Development of a novel β-1,6-glucan-specific detection system using functionally-modified recombinant endo-β-1,6-glucanase.

The Journal of Biological Chemistry
Daisuke YamanakaNaohito Ohno

Abstract

β-1,3-d-Glucan is a ubiquitous glucose polymer produced by plants, bacteria, and most fungi. It has been used as a diagnostic tool in patients with invasive mycoses via a highly-sensitive reagent consisting of the blood coagulation system of horseshoe crab. However, no method is currently available for measuring β-1,6-glucan, another primary β-glucan structure of fungal polysaccharides. Herein, we describe the development of an economical and highly-sensitive and specific assay for β-1,6-glucan using a modified recombinant endo-β-1,6-glucanase having diminished glucan hydrolase activity. The purified β-1,6-glucanase derivative bound to the β-1,6-glucan pustulan with a KD of 16.4 nm We validated the specificity of this β-1,6-glucan probe by demonstrating its ability to detect cell wall β-1,6-glucan from both yeast and hyphal forms of the opportunistic fungal pathogen Candida albicans, without any detectable binding to glucan lacking the long β-1,6-glucan branch. We developed a sandwich ELISA-like assay with a low limit of quantification for pustulan (1.5 pg/ml), and we successfully employed this assay in the quantification of extracellular β-1,6-glucan released by >250 patient-derived strains of different Candida species (includ...Continue Reading

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Methods Mentioned

BETA
NMR
ELISA
bio-layer
chip
biosensor
isothermal titration calorimetry
FACE
dot-blot
electrophoresis
flow cytometry

Key Resources (RRID) Mentioned

SCR_012909

Software Mentioned

BLItz Pro
GraphPad
FlowJo
BD CSampler
NanoAnalyze
Diva
GraphPad Prism

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