Combined replacement effects of human modified β-hexosaminidase B and GM2 activator protein on GM2 gangliosidoses fibroblasts

Abstract

GM2 gangliosidoses are autosomal recessive lysosomal storage diseases (LSDs) caused by mutations in the HEXA, HEXB and GM2A genes, which encode the human lysosomal β-hexosaminidase (Hex) α- and β-subunits, and GM2 activator protein (GM2A), respectively. These diseases are associated with excessive accumulation of GM2 ganglioside (GM2) in the brains of patients with neurological symptoms. Here we established a CHO cell line overexpressing human GM2A, and purified GM2A from the conditioned medium, which was taken up by fibroblasts derived from a patient with GM2A deficiency, and had the therapeutic effects of reducing the GM2 accumulated in fibroblasts when added to the culture medium. We also demonstrated for the first time that recombinant GM2A could enhance the replacement effect of human modified HexB (modB) with GM2-degrading activity, which is composed of homodimeric altered β-subunits containing a partial amino acid sequence of the α-subunit, including the GSEP loop necessary for binding to GM2A, on reduction of the GM2 accumulated in fibroblasts derived from a patient with Tay-Sachs disease, a HexA (αβ heterodimer) deficiency, caused by HEXA mutations. We predicted the same manner of binding of GM2A to the GSEP loop located in the modified HexB β-subunit to that in the native HexA α-subunit on the basis of the x-ray crystal structures. These findings suggest the effectiveness of combinational replacement therapy involving the human modified HexB and GM2A for GM2 gangliosidoses.

Keywords: CI-M6PR, cation-independent M6P receptor; CM, conditioned medium; ERT, enzyme replacement therapy; Enzyme replacement therapy; GM2, GM2 ganglioside; GM2A, GM2 activator protein; Gm2 activator protein; Gm2 gangliosidosis; Hex, β-hexosaminidase; LAMP-1, lysosomal associated membrane protein 1; LSD, lysosomal storage disease; Lysosomal storage disease; M6P, mannose-6-phosphate; SD, Sandhoff disease; TSD, Tay-Sachs disease; modB, modified HexB; β-hexosaminidase.

Fig. 1

Expression and purification of GM2A-His with CHO cell lines. (A) Schematic drawing of the GM2A construct. GLAsig: α-galactosidase A signal sequence, GM2A (sig–): GM2A without a signal sequence. (B) Immunoblotting of CM with anti-hGM2A antibodies. Each lane contained 20 μL of CM. (C) GM2A-immunoreactivity indicated as relative signal intensity of GM2A. 1 V: one vector, 2 V: two vectors transfected. (D) Immunoblotting of N-glycosylated GM2A and the digested product with PNGase F. Each lane contained 50 μL of CM. (E) Purification of GM2A by Ni-column chromatography. Each fraction was separated by SDS-PAGE, and then silver staining was performed. Each lane contained 5 μg of protein. CM: conditioned medium. Elu: eluted fraction.

Fig. 2

GM2A replacement effect on variant AB fibroblasts. (A) Immunoblotting of cell extracts with anti-hGM2A antibodies after GM2A replacement. Each lane contained 9 μg protein. (B) Relative signal intensities of GM2A. Error bars show means ±SEM (n=3). Unpaired t-test,*P<0.05. (C) Variant AB fibroblasts treated with GM2A were immunostained for GM2 (green) and LAMP-1 (red), and then examined by confocal laser scanning microscopy. Scale bars indicate 20 µm.

Fig. 3

Combined replacement effect of modified HexB and GM2A on TSD fibroblasts. Reduction of the GM2 accumulated in TSD fibroblasts was evaluated by GM2-ELISA after treatment with modB or GM2A. Error bars show means ±SEM (n=6–7)., ANOVA with a Tukey post-hoc test,*P<0.05.

Fig. 4

Homology model of the modified HexB and GM2A complex. Proposed model for the modB/GM2A complex. (A) Overview of the model. Amino acid substitution regions (RQNK 312–315 GSEP and DL 452–453 NR) in modB are shown as sphere models with labels (GSEP and NR). (B) Close-up view of the substitution region of GSEP in the A chain of modB. The GSEP-interacting loop region (I66–C75) of GM2A is shown in red.