Structure-Function Relationships in Class CA1 Cysteine Peptidase Propeptides*

Vol. 50 No. 3/2003 691–713 QUARTERLY Review Structure-function relationships in class CA1 cysteine peptidase propeptides* Bernd Wiederanders½ Institute of Biochemistry, Klinikum, Friedrich-Schiller-University Jena, Nonnenplan 2, D-07743 Jena, Germany Received: 30 May, 2003; revised: 18 July, 2003; accepted: 29 August, 2003 Key words: papain family, cathepsin L-like peptidases, propeptide inhibition, processing, foldase Regulation of proteolytic enzyme activity is an essential requirement for cells and tissues because proteolysis at a wrong time and location may be lethal. Proteases are synthesized as inactive or less active precursor molecules in order to prevent such in- appropriate proteolysis. They are activated by limited intra- or intermolecular proteolysis cleaving off an inhibitory peptide. These regulatory proenzyme regions have at- tracted much attention during the last decade, since it became obvious that they har- bour much more information than just triggering activation. In this review we summarize the structural background of three functions of clan CA1 cysteine peptidase (papain family) proparts, namely the selectivity of their inhibitory potency, the participation in correct intracellular targeting and assistance in fold- ing of the mature enzyme. Today, we know more than 500 cysteine peptidases of this family from the plant and animal kingdoms, e.g. papain and the lysosomal cathepsins L and B. As it will be shown, the propeptide functions are determined by certain structural motifs conserved over millions of years of evolution. Cysteine peptidases of clan CA family C1 and prokaryotes. Five hundred and fifty nine (papain family) can be found in the animal members of this peptidase family are actually and plant kingdoms as well as in some viruses registered in the respective tree of the *Presented at the XXX Winter School of Faculty of Biotechnology, Jagiellonian University, Koœcielisko, Poland, 28th February–4th March, 2003. ½ To whom correspondence should be addressed: tel.: (49 3641) 938 611; fax: (49 3641) 938612; e-mail: [email protected] Abbreviations: BCP, Bombyx cysteine protease; BCPI, Bombyx cysteine protease inhibitor; DPPI, dipeptydyl peptidase I; ER, endoplasmic reticulum; GlcNAc, N-acetylglucosamine; MMPs, matrix metalloproteinases; PPIV, papaya protease IV. The suffix E70p means that this residue is located in the propeptide part of the sequence. 692 B. Wiederanders 2003 MEROPS protease database (http: //merops. The functions of CA1 peptidases are differ- sanger.ac.uk) (last entry from June 12, 2003). ent in various organisms. Only few examples The enzymes share their general architecture of viral, prokaryotic and yeast CA1 peptidases but also the micro-arrangement of the three are listed in the MEROPS protease database. catalytic residues Cys 25, His 159 and Asn 175 There is only little knowledge of their physio- (according to the papain numbering). The ion- logical functions. ized state of the nucleophilic cysteine residue Plant proteinases of this class are mainly in the active site is independent of substrate used to mobilize storage proteins in seeds. binding making these and other cysteine pro- Protein bodies of seeds contain both storage teases a priori active (Polgar & Halasz, 1982). proteins and protease precursors. The latter This catalytic mechanism is basically differ- become activated after germination and start ent from that of serine proteases whose serine degradation of the stored proteins (Schlereth residue in the catalytic triad becomes ionized et al., 2001). Some of these enzymes have only upon substrate binding. medical significance because they are Eukaryotic papain family peptidases com- resorbed in the gut as active enzymes and ex- prise three parts: an N-terminal signal se- ert an immunogenic potential (Furmona- quence (10–20 amino acids) is followed by the viciene et al., 2000; Nettis et al., 2001). prosequence (between 38 and 250 amino ac- Most parasitic cysteine peptidases act ids), the third part represents the mature en- extracellularly. They help the parasites to in- zyme, generally 220–260 amino acids long. vade tissues and cells, to gain nutrients, to The tertiary structure of the enzyme part is hatch, to enter and to leave cysts, or even to characterized by two domains (R and L) of evade the host immune system. For details see comparable size with the active site cleft in be- the review by Sajid & McKerrow (2002). Primi- tween. The catalytic site is already preformed tive organisms depending on phagocytosis use in the precursor. It is localized at the bottom cysteine proteases to digest phagocytosed pro- of the active site cleft and involves the three teins. The enzymes of these organisms are al- residues mentioned above. For more details ready packed in lysosomes or acidified see the recent review by McGrath (1999). lysosome-like structures (Volkel et al., 1996; Most CA1 peptidases act as endopeptidases. Krasko et al., 1997; Gotthardt et al., 2002). Some peculiarities and exceptions from this