Family of peptides known as xenoxins

Abstract

The present invention relates to a peptide family designated as xenonins, wherein the representative members can be isolated from the substance secreted from the skin of Xenopus laevis , and having precious pharmacological properties. The present invention further relates to a method for preparing a peptide of the invention comprising transforming a cell with an isolated DNA fragment encoding the peptide, culturing the cell in a suitable medium and recovering the peptide of the invention from the culture.

1 . Peptide, characterized in that it comprises an amino acid sequence which: 
 a. contains at least 8 cysteines (Cys) which are linked through 4 disulfide bridges according to the arrangement Cys 1  with CyS 3 , Cys 2  with Cys 4 , Cys 5  with Cys 6  and Cys 7  with Cys 8 ;    b. contains 0 to 3 amino acids on the N-terminal side of Cys 1 , 9 to 14 amino acids between Cys 1  and Cys 2 , 3 to 7 amino acids between Cys 2  and Cys 3 , 11 to 18 amino acids between Cys 3  and sCys 4 , 1 to 6 amino acids between Cys 4  and Cys 5 , 7 to 15 amino acids between Cys 5  and Cys 6 , no amino acid between Cys 6  and Cys 7 , 3 to 5 amino acids between Cys 7  and Cys 8  and 0 to 10 amino acids on the C-terminal side of Cys 8 ; and    c. displays an identity of amino acids, after alignment, of at least 40% with the amino acid sequence identified under the number SEQ ID NO:  3 ,    as well as a fragment derived from this sequence:    
     
     
         2 . Peptide, characterized in that it comprises an amino acid sequence which: 
 a. contains at least 8 cysteines which, when the peptide adopts its folded conformation, are linked through 4 disulfide bridges according to the arrangement Cys 1  with Cys 3 , Cys 2  with Cys 4 , Cys 5  with Cys 6  and Cys 7  with Cys 8 ;    b. contains 0 to 3 amino acids on the N-terminal side of Cys 1 , 9 to 14 amino acids between Cys 1  and Cys 2 , 3 to 7 amino acids between Cys 2  and Cys 3 , 11 to 18 amino acids between Cys 3  and Cys 4 , 1 to 6 amino acids between Cys 4  and Cys 5 , 7 to 15 amino acids between Cys 5  and Cys 6 , no amino acid between Cys 6  and Cys 7  , 3 to 5 amino acids between Cys 7  and Cys 8  and 0 to 10 amino acids on the C-terminal side of Cys 8 ; and    c. displays an identity of amino acids, after alignment, of at least 40% with the amino acid sequence identified under the number SEQ ID NO: 3,    or a fragment derived from this sequence.    
     
     
         3 . A peptide according to  claim 1  or  2 , characterized in that the amino acid sequence contains 2 amino acids on the N-terminal side of Cys 1 , 10 to 13 amino acids between Cys 1  and Cys 2 , 4 to 6 amino acids between Cys 2  and Cys 3 , 12 to 17 amino acids between Cys 3  and Cys 4 , 1 to 5 amino acids between Cys 4  and Cys 5 , 8 to 14 amino acids between Cys 5  and Cys 6 , no amino acid between Cys 6  and Cys 7 , 4 amino acids between Cys 7  and Cys 8  and 2 amino acids on the C-terminal side of Cys 8 .  
     
     
         4 . A peptide according to one of  claims 1  to  3 , characterized in that the amino acid sequence displays an identity of amino acids of at least 50%, and preferably at least 60%, with the sequence of the amino [lacuna] identified under the number SEQ ID NO: 3.  
     
     
         5 . A peptide according to  claim 4 , characterized in that the identity of the amino acids is at least 70%.  
     
     
         6 . A peptide according to  claim 1  or  2 , characterized in that it comprises an amino acid sequence which contains 0 to 3 amino acids on the N-terminal side of Cys 1 , 13 amino acids between Cys 1  and Cys 2 , 6 amino acids between Cys 2  and Cys 3 , 12 amino acids between Cys 3  and Cys 4 , 5 amino acids between Cys 4  and Cys 5 , 14 amino acids between Cys 5  and Cys 6 , no amino acid between Cys 6  and Cys 7 , 4 amino acids between Cys 7  and Cys 8  and 0 to 10 amino acids on the C-terminal side of Cys 8 , and which displays an identity of amino acids, after alignment, of at least 80%, and preferably 90%, with the amino acid sequence identified under the number SEQ ID NO: 3.  
     
