IDUA

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Background

Asparagine-linked protein glycosylation is one of the major post-translational modifications in eukaryotes, this results in the oligosaccharide chain being linked to the Nδ atom of asparagine (Asn-Xaa-Ser/Thr, Xaa can be any amino acid except proline). N-glycosylation is very important in organisms, it confers a variety of abilities to proteins, including immune recognition, cell signal transduction, ligand-receptor binding, folding and stability. For some lysosomal enzymes, such as α-galactosidase A and glucocerebrosidase, deglycosylation may reduce protein stability, which in turn reduces enzyme activity. However, there is still a lack of research data on the exact relationship between N-glycan and enzyme activity.

α- L- iduronidase (IDUA; EC 3.2.1.76) is an important lysosomal enzyme. The decrease or loss of IDUA activity will lead to the accumulation of glycosaminoglycans in lysosomes, which in turn leads to mucopolysaccharidosis type I (MPS I). Human IDUA is translated into 653 amino acids and has N-glycosylation at six potential sites (N110, N190, N336, N372, N415, and N451). Subsequently, the 26 residues at N-terminus are removed and processed into the mature form in lysosome.

IDUA hydrolyzes the glycosidic bond between the terminal of L-iduronic acid (IdoA) and the second sugar of N-acetylgalactosamine (GalNAc)-4-sulfate/N-sulfo-D-glucosamine (GlcNS)-6-sulfate. Therefore, IDUA defects can lead to the accumulation of dermatan/heparan sulfate and lead to systemic disorder, MPS I, including progressive mental retardation, small stature, enlarged and deformed skull, corneal opacity, joint contractures, and hepatosplenomegaly, etc. From 1981 to 2003, the prevalence of MPS I in England and Wales was 1.07 per 100,000 births. At present, the use of Chinese hamster ovary (CHO) cells to express recombinant human IDUA (hIDUA) has been used to develop enzyme replacement therapy, which is widely used in MPS I treatment.

IDUA belongs to glycoside hydrolase (GH) family 39. So far, the crystal structure of the bacterial GH39 β-xylosidase (XynB) has been obtained. In addition, studies have reported the hIDUA homology model constructed from Thermoanaerobacterium saccharolyticum XynB (PDB ID code 1PX8), and the crystal structure of apo-hIDUA expressed in plant seeds has also been obtained. However, the structural data of hIDUA expressed in mammalian cells is still needed to gain a deeper understanding of the basis of MPS I and develop new therapies.

Figure 1. Reaction scheme of dermatan sulfate hydrolysis catalyzed by Hidua (Maita, N.; et al. 2013)

Overall Structure of hIDUA

The crystal structure of apo- and IdoA hIDUA have been obtained to determine the structural basis of the relationship between hIDUA enzyme activity and N-glycans. There are two hIDUA subunits in each asymmetric unit. hIDUA consists of three domains: residues 42-396 form a (β/α)8 triosephosphate isomerase (TIM) barrel fold; residues 27-42 and 397-545 form a β-sandwich domain with a short helix-loop-helix (482-508), and residues 546-642 form an Ig(Ig)-like domain. The latter two domains are linked by a disulfide bond located between C541 and C577. After comparison, the topological structure of the TIM barrel and β-sandwich domain of hIDUA is almost the same as that of XynB, but the amino acid length of XynB is shorter than hIDUA, and the C-terminal lacks the Ig-like domain. The tip of the mannose residue (Man7) reaches the active site and forms part of the substrate binding pocket. A hydrophobic interaction between Y355 and Man3 was also observed.

Figure 2. Domain organization of hIDUA (Maita, N.; et al. 2013)

Recently, two crystal structures of hIDUA have been published in the Protein Data Base, the space groups are R3 (PDB ID code 4JXO) and P21 (PDB ID code 4JXP). But they contain up to 6 sugars and lack Man7, which may be due to the use of plant seed expression system. In the IdoA-bound hIDUA structure, we clearly observe the electron density of the molecule IdoA in the center of the TIM barrel. The structural difference between the apo and IdoA-bound forms is very small; only the side chain of D187 flips to the active site, a hydrogen bond is formed between the Oδ2 atom and the backbone oxygen and Nδ atom of N181. This change may create a tight hydrogen network between the O2 atom of IdoA and the Nδ of N181. In the structure of hIDUA bound by IdoA, we clearly observe the electron density of the IdoA molecule located in the center of the TIM barrel. After comparison, the structural difference between the binding forms of apo and IdoA is very small, only the side chain of D187 is flipped a little toward the active site, thus forming a hydrogen bond. This change may create a tight hydrogen network between the O2 atom of IdoA and the Nδ of N181.

Figure 3. Molecular surface representation around the substrate-binding pocket of hIDUA (subunit B) (Maita, N.; et al. 2013)