Purification, molecular cloning and mechanism of action of graminelysin I, a snake-venom-derived metalloproteinase that induces apoptosis of human endothelial cells.
Apoptosis, a programmed, physiological mode of cell death, is important in tissue homoeostasis. Here we report that a new metalloproteinase, graminelysin I, purified from Trimeresurus gramineus venom, induced apoptosis of human endothelial cells as examined by electrophoresis and flow cytometry. Graminelysin I contains only a metalloproteinase domain. It is a single-chain proteinase with a molecular mass of 27020 Da. cDNA sequence analysis revealed that the disintegrin-like and cysteine-rich domains of the putative precursor protein of graminelysin I are likely to be processed post-translationally, producing the proteinase domain (graminelysin I). Graminelysin I cleaved the alpha chain of fibrinogen preferentially and cleaved the beta chain either on longer incubation or at higher concentration. Graminelysin I inhibited the adhesion of human umbilical-vein endothelial cells (HUVECs) to immobilized fibrinogen and induced HUVECs detachment in a dose-dependent manner. These effects on HUVECs were abolished when graminelysin I was pretreated with EDTA. However, graminelysin I did not inhibit the adhesion of HUVECs to immobilized collagen. HUVECs were susceptible to death after treatment with graminelysin I when they were cultured on immobilized fibrinogen. In contrast, HUVECs were rather resistant to treatment with graminelysin I if they were cultured on immobilized collagen. Furthermore, graminelysin I induced apoptosis of HUVECs in a dose-dependent manner. Similarly, its apoptosis-inducing activity was blocked if it was treated with EDTA. These results suggest that the catalytic activity of graminelysin I on matrix proteins contributes to its apoptosis-inducing activity.
Wu
W B
WB
Department of Pharmacology, College of Medicine, National Taiwan University, No. 1, Sec. 1, Jen-Ai Rd., Taipei, Taiwan.
Chang
S C
SC
Liau
M Y
MY
Huang
T F
TF
D000595:Amino Acid Sequence; D017209:Apoptosis; D001483:Base Sequence; D002448:Cell Adhesion / Q000187:drug effects; D002478:Cells, Cultured; D003001:Cloning, Molecular; D003094:Collagen / Q000378:metabolism / Q000494:pharmacology; D003435:Crotalid Venoms / Q000201:enzymology*; D018076:DNA, Complementary / Q000032:analysis; D004347:Drug Interactions; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects*; D005340:Fibrinogen / Q000378:metabolism; D006801:Humans; D008666:Metalloendopeptidases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D012910:Snake Venoms / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology
D003435:Crotalid Venoms; D018076:DNA, Complementary; D012910:Snake Venoms; C029748:Trimeresurus venoms; C433504:graminelysin I; D005340:Fibrinogen; D003094:Collagen; D008666:Metalloendopeptidases