title: Functional Properties of Mouse Chitotriosidase Expressed in the Periplasmic Space of Escherichia coli.
abstract: 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.
authors.affiliation: Department of Chemistry and Life Science, Kogakuin University, Hachioji, Tokyo, 192-0015, Japan.
authors.forename: Masahiro
authors.initials: M
authors.lastname: Kimura
authors.affiliation: Department of Chemistry and Life Science, Kogakuin University, Hachioji, Tokyo, 192-0015, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Wakita
authors.affiliation: Department of Chemistry and Life Science, Kogakuin University, Hachioji, Tokyo, 192-0015, Japan.
authors.forename: Kotarou
authors.initials: K
authors.lastname: Ishikawa
authors.affiliation: Department of Chemistry and Life Science, Kogakuin University, Hachioji, Tokyo, 192-0015, Japan.
authors.forename: Kazutaka
authors.initials: K
authors.lastname: Sekine
authors.affiliation: Department of Chemistry and Life Science, Kogakuin University, Hachioji, Tokyo, 192-0015, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Yoshikawa
authors.affiliation: Department of Chemistry and Life Science, Kogakuin University, Hachioji, Tokyo, 192-0015, Japan.
authors.forename: Akira
authors.initials: A
authors.lastname: Sato
authors.affiliation: Department of Chemistry and Life Science, Kogakuin University, Hachioji, Tokyo, 192-0015, Japan.
authors.forename: Kazuaki
authors.initials: K
authors.lastname: Okawa
authors.affiliation: Department of Chemistry and Life Science, Kogakuin University, Hachioji, Tokyo, 192-0015, Japan.
authors.forename: Akinori
authors.initials: A
authors.lastname: Kashimura
authors.affiliation: Department of Chemistry and Life Science, Kogakuin University, Hachioji, Tokyo, 192-0015, Japan.
authors.forename: Masayoshi
authors.initials: M
authors.lastname: Sakaguchi
authors.affiliation: Department of Chemistry and Life Science, Kogakuin University, Hachioji, Tokyo, 192-0015, Japan.
authors.forename: Yasusato
authors.initials: Y
authors.lastname: Sugahara
authors.affiliation: Laboratory for Immunopharmacology of Microbial Products, Tokyo University of Pharmacy and Life Sciences, Hachioji, Tokyo, 192-0392, Japan.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Yamanaka
authors.affiliation: Laboratory for Immunopharmacology of Microbial Products, Tokyo University of Pharmacy and Life Sciences, Hachioji, Tokyo, 192-0392, Japan.
authors.forename: Naohito
authors.initials: N
authors.lastname: Ohno
authors.affiliation: Department of Neuroscience, Mayo Clinic, Jacksonville, FL, 32224, United States of America.
authors.forename: Peter O
authors.initials: PO
authors.lastname: Bauer
authors.affiliation: Department of Chemistry and Life Science, Kogakuin University, Hachioji, Tokyo, 192-0015, Japan.
authors.forename: Fumitaka
authors.identifier: http://orcid.org/0000-0002-1095-8306
authors.initials: F
authors.lastname: Oyama
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D002686:Chitin / Q000378:metabolism; D003412:Cricetulus; D004926:Escherichia coli / Q000378:metabolism*; D006596:Hexosaminidases / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D051379:Mice; D019897:Periplasm / Q000378:metabolism*; D013205:Staphylococcal Protein A / Q000378:metabolism
chemical_list: D013205:Staphylococcal Protein A; D002686:Chitin; D006596:Hexosaminidases; C086400:chitotriosidase