Title : Investigations on therapeutic glucocerebrosidases through paired detection with fluorescent activity-based probes
Abstract :
- Deficiency of glucocerebrosidase ( GBA ) causes Gaucher disease (GD)
- In the common non-neuronopathic GD type I variant , glucosylceramide accumulates primarily in the lysosomes of visceral macrophages
- Supplementing storage cells with lacking enzyme is accomplished via chronic intravenous administration of recombinant GBA containing mannose-terminated N-linked glycans, mediating the selective uptake by macrophages expressing mannose-binding lectin(s)
- Two recombinant GBA preparations with distinct N-linked glycans are registered in Europe for treatment of type I GD: imiglucerase ( Genzyme ), contains predominantly Man(3) glycans, and velaglucerase ( Shire PLC ) Man(9) glycans
- Activity-based probes (ABPs) enable fluorescent labeling of recombinant GBA preparations through their covalent attachment to the catalytic nucleophile E340 of GBA
- We comparatively studied binding and uptake of ABP-labeled imiglucerase and velaglucerase in isolated dendritic cells, cultured human macrophages and living mice, through simultaneous detection of different GBAs by paired measurements
- Uptake of ABP-labeled rGBAs by dendritic cells was comparable, as well as the bio-distribution following equimolar intravenous administration to mice
- ABP-labeled rGBAs were recovered largely in liver, white-blood cells, bone marrow and spleen
- Lungs, brain and skin, affected tissues in severe GD types II and III, were only poorly supplemented
- Small , but significant differences were noted in binding and uptake of rGBAs in cultured human macrophages, in the absence and presence of mannan
- Mannan-competed binding and uptake were largest for velaglucerase, when determined with single enzymes or as equimolar mixtures of both enzymes
- Vice versa, imiglucerase showed more prominent binding and uptake not competed by mannan
- Uptake of recombinant GBAs by cultured macrophages seems to involve multiple receptors , including several mannose-binding lectins
- Differences among cells from different donors (n = 12) were noted, but the same trends were always observed
- Our study suggests that further insight in targeting and efficacy of enzyme therapy of individual Gaucher patients could be obtained by the use of recombinant GBA , trace-labeled with an ABP , preferably equipped with an infrared fluorophore or other reporter tag suitable for in vivo imaging
Output (sent_index, trigger,
protein,
sugar,
site):
- 3. containing, , GBA, mannose-terminated N-linked glycans, -
- 4. PLC, , Shire PLC, 9, -
- 4. PLC, , Shire PLC, velaglucerase (Shire PLC) Man, -
- 4. Shire, , Shire PLC, 9, -
- 4. Shire, , Shire PLC, velaglucerase (Shire PLC) Man, -
- 4. contains, , GD: imiglucerase, Man(3) glycans, -
- 4. contains, , Genzyme, Man(3) glycans, -
Output(Part-Of) (sent_index,
protein,
site):
*Output_Site_Fusion* (sent_index,
protein,
sugar,
site):