Source: http://www.google.com/patents/US7923535?ie=ISO-8859-1
Timestamp: 2015-08-30 18:44:53
Document Index: 594690042

Matched Legal Cases: ['Application No. 60', 'ART2', 'ART2', 'ART2', 'ART2', 'ART2', 'ART2', 'ART2', 'ART2', 'ART2', 'ART2', 'ART2', 'ART2', 'ART2', 'ART2', 'ART2', 'ART2', 'ART2', 'ART2', 'ART2', 'ART2', 'ART2', 'ART2', 'ART2', 'ART2', 'ART2', 'ART2', 'ART2', 'ART2', 'ART2', 'ART2', 'ART2', 'ART2', 'ART2', 'ART2']

Patent US7923535 - Tryptophan as a functional replacement for ADP-ribose-arginine in ... - Google PatentsSearch Images Maps Play YouTube News Gmail Drive More »Sign inAdvanced Patent SearchPatentsA method is disclosed for producing a polypeptide with a modified activity or stability, by replacing an arginine residue capable of being ADP-ribosylated with a tryptophan or a phenylalanine. In one embodiment, compositions are provided that include polypeptides, such as alpha defensin, with arginine-to-tryptophan...http://www.google.com/patents/US7923535?utm_source=gb-gplus-sharePatent US7923535 - Tryptophan as a functional replacement for ADP-ribose-arginine in recombinant proteinsAdvanced Patent SearchPublication numberUS7923535 B2Publication typeGrantApplication numberUS 12/430,023Publication dateApr 12, 2011Filing dateApr 24, 2009Priority dateJun 28, 2002Fee statusPaidAlso published asCA2490290A1, EP1549746A1, EP1549746A4, US7541139, US20060074037, US20090203878, WO2004003195A1Publication number12430023, 430023, US 7923535 B2, US 7923535B2, US-B2-7923535, US7923535 B2, US7923535B2InventorsJoel Moss, Linda Stevens, Christelle Bourgeois, Rita BortellOriginal AssigneeThe United States Of America As Represented By The Secretary Of The Department Of Health And Human Services, University Of MassachusettsExport CitationBiBTeX, EndNote, RefManPatent Citations (10), Non-Patent Citations (57), Classifications (29), Legal Events (3) External Links: USPTO, USPTO Assignment, EspacenetTryptophan as a functional replacement for ADP-ribose-arginine in recombinant proteins
US 7923535 B2Abstract
1. A substituted human defensin polypeptide, comprising :
(i) a tryptophan residue or a phenylalanine residue substituted for at least one arginine residue in a corresponding non-substituted human alpha-defensin or human beta-defensin polypeptide;
(ii) six conserved cysteine residues that form three disulfide bonds;
(iii) decreased cytotoxic activity, compared to the corresponding non-substituted human defensin polypeptide; and
(iv) increased polypeptide stability, compared to the corresponding non-substituted human defensin polypeptide, wherein the at least one arginine residue in the corresponding non-substituted human defensin polypeptide is ADP-ribosylated,
wherein the polypeptide is no more than 94 amino acids in length and retains anti-microbial activity.
2. The substituted human defensin polypeptide of claim 1, wherein the polypeptide is no more than 50 amino acids in length.
3. The substituted human defensin polypeptide of claim 1, wherein a tryptophan residue is substituted for the at least one arginine residue.
4. The substituted human defensin polypeptide of claim 1, wherein a phenylalanine residue is substituted for the at least one arginine residue.
5. The substituted human defensin polypeptide of claim 2, wherein the polypeptide is between 29 and 34 or between 34 and 37 amino acids in length.
6. The substituted human defensin polypeptide of claim 1, wherein the corresponding non-substituted human defensin is an alpha defensin.
7. The substituted human defensin polypeptide of claim 6, wherein the alpha defensin is human neutrophil peptide (HNP)-1, HNP-2, HNP-3, HNP-4, human defensin (HD)-5, HD-6, or defensin-X.
