title: Specific substrate for CLN2 protease/tripeptidyl-peptidase I assay.
abstract: 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.
authors.affiliation: Department of Developmental Biochemistry, New York State Institute for Basic Research in Developmental Disabilities, 1050 Forest Hill Road, Staten Island, NY 10314, USA. majunaid@aol.com
authors.forename: M A
authors.initials: MA
authors.lastname: Junaid
authors.forename: S S
authors.initials: SS
authors.lastname: Brooks
authors.forename: R K
authors.initials: RK
authors.lastname: Pullarkat
mesh_terms: D000626:Aminopeptidases; D002624:Chemistry, Clinical / Q000379:methods*; D015193:Chorionic Villi Sampling; D002855:Chromatography, Thin Layer; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; D010450:Endopeptidases; D005260:Female; D006580:Genetic Carrier Screening; D006801:Humans; D007223:Infant; D009472:Neuronal Ceroid-Lipofuscinoses / Q000175:diagnosis* / Q000201:enzymology*; D010447:Peptide Hydrolases / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D011247:Pregnancy; D012680:Sensitivity and Specificity; D057057:Serine Proteases; D013379:Substrate Specificity; D000091346:Tripeptidyl-Peptidase 1
chemical_list: D000091346:Tripeptidyl-Peptidase 1; D010450:Endopeptidases; D010447:Peptide Hydrolases; D057057:Serine Proteases; D000626:Aminopeptidases; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; C000717267:TPP1 protein, human