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 prote- olysis 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 inhibi- tory 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 struc- tural 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 pre- cursors 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). This particular fea- subgroup (Wex et al., 1999). ture is highly conserved amongst cysteine pro- Another highly conserved motif is the teases of this type in all kingdoms (Musil et al., GxNxFxD heptapeptide motif (GNFD in brief) 1991; McGrath et al., 1995; Roche et al., 1999). which can also be found in most of the cys- 694 B. Wiederanders 2003 teine protease propeptides. The motif is lo- low Mr inhibitors mentioned below. Table 1 cated at the kink of the b-sheet immediately shows details of kinetic experiments in which before the chain builds the third helix turning the propeptides act in trans. down into the active site cleft. The Asp resi- An interesting observation was the existence due in the GNFD motif seems to be essential of proteins with homology to the propeptide for the correct processing of the protease pre- regions of cysteine proteases, however, ex- cursors since replacement of this Asp by Asn, pressed independently from the co-expressed Tyr, Met, Val or Glu resulted in non functional enzyme. The occurrence of such proteins has papain mutants (Vernet et al., 1995). been reported in activated mouse T lympho- Procathepsin F contains a propeptide which cytes and mast cells, they were named CTLA-1 is more than twice as long as the propeptides and -2 (Denizot et al., 1989; Delaria et al., of a closely related ERFNAQ subfamily mem- 1994), and in the hemolymph of the silkmoth ber, cathepsin W. Besides this distinctive fea- Bombyx mori, named BCPI a and b (Bombyx ture it shows a cystatin-like domain which cysteine protease inhibitor (Yamamoto et al., makes the propeptide of cathepsin F unique 1999a; 1999b; Kurata et al., 2001; Yamamoto amongst the CA1 cysteine peptidases (Nagler et al., 2002). Moreover, a search in the et al., 1999). It is still unclear whether or not SwissProt databank revealed the occurrence the cystatin-structure element contributes of further sequences homologous to CTLA-2 much to the inhibitory potency of the in rat (R/CTLA-2) as well in the Drosophila ge- cathepsin F propeptide in vivo. nome (D/CTLA-2). Whereas the kinetic con- stants of BCPIs and CTLAs have been mea- sured, the rat and the Drosophila proteins Inhibition type and constants have not been characterized so far. Yamamoto Human papain like proteases are involved in et al. (2002) suggested the origin of this class a variety of pathological processes, such as of inhibitory peptides by partial gene duplica- malignant tumour invasion and chronic de- tion of an ancestor cysteine protease gene structive processes. Moreover, proteases of thus leading to a new class of endogenous in- this type are also important for the life cycle hibitors without relation to cystatins and and infectivity of parasitic pathogens like other endogenous inhibitors. A biological role Fasciola hepatica, Trypanosoma species and of these proteins has not been reported so far. others. Therefore, enzymes of this class repre- sent attractive targets for the development of Selectivity of inhibition therapeutic inhibitors. However, since the proteases play important roles in normal pro- Selectivity of propeptide inhibition has to tein turnover and protein processing, and due consider two different meanings: 1st how se- to the broad substrate specificity of cysteine lective is an effect between two members of proteases, the development of inhibitors with the family within a given species (e.g. human high selectivity is a great challenge. cathepsin L vs human cathepsin K), and 2nd The structural details of the interaction be- how selective is the effect in an interspecies tween the propeptides of cysteine proteases comparison (e.g. human cathepsin L vs. F. he- and their cognate enzymes suggest a selectiv- patica cathepsin L). This is important if ity of inhibitory propeptide action. Kinetic propeptide derived inhibitors should be taken constants of this inhibition have been studied as leading structures for the development of by several groups. The selectivity has various pharmaceuticals. Numerous experiments reasons and it is by far not as good as might be have been performed to characterize the type expected. Nevertheless, the studies were the of propeptide inhibition toward different cys- basis for the development of peptide derived teine peptidases, moreover, truncated and Vol. 50 Propeptide functions of cysteine peptidases 695
Table 1. Inhibition constants of cysteine protease propeptides toward different proteases, either the cognate or a non-cognate enzyme, using low MW substrates
*P.c. Paramecium caudatum; **B.m. Bombyx mori 696 B. Wiederanders 2003 chimeric propeptides were involved in these and Leu78p (procathepsin K numbering) studies in order to determine which part of which cover the active site cleft. Whereas the the propeptide is of importance for selectivity. cathepsins S, K and L share 10 of the 24 The results can be summarized as follows: amino acids, papain shares only 4, and papain uMammalian cathepsin propeptides with a shows an additional insertion between the res- complete a2 helix are poor inhibitors of idues binding to S1 and S2 (Guay et al., 2000). cathepsin B and of papain (Delaria et al., From some data one might speculate that 1994; Carmona et al., 1996; Guay et al., propeptides of cysteine peptidases have ac- 2000; Billington et al., 2000; Kurata et al., quired increasing selectivity and increasing 2001); inhibitory potency during evolution: uThe cathepsin B propeptide is a poor inhib- uMammalian propeptides are by far the itor of papain (Fox et al., 1992); best inhibitors of their cognate enzymes in uThe species differences between inhibitory comparison to the propeptide mediated in- propeptide and enzyme do not follow a hibition of Paramecium tetraurelia or plant general rule (Roche et al., 1999; Guo et al., enzymes (Taylor et al., 1995; Roche et al., 2000; Kurata et al., 2001); 1999; Guo et al., 2000); uThere is only little selectivity of propeptide uThe Paramecium tetraurelia cathepsin L inhibition between cathepsins S, L and K propeptide inhibits the parental enzyme in (Guay et al., 2000); a two step reaction (Guo et al., 2000) (see