Role of glutamine-169 in the substrate recognition of human aminopeptidase B

https://doi.org/10.1016/j.bbagen.2014.01.002Get rights and content

Highlights

  • Substitution of Gln169 caused a significant change of APB enzymatic properties.

  • The EC50 value of chloride ion binding increased with the Asn mutant.

  • Gln169 was required for maximal cleavage of peptide substrates.

  • Modeling suggests that Gln169 bridges with N-terminal Arg of substrate.

Abstract

Background

Aminopeptidase B (EC 3.4.11.6, APB) preferentially hydrolyzes N-terminal basic amino acids of synthetic and peptide substrates. APB is involved in the production and maturation of peptide hormones and neurotransmitters such as miniglucagon, cholecystokinin and enkephalin by cleaving N-terminal basic amino acids in extended precursor proteins. Therefore, the specificity for basic amino acids is crucial for the biological function of APB.

Methods

Site-directed mutagenesis and molecular modeling of the S1 site were used to identify amino acid residues of the human APB responsible for the basic amino acid preference and enzymatic efficiency.

Results

Substitution of Gln169 with Asn caused a significant decrease in hydrolytic activity toward the fluorescent substrate Lys-4-methylcoumaryl-7-amide (MCA). Substantial retardation of enzyme activity was observed toward Arg-MCA and substitution with Glu caused complete loss of enzymatic activity of APB. Substitution with Asn led to an increase in IC50 values of inhibitors that interact with the catalytic pocket of APB. The EC50 value of chloride ion binding was also found to increase with the Asn mutant. Gln169 was required for maximal cleavage of the peptide substrates. Molecular modeling suggested that interaction of Gln169 with the N-terminal Arg residue of the substrate could be bridged by a chloride anion.

Conclusion

Gln169 is crucial for obtaining optimal enzymatic activity and the unique basic amino acid preference of APB via maintaining the appropriate catalytic pocket structure and thus for its function as a processing enzyme of peptide hormones and neurotransmitters.

Introduction

Aminopeptidases hydrolyze N-terminal amino acids of proteins or peptide substrates [1]. Among them, the M1 family of zinc aminopeptidases shares GXMEN and HEXXH(X)18E motifs essential for enzymatic activity. This family, which comprises 11 human enzymes, plays important roles in several pathophysiological processes including angiogenesis, cell cycle regulation, reproduction, memory retention, blood pressure regulation and antigen presentation to major histocompatibility complex class I molecules [2].

Aminopeptidase B (EC 3.4.11.6, APB) is a monomeric secretory protein first detected in several rat tissues which preferentially removes basic amino acids (Arg and Lys) from the N-terminus of several peptide and fluorogenic substrates [3]. Characterization of the enzymatic properties of APB showed that the hydrolytic activity of the enzyme was enhanced in the presence of physiological concentrations of chloride anions [4].

cDNA cloning of the rat APB revealed that the enzyme belongs to the M1 family and is closely related to leukotriene A4 hydrolase (LTA4H), which converts LTA4 to LTB4 [5], [6]. LTA4H contains both GXMEN and HEXXH(X)18E motifs and also has aminopeptidase activity [7]. Similarly, it was reported that human APB also shows hydrolytic activity toward LTA4, suggesting that the enzyme plays a role in certain inflammatory processes; however, another group could not detect this activity [6], [8].

Since APB shows preference for basic amino acids, this enzyme has been suggested to be involved in the proteolytic processing and maturation of peptide hormones and neurotransmitters [3], [9]. APB has been reported to be involved in the generation of active peptides through cleavage of extended N-terminal Arg and Lys residues in intermediate propeptides produced by endopeptidases such as cathepsin L and N-arginine dibasic convertase (NRD)/nardilysin [9], [10], [11]. Moreover, APB co-localizes with cathepsin L in neuropeptide-containing secretary vesicles [9]. Given that the hydrolytic activity of APB toward Arg and Lys residues is essential for processing and maturation of hormones and neurotransmitters, it is important to elucidate the structural aspects underlying APB activity. In this context, Fukasawa et al. reported that replacement of Asp405 with Ala or Asn in rat APB caused a change of substrate specificity toward fluorogenic substrates [12].

