Carbohydrate ligands specific for MHC molecules

Abstract

The present invention provides a substantially purified carbohydrate ligand that specifically binds to a leczyme. The invention also provides methods to identify a carbohydrate ligand that specifically binds to a leczyme or a leczyme that specifically binds to a carbohydrate ligand. The invention further provides methods to identify a peptide that binds to the carbohydrate ligand binding site of a leczyme. The present invention provides methods to isolate a carbohydrate ligand or a leczyme and to identify a carbohydrate ligand or a leczyme that modifies the function of a cell and to obtain such functionally modified cells. The invention further provides methods to modify a cell to express a carbohydrate ligand by introducing an expression vector encoding a leczyme into the cell. The invention also provides methods to modulate the immune response to an antigen by administering the antigen and a carbohydrate ligand. In addition, the invention further provides methods to treat a disease state involving a leczyme by administering a carbohydrate ligand that binds the leczyme or by administering a leczyme that has a similar binding specificity to the leczyme involved in the disease state. The invention further provides methods to diagnose a genetic basis for hemochromatosis by detecting a mutation in a class I MHC molecule that reduces it's ability to associate with β 2 microglobulin.

I claim:  
     
         1 . A composition, comprising a substantially purified carbohydrate ligand that specifically binds to a leczyme.  
     
     
         2 . The composition of  claim 1 , further comprising a polypeptide or lipid moiety bonded to said carbohydrate ligand.  
     
     
         3 . A method of identifying a carbohydrate ligand that binds to a leczyme, comprising the steps of: 
 a. contacting a sample containing a carbohydrate ligand with a leczyme suspected to bind such a ligand; and    b. detecting whether binding of the ligand and the leczyme has occurred.    
     
     
         4 . The method of  claim 3 , wherein said leczyme is an MHC-derived gene product.  
     
     
         5 . The method of  claim 4 , wherein said MHC-derived gene product is a class I molecule.  
     
     
         6 . The method of  claim 4 , wherein said MHC-derived gene product is a class II molecule.  
     
     
         7 . The method of  claim 4 , wherein said MHC-derived gene product is an nonclassical class I molecule.  
     
     
         8 . The method of  claim 3 , wherein said leczyme is a non-MHC derived gene product.  
     
     
         9 . The method of  claim 3 , wherein said carbohydrate ligand is bonded to a polypeptide or lipid moiety.  
     
     
         10 . The method of  claim 3 , wherein said carbohydrate ligand or said leczyme contains a detectable label.  
     
     
         11 . The method of  claim 3 , wherein said carbohydrate ligand or said leczyme is associated with a cell.  
     
     
         12 . A method of identifying a leczyme that binds to a carbohydrate ligand, comprising the steps of: 
 a. contacting a sample containing a leczyme with a carbohydrate ligand suspected to bind such a leczyme; and    b. detecting whether binding of the leczyme and the ligand has occurred.    
     
     
         13 . The method of  claim 12 , wherein said leczyme is an MHC-derived gene product.  
     
     
         14 . The method of  claim 12 , wherein said leczyme is a non-MHC derived gene product.  
     
     
         15 . The method of  claim 12 , wherein said carbohydrate ligand is bonded to a polypeptide or lipid moiety.  
     
     
         16 . The method of  claim 12 , wherein said carbohydrate ligand or said leczyme contains a detectable label.  
     
     
         17 . The method of  claim 12 , wherein said carbohydrate ligand or said leczyme is associated with a cell.  
     
     
         18 . A method of purifying a carbohydrate ligand that specifically binds to a reagent, comprising the steps of: 
 a. contacting a sample containing a carbohydrate ligand with a reagent capable of binding such a ligand to form a ligand-reagent complex;    b. separating the complex from the rest of the sample; and    c. dissociating the ligand from the complex to obtain the purified ligand.    
     
     
         19 . The method of  claim 18 , wherein said reagent is a leczyme.  
     
