Glycosylation of peptides via O-linked glycosylation sequences

1. A method comprising expressing a sequon polypeptide in a host cell, wherein said sequon polypeptide corresponds to a parent polypeptide and comprises an exogenous O-linked glycosylation sequence comprising the amino acid sequence of SEQ ID NO: 1:

(X) m PO*U(B) p (Z) r (J) s (O) t (P) n   (SEQ ID NO: 1)

wherein

m, n, and p are integers independently selected from the group consisting of 0 and 1;

r, s, and t are 0;

P is proline;

O* is serine (S) or threonine (T);

U is a member selected from the group consisting of proline (P), glutamic acid (E), glutamine (Q), asparagine (N), threonine (T), glycine (G), and alanine (A);

X and B are members independently selected from the group consisting of glutamic acid (E), glutamine (Q), aspartic acid (D), asparagine (N), threonine (T), and serine (S); and

Z, J and O are members independently selected from the group consisting of glutamic acid (E), glutamine (Q), aspartic acid (D), asparagine (N), threonine (T), serine (S), tyrosine (Y), and methionine (M),

wherein the O-linked glycosylation is at O*, and

wherein the parent polypeptide is selected from the group consisting of bone morphogenetic protein 2 (BMP-2), bone morphogenetic protein 7 (BMP-7), bone morphogenetic protein 15 (BMP-15), neurotrophin-3 (NT-3), von Willebrand factor (vWF) protease, α1-antitrypsin (α-1 protease inhibitor), tissue-type plasminogen activator (TPA), human chorionic gonadotropin (hCG), anti-thrombin III (AT III), follicle stimulating hormone (FSH), glucagon-like peptide-2 (GLP-2), Factor VII, Factor VIII, B-domain deleted Factor VIII, Factor IX, Factor X, and Factor XIII.

2. The method according to claim 1 , further comprising isolating said sequon polypeptide.

3. The method according to claim 1 , further comprising enzymatically glycosylating said sequon polypeptide at said O-linked glycosylation sequence.

4. The method according to claim 3 , wherein said enzymatically glycosylating is accomplished using a glycosyltransferase.

5. The method according to claim 4 , wherein said glycosyltransferase is N-acetylgalactosamine transferase 2 (GalNAc-T2).

6. The method of claim 5 , wherein said GalNAc-T2 is a member selected from the group consisting of lectin-domain deleted GalNAc-T2 and lectin domain truncated GalNAc-T2.

7. The method according to claim 1 , further comprising generating an expression vector comprising a nucleic acid sequence encoding said sequon polypeptide.

8. The method according to claim 7 , further comprising transfecting said host cell with said expression vector.

9. A method for making a library of sequon polypeptides,

wherein said library of sequon polypeptides comprises a plurality of different members,

wherein each member of said library corresponds to a common parent polypeptide,

wherein each member of said library comprises an exogenous O-linked glycosylation sequence,

wherein each of said O-linked glycosylation sequence comprises the amino acid sequence of SEQ ID NO: 1:

(X) m PO*U(B) p (Z) r (J) s (O) t (P) n   (SEQ ID NO: 1)

wherein

m, n, and p are integers independently selected from the group consisting of 0 and 1;

r, s, and t are 0;

P is proline;

O* is serine (S) or threonine (T);

U is a member selected from the group consisting of proline (P), glutamic acid (E), glutamine (Q), asparagine (N), threonine (T), glycine (G), and alanine (A);

X and B are members independently selected from the group consisting of glutamic acid (E), glutamine (Q), aspartic acid (D), asparagine (N), threonine (T), and serine (S); and

Z, J and O are members independently selected from the group consisting of glutamic acid (E), glutamine (Q), aspartic acid (D), asparagine (N), threonine (T), serine (S), tyrosine (Y), and methionine (M),

wherein the O-linked glycosylation is at O*,

wherein the parent polypeptide is selected from the group consisting of bone morphogenetic protein 2 (BMP-2), bone morphogenetic protein 7 (BMP-7), bone morphogenetic protein 15 (BMP-15), neurotrophin-3 (NT-3), von Willebrand factor (vWF) protease, α1-antitrypsin (α-1 protease inhibitor), tissue-type plasminogen activator (TPA), human chorionic gonadotropin (hCG), anti-thrombin III (AT III), follicle stimulating hormone (FSH), glucagon-like peptide-2 (GLP-2), Factor VII, Factor VIII, B-domain deleted Factor VIII, Factor IX, Factor X, and Factor XIII, and

wherein said parent polypeptide has y amino acids, each amino acid corresponding to an amino acid position, said library comprising:

(a) a first sequon polypeptide having said O-linked glycosylation sequence at a first amino acid position (AA) z , wherein z is a member selected from 1 to y; and

(b) at least one additional sequon polypeptide, each additional sequon polypeptide having said O-linked glycosylation sequence at an additional amino acid position, which is a member selected from (AA) z+x and (AA) z−x , wherein x is a member selected from 1 to (y−z),

said method comprising:

(i) recombinantly producing a first sequon polypeptide by introducing said O-linked glycosylation sequence at a first amino acid position (AA) z ; and

(ii) recombinantly producing at least one additional sequon polypeptide by introducing said O-linked glycosylation sequence at an additional amino acid position selected from (AA) z+x and (AA) z−x , wherein x is a member selected from 1 to (y−z).

