BanLec-eGFP Chimera as a Tool for Evaluation of Lectin Binding to High-Mannose Glycans on Microorganisms

Abstract

Fluorescently labeled lectins are useful tools for in vivo and in vitro studies of the structure and function of tissues and various pathogens such as viruses, bacteria, and fungi. For the evaluation of high-mannose glycans present on various glycoproteins, a three-dimensional (3D) model of the chimera was designed from the crystal structures of recombinant banana lectin (BanLec, Protein Data Bank entry (PDB): 5EXG) and an enhanced green fluorescent protein (eGFP, PDB 4EUL) by applying molecular modeling and molecular mechanics and expressed in Escherichia coli. BanLec-eGFP, produced as a soluble cytosolic protein of about 42 kDa, revealed β-sheets (41%) as the predominant secondary structures, with the emission peak maximum detected at 509 nm (excitation wavelength 488 nm). More than 65% of the primary structure was confirmed by mass spectrometry. Competitive BanLec-eGFP binding to high mannose glycans of the influenza vaccine (Vaxigrip®) was shown in a fluorescence-linked lectin sorbent assay (FLLSA) with monosaccharides (mannose and glucose) and wild type BanLec and H84T BanLec mutant. BanLec-eGFP exhibited binding to mannose residues on different strains of Salmonella in flow cytometry, with especially pronounced binding to a Salmonella Typhi clinical isolate. BanLec-eGFP can be a useful tool for screening high-mannose glycosylation sites on different microorganisms.

H84T BanLec has broad spectrum antiviral activity against human herpesviruses in cells, skin, and mice

“…More recently, computer modeling showed that H84T BanLec is expected to bind the SARS-CoV-2 S-glycoprotein with high affinity 23 . Wild-type BanLec is active against additional pathogens, including bovine viral diarrheal virus (BVDV) and bovine alphaherpesvirus 1 (BoHV-1) 24 and Salmonella 25 . Interestingly, when a change similar to that which converted BanLec to H84T BanLec was made in the related Malaysian banana lectin, producing F84T Malay BanLec, broad-spectrum antiviral activity was retained and mitogenicity was reduced in a manner comparable to H84T BanLec 26 .…”

Banana Lectin from Musa paradisiaca Is Mitogenic for Cow and Pig PBMC via IL-2 Pathway and ELF1

“…This BanLec-HIV recognition is thought to be mediated through mannose epitopes on high-mannose N-glycans [19]. BanLec is one of the most potent anti-viral lectins, but also recognizes bacteria with high-mannose type structures [19,20]. This makes BanLec very interesting as a very potent anti-infective agent with additional immune modulatory capacities [20][21][22][23].…”

“…BanLec is one of the most potent anti-viral lectins, but also recognizes bacteria with high-mannose type structures [19,20]. This makes BanLec very interesting as a very potent anti-infective agent with additional immune modulatory capacities [20][21][22][23]. Further information about the intervention with the immune system is needed to understand respective processes.…”