uN-terminal truncation of the propeptide re- Scheme 2 below and the respective discus- duces the inhibitory potency more than sion). This is a unique observation in the truncation at the C-terminus (Carmona et papain family up to now; u al., 1996; Kurata et al., 2001); The Ki values of the inhibition of plant en- uThe N-terminal part of the propeptide zymes by their propeptides have been de- shows a greater influence on selectivity duced from steady-state measurements. than the C-terminal part (Guo et al., 2000). Pre-steady-state was not recorded at all Some of the findings from different laborato- (Taylor et al., 1995). ries are not compatible, e.g. the inhibition of Measuring the pre-steady-state inhibition ki- cathepsin L by the propeptide of cathepsin S netics of plant peptidases by their respective showed a difference in Ki of one order of mag- propeptides would be the key experiment in nitude (Maubach et al., 1997 and Guay et al., order to support the optimisation hypothesis. 2000) vs (Guo et al., 2000). Most of the pro- A closer view on the active site of cathepsins peptides are very hydrophobic. We observed a L, K and S shows that the two bulky aromatic remarkable adsorption of the propeptides to residues Phe63p and Phe71p of the cathepsin the surface of assay tubes. This resulted in in- L propeptide do not fit well into the respective correctly high Ki values (Maubach et al., 1997) binding pocket of cathepsin S due to steric which we later corrected on the basis of a hindrance by Phe146 (cathepsin S). In more careful analysis (Guo et al., 2000). Some cathepsin L, Leu144 is in this position which of the deviating data may be caused by such allows a tight contact of the propeptide with experimental details, but others may also be the mature enzyme. This may explain the dif- explained by different substrate and pH condi- ferences in Ki of cathepsin L propeptide inhi- tions as the authors already discussed (Guay bition toward cathepsins L and S (Guay et al., et al., 2000). 2000; Guo et al., 2000). The propeptide of Guay et al. (2000) discussed the inhibition of cathepsin K shows in this position two cathepsins S, K and L vs. papain by the respec- branched amino acids, Leu63p and Val71p. tive propeptides on the basis of homologies of The hydrophobic interactions of these resi- the propeptide sequences between Thr55p dues with Phe146 and Leu144 in cathepsins S Vol. 50 Propeptide functions of cysteine peptidases 697 and L, respectively, are obviously much more tight than with Gln143 in the cognate kK PESSEI¬¾¾¾¬®cat¾¾++ m cathepsin K. The different kinetic constants k for this inhibition detected by two independ- ¾®¾¾on ¬®¾ EI¬¾¾¾ EI * ent groups may be explained by the different koff conditions: whereas Guay et al. (2000) deter- mined the activity at pH 5.5 toward Scheme 2 Z-LR-AMC, the activity of cathepsins L and K was measured with Z-FR-AMC at pH 6.0 (Billington et al., 2000). Propeptide derived peptidomimetic inhibi- In cathepsin B, the occluding loop prevents tors the propeptide of other cathepsins from bind- The development of protease inhibitors is an ing to the active enzyme. obvious challenge, either for the treatment of The electrostatic surface potentials of diseases whose progress is caused by uncon- papain, various cathepsins and their propep- trolled protease activity, or for intervention tides reveal significant differences amongst into the life cycle of parasites. Most inhibitors them (see Fig. 1). Thus, the overall surface blocking the active site of a protease react co- charge besides the atomic interactions may in valently. They are directed to the catalytic some cases also contribute to the selectivity of cysteine residue common within all members propeptide inhibition. of the family — the basis of the clan classifica- Two different types of inhibition can be ob- tion — and act, therefore, with low selectivity. served when propeptides are incubated with However, the CA1 cysteine peptidase precur- various enzymes. The general mechanism by sors provide examples of non-covalent inhibi- which the propeptides react with their cog- tion with relatively high selectivity as men- nate peptidases is tight binding. It is a one tioned above, and there are some approaches step reaction according to Scheme 1. to use their structural features for the devel- opment of effective medicine. Furthermore, modifications stabilizing the peptide moieties against degradation are also necessary, as e.g. ¾®¾k¾on kKcat m introduction of D-amino acids. PESSEIEI¬¾¾¾¬®¾¾++¬¾¾¾ koff Carmona et al. (1996) used already trun- cated forms of recombinant cathepsin L pro- peptides in order to study the contributions Scheme 1 of defined segments to the inhibitory po- Another type of inhibition can also be ob- tency. However, truncation led to a dramatic served in which the enzyme first forms a loose loss of affinity suggesting that extensive mo- contact to the propeptide (Scheme 2). The ini- lecular contacts are necessary for efficient tial contact induces in both the propeptide inhibition. Chowdhury et al. (2002) carefully and the enzyme a conformational shift result- analysed the binding mode of the penta- ing in koff values of the complexes resembling peptide moiety MNGFQ (residues 75p to 79p those of the cognate propeptide/enzyme of the cathepsin L propeptide) spanning the pairs. This was observed with mutants desta- active site groove of cathepsin L from the S2¢ bilizing the mini domain between helices a1 to S3 subsites in order to synthesize an opti- and a2 in procathepsin S (Schilling et al., mized non-covalent selective cathepsin L in- 2001) and also for the inhibition of cathepsin hibitor. They ended up with a series of com- H by the cathepsin L propeptide (own unpub- pounds, the best of which showed a Ki of 19 lished result). nM toward cathepsin L, the Ki values toward 698 B. Wiederanders 2003