10. A method for identifying a lead polypeptide, said method comprising:

(i) generating a library of sequon polypeptides, wherein said library of sequon polypeptides comprises a plurality of different members, wherein each member of said library corresponds to a common parent polypeptide, wherein each member of said library comprises an exogenous O-linked glycosylation sequence, and wherein each of said O-linked glycosylation sequence comprises the amino acid sequence of SEQ ID NO: 1:

(X) m PO*U(B) p (Z) r (J) s (O) t (P) n   (SEQ ID NO: 1)

wherein

m, n, and pare integers independently selected from the group consisting of 0 and 1;

r, s, and t are 0;

P is proline;

O* is serine (S) or threonine (T);

U is a member selected from the group consisting of proline (P), glutamic acid (E), glutamine (Q), asparagine (N), threonine (T), glycine (G), and alanine (A);

X and B are members independently selected from the group consisting of glutamic acid (E), glutamine (Q), aspartic acid (D), asparagine (N), threonine (T), and serine (S); and

Z, J and O are members independently selected from the group consisting of glutamic acid (E), glutamine (Q), aspartic acid (D), asparagine (N), threonine (T), serine (S), tyrosine (Y), and methionine (M),

wherein the O-linked glycosylation is at O*,

wherein the parent polypeptide is selected from the group consisting of bone morphogenetic protein 2 (BMP-2), bone morphogenetic protein 7 (BMP-7), bone morphogenetic protein 15 (BMP-15), neurotrophin-3 (NT-3), von Willebrand factor (vWF) protease, α1-antitrypsin (α-1 protease inhibitor), tissue-type plasminogen activator (TPA), human chorionic gonadotropin (hCG), anti-thrombin III (AT III), follicle stimulating hormone (FSH), glucagon-like peptide-2 (GLP-2), Factor VII, Factor VIII, B-domain deleted Factor VIII, Factor IX, Factor X, and Factor XIII; and

(ii) subjecting at least one member of said library to an enzymatic glycosylation reaction, transferring a glycosyl moiety from a glycosyl donor molecule onto at least one of said O-linked glycosylation sequences, wherein said glycosyl moiety is optionally derivatized with a modifying group,

thereby identifying said lead polypeptide.

11. The method according to claim 10 , further comprising measuring yield for said enzymatic glycosylation reaction for at least one member of said library.

12. The method according to claim 11 , wherein said measuring is accomplished by a member selected from the group consisting of mass spectroscopy, gel electrophoresis, nuclear magnetic resonance (NMR) and HPLC.

13. The method according to claim 11 , wherein said yield for said lead polypeptide is between about 50% and about 100%.

14. The method according to claim 10 , further comprising, prior to step (ii), purifying at least one member of said library.

15. The method according to claim 10 , wherein said glycosyl moiety of step (ii) comprises a member selected from the group consisting of a galactose moiety and a GalNAc moiety.

16. The method according to claim 10 , wherein said enzymatic glycosylation reaction of step (ii) occurs within a host cell, in which said at least one member of said library is expressed.

17. The method according to claim 10 , further comprising:

(iii) subjecting the product of step (ii) to a PEGylation reaction, wherein said PEGylation reaction is a member selected from the group consisting of a chemical PEGylation reaction and an enzymatic glycoPEGylation reaction.

18. The method according to claim 17 , wherein step (ii) and step (iii) are performed in a single reaction vessel.

19. The method according to claim 17 , further comprising measuring yield of said PEGylation reaction.

20. The method according to claim 19 , wherein said measuring is accomplished by a member selected from the group consisting of mass spectroscopy, gel electrophoresis, nuclear magnetic resonance (NMR) and HPLC.

21. The method according to claim 19 , wherein said yield of said PEGylation reaction for said lead polypeptide is between about 50% and about 100%.

22. The method according to claim 17 , wherein said lead polypeptide upon said PEGylation reaction has a therapeutic activity essentially the same as the therapeutic activity of said parent polypeptide.

23. The method according to claim 17 , wherein said lead polypeptide upon said PEGylation reaction has a therapeutic activity distinct from the therapeutic activity of said parent polypeptide.

24. The method according to claim 10 , further comprising generating an expression vector comprising a nucleic acid sequence encoding said sequon polypeptide.

25. The method according to claim 24 , further comprising transfecting said host cell with said expression vector.