Abstract
The cysteine-rich secretory proteins (CRISPs) are widely distributed in mammals, reptiles and amphibians and have been identified in diverse organisms, including epididymis, salivary glands, and snake venoms. These proteins containing an N-terminal domain which is also name pathogenesis-related proteins of group 1 (PR-1) domain and a C-terminal cysteine-rich domain (CRD). We have purified natrin from Naja atra venom with molecule mass of 25KD and belong to CRISP family. In the present study, we presented the X-ray structure of natrin and investigated the physiological role in modulating the inflammatory function of vascular endothelial cells (ECs) and its underlying mechanisms. The 3D structures of natrin without and with zinc soaking are also reported at resolution 1.58 and 2.54 Å in, respectively. In addition to the presence of an ion channel-blocking BgK toxin fold (C-terminal), it comprises an α-β-α sandwich fold (N-terminal) with a Ser76-His115-Glu96 enzymatic triad. Biophysical characterization of natrin by using fluorescence, circular-dichroism, and X-ray crystallographic methods further reveals the presence of two Zn2+-binding sites for natrin. The strong Zn2+ binding site is near the putative Ser76-His115-Glu96 catalytic triad and binds to the Nε2 atoms of His60, His115 and a water molecule in tetrahedral coordination geometry. The weak binding site remains to be characterized, but it may modulate HS binding by enhancing its interaction with long chain HS. Natrin induced activation of mitogen-activated protein kinases (MAPKs; i.e., extracellular signal-regulated kinase [ERK], c-Jun-NH2-terminal kinase [JNK], and p38 MAPK) and nuclear factor-□B (NF-kB) and expression of adhesion molecules (i.e., intercellular adhesion molecule-1 [ICAM-1], vascular adhesion molecule-1 [VCAM-1], and E-selectin) in ECs and hence monocytic cell adhesion. Pre-treatment of ECs with a specific inhibitor for ERK (PD98059), JNK (SP600125), p38 MAPK (SB203580), or NF-kB (lactacystin), or the MAPK-specific small interfering RNAs (siRNAs) inhibited natrin-induced adhesion molecule expression. Surface plasmon resonance results showed that natrin exhibits strong binding activity to heparin. Pre-treating ECs with heparinase, an enzyme that cleaves the sulfation sites of heparan sulfate on the EC surfaces, resulted in reductions in natrin-induced activations of MAPKs and NF-□B and expressions of adhesion molecules in ECs, suggesting that heparan sulfate on the EC surface plays important roles in EC responses to natrin. Our findings provide insights into the role of CRISP functions as an inflammatory modulator via a novel Zn2+- and heparan sulfate- dependent transcriptional regulation of endothelial cell adhesion molecules