Cargando…

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

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-Hi...

Descripción completa

Detalles Bibliográficos
Autores principales: Kashimura, Akinori, Kimura, Masahiro, Okawa, Kazuaki, Suzuki, Hirotaka, Ukita, Atsushi, Wakita, Satoshi, Okazaki, Kana, Ohno, Misa, Bauer, Peter O., Sakaguchi, Masayoshi, Sugahara, Yasusato, Oyama, Fumitaka
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2015
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4346942/
https://www.ncbi.nlm.nih.gov/pubmed/25689423
http://dx.doi.org/10.3390/ijms16024028
_version_ 1782359765915009024
author Kashimura, Akinori
Kimura, Masahiro
Okawa, Kazuaki
Suzuki, Hirotaka
Ukita, Atsushi
Wakita, Satoshi
Okazaki, Kana
Ohno, Misa
Bauer, Peter O.
Sakaguchi, Masayoshi
Sugahara, Yasusato
Oyama, Fumitaka
author_facet Kashimura, Akinori
Kimura, Masahiro
Okawa, Kazuaki
Suzuki, Hirotaka
Ukita, Atsushi
Wakita, Satoshi
Okazaki, Kana
Ohno, Misa
Bauer, Peter O.
Sakaguchi, Masayoshi
Sugahara, Yasusato
Oyama, Fumitaka
author_sort Kashimura, Akinori
collection PubMed
description 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.
format Online
Article
Text
id pubmed-4346942
institution National Center for Biotechnology Information
language English
publishDate 2015
publisher MDPI
record_format MEDLINE/PubMed
spelling pubmed-43469422015-04-03 Functional Properties of the Catalytic Domain of Mouse Acidic Mammalian Chitinase Expressed in Escherichia coli Kashimura, Akinori Kimura, Masahiro Okawa, Kazuaki Suzuki, Hirotaka Ukita, Atsushi Wakita, Satoshi Okazaki, Kana Ohno, Misa Bauer, Peter O. Sakaguchi, Masayoshi Sugahara, Yasusato Oyama, Fumitaka Int J Mol Sci Article 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. MDPI 2015-02-13 /pmc/articles/PMC4346942/ /pubmed/25689423 http://dx.doi.org/10.3390/ijms16024028 Text en © 2015 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Kashimura, Akinori
Kimura, Masahiro
Okawa, Kazuaki
Suzuki, Hirotaka
Ukita, Atsushi
Wakita, Satoshi
Okazaki, Kana
Ohno, Misa
Bauer, Peter O.
Sakaguchi, Masayoshi
Sugahara, Yasusato
Oyama, Fumitaka
Functional Properties of the Catalytic Domain of Mouse Acidic Mammalian Chitinase Expressed in Escherichia coli
title Functional Properties of the Catalytic Domain of Mouse Acidic Mammalian Chitinase Expressed in Escherichia coli
title_full Functional Properties of the Catalytic Domain of Mouse Acidic Mammalian Chitinase Expressed in Escherichia coli
title_fullStr Functional Properties of the Catalytic Domain of Mouse Acidic Mammalian Chitinase Expressed in Escherichia coli
title_full_unstemmed Functional Properties of the Catalytic Domain of Mouse Acidic Mammalian Chitinase Expressed in Escherichia coli
title_short Functional Properties of the Catalytic Domain of Mouse Acidic Mammalian Chitinase Expressed in Escherichia coli
title_sort functional properties of the catalytic domain of mouse acidic mammalian chitinase expressed in escherichia coli
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4346942/
https://www.ncbi.nlm.nih.gov/pubmed/25689423
http://dx.doi.org/10.3390/ijms16024028
work_keys_str_mv AT kashimuraakinori functionalpropertiesofthecatalyticdomainofmouseacidicmammalianchitinaseexpressedinescherichiacoli
AT kimuramasahiro functionalpropertiesofthecatalyticdomainofmouseacidicmammalianchitinaseexpressedinescherichiacoli
AT okawakazuaki functionalpropertiesofthecatalyticdomainofmouseacidicmammalianchitinaseexpressedinescherichiacoli
AT suzukihirotaka functionalpropertiesofthecatalyticdomainofmouseacidicmammalianchitinaseexpressedinescherichiacoli
AT ukitaatsushi functionalpropertiesofthecatalyticdomainofmouseacidicmammalianchitinaseexpressedinescherichiacoli
AT wakitasatoshi functionalpropertiesofthecatalyticdomainofmouseacidicmammalianchitinaseexpressedinescherichiacoli
AT okazakikana functionalpropertiesofthecatalyticdomainofmouseacidicmammalianchitinaseexpressedinescherichiacoli
AT ohnomisa functionalpropertiesofthecatalyticdomainofmouseacidicmammalianchitinaseexpressedinescherichiacoli
AT bauerpetero functionalpropertiesofthecatalyticdomainofmouseacidicmammalianchitinaseexpressedinescherichiacoli
AT sakaguchimasayoshi functionalpropertiesofthecatalyticdomainofmouseacidicmammalianchitinaseexpressedinescherichiacoli
AT sugaharayasusato functionalpropertiesofthecatalyticdomainofmouseacidicmammalianchitinaseexpressedinescherichiacoli
AT oyamafumitaka functionalpropertiesofthecatalyticdomainofmouseacidicmammalianchitinaseexpressedinescherichiacoli