title: 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.
authors: Institute of Human Virology, University of Maryland School of Medicine , Baltimore, Maryland 21201, United States.
authors: Takahiro
authors: T
authors: Yamaguchi
authors: Institute of Human Virology, University of Maryland School of Medicine , Baltimore, Maryland 21201, United States.|Department of Chemistry and Biochemistry, University of Maryland , College Park, Maryland 20742, United States.
authors: Mohammed N
authors: MN
authors: Amin
authors: Department of Chemistry and Biochemistry, University of Maryland , College Park, Maryland 20742, United States.
authors: Christian
authors: C
authors: Toonstra
authors: Institute of Human Virology, University of Maryland School of Medicine , Baltimore, Maryland 21201, United States.|Department of Chemistry and Biochemistry, University of Maryland , College Park, Maryland 20742, United States.
authors: Lai-Xi
authors: LX
authors: Wang
mesh_terms: D000818:Animals; D002236:Carbohydrate Conformation; D002417:Cattle; D006023:Glycoproteins / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism; D008360:Mannosephosphates / Q000737:chemistry*; D009154:Mutation; D010766:Phosphorylation; D011485:Protein Binding; D017527:Receptor, IGF Type 2 / Q000737:chemistry / Q000378:metabolism*; D012260:Ribonucleases / Q000737:chemistry / Q000378:metabolism*
chemical_list: D006023:Glycoproteins; D008360:Mannosephosphates; D017527:Receptor, IGF Type 2; C027693:mannose-6-phosphate; D012260:Ribonucleases; C029600:ribonuclease B