Structure aided design of a Neu5Gc specific lectin

Area of Science:

• Biochemistry
• Molecular Biology
• Oncology

Background:

• Subtilase cytotoxin (SubAB) from Escherichia coli is an AB5 toxin.
• Its B subunit (SubB) binds N-glycolylneuraminic acid (Neu5Gc), a molecule present on cancer cells but not normal human cells.
• Elevated Neu5Gc is linked to various cancers and indicates poor prognosis.

Purpose of the Study:

• To engineer SubB variants with enhanced specificity for Neu5Gc over Neu5Ac.
• To explore the utility of modified SubB as a diagnostic tool for detecting Neu5Gc tumor antigens.

Main Methods:

• Utilized molecular modeling to design site-directed mutations in SubB.
• Created SubB mutants to alter α2-3/α2-6 linkage preference and improve Neu5Gc selectivity.
• Employed surface plasmon resonance, glycan array analysis, and ELISA to assess mutant binding and specificity.

Main Results:

• The SubBΔS106/ΔT107 mutant showed improved specificity for Neu5Gc.
• This mutant also demonstrated binding to α2-6-linked Neu5Gc and could discriminate between Neu5Gc and Neu5Ac in assays.
• Native SubB exhibits significantly lower binding to Neu5Ac and α2-6-linked Neu5Gc.

Conclusions:

• Engineered SubB mutants offer enhanced specificity for the cancer-associated biomarker Neu5Gc.
• These modified SubB proteins represent a promising new tool for cancer diagnostics.
• Potential applications include testing biological samples like serum for cancer detection.