Chemoenzymatic Synthesis and Receptor Binding of Mannose-6-Phosphate (M6P)-Containing Glycoprotein Ligands Reveal Unusual Structural Requirements for M6P Receptor Recognition

Abstract

Mannose-6-phosphate (M6P)-terminated oligosaccharides are important signals for M6P-receptor-mediated targeting of newly synthesized hydrolases from Golgi to lysosomes, but the precise structural requirement for the M6P ligand–receptor recognition has not been fully understood due to the difficulties in obtaining homogeneous M6P-containing glycoproteins. We describe here a chemoenzymatic synthesis of homogeneous phosphoglycoproteins carrying natural M6P-containing N-glycans. The method includes the chemical synthesis of glycan oxazolines with varied number and location of the M6P moieties and their transfer to the GlcNAc-protein by an endoglycosynthase to provide homogeneous M6P-containing glycoproteins. Simultaneous attachment of two M6P-oligosaccahrides to a cyclic polypeptide was also accomplished to yield bivalent M6P-glycopeptides. Surface plasmon resonance binding studies reveal that a single M6P moiety located at the low α-1,3-branch of the oligomannose context is sufficient for a high-affinity binding to receptor CI-MPR, while the presence of a M6P moiety at the α-1,6-branch is dispensable. In addition, a binding study with the bivalent cyclic and linear polypeptides reveals that a close proximity of two M6P-oligosaccharide ligands is critical to achieve high affinity for the CI-MPR receptor. Taken together, the present study indicates that the location and valency of the M6P moieties and the right oligosaccharide context are all critical for high-affinity binding with the major M6P receptor. The chemoenzymatic method described here provides a new avenue for glycosylation remodeling of recombinant enzymes to enhance the uptake and delivery of enzymes to lysosomes in enzyme replacement therapy for the treatment of lysosomal storage diseases.

Synthesis of Glycosides by Glycosynthases

“…The mutant was capable of synthesizing asialo biantennary and complex triantennary core-fucosylated glycoforms of rituximab (intact antibody) in yields over 95%. Further applications, indicating the high potential of the method for the synthesis of pharmaceutical relevant compounds were the chemoenzymatic production of vaccine candidates [ 72 ]; the site-selective glycosylation of HIV-1 polypeptide antigen bearing two different glycans (yields up to 95%) [ 73 ]; the glycan remodeling of human erythropoietin (EPO) [ 74 ]; and the synthesis of mannose-6-phosphate-containing glycoproteins [ 75 ]. Tang et al impressively demonstrated a one-pot N -glycan remodeling of IgG proteins by combining the wild type ( wt ) Endo-M glycosidase with the synthase variant Endo-S D322S [ 76 ].…”

Allosteric regulation of lysosomal enzyme recognition by the cation-independent mannose 6-phosphate receptor

“…5e-f). Bidentate binding to two arms of a single oligosaccharide was ruled out due to previously reported findings by Yamaguchi et al who demonstrated that a cyclic glycopeptide had a more than 10-fold higher affinity for CI-MPR than a phosphoglycoprotein ligand harboring a single M6P-phosphoglycan and that linearization of the cyclic glycopeptide through reduction of the disulfide resulted in an ~20-fold loss in affinity 48 . However, inspection of the X-ray structure (PDB 1EI9) revealed that PPT1's three N-glycans were grouped in close proximity to each other and molecular dynamics simulations demonstrate the inherent flexibility of glycans (Fig 6b).…”

Synthetic and semi-synthetic approaches to unprotected N-glycan oxazolines

“…Although in later papers Shoda has published alternative reagents that may be used to achieve the same transformation, such as 2-chloro-1,3-dimethyl-1 H -benzimidazol-3-ium chloride (CDMBI) [ 43 ], DMC remains the most popular reagent for glycosyl oxazoline production. DMC is remarkably tolerant of other functional groups in the oligosaccharide, for example sialic acids [ 44 ] and phosphates [ 45 – 46 ] are completely unaffected; the former is perhaps rather surprising since DMC was first developed as a carboxylic acid activating agent for peptide synthesis by Ishikawa [ 47 ]! One caveat to the procedure is that it is considerably less efficient for GalNAc; in this case the corresponding oxazoline is only produced in ≈50% yield.…”