Construction of a hybrid β-hexosaminidase subunit capable of forming stable homodimers that hydrolyze GM2 ganglioside in vivo

Michael B. Tropak, Sayuri Yonekawa, Subha Karumuthil-Melethil, Patrick Thompson, Warren Wakarchuk, Steven J. Gray, Jagdeep S. Walia, Brian L. Mark, Don Mahuran

Research output: Contribution to journalArticle

9 Citations (Scopus)

Abstract

Tay-Sachs or Sandhoff disease result from mutations in either the evolutionarily related HEXA or HEXB genes encoding respectively, the α- or β-subunits of β-hexosaminidase A (HexA). Of the three Hex isozymes, only HexA can interact with its cofactor, the GM2 activator protein (GM2AP), and hydrolyze GM2 ganglioside. A major impediment to establishing gene or enzyme replacement therapy based on HexA is the need to synthesize both subunits. Thus, we combined the critical features of both α- and β-subunits into a single hybrid µ-subunit that contains the α-subunit active site, the stable β-subunit interface and unique areas in each subunit needed to interact with GM2AP. To facilitate intracellular analysis and the purification of the µ-homodimer (HexM), CRISPR-based genome editing was used to disrupt the HEXA and HEXB genes in a Human Embryonic Kidney 293 cell line stably expressing the µ-subunit. In association with GM2AP, HexM was shown to hydrolyze a fluorescent GM2 ganglioside derivative both in cellulo and in vitro. Gene transfer studies in both Tay-Sachs and Sandhoff mouse models demonstrated that HexM expression reduced brain GM2 ganglioside levels.

Original languageEnglish (US)
Number of pages1
JournalMolecular Therapy - Methods and Clinical Development
Volume3
DOIs
StatePublished - Mar 16 2016

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G(M2) Ganglioside
G(M2) Activator Protein
Hexosaminidases
Hexosaminidase A
Genes
Sandhoff Disease
Clustered Regularly Interspaced Short Palindromic Repeats
Tay-Sachs Disease
Enzyme Replacement Therapy
HEK293 Cells
Isoenzymes
Catalytic Domain
Mutation
Brain

ASJC Scopus subject areas

  • Molecular Medicine
  • Molecular Biology
  • Genetics

Cite this

Construction of a hybrid β-hexosaminidase subunit capable of forming stable homodimers that hydrolyze GM2 ganglioside in vivo. / Tropak, Michael B.; Yonekawa, Sayuri; Karumuthil-Melethil, Subha; Thompson, Patrick; Wakarchuk, Warren; Gray, Steven J.; Walia, Jagdeep S.; Mark, Brian L.; Mahuran, Don.

In: Molecular Therapy - Methods and Clinical Development, Vol. 3, 16.03.2016.

Research output: Contribution to journalArticle

Tropak, Michael B. ; Yonekawa, Sayuri ; Karumuthil-Melethil, Subha ; Thompson, Patrick ; Wakarchuk, Warren ; Gray, Steven J. ; Walia, Jagdeep S. ; Mark, Brian L. ; Mahuran, Don. / Construction of a hybrid β-hexosaminidase subunit capable of forming stable homodimers that hydrolyze GM2 ganglioside in vivo. In: Molecular Therapy - Methods and Clinical Development. 2016 ; Vol. 3.
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