridines. The availability of such compounds should prove to be of great value for diagnosis of inherited disease like GM1 Generation of fluorescent compound 2 and labelling of β-D-galactosidases gangliosidosis, MPS IVB, galactosialidosis and Krabbe disease as well as investigations on lactose Given the positive findings with compound 1 we used click chemistry with BCN-based fluorophore intolerance. These tools might assist in obtaining better understanding of the remarkable variable (1-(1-((1R,8S,9R)-bicyclo[6.1.0]non-4-yn-9-yl)-3,14-dioxo-2,7,10-trioxa-4,13-diazanonadecan-19- consequences of GLB1 deficiency, ranging from neuronopathic GM1 gangliosidosis to non- yl)-3,3-dimethyl-2-((1E,3E)-5-((E)-1,3,3-trimethylindolin-2-ylidene)penta-1,3-dien-1-yl)-3H-indol-1- neuropathic MPS IVB. Moreover, it may help to shed more light on the puzzling influence of GLB1 ium) to generate Cy5 fluorophore engrafted β-galactoside-configured cyclophellitol-aziridine gene dosage on the phenotype of GALC-deficient Twitcher mice (34). Twitcher animals with an (compound 2). Culture medium of cells overexpressing GALC and fibroblasts (expressing additional complete deficiency of GLB1 show the mildest phenotype and those with a single endogenous GLB1 and low levels of GALC) were incubated with compound 2 freshly generated by functional GLB1 gene have the most severe disease. Compared to the other genotypes, the latter click chemistry of compound 1 with the BCN-based fluorophore. Labelling of both GLB1 and GALC animals show the highest levels of toxic galactosylsphingosine (psychosine) in brain. ABPs in could be detected in this way (Figure 3A). Some out of class labelling of the β-glucosidase GBA was combination with a thorough analysis of metabolites may reveal the mechanism underlying the cross also observed. Labeling of murine GLB1 and GALC was also evident in mouse tissue homogenates talk between GLB1 and GALC. (Figure 3B). (Figure also observed.also could becould detected click chemistry compound of endogenous GLB1 and as loading control. (CBB) Blue withBrilliant Coomassie weregelsstained LPH (50 µg (50 LPH (compound by ABP ABP by ium) (A) and brain (B) yl) ( Given the positive findings with compound Generation of fluorescent compound 2 and labelling β of Figure specificwith cyclophellitol β lysosomal of labeling interfering configured cyclophellitol cyclophellitol labelling 1- - (1 3,3

- ((1 to generateto - 2 dimethyl 4 3B).

R of GBA by ABP by GBA ABP of . . was GBA withABP specific labelled | C ,8S,9R

of protein of ompound 1 1 ompound

). 2). -

aziridine Labeling - homogenates (50 µg homogenates (50 ) 2 - Culture medium in thisway in bicyclo[6.1.0]non - ((1 ) with with ) Cy5 Cy5 E

blocks the labelling of of labelling the blocks ,3E

low levels 1 of murineofGLB1 GALC and was also evident mousein homogenates tissue - 5. inhibitor epoxide fluorophore engrafted ) (Figure - - 5 epoxide epoxide 1 - (Figure ((E with )

- 1

-

of GALC)of were incubatedwith compound 1,3,3- of protein glucosidase GBA 3 , - 1

4- of cells overexpressing cells of GALC and fibroblasts (expressing the the but not with inhibitor with not but ) ABP ABP 3A . (Figure yn smlr hnmnn a osre wt β with observed was phenomenon similar A BCN ). Some out of class labelling of the β the of labelling class of out Some ). trimethylindolin - 9 - 5 yl) 5 ) with with ) . Unlike. compound 1 . C) Pre β - based fluorophorebased - - weused click chemistry 1 galactosidases by ABP 4 ABP by galactosidases 3,14 ).

inhibitors For more convenient analysis the of gels, the β - - dioxo incubation of lysates of HEK293 cells overexpressing cells overexpressing HEK293 of of lysates incubation - g with ala

3 - ctoside 2 , -

2,7,10 - prevents labelling of of prevents labelling - ABP ABP 1 ylidene)penta D

or or - galactosidases 3 . Labelling both of GLB1GALC and - 4

- confi 3, compound prevents labelling of GALC and GLB1 GLB1 prevents of GALCand labelling trioxa

was prevented bypre its gured cyclophellitol .

