Engineering glycosyltransferases into glycan binding proteins using a mammalian surface display platform

Area of Science:

• Glycobiology
• Protein Engineering
• Biochemistry

Background:

• Traditional lectins have broad specificity, complicating targeted glycan analysis.
• Developing specific anti-glycan antibodies is difficult due to low immunogenicity.
• There is a need for novel glycan-binding proteins for single-cell glycosylation pathway studies.

Purpose of the Study:

• To test if engineering mammalian glycosyltransferases can create specific glycan-binding proteins.
• To develop a method for generating targeted lectins from enzymes.

Main Methods:

• Rational design and mutation (H302A) of porcine ST3Gal1 to abolish enzymatic activity.
• Development of a mammalian cell-surface display platform for variant screening.
• Characterization of a ST3Gal1 mutant (sCore2) with enhanced binding specificity.

Main Results:

• The H302A mutation yielded a lectin binding sialylated core-2 O-linked glycans.
• A triple mutant (sCore2) showed improved binding specificity for these glycans.
• Spectral flow cytometry and tissue microarray analysis revealed distinct sialyl core-2 staining patterns in human cells and tissues.

Conclusions:

• Mammalian glycosyltransferases can be successfully engineered into specific glycan-binding proteins.
• This protein engineering strategy offers a new avenue for creating custom lectins.
• The approach holds potential for application to other glycosylation enzymes.