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The milk acid proteinase cathepsin D: a review
International Dairy Journal 10 (2000) 673}681
The milk acid proteinase cathepsin D: a review M.J. Hurley , L.B. Larsen, A.L. Kelly *, P.L.H. McSweeney Department of Food Science and Technology, University College, Cork, Ireland Department of Animal Product Quality, Danish Institute of Agricultural Sciences, P.O. Box 50, DK-8830 Tjele, Denmark Received 23 February 2000; accepted 23 September 2000
Abstract Cathepsin D is a lysosomal aspartic proteinase with a low pH optimum, found in many tissues and in bovine milk. It is synthesised as its inactive zymogen, procathepsin D. Procathepsin D can convert itself, by an autoproteolytic pathway, into an active intermediate, pseudocathepsin D, while other proteases are involved in the processing to mature cathepsin D. Cathepsin D is known to participate in a range of physiological processes. Pro-, pseudo- and mature cathepsin D have been puri"ed from bovine milk, but procathepsin D is the major form present in milk. Cathepsin D activity in milk appears correlated with somatic cell count. The active forms of the enzyme readily hydrolyse a -, a - and b-caseins, in the case of a -casein and b-casein with a speci"city similar to that of 1 1 1 chymosin. Cathepsin D can also produce para-i-casein from i-casein and, at high concentrations, can coagulate milk. Cathepsin D appears to be able to at least partially survive commercial pasteurisation processes. In recent years, increasing evidence has been documented indicating a role for this enzyme in proteolysis in cheese during ripening, most clearly in cheese where rennet activity is low, such as Swiss cheese, Quarg and Feta. Further research is required to evaluate the signi"cance of this enzyme in more detail. 2001 Elsevier Science Ltd. All rights reserved. Keywords: Cathepsin D; Milk; Proteolysis
1. Introduction The presence of proteolytic activity in tissue extracts at mildly acidic pH has been recognised since the early part of this century. Initially, the proteinase was referred to as `kathepsina (meaning, `to digesta in Greek), which was later modi"ed to `catepsinea, and "nally to the modern version of `cathepsina (Barrett, 1977). Following the isolation of an enzyme which accounted for over two-thirds of the total proteolytic activity in bovine spleen, Press, Porter, and Cebra (1960) designated the proteinase `cathepsin Da. Cathepsin D (EC 3.4.23.5) is an aspartic proteinase located in the lysosomes of all mammalian cells, and is the predominant proteolytic enzyme within such organelles (Barrett, 1977), which in addition contain a range of other cathepsins, such as B, H, L and S, which are cysteine proteases. An increasing range of proteolytic enzymes have been identi"ed in bovine milk. The pres-
* Corresponding author. Tel.: #353-21-903405; fax: #353-21270213. E-mail address: [email protected] (A.L. Kelly).
ence of an indigenous `acid proteasea in bovine milk was "rst reported nearly 30 years ago by Kaminogawa and Yamauchi (1972). They suggested that the proteinase was cathepsin D and this has been con"rmed by recent investigations.
2. Aspartic proteinases Aspartic proteinases, formerly called acid proteases, comprise a large group of proteolytic enzymes. Members of the family include pepsin, chymosin and HIV protease. The group exhibits a high degree of similarity in amino acid sequence and three-dimensional structure (Tang & Wong, 1987; Davies, 1990). The aspartic proteinases are bi-lobed molecules each lobe contributing an active site Asp residue to the substrate-binding cleft formed between the two. A group inhibitor is the pentapeptide, pepstatin, which binds reversibly to the active site cleft. The enzymes are mostly synthesised as proenzymes with a N-terminal propeptide which is removed to form the mature enzyme.
0958-6946/01/$ - see front matter 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 9 5 8 - 6 9 4 6 ( 0 0 ) 0 0 1 0 0 - X
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3. Structure and synthesis Cathepsin D, like all lysosomal enzymes, is synthesised at the rough endoplasmic reticulum with a 20-residue pre-sequence (signal peptide) (Erickson & Blobel, 1979). Following the initial cleavage of the signal peptide, the molecular mass of porcine procathepsin D increases from 43 to 46 kDa due to glycosylation (Erickson, Connor, & Blobel, 1981). The amino terminal propart of procathepsin D consists of 44 residues (Erickson et al., 1981). Procathepsin D is proteolytically inactive (Hasilik, von Figura, Conzelman, Nehrkorn, & Sadho!, 1982; Briozzo, Morisset, Capony, Rougeot, & Rochefort, 1988). Inside the cell, procathepsin D is very short lived, as it is processed rapidly into a catalytically active single-chain form of cathepsin D (M "44 kDa) by removal of the propeptide, presumably in prelysosomal compartments (Erickson et al., 1981; Gieselmann, Pohlmann, Hasilik, & von Figura, 1983; Rijnboutt, Stoorvogel, Geuze, & Strous, 1992). Studies have indicated this main proteolytic activation event to be mediated by cysteine protease(s) (Samarel, Ferguson, Decker, & Lesch, 1989). Depending on species, this single-chain form of cathepsin D may undergo further cleavage after arrival at the lysosome, most likely also by cysteine proteinase(s) (Gieselmann, Hasilik, & von Figura, 1985), involving the removal of two residues from bovine cathepsin D, while "ve residues are released in the processing of porcine cathepsin D (Yonezawa et al., 1988). Two chains are produced from the single-chain form, a light chain derived from the N-terminus and a heavy chain from the C-terminus, with M values of 31,000 and 13,000}14,000 Da, respectively (Hasilik & Neufeld, 1980a; Gieselmann et al., 1983). For unknown reasons, this proteolytic processing of bovine cathepsin D is not complete, as mature bovine cathepsin D exists in the lysosomes as an approximately equal mixture of singleand two-chain forms, while porcine cathepsin D consists primarily of the two-chain form (Takahashi & Tang, 1981). The activation of puri"ed procathepsin D has been studied and the formation of a catalytically active intermediate at acidic pH values was observed (Hasilik, von Figura, Conzelman, Nehrkorn, & Sadho!, 1982; Samarel, Worobec, Ferguson, Decker, & Lesch, 1986; Briozzo et al., 1988). This form was produced by a pHdependent reduction in molecular mass of 1}2 kDa, with the conversion being inhibited by pepstatin (Hasilik et al., 1982; Samarel et al., 1986). Procathepsin D can be converted into an active form by means of an autocatalytic cleavage between residues LeuP26 and IleP27 of the propeptide, yielding a protein with an N-terminal extension of 18 residues compared to mature cathepsin D (Conner & Richo, 1992; Larsen, Boisen, & Petersen, 1993). The active intermediate was termed pseudocathepsin D. However, unlike pepsinogen and
most other zymogens of aspartic proteinases, procathepsin D appears unable to autoactivate to mature cathepsin D and the formation of cathepsin D with mature N-terminus therefore seems to require the involvement of other enzymes. It also remains to be determined whether procathepsin D, the autoactivation product, is formed in vivo as part of the activation process. Cathepsin D is a glycoprotein with mannose-containing oligosaccharides attached at positions Asn and Asn (Hasilik & Neufeld, 1980b; Chitpinityol & Crabbe, 1998). Within the cis-Golgi compartment, the mannosecontaining oligosaccharides are phosphorylated and these mannose-6-phosphate residues are required for sorting to the lysosomes via mannose-6-phosphate receptors (MPR) (von Figura & Hasilik, 1986). Following binding to MPR in the trans-Golgi network the cathepsin D precursor is targeted to a prelysosomal compartment, where it is released from the MPR due to the acidic environment (von Figura & Hasilik, 1986). Mannose-6phosphate-independent sorting may, however, contribute to the targeting of procathepsin D in some cell types through transient mannose-6-phosphate-independent association of procathepsin D with intracellular membranes (Rijnboutt, Kal, Geuze, Aerts, & Strous, 1991). Press et al. (1960) were the "rst to isolate bovine cathepsin D (from spleen) and identi"ed the N-terminal amino acid as glycine. Following this initial work, the crystal structures of native and pepstatin-inhibited cathepsin D from human liver (Baldwin et al., 1993), native human spleen cathepsin D (Fusek, Baudys\ , & Metcalf, 1992) and bovine liver cathepsin D complexed with pepstatin (Metcalf & Fusek, 1993) have been determined. Like all aspartic proteinases, cathepsin D is a bilobed molecule and its secondary structure consists mainly of b-sheets. The two structurally similar domains both contain a single carbohydrate group along with two disulphide bonds (Metcalf & Fusek, 1993; Chitpinityol & Crabbe, 1998). Cathepsin D is expressed in all tissues studied, but the level of expression varies considerably (Reid, Valler, & Kay, 1986). The human cathepsin D gene has been found to contain a mixed promoter and can be expressed from two start points, constitutive and oestrogen-regulated, which indicate a range of physiological functions of the enzyme (Cavailles, Augereau, & Rochefort, 1993; May, Smith, & Westley, 1993).
