Gal f-Specific Neolectins: Towards Promising Diagnostic Tools

Abstract

In the absence of naturally available galactofuranose-specific lectin, we report herein the bioengineering of GalfNeoLect, from the first cloned wild-type galactofuranosidase (Streptomyces sp. strain JHA19), which recognises and binds a single monosaccharide that is only related to nonmammalian species, usually pathogenic microorganisms. We kinetically characterised the GalfNeoLect to confirm attenuation of hydrolytic activity and used competitive inhibition assay, with close structural analogues of Galf, to show that it conserved interaction with its original substrate. We synthetised the bovine serum albumin-based neoglycoprotein (GalfNGP), carrying the multivalent Galf units, as a suitable ligand and high-avidity system for the recognition of GalfNeoLect which we successfully tested directly with the galactomannan spores of Aspergillus brasiliensis (ATCC 16404). Altogether, our results indicate that GalfNeoLect has the necessary versatility and plasticity to be used in both research and diagnostic lectin-based applications.

Keywords: galactofuranosidase; neoglycoprotein; neolectin bioengineering; site-directed mutagenesis.

Figure 1

A graphical abstract depicting the bioengineering of NeoLectins from galactofuranosidase by the site-directed mutagenesis method. The glutamic acid/base catalytic residue (E464) was specifically changed to glutamine (Q), resulting in galactofuranosidase E464Q mutant with attenuated hydrolytic activity that can act as galactofuranose-binding neolectin (GalfNeoLect).

Figure 2

Alignment of partial ORF of Galf-ase (Streptomyces spp.) amino acid sequence and corresponding regions of its homologs. The conserved E464 and E573 amino acid residues are indicated with asterisks.

Figure 3

(A) Structural formulas of amino acids selected for the generation of E464X Galf-ase mutant variants. (B) SDS–PAGE analysis of wild-type Galf-ase after IPTG induction and Ni-NTA chromatography purification, as well as Galf-ase E464A, E464C, E464Q, and E464S mutants. Lane 1: protein marker. Lanes 2 and 3: cells’ extract before and after IPTG’s induction of wild-type Galf-ase. Lane 4: protein marker. Lane 5: purified wild-type Galf-ase. Lane 6: protein marker. Lane 7: traces of E464A mutant (not obtained in purified form). Lane 8: protein marker. Lane 9 to 11: purified E464C, E464Q, and E464S mutants.

Figure 4

Binding interaction of biotinylated GalfNGP to immobilised hIntL-1, wild-type Galf-ase, and GalfNeoLect. Galf-ase E464Q mutant variant was selected as a novel Galf-binding lectin (GalfNeoLect).

Figure 5

Structural formulas of pNP-pyranosyl, -furanosyl, azido, or thioaryl substrates used for wild-type Galf-ase and GalfNeoLect substrate specificity screening.

Figure 6

Inhibition profile of Aspergillus brasiliensis (ATCC 16404) spores with GalfNeoLect. Biotinylated GalfNGP (C = 2 μg/mL) was used as a tracer. Briefly, the A. brasiliensis (ATCC 16404) spores naturally carry on its surface Galf-containing galactomannan which is in competition for binding to GalfNeoLect with biotinylated GalfNGP.