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.
Subject(s)
Acetylglucosaminidase/genetics , Fibroblasts/metabolism , Insulin-Like Growth Factor II/genetics , Lysosomes/genetics , Mucopolysaccharidosis III/metabolism , Acetylglucosaminidase/biosynthesis , Acetylglucosaminidase/metabolism , Amino Acid Motifs/genetics , Animals , Binding Sites/genetics , CHO Cells , Cell Line, Tumor , Cricetinae , Cricetulus , Endocytosis/genetics , Fibroblasts/enzymology , Fibroblasts/pathology , Humans , Lysosomes/enzymology , Lysosomes/metabolism , Mucopolysaccharidosis III/enzymology , Mucopolysaccharidosis III/genetics , Protein Binding/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Up-Regulation/geneticsABSTRACT
There is currently great interest in the development of methods to modulate the function of diverse classes of target proteins with chemicals (agonists or antagonists). These would be valuable reagents for biomedical research and some might serve as potential drug leads. Traditionally, most chemicals that modulate protein function have been enzyme inhibitors isolated in functional screens specific for the enzyme of interest. However, recent efforts from many laboratories have suggested that relatively simple binding assays may provide a more convenient and general route to chemical modulators. We review here this work with a particular emphasis on peptide modulators.
