Expression and functional analysis of mouse chitinases without the ZZ domain of Staphylococcus aureus Protein A

doi:10.1016/j.ijbiomac.2025.139932

International Journal of Biological Macromolecules

Volume 290, February 2025, 139932

https://doi.org/10.1016/j.ijbiomac.2025.139932 Get rights and content

Highlights

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A chitinase lacking the ZZ domain (Chit1-V5-His) was obtained using pET21a vector.
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Chit1-V5-His showed properties similar to those of natural Chit1.
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This system could successfully express another mouse chitinase, AMCase.
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Proteins expressed using the system are useful in immunological analysis.

Abstract

Chitinase plays a role in mammalian immune responses, particularly in the degradation of fungal cell walls. The aim of the present study was to express and characterize recombinant mouse chitotriosidase (Chit1) and acidic mammalian chitinase (AMCase) without the ZZ domain, a domain that may interfere with immunological analyses. We successfully expressed recombinant chitinases without the ZZ domain (Chit1-V5-His and AMCase-V5-His) as a soluble protein from an expression vector pET21a in the Escherichia coli Rosetta-gami B (DE3) strain. Chit1-V5-His exhibited chitinolytic activity similar to that of ProteinA-Chit1-V5-His (a recombinant Chit1 with the ZZ domain) and natural Chit1, both with synthetic and natural substrates. Differential scanning fluorimetry and thermal stability assays revealed that Chit1-V5-His retained functional stability comparable to that of ProteinA-Chit1-V5-His, although ProteinA-Chit1-V5-His was more thermally stable. AMCase-V5-His demonstrated prominent chitinolytic activity at pH 2.0, aligning with the properties of natural AMCase. Owing to the lack of the ZZ domain that potentially binds to immunoglobulin G Fc region, Chit1-V5-His and AMCase-V5-His are advantageous tools for immunological analyses, as they do not block the Fc receptor-mediated phagocytosis of fungi by polymorphonuclear neutrophils and macrophages. Thus, this expression system effectively produces functional chitinases, facilitating further studies on their roles in mammalian immunity.

Introduction

Chitinase (EC 3.2.1.14) hydrolyzes chitin, a polymer of N-acetyl-d-glucosamine (GlcNAc) [[1], [2], [3]]. The CAZy database [4] classifies chitinases into two glycoside hydrolase (GH) families: GH18 and GH19. The catalytic domain (CatD) of GH18 chitinases is characterized by a folded (β/α)₈ barrel (TIM barrel) and conserved DXXDXDXE motif containing a catalytic Glu (E) residue [[5], [6], [7], [8]]. Most GH18 chitinases have a chitin-binding domain(s) (CBD(s)) that increases their affinity for the substrate and contributes to the efficient degradation of crystalline chitin. Mammals express two types of chitinases belonging to the GH18 family, chitotriosidase (Chit1) and acidic mammalian chitinase (AMCase) [[9], [10], [11]]. Both mammalian chitinases have a molecular mass of approximately 50 kDa and contain an N-terminal CatD and a C-terminal CBD [12,13].

There is increasing evidence that mammalian chitinase is involved in providing immunity to mammals. The expressions of Chit1 and AMCase is upregulated in inflammatory diseases [11,14], and is associated with the expression levels of transforming growth factor-β and interleukin-13, respectively. These findings suggest the possible involvement of mammalian chitinases in immunological disorders [15,16]. Both human polymorphonuclear neutrophils (PMNs) and macrophages (Mɸs) contain chitotriosidase [17]. A previous study reported that administration of human chitinase to neutropenic mice with representative fungal infections, including candidiasis and aspergillosis, increased the survival rate of the affected mice [17]. Considering that chitin is a major component of the cell wall of fungi, it is possible to hypothesize that chitinases are involved in the degradation process of fungi phagocytosed by PMNs and Mɸs. Thus, functional chitinases are necessary for analyzing the role of chitinases in the mammalian immune system.

Previously, we succeeded in purifying functional recombinant chitinases, ProteinA-Chit1-V5-His and ProteinA-AMCase-V5-His, using a pEZZ18 vector from Escherichia coli [[18], [19], [20]]. The pEZZ18 vector is designed to express a target protein that possesses a signal sequence and the ZZ domain of Staphylococcus aureus Protein A at the N-terminus [21,22]. ProteinA-Chit1-V5-His and ProteinA-AMCase-V5-His showed properties identical to the natural Chit1 and AMCase expressed in Chinese hamster ovary (CHO) cells, respectively [[18], [19], [20]]. These observations were consistent with those of a previous study, which reported that the ZZ domain does not interfere with the proper folding of fused target proteins [22]. However, the ZZ domain at the N-terminus of ProteinA-Chit1-V5-His or ProteinA-AMCase-V5-His does not appear to be suitable for immunological analysis.

