Cobra CRISP functions as an inflammatory modulator via a novel Zn2+- and heparan sulfate-dependent transcriptional regulation of endothelial cell adhesion molecules.
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.
Wang
Yu-Ling
YL
From the Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu 30013, Taiwan.
Kuo
Je-Hung
JH
Lee
Shao-Chen
SC
Liu
Jai-Shin
JS
Hsieh
Yin-Cheng
YC
Shih
Yu-Tsung
YT
Chen
Chun-Jung
CJ
Chiu
Jeng-Jiann
JJ
Wu
Wen-Guey
WG
D000818:Animals; D001665:Binding Sites; D002448:Cell Adhesion; D015815:Cell Adhesion Molecules / Q000235:genetics* / Q000276:immunology; D002460:Cell Line; D002478:Cells, Cultured; D004546:Elapid Venoms / Q000737:chemistry / Q000276:immunology / Q000494:pharmacology*; D017815:Elapidae; D042783:Endothelial Cells / Q000187:drug effects / Q000276:immunology*; D015870:Gene Expression / Q000187:drug effects; D006497:Heparan Sulfate / Q000276:immunology*; D006801:Humans; D018836:Inflammation Mediators / Q000737:chemistry / Q000276:immunology / Q000494:pharmacology*; D008968:Molecular Conformation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D014158:Transcription, Genetic / Q000187:drug effects*; D014961:X-Ray Diffraction; D015032:Zinc / Q000276:immunology*
D015815:Cell Adhesion Molecules; D004546:Elapid Venoms; D006497:Heparan Sulfate; D018836:Inflammation Mediators; D015032:Zinc; C495581:natrin protein, Naja atra