Carbohydrate-binding protein
View Patent ↗The carbohydrate processing enzyme PNGase F was catalytically inactivated through mutation. Additional mutations yielded a catalytically inactive carbohydrate-binding protein with lectin-like properties including high affinity and specificity N-linked glycans, O-linked glycans, or both.
1. An isolated catalytically inactive carbohydrate-binding PNGase F protein with binding specificity and affinity to an O-linked glycan, said protein having a plurality of amino acid mutations compared to a corresponding wild-type PNGase F protein, said plurality of mutations comprising
(a) at least one first mutation that reduces or eliminates the catalytic activity of the PNGase F protein; and
(b) at least one second mutation that provides binding specificity and affinity to an O-linked glycan;
wherein the at least one second mutation comprises an amino acid substitution at position D57 of SEQ ID NO: 1, or a corresponding position in a homologous PNGase F sequence, with leucine, alanine, methionine, arginine, lysine, cysteine, or tryptophan.
2. The catalytically inactive carbohydrate-binding PNGase F protein of claim 1 wherein the O-linked glycan comprises an O-linked GlcNAc.
3. The catalytically inactive carbohydrate-binding PNGase F protein of claim 1 wherein the corresponding wild-type protein comprises a protein selected from the group consisting of PNGase F from Elizabethkingia meningosepticum (SEQ ID NO:1), PNGase F-II from Elizabethkingia meningosepticum (SEQ ID NO:3), PNGase F from Bacteroides fragilis (SEQ ID NO:4), and PNGase F from Elizabethkingia miricola (SEQ ID NO:5).
4. The catalytically inactive carbohydrate-binding PNGase F protein of claim 1 comprising mutations at amino acid positions 60, 156, 192, 206, and/or 248 in E. meningosepticum PNGase F (SEQ ID NO: 1), or a corresponding position in a homologous PNGase F sequence.
5. The catalytically inactive carbohydrate-binding PNGase F protein of claim 4 further comprising a mutation at amino acid position 61, 85, 118, 119, 123, 125, 190, and/or 191 in E. meningosepticum PNGase F (SEQ ID NO:1), or a corresponding position in a homologous PNGase F sequence.
6. The catalytically inactive carbohydrate-binding PNGase F protein of claim 5 , wherein the amino acid substitution at position 118 of SEQ ID NO:1, or a corresponding position in a homologous PNGase F sequence, comprises alanine, glutamine, threonine, or cysteine.
7. The catalytically inactive carbohydrate-binding PNGase F protein of claim 4 , wherein:
the amino acid substitution at position 60 of SEQ ID NO: 1, or a corresponding position in a homologous PNGase F sequence, comprises alanine, cysteine, valine, serine, glycine, or tryptophan;
the amino acid substitution at position 156 of SEQ ID NO: 1, or a corresponding position in a homologous PNGase F sequence, comprises leucine, threonine, methionine, glycine, tryptophan, or histidine;
the amino acid substitution at position 192 of SEQ ID NO: 1, or a corresponding position in a homologous PNGase F sequence, comprises isoleucine, tryptophan, alanine, histidine, threonine, cysteine, or serine;
the amino acid substitution at position 206 of SEQ ID NO: 1, or a corresponding position in a homologous PNGase F sequence, comprises serine, tryptophan, histidine, cysteine, or arginine; and/or
the amino acid substitution at position 248 of SEQ ID NO: 1, or a corresponding position in a homologous PNGase F sequence, comprises tryptophan, serine, proline, valine, aspartate, tyrosine, phenylalanine, or lysine.
8. The catalytically inactive carbohydrate-binding PNGase F protein of claim 7 , wherein the a homologous PNGase F sequence comprises SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO:5.
9. The catalytically inactive carbohydrate-binding PNGase F protein of claim 1 , comprising SEQ ID NO:1 with a D57L amino acid substitution at position 57, a D60C amino acid substitution at position 60, an I156L amino acid substitution at position 156, a G192I amino acid substitution at position 192, an E206S amino acid substitution at position 206, and/or a R248W amino acid substitution at position 248.
10. The catalytically inactive carbohydrate-binding PNGase F protein of claim 9 , further comprising an E118Q amino acid substitution at position 118.
11. The catalytically inactive carbohydrate-binding PNGase F protein of claim 1 , which binds to an O-linked glycan, O-linked glycoconjugate, O-linked glycopeptide, O-linked glycoprotein, or free O-glycan.
12. A conjugate comprising a first component comprising a catalytically inactive carbohydrate-binding PNGase F protein of claim 1 covalently linked to a second component.
13. A diagnostic or therapeutic composition comprising a catalytically inactive carbohydrate-binding PNGase F protein of claim 1 .
14. An isolated polynucleotide encoding a catalytically inactive carbohydrate-binding PNGase F protein of claim 1 .
15. A method for detecting a glycan comprising:
contacting a biological or laboratory sample with a catalytically inactive carbohydrate-binding PNGase F protein of claim 1 under conditions to allow binding of PNGase F protein to a glycan; and
detecting the glycan;
wherein the glycan is selected from the group consisting of an O-linked glycan, and a free O-glycan.
16. A method for enriching, isolating or purifying an O-linked glycan or free O-glycan, the method comprising:
contacting a biological or laboratory sample with a catalytically inactive carbohydrate-binding PNGase F protein of claim 1 under conditions to allow binding of PNGase F protein to an O-glycan so as to yield an enriched, isolated or purified O-linked glycan or free O-glycan.
17. The method of claim 16 further comprising identifying a constituent saccharide, determining saccharide composition, determining linkage positions, or determining stereochemistry of the enriched, isolated or purified O-linked glycan or free O-glycan.