pH-induced Conformational Transitions of the Propeptide of Human Cathepsin L

Abstract

Synthesis of proteases as inactive zymogens is a very important mechanism for the regulation of their activity. For lysosomal proteases proteolytic cleavage of the propeptide is triggered by the acidic pH. By using fluorescence, circular dichroism, and NMR spectroscopy, we show that upon decreasing the pH from 6.5 to 3 the propeptide of cathepsin L loses most of the tertiary structure, but almost none of the secondary structure is lost. Another partially structured intermediate, prone to aggregation, was identified between pH 6.5 and 4. The conformation, populated below pH 4, where the activation of cathepsin L occurs, is not completely unfolded and has the properties of molten globule, including characteristic binding of the 1-anilinonaphthalene-8-sulfonic acid. This pH unfolding of the propeptide parallels a decrease of its affinity for cathepsin L and suggests the mechanism for the acidic zymogen activation. Addition of anionic polysaccharides that activate cathepsin L already at pH 5.5 unfolds the tertiary structure of the propeptide at this pH. Propeptide of human cathepsin L which is able to fold independently represents an evolutionary intermediate in the emergence of novel inhibitors originating from the enzyme proregions.All lysosomal and most other proteases are synthesized in the form of inactive precursors (1, 2). Propeptides are generally located N-terminal to the mature enzyme, and activation of the enzyme is accomplished by cis-or trans-cleavage of the propeptide. Propeptides vary from a few (e.g. trypsin) to more than 200 residues (e.g. cathepsin C). Longer propeptides are generally strong and specific inhibitors of their mature enzymes (3-7). In most cases propeptides are also indispensable for correct folding of the enzymes (8). In some enzymes folding of the mature form is extremely slow, and the propeptide assists in overcoming the kinetic barrier (9), which may also be overcome by physicochemical factors such as high ionic strength in subtilisin, for example (10). Proenzymes are quite often more stable than mature enzymes (11, 12) and can represent a pool of latent enzyme until the activation occurs in the proper conditions. Propeptides are also involved in targeting to specific organelles (13, 14); they can affect posttranslational modification such as glycosylation (15) and mediate interactions with other molecules (16,17). Propeptides can be cleaved either by other proteases or by intra-or intermolecular autocatalysis. pH change is one of the most common environmental parameters responsible for triggering the activation of proteases, occurring in cysteine, aspartic acid, and metalloproteases (1,18,19). Low pH is thought either to increase the susceptibility of the propeptide as a substrate due to the protonation of groups close to the cleavage site or to cause a conformational change in the propeptide or enzyme.Cathepsin L is one of the most active cysteine proteases and accounts for most of the lysosomal cysteine protease activity (20). It has been implicated in a range of processes incl...

Glycosaminoglycans Modulate Activation, Activity, and Stability of Tripeptidyl-peptidase I in Vitro and in Vivo

“…For cathepsin L, it was proposed that DS unfolds the tertiary structure of the propeptide at pH values below ϳ5.5, thereby increasing its susceptibility to proteolysis (65). However, no conformation changes of pro-cathepsin L during autoprocessing could be found by further studies (66), and similarly, we could not detect any major conformation changes during pro-TPP I autoactivation (25).…”

Relationship between Propeptide pH Unfolding and Inhibitory Ability during ProDer p 1 Activation Mechanism

“…On the other hand, at pH 4, it lost a large part of its tertiary structure but retained its secondary structure content. 25 In these conditions, the obtained propeptide intermediate was able to bind ANS. Our data showed that, as for the cathepsin L propeptide, the Der p 1 propeptide and its C-truncated form were folded as compact globular domains at neutral pH.…”

“…21 For the cathepsin L propeptide, lack of inhibition has been associated with partial unfolding of the propeptide and formation of a molten globule state under acidic conditions. 25 Moreover, under these conditions, it has been demonstrated that the cathepsin S propeptide is slowly degraded by mature cathepsin L. 21 To date, the interaction between mature Der p 1 and its propeptide has not been investigated due to the difficulty of obtaining correctly matured recombinant Der p 1 (rDer p 1). In order to characterize this interaction, we studied the activation steps involved in the zymogen maturation mechanism.…”

Characterization of Native and Recombinant Falcipain-2, a Principal Trophozoite Cysteine Protease and Essential Hemoglobinase ofPlasmodium falciparum

“…Surprisingly, the processing occurred in refolding buffer at an alkaline pH (8.0) at which mature falcipain-2 has minimal activity against peptide substrates or hemoglobin. For other papain family enzymes, acidic conditions appear to be required to unfold the prodomain and allow autoactivation (62). Processing of profalcipain-2 may have been expedited by the fact that our expression construct included a truncated prodomain.…”