title: Functional properties of the catalytic domain of mouse acidic mammalian chitinase expressed in Escherichia coli.
abstract: Mouse acidic mammalian chitinase (AMCase) plays important physiological roles in defense and nutrition. AMCase is composed of an N-terminal catalytic domain (CatD) and a C-terminal chitin-binding domain (CBD). We expressed CatD of mouse AMCase as a recombinant fusion protein with Protein A and V5-His in Escherichia coli (Protein A-CatD-V5-His), evaluated its functional properties and compared them to the full-length AMCase (Protein A-AMCase-V5-His). Under our experimental conditions, the chitinolytic activity of both proteins against 4-nitrophenyl N,N'-diacetyl-β-D-chitobioside was equivalent with regard to their specific enzymatic activities, optimal pH and temperature as well as to the pH and temperature stability. CatD bound to chitin beads and cleaved the N-acetylglucosamine hexamer, colloidal and crystalline chitin as well as the shrimp shell, and released primarily N,N'-diacetylchitobiose fragments at pH 2.0. These results indicate that the primary structure of CatD is sufficient to form a proper tertiary structure required for chitinolytic activity, recognize chitin substrates and degrade them in the absence of a CBD. Our recombinant proteins can be used for further studies evaluating pathophysiological roles of AMCase in different diseases.
authors.affiliation: Department of Applied Chemistry, Kogakuin University, Hachioji, Tokyo 192-0015, Japan. bd12001@ns.kogakuin.ac.jp.
authors.forename: Akinori
authors.initials: A
authors.lastname: Kashimura
authors.affiliation: Department of Applied Chemistry, Kogakuin University, Hachioji, Tokyo 192-0015, Japan. b411050@ns.kogakuin.ac.jp.
authors.forename: Masahiro
authors.initials: M
authors.lastname: Kimura
authors.affiliation: Department of Applied Chemistry, Kogakuin University, Hachioji, Tokyo 192-0015, Japan. bd14001@ns.kogakuin.ac.jp.
authors.forename: Kazuaki
authors.initials: K
authors.lastname: Okawa
authors.affiliation: Department of Applied Chemistry, Kogakuin University, Hachioji, Tokyo 192-0015, Japan. hirotaka.suzuki@ssd.hpk.co.jp.
authors.forename: Hirotaka
authors.initials: H
authors.lastname: Suzuki
authors.affiliation: Department of Applied Chemistry, Kogakuin University, Hachioji, Tokyo 192-0015, Japan. atsushi.ukita@gmail.com.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Ukita
authors.affiliation: Department of Applied Chemistry, Kogakuin University, Hachioji, Tokyo 192-0015, Japan. bm13028@ns.kogakuin.ac.jp.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Wakita
authors.affiliation: Department of Applied Chemistry, Kogakuin University, Hachioji, Tokyo 192-0015, Japan. kanaokazaki@eurofins.com.
authors.forename: Kana
authors.initials: K
authors.lastname: Okazaki
authors.affiliation: Department of Applied Chemistry, Kogakuin University, Hachioji, Tokyo 192-0015, Japan. bd13001@ns.kogakuin.ac.jp.
authors.forename: Misa
authors.initials: M
authors.lastname: Ohno
authors.affiliation: Department of Neuroscience, Mayo Clinic, Jacksonville, FL 32224, USA. Bauer.Peter@mayo.edu.
authors.forename: Peter O
authors.initials: PO
authors.lastname: Bauer
authors.affiliation: Department of Applied Chemistry, Kogakuin University, Hachioji, Tokyo 192-0015, Japan. bt11532@ns.kogakuin.ac.jp.
authors.forename: Masayoshi
authors.initials: M
authors.lastname: Sakaguchi
authors.affiliation: Department of Applied Chemistry, Kogakuin University, Hachioji, Tokyo 192-0015, Japan. bt79310@ns.kogakuin.ac.jp.
authors.forename: Yasusato
authors.initials: Y
authors.lastname: Sugahara
authors.affiliation: Department of Applied Chemistry, Kogakuin University, Hachioji, Tokyo 192-0015, Japan. f-oyama@cc.kogakuin.ac.jp.
authors.forename: Fumitaka
authors.initials: F
authors.lastname: Oyama
mesh_terms: D000818:Animals; D020134:Catalytic Domain; D002686:Chitin / Q000737:chemistry / Q000378:metabolism; D002688:Chitinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D051379:Mice; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D013696:Temperature
chemical_list: D011993:Recombinant Fusion Proteins; D002686:Chitin; C499255:AMCase, mouse; D002688:Chitinases