8. A pharmaceutical composition comprising a therapeutically effective amount of the substituted human defensin polypeptide of claim 1.
9. The substituted human defensin polypeptide of claim 1, wherein the antimicrobial activity comprises chemotaxis of T cells, neutrophil recruitment or cytokine release.
10. The pharmaceutical composition of claim 8, wherein the antimicrobial activity comprises chemotaxis of T cells, neutrophil recruitment or cytokine release.
11. The substituted human defensin polypeptide of claim 7, wherein the substituted human defensin polypeptide comprises an amino acid sequence set forth as SEQ ID NO: 3 and wherein at least one arginine residue at position 4, 13, 14, or 23 of SEQ ID NO: 3 is substituted with a tryptophan residue or a phenylalanine residue.
12. The substituted human defensin polypeptide of claim 7, wherein the substituted human defensin polypeptide comprises an amino acid sequence set forth as SEQ ID NO: 6 and wherein at least one arginine residue at position 5, 10, 11, 15, or 32 of SEQ ID NO: 6 is substituted with a tryptophan residue or a phenylalanine residue.
13. The substituted human defensin polypeptide of claim 7, wherein the substituted human defensin polypeptide comprises an amino acid sequence set forth as SEQ ID NO: 8 and wherein at least one arginine residue at position 5, 8, 12, 24, 27, or 31 of SEQ ID NO: 8 is substituted with a tryptophan residue or a phenylalanine residue.
14. The substituted human defensin polypeptide of claim 7, wherein the substituted human defensin polypeptide comprises an amino acid sequence set forth as SEQ ID NO: 10 and wherein at least one arginine residue at position 5, 6, or 26 of SEQ ID NO: 10 is substituted with a tryptophan residue or a phenylalanine residue.
15. The pharmaceutical composition of claim 8, wherein the substituted human defensin polypeptide comprises an amino acid sequence set forth as SEQ ID NO: 3 and wherein at least one arginine residue at position 4, 13, 14, or 23 of SEQ ID NO: 3 is substituted with a tryptophan residue or a phenylalanine residue.
16. The pharmaceutical composition of claim 8, wherein the substituted human defensin polypeptide comprises an amino acid sequence set forth as SEQ ID NO: 6 and wherein at least one arginine residue at position 5, 10, 11, 15, or 32 of SEQ ID NO: 6 is substituted with a tryptophan residue or a phenylalanine residue.
17. The pharmaceutical composition of claim 8, wherein the substituted human defensin polypeptide comprises an amino acid sequence set forth as SEQ ID NO: 8 and wherein at least one arginine residue at position 5, 8, 12, 24, 27, or 31 of SEQ ID NO 8 is substituted with a tryptophan residue or a phenylalanine residue.
18. The pharmaceutical composition of claim 8, wherein the substituted human defensin polypeptide comprises an amino acid sequence set forth as SEQ ID NO: 10 and wherein at least one arginine residue at position 5, 6, or 26 of SEQ ID NO: 10 is substituted with a tryptophan residue or a phenylalanine residue.
19. The substituted human defensin polypeptide of claim 1, wherein the corresponding non-substituted human defensin is a beta defensin.
20. The substituted human defensin polypeptide of claim 19, wherein the beta defensin is human beta defensinl (hBD1), hBD2, hBD3, or hBD4.
21. The substituted human defensin polypeptide of claim 11, wherein the substituted human defensin polypeptide comprises an amino acid sequence set forth as SEQ ID NO: 2 and wherein at least one arginine residue at position 5, 14, 15, or 24 of SEQ ID NO: 2 is substituted with a tryptophan residue or a phenylalanine residue.
22. The substituted human defensin polypeptide of claim 11, wherein the substituted human defensin polypeptide comprises an amino acid sequence set forth as SEQ ID NO: 4 and wherein at least one arginine residue at position 5, 14, 15, or 24 of SEQ ID NO: 4 is substituted with a tryptophan residue or a phenylalanine residue.