Figure 1. Model of electrostatic potentials on the surfaces of three cysteine protease propeptides and of three mature cysteine proteases. The models were calculated by Swiss-PdbViewer (Guex & Peitsch, 1997) using atomic partial charges for each mole- cule. The colour of the electrostatic potential maps ranges from red (–8 kT/mol) via white (neutral) to blue (+8 kT/mol). Upper row: propeptides of cathepsins S (Kaulmann, PhD thesis), K (PDB entry 1BY8), and L (PDB entry 1CS8). The mature enzymes are shown as thin white sequence chains, i.e. the model does not show the contact area with the mature enzyme. Lower rows: papain (PDB entry 9PAP), procathepsin L (PDB entry 1CS8) and procathepsin K (PDB entry 1BY8). View on the active-site cavity and on the contact area of the mature enzyme with the propeptide. The proparts of cathepsins K and L are shown as thin white sequence chains. Vol. 50 Propeptide functions of cysteine peptidases 699 cathepsins K and B being 5.9 and 4.1 mM, re- thus showed a satisfying degree of selectivity. spectively, which is remarkable since the full However, the best Ki values were around 5 mM length propeptide of cathepsin L shows only and, therefore, were too high to make these a 2-fold better selectivity toward cathepsin K. peptides efficient inhibitors (Lalmanach et al., The propeptide of cathepsin B was also sys- 1998). Nevertheless, the pentapeptide YHN- tematically truncated from the N- as well as GA was present in all four peptides and consti- from the C-terminus in steps of 5 amino acids, tuted an essential element for selectivity and and the change of Ki toward cathepsin B was inhibition of parasite proteases and, there- recorded. The stepwise truncation from either fore, was discussed as a promising leading the N- or the C-terminus increased the Ki mod- structure for the development of anti-para- erately (Chen et al., 1996). sitic therapeutic drugs. Schaschke et al. (1998) in a vary sophisti- It must be mentioned that the development cated approach used the Leu-Gly-Gly of substrate derived low Mr inhibitors re- (44p–46p of cathepsin B propeptide) motif to sulted in very promising substances. They develop selective trans-epoxysuccinyl deriva- represent a much greater group which, how- tive based cathepsin B inhibitors. This se- ever, will not be reviewed here. quence binds in the anti-substrate orientation to the S subsite thus mimicking the binding mode of the propeptide, whereas the PROCESSING Leu-Pro-OH moiety occupies the S¢ subsite. The inhibitor MeO- GGL-trans-epoxysuc- Here, we summarize the current knowledge cinyl-LP-OH showed remarkable selectivity of about the mechanistic and general aspects of cathepsin B inhibition: the k2/Ki ratios (which the cysteine protease precursor activation is the apparent second-order rate constant cal- processes. The processing of protease precur- culated from kobs/[I] for [I]<
FOLDING ASSISTANCE Such small loops are considered to be benefi- cial for folding (Cunningham et al., 1999). It has been frequently stated that cysteine These results showed that not all cathepsins proteases need their propeptide parts for need their propetide parts for correct folding. proper folding, however, experimental verifi- The answer to the second question came cations are rare. The first strong hint for from experiments with exogenously added propeptide assisted folding of mature propeptides to refolding assays of denatured papain-like cysteine proteases was the very proteases similar to those reported earlier for low specific activity of in vitro refolded recom- other protease classes (Ohta et al., 1991; binant procathepsin L with N-terminal trunca- Baker et al., 1992; Ogino et al., 1999). Experi- tions. The loss of the specific activity was pro- ments like that with cysteine peptidases were portional to the extent of propeptide trunca- lacking until recently. The propeptides of tion (Smith & Gottesman, 1989). This result cysteine proteases fold correctly when ex- was later confirmed by other groups which pressed as recombinant proteins. They seem also showed that not any but the complete cog- to retain their secondary structure over a nate propeptide was necessary for the synthe- wide pH range from 6.5 to 3.0 (Jerala et al., sis of correctly folded procathepsin L (Tao et 1998). Several experiments have shown that al., 1994; Ogino et al., 1999). This function of addition of recombinant propeptides can effi- the propeptide cannot be replaced even under ciently catalyze the refolding of denatured ma- optimized folding conditions (Tobbell et al., ture cysteine proteinases (Pietschmann et al., 2002). Another set of hints came from muta- 2002; Yamamoto et al., 2002; Capetta et al., tions of the propeptide affecting its structure 2002). With respect to cathepsin S, this effect and, as an obvious consequence of the was strongly dependent on the three-dimen- destabilized structure, also its function to sup- sional structure of the propeptide. Mutants af- port folding (Yamamoto et al., 1999; Hou et fecting the aromatic Trp core of the al., 1999; Kreusch et al., 2000). The questions propeptide showed a much weaker foldase ef- arising from these results were whether fold- fect than the wild type propeptide (Piets- ing assistance by the propeptide is a general chmann et al., 2002). A mutation in the con- phenomenon valid for all members of the CA1 served GNFD motif (N70pI/F72pI) of the peptidase family, and whether the propeptide F. hepatica cathepsin L propeptide also dimin- has to be bound covalently to the mature en- ished the foldase function of the propeptide zyme in order to exert the foldase effect. (Capetta et al., 2002). Thus, a strong correla- The answer to the first question came from tion exists between the structural integrity of experiments with cathepsin B (Mehtani et al., the propeptide, its inhibitory potency and its 1998) and from a general observation: ability to catalyze correct folding of the ma- Cathepsin B was synthesized as active enzyme ture enzyme (see Fig. 2). from a truncated mRNA lacking exons 2 and The recently described nucleation-conden- 3. Exon 3 codes for a part of the cathepsin B sation mechanism of protein folding is a com- propeptide. A D 51 splice variant of cathepsin bination of the framework model and the hy- B is expressed by some malignant tumours, drophobic collapse model. It implies the possi- and the resulting active enzyme is correctly bility of shifting to either of the models folded. depending on the stability of secondary and Cathepsins X and B show shorter propep- tertiary structures (Dagett & Fersht, 2003). tides than all other mammalian cathepsins, This fits very well with the phenomenon seen but they show also small extra loops in the ma- in some peptidases where N-terminal folded ture parts which may facilitate the folding. structures can provide a scaffold for further 704 B. Wiederanders 2003 folding and thus facilitate the process in vivo ENZYME TARGETING (Frydman, 2001). In cathepsin