The Glce gene was chemically synthesized according to the codon preference of Escherichia coli. The Glce (G102-N617) and Glce (Y167-N617) gene fragments were amplified from full-length Glce cDNA with a 5′ overhang containing the EcoRI site and a 3′ overhang containing the XhoI site. The PCR products were subsequently inserted into the pET-15b-SUMO vector (Invitrogen) using the EcoRI and XhoI restriction sites, producing a SUMO and His₆ tag at the N-terminus. The pET-15b-SUMO-Glce^102-617 and pET-15b-SUMO-Glce^167-617 constructs were confirmed by double restriction enzyme digestion and sequencing.

The recombinant plasmids were transformed into C2566H E. coli competent cells (New England Biolabs) for expression of SUMO-Glce^102-617 and SUMO-Glce^167-617 fusion proteins. Cells containing the Glce gene expression plasmid were initially grown in LA (Luria-Bertani Agar) medium with 100 μg/mL ampicillin at 37°C. Selected colonies were inoculated into Luria Bertani (LB) medium containing 100 μg/mL ampicillin and incubated at 37°C, 180 rpm for overnight. The seed culture was subsequently transferred to LB medium (0.1% inoculation volume) and grown under identical conditions until the cell density reached an OD₆₀₀ of 0.8–1.2. Protein induction was initiated by reducing the temperature was lowered to 20°C, followed by addition of 0.15 mM IPTG and incubation for 16 h at 18°C, 180 rpm.

Bacterial cells were collected by centrifugation and resuspended in lysis buffer containing 20 mM Tris–HCl, pH 8.0, 500 mM NaCl, 5 mM imidazole and 5% glycerol (v/v). Following sonication, the lysate was centrifuged at 12,000 rpm for 1 h. The supernatant was loaded onto a Ni²⁺ charged immobilized metal affinity chromatography (IMAC) column (GE Healthcare), followed by sequential washing with lysis buffer and wash buffer [20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 20 mM imidazole and 5% glycerol (v/v)]. Target proteins were eluted with elution buffer [20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 200 mM imidazole and 5% glycerol (v/v)]. Proteolytic cleavage of the N-terminal SUMO and His₆ tag was removed by incubation the target proteins with SUMO protease at a molar ratio of 250:1 for overnight at 4°C. The tag-free target proteins were diluted in buffer [20 mM Tris–HCl, pH 8.0, 5% glycerol (v/v), 1 mM 1, 4-dithioerythritol (DTT)] and loaded onto a 1 mL HiTrap SP column (GE Healthcare) at 18°C. Bound proteins were eluted using a linear NaCl gradient to 1 M, and the target protein-containing fractions were subsequently diluted in buffer (20 mM Tris-HCl, pH 8.0, 200 mM NaCl, 1 mM DTT). Final the target protein-containing fractions were concentrated to 8 mg/mL using ultrafiltration devices and stored at −80°C.

The aggregation state of Glce^167-617 protein in solution was analyzed using static- and dynamic-light scattering. For static light scattering experiment, which was performed on a Superdex 200 10/300 GL column (GE Healthcare) using a fast protein liquid chromatography (FPLC) system (ÄKTA Purifier, GE Healthcare) at 18°C. The column was equilibrated with buffer (20 mM Tris-HCl, pH 8.0, 200 mM NaCl, 1 mM DTT) and calibrated using gel filtration standard (Bio-Rad) at a flow-rate of 0.2 mL/min. Subsequently, 100 μg of Glce^167-617 was loaded onto the column, and elution profiles were monitored via UV absorbance at 280 nm. Molecular weight determination of Glce^167-617 protein was calculated with the fitted standard curve of ln(M_w) = −8.4755Kav + 15.145 (R² = 0.9841).

Dynamic light scattering (DLS) was performed at room temperature using a DynaPro NanoStar instrument (Wyatt Technology Corporation, United States) with a 20 μL micro-cuvette (Liu et al., 2015). All buffers and samples were filtered with 0.22 μm filter membranes and centrifuged at 12,000 rpm for 30 min. A preparation 2 μM Glce^167-617 protein was used for the DLS experiment. Following the instrument equilibration, data acquisition durations were maintained between 3 and 5 min, and the collected data were analyzed using Dynamics 7.0 software.

Crystallization experiments were conducted using commercially available crystallization screens with the sitting-drop vapour diffusion method at 20°C. During preliminary screening, crystals of human Glce were grown in drops containing 0.25 μL protein solution (8 mg/mL) and 0.25 μL reservoir solution composed of 0.2 M ammonium sulfate, 0.1 M Tris, pH 6.5–8.5, and 25% (w/v) polyethylene glycol 3,350. Following optimization, rod-shaped crystals were successfully obtained with a reservoir solution containing 0.2 M ammonium sulfate, 0.1 M Tris, pH 8.5, and 22% (w/v) polyethylene glycol 3,350.

All X-ray diffraction data were collected at beamline BL19U1 of the Shanghai Synchrotron Radiation Facility (SSRF, Shanghai, China) using a Pilatus 6M detector (Dectris) and processed using X-ray Detector Software (XDS) (Kabsch, 2010). Date collection and processing statistics are summarized in Table 1.

To enhance the soluble fragment of human Glce protein and simplify the purification steps, the coding sequence was fused to the C-terminus of the SUMO tag. This fusion provides a simple and rapid purification strategy (Zhou et al., 2020). The recombination protein bearing the SUMO tag could be cleaved by SUMO protease. In addition, a His₆ tag was incorporated at the N-terminus of the SUMO tag, thereby enabling protein purification via Ni²⁺-IMAC affinity chromatography.

