Monitoring intracellular activity of Arylsulfatase B on its natural substrates in a functional bioassay using LIF-CZE Erno Pungor Jr; Charles M. Hague; Ginger Chen; Jeffrey F. Lemontt; William S. Prince
Arylsulfatase B (ASB) Arylsulfatase B (ASB, N-acetylgalactosamine-4-sulfatase) a lysosomal sulfatase participating in the lysosomal degradation of Dermatan Sulfate (DS) and Chondroitin Sulfate (CS)... [IduA GalNAc] N1 [GlcA GalNAc] M1 [IduA GalNAc] N2... hydrolyzing sulfate from N-acetylgalactosamine-4-sulfate on the non-reducing end of the oligosaccharides Biological role with variable sulfation on 3 sites DS and CS are synthesized and secreted by cells as components of glycosaminoglycans (GAGs) in the extracellular matrix DS and CS internalized by cells for degradation through specific receptors to the lysosome to supply building blocks for synthetic pathways ASB is one of the enzymes degrading DS and CS (in collaboration with β- hexosaminidase, iduronate-2-sulfatase, glucuronate-2-sulfatase, α-iduronidase, α- glucuronidase N-acetylgalactosamine-6-sulfatase, etc.)
Functional (efficacy indicating) bioassay The regulatory expectation: an assay that describes the specific ability or capacity of a product to achieve a defined biological effect (ICH Q6B) Utility of the a functional bioassay in vitro product comparability, in lieu of demonstrating efficacy in animal or human studies demonstrates cellular activity of rhasb (the active ingredient in Naglazyme, Galsulfase) enzyme needs to be taken up by the target cell locate to the lysosome show activity at the site on the natural substrate
Indicator cell for an efficacy indicating bioassay First choice: human cells several published studies note differences in metabolic activities between human and animal disease model cells in lysosomal storage disorder Cells with a single (ASB) deficiency in the lysosomal enzyme repertoire and the ability to take up rhasb if the single deficiency in the lysosome is corrected by the uptake of the enzyme of interest (partially or fully), the entire pathway will be turned on amplification system by sequentially degrading the natural substrate GAGs Cells should be able to synthesize, secrete and re-import DS and/or CS to avoid the need to supplement the natural substrate into the culture medium cells with lysosomal degradation deficiency tend to accumulate GAGs from the medium more than normal cells imported GAGs will be degraded until the degradation reaches the deficient step partially degraded GAGs will be stored natural substrate for the missing enzyme Selected a fibroblast line (GM00519) from an MPS VI patient for the study
Monitoring options accumulation of degradation intermediates or end products depletion of intracellular GAG pool avoids the potential problem of the transient nature of intermediates or degradation end products being re-utilized by the cell (monosaccharides, oligosaccharides and inorganic sulfate)
Monitoring accumulation and depletion of DS and CS Digestion with Chondroitin ABC lyase (EC 4.2.2.4) breaks down larger DS and CS oligosaccharides to disaccharides possibly one trisaccharide or tetrasaccharide/chain All these disaccharides are negatively charged and only 1 of 8 is not sulfated Detection options LC/MS/MS Fluorescent labeling + capillary zone electrophoresis with laser induced fluorescence (CZE-LIF) labeling disaccharides with a neutral dye can allow a highly selective detection through CZE without complicated sample treatments
Monitoring DS and CS by LIF-CZE Δ4,5 UA-GalNAc-4S Δ4,5 UA-GalNAc-6S GM00519 Cell Δ4,5 UA-GalNAc-4,6S lysate + Chondroitin ABC lyase digestion + IS GlcNAc-6S DS in water + Chondroitin ABC lyase digestion + IS GM00519 Cell lysate + IS CZE conditions: pressure injection (0.5 p.s.i., 10 s) capillary: N-CHO Coated Capillary, 40 cm, 50 mm, I.D buffer: ProteomeLab Carbohydrate Separation Gel Buffer-N separation at 18 kv for 20 minutes (0.18 minute ramp, reverse polarity). GlcNAc6S (IS) GM00519 Cell lysate Very simple sample preparation from lysis to LIF-CZE lysate centrifuged, supernatant digested, dried, labeled, reconstituted, centrifuged taking advantage of the selectivity of CZE for negative targets with neutral dye
