title: Engineering of a human kringle domain into agonistic and antagonistic binding proteins functioning in vitro and in vivo.
abstract: Here, we report the development of target-specific binding proteins based on the kringle domain (KD) ( approximately 80 residues), a ubiquitous modular structural unit occurring across eukaryotic species. By exploiting the highly conserved backbone folding by core residues, but using extensive sequence variations in the seven loop regions of naturally occurring human KDs, we generated a synthetic KD library on the yeast cell surface by randomizing 45 residues in the loops of a human KD template. We isolated KD variants that specifically bind to anticancer target proteins, such as human death receptor 4 (DR4) and/or DR5, and that function as agonists to induce apoptotic cell death in several cancer cell lines in vitro and inhibit tumor progression in mouse models. Combined treatments with KD variants possessing different recognition sites on the same target protein exerted synergisitic tumoricidal activities, compared to treatment with individual variants. In addition to the agonists, we isolated an antagonistic KD variant that binds human tumor necrosis factor-alpha (TNFalpha) and efficiently neutralizes TNFalpha-induced cytotoxicity in vitro and in vivo. The KD scaffold with seven flexible loops protruding from the central core was strongly sequence-tolerant to mutations in the loop regions, offering a potential advantage of distinct binding sites for target recognition on the single domain. Our results suggest that the KD scaffold can be used to develop target-specific binding proteins that function as agonists or antagonists toward given target molecules, indicative of their potential use as biotherapeutics.
authors.affiliation: Department of Molecular Science and Technology, Ajou University, Yeongtong-gu, Suwon 443-749, Korea.
authors.forename: Chang-Han
authors.initials: CH
authors.lastname: Lee
authors.forename: Kyung-Jin
authors.initials: KJ
authors.lastname: Park
authors.forename: Eun-Sil
authors.initials: ES
authors.lastname: Sung
authors.forename: Aeyung
authors.initials: A
authors.lastname: Kim
authors.forename: Ji-Da
authors.initials: JD
authors.lastname: Choi
authors.forename: Jeong-Sun
authors.initials: JS
authors.lastname: Kim
authors.forename: Soo-Hyun
authors.initials: SH
authors.lastname: Kim
authors.forename: Myung-Hee
authors.initials: MH
authors.lastname: Kwon
authors.forename: Yong-Sung
authors.initials: YS
authors.lastname: Kim
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D045744:Cell Line, Tumor; D005260:Female; D006801:Humans; D018082:Kringles; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019151:Peptide Library; D011485:Protein Binding; D015202:Protein Engineering; D017510:Protein Folding; D054730:Protein Interaction Domains and Motifs; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D023041:Xenograft Model Antitumor Assays
chemical_list: D019151:Peptide Library; D011506:Proteins
references.citation: Cell. 1997 Jan 24;88(2):277-85
references.pmid: 9008168
references.citation: J Mol Evol. 1987;26(4):358-69
references.pmid: 3131537
references.citation: Biochem Biophys Res Commun. 2006 Aug 4;346(3):896-903
references.pmid: 16777066
references.citation: Biochemistry. 1999 Nov 30;38(48):15741-55
references.pmid: 10625440
references.citation: Curr Opin Biotechnol. 2007 Aug;18(4):295-304
references.pmid: 17643280
references.citation: Curr Med Chem Anticancer Agents. 2002 Nov;2(6):667-81
references.pmid: 12678719
references.citation: Nat Biotechnol. 2005 Dec;23(12):1556-61
references.pmid: 16299519
references.citation: Nat Biotechnol. 2005 Oct;23(10):1257-68
references.pmid: 16211069
references.citation: Proc Natl Acad Sci U S A. 1999 Mar 2;96(5):1898-903
references.pmid: 10051566
references.citation: J Mol Biol. 2007 Dec 14;374(5):1374-88
references.pmid: 17996896
references.citation: Oncogene. 2001 Aug 23;20(37):5186-94
references.pmid: 11526507
references.citation: Mol Cancer Ther. 2009 Aug;8(8):2276-85
references.pmid: 19638452
references.citation: J Histochem Cytochem. 1998 Oct;46(10):1175-83
references.pmid: 9742073
references.citation: Proc Natl Acad Sci U S A. 2009 May 19;106(20):8198-203
references.pmid: 19416843
references.citation: Exp Mol Med. 2008 Feb 29;40(1):35-42
references.pmid: 18305396
references.citation: Pharmacol Ther. 2008 Feb;117(2):244-79
references.pmid: 18155297
references.citation: J Clin Invest. 2008 Jun;118(6):1979-90
references.pmid: 18523647
references.citation: FEBS J. 2008 Jun;275(11):2684-90
references.pmid: 18435757
references.citation: J Clin Invest. 1999 Jul;104(2):155-62
references.pmid: 10411544
references.citation: Nat Biotechnol. 1997 Aug;15(8):772-7
references.pmid: 9255793
references.citation: Nat Biotechnol. 1999 Feb;17(2):170-5
references.pmid: 10052354
references.citation: Nucleic Acids Res. 2006 Jan 1;34(Database issue):D257-60
references.pmid: 16381859
references.citation: J Mol Biol. 2001 May 11;308(4):705-19
references.pmid: 11350170
references.citation: Angew Chem Int Ed Engl. 2006 Dec 11;45(48):8106-25
references.pmid: 17120282
references.citation: Cancer Res. 2007 Aug 1;67(15):7327-34
references.pmid: 17671202
references.citation: J Mol Biol. 2003 Jan 10;325(2):337-54
references.pmid: 12488099
references.citation: Nat Biotechnol. 2004 May;22(5):575-82
references.pmid: 15097997