4. Physiological function The location and the acidic pH optimum of cathepsin D suggests that it is adapted for the role of intracellular digestion of proteins in the acidi"ed environment provided by the lysosomal system (Barrett, 1977). The enzyme preferentially cleaves between two hydrophobic residues, but there is also a strong preference for
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hydrophobic amino acids in the P position of the sub strate. More distant residues, up to the P position, also in#uence the e$ciency of hydrolysis (Scarborough et al., 1993). One role of the enzyme is degradation of haemoglobin in the lysosomes of the spleen, permitting the reutilisation of the heme group (Barrett, 1977). Cathepsin D has also been suggested to be involved in the degradation of internalised "brinogen (Simon, Ezratty, & Loscalzo, 1994) and in the release of acid phosphatase from its membrane-bound form into the lysosomal matrix (Tanaka, Himeno, & Kato, 1990). Cathepsin D is a major lysosomal proteinase of white blood cells (somatic cells) and is present in phagocytic cells such as macrophages and polymorphonuclear leucocytes, particularly the former (Cohn, 1975). Cathepsin D has also been implicated in the processing of prohormones (Krieger & Hook, 1992), procollagen (Helseth & Veis, 1984), in processing of antigens in immune cells (Rhodes & Andersen, 1992) and in the regulation of the activity of insulin-like growth factor-binding protein-3 under acidic conditions (Conover & De Leon, 1994). Studies of mice lacking cathepsin D have suggested that a vital function of cathepsin D is in limited proteolysis of regulating proteins. The cathepsin-D-de"cient mice were short-lived and exhibited atrophy of the intestinal mucosa and destruction of lymphoid cells, while the contribution of cathepsin D to proteolysis in lysosomes appeared to be less vital (Saftig et al., 1995). In recent years, the association of cathepsin D with development of breast cancer has received increased attention. Cathepsin D is overexpressed (2}50)-fold in most breast cancer tumor cytosols, when compared with its concentration in normal mammary glands (Rochefort & Capony, 1993). Procathepsin D is known to be overexpressed and secreted in oestrogen receptor-positive breast cancer cells and procathepsin D has been suggested to facilitate metastasis of tumour cells (Rochefort, Capony, & Garcia, 1990; Sloane & Berquin, 1993). Importantly, procathepsin D has been shown to have mitogenic e!ect on breast cancer cell lines (Vignon et al., 1986; Vetvicka, Vetvickova, & Fusek, 1994). The major mechanism involved in the stimulation of metastasis has been suggested to be increased cell proliferation (Rochefort & Liaudet-Coopman, 1999). The mitogenic e!ect was speci"c for the pro-form and a synthetic peptide corresponding to the procathepsin D propeptide had the same e!ect (Fusek & Vetvicka, 1994), through interaction with a previously unknown receptor of breast cancer cells (Vetvicka, Vetvickova, Hilgert, Voburka, & Fusek, 1997). The use of cathepsin D and procathepsin D levels as a prognostic tool in breast cancer is, however, controversial (Rochefort et al., 1990; Brouillet et al., 1993; Losch et al., 1998). Cathepsin D has also been proposed to play a pivotal role as a growth modulator in androgen-dependent prostate cancer (Mordente, Choudhury, Tazaki, Mallouh, & Konna, 1998). Furthermore, cathep-
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sin D may also play a role in certain neurological disorders, such as Alzheimer's disease (Masters et al., 1985; Kang et al., 1987) and scrapie, a transmissible neurodegenerative disease caused by prions and common to sheep (Diedrich et al., 1991). The participation of degradation of meat proteins and post-mortem changes is well characterised (Dutson, 1983) and it has been implicated in the proteolysis of collagen (Scott & Pearson, 1978) and of myo"brillar proteins (Matsumoto, Okitani, Kitamura, & Kato, 1983; Zeece, Katoh, Robson, & Parrish, 1986).
5. Cathepsin D in milk The occurrence of an acid protease in bovine milk was "rst demonstrated by Kaminogawa & Yamauchi (1972). This protease was considered to be similar to the lysosomal enzyme cathepsin D. Larsen et al. (1993) con"rmed that this aspartic proteinase was actually procathepsin D. Incubation of procathepsin D puri"ed from bovine milk at pH 3.5 and 5.0 indicated autoactivation to the proteolytically active form, pseudocathepsin D, but apparently not into mature cathepsin D (Larsen et al., 1993). Larsen and Petersen (1995) subsequently puri"ed "ve molecular forms of cathepsin D from bovine milk by ammonium sulphate precipitation of acid whey, followed by separation on DEAE-Sepharose. The "nal puri"cation step was a$nity chromatography on pepstatin}Sepharose, which permitted separation of the di!erent forms by exploiting pH-dependent di!erences in binding to pepstatin. The "ve forms isolated from milk had apparent molecular masses of 46, 45, 43, 39 and 31 kDa (Fig. 1). Both the 46 and 45 kDa bands corresponded to procathepsin D, but the reason for the variability in molecular mass is not known. The 43 kDa form corresponded to pseudocathepsin D, while the 39 and 31 kDa forms were identi"ed as mature single- and heavy-chain cathepsin D, respectively. The cathepsin D light chain, which was most likely also present, was not detected in the experiment, probably due to lack of sensitivity. Procathepsin D (45 kDa) appeared to be the major form present in milk (Larsen & Petersen, 1995). The detection of the mature forms of cathepsin D (39 and 31 kDa) in the puri"ed preparation implies either that milk contains mature cathepsin D or alternatively that mature cathepsin D was formed during the puri"cation procedure by other enzymes capable of activating procathepsin D or pseudocathepsin D. The observation that mature cathepsin D was present in the eluate from DEAE-Sepharose (Larsen & Petersen, 1995) may indicate, however, that the mature forms are present in milk, although in small amounts. Cathepsin D concentrations in skimmed milk and acid whey were estimated by a competitive enzyme-linked
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Fig. 1. Molecular forms of cathepsin D identi"ed in bovine milk. Prefix P denotes residues in the propart (adapted from Larsen & Petersen, 1995). The procathepsin D forms are inactive, while other forms are proteolytically active.
