Tryptophan as a functional replacement for ADP-ribose-arginine in recombinant proteins
A 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 or arginine-to-phenylalanine substitutions, where the arginine residue is capable of being ADP-ribosylated. In another embodiment, methods are disclosed for modifying an immune response in a subject.
1. A method of producing a protein with an increased antimicrobial activity or polypeptide stability, comprising:
replacing an arginine residue in a polypeptide of interest with a tryptophan residue or a phenylalanine residue to produce a tryptophan-substituted or phenylalanine-substituted polypeptide; and
comparing the antimicrobial activity or polypeptide stability of the polypeptide of interest with the tryptophan-substituted or phenylalanine-substituted polypeptide, wherein the tryptophan-substituted or phenylalanine-substituted polypeptide has increased antimicrobial activity or polypeptide stability compared to the polypeptide of interest, and wherein the tryptophan-substituted or phenylalanine-substituted polypeptide has similar antimicrobial activity or increased polypeptide stability compared to the polypeptide of interest wherein the arginine residue is ADP-ribosylated, thereby producing the protein with increased antimicrobial activity or polypeptide stability.
2. The method of claim 1 , wherein the tryptophan-substituted or phenylalanine-substituted polypeptide has an increased antimicrobial activity.
3. The method of claim 2 , wherein the antimicrobial activity comprises chemotaxis of T cells, neutrophil recruitment, or cytokine release.
4. The method of claim 3 , wherein the cytokine release comprises interleukin-8 release.
5. The method of claim 2 , wherein the protein is a defensin.
6. The method of claim 5 , wherein the defensin is an alpha defensin.
7. The method of claim 2 , wherein the arginine residue is substituted with a tryptophan residue.
8. The method of claim 2 , wherein the arginine residue is substituted with a phenylalanine residue.
9. The method of claim 2 , wherein the activity is increased as compared to the polypeptide of interest.
10. The method of claim 2 , wherein the stability is increased as compared to the polypeptide of interest.
11. The method of claim 2 , wherein the increased activity or stability is a 100% increase, as compared to a control polypeptide.
12. The method of claim 2 , wherein the increased activity or stability is a 50% increase, as compared to a control polypeptide.
13. A method of increasing antimicrobial activity or polypeptide stability of a defensin polypeptide of interest, comprising:
substituting an arginine residue in the defensin polypeptide of interest with a tryptophan or a phenylalanine to produce a tryptophan-substituted or phenylalanine-substituted defensin polypeptide;
comparing the antimicrobial activity or polypeptide stability of the defensin polypeptide of interest with the tryptophan-substituted or phenylalanine-substituted defensin polypeptide, wherein the tryptophan-substituted or phenylalanine-substituted defensin polypeptide has increased antimicrobial activity or polypeptide stability compared to the defensin polypeptide of interest, and wherein the tryptophan-substituted or phenylalanine-substituted defensin polypeptide has similar antimicrobial activity or increased polypeptide stability compared to the defensin polypeptide of interest wherein the arginine residue is ADP-ribosylated,
thereby increasing the antimicrobial activity or the polypeptide stability of the defensin polypeptide.
14. The method of claim 13 , wherein the defensin polypeptide is an alpha defensin.
15. The method of claim 13 , wherein the antimicrobial activity comprises T cell chemotaxis, neutrophil recruitment, or cytokine release.
16. A method of increasing an antimicrobial immune response in a subject infected with or at risk of being infected with a microbe, comprising administering to the subject a therapeutically effective amount of a defensin polypeptide comprising an amino acid substitution, wherein the amino acid substitution is a replacement of an arginine in a defensin polypeptide of interest with a tryptophan or a phenylalanine to produce a tryptophan-substituted or phenylalanine-substituted defensin polypeptide, wherein the tryptophan-substituted or phenylalanine-substituted defensin polypeptide has similar antimicrobial activity or increased polypeptide stability, compared to the defensin polypeptide of interest wherein the at least one arginine residue is ADP-ribosylated,
thereby increasing the antimicrobial immune response in the subject infected with or at risk of being infected with a microbe.
17. The method of claim 16 , wherein the immune response comprises T cell chemotaxis, neutrophil recruitment, or cytokine release.
18. The method of claim 16 , wherein the subject has an immune disorder.