Mammalian CA1 cysteine peptidases are general rule can be explained by structural de- considered as primarily lysosomal enzymes. tails of the catalytic domains as the “occluding Only cathepsin W seems to be retained in the loop” in cathepsin B which favours the bind- endoplasmic reticulum (ER) (Wex et al., ing of protein C-termini thus enabling its 2001). Some cathepsins are found in nearly all peptidyl dipeptidase activity (Musil et al., tissues and cells (cathepsins B, C, H, L, O), 1991), the Cys 331 of cathepsin C which is nec- thus probably fulfilling housekeeping func- essary for tetramerization and thus for tions. Others show a restricted organ distribu- dipeptidyl peptidase activity (Horn et al., tion (cathepsins S, K, V, F, X, W) suggesting 2002), or the “mini-chain” of cathepsin H an- specific functions. Recent information about choring the positively charged amino group of cathepsin functions came from modern ge- the substrate N-terminus (Guncar et al., netic approaches as mutational analyses and 1998). Reasons of other features, such as the gene knock out animals. A recent review of carboxypeptidase activity of cathepsin X, the the physiological and pathological roles of lack of the activation of procathepsin W or the mammalian and parasitic CA1 peptidases is functions of C-terminal extensions of parasite recommended for interested readers (Lecaille derived enzymes still remain to be elucidated. et al., 2002). Vol. 50 Propeptide functions of cysteine peptidases 693 PROPEPTIDE INHIBITION The first structures of cysteine protease precursors were published by Cygler et al. (1996), Turk et al. (1996) and Coulombe et al. (1996). Structural background Their studies revealed how the propeptide is The inhibition of CA1 cysteine proteases by attached to the mature enzyme (Coulombe et their respective propeptide parts was first ob- al., 1996). The most striking result was the served by Fox et al. (1992). They observed a elucidation of the mode of propeptide inhibi- strong inhibition of cathepsin B by a 56 amino tion. The authors clearly showed that the acids long synthetic peptide corresponding to propeptide covers the active site cleft in a residues -62 to -7 of rat liver procathepsin B. non-productive orientation. The S subsite is The inhibition was pH dependent. At pH 6.0, occupied by the C-terminal residues (e.g. the inhibition was a slow binding one step re- Gly77p, Leu78p and Gln79p in procathe- action, whereas the inhibition at pH 4.0 fol- psin L) whereas the S¢ subsites are mainly oc- lowed the classical scheme. The propeptide cupied by the N-terminal residues. Such an was also slowly degraded by the enzyme at pH orientation does not allow the hydrolysis of 4.0. The authors suggested a loose complex be- the peptide bond, however, the tightly bound tween the pro and the mature domain at molecule hinders the access of substrate mole- acidic pH. cules to the active site. This mode of inhibition Taylor et al. (1995a) expressed the pro-re- is found in all class CA1 cysteine peptidases gions of two proteases from Carica papaya, whose zymogen structures have been re- papain and PPIV (papaya protease IV), as re- solved. combinant proteins in Escherichia coli and The propeptides contain some characteristic studied the inhibitory activity of the peptides elements which are highly conserved in evolu- toward papain, caricain, chymopapain and tion. Karrer et al. (1993) compared the N-ter- PPIV. They found different Ki values being minal amino-acid sequences of 15 cysteine three orders of magnitudes higher for PPIV protease zymogens. They found a consensus than for the others. They discussed this selec- sequence known as ERFNIN motif present in tivity for the first time on the basis of struc- the a2 helix of a great number of cysteine pro- tural differences. Numerous reports by differ- tease propeptides of numerous species, in- ent groups confirmed later the general fact cluding Tetrahymena. However, in cathepsin that the inhibitory propeptide parts are regu- B, the a2 helix is much shorter and does not latory elements of cysteine protease activity contain the ERFNIN motif. On this basis, the (Volkel et al., 1996; Maubach et al., 1997; Visal authors defined two subfamilies of cysteine et al., 1998; Guay et al., 2000; Billington et al., proteases, the cathepsin L like containing the 2000). ERFNIN motif, and the cathepsin B like lack- The structural background of this inhibition ing this motif. In cathepsins F (Wang et al., was elucidated by X-ray structure analyses. 1998; Nagler et al., 1999) and W (Linnevers et Papain was the first mature cysteine protease al., 1997; Brown et al., 1998; Wex et al., 1998), whose structure was published (Drenth et al., the Ile and Asn residues of the ERFNIN motif 1968). A 1.65 Å resolution revealed later a two are replaced by Ala and Gln, defining a third domain fold of papain with the active site lo- subgroup of cysteine proteases characterized cated in a groove between the two domains by the ERFNAQ motif, called cathepsin F like (Kamphuis et al., 1984).

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