     
         7 . A peptide according to  claim 6  characterized in that the identity of amino acids is at least a sequence as defined under the number SEQ ID NO: 4 or SEQ ID NO: 5.  
     
     
         8 . A peptide according to  claim 6 , characterized in that the identity of amino acids is at least 95% with the amino acid sequence identified under the number SEQ ID NO: 3.  
     
     
         9 . A peptide according to  claim 8 , characterized in that the amino acid sequence comprises a sequence as defined under the number SEQ ID NO: 3.  
     
     
         10 . A method for preparing a peptide according to one of  claims 1  to  9 , characterized in that, in order to isolate said peptide from the matter secreted by the skin of a frog, preferably  Xenopus laevis,  
 a. the matter secreted by frog's skin is divided into fractions by precipitation using ammonium sulfate or an equivalent precipitating agent,  
 b. the precipitates which form at between 30% and 70% saturation with ammonium sulfate, and preferably between 30% and 60%, or under equivalent conditions, are subjected to gel permeation chromatography, and  
 c. the peptide fraction which elutes late is recovered,  
 and if necessary,  
 d. this peptide fraction is subjected to cation exchange HPLC chromatography optionally followed by reversed-phase HPLC chromatography.  
 
     
     
         11 . An isolated DNA fragment, characterized in that it comprises a sequence coding for a peptide according to one of  claims 1  to  9 .  
     
     
         12 . A DNA fragment according to  claim 11 , characterized in that it comprises a sequence coding for the precursor of a peptide according to one of  claims 1  to  9 .  
     
     
         13 . A DNA fragment according to  claim 11 , characterized in that the sequence codes for an amino acid sequence as identified under the number SEQ ID NO:3, SEQ ID NO: 4 or SEQ ID NO:5.  
     
     
         14 . A DNA fragment according to  claim 11 , characterized in that it comprises a nucleic acid sequence coding for an amino acid sequence as identified under the number SEQ ID NO: 1 and extending from the base at position 93 to the base at position 290.  
     
     
         15 . A DNA fragment according to  claim 12 , characterized in that it comprises a nucleic acid sequence coding for an amino acid sequence as defined under the number SEQ ID NO: 1 and extending from the nucleic acid at the position 39 to the nucleic acid at position 290.  
     
     
         16 . A cassette for the expression of an isolated DNA fragment according to one of  claims 11  to  15 , characterized in that said DNA fragment is placed under the control of elements permitting its transcription and/or translation in the transformed cell in question.  
     
     
         17 . An expression cassette according to  claim 16 , characterized in that it comprises, in addition, a pre sequence and a pro sequence, in particular the pro sequence of  Phormia terranovae  defensin A.  
     
     
         18 . An expression cassette according to  claim 16  or  17 , characterized in that it comprises a DNA fragment according to  claim 12 .  
     
     
         19 . A cell transformed with an expression cassette according to one of  claims 16  to  18 , characterized in that said expression cassette is either integrated in the genome of the transformed cell or carried by an expression vector.  
     
     
         20 . A transformed cell according to  claim 19 , characterized in that it is chosen from the group consisting of  S. cerevisiae  and  E. coli.    
     
     
         21 . A transformed cell according to  claim 19 , characterized in that the cell is of mammalian origin.  
     
     
         22 . Method for preparing a peptide according to one of  claims 1  to  9 , characterized in that a transformed cell according to one of  claims 19  to  21  is cultured on a suitable medium and the peptide is recovered from said culture.  
     
     
         23 . Pharmaceutical composition for human or veterinary use, characterized in that it comprises as active agent at least one peptide according to one of  claims 1  to  9 , one vector containing an expression cassette according to one of  claims 16  to  18  or one transformed cell according to one of  claims 19  to  21 .  
     
     
         24 . A monoclonal or polyclonal antibody, characterized in that it recognizes a peptide according to one of  claims 1  to  9 .  
     
     
         25 . Diagnostic kit comprising a peptide according to one of  claims 1  to  9  or an antibody according to claim  24 .