Production and characterization of recombinant human CLN2 protein for enzyme-replacement therapy in late infantile neuronal ceroid lipofuscinosis

Author:

LIN Li12,LOBEL Peter13

Affiliation:

1. Center for Advanced Biotechnology and Medicine, 679 Hoes Lane, Piscataway, NJ 08854, U.S.A.

2. Graduate Program in Cell and Developmental Biology, Rutgers University, 679 Hoes Lane, Piscataway, NJ 08854, U.S.A.

3. Department of Pharmacology, Robert Wood Johnson Medical School–University of Medicine and Dentistry of New Jersey, 679 Hoes Lane, Piscataway, NJ 08854, U.S.A.

Abstract

Late infantile neuronal ceroid lipofuscinosis (LINCL) is a fatal recessive childhood disease caused by mutations in the CLN2 gene, which encodes the lysosomal enzyme tripeptidyl peptidase I. As a step towards understanding the protein and developing therapeutics for the disease, we have produced and characterized recombinant human CLN2 (ceroid lipofuscinosis, neuronal 2) protein from Chinese-hamster ovary cells engineered to secrete high levels of the enzyme. The protein was secreted as an inactive soluble proenzyme of ≈ 65kDa that appears as a monomer by gel filtration. Upon acidification, the protein is processed to mature form and acquires activity. The enzyme is efficiently delivered to the lysosomes of LINCL fibroblasts by mannose 6-phosphate-receptor-mediated endocytosis (EC50≈ 2nM), where it remains active for long periods of time (t1/2≈ 12 days). In addition, the enzyme is taken up by rat cerebellar granule neurons by mannose 6-phosphate-dependent and -independent mechanisms. Treatment of LINCL fibroblasts with recombinant CLN2 protein restores normal enzyme activity and ameliorates accumulation of the major storage protein, mitochondrial ATP synthase subunit c.

Publisher

Portland Press Ltd.

Subject

Cell Biology,Molecular Biology,Biochemistry

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