A.J. BARRETT
Biochemistry Department
Strangeways Research Laboratory
Cambridge CB1 4RN, UK
Cathepsin L
Histolysin
Inhibitors of Cysteine Proteinases
References
I shall review the ability of two particular proteinases to degrade extracellular connective tissue matrix components. Such activity is an essential aspect of the invasion of the tissues of the mammalian organism either by tumours or by parasites. Other pathophysiological situations in which such degradation is important include the remodelling of tissue structures in development and wound healing. The enzymes I shall consider are two cysteine endopeptidases.
The terms "endopeptidase" and "proteinase" are completely synonymous; "proteinase" is more familiar to most people, but "endopeptidase" is more rational, and is the term that we should use in the future.1 Biochemists divide endopeptidases into four classes on the basis of the chemical groups responsible for their catalytic activity: these are the serine, cysteine, aspartic and metallo-endopeptidases.² It happens that representatives of all four groups are involved to a greater or lesser extent in the degradation of extracellular matrix components. Much recent work has emphasized the role of metallo-endopeptidases such as collagenase and serine proteinases such as leukocyte elastase. These enzymes have neutral pH optima, which obviously suit them well to extracellular activity, but the cysteine and aspartic endopeptidases have their part to play also.
One of the cysteine endopeptidases that I shall deal with is one from the Lysosomes of human cells, cathepsin L, and another is from the parasitic protozoan, Entamoeba histolytica, that we have called histolysin. I shall also mention the endogenous inhibitors that control the activities of these enzymes in the human body.
Human cathepsin L was first purified from liver, by Dr Rob Mason, in our laboratory.³ The amino acid sequence of the N-terminal part of the molecule shows a close evolutionary relationship with papain.4
One of the first natural substrates of cathepsin L to be discovered was Type I collagen.5 Much more recently we have discovered that cathepsin L also degrades elastin.6 Perhaps still more relevant to the issue of tissue invasion is the finding that cathepsin L is active on basement membrane collagen.7 There is good reason to think that the ability to penetrate basement membranes is an important property in invasion.
I have broached the subject of the activity of cathepsin L against extracellular proteins without considering the question of whether the enzyme appears outside cells. Although usually considered an intracellular enzyme, cathepsin has been shown to be secreted from cells under conditions in which the degradation of matrix components occurs. Cells particularly active in the secretion of this and other Lysosomal enzymes include macrophages and osteoclasts. These cells are also known to create pericellular regions of acidic pH that are well suited for the extracellular action of the enzymes. It is therefore our view that cathepsin L is one of the battery of enzymes that play a part in the remodelling of connective tissue elements in a variety of pathophysiological processes.
Because of our interest in the contribution of enzymes such as cathepsin L to the breakdown of connective tissue elements, we were interested to read about a cysteine proteinase of Entamoeba histolytica that was thought to act in the same way. Entamoeba histolytica is the organism that causes amoebic dysentry, but it does not always confine its activities to the intestinal lumen. Sometimes it causes ulceration of the intestinal wall, invades the circulation and metastasises to other organs such as the liver, where it is able to set up secondary foci or infection. The escape from the intestine involves the disruption of the tissue structure, or "histolysis" - hence the specific name of the organism. It has been believed for a long time that endopeptidases secreted by E. histolytica play an important part in this process, but there has been little agreement on the properties of these enzymes. We were therefore delighted when a Cuban scientist, Alfredo Luaces, was able to join us for a year to do some work on the cysteine proteinase of E. histolytica.
Luaces grew the trophozoites of the axenic HM 1 strain of E. histolytica in culture and found that both the organisms and the culture medium contained cysteine endopeptidase activity. The activity was greatest in the organisms, so these formed the source used for the purification of the enzyme. We were fortunate to discover a method for affinity chromatography of the enzyme that led to a virtual one-step purification.8 There was not much of the purified enzyme to spare, but we got a single automated sequencing run on the N terminus. A high proportion of the residues that were identified were identical with the corresponding residues in papain and cathepsin L, showing that all three enzymes are closely related in evolution.