- with Pre 4,13- - 1,3 - incubation of mouse kidney kidney mouse of incubation BCN -

diazanonadecan dien - LPH 2 glucosidase GBA was

1 freshly generatedby

- - partly blockedpartly the

based fluorophorebased 1 by ABP ABP by - yl) - - 3H g alactoside 4 - - - . labelling aziridine indol

The slab slab The - 19- - 1

- - mouse kidney homogenate mouse kidney medium of culture in cell GALC precursor slabs were stained with Coomassie Brilliant Blue (CBB) as loading control. control. as loading (CBB) Blue Brilliant Coomassie with werestained slabs based fluorophore g neuropathicIVB MPS GLB1consequences deficiency, of intolerance. gangliosid compoun also hold fluorescentalso for applicabilit talk between GLB1 and GALC. combination withthorough a metabolitesof analysis maymechanism revealthe under animalsshow thehighest levels functional additional completedeficiency GLB1 of show the mildest phenotypeand those withsingle a permea D encompassthe synthesisof compound pure visualization of aziridineshold great potential as The investigations thisin provide chapter evidence that Discussion 3 Figure ABPs for retaining β-galactosidases ene dosageene on - galactoside, ble and label their lysosomal target enzymes | ds should proveshould greatds of be to

Compound 2 labels β labels 2 Compound osis, MPS IVB, galactosialidosisand Krabbedisease wellas as investigationson lactose y GLB1

These These toolsmight assist in obtaining better understanding of the remarkable for labelling of enzymes in

α -

the the L resulted in compound compound resulted in β gene have most the severe disease. Compared to other genotypes,the the latter - - D fucosi - phenotype of g . Moreover, it. Moreover, maymore shed to on light help puzzling the alactosidases. alactosidases.

(left lanes) (left de β - g - configured cyclophellitolconfigured - alactoside -

galactosidases , and in and , of of toxicgalactosylsphingosine (psychosine) brain. in ABPs in GALC scaffold 2 Follow - , which which , ranging from configured cyclophellitol aziridines. The availability of such

intact cells.

HEK293 cells cells HEK293 mouse brain homogenate both GALC and GLB1 GALCand both homogenate brain mouse - deficient 2

-

in cells and tissues and in cells valuefor diagnosis inheritedof like disease GM1 up of thisinvestigation up of (work progress) in and labels for thedevelopment of fluorescent ABP allowing

in situ

Earlier it has been GLB1 and GBA in fibroblast lysatefibroblast GBA and in GLB1 T witcher aziridines neuronopathic GM1 gang with GALC overexpression with GALC . It seems β - g mice mice . alactoside A) Pre e ngrafted f with therefore (34) subsequent B) B) - noticed that clicking compound compound clicking is needed is and . Compound Compound - Twitcher animalswith an configured cyclophellitolconfigured very likely that that likely very

l investigations (right lanes) uorophores are liosidosis influence of β 2 -

D (right lanes).

labels lyingcross the (left lanes) (left - will haveto g lucoside, lucoside, 1

and and GALC in in GALC . to this variable The gel gel The GLB1

n its its on BCN , and and , non

will cell cell 93 α

- - - -

Chapter 5 94 Chapter 5

Author affiliations Human beta-galactosidase gene mutations in GM1-gangliosidosis: a common mutation among Japanese 1 adult/chronic cases. Am. J. Hum. Genet. 49: 435–442. Department of Medical Biochemistry, Academic Medical Center, University of Amsterdam, 1105 16. Santamaria, R., M. Blanco, A. Chabás, D. Grinberg, and L. Vilageliu. 2007. Identification of 14 novel GLB1 AZ Amsterdam, The Netherlands 2 mutations, including five deletions, in 19 patients with GM1 gangliosidosis from South America. Clin. Department of Bio-organic Synthesis, Leiden Institute of Chemistry, Leiden University, 2333 CC Genet. 71: 273–279. Leiden, The Netherlands 17. Morrone, A., T. Bardelli, M. A. Donati, M. Giorgi, M. Di Rocco, R. Gatti, R. Parini, R. Ricci, G. Taddeucci, 3 Department of Medical Biochemistry, Leiden Institute of Chemistry, Leiden University, 2333 CC A. D’Azzo, and E. Zammarchi. 2000. beta-galactosidase gene mutations affecting the lysosomal enzyme Leiden, The Netherlands and the elastin-binding protein in GM1-gangliosidosis patients with cardiac involvement. Hum. Mutat. 15: 354–366. 18. Nishimoto, J., E. Nanba, K. Inui, S. Okada, and K. Suzuki. 1991. GM1-gangliosidosis (genetic beta- References galactosidase deficiency): identification of four mutations in different clinical phenotypes among Japanese patients. Am. J. Hum. Genet. 49: 566–574. 19. Callahan, J. W.. 1999. Molecular basis of GM1 gangliosidosis and Morquio disease, type B. Structure- 1. Sips, H. J., H. A. de Wit-Verbeek, J. de Wit, A. Westerveld, and H. Galjaard. 1985. The chromosomal function studies of lysosomal beta-galactosidase and the non-lysosomal beta-galactosidase-like protein. localization of human beta-galactosidase revisited: a locus for beta-galactosidase on human Biochim. Biophys. Acta. 1455: 85–103. chromosome 3 and for its protective protein on human chromosome 22. Hum. 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