immunosorbent assay to be 0.4 and 0.3 lg mL\, respectively, indicating that cathepsin D is mainly present in the whey (Larsen, Benfeldt, Rasmussen, & Petersen, 1996). The origin of procathepsin D and cathepsin D in milk is not known. Procathepsin D can be secreted both by mammary epithelial cells (Capony et al., 1989) and by milk-secreting cells, as activated lymphocytes can secrete lysosomal enzymes (Hasilik, 1992). Cathepsin D activity in milk is correlated with somatic cell count (O'Driscoll, Rattray, McSweeney, & Kelly, 1999), but whether this re#ects an increased amount of cathepsin D and/or procathepsin D and/or increased activation of the indigenous procathepsin D pool remains to be determined. The physiological function of procathepsin D and cathepsin D in milk is unknown. Procathepsin D has also been found in human breast milk (Vetvicka et al., 1993) and can activate neutrophils and lymphocytes through the regulation of surface receptors. Thus, a possible function has been suggested in the immune defence of the breast-fed newborn (Vetvicka, Vetvickova, & Fusek, 1995). Interestingly, Saftig et al. (1995) suggested that one reason that mice de"cient for cathepsin D could survive up to four weeks after birth may be that the neonates received cathepsin D with the maternal milk. 5.1. Factors awecting enzyme activity A commonly-used substrate for measuring cathepsin D activity is haemoglobin, usually after denaturation by acid or urea (Takahashi & Tang, 1981; Sarath, De La Motte, & Wagner, 1989). O'Driscoll et al. (1999) showed that a synthetic heptapeptide substrate (Pro}Thr}Glu}
Phe}[NO }Phe]}Arg}Leu) was a good substrate for measuring cathepsin D activity in milk. Cathepsin D displays greatest activity against haemoglobin in the pH range 3.0}3.5 (Press et al., 1960; Barrett, 1972). The optimum pH for partly isolated milk cathepsin D on haemoglobin was found to be pH 4.0 (Kaminogawa & Yamauchi, 1972) and the optimum temperature of cathepsin D activity was determined to be 373C (Barrett, 1972). Draper and Zeece (1989) showed that the heat stability of bovine cathepsin D increased with decreasing pH, with a corresponding increase in enzyme activity. Complete inactivation of cathepsin D in milk on heating at 703C for 10 min has been observed (Kaminogawa & Yamauchi, 1972). Recent studies have indicated that cathepsin D at least partially survives pasteurisation treatments (72.53C for 15 s) (Larsen et al., 2000; Hayes et al., in press). 5.2. Hydrolysis of milk proteins by cathepsin D Kaminogawa, Yamauchi, Miyazawa and Koga (1980) investigated the degradation of individual caseins by partially puri"ed cathepsin D. In recent years, the degradation of caseins in vitro by cathepsin D has received closer examination and the proteolysis products have been compared to those produced by chymosin (McSweeney, Fox, & Olson, 1995; Larsen et al., 1996). As procathepsin D is the major form of cathepsin D found in milk, Larsen et al. (1996) investigated the degradation of milk proteins both with cathepsin D from bovine spleen and with procathepsin D puri"ed from milk. The hydrolysates were performed at pH 5.0, at which pH the
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Fig. 2. Speci"city of cathepsin D against (a) a - and (b) b-casein. Arrows indicate identi"ed cleavage sites (McSweeney et al., 1995; Larsen et al., 1996). 1
added procathepsin D autoactivated to proteolytically active pseudocathepsin D. Analysis by SDS-PAGE showed that the degradation patterns of the two enzymes were similar, although the hydrolysis was slower with pseudocathepsin D than with mature cathepsin D. The primary cleavage sites of cathepsin D on a - and 1 b-casein are summarised in Fig. 2. Kaminogawa et al. (1980) and McSweeney et al. (1995) showed that partially puri"ed cathepsin D from milk degraded a -casein to a peptide with the same molecular 1 mass as a -CN f24-199 (a -I-casein), which is known to 1 1 be produced from a -casein by the action of chymosin 1 (Hill, Lahav, & Givol, 1974). The a -I-casein peptide is 1 further degraded by cathepsin D in vitro (Larsen et al., 1996). Proteolysis of b-casein by cathepsin D is also similar to the action of chymosin (Fig. 2). Cathepsin D hydrolysates of a -casein di!er markedly from those 1 produced by chymosin, with very few peptides in common (McSweeney et al., 1995). The "rst bond cleaved in the a -casein monomer by cathepsin D is Leu } 1 Tyr , followed by further degradation of the C-ter minal fragment (Larsen et al., 1996). The Leu }Tyr bond is the only bond cleaved in dimeric a -casein. 1 i-Casein is cleaved by partially puri"ed cathepsin D to produce para-i-casein (Kaminogawa et al., 1980; McSweeney et al., 1995; Larsen et al., 1996). Larsen et al. (1996) identi"ed two subsequent cleavage sites of cathepsin D in i-casein, Leu }Ser and Leu }Ser . Two cleavage sites of cathepsin D on a-lactalbumin have been identi"ed (Leu }Phe and Trp }Leu ), while native b-lactoglobulin is resistant to cleavage (Larsen et al., 1996).
5.3. Coagulation of milk As cathepsin D is an aspartic proteinase with a proteolytic speci"city similar to that of chymosin, one might expect that it possesses the ability to coagulate milk. McSweeney et al. (1995) and Larsen et al. (1996) both showed that cathepsin D generated the caseinomacropeptide (i-CN 106-169) when incubated with isolated i-casein. McSweeney et al. (1995) examined the in#uence of cathepsin D addition on the rennetability of skim milk at pH 6.4 and 303C. Aliquots were withdrawn periodically after enzyme addition and their rennet clotting time (RCT) determined. Incubation of milk with cathepsin D reduced RCT, presumably as a result of slow hydrolysis of the micelle-stabilising protein, i-casein, by cathepsin D. Larsen et al. (1996) investigated the ability of cathepsin D to coagulate acidi"ed skim milk (at pH 5.0) at 373C using a Formagraph. The pH dependence of milk coagulation by cathepsin D was also investigated by adding a "xed amount of enzyme to bovine milk, adjusted to pH values in the range 5.0}6.5. Coagulation was observed at pH 5.0 with concentrations of cathepsin D above 3.1 lg mL\, or approximately 10 times the indigenous amount in milk. Cathepsin D was capable of coagulating milk over all pH values examined with RCT decreasing with decreasing pH, presumably due to acidic pH optimum of the enzyme. Thus, although milk possesses an indigenous milkclotting enzyme, the level present (0.4 lg mL\) in pooled raw milk may be too low to signi"cantly in#uence milk
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coagulation in cheese production, but may contribute to degradation of i-casein during long-term storage of some milk products. 5.4. Contribution to proteolysis in cheese As cathepsin D has the ability to degrade all four caseins, it may be expected that the enzyme could play a role in cheese ripening. The speci"city of cathepsin D is quite similar to that of the milk coagulant, chymosin, which is added to milk at a far greater concentration compared with the level of cathepsin D already present; the level of coagulant retained in the curd being up to 15% (Ohmiya & Sato, 1972). This, along with the fact that cathepsin D, at least in unpasteurized milk, is predominantly a whey protein (Larsen et al., 1996), suggests that chymosin is the principal aspartic proteinase in cheese curd. Thus, in many cases, cathepsin D activity may be masked by that of chymosin (McSweeney et al., 1995). To investigate the contribution of various agents to proteolysis, rennet-free cheeses have been developed. Visser (1976) developed a method based on the thermal inactivation of rennet (after it had completed its primary cleavage of the Phe }Met bond of i-casein) by pasteurisation (723C for 20 s) of milk in which the calcium concentration had been decreased by means of ion exchange, thereby preventing coagulation. After pasteurisation, the milk was cooled (to 4}53C) and a rennet-free coagulum formed by the addition of a solution of calcium chloride and lactic acid and warming to 303C. Due to the complexity of this procedure, alternative methods have been sought. O'Kee!e, Fox, and Daly (1977) and Lane, Fox, Johnston, and McSweeney (1997) exploited the very low stability of porcine pepsin (EC 2.4.23.1) at neutral pH in the production of rennet-free cheese. The above methods are designed to eliminate rennet activity, thus allowing the examination of role of indigenous proteinase activity in casein degradation. Noomen (1978), employing the method of Visser (1976), observed production of a -I-casein in simulated Noordhollandse 1 Meshanger cheese. Lane et al. (1997) observed limited hydrolysis of a -casein in Cheddar, again with produc1 tion of a -I-casein. Presuming the successful inactiva1 tion of the incorporated coagulant (which may not have been the case in all studies), production as a -I-casein 1 may be attributed to cathepsin D activity, thereby indicating that cathepsin D may have a role to play in softening of cheese texture, which is related to production of this peptide (Creamer, Zoerb, Olson, & Richardson, 1982). To determine the contribution of cathepsin D to casein degradation, a more conclusive approach would be to manufacture cheese entirely without coagulant, thereby eliminating any speculation as to whether the rennet was totally inactivated. Wium, Kristiansen, and Qvist (1998)