     
         20 . The method of  claim 18 , wherein said reagent is an antibody.  
     
     
         21 . The method of  claim 18 , wherein said leczyme is an MHC-derived gene product.  
     
     
         22 . The method of  claim 18 , wherein said leczyme is a non-MHC derived gene product.  
     
     
         23 . The method of  claim 18 , wherein said carbohydrate ligand is bonded to a polypeptide or lipid moiety.  
     
     
         24 . A method of purifying a leczyme that specifically binds to a carbohydrate ligand, comprising the steps of: 
 a. contacting a sample containing a leczyme with a carbohydrate ligand capable of binding such a leczyme to form a ligand-leczyme complex;    b. separating the complex from the rest of the sample; and    c. dissociating the leczyme from the complex to obtain the purified leczyme.    
     
     
         25 . The method of  claim 24 , wherein said leczyme is an MHC-derived gene product.  
     
     
         26 . The method of  claim 24 , wherein said leczyme is a non-MHC derived gene product.  
     
     
         27 . The method of  claim 24 , wherein said carbohydrate ligand is bonded to a polypeptide or lipid moiety.  
     
     
         28 . A method of identifying a carbohydrate ligand that modifies the function of a leczyme-expressing cell, comprising the steps of: 
 a. contacting a sample containing such a carbohydrate ligand with a leczyme-expressing cell; and    b. subsequently assaying the cell to determine it's function.    
     
     
         29 . The method of  claim 28 , wherein said leczyme is an MHC-derived gene product.  
     
     
         30 . The method of  claim 28 , wherein said leczyme is a non-MHC derived gene product.  
     
     
         31 . The method of  claim 28 , wherein said carbohydrate ligand is bonded to a polypeptide or lipid moiety.  
     
     
         32 . A method of identifying a leczyme that modifies the function of a carbohydrate ligand-expressing cell, comprising the steps of: 
 a. contacting a sample containing such a leczyme with a carbohydrate ligand-expressing cell; and    b. subsequently assaying the cell to determine it's function.    
     
     
         33 . The method of  claim 32 , wherein said leczyme is an MHC-derived gene product.  
     
     
         34 . The method of  claim 32 , wherein said leczyme is a non-MHC derived gene product.  
     
     
         35 . A method of modifying the function of a leczyme-expressing cell, comprising contacting the cell with a carbohydrate ligand that binds the leczyme.  
     
     
         36 . The method of  claim 35 , wherein said leczyme is an MHC-derived gene product.  
     
     
         37 . The method of  claim 35 , wherein said leczyme is a non-MHC derived gene product.  
     
     
         38 . The method of  claim 35 , wherein said carbohydrate ligand is bonded to a polypeptide or lipid moiety.  
     
     
         39 . A method of modifying the function of a carbohydrate ligand-expressing cell, comprising contacting the cell with a leczyme that binds the ligand.  
     
     
         40 . The method of  claim 39 , wherein said leczyme is an MHC-derived gene product.  
     
     
         41 . The method of  claim 39 , wherein said leczyme is a non-MHC derived gene product.  
     
     
         42 . A method of identifying a peptide that binds to a carbohydrate ligand binding site of a leczyme, comprising the steps of: 
 a. contacting a leczyme and a carbohydrate ligand known to bind the leczyme with a test sample containing a peptide to be identified;    b. determining the amount of carbohydrate ligand bound to the leczyme after said reacting; and    c. comparing the amount of carbohydrate ligand bound in the test sample with the amount of carbohydrate ligand bound in a control sample, wherein a decreased amount of carbohydrate ligand bound in the test sample relative to the amount of carbohydrate ligand bound in the control sample indicates binding of the peptide to the leczyme.    
     
     
         43 . The method of  claim 42 , wherein said peptide and said leczyme are contacted prior to contacting said carbohydrate ligand.  
     
     
         44 . The method of  claim 42 , wherein said carbohydrate ligand or said leczyme contains a detectable label.  
     
