Cobra CRISP Functions as an Inflammatory Modulator via a Novel Zn2+- and Heparan Sulfate-dependent Transcriptional Regulation of Endothelial Cell Adhesion Molecules

Yu-Ling Wang,Je-Hung Kuo,Shao-Chen Lee,Jai-Shin Liu,Yin-Cheng Hsieh,Yu-Tsung Shih,Chun-Jung Chen,Jeng-Jiann Chiu,Wen-Guey Wu

doi:10.1074/jbc.m110.146290

Abstract

Cysteine-rich secretory proteins (CRISPs) have been identified as a toxin family in most animal venoms with biological functions mainly associated with the ion channel activity of cysteine-rich domain (CRD). CRISPs also bind to Zn(2+) at their N-terminal pathogenesis-related (PR-1) domain, but their function remains unknown. Interestingly, similar the Zn(2+)-binding site exists in all CRISP family, including those identified in a wide range of organisms. Here, we report that the CRISP from Naja atra (natrin) could induce expression of vascular endothelial cell adhesion molecules, i.e. intercellular adhesion molecule-1, vascular adhesion molecule-1, and E-selectin, to promote monocytic cell adhesion in a heparan sulfate (HS)- and Zn(2+)-dependent manner. Using specific inhibitors and small interfering RNAs, the activation mechanisms are shown to involve both mitogen-activated protein kinases and nuclear factor-κB. Biophysical characterization of natrin by using fluorescence, circular dichroism, and x-ray crystallographic methods further reveals the presence of two Zn(2+)-binding sites for natrin. The strong binding site is located near the putative Ser-His-Glu catalytic triad of the N-terminal domain. The weak binding site remains to be characterized, but it may modulate HS binding by enhancing its interaction with long chain HS. Our results strongly suggest that natrin may serve as an inflammatory modulator that could perturb the wound-healing process of the bitten victim by regulating adhesion molecule expression in endothelial cells. Our finding uncovers a new aspect of the biological role of CRISP family in immune response and is expected to facilitate future development of new therapeutic strategy for the envenomed victims.

Highlights

The three-dimensional structures of snake Cysteine-rich secretory proteins (CRISPs) from several species have been determined by x-ray crystallography to consist of two domains: the N-terminal conserved fold of pathogenesis-related (PR-1) domain and the C-terminal cysteine-rich domain (CRD)
The results that natrin regulations of adhesion molecule expression in endothelial cells (ECs) are transcriptional events were confirmed by the transfections of ECs with the promoter constructs containing the promoter regions of intercellular adhesion molecule-1 (ICAM-1) (Ϫ850 bp) or E-selectin (Ϫ540) and the reporter gene luciferase [40], which showed that ECs treated with natrin significantly increase the promoter activities of these adhesion molecules in ECs compared with static control cells
mitogen-activated protein kinases (MAPKs) and nuclear factor (NF)-␬B Are Involved in Natrin-induced Adhesion Molecule Expression in ECs—Given our findings that natrin induces the expressions of ICAM-1, vascular adhesion molecule-1 (VCAM-1), and E-selectin and activations of MAPKs and NF-␬B in ECs, we investigated whether MAPKs and NF-␬B are involved in natrin-induced EC ICAM-1, VCAM-1, and E-selectin expressions

Summary

EXPERIMENTAL PROCEDURES

Materials—Mouse monoclonal antibodies (mAbs) against ICAM-1, VCAM-1, and E-selectin were purchased from R&D Systems (Minneapolis, MN). The mRNA transcription levels were initially down-regulated within 1 h after actinomycin D treatment; the expressions became stabilized from 1– 4 h (Fig. 3B), indicating that natrin-induced ICAM-1, VCAM-1, and E-selectin expressions are stable and regulated at the transcriptional level in ECs. The results that natrin regulations of adhesion molecule expression in ECs are transcriptional events were confirmed by the transfections of ECs with the promoter constructs containing the promoter regions of ICAM-1 (Ϫ850 bp) or E-selectin (Ϫ540) and the reporter gene luciferase [40], which showed that ECs treated with natrin significantly increase the promoter activities of these adhesion molecules in ECs compared with static control cells (data not shown). The EC expressions of ICAM-1, VCAM-1, and E-selectin induced by natrin were inhibited by pretreating the cells with NF-␬B inhibitor lactacystin (20 ␮M), indicating that NF-␬B is involved in natrin-induced ICAM-1, VCAM-1, and E-selectin expressions in ECs (Fig. 5C)

ECs were treated with heparinase

DISCUSSION

Findings

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