L like cysteine proteases, a mini-domain in the propeptide Mannose-6-phosphate dependent sorting represents this nucleation centre and is sug- mechanism gested to be the structural correlate of its foldase function (Schilling et al., 2001). Mannose-6-phosphate signals for delivery of enzymes to the lysosomes or acidified vesicles can be found in the propeptide parts as well as in the mature catalytic domains. Here, we re- fer only to the sorting signals located in the propeptide parts. The transfer of phospho-Glc-NAc to man- nose residues of N-linked oligosaccharides of lysosomal proteins is catalyzed by the UDP-GlcNAc: lysosomal enzyme N-acetyl- glucosamine-1-phosphotransferase. The re- moval of terminal GlcNAc results in the gener- ation of mannose-6-phosphate which then at- taches the lysosomal proteins to the respec- tive receptors for further transport to the lysosomes. The selectivity of the mannose phosphorylation of lysosomal enzymes has its structural basis in at least two Lys residues on Figure 2. Effect of mutagenesis of the highly con- the enzymes’ outer surface which are about served prodomain residues on various structural 34 Å apart. Introduction of putative phospho- and functional properties of human cathepsin S transferase recognition sequences into a se- propeptide. cretory non-glycosylated protein, pepsinogen, Renaturation rate (left ordinate, l), compactness of resulted in its proper glycosylation (Baranski tertiary structure (abscissa) and inhibitory function et al., 1990). (right ordinate, o). Taken from Pietschmann et al. In cathepsin L, this motif is located in the (2002). propeptide. Mutation of only one of the two residues in procathepsin L prevented the molecule phosphorylation (Cuozzo et al., The name foldase was introduced by 1995; 1998). However, the recognition motifs Inouye’s group for specific folding assistance for the phosphotransferase are not located in (Zhu et al., 1989). It describes a real en- the propeptide part in all cathepsins, as has zyme-like catalysis as the name suggests. It is been shown for cathepsin B (Cuozzo et al., noteworthy that acceleration of protease fold- 1998; Lukong et al., 1999). In cathepsin S, ing can be achieved in cis and in trans, i.e. the the only potential glycosylation site is lo- propeptide acts either covalently bound to the cated in the propeptide 11 amino acids up- protein in statu nascendi or it is added as a re- stream from the N-terminus of the mature combinant protein to renaturation assays. enzyme (Wiederanders et al., 1992). In rat The first approach to study the pH-dependent hepatocytes, procathepsin B was glyco- denaturation/renaturation cycle of mature sylated only at one of the two potential cathepsin B was performed by NMR-spectros- glycosylation sites, namely that within the copy (Song et al., 2000). propeptide (Tanaka et al., 2000). These exam- Vol. 50 Propeptide functions of cysteine peptidases 705 ples seem to be rather the exceptions, as evolved early in evolution and is maintained most other lysosomal proteins are glyco- until today. sylated in both the pro- and mature regions. The apparent structural background of man- In summary, although the correct fold of the nose-6-phosphate independent sorting is obvi- propeptide is a prerequisite for proper glyco- ously a conserved 9 amino acid long peptide sylation in the ER, the recognition motifs for motif which was identified to be implicated in glycosylation and the glycosylation sites per the alternative trafficking process (Huete- se are not exclusively restricted to the pro- Perez et al., 1999). It is located close to the peptide part of cathepsins. N-terminal tail of the propeptides. The motif is highly conserved as examplified by enzymes from plant, parasite, crustacean and human Mannose-6-phosphate independent sorting systems (see Fig. 3). It has to be mentioned, mechanisms however, that some cathepsins lack this cryp- The existence of mannose-6-phosphate inde- tic and ancient sorting motif, e.g. cathepsins pendent trafficking of lysosomal proteins has B, F, and X, and also baculovirus cathepsin. long been suggested by various lines of evi- dence. uSite directed mutagenesis experiments in which the essential Asn residues of poten- tial glycosylation sites in lysosomal proteins have been replaced by Gly or Gln. The mass of such non glycosylated enzymes was se- creted, however, a small fraction remained intracellular (Nissler et al., 1998; Tanaka et al., 2000). uLysosomal membrane attachment of non- glycosylated procathepsin S has been dem- Figure 3. Homologous nonapeptide sequences in onstrated (Nissler et al., 1998). class CA1 peptidase precursors of various spe- uLymphocytes of patients with I-cell disease cies. show normal cellular levels of lysosomal en- This motif is suggested to mediate “primitive” manno- zymes despite severely reduced phospho- se-6-phosphate independent targeting to lysosomes and transferase activity and consequently a lack lysosome-like vesicles. Numbering starts from Met 1, of mannose phosphorylation (Glickman & data are taken from the MEROPS database. *Giardia Kornfeld, 1993). lamblia cysteine proteases 1, 2 and 3; **Drosophila u melanogaster cathepsin L; ***Homerus americanus Some parasite cathepsins are not glyco- cathepsin L sylated due to mutations of the essential Asn residues (Sajid & McKerrow, 2002); Dictyostelium discoideum does not express A receptor recognizing the motif has not detectable amounts of mannose-6-phos- been identified so far, although a 43 kD inte- phate receptors (Cardelli et al., 1986); and gral lysosomal membrane protein was de- murine cell lines defective in manno- scribed binding mouse procathepsin L in a pH se-6-phosphate receptor are also known dependent manner (McIntyre & Erickson, (Gabel et al., 1983). Nevertheless, the 1993). The binding of procathepsin L to this lysosomal cathepsins take their correct way putative “receptor” could be blocked by a syn- into these organisms/cells suggesting that a thetic peptide comprising the first 24 kind of “primitive targeting” independent of N-terminal amino acids of procathepsin L mannose-6-phosphate and its receptor has (counted without the signal sequence) con- 706 B. Wiederanders 2003 taining the respective nonapeptide motif Cardelli JA, Golumbeski GS, Dimond RL. (1986) (McIntyre et al., 1994). Lysosomal enzymes in Dictyostelium discoideum are transported to lysosomes at distinctly different rates. J Cell Biol.; 102: 1264–70. REFERENCES Carmona E, Dufour E, Plouffe C, Takebe S, Ma- Baker D, Silen JL, Agard DA. (1992) Protease son P, Mort JS, Menard R. (1996) Potency pro region required for folding is a potent in- and selectivity of the cathepsin L propeptide hibitor of the mature enzyme. Proteins.; 12: as an inhibitor of cysteine proteases. Bio- 339–44. chemistry.; 35: 8149–57. Baker KC, Taylor MA, Cummings NJ, Tunon Chen Y, Plouffe C, Menard R, Storer AC. (1996) MA, Worboys KA, Connerton IF. (1996) Delineating functionally important regions Autocatalytic processing of pro-papaya and residues in the cathepsin B propeptide proteinase IV is prevented by crowding of for inhibitory activity. FEBS Lett.; 393: the active-site cleft. Protein Eng.; 9: 525–9. 