title: Tripeptidyl peptidase-I is essential for the degradation of sulphated cholecystokinin-8 (CCK-8S) by mouse brain lysosomes.
abstract: Tripeptidyl peptidase-I (TPP-I) is a lysosomal exopeptidase which removes tripeptides from the N-terminus of small proteins. Mutations in the TPP-I gene result in a lethal neurodegenerative disease, late infantile neuronal ceroid lipofuscinosis. The pathological consequences of loss of activity are only manifested in neuronal cells suggesting that TPP-I may be involved in the lysosomal degradation of neuropeptides. We have investigated the degradation of the C-terminal octapeptide of sulphated cholecystokinin (CCK-8S) by a lysosomal fraction purified from mouse brain. Degradation products were characterised by reversed phase HPLC and mass spectrometry. Incubation of CCK-8S with brain lysosomes results in the sequential removal of the tripeptides DY(SO(3)H)M and Glycl-Tryptophanyl-Methionine from the N-terminus of CCK-8S. Degradation of CCK-8S in the isolated lysosomal fraction is completely prevented by Ala-Ala-Phe-chloromethyl ketone, an inhibitor of TPP-I. Butabindide, a specific inhibitor of TPP-II, a cell surface peptidase which also cleaves CCK-8S, inhibits TPP-I but kinetic studies indicate that the Ki for inhibition of TPP-I is 1000-fold higher than the Ki for the inhibition of TPP-II. Consequently, higher concentrations of butabindide are required for the inhibition of CCK-8S degradation by TPP-I than by TPP-II. These results indicate that whereas cell surface TPP-II is responsible for regulating extracellular CCK-8S levels, lysosomal TPP-I is largely responsible for the degradation of CCK-8S which enters the cell by receptor-mediated endocytosis.
mesh_terms: D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000626:Aminopeptidases; D000818:Animals; D001921:Brain / Q000201:enzymology*; D002851:Chromatography, High Pressure Liquid; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; D010450:Endopeptidases / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007211:Indoles / Q000494:pharmacology; D008247:Lysosomes / Q000201:enzymology*; D051379:Mice; D057057:Serine Proteases; D012844:Sincalide / Q000031:analogs & derivatives* / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013997:Time Factors; D000091346:Tripeptidyl-Peptidase 1
chemical_list: C090019:8-sulfocholecystokinin octapeptide; D000590:Amino Acid Chloromethyl Ketones; D004791:Enzyme Inhibitors; D007211:Indoles; C000717268:Tpp1 protein, mouse; D000091346:Tripeptidyl-Peptidase 1; C098807:butabindide; C055604:alanyl-alanyl-phenylalanine chloromethyl ketone; D010450:Endopeptidases; D057057:Serine Proteases; D000626:Aminopeptidases; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; D012844:Sincalide