Crystal structure of human α-N-acetylglucosaminidase
View Patent ↗The present invention provides the three-dimensional structure of human α-N-acetylglucosaminidase (NAGLU) protein. This crystallographic information is useful in the identification and development of novel binding compounds of NAGLU, NAGLU mutants, for example, those associated with Sanfilippo syndrome type B (mucopolysaccharidosis III B (MPS III-B)), and other NAGLU family members (family 89 α-N-acetylglucosaminidase) which may modulate the activity and/or stability of mutated NAGLU. Such compounds may be useful for the treatment of Sanfilippo syndrome type B (mucopolysaccharidosis III B (MPS III-B)).
1. A method of obtaining a purified recombinant a-N-acetylglucosaminidase (NAGLU) polypeptide with an altered glycosylation pattern compared to native NAGLU, the method comprising:
a) expressing a recombinant NAGLU polypeptide in a host cell,
b) treating the host cell with a glycosidase inhibitor, and
c) purifying the recombinant NAGLU polypeptide in a solution to obtain a purified recombinant NAGLU polypeptide with an altered glycosylation pattern compared to native NAGLU;
wherein the recombinant NAGLU polypeptide is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 1 or 3; and
wherein the recombinant NAGLU polypeptide comprises one or more glycans at each of the following asparagine (N) residues: N261, N272, N435, N503, N526, and N532 corresponding to SEP ID NO: 1 or 3.
2. The method of claim 1 , wherein the glycosidase inhibitor is a mannosidase I inhibitor.
3. The method of claim 2 , wherein the mannosidase I inhibitor is kifunensine.
4. The method of claim 1 , wherein the recombinant NAGLU polypeptide is expressed in mammalian cells.
5. The method of claim 1 , wherein the recombinant NAGLU polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 3.
6. The method of claim 1 , wherein the recombinant NAGLU polypeptide comprises one or more amino acid substitutions, deletions, or additions as set forth in Table 4 compared to wild-type human NAGLU polypeptide.
7. The method of claim 1 further comprising crystallizing the recombinant NAGLU polypeptide from a solution with:
a pH ranging from about 6.5 to about 9.0;
a precipitant selected from the group consisting of Li 2 SO 4 , monomethyl ether (MME), polyethylene glycol (PEG), 2-methyl-2,4-pentanediol (MPD), and combinations thereof; and
optionally a salt selected from the group consisting of ZnSO 4 , Zn(OAc) 2 , NaCl, (NH 4 ) 2 SO 4 , NiCl 2 , and combinations thereof,
wherein the recombinant NAGLU polypeptide is identical to the amino acid of SEQ ID NO: 1 or 3.
8. The method of claim 7 , wherein the crystallized NAGLU is characterized with space group P6 3 and has unit cell parameters of a=b=205.66 Å, c=78.69 Å, a=b=207.5 Å, c=79.6 Å, or a=b=205.13 Å, c=78.44 Å and bond angles of α=β=90°, γ=120°.
9. The method of claim 7 , comprising crystallizing the recombinant NAGLU polypeptide by concentrating a solution of the purified recombinant NAGLU polypeptide to a polypeptide concentration at which the NAGLU polypeptide precipitates and forms crystals.
10. The method of claim 9 , wherein the recombinant NAGLU polypeptide concentration in solution at which the NAGLU polypeptide precipitates and forms crystals is about 1 mg/ml.
11. The method of claim 7 , wherein the solution has a pH of 8.5 and comprises Li 2 SO 4 and NiCl 2 .
12. The method of claim 1 , wherein the recombinant NAGLU polypeptide is a mutant recombinant NAGLU.
13. The method of claim 12 , wherein the recombinant mutant NAGLU polypeptide is a-N-acetylglucosaminidase-kifunensine (NAGLU-kif).
14. The method of claim 1 , wherein the recombinant NAGLU polypeptide comprises an amino acid sequence having at least 98% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 3.
15. The method of claim 1 , wherein the recombinant NAGLU polypeptide comprises one or more sites of amino acid substitutions, deletions or duplications associated with Mucopolysaccharidosis type III-B (MPS III-B).
16. The method of claim 1 , wherein the recombinant NAGLU polypeptide comprises at least one N-acetylglucosamine molecule each on N261, N503, N526 and N532, and at least two N-acetylglucosamine molecules each on N272 and N435.
17. The method of claim 1 , wherein the recombinant NAGLU polypeptide is expressed in HT1080 cells.
18. The method of claim 1 , wherein the recombinant NAGLU polypeptide is secreted from mammalian cells.
19. The method of claim 1 , wherein purifying the recombinant NAGLU polypeptide comprises using column purification.
20. The method of claim 1 , wherein the recombinant NAGLU polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1.