title: Insulin-like growth factor II peptide fusion enables uptake and lysosomal delivery of α-N-acetylglucosaminidase to mucopolysaccharidosis type IIIB fibroblasts.
abstract: Enzyme replacement therapy for MPS IIIB (mucopolysaccharidosis type IIIB; also known as Sanfilippo B syndrome) has been hindered by inadequate mannose 6 phosphorylation and cellular uptake of rhNAGLU (recombinant human α-N-acetylglucosaminidase). We expressed and characterized a modified rhNAGLU fused to the receptor-binding motif of IGF-II (insulin-like growth factor 2) (rhNAGLU-IGF-II) to enhance its ability to enter cells using the cation-independent mannose 6-phosphate receptor, which is also the receptor for IGF-II (at a different binding site). RhNAGLU-IGF-II was stably expressed in CHO (Chinese-hamster ovary) cells, secreted and purified to apparent homogeneity. The Km and pH optimum of the fusion enzyme was similar to those reported for rhNAGLU. Both intracellular uptake and confocal microscopy suggested that MPS IIIB fibroblasts readily take up the fusion enzyme via receptor-mediated endocytosis that was inhibited significantly (P<0.001) by the monomeric IGF-II peptide. Glycosaminoglycan storage was reduced by 60% (P<0.001) to near background levels in MPS IIIB cells after treatment with rhNAGLU-IGF-II, with half-maximal correction at concentrations of 3-12 pM. A similar cellular uptake mechanism via the IGF-II receptor was also demonstrated in two different brain tumour-derived cell lines. Fusion of rhNAGLU to IGF-II enhanced its cellular uptake while maintaining enzymatic activity, supporting its potential as a therapeutic candidate for treating MPS IIIB.
authors.affiliation: *Department of Pediatrics, Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, Torrance, CA 90502, U.S.A.
authors.forename: Shih-hsin
authors.initials: SH
authors.lastname: Kan
authors.affiliation: *Department of Pediatrics, Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, Torrance, CA 90502, U.S.A.
authors.forename: Larisa A
authors.initials: LA
authors.lastname: Troitskaya
authors.affiliation: *Department of Pediatrics, Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, Torrance, CA 90502, U.S.A.
authors.forename: Carolyn S
authors.initials: CS
authors.lastname: Sinow
authors.affiliation: *Department of Pediatrics, Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, Torrance, CA 90502, U.S.A.
authors.forename: Karyn
authors.initials: K
authors.lastname: Haitz
authors.affiliation: *Department of Pediatrics, Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, Torrance, CA 90502, U.S.A.
authors.forename: Amanda K
authors.initials: AK
authors.lastname: Todd
authors.affiliation: *Department of Pediatrics, Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, Torrance, CA 90502, U.S.A.
authors.forename: Ariana
authors.initials: A
authors.lastname: Di Stefano
authors.affiliation: *Department of Pediatrics, Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, Torrance, CA 90502, U.S.A.
authors.forename: Steven Q
authors.initials: SQ
authors.lastname: Le
authors.affiliation: *Department of Pediatrics, Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, Torrance, CA 90502, U.S.A.
authors.forename: Patricia I
authors.initials: PI
authors.lastname: Dickson
authors.affiliation: *Department of Pediatrics, Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, Torrance, CA 90502, U.S.A.
authors.forename: Brigette L
authors.initials: BL
authors.lastname: Tippin
mesh_terms: D000118:Acetylglucosaminidase / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D020816:Amino Acid Motifs / Q000235:genetics; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D016466:CHO Cells; D045744:Cell Line, Tumor; D006224:Cricetinae; D003412:Cricetulus; D004705:Endocytosis / Q000235:genetics; D005347:Fibroblasts / Q000201:enzymology / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D007335:Insulin-Like Growth Factor II / Q000235:genetics*; D008247:Lysosomes / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D009084:Mucopolysaccharidosis III / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015854:Up-Regulation / Q000235:genetics
chemical_list: D011993:Recombinant Fusion Proteins; D007335:Insulin-Like Growth Factor II; C033079:alpha-N-acetyl-D-glucosaminidase; D000118:Acetylglucosaminidase