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.
Highlights
Chitinases are enzymes that digest chitin, a polymer of (β-1,4)-linked N-acetyl-D-glucosamine (GlcNAc)
For production in E. coli, mature acidic mammalian chitinase (AMCase)-V5 epitope and (His)6 tag (V5-His) cDNA or its catalytic domain (CatD) region was cloned into pEZZ18 vector, containing the signal sequence of Staphylococcus aureus Protein A to express precursor forms of recombinant proteins of pre-Protein A-AMCase-V5-His and pre-Protein A-CatD-V5-His
We constructed pre-Protein A-V5-His (Figure 1C). Expression of these constructs in E. coli led to secretion of the mature forms of the recombinant proteins Protein A-AMCase-V5-His, Protein A-CatD-V5-His (Figure S1B) and Protein A-V5-His to the periplasmic space of E. coli
Summary
Chitinases are enzymes that digest chitin, a polymer of (β-1,4)-linked N-acetyl-D-glucosamine (GlcNAc). Chitinases are produced by various organisms, including bacteria, fungi, plants, nematodes, and arthropods [1,2,3] and are thought to have important roles in chitin digestion for organism defense or for providing a source of carbon and energy [4]. Despite the absence of endogenous chitin, humans and mice express two active chitinases, designated as chitotriosidase (Chit1) and acidic mammalian chitinase (AMCase). Both enzymes show sequence homology to bacterial chitinases and belong to the family 18 of glycoside hydrolases [3,5,6]. Mouse AMCase has been shown to be most active at pH 2.0 and is acid-stable, whereas Chit is inactivated at low pH conditions [9,10]
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