Functionally modified chitotriosidase catalytic domain for chitin detection based on split-luciferase complementation. (15th April 2022)

• Record Type: Journal Article
• Authors: Yamanaka, Daisuke; Suzuki, Kento; Kimura, Masahiro; Oyama, Fumitaka; Adachi, Yoshiyuki
• Is Part Of: Carbohydrate polymers. Volume 282(2022)
• Publication Date: 2022-04-15
• Subjects: ANTS 8-aminonaphthalene-1, 3, 6-trisulfonic acid -- BGRP β-1, 3-glucan recognition protein -- BLI bio-layer interferometry -- BSA bovine serum albumin -- CatD catalytic domain -- CBD chitin-binding domain -- CBM carbohydrate binding module -- CFW calcofluor-white -- Chit1 chitotriosidase-1 -- EGC ethylene glycol-chitin -- ELISA enzyme-linked immunosorbent assay -- FACE fluorophore-assisted carbohydrate electrophoresis -- HKCA heat-killed Candida albicans -- PAGE polyacrylamide gel electrophoresis -- PBS phosphate-buffered saline -- SDS sodium dodecyl sulfate -- WGA wheat germ agglutinin; Chitin -- Chitosan -- Chitin binding-module -- Chitinase -- Chit1 -- NanoBiT; Polysaccharides -- Periodicals; Polysaccharides -- Periodicals; Polysaccharides -- Périodiques; Electronic journals; 547.78
• DOI: 10.1016/j.carbpol.2022.119125
• Languages: English
• ISSNs: 0144-8617

Abstract

Abstract: In this study, we applied a luciferase-fragment complementation assay for chitin detection. When luciferase-fragment fused chitin-binding proteins were mixed with chitin, the reconstituted luciferase became active. The recombinant chitin-binding domain (CBD) and a functionally modified catalytic domain (CatD) of human chitotriosidase were employed for this method. We designed the CatD mutant as a chitin-binding protein with diminished chitinolytic activity. The non-wash assay using the CatD mutant had higher sensitivity than CBD for chitin detection and proved to be a structure-specific biosensor for chitin, including crude biomolecules (from fungi, mites, and cockroaches). The CatD mutant recognized a chitin-tetramer as the minimal binding unit and bound chitin at K D 99 nM. Furthermore, a sandwich ELISA using modified CatD showed a low limit of quantification for soluble chitin (13.6 pg/mL). Altogether, our work shows a reliable method for chitin detection using the potential capabilities of CatD. Graphical abstract: Unlabelled Image