Functional Properties of the Catalytic Domain of Mouse Acidic Mammalian Chitinase Expressed in Escherichia coli

International Journal of Molecular Sciences

Masahiro Kimura¹  Akinori Kashimura¹  Misa Ohno¹  Yasusato Sugahara¹  Kana Okazaki¹  Hirotaka Suzuki¹  Satoshi Wakita¹  Atsushi Ukita¹  Masayoshi Sakaguchi¹  Fumitaka Oyama¹  Kazuaki Okawa¹  Peter O. Bauer² 

[1] Department of Applied Chemistry, Kogakuin University, Hachioji, Tokyo 192-0015, Japan;Department of Neuroscience, Mayo Clinic, Jacksonville, FL 32224, USA;

关键词: acidic mammalian chitinase;    allergy;    asthma;    catalytic domain;    chitin-binding activity;    chitinolytic activity;    colloidal and crystalline chitin;    food processing;    innate immunity;    mouse;   

DOI  :  10.3390/ijms16024028

来源: DOAJ

【 摘 要 】

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 thefull-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 theN-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 absenceof a CBD. Our recombinant proteins can be used for further studies evaluating pathophysiological roles of AMCase in different diseases.

【 授权许可】

Unknown