Role of unique basic residues in cytotoxic, antibacterial and antiparasitic activities of human eosinophil cationic protein

AbstractEosinophil granule proteins, eosinophil cationic protein (ECP) and eosinophil-derived neurotoxin are members of the RNase A superfamily, which play a crucial role in host defense against various pathogens as they are endowed with several biological activities. Some of the biological activities possessed by ECP have been attributed to its strong basic character. In the current study, we have investigated the role of five unique basic residues, Arg22, Arg34, Arg61, Arg77 and His64 of ECP in its catalytic, cytotoxic, antibacterial and antiparasitic activities. These residues were changed to alanine to generate single and double mutants. None of the selected residues was found to be involved in the RNase activity of ECP. The substitution of all five residues individually was detrimental for the cytotoxic, antibacterial and antiparasitic activities of ECP; however, mutation of Arg22 and Arg34 resulted in the most significant effects. The double mutants also had reduced biological activities. All ECP mutants that had significantly reduced toxicity also had reduced membrane destabilization activity. Our study demonstrates that Arg22, Arg34, Arg61, Arg77 and His64 of ECP are crucial for its membrane destabilization activity, which appears to be the underlying mechanism of its cytotoxic, antibacterial and antiparasitic activities.

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Role of catalytic and non-catalytic subsite residues in ribonuclease activity of human eosinophil-derived neurotoxin

Biological Chemistry ◽

10.1515/bc.2009.025 ◽

2009 ◽

Vol 390 (3) ◽

Cited By ~ 8

Author(s):

Deepa Sikriwal ◽

Divya Seth ◽

Janendra K. Batra

Keyword(s):

Catalytic Activity ◽

Biological Activities ◽

Secretory Protein ◽

Rnase A ◽

Phosphate Binding ◽

Human Eosinophil ◽

Ribonucleolytic Activity ◽

Phosphoryl Groups ◽

Binding Subsites

Abstract Human eosinophil-derived neurotoxin (EDN), a secretory protein from eosinophils, is a member of the RNase A superfamily. The ribonucleolytic activity of EDN is central to its biological activities. EDN binds RNA in a cationic cleft, and the interaction between EDN and RNA substrate extends beyond the scissile bond. Based on its homology with RNase A, putative substrate binding subsites have been identified in EDN. The B1 and B2 subsites interact specifically with bases, whereas P0, P1, and P2 subsites interact with phosphoryl groups. In this study, we evaluated the role of putative residues of these subsites in the ribonucleolytic activity of EDN. We demonstrate that of the two base binding subsites, B1 is critical for the catalytic activity of EDN, as the substrate cleavage was dramatically reduced upon substitution of B1 subsite residues. Among the phosphate-binding subsites, P1 is the most crucial as mutations of its constituting residues totally abolished the catalytic activity of EDN. Mutation of P0 and P2 subsite residues only affected the catalytic activity on the homopolymer Poly(U). Our study demonstrates that P1 and B1 subsites of EDN are critical for its catalytic activity and that the other phosphate-binding subsites are involved in the activity on long homopolymeric substrates.

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Effects of human eosinophil granule-derived cationic proteins on C-fiber afferents in the rat lung

Journal of Applied Physiology ◽

10.1152/jappl.2001.91.3.1318 ◽

2001 ◽

Vol 91 (3) ◽

pp. 1318-1326 ◽

Cited By ~ 35

Author(s):

Lu-Yuan Lee ◽

Qihai Gu ◽

Gerald J. Gleich

Keyword(s):

Eosinophil Cationic Protein ◽

Eosinophil Infiltration ◽

Cationic Protein ◽

Human Eosinophil ◽

Lung Inflation ◽

Cationic Proteins ◽

C Fibers ◽

Arterial Blood ◽

C Fiber ◽

Eosinophil Granule

Experiments were performed to test the hypothesis that human eosinophil granule-derived cationic proteins stimulate vagal C-fiber afferents in the lungs and elicit pulmonary chemoreflex responses in anesthetized Sprague-Dawley rats. Intratracheal instillation of eosinophil cationic protein (ECP; 1–2 mg/ml, 0.1 ml) consistently induced an irregular breathing pattern, characterized by tachypnea (change in breathing frequency of 44.7%) and small unstable tidal volume (Vt). The tachypnea, accompanied by decreased heart rate and arterial blood pressure, started within 30 s after the delivery of ECP and lasted for >30 min. These ECP-induced cardiorespiratory responses were completely prevented by perineural capsaicin treatment of both cervical vagi, which selectively blocked C-fiber conduction, suggesting the involvement of these afferents. Indeed, direct recording of single-unit activities of pulmonary C-fibers further demonstrated that the same dose of ECP evoked a pronounced and sustained (>30-min) stimulatory effect on pulmonary C-fibers. Furthermore, the sensitivity of these afferents to lung inflation was also markedly elevated after the ECP instillation, whereas the vehicle of ECP administered in the same manner had no effect. Other types of eosinophil granule cationic proteins, such as major basic protein and eosinophil peroxidase, induced very similar respiratory and cardiovascular reflex responses. In conclusion, these results show that eosinophil granule-derived cationic proteins induce a distinct stimulatory effect on vagal pulmonary C-fiber endings, which may play an important role in the airway hyperresponsiveness associated with eosinophil infiltration in the airways.

