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

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

Biological Chemistry ◽

10.1515/bc.2011.037 ◽

2011 ◽

Vol 392 (4) ◽

Cited By ~ 17

Author(s):

Anubha Singh ◽

Janendra K. Batra

Keyword(s):

Biological Activities ◽

Eosinophil Cationic Protein ◽

Underlying Mechanism ◽

Rnase A ◽

Cationic Protein ◽

Human Eosinophil ◽

Eosinophil Granule ◽

Reduced Toxicity ◽

Membrane Destabilization

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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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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Biochemical and functional similarities between human eosinophil-derived neurotoxin and eosinophil cationic protein: homology with ribonuclease.

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.83.10.3146 ◽

1986 ◽

Vol 83 (10) ◽

pp. 3146-3150 ◽

Cited By ~ 177

Author(s):

G. J. Gleich ◽

D. A. Loegering ◽

M. P. Bell ◽

J. L. Checkel ◽

S. J. Ackerman ◽

...

Keyword(s):

Eosinophil Cationic Protein ◽

Protein Homology ◽

Cationic Protein ◽

Human Eosinophil

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Cellular side of acquired immunity (B cells)

Oxford Textbook of Rheumatology ◽

10.1093/med/9780199642489.003.0050_update_001 ◽

2013 ◽

pp. 371-378

Author(s):

Thomas Dörner ◽

Peter E. Lipsky

Keyword(s):

B Cells ◽

B Cell ◽

T Cell Activation ◽

Cell Activation ◽

Plasma Cells ◽

Adaptive Immune System ◽

Adaptive Immune ◽

B Cell Homeostasis ◽

Anti Cd20 ◽

Antigen Presenting

B cells have gained interest in rheumatoid arthritis (RA) beyond being the precursors of antibody-producing plasma cells since they are also a broader component of the adaptive immune system. They are capable of functioning as antigen-presenting cells for T-cell activation and can produce an array of cytokines. Disturbances of peripheral B-cell homeostasis together with the formation of ectopic lymphoid neogenesis within the inflamed synovium appears to be a characteristic of patients with RA. Enhanced generation of memory B cells and autoreactive plasma cells producing IgM-RF and ACPA-IgG antibodies together with formation of immune complexes contribute to the maintenance of RA, whereas treatment with B-cell-directed anti-CD20 and CLTA4-Ig therapy provides clinical benefit.

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