Anti-inflammatory proteins and methods of preparation and use thereof

Abstract

The present disclosure relates to anti-inflammatory proteins, their uses, methods of preparation and methods of their detection. In particular, the invention relates to major royal jelly proteins modified by methyglyoxal and fragments thereof from manuka honey.

1 .- 29 . (canceled) 
     
     
         30 . A method of:
 a) reducing inflammation in a cellular tissue;   b) reducing the rate of phagocytosis by immune system cells;   c) inhibiting the receptors for phagocytosis on immune system cells; or   d) reducing the respiratory burst and release of reactive oxygen species in inflammatory cells;   comprising the step of contacting a composition to the cellular tissue or the cells, wherein the composition consists essentially of an isolated apalbumin protein or fragment thereof, which has been chemically modified by methylglyoxal (MGO).   
     
     
         31 . The method of  claim 30 , wherein the apalbumin protein or fragment thereof comprises between 17 and 32 amino acid residues modified by MGO. 
     
     
         32 . The method of  claim 31 , wherein the amino acid residues that are modified are either lysine or arginine. 
     
     
         33 . An apalbumin protein fragment, which has been chemically modified by methylglyoxal (MGO), which has anti-inflammatory capacity, which is not naturally occurring, which is not identical to the amino acid sequence set forth in SEQ ID NO:1, and which comprises at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 . 
     
     
         34 . The protein fragment of  claim 33 , wherein the protein fragment comprises between 17 and 32 amino acid residues modified by MGO. 
     
     
         35 . The protein fragment of  claim 34 , wherein the amino acid residues that are modified are either lysine or arginine. 
     
     
         36 . A composition comprising the protein fragment of  claim 33 . 
     
     
         37 . A method of:
 a) reducing inflammation in a cellular tissue;   b) reducing the rate of phagocytosis by immune system cells;   c) inhibiting the receptors for phagocytosis on immune system cells; or   d) reducing the respiratory burst and release of reactive oxygen species in inflammatory cells;   comprising the step of contacting the composition of  claim 36  to the cellular tissue or the cells.   
     
     
         38 . An apalbumin protein variant, which has been chemically modified by methylglyoxal (MGO), which has anti-inflammatory capacity, which is not naturally occurring, which is not identical to the amino acid sequence set forth in SEQ ID NO:1, and which comprises at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO:1. 
     
     
         39 . The protein variant of  claim 38 , comprising between 17 and 32 amino acid residues modified by MGO. 
     
     
         40 . The protein variant of  claim 39 , wherein the amino acid residues that are modified are either lysine or arginine. 
     
     
         41 . A composition comprising the protein variant of  claim 38 . 
     
     
         42 . A method of:
 a) reducing inflammation in a cellular tissue;   b) reducing the rate of phagocytosis by immune system cells;   c) inhibiting the receptors for phagocytosis on immune system cells; or   d) reducing the respiratory burst and release of reactive oxygen species in inflammatory cells;   comprising the step of contacting the composition of  claim 41  to the cellular tissue or the cells.   
     
     
         43 . A method of producing an anti-inflammatory molecule by modifying royal jelly, the method including the step of reacting royal jelly with MGO at a concentration of at least 0.1%, 0.5%, or 1.0% MGO, thereby producing an MGO-modified apalbumin or fragment thereof, wherein the MGO-modified apalbumin or fragment thereof is an anti-inflammatory molecule. 
     
     
         44 . The method of  claim 43 , which further includes the step of isolating the MGO-modified apalbumin protein or fragment thereof from the royal jelly. 
     
     
         45 . A method of:
 a) reducing inflammation in a cellular tissue;   b) reducing the rate of phagocytosis by immune system cells;   c) inhibiting the receptors for phagocytosis on immune system cells; or   d) reducing the respiratory burst and release of reactive oxygen species in inflammatory cells;   comprising the step of contacting the cellular tissue or the cells with a composition comprising the MGO-modified apalbumin protein or fragment thereof of  claim 44 .   
     
     
         46 . A method of increasing the anti-inflammatory capacity of an apalbumin protein or fragment thereof, by chemically treating the apalbumin protein or fragment thereof with MGO, formaldehyde, glyoxal, and/or glutaraldehyde, thereby increasing the anti-inflammatory capacity of the apalbumin protein or fragment thereof. 
     
     
         47 . The method of  claim 46 , wherein the apalbumin protein or fragment thereof is an apalbumin 1 protein or fragment thereof. 
     
     
         48 . A method of increasing the anti-inflammatory capacity and MGO-modified apalbumin protein content of a honey sample, comprising the step of adding MGO or a MGO-precursor molecule to the honey sample, thereby increasing the anti-inflammatory capacity and the content of MGO-modified apalbumin protein of the honey sample. 
     
     
         49 . The method of  claim 48 , wherein the MGO-modified apalbumin protein is an MGO-modified apalbumin 1 protein.