title: Glycan structure determinants for cation-independent mannose 6-phosphate receptor binding and cellular uptake of a recombinant protein.
abstract: The cation-independent mannose 6-phosphate receptor (CI-MPR) plays a critical role in intracellular transport of lysosomal enzymes as well as the uptake of recombinant proteins. To define the minimal glycan structure determinants necessary for receptor binding and cellular uptake, we synthesized a series of glycans containing mono-, di-, tri-, tetra-, and hexamannoses terminated with either one or two phosphates for conjugating to a model protein, recombinant human acid α-glucosidase. A high affinity interaction with the CI-MPR can be achieved for the enzyme conjugated to a dimannose glycan with a single phosphate. However, tightest binding to a CI-MPR affinity column was observed with a hexamannose structure containing two phosphates. Moreover, maximal cellular uptake and a 5-fold improvement in in vivo potency were achieved when the bisphosphorylated hexamannose glycan is conjugated to the protein by a β linker. Nevertheless, even a monophosphorylated dimannose glycan conjugate showed stronger binding to the receptor affinity column, higher cellular uptake, and significantly greater in vivo efficacy compared to the unconjugated protein which contains a low level of high affinity glycan structure. These results demonstrate that the phosphorylated dimannose moiety appears to be the minimal structure determinant for enhanced CI-MPR binding and that the orientation of the glycan is critical for maximum receptor interaction. In summary, we have improved the understanding of the mechanism of CI-MPR binding and developed a simple alternative for CI-MPR targeting.
authors.affiliation: Genzyme Corporation, A Sanofi Company , Framingham, Massachusetts 01701, United States.
authors.forename: Qun
authors.initials: Q
authors.lastname: Zhou
authors.forename: Luis Z
authors.initials: LZ
authors.lastname: Avila
authors.forename: Paul A
authors.initials: PA
authors.lastname: Konowicz
authors.forename: John
authors.initials: J
authors.lastname: Harrahy
authors.forename: Patrick
authors.initials: P
authors.lastname: Finn
authors.forename: Jennifer
authors.initials: J
authors.lastname: Kim
authors.forename: Michael R
authors.initials: MR
authors.lastname: Reardon
authors.forename: Josephine
authors.initials: J
authors.lastname: Kyazike
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Brunyak
authors.forename: Xiaoyang
authors.initials: X
authors.lastname: Zheng
authors.forename: Scott M Van
authors.initials: SM
authors.lastname: Patten
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Miller
authors.forename: Clark Q
authors.initials: CQ
authors.lastname: Pan
mesh_terms: D000818:Animals; D006801:Humans; D042541:Intracellular Space / Q000378:metabolism; D008358:Mannose / Q000737:chemistry; D032446:Myoblasts / Q000166:cytology; D011134:Polysaccharides / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D021381:Protein Transport; D051381:Rats; D017527:Receptor, IGF Type 2 / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism*; D000520:alpha-Glucosidases / Q000378:metabolism*
chemical_list: D011134:Polysaccharides; D017527:Receptor, IGF Type 2; D011994:Recombinant Proteins; C509951:GAA protein, human; D000520:alpha-Glucosidases; D008358:Mannose