Cleavage of fucose in N-glycans
View Patent ↗Provided herein is an α-fucosidase that can cleave a conjugate comprising an N-glycan and a label where the label is added by amine reactive chemistry. The α-fucosidase also has an accelerated reaction time using Schiff base labeled N-glycans compared with bovine kidney fucosidase. A reaction mix, enzyme mix and kit comprising the α-fucosidase are provided, as well as a method for analyzing glycoproteins. The α-fucosidase finds particular use in analyzing the N-glycans of therapeutic glycoproteins.
1. A reaction mix comprising:
(a) an α-L-fucosidase comprising an amino acid sequence that is identical to amino acids 23-359 of the sequence of SEQ ID NO:1; and
(b) a conjugate comprising an N-glycan and a label that are joined by an carbamide bond.
2. The reaction mix of claim 1 , wherein the label comprises a fluorophore and/or a charge tag.
3. The reaction mix of claim 1 , wherein the N-glycan of the conjugate comprises a core fucose.
4. The reaction mix of claim 1 , wherein the N-glycan is linked to the label via the reducing end of the N-glycan.
5. The reaction mix of claim 1 , wherein the N-glycan is from a therapeutic glycoprotein.
6. The reaction mix of claim 1 , wherein the reaction mix further comprises:
(c) one or more exoglycosidases selected from the group consisting of:
α2-3 neuraminidase S;
α2-3,6,8,9 neuraminidase A;
α1-3,4,6 galactosidase;
β1-4 galactosidase;
β-N-acetylglucosaminidase S; and
α1-2,3,6 mannosidase.
7. The reaction mix of claim 1 , wherein the reaction mix has a pH in the range of pH 3.0 to pH 5.5.
8. An enzyme mix comprising:
(a) an α-fucosidase comprising an amino acid sequence that is identical to amino acids 23-359 of the sequence of SEQ ID NO:1; and
(b) one or more exoglycosidases that are not encoded by the genome of Omnitrophica , selected from the group consisting of:
α2-3 neuraminidase S;
α2-3,6,8,9 neuraminidase A;
α1-3,4,6 galactosidase;
β1-4 galactosidase;
β-N-acetylglucosaminidase S; and
α1-2,3,6 mannosidase.
9. The enzyme mix according to claim 8 , wherein none of the one or more exoglycosidases in the enzyme mix is glycosylated.
10. The enzyme mix of claim 8 , wherein the enzyme mix comprises two or more of the exoglycosidases.
11. The enzyme mix of claim 8 , wherein the enzyme mix comprises three or more of the exoglycosidases.
12. The enzyme mix of claim 8 , wherein the enzyme mix comprises α2-3 neuraminidase S, α2-3,6,8,9 neuraminidase A, α1-3,4,6 galactosidase, β1-4 galactosidase and β-N-acetylglucosaminidase S.
13. A kit, comprising:
a. an α-fucosidase comprising an amino acid sequence that is identical to amino acids 23-269 of the sequence of SEQ ID NO:1; and
b. a label comprising a reactive carbamate.
14. The kit of claim 13 , wherein the kit further comprises peptide:N-glycosidase F (PNGase F).
15. The kit of claim 13 , further comprising one or more exoglycosidases selected from the group consisting of:
α2-3 neuraminidase S;
α2-3,6,8,9 neuraminidase A;
α1-3,4,6 galactosidase;
β1-4 galactosidase;
β-N-acetylglucosaminidase S; and
α1-2,3,6 mannosidase.
16. The kit of claim 15 , wherein at least one of the one or more exoglycosidases is combined with the α-fucosidase in a reaction mixture.
17. The kit of claim 13 , wherein the label comprises a fluorophore and/or a charge tag.