Purification and characterization of Moschatin, a novel type I ribosome-inactivating protein from the mature seeds of pumpkin (Cucurbita moschata), and preparation of its immunotoxin against human melanoma cells.
A novel ribosome-inactivating protein designated Moschatin from the mature seeds of pumpkin (Cucurbita moschata) has been successively purified to homogeneity, using ammonium sulfate precipitation, CM-cellulose 52 column chromatography, Blue Sepharose CL-6B Affinity column chromatography and FPLC size-exclusion column chromatography. Moschatin is a type 1 RIP with a pI of 9.4 and molecular weight of approximately 29 kD. It is a rRNA N-glycosidase and potently blocked the protein synthesis in the rabbit reticulocyte lysate with a IC50 of 0.26 nM. Using the anti-human melanoma McAb Ng76, a novel immunotoxin Moschatin-Ng76 was prepared successfully and it efficiently inhibited the growth of targeted melanoma cells M21 with a IC50 of 0.04 nM, 1500 times lower than that of free Moschatin. The results implied that Moschatin could be used as a new potential anticancer agent.
Xia
Heng Chuan
HC
Key Laboratory of Proteomics, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.
Li
Feng
F
Li
Zhen
Z
Zhang
Zu Chuan
ZC
D000818:Animals; D000972:Antineoplastic Agents, Phytogenic / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D045744:Cell Line, Tumor; D028464:Cucurbita / Q000737:chemistry*; D004354:Drug Screening Assays, Antitumor; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology; D000922:Immunotoxins / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification / Q000494:pharmacology*; D008545:Melanoma / Q000188:drug therapy*; D008970:Molecular Weight; D009699:N-Glycosyl Hydrolases / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000737:chemistry / Q000494:pharmacology*; D011817:Rabbits; D054788:Ribosome Inactivating Proteins; D012270:Ribosomes / Q000187:drug effects; D012639:Seeds / Q000737:chemistry*
D000972:Antineoplastic Agents, Phytogenic; D007074:Immunoglobulin G; D000922:Immunotoxins; D009699:N-Glycosyl Hydrolases; D054788:Ribosome Inactivating Proteins