24–6. Baranski TJ, Faust PL, Kornfeld S. (1990) Gen- Chowdhury SF, Sivaraman J, Wang J, eration of a lysosomal enzyme targeting sig- Devanathan G, Lachance P, Qi H, Menard R, nal in the secretory protein pepsinogen. Lefebvre J, Konishi Y, Cygler M, Sulea T, Cell.; 63: 281–91. Purisima EO. (2002) Design of noncovalent inhibitors of human cathepsin L. From the Billington CJ, Mason P, Magny MC, Mort JS. 96-residue proregion to optimized (2000) The slow-binding inhibition of tripeptides. J Med Chem.; 45: 5321–9. cathepsin K by its propeptide. Biochem Biophys Res Commun.; 276: 924–9. Cigic B, Krizaj I, Kralj B, Turk V, Pain RH. (1998) Stoichiometry and heterogeneity of Bossard MJ, Tomaszek TA, Thompson SK, the pro-region chain in tetrameric human Amegadzie BY, Hanning CR, Jones C, cathepsin C. Biochim Biophys Acta.; 1382: Kurdyla JT, McNulty DE, Drake FH, Gowen 143–50. M, Levy MA. (1996) Proteolytic activity of human osteoclast cathepsin K. Expression, Cigic B, Dahl SW, Pain RH. (2000) The residual purification, activation, and substrate identi- pro-part of cathepsin C fulfills the criteria fication. J Biol Chem.; 271: 12517–24. required for an intramolecular chaperone in folding and stabilizing the human Bromme D, Bonneau PR, Lachance P, proenzyme. Biochemistry.; 39: 12382–90. Wiederanders B, Kirschke H, Peters C, Thomas DY, Storer AC, Vernet T. (1993) Claveau D, Riendeau D. (2001) Mutations of the Functional expression of human cathepsin S C-terminal end of cathepsin K affect in Saccharomyces cerevisiae. Purification and proenzyme secretion and intracellular matu- characterization of the recombinant enzyme. ration. Biochem Biophys Res Commun.; 281: J Biol Chem.; 268: 4832–8. 551–7. Brown J, Matutes E, Singleton A, Price C, Coulombe R, Grochulski P, Sivaraman J, Molgaard H, Buttle D, Enver T. (1998) Menard R, Mort JS, Cygler M. (1996) Struc- Lymphopain, a cytotoxic T and natural killer ture of human procathepsin L reveals the cell-associated cysteine proteinase. Leuke- molecular basis of inhibition by the mia.; 12: 1771–81. prosegment. EMBO J.; 15: 5492–503. Cappetta M, Roth I, Diaz A, Tort J, Roche L. Cunningham EL, Jaswal SS, Sohl JL, Agard DA. (2002) Role of the prosegment of Fasciola he- (1999) Kinetic stability as a mechanism for patica cathepsin L1 in folding of the cata- protease longevity. Proc Natl Acad Sci U S lytic domain. Biol Chem.; 383: 1215–21. A.; 96: 11008–14. Vol. 50 Propeptide functions of cysteine peptidases 707
Cuozzo JW, Tao K, Wu QL, Young W, Sahagian cathepsin K is processed intracellularly prior GG. (1995) Lysine-based structure in the to attachment and bone resorption. J Bone proregion of procathepsin L is the recogni- Miner Res.; 16: 478–86. tion site for mannose phosphorylation. J Drenth J, Jansoniu Jn, Koekoek R, Swen HM, Biol Chem.; 270: 15611–9. Wolthers BG. (1968) Structure of papain. Cuozzo JW, Tao K, Cygler M, Mort JS, Nature.; 218: 929–31. Sahagian GG. (1998) Lysine-based structure Fox T, Demiguel E, Mort JS, Storer AC. (1992) responsible for selective mannose Potent slow-binding inhibition of cathepsin-B phosphorylation of cathepsin D and by its propeptide. Biochemistry.; 31: cathepsin L defines a common structural 12571–6. motif for lysosomal enzyme targeting. J Biol Chem.; 273: 21067–76. Frydman J. (2001) Folding of newly translated proteins in vivo: the role of molecular Cygler M, Sivaraman J, Grochulski P, Coulombe chaperones. Annu Rev Biochem.; 70: 603–47. R, Storer AC, Mort JS. (1996) Structure of rat procathepsin B: model for inhibition of Furmonaviciene R, Sewell HF, Shakib F. (2000) cysteine protease activity by the proregion. Comparative molecular modelling identifies Structure.; 4: 405–16. a common putative IgE epitope on cysteine protease allergens of diverse sources. Clin Daggett V, Fersht AR. (2003) Is there a unifying Exp Allergy.; 30: 1307–13. mechanism for protein folding? Trends Biochem Sci.; 28: 18–25. Gabel CA, Goldberg DE, Kornfeld S. (1983) Identification and characterization of cells Dahl SW, Halkier T, Lauritzen C, Dolenc I, deficient in the mannose 6-phosphate recep- Pedersen J, Turk V, Turk B. (2001) Human tor: evidence for an alternate pathway for recombinant pro-dipeptidyl peptidase I. lysosomal enzyme targeting. Proc Natl Acad (cathepsin C) can be activated by cathepsins SciUSA.; 80: 775–9. L and S but not by autocatalytic processing. Biochemistry.; 40: 1671–8. Glickman JN, Kornfeld S. (1993) Mannose 6-phosphate-independent targeting of Dalet-Fumeron V, Boudjennah L, Pagano M. lysosomal enzymes in I-cell disease B (1996) Competition between plasminogen lymphoblasts. J Cell Biol.; 123: 99–108. and procathepsin B as a probe to demon- strate the in vitro activation of procathepsin Gotthardt D, Warnatz HJ, Henschel O, Bruckert B by the tissue plasminogen activator. Arch F, Schleicher M, Soldati T. (2002) Biochem Biophys.; 335: 351–7. High-resolution dissection of phagosome maturation reveals distinct membrane traf- Delaria K, Fiorentino L, Wallace L, Tamburini ficking phases. Mol Biol Cell.; 13: 3508–20. P, Brownell E, Muller D. (1994) Inhibition of cathepsin L-like cysteine proteases by Guay J, Falgueyret JP, Ducret A, Percival MD, cytotoxic T-lymphocyte antigen-2 beta. J Biol Mancini JA. (2000) Potency and selectivity Chem.; 269: 25172–7. of inhibition of cathepsin K, L and S by their respective propeptides. Eur J Biochem.; Denizot F, Brunet JF, Roustan P, Harper K, 267: 6311–8. Suzan M, Luciani MF, Mattei MG, Golstein P. (1989) Novel structures CTLA-2 alpha and Guex N, Peitsch MC. (1997) SWISS-MODEL and CTLA-2 beta expressed in mouse activated T the Swiss-PdbViewer: an environment for cells and mast cells and homologous to comparative protein modeling. Electrophore- cysteine proteinase proregions. Eur J sis.; 18: 2714–23. Immunol.; 19: 631–5. Guncar G, Podobnik M, Pungercar J, Strukelj B, Dodds RA, James IE, Rieman D, Ahern R, Turk V, Turk D. (1998) Crystal structure of Hwang SM, Connor JR, Thompson SD, porcine cathepsin H determined at 2.1 Å res- Veber DF, Drake FH, Holmes S, Lark MW, olution: location of the mini-chain C-terminal Gowen M. (2001) Human osteoclast carboxyl group defines cathepsin H 708 B. Wiederanders 2003