produced Feta without addition of coagulant. The cheesemilk was homogenised, pasteurised at 723C for 15 s, ultra"ltered at 503C, pasteurised again and stored at 43C until the next day. The ultra"ltered milk was then subjected to a "nal pasteurisation and homogenised, followed by the addition of gluconic acid-d-lactone, causing a drop in pH which induced coagulation (Wium & Qvist, 1998). By using gluconic acid-d-lactone as acidulent, enzymes from lactic acid bacteria were eliminated, thereby simplifying the model system. a -Casein, 1 a -I-casein (produced from a -casein) and i-casein 1 1 were all degraded over time. This, along with the absence of rennet, strongly suggested the involvement of an indigenous milk proteinase capable of acting under acidic conditions. i-Casein was degraded with concomitant production of para-i-casein, which is in accordance with the speci"city of cathepsin D (McSweeney et al., 1995; Larsen et al., 1996). There was also a preference for the hydrolysis of a -casein, while b-casein remained undeg1 raded, indicating a lack of plasmin activity in the cheese, which was attributed to its low pH (pH"4.6), far from the alkaline pH optimum of plasmin (Bastian & Brown, 1996). This was, however, characteristic of cathepsin D activity, which preferentially hydrolyses a -casein 1 (Kaminogawa & Yamauchi, 1972). These observations strongly indicated the presence of cathepsin D-like activity in this cheese, which was recently con"rmed by the demonstration of cathepsin D activity in rennet-free Feta cheese extracts and furthermore the presence of procathepsin D was demonstrated immunologically (Larsen et al., 2000). The question of cathepsin D activity has also arisen in respect to cheese varieties such as Swiss cheese, in which chymosin is believed to be largely inactivated during cooking at 53}553C for up to 1 h (Matheson, 1981; Garnot & Molle, 1987). With full inactivation of the heatlabile coagulant, the absence of rennet-like activity would be expected, with little or no degradation of a -casein. 1 However, this does not appear to be the case. Igoshi and Arima (1993) isolated an active acid proteinase from Swiss-type cheese (Emmental and Gruye`re) which was deemed not to be chymosin, due to the observation that it did not decompose i-casein at a greater rate than a -casein (as chymosin would) but rather it was con1 sidered to be cathepsin D. Subsequently, production of a -I-casein has been again identi"ed in Swiss cheese 1 (Beuvier et al., 1997; Cooney, Tiernan, Joyce, & Kelly, 2000), and attributed to the action of cathepsin D. Furthermore, Cooney et al. (1999) found breakdown of a 1 casein in Swiss cheese to be proportional to milk somatic cell count, which again would be consistent with cathepsin D being the enzyme responsible for this degradation of a -casein. 1 Recently, Hurley et al. (2000) identi"ed, by both speci"c enzyme assay and immunological methods, cathepsin D activity in Quarg cheese made from raw,
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pasteurised and ultra"ltrated milk and demonstrated that the enzyme contributed to proteolysis during storage.
6. Conclusions and research requirements Cathepsin D appears to be quite stable in milk, capable of at least partially surviving three cycles of pasteurisation (723C for 15 s) (Wium & Qvist, 1998; Larsen et al., 2000). This would suggest that this enzyme may play a role in the ripening of high-cooked cheese varieties, such as Swiss cheese. Cathepsin D activity is obvious in cheese varieties where no rennet is added (such as rennet-free UF Feta). However, it is di$cult to quantify the contribution of cathepsin D to the ripening of cheese varieties such as Cheddar, where the activity may be masked by chymosin activity. To fully evaluate the potential signi"cance of this enzyme for dairy product quality, much study remains. In comparison to the extensive literature concerning the plasmin system in milk, the stability of its various components and the e!ects of a wide range of production and processing variables on the activity of the enzyme, cathepsin D is very poorly understood. As a primary goal, attention must be focussed on the activity and stability of this enzyme in milk and sources of variation in this activity (such as stage of lactation). In particular, the relationship between the various forms of the enzyme and the activation pathways in milk need to be elucidated. Also, little is known regarding the activity of this enzyme in most dairy products, bar those discussed in this review. Finally, as cathepsin D is only one of a large number of lysosomal enzymes, the presence and activity of other cell-derived proteolytic enzymes in milk, although widely speculated, requires further characterisation.
Acknowledgements This authors wish to acknowledge grant aid under the Food Sub-Programme of the Operational Programme for Industrial Development which is administered by the Department of Agriculture, Food and Forestry and supported by National and EU funds and the F"TEK programme, which is supported by the Danish Dairy Research Foundation (Danish Dairy Board) and the Danish Government.
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M.J. Hurley et al. / International Dairy Journal 10 (2000) 673}681 Rochefort, H., & Liaudet-Coopman, E. (1999). Cathepsin D in cancer metastasis: A protease and a ligand. APMIS, 107, 86}95. Saftig, P., Hetman, M., Schmahl, W., Weber, K., Heine, L., Mossmann, H., KoK ster, A., Hess, B., Evers, M., von Figura, K., & Peters, C. (1995). Mice de"cient for the lysosomal proteinase cathepsin D exhibit progressive atrophy of the intestinal mucosa and profound destruction of lymphoid cells. EMBO Journal, 14, 3599}3608. Samarel, A. M., Worobec, S. W., Ferguson, A. G., Decker, R. S., & Lesch, M. (1986). Limited proteolysis of rabbit cardiac procathepsin D in a cell-free system. American Journal of Physiology, 250, C589}C596. Samarel, A. S., Ferguson, A. G., Decker, R. S., & Lesch, M. (1989). E!ects of cysteine inhibitors on rabbit cathepsin D maturation. American Journal of Physiology, 257, C1069}C1079. Sarath, G., De La Motte, R. S., & Wagner, F. W. (1989). Protease assay methods. In R. J. Beynon, & J. S. Bond (Eds.), Proteolytic enzymes * a practical approach (pp. 25}55). Oxford: IRL Press. Scarborough, P. E., Guruprasad, K., Topham, C., Richo, G. R., Conner, G. E., Blundell, T. L., & Dunn, B. M. (1993). Exploration of subsite binding speci"city of human cathepsin D through kinetics and rule-based molecular modeling. Protein Science, 2, 264}276. Scott, P. G., & Pearson, C. H. (1978). Cathepsin D: Cleavage of soluble collagen and crosslinked peptides. FEBS Letters, 88, 41}45. Simon, D. I., Ezratty, A. M., & Loscalzo, J. (1994). The "brin(ogen)olytic properties of cathepsin D. Biochemistry, 33, 6555}6563. Sloane, B. F., & Berquin, I. M. (1993). Proteases and cancer: An introduction. In J. S. Bond, & A. J. Barrett (Eds.), Proteolysis and protein turnover (pp. 225}231). London: Portland Press. Takahashi, T., & Tang, J. (1981). Cathepsin D from porcine and bovine spleen. Methods in Enzymology, 80C, 565}581. Tanaka, Y., Himeno, M., & Kato, K. (1990). Release of acid phosphatase from lysosomal membranes by cathepsin D. Journal of Biochemistry, 108, 287}291. Tang, J., & Wong, R. N. S. (1987). Evolution in the structure and function of aspartic proteinases. Journal of Cellular Biochemistry, 33, 53}63.
