Carnosine (␤-alanyl-L-histidine) and homocarnosine (␥-aminobutyric acid-L-histidine) are two naturally occurring dipeptides with potential neuroprotective and neurotransmitter functions in the brain. Peptidase activities degrading both carnosine and homocarnosine have been described previously, but the genes linked to these activities were unknown. Here we present the identification of two novel cDNAs named CN1 and CN2 coding for two proteins of 56.8 and 52.7 kDa and their classification as members of the M20 metalloprotease family. Whereas human CN1 mRNA and protein are brain-specific, CN2 codes for a ubiquitous protein. In contrast, expression of the mouse and rat CN1 orthologues was detectable only in kidney. The recombinant CN1 and CN2 proteins were expressed in Chinese hamster ovary cells and purified to homogeneity. CN1 was identified as a homodimeric dipeptidase with a narrow substrate specificity for Xaa-His dipeptides including those with Xaa ‫؍‬ ␤Ala (carnosine, K m 1.2 mM), N-methyl ␤Ala, Ala, Gly, and ␥-aminobutyric acid (homocarnosine, K m 200 M), an isoelectric point of pH 4.5, and maximal activity at pH 8.5. CN2 protein is a dipeptidase not limited to Xaa-His dipeptides, requires Mn 2؉ for full activity, and is sensitive to inhibition by bestatin (IC 50 7 nM). This enzyme does not degrade homocarnosine and hydrolyzes carnosine only at alkaline pH with an optimum at pH 9.5. Based on their substrate specificity and biophysical and biochemical properties CN1 was identified as human carnosinase (EC 3.4.13.20), whereas CN2 corresponds to the cytosolic nonspecific dipeptidase (EC 3.4.13.18).

Formation of the 4-kDa peptides, which are essential constituents of the extracellular plaques in Alzheimer's disease, involves the sequential cleavage of the amyloid precursor protein (APP) by p-and y-secretases. The carboxy-terminal 99-amino-acid peptide which is liberated from APP by p-secretase was used as a potential native substrate of the y-secretase(s). With the addition of an initiator Met and a FLAG sequence at the C-terminus (PAPP100-FLAG), it was expressed in Escherichia coli under the control of the T7 promotor. The preferred site(s) of cleavage in the N-terminal 40-amino-acid P-amyloid peptide and PAPPI 00-FLAG by potential y-secretase(s) were rapidly identified using matrix-assisted Iaser-desorptionlionization time-of-flight mass spectroscopy in addition to peptide mapping followed by protein sequence analysis. Since y-secretases seem to be active at acidic pH, three cathepsins (D, E and B) were selected for testing. Studies using different detergents indicated that the cleavage preference of cathepsin D for the PAPP100-FLAG is highly dependent on the surfactant used to solubilize this substrate. All three cathepsins were found to be capable of catabolizing both p-amyloid peptides and the PAPPIOO-FLAG. As cathepsin D was found to cleave the PAPP100-FLAG in the vicinity of the C-terminus of the /l-aniyloid peptides and cathepsin B has a high carboxypeptidase activity at low pH, the possibility cannot be excluded that cathepsins D and B are involved in the amyloidogenic processing of APP.

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