Peptide Analogues of PA-IL and Their Utility for Glycan and Glycoconjugate Analysis and Purification

Abstract

Provided are peptide analogues of PA-IL and compositions containing them. The PA-IL peptide analogues have altered carbohydrate binding specificity relative to a PA-IL of SEQ ID NO:1, and thus the analogues contain amino acid substitutions in SEQ ID NO:1. The substitutions can be at positions 50, 52 and 53 of SEQ ID NO:1 and can include combinations of amino acid substitutions at those positions. Also included are methods for detecting changes in the glycosylation of carbohydrates and for separating biomolecules which contain glycoproteins or glycoconjugates.

1 . A peptide analogue of PA-IL of SEQ ID NO: 1 comprising an amino acid substitution at one, two or three of positions 50, 52 and 53;
 wherein the amino acid substitution at position 50 is selected from the group consisting of Ala, Val, Leu, Ile, Met, Phe, Trp, Pro, Ser, Thr, Cys, Tyr, Gly, Asn, Asp, Gln, Glu, Lys, and Arg;   wherein the amino acid substitution at position 52 is selected from the group consisting of Ser, Thr, Cys, Asn, Gln, Asp, Glu, Lys, Arg and His; and   wherein the amino acid substitution at position 53 is selected from the group consisting of Ala, Val, Leu, Ile, Met, Phe, Trp, Pro, Gly, Ser, Thr, Cys, Tyr, Asn, Asp, Glu, Lys, Arg and His.   
     
     
         2 . The peptide analogue of  claim 1  comprising only one substitution, wherein the only one substitution is at position 53 or at position 50. 
     
     
         3 . The peptide analogue of  claim 2  wherein the only one substitution is at position 53. 
     
     
         4 . The peptide analogue of  claim 3 , wherein the only one substitution at position 53 is Q53E. 
     
     
         5 . The peptide analogue of  claim 2 , wherein the only one substitution is at position 50. 
     
     
         6 . The peptide analogue of  claim 5 , wherein the only one substitution at position 50 is H50Q. 
     
     
         7 . The peptide analogue of  claim 1  comprising only two substitutions, wherein the only two substitutions are at positions 50 and 53. 
     
     
         8 . The peptide analogue of  claim 7 , wherein the substitution at position 50 is H50V. 
     
     
         9 . The peptide analogue of  claim 7 , the substitution at position at position 53 is Q53R. 
     
     
         10 . The peptide analogue of  claim 7 , wherein the only two substitutions are H50V and Q53R. 
     
     
         11 . The peptide analogue of  claim 1  comprising substitutions at positions 50, 52 and 53. 
     
     
         12 . The peptide analogue of  claim 11 , wherein the substitution at position 50 is H50N or H50P. 
     
     
         13 . The peptide analogue of  claim 11 , wherein the substitution at position 52 is D52T or D52R. 
     
     
         14 . The peptide analogue of  claim 11 , wherein the substitution at position 53 is Q53S or Q53L. 
     
     
         15 . The peptide analogue of  claim 11  comprising H50N, D52T, and Q53S. 
     
     
         16 . The peptide analogue of  claim 11  comprising H50P, D52R and Q53L. 
     
     
         17 . A method for detecting changes in the glycosylation of a biomolecule, the method comprising qualitatively or quantitatively assessing terminal galactosylation by exposing the biomolecule to the peptide analogue of  claim 1 , determining affinity of the biomolecule for the peptide, and comparing the affinity to a reference, wherein a difference in the affinity of the biomolecule relative to the reference indicates a change in glycosylation of the biomolecule. 
     
     
         18 . A method of separating and isolating biomolecules, cells or a combination thereof comprising a glycoprotein or a glycoconjugate, the method comprising contacting the peptide analogue of  claim 1  with a solution or suspension containing the biomolecules, cells or a combination thereof; and separating the biomolecules, the cells or the combination thereof not bound by the peptide analogue. 
     
     
         19 . A peptide of  claim 1 , wherein the peptide is non-covalently bound to a carbohydrate. 
     
     
         20 . The peptide of  claim 19 , wherein the carbohydrate comprises a terminal α-linked galactose or a terminal β-linked galactose.