Enzyme replacement for GM1-gangliosidosis: Uptake, lysosomal activation, and cellular disease correction using a novel β-galactosidase:RTB lectin fusion

• Published in: Molecular genetics and metabolism Vol. 117; no. 2; pp. 199 - 209
• Main Authors: Condori, Jose, Acosta, Walter, Ayala, Jorge, Katta, Varun, Flory, Ashley, Martin, Reid, Radin, Jonathan, Cramer, Carole L., Radin, David N.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.02.2016
• Subjects: beta-Galactosidase - chemistry; beta-Galactosidase - genetics; beta-Galactosidase - metabolism; Cells, Cultured; Enzyme Replacement Therapy; Fibroblasts - enzymology; Gangliosidosis, GM1 - drug therapy; GM1-gangliosidosis; Humans; Kinetics; Lectin-mediated delivery; Lysosomal β-galactosidase; Lysosomes - metabolism; Nicotiana; Plant Lectins - chemistry; Plant Lectins - genetics; Plant Lectins - metabolism; Plant-based bioproduction; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; RTB lectin; RTB; BBB; ERT; CNS; GM1; Plant-based bioproduction; Lectin-mediated delivery; Lysosomal β-galactosidase; RTB lectin; Neu1; PPCA; LSD; GM1-gangliosidosis; β-gal
• ISSN: 1096-7192; 1096-7206
• DOI: 10.1016/j.ymgme.2015.12.002

New enzyme delivery technologies are required for treatment of lysosomal storage disorders with significant pathologies associated with the so-called “hard-to-treat” tissues and organs. Genetic deficiencies in the GLB1 gene encoding acid β-galactosidase lead to GM1-gangliosidosis or Morquio B, lysosomal diseases with predominant disease manifestation associated with the central nervous system or skeletal system, respectively. Current lysosomal ERTs are delivered into cells based on receptor-mediated endocytosis and do not effectively address several hard-to-treat organs including those critical for GM1-gangliosidosis patients. Lectins provide alternative cell-uptake mechanisms based on adsorptive-mediated endocytosis and thus may provide unique biodistribution for lysosomal disease therapeutics. In the current study, genetic fusions of the plant galactose/galactosamine-binding lectin, RTB, and the human acid β-galactosidase enzyme were produced using a plant-based bioproduction platform. β-gal:RTB and RTB:β-gal fusion products retained both lectin activity and β-galactosidase activity. Purified proteins representing both fusion orientations were efficiently taken up into GM1 patient fibroblasts and mediated the reduction of GM1 ganglioside substrate with activities matching mammalian cell-derived β-galactosidase. In contrast, plant-derived β-gal alone was enzymatically active but did not mediate uptake or correction indicating the need for either lectin-based (plant product) or mannose-6-phosphate-based (mammalian product) delivery. Native β-galactosidase undergoes catalytic activation (cleavage within the C-terminal region) in lysosomes and is stabilized by association with protective protein/cathepsin A. Enzymatic activity and lysosomal protein processing of the RTB fusions were assessed following internalization into GM1 fibroblasts. Within 1–4h, both β-gal:RTB and RTB:β-gal were processed to the ~64kDa “activated” β-gal form; the RTB lectin was cleaved and rapidly degraded. The activated β-gal was still detected at 48h suggesting interactions with protective protein/cathepsin A. Uptake-saturation analyses indicated that the RTB adsorptive-mediated mechanisms of β-gal:RTB supported significantly greater accumulation of β-galactose activity in fibroblasts compared to the receptor-mediated mechanisms of the mammalian cell-derived β-gal. These data demonstrate that plant-made β-gal:RTB functions as an effective replacement enzyme for GM1-gangliosidosis — delivering enzyme into cells, enabling essential lysosomal processing, and mediating disease substrate clearance at the cellular level. RTB provides novel uptake behaviors and thus may provide new receptor-independent strategies that could broadly impact lysosomal disease treatments. •The RTB plant lectin is proposed as a carrier for lysosomal replacement enzymes.•Plant-made β-gal:RTB fusions retain galactosidase and lectin-binding activities.•β-gal:RTB reduces lysosomal GM1 ganglioside levels in GM1 patient fibroblasts.•β-gal:RTB undergoes lysosomal β-gal proteolytic activation in GM1 fibroblasts.•Dynamics of GM1 correction of β-gal:RTB differs from mammalian cell-derived β-gal.