aminopeptidase function. Structure.; 6: Kominami E. (1999) Analysis of where and 51–61. which types of proteinases participate in lysosomal proteinase processing using Guo YL, Kurz U, Schultz JE, Lim CC, bafilomycin A1 and Helicobacter pylori Vac A Wiederanders B, Schilling K. (2000) The toxin. J Biochem (Tokyo).; 125: 770–9. alpha1/2 helical backbone of the prodomains defines the intrinsic inhibitory specificity in Jerala R, Zerovnik E, Kidric J, Turk V. (1998) the cathepsin L-like cysteine protease pH-Induced conformational transitions of subfamily. FEBS Lett.; 469: 203–7. the propeptide of human cathepsinL—A role for a molten globule state in zymogen Hara K, Kominami E, Katunuma N. (1988) Ef- activation. J Biol Chem.; 273: 11498–504. fect of proteinase inhibitors on intracellular processing of cathepsin B, H and L in rat Kamphuis IG, Kalk KH, Swarte MBA, Drenth J. macrophages. FEBS Lett.; 231: 229–31. (1984) Structure of papain refined at 1.65 Å resolution. J Mol Biol.; 179: 233–56. Horn M, Baudys M, Voburka Z, Kluh I, Vondrasek J, Mares M. (2002) Free-thiol Karrer KM, Pfeifer SL, DiTomas ME. (1993) Cys331 exposed during activation process is Two distinct gene subfamilies within the critical for native tetramer structure of family of cysteine protease genes. Proc Natl cathepsin C (dipeptidyl peptidase I). Protein AcadSciUSA.; 90: 3063–7. Sci.; 11: 933–43. Kawabata T, Nishimura Y, Higaki M, Kato K. Hou WS, Bromme D, Zhao Y, Mehler E, Dushey (1993) Purification and processing of rat C, Weinstein H, Miranda CS, Fraga C, Greig liver procathepsin B. J Biochem (Tokyo).; F, Carey J, Rimoin DL, Desnick RJ, Gelb 113: 389–94. BD. (1999) Characterization of novel Kihara M, Kakegawa H, Matano Y, Murata E, cathepsin K mutations in the pro and ma- Tsuge H, Kido H, Katunuma N. (2002) ture polypeptide regions causing Chondroitin sulfate proteoglycan is a potent pycnodysostosis. J Clin Invest.; 103: 731–8. enhancer in the processing of procathepsin Hou WS, Li W, Keyszer G, Weber E, Levy R, L. Biol Chem.; 383: 1925–9. Klein MJ, Gravallese EM, Goldring SR, Konttinen YT, Mandelin J, Li TF, Salo J, Lassus Bromme D. (2002) Comparison of cathepsins J, Liljestrom M, Hukkanen M, Takagi M, K and S expression within the rheumatoid Virtanen I, Santavirta S. (2002) Acidic and osteoarthritic synovium. Arthritis cysteine endoproteinase cathepsin K in the Rheum.; 46: 663–74. degeneration of the superficial articular Huete-Perez JA, Engel JC, Brinen LS, Mottram hyaline cartilage in osteoarthritis. Arthritis JC, McKerrow JH. (1999) Protease traffick- Rheum.; 46: 953–60. ing in two primitive eukaryotes is mediated Krasko A, Gamulin V, Seack J, Steffen R, by a prodomain protein motif. J Biol Chem.; Schroder HC, Muller WE. (1997) Cathepsin, 274: 16249–56. a major protease of the marine sponge Ishidoh K, Kominami E. (1995) Procathepsin L Geodia cydonium: purification of the enzyme degrades extracellular matrix proteins in the and molecular cloning of cDNA. Mol Mar presence of glycosaminoglycans in vitro. Biol Biotechnol.; 6: 296–307. Biochem Biophys Res Commun.; 217: 624–31. Kreusch S, Fehn M, Maubach G, Nissler K, Ishidoh K, Saido TC, Kawashima S, Hirose M, Rommerskirch W, Schilling K, Weber E, Watanabe S, Sato N, Kominami E. (1998) Wenz I, Wiederanders B. (2000) An Multiple processing of procathepsin L to evolutionarily conserved tripartite cathepsin L in vivo. Biochem Biophys Res tryptophan motif stabilizes the prodomains Commun.; 252: 202–7. of cathepsin L-like cysteine proteases. Eur J Biochem.; 267: 2965–72. Ishidoh K, Takeda-Ezaki M, Watanabe S, Sato N, Aihara M, Imagawa K, Kikuchi M, Vol. 50 Propeptide functions of cysteine peptidases 709
Kurata M, Yamamoto Y, Watabe S, Makino Y, Maubach G, Schilling K, Rommerskirch W, Ogawa K, Takahashi SY. (2001) Bombyx Wenz I, Schultz JE, Weber E, Wiederanders cysteine proteinase inhibitor. (BCPI) homolo- B. (1997) The inhibition of cathepsin S by its gous to propeptide regions of cysteine pro- propeptide — specificity and mechanism of teinases is a strong, selective inhibitor of action. Eur J Biochem.; 250: 745–50. cathepsin L-like cysteine proteinases. J McDonald JK, Emerick JM. (1995) Purification Biochem (Tokyo).; 130: 857–63. and characterization of procathepsin L, a Lalmanach G, Lecaille F, Chagas JR, Authie E, self-processing zymogen of guinea pig sper- Scharfstein J, Juliano MA, Gauthier F. matozoa that acts on a cathepsin D assay (1998) Inhibition of trypanosomal cysteine substrate. Arch Biochem Biophys.; 323: proteinases by their propeptides. J Biol 409–22. Chem.; 273: 25112–6. McGrath ME. (1999) The lysosomal cysteine pro- Lecaille F, Kaleta J, Bromme D. (2002) Human teases. Annu Rev Biophys Biomol Struct.; 28: and parasitic papain-like cysteine proteases: 181–204. their role in physiology and pathology and McGrath ME, Eakin AE, Engel JC, Mckerrow recent developments in inhibitor design. JH, Craik CS, Fletterick RJ. (1995) The crys- Chem Rev.; 102: 4459–88. tal-structure of Cruzain — a therapeutic tar- Linke M, Jordans S, Mach L, Herzog V, Brix K. get for Chagas-disease. J Mol Biol.; 247: (2002) Thyroid stimulating hormone 251–9. upregulates secretion of cathepsin B from McIntyre GF, Erickson AH. (1993) The thyroid epithelial cells. Biol Chem.; 383: lysosomal proenzyme receptor that binds 773–84. procathepsin L to microsomal membranes at Linnevers C, Smeekens SP, Bromme D. (1997a) pH 5 is a 43–kDa integral membrane pro- Human cathepsin W, a putative cysteine pro- tein. ProcNatlAcadSciUSA.; 90: tease predominantly expressed in CD8+ 10588–92. T-lymphocytes. FEBS Lett.; 405: 253–9. McIntyre GF, Godbold GD, Erickson AH. (1994) Linnevers CJ, McGrath ME, Armstrong R, The pH-dependent membrane association of Mistry FR, Barnes MG, Klaus JL, Palmer procathepsin L is mediated by a 9-residue se- JT, Katz BA, Bromme D. (1997b) Expression quence within the propeptide. J Biol Chem.; of human cathepsin K in Pichia pastoris