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Vetvicka, V., Vagner, J., Baudys, M., Tang, J., Foundling, S. I., & Fusek, M. (1993). Human breast milk contains procathepsin D * detection by speci"c antibodies. Biochemistry and Molecular Biology International, 30, 921}928. Vetvicka, V., Vetvickova, J., & Fusek, M. (1994). E!ect of human procathepsin D on proliferation of human cell lines. Cancer Letters, 79, 131}135. Vetvicka, V., Vetvickova, J., & Fusek, M. (1995). Participation of the propeptide on procathepsin D activation of human peripheral lymphocytes and neutrophils. Archives of Biochemistry and Biophysics, 322, 295}298. Vetvicka, V., Vetvickova, J., Hilgert, I., Voburka, Z., & Fusek, M. (1997). Analysis of the interaction of procathepsin D activation peptide with breast cancer cells. International Journal of Cancer, 73, 403}409. Vignon, F., Capony, F., Chambon, M., Freiss, G., Garcia, M., & Rochefort, H. (1986). Autocrine growth stimulation of the MCF 7 breast cancer cells by the estrogen-regulated 52 k protein. Endocrinology, 118, 1537}1545. Visser, F. M. W. (1976). Method for the manufacture of rennet-free cheese. Netherlands Milk and Dairy Journal, 30, 41}54. von Figura, K., & Hasilik, A. (1986). Lysosomal enzymes and their receptors. Annual Reviews in Biochemistry, 55, 167}193. Wium, H., & Qvist, K. B. (1998). E!ect of rennet concentration and method of coagulation on the texture of Feta cheese made from ultra"ltered bovine milk. Journal of Dairy Research, 65, 653}663. Wium, H., Kristiansen, K. R., & Qvist, K. B. (1998). Proteolysis and its role in relation to texture of Feta cheese made from ultra"ltered milk with di!erent amounts of rennet. Journal of Dairy Research, 65, 665}674. Yonezawa, S., Takahashi, T., Wang, X., Wong, R. N. S., Hartsuck, J. A., & Tang, J. (1988). Structures at the proteolytic processing region of cathepsin D. Journal of Biological Chemistry, 263, 16504}16511. Zeece, M. G., Katoh, K., Robson, R. M., & Parrish Jr., F. C. (1986). E!ect of cathepsin D on bovine myo"brils under di!erent conditions of pH and temperature. Journal of Food Science, 51, 769}773.
The milk acid proteinase cathepsin D: a review M.J. Hurley , L.B. Larsen, A.L. Kelly *, P.L.H. McSweeney Department of Food Science and Technology, University College, Cork, Ireland Department of Animal Product Quality, Danish Institute of Agricultural Sciences, P.O. Box 50, DK-8830 Tjele, Denmark Received 23 February 2000; accepted 23 September 2000
Abstract Cathepsin D is a lysosomal aspartic proteinase with a low pH optimum, found in many tissues and in bovine milk. It is synthesised as its inactive zymogen, procathepsin D. Procathepsin D can convert itself, by an autoproteolytic pathway, into an active intermediate, pseudocathepsin D, while other proteases are involved in the processing to mature cathepsin D. Cathepsin D is known to participate in a range of physiological processes. Pro-, pseudo- and mature cathepsin D have been puri"ed from bovine milk, but procathepsin D is the major form present in milk. Cathepsin D activity in milk appears correlated with somatic cell count. The active forms of the enzyme readily hydrolyse a -, a - and b-caseins, in the case of a -casein and b-casein with a speci"city similar to that of 1 1 1 chymosin. Cathepsin D can also produce para-i-casein from i-casein and, at high concentrations, can coagulate milk. Cathepsin D appears to be able to at least partially survive commercial pasteurisation processes. In recent years, increasing evidence has been documented indicating a role for this enzyme in proteolysis in cheese during ripening, most clearly in cheese where rennet activity is low, such as Swiss cheese, Quarg and Feta. Further research is required to evaluate the signi"cance of this enzyme in more detail. 2001 Elsevier Science Ltd. All rights reserved. Keywords: Cathepsin D; Milk; Proteolysis
1. Introduction The presence of proteolytic activity in tissue extracts at mildly acidic pH has been recognised since the early part of this century. Initially, the proteinase was referred to as `kathepsina (meaning, `to digesta in Greek), which was later modi"ed to `catepsinea, and "nally to the modern version of `cathepsina (Barrett, 1977). Following the isolation of an enzyme which accounted for over two-thirds of the total proteolytic activity in bovine spleen, Press, Porter, and Cebra (1960) designated the proteinase `cathepsin Da. Cathepsin D (EC 3.4.23.5) is an aspartic proteinase located in the lysosomes of all mammalian cells, and is the predominant proteolytic enzyme within such organelles (Barrett, 1977), which in addition contain a range of other cathepsins, such as B, H, L and S, which are cysteine proteases. An increasing range of proteolytic enzymes have been identi"ed in bovine milk. The pres-
* Corresponding author. Tel.: #353-21-903405; fax: #353-21270213. E-mail address: [email protected] (A.L. Kelly).
ence of an indigenous `acid proteasea in bovine milk was "rst reported nearly 30 years ago by Kaminogawa and Yamauchi (1972). They suggested that the proteinase was cathepsin D and this has been con"rmed by recent investigations.
2. Aspartic proteinases Aspartic proteinases, formerly called acid proteases, comprise a large group of proteolytic enzymes. Members of the family include pepsin, chymosin and HIV protease. The group exhibits a high degree of similarity in amino acid sequence and three-dimensional structure (Tang & Wong, 1987; Davies, 1990). The aspartic proteinases are bi-lobed molecules each lobe contributing an active site Asp residue to the substrate-binding cleft formed between the two. A group inhibitor is the pentapeptide, pepstatin, which binds reversibly to the active site cleft. The enzymes are mostly synthesised as proenzymes with a N-terminal propeptide which is removed to form the mature enzyme.
