Insulin-like growth factor II peptide fusion enables uptake and lysosomal delivery of α-N-acetylglucosaminidase to mucopolysaccharidosis type IIIB fibroblasts.

NIH Public Access Author Manuscript Biochem J. Author manuscript; available in PMC 2015 March 01. NIH-PA Author Manuscript Published in final edited form as: Biochem J. 2014 March 1; 458(2): 281–289. doi:10.1042/BJ20130845. Insulin-like growth factor II peptide fusion enables uptake and lysosomal delivery of α-N-acetylglucosaminidase to mucopolysaccharidosis type IIIB fibroblasts Shih-hsin Kan, Larisa A. Troitskaya, Carolyn S. Sinow, Karyn Haitz, Amanda K. Todd, Ariana Di Stefano, Steven Q. Le, Patricia I. Dickson*, and Brigette L. Tippin Department of Pediatrics, Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, Torrance, CA, 90502 USA Abstract NIH-PA Author Manuscript NIH-PA Author Manuscript Enzyme replacement therapy for mucopolysaccharidosis type IIIB (MPS IIIB; Sanfilippo B syndrome) has been hindered by inadequate mannose 6-phosphorylation and cellular uptake of recombinantly produced human α-N-acetyl-glucosamindase (rhNAGLU). We expressed and characterized a modified, recombinant human NAGLU fused to the receptor binding motif of insulin-like growth factor-II (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 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 MPS IIIB fibroblasts readily take up the fusion enzyme via receptor-mediated endocytosis that was significantly inhibited (p<0.001) by 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. Similar cellular uptake mechanism via the IGF-II receptor was also demonstrated in two different brain tumor-derived cell lines. Fusion of NAGLU to IGF-II enhanced its cellular uptake while maintaining enzymatic activity, supporting its potential as a therapeutic candidate for MPS IIIB. To whom correspondence should be addressed: Patricia I. Dickson, Division of Medical Genetics, Department of Pediatrics, LA BioMed at Harbor-UCLA, 1124 W. Carson Street, Torrance, CA 90502, USA, Tel: 310-781-1399; Fax: 310-782-2999; . Treatment of mucopolysaccharidosis type IIIB is hindered by inadequate intracellular uptake of recombinantly-produced alpha-Nacetylglucosamindase (NAGLU). We generated an improved form of NAGLU by fusing it to insulin-like growth factor II, which is a natural ligand for the mannose 6-phosphate receptor. Authors Contribution: Brigette L. Tippin and Patricia I. Dickson developed the original concept and supervised the project. Shih-hsin Kan and Larisa A. Troitskaya conceived and designed the experiments. Shihhsin Kan, Carolyn S. Sinow, Karyn Haitz, Amanda K. Todd and Ariana Di Stefano made the construct and screened the expression clones. Shih-hsin Kan, Larisa A. Troitskaya, Carolyn S. Sinow and Steven Q. Le purified the protein and performed the biochemical assays. Shih-hsin Kan and Steven Q. Le performed and analyzed the in vitro studies. Shih-hsin Kan wrote the paper. Shih-hsin Kan, Brigette L. Tippin, Larisa A. Troitskaya, and Patricia I. Dickson proofread and edited the paper. Kan et al. Page 2 Keywords NIH-PA Author Manuscript Enzyme replacement therapy; lysosomal storage disease; Sanfilippo; mucopolysaccharidosis; insulin-like growth factor Introduction Sanfilippo syndrome type B (MIM 252920; mucopolysaccharidosis IIIB, MPS IIIB) is a rare autosomal recessive lysosomal storage disorder caused by the deficiency of α-Nacetylglucosaminidase (NAGLU; EC 3.2.1.50), an enzyme in the heparan sulfate degradation pathway. Cells lacking NAGLU accumulate heparan sulfate glycosaminoglycan (GAG) in their lysosomes, and the accumulation of undegraded GAG gives rise to adverse cellular and devastating clinical consequences. Individuals affected by this fatal disease exhibit severe central nervous system (CNS) degeneration with progressive cognitive impairment and aggressive behavioral problems, in addition to milder somatic symptoms [1]. There is currently no effective treatment for MPS IIIB. NIH-PA Author Manuscript NIH-PA Author Manuscript Enzyme replacement therapy (ERT) is a therapeutic approach to lysosomal storage disorders in which the deficient enzyme is administered intravenously. ERT has been successfully applied to treat patients in the clinical setting for some storage disorders, including MPS I, II, and VI, Fabry, Pompe, and Gaucher diseases [2–7]. Enzyme can be administered into the cerebrospinal fluid to correct brain storage in animal models [8–13]. However, ERT for MPS IIIB was effective only in cells of the macrophage lineage in liver and spleen [14]. Uptake of exogenous ERT relies in most cases on binding of the mannose 6-phosphate residues on the enzyme to the 300 kDa, cationin-dependent mannose 6-phosphate receptor. Most cells, including neurons, depend on the M6P receptor system for uptake of exogenous lysosomal enzymes, whereas cells of the macrophage lineage use the mannose receptor. Previous attempts by several independent laboratories to generate recombinant NAGLU for ERT in multiple cell types were not successful, due in large part to inadequate mannose 6phosphorylation of the enzyme and resultant poor intracellular uptake [15, 16]. Retroviralmediated gene therapy and transduced hematopoietic stem cells have both shown improved pathology and lifespan in MPS IIIB mice [17–22]. However, cells transfected with vectors containing NAGLU cDNA are expected to secrete poorly phosphorylated enzyme, hampering their ability to cross-correct tissues away from the injection and implantation sites. The M6P receptor also binds insulin-like growth factor-II and is henceforth referred to as the mannose 6-phosphate/insulin-like growth factor-II receptor (M6P/IGF-IIR) [23]. Here, we have employed a fusion protein strategy to enable recombinantly produced NAGLU to gain entry to cells through M6P/IGF-IIR which recognizes multiple ligands on its extracellular domain, including M6P-containing lysosomal enzymes, retinoic acid, and IGF-II via distinct binding sites on the receptor surface [24, 25]. Previous work by others showed that fusion proteins of lysosomal enzymes and IGF-II entered cells and lysosomes via the M6P/IGF-IIR on the cell membrane [26, 27]. We generated an expression construct of NAGLU and the receptor binding domain of IGF-II on the C-terminus to produce recombinant human Biochem J. Author manuscript; available in PMC 2015 March 01. Kan et al. Page 3 NIH-PA Author Manuscript NAGLU-IGF-II (rhNAGLUIGF-II). In this study, rhNAGLU-IGF-II was expressed and purified from Chinese hamster ovary (CHO) cells for biochemical characterization and further tested for f (...truncated)