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.

Abbreviations used: LINCL, late infantile neuronal ceroid lipofuscinosis; TPP-I, tripeptidyl peptidase I; CLN2p, CLN2 (ceroid lipofuscinosis, neuronal 2) protein; LAMP, lysosome-associated membrane protein; 4-MU, 4-methylumbelliferyl; MTX, methotrexate; Man-6-P, mannose 6-phosphate; MPR, Man-6-P receptor; subunit c, mitochondrial ATP synthase subunit c; CHO, Chinese-hamster ovary; DME, Dulbecco's modified Eagle's; FBS, fetal-bovine serum.

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