Zinc-binding of endostatin is essential for its antiangiogenic activity.
Endostatin is a potent angiogenesis inhibitor in vitro and in vivo. We used the yeast Pichia pastoris to express and purify soluble endostatin. It was discovered that metal chelating agents can induce N-terminal degradation of endostatin. We theorized that a metal was removed from endostatin which changed the conformation and allowed a contaminating protease to degrade the N-terminus. Atomic absorption and amino acid analysis of endostatin purified from Pichia pastoris and mammalian cells showed a 1:1 molar ratio of Zn2+ to protein. Ding et al. have shown that histidines 1, 3, 11, and aspartic acid 76 coordinate the Zn2+ atom (1). An H1/3A double, an H11A, and a D76A single mutant of endostatin were not able to regress Lewis lung carcinoma. We conclude that the ability of endostatin to bind Zn2+ is essential for its antiangiogenic activity.
Boehm
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T
The Children's Hospital, and Departments of Surgery and Cellular Biology, Harvard Medical School, 300 Longwood Avenue, Boston, Massachusetts, 02115, USA. boehm_t@a1.tch.harvard.edu
O'reilly
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MS
Keough
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K
Shiloach
J
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Shapiro
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Folkman
J
J
D000595:Amino Acid Sequence; D000818:Animals; D000970:Antineoplastic Agents / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D001665:Binding Sites; D003001:Cloning, Molecular; D003094:Collagen / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology*; D043169:Endostatins; D007700:Kinetics; D008322:Mammals; D008546:Melanoma, Experimental; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009389:Neovascularization, Pathologic / Q000517:prevention & control*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology*; D010843:Pichia; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D014162:Transfection; D015032:Zinc / Q000378:metabolism*
D000970:Antineoplastic Agents; D043169:Endostatins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D003094:Collagen; D015032:Zinc