Probing the substrate specificity of Golgi alpha-mannosidase II by use of synthetic oligosaccharides and a catalytic nucleophile mutant

Abstract

Inhibition of Golgi alpha-mannosidase II (GMII), which acts late in the N-glycan processing pathway, provides a route to blocking cancer-induced changes in cell surface oligosaccharide structures. To probe the substrate requirements of GMII, oligosaccharides were synthesized that contained an alpha(1,3)- or alpha(1,6)-linked 1-thiomannoside. Surprisingly, these oligosaccharides were not observed in X-ray crystal structures of native Drosophila GMII (dGMII). However, a mutant enzyme in which the catalytic nucleophilic aspartate was changed to alanine (D204A) allowed visualization of soaked oligosaccharides and led to the identification of the binding site for the alpha(1,3)-linked mannoside of the natural substrate. These studies also indicate that the conformational change of the bound mannoside to a high-energy B 2,5 conformation is facilitated by steric hindrance from, and the formation of strong hydrogen bonds to, Asp204. The observation that 1-thio-linked mannosides are not well tolerated by the catalytic site of dGMII led to the synthesis of a pentasaccharide containing the alpha(1,6)-linked Man of the natural substrate and the beta(1,2)-linked GlcNAc moiety proposed to be accommodated by the extended binding site of the enzyme. A cocrystal structure of this compound with the D204A enzyme revealed the molecular interactions with the beta(1,2)-linked GlcNAc. The structure is consistent with the approximately 80-fold preference of dGMII for the cleavage of substrates containing a nonreducing beta(1,2)-linked GlcNAc. By contrast, the lysosomal mannosidase lacks an equivalent GlcNAc binding site and kinetic analysis indicates oligomannoside substrates without non-reducing-terminal GlcNAc modifications are preferred, suggesting that selective inhibitors for GMII could exploit the additional binding specificity of the GlcNAc binding site.

Figure 1

Structures of synthetic targets.

Figure 2

Binding of mannose in the active site of (A) D341A dGMII [PDB code 3BUP] or (B) D204A dGMII [PDB code 3BUQ]. F_o – F_c omit electron density maps are shown contoured at 3σ (0.2 e/ų). A magenta ball represents the active-site zinc. (C) Superposition of the bound mannosides. Superposition was based on the protein atoms, and the mannose was extracted from the fit structures. Mannose in the active site of D341N (yellow) is in a high-energy boat conformation, while in D204A (green) it is in a ⁴C₁ chair conformation.

Figure 3

Binding of 2 and 4 to D204A: stereoviews (divergent) of (A) 2 bound in F_o – F_c omit map contoured at 4σ (0.38 e/ų) or (B) 4 contoured at 1.6σ (0.15 e/ų) to visualize density in the +1 and +2 positions. The MPD displaced by GlcNAc binding is shown in panel A. The orientation in both panels is the same to illustrate the differences in position of each compound. (C, D) Interactions less than 3.2 Å are shown for (C) 2 or (D) 3. Distances are given in angstroms. Interacting waters are shown as orange spheres. Zinc is represented as a magenta sphere.

Figure 4

The presence of GlcNAc causes a radical rearrangement of oligosaccharides bound to Golgi mannosidase II. Shown is the binding of 3 (blue, from PDB structure 3BVV) and GlcNAc-modified 4 (yellow, from PDB structure 3BVW). For orientation purposes, one of the mannoside residues is colored green. This mannoside binds in the −1 site of D204A/3 complex and in the +3 site of the D204A/4 complex. The active-site zinc is colored magenta. Although 3 is a tetrasaccharide, only three sugars could be assigned to the electron density.

Figure 5

Comparison of binding of 2 (green) and 3 (slate) to D204A. Compound 2 has an α(1,6)-linked mannoside while 3 has an α(1,3)-linked mannoside bound in the −1 site. Compound 3 is a tetramannoside but only three mannosides are visible in the electron density; the terminal thio-linked mannose cannot be assigned. The thio bonds are colored orange, and zinc in the active site is represented by a magenta ball.

