The Ananas comosus stem extract (often improperly described as stem bromelain) is a complex extract containing various cysteine proteases (iso)forms of the papain family (CA clan, C1 family) and other partially characterized non-proteolytic compounds. One of the key challenges faced by researchers studying cysteine proteases, particularly those of plant origin, was the characterization of multiple enzyme (iso)forms, such as those found in A. comosus stem extract. These multiple proteases, despite having high homology in their primary sequences, show differences in substrate specificity and inhibitory properties. It is therefore interesting to identify the structural modifications that may be linked to such deviations. Cysteine proteases in particular can easily be irreversibly oxidized, e.g. by air, making their separation from active forms very challenging. The preparation of fully active enzymes from mixtures containing inactivated material has been enabled by the use of affinity chromatography. However, affinity chromatography is not convenient for both practical and economic reasons for the production of the large quantities of pure proteases required for biophysical, mechanistic and structural investigations. Three different cysteine proteases of the C1A family were usually identified in A. comosus stem extracts: basic stem bromelain (the major component), ananain and comosain. We have recently purified to homogeneity and characterized several catalytically competent species from A. comosus stem extracts by using an efficient strategy based on the covalent grafting of an activated polyethylene glycol chain followed by purification on classical chromatographic gel media. This allowed a further separation of the extract into two acidic bromelains, three basic bromelains, two ananains and comosain. Basic bromelains represent the most abundant cysteine proteases fraction of the crude A. comosus stem extract. Interestingly, basic bromelains are scarcely inhibited by chicken cystatin and slowly inactivated by E64, unlike most cysteine proteinases of the papain family. A number of deletions and mutations have been proposed on the basis of sequence alignment, to explain such uncommon behavior when compared to the archetypal protease, papain. Comparatively, it has been shown that papain has a much better reactivity for iodoacetate than for iodoacetamide, in particular because imidazolium group of the active thiolate-imidazolium catalytic dyad interacts favorably with the negative charge of the carboxylate group of the alkylating agent. In the case of basic bromelains, the difference in reactivity towards these two compounds is very small compared to papain. These data clearly show that, on the one hand, basic bromelains have a low reactivity towards these alkylating agents and, on the other hand, low discrimination in favor of negatively charged alkylating agents. The fact that basic bromelains are only barely affected by cystatins was attributed to the modification of the structural organization of the catalytic site. However, this interpretation remains elusive in the absence of structural data. In contrast, ananain is distinguished from basic bromelains by both its catalytic specificity and its very high reactivity towards E64.
Therefore, a detailed comparative structural study of ananain, which behaves typically as the archetypal enzyme papain, and the basic bromelains may help understand the described dissimilarities at a molecular level.
Many studies have been conducted with A. comosus stem extracts, identifying a wide variety of biological systems affected. From these, the anticancer activities are perhaps the most attractive properties, deserving further studies. The complexity of A. comosus stem extracts does however not allow linking the observed effect to a particular constituent, consequently preventing a precise interpretation and understanding of the molecular mechanisms taking place. A recent study investigated the possible analgesic action of A. comosus stem extract by degrading the proenkephalin both, in vitro and in vivo, giving rise to multiple opioid peptides. The generated bio-active peptides were suggested to act in periphery where they can have an anti-inflammatory activity that has been recognized for many decades. This specificity of A. comosus stem extract to cleave proenkephalin mimics that of the nervous system prohormones convertases 1 and 2 which specifically and exclusively cleave proenkephalin after basic amino acids pairs. Assays of combinatorial peptide library and of solution-phase fluorogenic peptide microarrays indeed demonstrated the preference of A. comosus stem extract to cleave substrates after a pair of basic amino acids.