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Human eosinophil cationic protein manifests alarmin activity through its basicity and ribonuclease activity

10.1101/2021.03.24.436724 ◽

2021 ◽

Author(s):

Ayush Attery ◽

Irene Saha ◽

Prafullakumar Tailor ◽

Janendra K Batra

Keyword(s):

Eosinophil Cationic Protein ◽

Adaptive Immune System ◽

Rnase A ◽

Chemotactic Activity ◽

Rnase Activity ◽

Cationic Protein ◽

Human Eosinophil ◽

Adaptive Immune ◽

Antigen Presenting ◽

Dc Maturation

Eosinophil cationic protein (ECP), eosinophil derived neurotoxin (EDN), and human pancreatic ribonuclease (HPR) are members of the RNase A superfamily having similar catalytic residues and diverse functions. Alarmins are the endogenous mediators of innate immunity which activate or alarm the adaptive immune system by activating antigen presenting cells (APCs). EDN acts as an alarmin molecule and plays an important role in innate as well as adaptive immunity. EDN displays chemotactic activity for dendritic cells (DCs) and activates them, has antiviral and antiparasitic activities, and is rapidly released from immune cells. HPR only displays chemotactic activity while no such activity has been reported for ECP. In this study we show that ECP displays the chemotactic activity comparable to that of HPR and EDN. ECP also interacts with TLR-2 to activate NF-κB/AP-1 expression like EDN. The RNase activity of ECP, EDN and HPR, and basicity of ECP were found to be crucial determinants for their chemotactic activity for APCs, however for the DC maturation activity, RNase activity was not found to be essential. Bovine RNase A did not show any chemotactic activity despite having a very high RNase activity indicating that other determinants in addition to the RNase activity are involved in the chemotactic activity of ECP, EDN and HPR. The current study establishes that ECP also can act like an alarmin.

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Human eosinophil cationic protein. Molecular cloning of a cytotoxin and helminthotoxin with ribonuclease activity.

Journal of Experimental Medicine ◽

10.1084/jem.170.1.163 ◽

1989 ◽

Vol 170 (1) ◽

pp. 163-176 ◽

Cited By ~ 86

Author(s):

H F Rosenberg ◽

S J Ackerman ◽

D G Tenen

Keyword(s):

Amino Acid ◽

Cellular Localization ◽

Basic Protein ◽

Eosinophil Cationic Protein ◽

Ribonuclease Activity ◽

Monocytic Differentiation ◽

Cationic Protein ◽

Human Eosinophil ◽

Reading Frame ◽

The Matrix

We have isolated a 725-bp full-length cDNA clone for the human eosinophil cationic protein (ECP). ECP is a small, basic protein found in the matrix of the eosinophil's large specific granule that has cytotoxic, helminthotoxic, and ribonuclease activity, and is a member of the ribonuclease multigene family. The cDNA sequence shows 89% sequence identity with that reported for the related granule protein, eosinophil-derived neurotoxin (EDN). The open reading frame encodes a previously unidentified 27-amino acid leader sequence preceding a 133-residue mature ECP polypeptide with a molecular mass of 15.6 kD. The encoded amino acid sequence of ECP shows 66% identity to that of EDN and 31% identity to that of human pancreatic ribonuclease, including conservation of the essential structural cysteine and cataytic lysine and histidine residues. mRNA for ECP was detected in eosinophil-enriched peripheral granulocytes and in a subclone of the promyelocytic leukemia line, HL-60, induced toward eosinophilic differentiation with IL-5. No ECP mRNA was detected in uninduced HL-60 cells, or in HL-60 cells induced toward monocytic differentiation with vitamin D3 or toward neutrophilic differentiation with DMSO. In contrast, mRNA for EDN was detected in uninduced HL-60 cells and was upregulated in HL-60 cells induced with DMSO. Despite similarities in sequence and cellular localization, these results suggest that ECP and EDN are subject to different regulatory mechanisms.

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