Figure 6

In Vitro digestion time course of Man₅GlcNAc₂-PA and GlcNAcMan₅GlcNAc₂-PA by dGMII and hLM. Purified recombinant dGMII (A, C) and hLM (B, D) were used in digestion time course studies with GlcNAcMan₅GlcNAc₂-PA (A, B) or Man₅GlcNAc₂-PA (C, D) as substrates. Cleavage of the substrates (GlcNAcMan₅GlcNAc₂-PA or Man₅GlcNAc₂-PA, ◆) to smaller glycan structures (GlcNAcMan₄GlcNAc₂-PA or Man₄GlcNAc₃-PA, ■; GlcNAcMan₃GlcNAc₂-PA or Man₃GlcNAc₃-PA, ▴; GlcNAcMan₂GlcNAc₂-PA or Man₂GlcNAc₃-PA, ●) were quantitated by HPLC. dGMII cleaved GlcNAcMan₅GlcNAc₂-PA ~80-fold faster than Man₅GlcNAc₂-PA at equivalent enzyme concentrations. Minimal digestion of Man₅GlcNAc₂-PA or GlcNAcMan₅GlcNAc₂-PA by hLM was detected when equivalent enzyme activity units (based on 4MU-α-Man activity) of hLM and dGMII were employed (not shown). Increasing the enzyme concentration of hLM in the in vitro assays by 100-fold (B, D) resulted in detectable cleavage of Man₅GlcNAc₂-PA, but cleavage of GlcNAcMan₅GlcNAc₂-PA remained ~16-fold slower.

Figure 7

Spatial clash between Asp204 and sugar bound in the low-energy ⁴C₁ conformation. Coordinates for the D204A/5 complex (PDB code 3BVX) were superimposed with those of the unliganded native enzyme (PDB code 3BVT). If the pentasaccharide bound to the native enzyme in an identical manner to which it binds to D204A, the distance between the Asp204 Oδ2 and O2 of 5 would be only 1.2 Å. The pentasaccharide is colored in cyan, while amino acid side chains of the native protein are shown in green, except for Asp204, which is highlighted by coloring it magenta.

Figure 8

The swivel sugar is observed in a large number of positions. The two mannosides closest to the active-site zinc are shown for the α(1,6)-linked compounds 2 (green) and 5 (yellow) and for the α(1,3)-linked compounds 1 (cyan) and 3 (slate). In all cases, the position of the zinc-bound −1 mannoside is almost invariant, whereas the mannoside in the +1 site, which is described as the swivel residue, is highly variable. The oxygen at the cleavage site, indicated by an asterisk, is in almost the same position in all complexes.

Scheme 1a

a Reagents and conditions: (i) NIS, TfOH, DCM, 0 °C (80%); (ii) NaOMe, MeOH (89%); (iii) NIS, TfOH, DCM, 0 °C (76%); (iv) BH₃ in THF, Bu₂BOTf in DCM, 0 °C (67%).

Scheme 2a

a Reagents and conditions: (i) TrCl, pyridine, 80 °C, and then Ac₂O, pyridine (96%); (ii) FeCl₃ · 6H₂O, DCM (82%); (iii) Tf₂O, 2,6-lutidine, DCM, −40 °C; (iv) NIS, TfOH, DCM, 0 °C (76% for 17, 86% for 22); (v) diethylamine, DMF, 0 °C (73%); (vi) TFA, DCM, and then trichloroacetonitrile, DBU, DCM; (vii) TMSOTf, DCM, (80% for 23, 83% for 25); (viii) H₂NNH₂ · H₂O, EtOH, 90 °C, and then Ac₂O, pyridine; (ix) NaOMe, MeOH, and then Na (s), NH₃ (l), THF, −78 °C (86% for 4, 87% for 5).

Scheme 3a

a Reagents and conditions: (i) DMSO, 1:2 Ac₂O/DMSO, and then NaBH₄, 1:1 DCM/MeOH (75%); (ii) Tf₂O, 1:2 pyridine/DCM, and then 20, DMF, diethylamine, 0 °C (61%); (iii) BH₃ in THF, Bu₂BOTf (63%); (iv) 16, NIS, TfOH, DCM, 0 °C (77%); (v) NaOMe, MeOH, and then Na/NH₃(l), −78 °C (70%).