0958-6946/01/$ - see front matter 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 9 5 8 - 6 9 4 6 ( 0 0 ) 0 0 1 0 0 - X
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3. Structure and synthesis Cathepsin D, like all lysosomal enzymes, is synthesised at the rough endoplasmic reticulum with a 20-residue pre-sequence (signal peptide) (Erickson & Blobel, 1979). Following the initial cleavage of the signal peptide, the molecular mass of porcine procathepsin D increases from 43 to 46 kDa due to glycosylation (Erickson, Connor, & Blobel, 1981). The amino terminal propart of procathepsin D consists of 44 residues (Erickson et al., 1981). Procathepsin D is proteolytically inactive (Hasilik, von Figura, Conzelman, Nehrkorn, & Sadho!, 1982; Briozzo, Morisset, Capony, Rougeot, & Rochefort, 1988). Inside the cell, procathepsin D is very short lived, as it is processed rapidly into a catalytically active single-chain form of cathepsin D (M "44 kDa) by removal of the propeptide, presumably in prelysosomal compartments (Erickson et al., 1981; Gieselmann, Pohlmann, Hasilik, & von Figura, 1983; Rijnboutt, Stoorvogel, Geuze, & Strous, 1992). Studies have indicated this main proteolytic activation event to be mediated by cysteine protease(s) (Samarel, Ferguson, Decker, & Lesch, 1989). Depending on species, this single-chain form of cathepsin D may undergo further cleavage after arrival at the lysosome, most likely also by cysteine proteinase(s) (Gieselmann, Hasilik, & von Figura, 1985), involving the removal of two residues from bovine cathepsin D, while "ve residues are released in the processing of porcine cathepsin D (Yonezawa et al., 1988). Two chains are produced from the single-chain form, a light chain derived from the N-terminus and a heavy chain from the C-terminus, with M values of 31,000 and 13,000}14,000 Da, respectively (Hasilik & Neufeld, 1980a; Gieselmann et al., 1983). For unknown reasons, this proteolytic processing of bovine cathepsin D is not complete, as mature bovine cathepsin D exists in the lysosomes as an approximately equal mixture of singleand two-chain forms, while porcine cathepsin D consists primarily of the two-chain form (Takahashi & Tang, 1981). The activation of puri"ed procathepsin D has been studied and the formation of a catalytically active intermediate at acidic pH values was observed (Hasilik, von Figura, Conzelman, Nehrkorn, & Sadho!, 1982; Samarel, Worobec, Ferguson, Decker, & Lesch, 1986; Briozzo et al., 1988). This form was produced by a pHdependent reduction in molecular mass of 1}2 kDa, with the conversion being inhibited by pepstatin (Hasilik et al., 1982; Samarel et al., 1986). Procathepsin D can be converted into an active form by means of an autocatalytic cleavage between residues LeuP26 and IleP27 of the propeptide, yielding a protein with an N-terminal extension of 18 residues compared to mature cathepsin D (Conner & Richo, 1992; Larsen, Boisen, & Petersen, 1993). The active intermediate was termed pseudocathepsin D. However, unlike pepsinogen and
most other zymogens of aspartic proteinases, procathepsin D appears unable to autoactivate to mature cathepsin D and the formation of cathepsin D with mature N-terminus therefore seems to require the involvement of other enzymes. It also remains to be determined whether procathepsin D, the autoactivation product, is formed in vivo as part of the activation process. Cathepsin D is a glycoprotein with mannose-containing oligosaccharides attached at positions Asn and Asn (Hasilik & Neufeld, 1980b; Chitpinityol & Crabbe, 1998). Within the cis-Golgi compartment, the mannosecontaining oligosaccharides are phosphorylated and these mannose-6-phosphate residues are required for sorting to the lysosomes via mannose-6-phosphate receptors (MPR) (von Figura & Hasilik, 1986). Following binding to MPR in the trans-Golgi network the cathepsin D precursor is targeted to a prelysosomal compartment, where it is released from the MPR due to the acidic environment (von Figura & Hasilik, 1986). Mannose-6phosphate-independent sorting may, however, contribute to the targeting of procathepsin D in some cell types through transient mannose-6-phosphate-independent association of procathepsin D with intracellular membranes (Rijnboutt, Kal, Geuze, Aerts, & Strous, 1991). Press et al. (1960) were the "rst to isolate bovine cathepsin D (from spleen) and identi"ed the N-terminal amino acid as glycine. Following this initial work, the crystal structures of native and pepstatin-inhibited cathepsin D from human liver (Baldwin et al., 1993), native human spleen cathepsin D (Fusek, Baudys\ , & Metcalf, 1992) and bovine liver cathepsin D complexed with pepstatin (Metcalf & Fusek, 1993) have been determined. Like all aspartic proteinases, cathepsin D is a bilobed molecule and its secondary structure consists mainly of b-sheets. The two structurally similar domains both contain a single carbohydrate group along with two disulphide bonds (Metcalf & Fusek, 1993; Chitpinityol & Crabbe, 1998). Cathepsin D is expressed in all tissues studied, but the level of expression varies considerably (Reid, Valler, & Kay, 1986). The human cathepsin D gene has been found to contain a mixed promoter and can be expressed from two start points, constitutive and oestrogen-regulated, which indicate a range of physiological functions of the enzyme (Cavailles, Augereau, & Rochefort, 1993; May, Smith, & Westley, 1993).
4. Physiological function The location and the acidic pH optimum of cathepsin D suggests that it is adapted for the role of intracellular digestion of proteins in the acidi"ed environment provided by the lysosomal system (Barrett, 1977). The enzyme preferentially cleaves between two hydrophobic residues, but there is also a strong preference for
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hydrophobic amino acids in the P position of the sub strate. More distant residues, up to the P position, also in#uence the e$ciency of hydrolysis (Scarborough et al., 1993). One role of the enzyme is degradation of haemoglobin in the lysosomes of the spleen, permitting the reutilisation of the heme group (Barrett, 1977). Cathepsin D has also been suggested to be involved in the degradation of internalised "brinogen (Simon, Ezratty, & Loscalzo, 1994) and in the release of acid phosphatase from its membrane-bound form into the lysosomal matrix (Tanaka, Himeno, & Kato, 1990). Cathepsin D is a major lysosomal proteinase of white blood cells (somatic cells) and is present in phagocytic cells such as macrophages and polymorphonuclear leucocytes, particularly the former (Cohn, 1975). Cathepsin D has also been implicated in the processing of prohormones (Krieger & Hook, 1992), procollagen (Helseth & Veis, 1984), in processing of antigens in immune cells (Rhodes & Andersen, 1992) and in the regulation of the activity of insulin-like growth factor-binding protein-3 under acidic conditions (Conover & De Leon, 1994). Studies of mice lacking cathepsin D have suggested that a vital function of cathepsin D is in limited proteolysis of regulating proteins. The cathepsin-D-de"cient mice were short-lived and exhibited atrophy of the intestinal mucosa and destruction of lymphoid cells, while the contribution of cathepsin D to proteolysis in lysosomes appeared to be less vital (Saftig et al., 1995). In recent years, the association of cathepsin D with development of breast cancer has received increased attention. Cathepsin D is overexpressed (2}50)-fold in most breast cancer tumor cytosols, when compared with its concentration in normal mammary glands (Rochefort & Capony, 1993). Procathepsin D is known to be overexpressed and secreted in oestrogen receptor-positive breast cancer cells and procathepsin D has been suggested to facilitate metastasis of tumour cells (Rochefort, Capony, & Garcia, 1990; Sloane & Berquin, 1993). Importantly, procathepsin D has been shown to have mitogenic e!ect on breast cancer cell lines (Vignon et al., 1986; Vetvicka, Vetvickova, & Fusek, 1994). The major mechanism involved in the stimulation of metastasis has been suggested to be increased cell proliferation (Rochefort & Liaudet-Coopman, 1999). The mitogenic e!ect was speci"c for the pro-form and a synthetic peptide corresponding to the procathepsin D propeptide had the same e!ect (Fusek & Vetvicka, 1994), through interaction with a previously unknown receptor of breast cancer cells (Vetvicka, Vetvickova, Hilgert, Voburka, & Fusek, 1997). The use of cathepsin D and procathepsin D levels as a prognostic tool in breast cancer is, however, controversial (Rochefort et al., 1990; Brouillet et al., 1993; Losch et al., 1998). Cathepsin D has also been proposed to play a pivotal role as a growth modulator in androgen-dependent prostate cancer (Mordente, Choudhury, Tazaki, Mallouh, & Konna, 1998). Furthermore, cathep-
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sin D may also play a role in certain neurological disorders, such as Alzheimer's disease (Masters et al., 1985; Kang et al., 1987) and scrapie, a transmissible neurodegenerative disease caused by prions and common to sheep (Diedrich et al., 1991). The participation of degradation of meat proteins and post-mortem changes is well characterised (Dutson, 1983) and it has been implicated in the proteolysis of collagen (Scott & Pearson, 1978) and of myo"brillar proteins (Matsumoto, Okitani, Kitamura, & Kato, 1983; Zeece, Katoh, Robson, & Parrish, 1986).
