Mutant Glycoprotein Resistant to Modification with Asparagine-Linked Sugar Chain

Abstract

To obtain a mutant protein of an asparagine-linked glycoprotein, which has no N-linked sugar chain under ordinary circumstance, and remains a physiological activity of the glycoprotein before the mutation was introduced, at least one of the amino acids contained in the amino acid sequence motif (I) and/or (II) in the polypeptide of the asparagine-linked glycoprotein is substituted into another amino acid: (I) Asn Xa1 Xa2 (II) Xa3 Val Gly Asn Xa1 Xa2. In amino acid sequence motif (I) and (II), Xa1 represents an amino acid other than Pro, Xa2 represents Thr or Ser and Xa3 represents His or Asp.

1 . A mutant protein comprising an amino acid sequence of an asparagine-linked glycoprotein having the amino acid sequence motif (I) and/or (II) shown below, wherein at least one amino acid selected from the amino acids in the amino acid sequence motif (I) and/or (II) is substituted by other amino acid, and wherein the amino acid sequence containing the substituted amino acids is not subject to modification with an asparagine-linked sugar chain and an activity is maintained:
 (I) Asn Xa1 Xa2 (SEQ ID NO: 1); and   (II) Xa3 Val Gly Asn Xa1 Xa2 (SEQ ID NO: 2)   (Xa1 indicates an amino acid other than Pro, Xa2 indicates Thr or Ser, and Xa3 indicates His or Asp in the amino acid sequence motif (I) and (II)).   
     
     
         2 . The protein according to  claim 1 , wherein the amino acid substitution is one or more substitutions selected from (A) to (C):
 (A) substitution of Asn by Lys or Ser;   (B) substitution of Xa2 by Gln;   (C) substitution of Xa3 by Asp.   
     
     
         3 . The protein according to  claim 2 , wherein the asparagine-linked glycoprotein is an enzyme. 
     
     
         4 . The protein according to  claim 3 , wherein the enzyme is a glycosyltransferase. 
     
     
         5 . The protein according to  claim 4 , wherein the glycosyltransferase is a sialyltransferase. 
     
     
         6 . The protein according to  claim 5 , wherein the sialyltransferase is SAT-I, the amino acid sequence motif (I) is the amino acid sequence (i) and/or (ii) shown below, and the amino acid sequence motif (II) is the amino acid sequence (iii) shown below:
 (i) Asn Glu Ser;   (ii) Asn Val Thr;   (iii) His Val Gly Asn Lys Thr (SEQ ID NO: 43).   
     
     
         7 . The protein according to  claim 6 , wherein the amino acid substitution is one or more substitutions selected from (a) to (d):
 (a) substitution of Asn by Lys in the amino acid sequence (i);   (b) substitution of Thr by Gln in the amino acid sequence (ii);   (c) substitution of Asn by Ser in the amino acid sequence (iii); and   (d) substitution of His by Asp in the amino acid sequence (iii).   
     
     
         8 . The protein according to  claim 6 , wherein the amino acid sequence of SAT-I before the mutation is introduced is the amino acid sequence of SEQ ID NO: 4 or an amino acid sequence having homology of not less than 80% with the amino acid sequence of SEQ ID NO: 4. 
     
     
         9 . The protein according to  claim 8 , which comprises the amino acid sequence of SEQ ID NO: 6. 
     
     
         10 . The protein according to  claim 8 , which comprises the amino acid sequence of SEQ ID NO: 8. 
     
     
         11 . The protein according to  claim 8 , which comprises the amino acid sequence of SEQ ID NO: 10. 
     
     
         12 . The protein according to  claim 8 , which comprises the amino acid sequence of SEQ ID NO: 12. 
     
     
         13 . The protein according to  claim 8 , which comprises the amino acid sequence of SEQ ID NO: 14. 
     
     
         14 . The protein according to  claim 8 , which comprises the amino acid sequence of SEQ ID NO: 16. 
     
     
         15 . The protein according to  claim 8 , which comprises the amino acid sequence of SEQ ID NO: 18. 
     
     
         16 . A polynucleotide which encodes the protein according to  claim 1 . 
     
     
         17 . A method of producing a mutant protein that retains an activity, comprising:
 comparing, in an amino acid sequence of an asparagine-linked glycoprotein having the amino acid sequence motif (I) and/or (II) shown below, the amino acid sequence motif (I) and/or (II) described below or an amino acid sequence comprising the amino acid sequence motif (I) and/or (II) with a corresponding protein from another organism, or with a corresponding amino acid sequence of another protein which is from the same organism as the glycoprotein and which belongs to the same protein family as the glycoprotein to clarify differences between the amino acid sequences; and   substituting the different amino acids by corresponding amino acids of the target of comparison, wherein the amino acid sequence comprising the substituted amino acids undergoes no modification by an asparagine-linked sugar chain:   (I) Asn Xa1 Xa2 (SEQ ID NO: 1); and   (II) Xa3 Val Gly Asn Xa1 Xa2 (SEQ ID NO: 2)   (Xa1 indicates an amino acid other than Pro, Xa2 indicates Thr or Ser, and Xa3 indicates His or Asp in the amino acid sequence motif (I) and (II)).   
     
     
         18 . The method according to  claim 17 , wherein the amino acid substitution is one or more substitutions selected from (A) to (C):
 (A) substitution of Asn by Lys or Ser;   (B) substitution of Xa2 by Gln; and   (C) substitution of Xa3 by Asp.   
     
     
         19 . The method according to  claim 18 , wherein the asparagine-linked glycoprotein is an enzyme. 
     
     
         20 . The method according to  claim 19 , wherein the enzyme is glycosyltransferase. 
     
     
         21 . The method according to  claim 20 , wherein the glycosyltransferase is a sialyltransferase. 
     
     
         22 . The method according to  claim 21 , wherein the sialyltransferase is SAT-I, the amino acid sequence motif (I) is the amino acid sequence (i) and/or (ii) shown below, and the amino acid sequence motif (II) is the amino acid sequence (iii) shown below:
 (i) Asn Glu Ser;   (ii) Asn Val Thr; and   (iii) His Val Gly Asn Lys Thr (SEQ ID NO: 43).