and 269: 567–72. preliminary crystallographic studies of an in- McQueney MS, Amegadzie BY, D’Alessio K, hibitor complex. Protein Sci.; 6: 919–21. Hanning CR, McLaughlin MM, McNulty D, Lukong KE, Elsliger MA, Mort JS, Potier M, Carr SA, Ijames C, Kurdyla J, Jones CS. Pshezhetsky AV. (1999) Identification of (1997) Autocatalytic activation of human UDP-N-acetylglucosamine-phosphotransferas cathepsin K. J Biol Chem.; 272: 13955–60. e-binding sites on the lysosomal proteases, Mehtani S, Gong Q, Panella J, Subbiah S, cathepsins A, B, and D. Biochemistry.; 38: Peffley DM, Frankfater A. (1998) In vivo ex- 73–80. pression of an alternatively spliced human Mach L, Mort JS, Glossl J. (1994) Maturation of tumor message that encodes a truncated human procathepsin B. Proenzyme activa- form of cathepsin B. Subcellular distribution tion and proteolytic processing of the pre- of the truncated enzyme in COS cells. J Biol cursor to the mature proteinase, in vitro, are Chem.; 273: 13236–44. primarily unimolecular processes. J Biol Menard R, Carmona E, Takebe S, Dufour E, Chem.; 269: 13030–5. Plouffe C, Mason P, Mort JS. (1998) Mason RW, Massey SD. (1992) Surface activa- Autocatalytic processing of recombinant hu- tion of pro-cathepsin L. Biochem Biophys Res man procathepsin L. Contribution of both Commun.; 189: 1659–66. intermolecular and unimolecular events in 710 B. Wiederanders 2003
the processing of procathepsin L in vitro. J microsomal lumen: characterization of enzy- Biol Chem.; 273: 4478–84. matic activation and proteolytic processing in vitro. Arch Biochem Biophys.; 261: 64–71. Musil D, Zucic D, Turk D, Engh RA, Mayr I, Huber R, Popovic T, Turk V, Towatari T, Nishimura Y, Kawabata T, Furuno K, Kato K. Katunuma N, Bode W. (1991) The refined (1989) Evidence that aspartic proteinase is 2.15-Å X-ray crystal-structure of human liver involved in the proteolytic processing event cathepsin-B — the structural basis for its of procathepsin L in lysosomes. Arch specificity. EMBO J.; 10: 2321–30. Biochem Biophys.; 271: 400–6. Nagler DK, Sulea T, Menard R. (1999a) Nishimura Y, Kato K, Furuno K, Himeno M. Full-length cDNA of human cathepsin F pre- (1995) Inhibitory effect of leupeptin on the dicts the presence of a cystatin domain at intracellular maturation of lysosomal the N-terminus of the cysteine protease cathepsin L in primary cultures of rat zymogen. Biochem Biophys Res Commun.; hepatocytes. Biol Pharm Bull.; 18: 945–50. 257: 313–8. Nissler K, Kreusch S, Rommerskirch W, Strubel Nagler DK, Zhang R, Tam W, Sulea T, Purisima W, Weber E, Wiederanders B. (1998) EO, Menard R. (1999b) Human cathepsin X: Sorting of non-glycosylated human A cysteine protease with unique procathepsin S in mammalian cells. Biol carboxypeptidase activity. Biochemistry.; 38: Chem.; 379: 219–24. 12648–54. Nomura T, Fujisawa Y. (1997) Processing prop- Nettis E, Napoli G, Ferrannini A, Tursi A. erties of recombinant human procathepsin (2001) IgE-mediated allergy to bromelain. Al- L. Biochem Biophys Res Commun.; 230: lergy.; 56: 257–8. 143–6. Nishimura Y, Kato K. (1987a) Intracellular Oda K, Nishimura Y, Ikehara Y, Kato K. (1991) transport and processing of lysosomal Bafilomycin A1 inhibits the targeting of cathepsin B. Biochem Biophys Res Commun.; lysosomal acid hydrolases in cultured 148: 254–9. hepatocytes. Biochem Biophys Res Commun.; 178: 369–77. Nishimura Y, Kato K. (1987b) Intracellular transport and processing of lysosomal Ogino T, Kaji T, Kawabata M, Satoh K, Tomoo cathepsin H. Biochem Biophys Res Commun. K, Ishida T, Yamazaki H, Ishidoh K, 148: 329–34. Kominami E. (1999) Function of the propeptide region in recombinant expression Nishimura Y, Kato K. (1988) Identification of la- of active procathepsin L in Escherichia coli. J tent procathepsin H in microsomal lumen: Biochem (Tokyo).; 126: 78–83. characterization of proteolytic processing and enzyme activation. Arch Biochem Ohta Y, Hojo H, Aimoto S, Kobayashi T, Zhu X, Biophys.; 260: 712–8. Jordan F, Inouye M. (1991) Pro-peptide as an intramolecular chaperone: renaturation Nishimura Y, Amano J, Sato H, Tsuji H, Kato of denatured subtilisin E with a synthetic K. (1988a) Biosynthesis of lysosomal pro-peptide [corrected]. Mol Microbiol.; 5: cathepsins B and H in cultured rat 1507–10. hepatocytes. Arch Biochem Biophys.; 262: 159–70. Pietschmann S, Fehn M, Kaulmann G, Wenz I, Wiederanders B, Schilling K. (2002) Foldase Nishimura Y, Furuno K, Kato K. (1988b) function of the cathepsin S proregion is Biosynthesis and processing of lysosomal strictly based upon its domain structure. cathepsin L in primary cultures of rat Biol Chem.; 383: 1453–8. hepatocytes. Arch Biochem Biophys.; 263: 107–16. Polgar L, Halasz P. (1982) Current problems in mechanistic studies of serine and cysteine Nishimura Y, Kawabata T, Kato K. (1988c) Iden- proteinases. Biochem J.; 207: 1–10. tification of latent procathepsins B and L in Vol. 50 Propeptide functions of cysteine peptidases 711
Quraishi O, Nagler DK, Fox T, Sivaraman J, Schilling K, Pietschmann S, Fehn M, Wenz I, Cygler M, Mort JS, Storer AC. (1999) The Wiederanders B. (2001) Folding incompe- occluding loop in cathepsin B defines the pH tence of cathepsin L-like cysteine proteases dependence of inhibition by its propeptide. may be compensated by the highly con- Biochemistry.; 38: 5017–23. served, domain-building N-terminal exten- sion of the proregion. Biol Chem.; 382: Rieman DJ, McClung HA, Dodds RA, Hwang 859–65. SM, Holmes MW, James IE, Drake FH, Gowen M. (2001) Biosynthesis and process- Schlereth A, Standhardt D, Mock HP, Muntz K. ing of cathepsin K in cultured human osteo- (2001) Stored cysteine proteinases start clasts. Bone.; 28: 282–9. globulin mobilization in protein bodies of embryonic axes and cotyledons during vetch. Roche L, Tort J, Dalton JP. (1999) The (Vicia sativa L.) seed germination. Planta.; propeptide of Fasciola hepatica cathepsin L 212: 718–27. is a potent and selective inhibitor of the ma- ture enzyme. Mol Biochem Parasitol.; 98: Sivaraman J, Nagler DK, Zhang R, Menard R, 271–7. Cygler M. (2000) Crystal structure of human procathepsin X: a cysteine protease with the Rowan AD, Mason P, Mach L, Mort JS. (1992) proregion covalently linked to the active site Rat procathepsin B. Proteolytic processing cysteine. J Mol Biol.; 295: 939–51. to the mature form in vitro. J Biol Chem.; 267: 15993–9. Smith SM, Gottesman MM. (1989) Activity and deletion analysis of recombinant human Rozman