Mammalian Cell Surface Display as a Novel Method for Developing Engineered Lectins with Novel Characteristics

Area of Science:

• Biochemistry
• Molecular Biology
• Glycobiology

Background:

• Leguminous lectins, like peanut agglutinin (PNA), possess a conserved carbohydrate recognition site with four loops (A-D).
• Understanding lectin-carbohydrate interactions is crucial for various biological processes and therapeutic applications.

Purpose of the Study:

• To investigate the impact of random mutations in loops C and D of PNA on its glycan-binding specificity.
• To identify specific amino acid residues and loop structures that confer altered sugar-binding properties to PNA.

Main Methods:

• Random mutagenesis of PNA loops C and D, followed by expression on mouse green fluorescent protein (GFP)-reporter cells.
• Screening of mutated PNA variants using flow cytometry, limiting dilution, and cDNA cloning.
• Analysis of amino acid sequences of selected high-affinity lectin variants.

Main Results:

• Mutated PNAs exhibited a preference for sialylated glycans (NeuAcα2-6(Galβ1-3)GalNAc) over non-sialylated ones (Galβ1-3GlcNAc), unlike wild-type PNA.
• Key mutations involved loop C length (eight amino acids), specific residue substitutions (Asn at 127, Trp at 125, and hydrophobic residues at 130), and an unchanged loop D.
• Mutations significantly enhanced sugar-binding affinity, with Tyr125 to Trp increasing binding nine-fold and His130 to Tyr increasing it over 30-fold.

Conclusions:

• The study elucidates the structure-function relationship between amino acid sequences in the PNA carbohydrate recognition site and its sugar-binding abilities.
• Specific modifications in PNA loops C and D can engineer lectin specificity towards sialylated glycans.
• These findings provide valuable insights for designing lectin-based tools and therapeutics with tailored glycan-binding profiles.