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.
authors.affiliation: Department of Biochemistry, Leiden Institute of Chemistry, Leiden University, Leiden, The Netherlands.|Department of Medical Biochemistry, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.
authors.forename: Wouter W
authors.initials: WW
authors.lastname: Kallemeijn
authors.affiliation: Department of Medical Biochemistry, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.
authors.forename: Saskia
authors.initials: S
authors.lastname: Scheij
authors.affiliation: Department of Bio-organic Synthesis, Leiden Institute of Chemistry, Leiden University, Leiden, The Netherlands.
authors.forename: Sascha
authors.initials: S
authors.lastname: Hoogendoorn
authors.affiliation: Department of Bio-organic Synthesis, Leiden Institute of Chemistry, Leiden University, Leiden, The Netherlands.
authors.forename: Martin D
authors.initials: MD
authors.lastname: Witte
authors.affiliation: Department of Medical Biochemistry, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.
authors.forename: Daniela
authors.initials: D
authors.lastname: Herrera Moro Chao
authors.affiliation: Department of Medical Biochemistry, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.
authors.forename: Cindy P A A
authors.initials: CP
authors.lastname: van Roomen
authors.affiliation: Department of Medical Biochemistry, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.
authors.forename: Roelof
authors.initials: R
authors.lastname: Ottenhoff
authors.affiliation: Department of Bio-organic Synthesis, Leiden Institute of Chemistry, Leiden University, Leiden, The Netherlands.
authors.forename: Herman S
authors.initials: HS
authors.lastname: Overkleeft
authors.affiliation: Department of Biochemistry, Leiden Institute of Chemistry, Leiden University, Leiden, The Netherlands.|Department of Medical Biochemistry, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.
authors.forename: Rolf G
authors.initials: RG
authors.lastname: Boot
authors.affiliation: Department of Biochemistry, Leiden Institute of Chemistry, Leiden University, Leiden, The Netherlands.|Department of Medical Biochemistry, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.
authors.forename: Johannes M F G
authors.initials: JM
authors.lastname: Aerts
mesh_terms: D000818:Animals; D001572:Benzofurans / Q000737:chemistry; D002478:Cells, Cultured; D003713:Dendritic Cells / Q000201:enzymology*; D005455:Fluorescent Antibody Technique; D005456:Fluorescent Dyes / Q000737:chemistry*; D005962:Glucosylceramidase / Q000378:metabolism*; D006801:Humans; D008264:Macrophages / Q000201:enzymology*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D009000:Monocytes / Q000201:enzymology*; D011134:Polysaccharides / Q000378:metabolism
chemical_list: C493999:6-Aminobutylphthalide; D001572:Benzofurans; D005456:Fluorescent Dyes; D011134:Polysaccharides; D005962:Glucosylceramidase; C550184:Velaglucerase alfa, human; C090568:imiglucerase