Establishing assay conditions Spike recoveries, calibrations with the internal standard GlcNAc-6S Area of the GlcNAc-6S peak (RFU) 120000 100000 80000 60000 40000 20000 0 (RFU) 3000 2000 1000 0 0 1 2 3 (µm) 0 20 40 60 80 100 120 GlcNAc-6S concetration (µm) Area of the GlcNAc-6S peak (RFU) 2500 2000 1500 1000 500 0 In water In lysate + Chondroitin ABC lyase 0 50 100 150 200 250 300 350 GlcNAc-6S spike (picomoles/100 µl) recovery of commercial (porcine) DS spike Sample 4S disaccheride [nmol] Spike recovery In lysis buffer In cell lysate [%] Control 0.00 0.16 N/A 700 ng Dermatan Sulfate 0.44 0.59 98.8 350 ng Dermatan Sulfate 0.22 0.37 96.6 175 ng Dematan Sulfate 0.12 0.26 85.5
Accumulation of intracellular DS/CS in GM00519 cells GM00519 cells on 12 well plates 4S disaccharide [nmol/well 2.0 1.8 1.6 1.4 1.2 1.0 1 nmol (0.6 10 15 molecules) of 4S disaccharides in 200 µl of cell lysate 8.0 10 5 cells/well - 0.8 10 9 molecules/cell 0.8 2 3 4 5 6 Culture age past confluence [weeks] Protein content in wells remains constant (consistent with confluence) estimated number of cells (150 pg protein/cell) approximately 800,000 Faster growing cells accumulate DS/CS faster Same general profile (no plateau)
Localization of rhasb when taken up by GM00519 cells GM00519 cells cultured for 3 days in presence of variable concentrations of rhasb washed, permeabilized stained with primary antibodies against rhasb, lysosomal antigen LAMP1 or with DAPI (nuclear) A NO rhasb DAPI C LAMP1 co-localization B 1.25 nm rhasb LAMP1 DAPI D co-localization 2.5 nm rhasb LAMP1 5 nm rhasb LAMP1 DAPI co-localization DAPI co-localization
Depletion of intracellular DS/CS by supplementing cell culture medium with rhasb GM00519 cells on 12 well plates rhasb added 4 weeks post confluence for 5 days LIF - Channel 1 031109 1 LIF - Channel 1 031109 3 Δ4,5 UA-GalNAc-4S LIF - Channel 1 031109 5 LIF - Channel 1 031109 9 rep LIF - Channel 1 031109 11 r Δ4,5 UA-GalNAc 23.5 23.5 Relative Fluorescence RFU 23.0 22.5 22.0 21.5 21.0 Δ4,5 UA-GalNAc-4,6S Δ4,5 UA-GalNAc-6S GlcNAc-6S NO rhasb 0.3 pm rhasb 2.7 pm rhasb 8.1 pm rhasb 23.0 22.5 22.0 21.5 21.0 RFU 20.5 20.5 20.0 16.2 pm rhasb 20.0 6 7 8 9 10 11 12 13 14 15 16 17 18 Minutes
Depletion of intracellular DS/CS by supplementing cell culture medium with rhasb GM00519 cells on 12 well plates rhasb added 4 weeks post confluence 4S disaccharide [nmol in lysate] 1.20 1.00 0.80 0.60 0.40 0.20 0.00 4S+6S disaccharides [nanomoles in lysate] 1.20 1.00 5 day incubation with rhasb 1 day incubation 0.80 0.60 0.40 0.20 0.00 2 day incubation 5 day incubation 0 5 10 15 20 Incubation rhasb [pm] 0 5 10 15 20 Response dependent on rhasb dose and incubation time Residual CS/DS disaccharide can not be digested with increased dose likely transient in synthesis or export/import Incubation rhasb [pm]
Effect of an inactive rhasb mutant in the assay Mutant : C53S, active site mutation Cys - formylglycine conversion needed for catalytic activity can not take place 7.0 4S disaccharide [nmol] or rhasb [ng/ml] in cell lysate 6.0 5.0 4.0 3.0 2.0 1.0 0.0 No rhasb control 27 pm C53S rhasb 27 pm rhasb 27 pm rhasb + 27 pm C53S rhasb Mutant is taken up same as regular molecule No effect on the depletion of CS/DS
The functional bioassay is stability indicating rhasb samples were exposed to acid, base and peroxide treatment linear segments of profiles can be compared with parallel line analysis or hyperboloid segments can be compared after linear transformation 4Sdisaccharide [nmole in lysate] 8.00 7.00 6.00 5.00 4.00 3.00 2.00 1.00 0.00 control 0.00 5.00 10.00 15.00 20.00 Incubation rhasb [pm] 4S disaccharide [nmole in lysate] 8.00 7.00 6.00 5.00 4.00 3.00 2.00 1.00 0.00 acid treatment 0.00 5.00 10.00 15.00 20.00 Incubation rhasb [pm] 4S disaccharide [nmole in lysate] 9.00 8.00 7.00 6.00 5.00 4.00 3.00 2.00 1.00 0.00 0.00 5.00 10.00 15.00 20.00 Incubation rhasb [pm] base treatment 4S disaccharide [nmole in lysate] 8.00 7.00 6.00 5.00 4.00 3.00 2.00 1.00 0.00 peroxide treatment 0.00 5.00 10.00 15.00 20.00 Incubation rhasb [pm]
Summary Functional assay... describes the specific ability or capacity of a product to achieve a defined biological effect (ICH Q6B) Assay characteristics ex vivo setting (cells from MPS VI patient) natural (heterodisperse) substrates collaboration of taken-up rhasb with other lysosomal enzymes when degrading the natural substrates rate of the multi-enzyme process can be controlled by rhasb readily applicable to other lysosomal enzymes very simple sample preparation, utilizing selectivity and resolving power of LIF-CZE by labeling negatively charged targets with neutral dye