23. The substituted human defensin polypeptide of claim 11, wherein the substituted human defensin polypeptide comprises an amino acid sequence set forth as SEQ ID NO: 3 and wherein the arginine residue at position 13 of SEQ ID NO: 3 is substituted with a phenylalanine residue.
This is a continuation of U.S. application Ser. No. 10/517,565, filed Dec. 7, 2004 now U.S. Pat. No. 7,541,139, which is the �371 U.S. National Stage of International Application No. PCT/US2003/020498, filed Jun. 27, 2003, which was published in English under PCT Article 21(2), which in turn claims the benefit of U.S. Provisional Application No. 60/393,033, filed Jun. 28, 2002, all of which are incorporated by reference herein in their entirety.
FIG. 1 is a schematic drawing of the deduced amino acid sequences of rat ART2a (RT6.1) and rat ART2b (RT6.2). Identical amino acids are shaded. Arginine 204 and 81 are specific to ART2b. In ART2a, N58 and 58NKSE61 are in a putative consensus glycosylation site not present in ART2b. Regions I, II, and III, believed to participate in formation of the catalytic site in the bacterial toxin and mammalian ADP-ribosyltransferases, are indicated by solid lines and the putative catalytic amino acids by an asterisk. Dotted underlines indicate signal sequences, which are excised during the export into the endoplasmic reticulum (amino terminus) and attachment of the glycosylphosphotidylinositol (GPI) anchor (carboxy terminus).
FIG. 3 is a digital image of a set of blots demonstrating the auto-ADP-ribosyltransferase activity (FIG. 3A) and immunoreactivity (FIG. 3B), as well as data demonstrating the NAD glycohydrolase (NADase) activity (nmol per hour) (FIG. 3C) of ART2b, ART2a, and various mutant forms of these proteins. The left column shows data for the following wild-type and mutant ART2b proteins: ART2b (lane 1), ART2b(R204K) (lane 2), ART2b(R81K) (lane 3), ART2b(R204Y) (lane 4), ART2b(R204E) (lane 5), ART2b(R204W) (lane 6), ART2b(R81K,R204K) (lane 7). The right column shows data for the following wild-type and mutant ART2a proteins: ART2a (lane 1), ART2a(M81R) (lane 2), ART2a(Y204R) (lane 3), ART2a(M81R,Y204R) (lane 4), ART2a(N58A,Y204R) (lane 5), ART2a(59NMA61,Y204R) (lane 6). Data shown are representative of two experiments.
FIG. 4 is a digital image of a set of blots demonstrating the auto-ADP-ribosylation of ART2b (FIG. 4A), ART2a (FIG. 4B), and their various mutant forms. The gels contain samples from cells expressing ART2b wild-type (lane 1), ART2b(R81K) (lane 2), ART2b(R204W) (lane 3), ART2a(Y204R) (lane 4), ART2a(Y204R,M81R) (lane 5) and ART2a(59NMA61,Y204R) (lane 6). Data represent one of two experiments.
FIG. 6 is a digital image of a set of blots demonstrating the SDS-PAGE separation of proteins tested for their sensitivity to acid, hydroxylamine and mercuric chloride. Samples from cells expressing wild-type ART2b, ART2b(R204W), ART2a(M81R,Y204R) and ART2a(N58A,Y204R) were auto-ADP-ribosylated with 10 μM[32P]NAD followed by addition of 10% TCA (column II), or further incubation with 5 mM NAD at 30� C. for 1 hour before precipitation with 10% TCA (column I). Neutralized samples were suspended in 0.1M Tris-HCl pH 7.5 (lane 1), 0.2M HCl (lane 2), 10 mM HgCl2 (lane 3), 2M NH2OH (lane 4), or 0.2M NaCl (lane 5) for 2 hours at 37� C. The samples were separated by SDS-PAGE in 12% gels, transferred to nitrocellulose and analyzed by phosphorImager