Cathepsins thiol proteinases

Milk contains at least two proteinases, plasmin (alkaline milk proteinase) and cathepsin D (acid milk proteinase) and possibly several others, i.e. two thiol proteinases, thrombin and an aminopeptidase. In terms of activity and technological significance, plasmin is the most important of the indigenous proteinases and has been the subject of most attention. The relevant literature has been reviewed by Grufferty and Fox (1988) and Bastian and Brown (1996). 

Thiol proteinase inhibitor 90,000 Ficin, papain, cathepsin B and bromelain 45, 46... 

Thiol proteinase (papain) inhibitor 12,700 Papain, ficin, cathepsin B 22, 21... 

Quite recently, additional thiol proteinases have been found in lyso-somes. Cathepsin T, which catalyzed conversion of multiple forms of tyrosine aminotransferase, was demonstrated by Gk>hda and Pitot (20, 21). This enzyme does not cleave benzoylarginine-2-naphthylamide, a substrate for cathepsin B and cathepsin H. Melloni et al. (22, 23) reported the presence of a thiol peptidyl peptidase in a lysosomal membrane fraction of rabbit liver. In addition, cathepsin N with strong collagenolytic activity was found in bovine spleen and human placenta, although its lysosomal origin has not yet been demonstrated (24-26). 

Cathepsin B is the best known and most thoroughly investigated lysosomal thiol proteinase. It has been isolated from many mammalian species and tissues, including liver (33-35), spleen (5, 6,35), preputial gland (36), lung (15,16), parathyroid gland (37), brain (38), and breast (39). It is probably present in all mammalian cells. Crystals of cathepsin B from rat liver are shown in Fig. 1 (19). 

H, and L. Anti-cathepsin B and H sera quantitatively precipitated only the corresponding enzyme protein. Anti-cathepsin B serum reacted with cathepsin B, but not with cathepsin H or cathepsin L, and similarly anti-cathepsin H reacted only with cathepsin H, indicating that the three thiol proteinases are distinct. Immunological diffusions with antisera indicated that rat liver cathepsin B and H are immunologi-cally identical with cathepsin B and H from rat kidney, lung, spleen, brain, and heart. The immunological identity of cathepsin D from various tissues of rats was also demonstrated (60). 

Melloni et al. (22, 23) obtained three distinct enzymes from rabbit liver lysosomes that catalyze limited proteolysis of rabbit liver fructose-1,6-bisphosphatase, converting the neutral form to a form with an alkaline pH optimum. One of these proteinases (Mr = 70,000) is associated with the lysosomal membrane fraction. Since cathepsin B, H, and L are all present in the soluble fraction of lysosomes 61), this enzyme seems to be a new thiol proteinase of lysosomes. The enzyme is activated by cysteine, but not inhibited by leupeptin. Since it has only... 

Several lines of evidence indicate that neoplastic cells per se are the main source of extracellular thiol proteinase activity. Recent studies (39) have shown that malignant human breast tumors maintained in organ culture secrete high levels of a cathepsin B-like enzyme into the culture medium. Moreover high levels of cathepsin B-like enzyme are present in the serum of patients with a wide variety of cancers, and these levels decrease when the cancer tissue is removed or treated with therapeutic agents (64, 65). Cathepsin B-like enzyme from cultured cells of malignant tumors (39,66) possesses enzymic properties similar to those of cathepsin B with respect to specificity, affinity, and pH optima for synthetic substrates. It hydrolyzes Bz-Arg-Arg-2-naphthylamide and is inhibited by leupeptin. However, the tumor enzyme is much more stable than cathepsin B to inactivation above pH 7. It has a molecular weight of about 33,000-35,000. The distribution of cathepsin B-like activity was determined in fractions of control and neoplastic epithelial cells from human ectocervix (66). The activity is present mainly in the mitochondrial and lysosomal fractions of normal cells but mainly in the plasma membranes and nuclei of neoplastic cells. 

All the lysosomal thiol proteinases described in Section II have been isolated only recently and few structural studies have been carried out. The amino acid composition of only cathepsin B has been reported and is quite similar to those of cathepsin B from human liver (75) and rat liver (19) however Ouchterlony double difPiision analysis with antiserum against rat liver cathepsin B showed no immunological cross reactivity (40). All the lysosomal enzymes examined were shown to contain carbohydrate. The 111 of Asn is a glycosylation site in rat liver cathepsin B (128) but no detailed analysis of this carbohydrate has been reported. Since human liver cathepsin H can be purified on Con A-Sepharose (12), it may also contain a carbohydrate moiety. Rat liver cathepsin H and L also bind to Con A-Sepharose. 

Rat liver cathepsin B is the only mammalian thiol proteinase that has been sequenced (48, 78,128). Crystalline cathepsin B can be sepa-... 

Inhibition of cathepsin H by the inhibitor is rapid, reaching maximal levels within 15 seconds after addition of the inhibitor at 0°C. Kinetical studies indicate that the purified inhibitor inhibited papain noncom-petitively and pseudoirreversibly. The inhibitor inhibits cathepsin H by forming an enzyme- inhibitor complex in a molar ratio of 1 1. When cathepsin H is preincubated with E-64, formation of the enzyme-inhibitor complex is retarded. Conversely, when H-labeled E-64 is added to the reaction mixture after the enzyme-inhibitor complex is formed, incorporation of H-labeled E-64 into cathepsin H is slight. Those results suggest that the thiol proteinase inhibitor binds to the active site region of thiol proteinases. 

Proteinase inhibitors that inhibit cathepsin D and aminopeptidases slightly inhibit protein degradation in hepatocytes 63, 114) and cultured muscle 117). These findings also support the idea that lysosomal thiol proteinases play a major role in intracellular protein breakdown. In general, protein degradation is reduced by amounts of proteinase inhibitors that cause neither decrease of protein synthesis nor toxic effects 113). However, at higher concentrations or higher doses, they inhibit protein synthesis and have toxic effects 118). 

When it became apparent that the lysosomal thiol-proteinases were particularly important in the degradation of HPMA copolymer side-chains, a new series of side-chains was designed to meet known specificities of particular lysosomal enzymes.29 Two side-chains were synthesized (-Gly-Phe-Leu-Gly-Phe-NAp and -Gly-Gly-Phe-Leu-Gly-Phe-NAp) which contain a pentapeptide sequence previously shown O to be susceptible to the lysosomal proteinase cathepsin D, a thiol-independent enzyme. Five further sequences were prepared containing hydrophobic amino acids in the P2 and P positions in relation to the terminal bond (according to the terminology of... 

Schechter and Berger ). These side-chains were designed for cleavage by the lysosomal thiol-proteinase cathepsin L, a rat liver lysosomal enzyme 2 known to hydrolyse oxidized B-chain of insulin, such that hydrophobic residues are present in the 2 3... 

In cysteine proteinases (CP), activity depends upon a cysteine thiol group. Papain, actinidin, and a few lysosomal cathepsins are the best known members of this family. They hydrolyze peptides and esters in a generally similar fashion to serine proteinases. Two residues are directly involved in the catalytic process, Cys and His, with apparent pKa values of 4.2 and 8.6, respectively. This is the reverse of their normal pKa order, with His being more acidic than Cys. A bell-shaped relation of activity vs. pH for papain is spread out, with maximal activity around pH=6.5 and about half of the activity is retained near 4.5 and 8.5, dropping fast below and above these values. Thus, the active species has a zwitterion state, with a cysteine anion (thiolate) and a histidine cation. CP were discussed in a few reviews (Baker and Drenth, 1987 Fersht, 1985 Polgar and Halasz, 1982)... 

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