Multiple sequence alignment was conducted to demonstrate the sequence homology among human, mouse, zebrafish and Drosophila melanogaster. The alignment illustrated that Glce possesses a highly conserved protein sequence across species (Figure 1). AlphaFold structure prediction showed that the N-terminus of Glce composes one α-helical domain and disordered regions (Supplementary Figure 1). To achieve enhanced expression of Glce protein, two truncated constructs differing in N-terminal truncation positions were generated. In summary, the Glce^102-617 and Glce^167-617 gene fragments were cloned into the pET-15b-SUMO vector (Figures 2A,D), with construct validation performed through double restriction enzyme and Sanger sequencing (Figures 2B,E).

The E. coli C2566H strain was employed as the expression host for recombinant glucuronyl C5-epimerase. An engineered E. coli strain harboring Glce^102-617 or Glce^167-617 gene was cultured at 37°C to reach an OD₆₀₀ value of approximately 0.85. Following induction with 0.15 mM IPTG, SUMO-Glce^102-617 and SUMO-Glce^167-617 proteins were induced for efficient overexpression. SDS-PAGE analysis demonstrated that SUMO-Glce^167-617 protein exhibited a significantly higher expression level compared to SUMO-Glce^102-617 (Figures 2C,F).

An efficient purification protocol for the hGlce protein was established, demonstrating suitability for biochemical characterization and crystallization. The expressed Glce^102-617 and Glce^167-617 proteins underwent purification via a two-step chromatography method comprising Ni²⁺-IMAC column purification followed by HiTrap SP ion exchange chromatography. The first purification step using Ni²⁺-IMAC column eluted SUMO-Glce^102-617 or SUMO-Glce^167-617 fused proteins (Figures 3A,C). Subsequently, the eluted proteins underwent SUMO tag cleavage using SUMO protease at 4°C for overnight. The second purification step comprised HiTrap SP ion exchange column with elution conducted through a linear NaCl gradient (0.05–0.5 M) (Figures 3B,D). The purity of purified Glce^167-617 was exceeded 98%, whereas Glce^102-617 displayed a purity of approximately 50% (Figure 3E). SDS-PAGE analysis indicated that both proteins migrated with apparent molecular weights slightly smaller than their theoretical molecular weight (Mw). The purified Glce^102-617 (theoretical Mw: 58.9 kDa) migrated as ~55 kDa protein as determined by SDS-PAGE. Similarly, the purified Glce^167-617 (theoretical Mw: 52.1 kDa) migrated as ~50 kDa protein by SDS-PAGE. Purification procedures are detailed in Table 2. The yield of Glce^167-617 was observably higher than that of Glce^102-617.

The aggregation state of Glce^167-617 may be disrupted by denaturing conditions during SDS-PAGE. To further determine the homogenicity and aggregation state of Glce^167-617 in solution, DLS and size exclusion chromatography (SEC) analyses were conducted for Glce^167-617 characterization. DLS analysis revealed a hydrodynamic radius of 4.715 nm for Glce^167-617, which corresponded to an estimated molecular weight of 127 kDa (Figure 4A). These results demonstrate that Glce^167-617 exists as a dimer in solution. Similarly, the SEC analysis revealed a single peak at K_av = 0.425, yielding an apparent molecular weight of 102.5 kDa based on the calibration equation (Figure 4B).

The pure and homogenous Glce^167-617 protein was utilized for crystallization trails with different commercial screening kits. We succeeded in screening crystallization conditions, which grew a few single crystals with lamellar structures (Figure 5A). The crystal structure of Glce^167-617 was determined by molecular replacement and refined to 2.88 Å resolution with R-work/R-free values of 0.24/0.28, respectively. Comprehensive statistics for structure determination and refinement are summarized in Table 1.

Glce^167-617 crystallized in space group C 1 2 1 with two molecules in the asymmetric unit. The Glce^167-617 structure comprises two domains: an α-barrel domain (residues P167-I233 and D353-N617) and a β-sandwich domain (residues E234-R352). The overall dimeric structure adopts a butterfly-like shape (Figure 5B). Structure analysis reveals a high degree of structural conservation between Glce^167-617 and both human Glce^102-617 and zebrafish Glce^50-585. Structural superposition of hGlce^167-617 with hGlce^102-617 and zGlce^50-585 yielded root-mean-square deviations (RMSD) of 0.273 Å and 0.178 Å, respectively (Figure 5C). As previously reported, dimer formation in Glce involves N-terminal β-hairpin and C-terminal α-barrel domains (Debarnot et al., 2019; Qin et al., 2015). Our results found that deletion of the N-terminal β-hairpin did not disrupt the dimeric architecture in Glce. At the dimerization interface, the C-terminal α-barrel domain forms hydrophobic packing interactions with residues F490, M491, A547, L551, T554, and F565 (Supplementary Figure 2).

To identify the substrate-binding pocket, structural superposition was performed among three conformations: hGlce^167-617 apo, hGlce^102-617 in complex with (GlcA-GlcNS)₅, and zGlce^50-585 in complex with heparin. Structural superposition of hGlce^167-617 apo and the hGlce^102-617-(GlcA-GlcNS)₅ complex through alignment of α-barrel domain revealed that the (GlcA-GlcNS)₅ substrate occupies a cleft between the α-barrel and β-sandwich domains in hGlce^167-617 (Figure 6A). Similarly, superposition of hGlce^167-617 apo with zGlce^50-585-heparin complex via α-barrel domain alignment demonstrated that heparin substrate occupies the same cleft between α-barrel and β-sandwich domains in hGlce^167-617 (Figure 6B). Structural analysis suggests that hGlce^167-617 retains evolutionarily conserved substrate-binding sites.