Synthetic domains of cystatins linked to enkephalins are novel inhibitors of brain cathepsins L/B.
Cystatin domains or homologous sequences were synthesized and tested as inhibitors of papain, and rat brain cathepsins B and L. These domains included: I, an enzyme substrate binding site containing a -GG- cleavage site (YGGFL); II, known cystatin consensus sequences (-QVVAG- or -QLVSG-); and III, the proposed ancillary site for binding of chicken cystatin to papain (-IPWLN-). A Domain II analog QVVAG(K-NH2) inhibited cathepsin L and papain with Ki 1-4 X 10(-4) M but was inactive towards cathepsin B. A peptide containing Domains I and II, YGGFL-QVVAG(K-NH2), inhibited papain and cathepsin B with Ki 10(-4)-10(-5) M, and cathepsin L with Ki 10(-6) M. The presence of Domain III in the analog YGGFL-QVVAG-IPWLN(K-NH2) resulted in a 10-fold increase in potency towards papain. These data demonstrated that putative cystatin domains are: 1) probably proximal in the intact cystatins; 2) can be linked directly to each other to yield smaller peptides active as inhibitors; 3) showed some specificity towards the three cysteine proteinases.
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Nathan Kline Institute, Center for Neurochemistry, Wards Island, New York, NY 10035.
Berg
M J
MJ
Makofske
R C
RC
Danho
W
W
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000201:enzymology*; D002401:Cathepsin B / Q000037:antagonists & inhibitors*; D056668:Cathepsin L; D002403:Cathepsins / Q000037:antagonists & inhibitors*; D015891:Cystatins / Q000494:pharmacology*; D003546:Cysteine Endopeptidases; D010450:Endopeptidases; D004745:Enkephalins / Q000494:pharmacology*; D008969:Molecular Sequence Data; D051381:Rats; D012689:Sequence Homology, Nucleic Acid
D015891:Cystatins; D004745:Enkephalins; D002403:Cathepsins; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; D002401:Cathepsin B; D056668:Cathepsin L; C534280:Ctsl protein, rat