Residues responsible for substrate specificity of M1 aminopeptidases have been identified. Here, Gln181 of human endoplasmic reticulum aminopeptidase 1 (ERAP1) was found to be a residue important for ERAP1 substrate specificity [13]. Analysis of the recently resolved ERAP1 crystal structure revealed that Gln181 of human ERAP1 occupies the S1 site of the enzyme [14], [15]. Replacement of Gln181 with Asp demonstrated that the mutant enzyme has a preference for basic amino acids, suggesting that Asp in this position is important for basic amino acid preference. Moreover, ERAP2 shows a preference for basic amino acids and has an Asp residue in the corresponding site and substitution of this residue with Gln abrogated the basic amino acid preference of this enzyme [13], [16]. However, the corresponding residue of human APB is a Gln, despite the observation that this enzyme has a preference for basic amino acids.

Site-directed mutagenesis is a useful approach to elucidate mechanisms of enzymatic activity [17], [18], [19]. Using this approach, two consensus motifs, HEXXH(X)18E and GXMEN, were identified to be critical for the hydrolytic activities shared by the M1 family of aminopeptidases. Both His residues and the second Glu in the HEXXH(X)18E motif function as zinc ligands and are essential for the catalytic activity of M1 aminopeptidases, whereas the first Glu residue polarizes a water molecule and promotes nucleophilic attack of the carbonyl carbon of the peptide bond, forming a tetrahedral intermediate. On the other hand, Glu and Asn in the GXMEN motif are important for exopeptidase specificity through interaction with the N-terminal amide of substrates [17], [20], [21]. Moreover, it was recently reported that the Gly in GXMEN contributed to enzymatic activity and substrate specificity of M1 aminopeptidases [22], [23].

In this study, we have analyzed the role of human APB Gln169, which corresponds to Gln181 of ERAP1, by site-directed mutagenesis analysis. We found that Gln169 was required for the preferential cleavage of Arg- and Lys-MCA fluorogenic substrates, chloride anion sensitivity and the maximal enzymatic activity toward several peptide substrates with N-terminal basic amino acids. Our data suggest that Gln169 (together with Asp406) is important to maintain the basic amino acid preference of APB, which is essential for its function as a precursor processing enzyme of hormones and neurotransmitters.

Section snippets

Molecular modeling of the human APB

The recently published X-ray crystallographic structure of the human LTA4H with the RSR substrate (PDB ID: 3B7S) was used as a template for modeling the catalytic site of the human APB using the SWISS-MODEL Internet server (http://swissmodel.expasy.org/). To optimize the structure, hydrogen atoms were added to the initial model and the protonation states were assigned using the Protonate-3D tool within the MOE2012.10 software package (Chemical Computing Group, Montreal, QC, Canada). Energy

Modeling of the catalytic pocket of APB

To identify amino acid residues responsible for the substrate specificity of APB, we first modeled the catalytic pocket of APB using LTA4H (3B7S) as a template. Phylogenetic analysis indicates that APB and LTA4H are the most closely related among the M1 family of aminopeptidases, representing a distinct subfamily [5], [6], [24]. Fig. 1A shows the molecular modeling of the human APB catalytic pocket containing a putative tripeptide substrate (RSR) that has an N-terminal Arg. Asp406 has been

Discussion

In this study, the role of Gln169 in the enzymatic properties of human APB was elucidated. First, replacement of Gln169 with Asn changed the enzymatic activity of APB, resulting in either a total loss or substantial decrease in recognition of Lys- but not Arg-containing fluorogenic substrates. Second, Gln169 substitutions were found to attenuate chloride anion-dependent enhancement of the APB enzymatic activity. Finally, Gln169 is required for maximal activity toward peptide substrates, [Arg0

Acknowledgement

This work was supported in part by a Grant-in-aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

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