One of the most crucial processes of the host defense mechanism against bacteria and fungi is phagocytosis of the pathogens by phagocytes, such as PMNs and Mɸs. In this process, a pathogen-immunoglobulin G (IgG) complex is efficiently phagocytosed by PMNs and Mɸs through the binding of the Fc region of the IgG molecule to the Fc receptor on the phagocytes. The ZZ domain, a domain in which two Z domains are connected in series, can bind to the IgG Fc region [23]. Therefore, the results of phagocytosis analysis using ProteinA-Chit1-V5-His and ProteinA-AMCase-V5 may not be accurate as the ZZ domain at the N-terminus of the recombinants binds to the IgG Fc region of a pathogen-IgG complex, which blocks FcR-mediated phagocytosis.

Our research goal is to elucidate the roles of mammalian chitinases in the biological defense mechanism against fungi. For this purpose, a certain amount of recombinant chitinases is required. Therefore, in the present study, we attempted to obtain functional chitinases without the ZZ domain for immunological analysis.

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Section snippets

Construction of plasmid DNAs for protein expression

We constructed the expression plasmid pET21a/Chit1-V5-His as follows: mouse Chit1-V5-His cDNA was polymerase chain reaction (PCR)-amplified using pEZZ18/pre-Protein A-mouse Chit1-V5-His [19] as a template and a pair of primers containing the restriction sites for NheI (forward primer) and SalI (reverse primer) (Table S1). The PCR reaction was performed using KOD Plus DNA polymerase (Toyobo, Kyoto, Japan), with an initial denaturation at 94 °C for 120 s, followed by 35 cycles of 30 s at 94 °C,

Purification of Chit1-V5-His expressed in E. coli

To obtain Chit1-V5-His (Fig. 1B and Fig. S2B), an expression plasmid pET21a/Chit1-V5-His was constructed and used for the transformation of E. coli as described in 2.1 Construction of plasmid DNAs for protein expression, 2.2 Preparation of recombinant chitinases. Chit1-V5-His possesses three additional amino acids (methionine-alanine-serine) derived from the T7 tag of the vector at the N-terminus.

Chit1-V5-His expressed in E. coli was first purified using a Ni Sepharose column, and further

Discussion

In the present study, we succeeded in expressing recombinant mouse chitinases (Chit1-V5-His and AMCase-V5-His) in E. coli as a soluble protein. The enzymatic activity of Chit1-V5-His was similar to that of ProteinA-Chit1-V5-His (Fig. 3) and natural Chit1. AMCase-V5-His exhibited the highest activity at pH 2.0, followed by that at pH 5.0 and 7.0 (Fig. 6A), and the characteristics were comparable to those of mouse AMCase [18,20]. Thus, this expression system using the pET21a vector shows the

Code availability (software application or custom code)

Not applicable.

CRediT authorship contribution statement

Masahiro Kimura: Writing – review & editing, Writing – original draft, Validation, Methodology, Investigation, Funding acquisition, Formal analysis, Conceptualization. Takumi Sakoh: Methodology, Formal analysis. Masayoshi Sakaguchi: Writing – review & editing, Methodology, Formal analysis. Shizuma Ishikawa: Writing – review & editing, Methodology. Takashi Odagiri: Writing – review & editing, Methodology. Naoto Yoshino: Writing – review & editing, Validation, Methodology, Formal analysis.

Ethics approval

Not applicable.

Funding

This study was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI [grant number: JP21K20574] and Yakult Honsha Co., Ltd. The funders had no role in the study design, data collection, data analysis, data interpretation, or writing of the report.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could appear to influence the work reported in this paper.

Acknowledgments

We thank Kazumichi Furuyama and Sumiko Yaegashi (Iwate Medical University) for their suggestions and assistance. We thank Editage (www.editage.com) for English language editing.

Glossary

4-NP: 4-nitrophenyl
4-NP-(GlcNAc)₂: 4-nitrophenyl N,N′-diacetyl-β-d-chitobioside
Chit1: chitotriosidase
AMCase: acidic mammalian chitinase
CatD: catalytic domain
CBD: chitin-binding domain
DSF: differential scanning fluorimetry
FACE: fluorophore-assisted carbohydrate electrophoresis
GlcNAc: N-acetyl-d-glucosamine
V5-His: V5 epitope and 6 × His tag
PMN: polymorphonuclear neutrophil
Mɸ: macrophage

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