Functional Properties of Mouse Chitotriosidase Expressed in the Periplasmic Space of Escherichia coli

• Published in: PloS one Vol. 11; no. 10; p. e0164367
• Main Authors: Kimura, Masahiro, Wakita, Satoshi, Ishikawa, Kotarou, Sekine, Kazutaka, Yoshikawa, Satoshi, Sato, Akira, Okawa, Kazuaki, Kashimura, Akinori, Sakaguchi, Masayoshi, Sugahara, Yasusato, Yamanaka, Daisuke, Ohno, Naohito, Bauer, Peter O, Oyama, Fumitaka
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 07.10.2016; Public Library of Science (PLoS)
• Subjects: Alzheimer's disease; Amino Acid Sequence; Analysis; Animals; Bacteria; Biology and Life Sciences; C-Terminus; Cell Line; Chitin; Chitin - metabolism; Chitinase; CHO Cells; Chronic obstructive pulmonary disease; Colloid chitin; Cricetulus; Cystic fibrosis; Cytokinins; Cytoplasm; Diacetyl; Disease; E coli; Enzymes; Escherichia coli; Escherichia coli - metabolism; Gaucher's disease; Glucosamine; Hexosaminidases - metabolism; Hydrogen ions; Hydrogen-Ion Concentration; Laboratories; Life sciences; Lung diseases; Mammals; Mice; N-Acetylglucosamine; N-Terminus; Neurodegenerative diseases; Obstructive lung disease; Oligomers; Periplasm - metabolism; Periplasmic space; pH effects; Pharmacy; Physical Sciences; Physiological aspects; Protein A; Proteins; Research and Analysis Methods; Staphylococcal Protein A - metabolism; Substrates; Japan
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0164367

Chitotriosidase (Chit1) is an enzyme associated with various diseases, including Gaucher disease, chronic obstructive pulmonary disease, Alzheimer disease and cystic fibrosis. In this study, we first expressed mouse mature Chit1 fused with V5 and (His)6 tags at the C-terminus (Chit1-V5-His) in the cytoplasm of Escherichia coli and found that most of the expressed protein was insoluble. In contrast, Chit1 tagged with Protein A at the N-terminus and V5-His at the C-terminus, was expressed in the periplasmic space of E. coli as a soluble protein and successfully purified. We evaluated the chitinolytic properties of the recombinant enzyme using 4-nitrophenyl N,N'-diacetyl-β-D-chitobioside [4NP-chitobioside, 4NP-(GlcNAc)2] and found that its activity was comparable to CHO cells-expressed Chit1-V5-His. Optimal conditions for the E. coli-produced Chit1 were pH ~5.0 at 50°C. Chit1 was stable after 1 h incubation at pH 5.0~11.0 on ice and its chitinolytic activity was lost at pH 2.0, although the affinity to chitin remained unchanged. Chit1 efficiently cleaved crystalline and colloidal chitin substrates as well as oligomers of N-acetyl-D-glucosamine (GlcNAc) releasing primarily (GlcNAc)2 fragments at pH 5.0. On the other hand, (GlcNAc)3 was relatively resistant to digestion by Chit1. The degradation of 4NP-(GlcNAc)2 and (GlcNAc)3 was less evident at pH 7.0~8.0, while (GlcNAc)2 production from colloidal chitin and (GlcNAc)6 at these pH conditions remained strong at the neutral conditions. Our results indicate that Chit1 degrades chitin substrates under physiological conditions and suggest its important pathophysiological roles in vivo.