We used the pure enzyme to study its catalytic activity. Like cathepsin L, histolysin has maximal activity on several protein substrates at acidic pH, but it differs in being stable at neutral and alkaline pH values, which would facilitate its extracellular activity. We tested it against matrix components at neutral pH. Contrary to some previous reports, we did not find that the enzyme was active on native Type I collagen; it is very active on denatured collagen, but that is probably not of much physiological significance. More importantly, the enzyme was active on the Type IV collagen of glomerular basement membrane, which could well help the organism escape from the intestine.
Equally important for invasion could be the ability to dissociate cells, so disrupting tissue architecture. Human skin fibroblasts in culture were transferred into serum-free medium and treated with 0.5 m a/ml of histolysin overnight. The cells rounded up, but they remained viable; this was not a cytotoxic effect, as has been claimed previously, but only cell detachment. Presumably this is due to degradation of some of the "sticky" proteins that cells produce, such as fibronectin.
I have spoken about two endopeptidases that have the potential to break down tissue structure by action on extracellular components. Before we can come to any sound understanding of whether such processes are significant in vivo, however, we need to understand the systems that exist to control them. For cysteine endopeptidases, we specifically need to know about the cysteine endopeptidase inhibitor systems.
The first recognized inhibitor of cysteine endopeptidases in the human body was µ -macroglobulin This remarkable protein inhibits endopeptidases of all four catalytic classes by physically trapping the enzyme molecule within its own molecule. It is a very large molecule with a molecular weight of about 725,000. It exists in the plasma and to some extent in extravascular fluids.9
The second class of inhibitors relevant to cathepsin L and histolysin comprises the cystatins. About 1980 we followed up earlier reports of the existence of a cysteine proteinase inhibitor in chicken egg white by isolating the inhibitor by affinity chromatography. We named it "cystatin", because of the way it stopped the activities of cysteine proteinase. We then developed the same type of purification procedure to deal with the more difficult task of isolating similar inhibitors from human liver. As a result of work in several laboratories, we now have a large family, technically a superfamily, of related cystatins. 10,11 Cystatins present in the human body are powerful inhibitors of both cathepsin L and histolysin. They would be likely to prevent such enzymes from causing any large-scale destruction of the tissues, but might be overwhelmed in the immediate vicinity of cells actively synthesizing and secreting the enzymes. One of the cystatins, human cystatin A, is located specifically in polymorphonuclear leucocytes and epithelial cells, suggesting that it may play a protective role.12
1. BARRETT, A.J. and J.K. McDONALD. 1985. Biochem. J. 231: 807.
2. BARRETT, A.J. 1986. In Proteinase Inhibitors. A.J. Barrett and G. Salvesen, Eds.: 3-22. Elsevier Science Publishers, Amsterdam.
3. MASON, R.W., G. D. J. GREEN and A.J. BARRETT. 1985. Biochem. J. 226: 233-241.
4. MASON, R.W., J.E. WALKER and F.D. NORTHROP. 1986. Biochem. J. 240: 373-377.
5. KERSCHKE, H., A.A. KEMBHAVI, P. BOHLEY and A.J. BARRETT. 1982. Biochem. J. 201: 367-372.
6. MASON, R.W., D.A. JOHNSON, A.J. BARRETT and H.A. CHAPMAN. 1986. Biochem. J. 233: 925-927.
7. BARICOS, W.H., Y. ZHOU, R.W. MASON and A.J. BARRETT. 1988. Biochem. J. 252: 301-304.
8. LUACES, A.L. and A.J. BARRETT. In press. Biochem. J.
9. BARRETT, A.J. 1981. Methods Enzymol. 80: 737-754.
10. BARRETT, A.J. 1981. Trends Biochem. Sci. 12: 193-196.
11. BARRETT, A.J., N. D. RAWLINGS, M.E. DAVIES, W. MACHLEIDT, G. SALVESEN and V. TURK. 1986. In Proteinase Inhibitors. A.J. Barrett and G. Salvesen, Eds.: 515-569 Elsevier Science Publishers, Amsterdam.
12. DAVIES, M.E. and A.J. BARRETT. 1984. Histochemistry 80: 373-377.