5. Cathepsin D in milk The occurrence of an acid protease in bovine milk was "rst demonstrated by Kaminogawa & Yamauchi (1972). This protease was considered to be similar to the lysosomal enzyme cathepsin D. Larsen et al. (1993) con"rmed that this aspartic proteinase was actually procathepsin D. Incubation of procathepsin D puri"ed from bovine milk at pH 3.5 and 5.0 indicated autoactivation to the proteolytically active form, pseudocathepsin D, but apparently not into mature cathepsin D (Larsen et al., 1993). Larsen and Petersen (1995) subsequently puri"ed "ve molecular forms of cathepsin D from bovine milk by ammonium sulphate precipitation of acid whey, followed by separation on DEAE-Sepharose. The "nal puri"cation step was a$nity chromatography on pepstatin}Sepharose, which permitted separation of the di!erent forms by exploiting pH-dependent di!erences in binding to pepstatin. The "ve forms isolated from milk had apparent molecular masses of 46, 45, 43, 39 and 31 kDa (Fig. 1). Both the 46 and 45 kDa bands corresponded to procathepsin D, but the reason for the variability in molecular mass is not known. The 43 kDa form corresponded to pseudocathepsin D, while the 39 and 31 kDa forms were identi"ed as mature single- and heavy-chain cathepsin D, respectively. The cathepsin D light chain, which was most likely also present, was not detected in the experiment, probably due to lack of sensitivity. Procathepsin D (45 kDa) appeared to be the major form present in milk (Larsen & Petersen, 1995). The detection of the mature forms of cathepsin D (39 and 31 kDa) in the puri"ed preparation implies either that milk contains mature cathepsin D or alternatively that mature cathepsin D was formed during the puri"cation procedure by other enzymes capable of activating procathepsin D or pseudocathepsin D. The observation that mature cathepsin D was present in the eluate from DEAE-Sepharose (Larsen & Petersen, 1995) may indicate, however, that the mature forms are present in milk, although in small amounts. Cathepsin D concentrations in skimmed milk and acid whey were estimated by a competitive enzyme-linked
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Fig. 1. Molecular forms of cathepsin D identi"ed in bovine milk. Prefix P denotes residues in the propart (adapted from Larsen & Petersen, 1995). The procathepsin D forms are inactive, while other forms are proteolytically active.
immunosorbent assay to be 0.4 and 0.3 lg mL\, respectively, indicating that cathepsin D is mainly present in the whey (Larsen, Benfeldt, Rasmussen, & Petersen, 1996). The origin of procathepsin D and cathepsin D in milk is not known. Procathepsin D can be secreted both by mammary epithelial cells (Capony et al., 1989) and by milk-secreting cells, as activated lymphocytes can secrete lysosomal enzymes (Hasilik, 1992). Cathepsin D activity in milk is correlated with somatic cell count (O'Driscoll, Rattray, McSweeney, & Kelly, 1999), but whether this re#ects an increased amount of cathepsin D and/or procathepsin D and/or increased activation of the indigenous procathepsin D pool remains to be determined. The physiological function of procathepsin D and cathepsin D in milk is unknown. Procathepsin D has also been found in human breast milk (Vetvicka et al., 1993) and can activate neutrophils and lymphocytes through the regulation of surface receptors. Thus, a possible function has been suggested in the immune defence of the breast-fed newborn (Vetvicka, Vetvickova, & Fusek, 1995). Interestingly, Saftig et al. (1995) suggested that one reason that mice de"cient for cathepsin D could survive up to four weeks after birth may be that the neonates received cathepsin D with the maternal milk. 5.1. Factors awecting enzyme activity A commonly-used substrate for measuring cathepsin D activity is haemoglobin, usually after denaturation by acid or urea (Takahashi & Tang, 1981; Sarath, De La Motte, & Wagner, 1989). O'Driscoll et al. (1999) showed that a synthetic heptapeptide substrate (Pro}Thr}Glu}
Phe}[NO }Phe]}Arg}Leu) was a good substrate for measuring cathepsin D activity in milk. Cathepsin D displays greatest activity against haemoglobin in the pH range 3.0}3.5 (Press et al., 1960; Barrett, 1972). The optimum pH for partly isolated milk cathepsin D on haemoglobin was found to be pH 4.0 (Kaminogawa & Yamauchi, 1972) and the optimum temperature of cathepsin D activity was determined to be 373C (Barrett, 1972). Draper and Zeece (1989) showed that the heat stability of bovine cathepsin D increased with decreasing pH, with a corresponding increase in enzyme activity. Complete inactivation of cathepsin D in milk on heating at 703C for 10 min has been observed (Kaminogawa & Yamauchi, 1972). Recent studies have indicated that cathepsin D at least partially survives pasteurisation treatments (72.53C for 15 s) (Larsen et al., 2000; Hayes et al., in press). 5.2. Hydrolysis of milk proteins by cathepsin D Kaminogawa, Yamauchi, Miyazawa and Koga (1980) investigated the degradation of individual caseins by partially puri"ed cathepsin D. In recent years, the degradation of caseins in vitro by cathepsin D has received closer examination and the proteolysis products have been compared to those produced by chymosin (McSweeney, Fox, & Olson, 1995; Larsen et al., 1996). As procathepsin D is the major form of cathepsin D found in milk, Larsen et al. (1996) investigated the degradation of milk proteins both with cathepsin D from bovine spleen and with procathepsin D puri"ed from milk. The hydrolysates were performed at pH 5.0, at which pH the
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Fig. 2. Speci"city of cathepsin D against (a) a - and (b) b-casein. Arrows indicate identi"ed cleavage sites (McSweeney et al., 1995; Larsen et al., 1996). 1
added procathepsin D autoactivated to proteolytically active pseudocathepsin D. Analysis by SDS-PAGE showed that the degradation patterns of the two enzymes were similar, although the hydrolysis was slower with pseudocathepsin D than with mature cathepsin D. The primary cleavage sites of cathepsin D on a - and 1 b-casein are summarised in Fig. 2. Kaminogawa et al. (1980) and McSweeney et al. (1995) showed that partially puri"ed cathepsin D from milk degraded a -casein to a peptide with the same molecular 1 mass as a -CN f24-199 (a -I-casein), which is known to 1 1 be produced from a -casein by the action of chymosin 1 (Hill, Lahav, & Givol, 1974). The a -I-casein peptide is 1 further degraded by cathepsin D in vitro (Larsen et al., 1996). Proteolysis of b-casein by cathepsin D is also similar to the action of chymosin (Fig. 2). Cathepsin D hydrolysates of a -casein di!er markedly from those 1 produced by chymosin, with very few peptides in common (McSweeney et al., 1995). The "rst bond cleaved in the a -casein monomer by cathepsin D is Leu } 1 Tyr , followed by further degradation of the C-ter minal fragment (Larsen et al., 1996). The Leu }Tyr bond is the only bond cleaved in dimeric a -casein. 1 i-Casein is cleaved by partially puri"ed cathepsin D to produce para-i-casein (Kaminogawa et al., 1980; McSweeney et al., 1995; Larsen et al., 1996). Larsen et al. (1996) identi"ed two subsequent cleavage sites of cathepsin D in i-casein, Leu }Ser and Leu }Ser . Two cleavage sites of cathepsin D on a-lactalbumin have been identi"ed (Leu }Phe and Trp }Leu ), while native b-lactoglobulin is resistant to cleavage (Larsen et al., 1996).
