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Expression of Human β3GalT5-1 in Insect Cells as Active Glycoforms for the Efficient Synthesis of Cancer-Associated

Chih-Chuan Kung1, Jennifer M Lo1, Kuo-Shiang Liao1

  • 1Genomics Research Center, Academia Sinica, Taipei 11529, Taiwan.

Journal of the American Chemical Society
|March 25, 2025
PubMed

Insights

Researchers engineered a human enzyme, beta3GalT5-1, to efficiently synthesize globo-series glycans, crucial targets in cancer therapy. This engineered enzyme is 10-fold more effective than previous methods for producing these cancer-targeting molecules.

Area of Science:

  • Glycobiology
  • Enzymology
  • Cancer Therapeutics

Background:

  • Globo-series glycosphingolipids (GSLs) are cell surface markers found on various cancers, making them targets for cancer vaccines and therapeutics.
  • Current enzymatic synthesis of globo-series glycans, particularly the conversion of Gb4 to Gb5 (SSEA-3), using microbial galactosyltransferase LgtD, is inefficient.

Purpose of the Study:

  • To enhance the efficiency of globo-series glycan synthesis by exploring human galactosyltransferases.
  • To identify and engineer a highly efficient human enzyme for the synthesis of globo-series glycans for cancer therapeutic development.

Main Methods:

  • Screened human beta-1,3-galactosyltransferase (β3GalT5) isozymes (1 and 2) for improved Gb4 to Gb5 conversion efficiency.
  • Evaluated activity and substrate specificity of different glycoforms of a common soluble domain of β3GalT5.
  • Utilized site-specific alanine scanning to identify a highly active variant (S66A β3GalT5).
  • Determined the X-ray structure of β3GalT5-1 for molecular modeling and mechanistic insights.

Main Results:

  • Human β3GalT5 isozyme 1 (β3GalT5-1) demonstrated higher activity than isozyme 2 (β3GalT5-2) in the synthesis of globo-series glycans.
  • A S66A variant of β3GalT5-1 was identified with a 10-fold increase in efficiency compared to the microbial LgtD.
  • Structural and kinetic analyses provided rationale for the enhanced catalytic efficiency of the engineered enzyme.

Conclusions:

  • Engineered human β3GalT5-1 (S66A variant) offers a significantly more efficient enzymatic method for synthesizing globo-series glycans.
  • This improved synthesis provides a valuable tool for the development of novel cancer vaccines and therapeutics targeting GSLs.

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