     
         45 . The method of  claim 42 , wherein said leczyme or said carbohydrate ligand is associated with a cell.  
     
     
         46 . The method of  claim 42 , wherein said test sample contains a peptide library.  
     
     
         47 . A method of modifying a cell to produce a carbohydrate ligand, comprising introducing an expression vector encoding a leczyme into the cell to obtain expression of the leczyme, wherein said expression results in production of the carbohydrate ligand by the cell.  
     
     
         48 . The method of  claim 47 , wherein said leczyme is an MHC-derived gene product.  
     
     
         49 . The method of  claim 47 , wherein said leczyme is a non-MHC derived gene product.  
     
     
         50 . A method for modulating an immune response in an individual to an antigen, comprising administering the carbohydrate ligand of  claim 1  and the antigen.  
     
     
         51 . The method of  claim 50 , wherein said administering results in an increase in the immune response to the antigen.  
     
     
         52 . The method of  claim 50 , wherein said administering results in a decrease in the immune response to the antigen.  
     
     
         53 . The Method of  claim 50 , wherein said administering further comprises an immune suppressing agent.  
     
     
         54 . The method of  claim 50 , wherein said antigen and said carbohydrate ligand are covalently bonded.  
     
     
         55 . The method of  claim 50 , wherein an adjuvant is administered along with said antigen and said carbohydrate ligand.  
     
     
         56 . A method for treating a disease state involving a leczyme, comprising administering an effective amount of the carbohydrate ligand of  claim 1 .  
     
     
         57 . The method of  claim 56 , wherein said disease state is an MHC-linked disease.  
     
     
         58 . The method of  claim 56 , wherein said MHC-linked disease is an autoimmune disease.  
     
     
         59 . The method of  claim 56 , wherein said MHC-linked disease is hemochromatosis.  
     
     
         60 . The method of  claim 56 , wherein said disease state is an infection.  
     
     
         61 . The method of  claim 56 , wherein said disease state is transplantation rejection.  
     
     
         62 . A method for treating a disease state involving a leczyme, comprising administering an effective amount of a leczyme having a similar binding specificity for a carbohydrate ligand as the leczyme involved in the disease state.  
     
     
         63 . The method of  claim 62 , wherein said leczyme is an MHC-derived gene product.  
     
     
         64 . The method of  claim 62 , wherein said leczyme is a non-MHC derived gene product.  
     
     
         65 . The method of  claim 62 , wherein said disease. state is an MHC-linked disease.  
     
     
         66 . The method of  claim 65 , wherein said MHC-linked disease is an autoimmune disease.  
     
     
         67 . The method of  claim 65 , wherein said MHC-linked disease is hemochromatosis.  
     
     
         68 . The method of  claim 62 , wherein said disease state is an infection.  
     
     
         69 . The method of  claim 62 , wherein said disease state is transplantation rejection.  
     
     
         70 . A method for detecting a genetic predisposition for hemochromatosis, comprising detecting a mutation in the heavy chain of a class I MHC molecule that reduces the ability of said heavy chain to associate with β 2  microglobulin.  
     
     
         71 . The method of  claim 70 , wherein said mutation eliminates a signal for the addition of a phosphate group.  
     
     
         72 . The method of  claim 70 , wherein said mutation eliminates the ability of a phosphate group in said heavy chain to be de-phosphorylated in a cell.  
     
     
         73 . A method for diagnosing hemochromatosis resulting from a reduction in the ability of a heavy chain of an MHC class I molecule to bind β 2  microglobulin, comprising the steps of: 
 a. isolating a class I MHC heavy chain from an individual to be tested;  
 b. contacting the heavy chain with β 2  microglobulin under conditions suitable for associating a class I MHC heavy chain with β 2  microglobulin; and  
 c. detecting the association of said heavy chain with said β 2  microglobulin, wherein a reduced association of said heavy chain with β 2  microglobulin compared to the association of a control heavy chain with β 2  microglobulin is diagnostic for hemochromatosis.