Specific substrate for CLN2 protease/tripeptidyl-peptidase I assay

The classic late infantile neuronal ceroid lipofuscinosis (LINCL, CLN2) is a fatal neurodegenerative disorder that results from mutations in a gene encoding a lysosomal proteinase, known as CLN2 protease (CLN2p) or tripeptidyl peptidase I (TPP-I). Three different substrates, fluorescein isothiocyanate-labelled haemoglobin, A-F-F-7-amino-4-methylcoumarin (AAF-AMC) and G-F-F-L-7-amino-4-trifluoromethylcoumarin (GFFL-AFC) have been used for the CLN2p/TPP-I assay with varying degrees of residual activities in patients with LINCL. Further, conclusive identification of carriers are not possible with the first two substrates. An assay for the CLN2p/TPP-I based on the cleavage of amino terminal tripeptide from G-F-F-L-AFC was applied to prenatal and postnatal diagnosis of LINCL patients and heterozygote carriers. In leukocytes, the CLN2p/TPP-I activities in controls and heterozygote carriers were 1995 +/- 154 (n = 15) and 918 +/- 253 (n = 15) nmol/h/mg protein respectively. No CLN2p/TPP-I activity was detectable in all but two patients. These two patients had less than 2% residual activity, and had delayed clinical symptoms for LINCL. This shows that the G-F-F-L-AFC is a highly specific substrate for the CLN2p/TPP-I assay. The fact that with this substrate the enzyme cleaves a peptide bond between the two amino acids may be the reason for the high level of specificity.