J, Stojan J, Kuhelj R, Turk V, Turk B. cathepsin L expressed in Escherichia coli. J (1999) Autocatalytic processing of recombi- Biol Chem.; 264: 20487–95. nant human procathepsin B is a bimolecular process. FEBS Lett.; 459: 358–62. Song J, Xu P, Xiang H, Su Z, Storer AC, Ni F. (2000) The active-site residue Cys-29 is re- Sajid M, McKerrow JH. (2002) Cysteine proteas- sponsible for the neutral-pH inactivation and es of parasitic organisms. Mol Biochem the refolding barrier of human cathepsin B. Parasitol.; 120: 1–21. FEBS Lett.; 475: 157–62. Salminen A, Gottesman MM. (1990) Inhibitor Tanaka Y, Tanaka R, Kawabata T, Noguchi Y, studies indicate that active cathepsin L is Himeno M. (2000) Lysosomal cysteine prote- probably essential to its own processing in ase, cathepsin B, is targeted to lysosomes by cultured fibroblasts. Biochem J.; 272: 39–44. the mannose 6-phosphate-independent path- Santamaria I, Velasco G, Pendas AM, Fueyo A, way in rat hepatocytes: site-specific Lopez-Otin C. (1998) Cathepsin Z, a novel phosphorylation in oligosaccharides of the human cysteine proteinase with a short proregion. J Biochem (Tokyo).; 128: 39–48. propeptide domain and a unique chromo- Tao K, Stearns NA, Dong J, Wu QL, Sahagian somal location. J Biol Chem.; 273: GG. (1994) The proregion of cathepsin L is 16816–23. required for proper folding, stability, and ER Santilman V, Jadot M, Mainferme F. (2002) Im- exit. Arch Biochem Biophys.; 311: 19–27. portance of the propeptide in the Taylor MAJ, Baker KC, Briggs GS, Connerton biosynthetic maturation of rat cathepsin C. IF, Cummings NJ, Pratt KA, Revell DF, Eur J Cell Biol.; 81: 654–63. Freedman RB, Goodenough PW. (1995a) Re- Schaschke N, Assfalg-Machleidt I, Machleidt W, combinant pro-regions from papain and pa- Moroder L. (1998) Sub- paya proteinase IV are selective high-affinity strate/propeptide-derived endo-epoxysuccinyl inhibitors of the mature papaya enzymes. peptides as highly potent and selective Protein Eng.; 8: 59–62. cathepsin B inhibitors. FEBS Lett.; 421: Taylor MAJ, Briggs GS, Baker KC, Cummings 80–2. NJ, Pratt KA, Freedman RB, Goodenough PW. (1995b) Expression of the pro-regions of 712 B. Wiederanders 2003
papain and papaya proteinase-IV in Esche- Paramecium tetraurelia. Purification, cloning, richia coli and their inhibition of mature sequencing and specific inhibition by its ex- cysteine proteinases. Biochem Soc Trans.; pressed propeptide. Eur J Biochem.; 238: 23: S80–S80. 198–206. Tobbell DA, Middleton BJ, Raines S, Needham Wang B, Shi GP, Yao PM, Li Z, Chapman HA, MR, Taylor IW, Beveridge JY, Abbott WM. Bromme D. (1998) Human cathepsin F. Mo- (2002) Identification of in vitro folding condi- lecular cloning, functional expression, tissue tions for procathepsin S and cathepsin S us- localization, and enzymatic characterization. ing fractional factorial screens. Protein Expr J Biol Chem.; 273: 32000–8. Purif.; 24: 242–54. Wex T, Levy B, Smeekens SP, Ansorge S, Turk D, Podobnik M, Kuhelj R, Dolinar M, Turk Desnick RJ, Bromme D. (1998) Genomic V. (1996) Crystal structures of human structure, chromosomal localization, and ex- procathepsin B at 3.2 and 3.3 Ångstroms pression of human cathepsin W. Biochem resolution reveal an interaction motif be- Biophys Res Commun.; 248: 255–61. tween a papain-like cysteine protease and its Wex T, Levy B, Wex H, Bromme D. (1999) Hu- propeptide. FEBS Lett.; 384: 211–4. man cathepsins F and W: A new subgroup van der Stappen JW, Williams AC, Maciewicz of cathepsins. Biochem Biophys Res RA, Paraskeva C. (1996) Activation of Commun.; 259: 401–7. cathepsin B, secreted by a colorectal cancer Wex T, Buhling F, Wex H, Gunther D, cell line requires low pH and is mediated by Malfertheiner P, Weber E, Bromme D. cathepsin D. Int J Cancer.; 67: 547–4. (2001) Human cathepsin W, a cysteine prote- Vernet T, Tessier DC, Richardson C, Laliberte F, ase predominantly expressed in NK cells, is Khouri HE, Bell AW, Storer AC, Thomas DY. mainly localized in the endoplasmic reticu- (1990) Secretion of functional papain precur- lum. J Immunol.; 167: 2172–8. sor from insect cells. Requirement for Wiederanders B, Kirschke H. (1989) The pro- N-glycosylation of the pro-region. J Biol cessing of a cathepsin L precursor in vitro. Chem.; 265: 16661–6. Arch Biochem Biophys.; 272: 516–21. Vernet T, Khouri HE, Laflamme P, Tessier DC, Wiederanders B, Bromme D, Kirschke H, von Musil R, Gour-Salin BJ, Storer AC, Thomas Figura K, Schmidt B, Peters C. (1992) Phylo- DY. (1991) Processing of the papain precur- genetic conservation of cysteine proteinases. sor. Purification of the zymogen and charac- Cloning and expression of a cDNA coding terization of its mechanism of processing. J for human cathepsin S. J Biol Chem.; 267: Biol Chem.; 266: 21451–7. 13708–13. Vernet T, Berti PJ, de Montigny C, Musil R, Yamamoto Y, Watabe S, Kageyama T, Takahashi Tessier DC, Menard R, Magny MC, Storer SY. (1999a) A novel inhibitor protein for AC, Thomas DY. (1995) Processing of the Bombyx cysteine proteinase is homologous papain precursor. The ionization state of a to propeptide regions of cysteine protein- conserved amino acid motif within the Pro ases. FEBS Lett.; 448: 257–60. region participates in the regulation of intramolecular processing. J Biol Chem.; Yamamoto Y, Watabe S, Kageyama T, Takahashi 270: 10838–46. SY. (1999b) Proregion of Bombyx mori cysteine proteinase functions as an Visal S, Taylor MA, Michaud D. (1998) The intramolecular chaperone to promote proper proregion of papaya proteinase IV inhibits folding of the mature enzyme. Arch Insect Colorado potato beetle digestive cysteine Biochem Physiol.; 42: 167–78. proteinases. FEBS Lett.; 434: 401–5. Yamamoto Y, Watabe S, Kageyama T, Takahashi Volkel H, Kurz U, Linder J, Klumpp S, Gnau V, SY. (1999c) Purification and characterization Jung G, Schultz JE. (1996) Cathepsin L is of Bombyx cysteine proteinase specific inhib- an intracellular and extracellular protease in Vol. 50 Propeptide functions of cysteine peptidases 713
itors from the hemolymph of Bombyx mori. proregions of cysteine proteinases. Curr Pro- Arch Insect Biochem Physiol.; 42: 119–29. tein Pept Sci.; 3: 231–8. Yamamoto Y, Kurata M, Watabe S, Murakami Zhu XL, Ohta Y, Jordan F, Inouye M. (1989) R, Takahashi SY. (2002) Novel cysteine Pro-sequence of subtilisin can guide the proteinase inhibitors homologous to the refolding of denatured subtilisin in an intermolecular process. Nature.; 339: 483–84.