5.3. Coagulation of milk As cathepsin D is an aspartic proteinase with a proteolytic speci"city similar to that of chymosin, one might expect that it possesses the ability to coagulate milk. McSweeney et al. (1995) and Larsen et al. (1996) both showed that cathepsin D generated the caseinomacropeptide (i-CN 106-169) when incubated with isolated i-casein. McSweeney et al. (1995) examined the in#uence of cathepsin D addition on the rennetability of skim milk at pH 6.4 and 303C. Aliquots were withdrawn periodically after enzyme addition and their rennet clotting time (RCT) determined. Incubation of milk with cathepsin D reduced RCT, presumably as a result of slow hydrolysis of the micelle-stabilising protein, i-casein, by cathepsin D. Larsen et al. (1996) investigated the ability of cathepsin D to coagulate acidi"ed skim milk (at pH 5.0) at 373C using a Formagraph. The pH dependence of milk coagulation by cathepsin D was also investigated by adding a "xed amount of enzyme to bovine milk, adjusted to pH values in the range 5.0}6.5. Coagulation was observed at pH 5.0 with concentrations of cathepsin D above 3.1 lg mL\, or approximately 10 times the indigenous amount in milk. Cathepsin D was capable of coagulating milk over all pH values examined with RCT decreasing with decreasing pH, presumably due to acidic pH optimum of the enzyme. Thus, although milk possesses an indigenous milkclotting enzyme, the level present (0.4 lg mL\) in pooled raw milk may be too low to signi"cantly in#uence milk
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coagulation in cheese production, but may contribute to degradation of i-casein during long-term storage of some milk products. 5.4. Contribution to proteolysis in cheese As cathepsin D has the ability to degrade all four caseins, it may be expected that the enzyme could play a role in cheese ripening. The speci"city of cathepsin D is quite similar to that of the milk coagulant, chymosin, which is added to milk at a far greater concentration compared with the level of cathepsin D already present; the level of coagulant retained in the curd being up to 15% (Ohmiya & Sato, 1972). This, along with the fact that cathepsin D, at least in unpasteurized milk, is predominantly a whey protein (Larsen et al., 1996), suggests that chymosin is the principal aspartic proteinase in cheese curd. Thus, in many cases, cathepsin D activity may be masked by that of chymosin (McSweeney et al., 1995). To investigate the contribution of various agents to proteolysis, rennet-free cheeses have been developed. Visser (1976) developed a method based on the thermal inactivation of rennet (after it had completed its primary cleavage of the Phe }Met bond of i-casein) by pasteurisation (723C for 20 s) of milk in which the calcium concentration had been decreased by means of ion exchange, thereby preventing coagulation. After pasteurisation, the milk was cooled (to 4}53C) and a rennet-free coagulum formed by the addition of a solution of calcium chloride and lactic acid and warming to 303C. Due to the complexity of this procedure, alternative methods have been sought. O'Kee!e, Fox, and Daly (1977) and Lane, Fox, Johnston, and McSweeney (1997) exploited the very low stability of porcine pepsin (EC 2.4.23.1) at neutral pH in the production of rennet-free cheese. The above methods are designed to eliminate rennet activity, thus allowing the examination of role of indigenous proteinase activity in casein degradation. Noomen (1978), employing the method of Visser (1976), observed production of a -I-casein in simulated Noordhollandse 1 Meshanger cheese. Lane et al. (1997) observed limited hydrolysis of a -casein in Cheddar, again with produc1 tion of a -I-casein. Presuming the successful inactiva1 tion of the incorporated coagulant (which may not have been the case in all studies), production as a -I-casein 1 may be attributed to cathepsin D activity, thereby indicating that cathepsin D may have a role to play in softening of cheese texture, which is related to production of this peptide (Creamer, Zoerb, Olson, & Richardson, 1982). To determine the contribution of cathepsin D to casein degradation, a more conclusive approach would be to manufacture cheese entirely without coagulant, thereby eliminating any speculation as to whether the rennet was totally inactivated. Wium, Kristiansen, and Qvist (1998)
produced Feta without addition of coagulant. The cheesemilk was homogenised, pasteurised at 723C for 15 s, ultra"ltered at 503C, pasteurised again and stored at 43C until the next day. The ultra"ltered milk was then subjected to a "nal pasteurisation and homogenised, followed by the addition of gluconic acid-d-lactone, causing a drop in pH which induced coagulation (Wium & Qvist, 1998). By using gluconic acid-d-lactone as acidulent, enzymes from lactic acid bacteria were eliminated, thereby simplifying the model system. a -Casein, 1 a -I-casein (produced from a -casein) and i-casein 1 1 were all degraded over time. This, along with the absence of rennet, strongly suggested the involvement of an indigenous milk proteinase capable of acting under acidic conditions. i-Casein was degraded with concomitant production of para-i-casein, which is in accordance with the speci"city of cathepsin D (McSweeney et al., 1995; Larsen et al., 1996). There was also a preference for the hydrolysis of a -casein, while b-casein remained undeg1 raded, indicating a lack of plasmin activity in the cheese, which was attributed to its low pH (pH"4.6), far from the alkaline pH optimum of plasmin (Bastian & Brown, 1996). This was, however, characteristic of cathepsin D activity, which preferentially hydrolyses a -casein 1 (Kaminogawa & Yamauchi, 1972). These observations strongly indicated the presence of cathepsin D-like activity in this cheese, which was recently con"rmed by the demonstration of cathepsin D activity in rennet-free Feta cheese extracts and furthermore the presence of procathepsin D was demonstrated immunologically (Larsen et al., 2000). The question of cathepsin D activity has also arisen in respect to cheese varieties such as Swiss cheese, in which chymosin is believed to be largely inactivated during cooking at 53}553C for up to 1 h (Matheson, 1981; Garnot & Molle, 1987). With full inactivation of the heatlabile coagulant, the absence of rennet-like activity would be expected, with little or no degradation of a -casein. 1 However, this does not appear to be the case. Igoshi and Arima (1993) isolated an active acid proteinase from Swiss-type cheese (Emmental and Gruye`re) which was deemed not to be chymosin, due to the observation that it did not decompose i-casein at a greater rate than a -casein (as chymosin would) but rather it was con1 sidered to be cathepsin D. Subsequently, production of a -I-casein has been again identi"ed in Swiss cheese 1 (Beuvier et al., 1997; Cooney, Tiernan, Joyce, & Kelly, 2000), and attributed to the action of cathepsin D. Furthermore, Cooney et al. (1999) found breakdown of a 1 casein in Swiss cheese to be proportional to milk somatic cell count, which again would be consistent with cathepsin D being the enzyme responsible for this degradation of a -casein. 1 Recently, Hurley et al. (2000) identi"ed, by both speci"c enzyme assay and immunological methods, cathepsin D activity in Quarg cheese made from raw,
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pasteurised and ultra"ltrated milk and demonstrated that the enzyme contributed to proteolysis during storage.
6. Conclusions and research requirements Cathepsin D appears to be quite stable in milk, capable of at least partially surviving three cycles of pasteurisation (723C for 15 s) (Wium & Qvist, 1998; Larsen et al., 2000). This would suggest that this enzyme may play a role in the ripening of high-cooked cheese varieties, such as Swiss cheese. Cathepsin D activity is obvious in cheese varieties where no rennet is added (such as rennet-free UF Feta). However, it is di$cult to quantify the contribution of cathepsin D to the ripening of cheese varieties such as Cheddar, where the activity may be masked by chymosin activity. To fully evaluate the potential signi"cance of this enzyme for dairy product quality, much study remains. In comparison to the extensive literature concerning the plasmin system in milk, the stability of its various components and the e!ects of a wide range of production and processing variables on the activity of the enzyme, cathepsin D is very poorly understood. As a primary goal, attention must be focussed on the activity and stability of this enzyme in milk and sources of variation in this activity (such as stage of lactation). In particular, the relationship between the various forms of the enzyme and the activation pathways in milk need to be elucidated. Also, little is known regarding the activity of this enzyme in most dairy products, bar those discussed in this review. Finally, as cathepsin D is only one of a large number of lysosomal enzymes, the presence and activity of other cell-derived proteolytic enzymes in milk, although widely speculated, requires further characterisation.
Acknowledgements This authors wish to acknowledge grant aid under the Food Sub-Programme of the Operational Programme for Industrial Development which is administered by the Department of Agriculture, Food and Forestry and supported by National and EU funds and the F"TEK programme, which is supported by the Danish Dairy Research Foundation (Danish Dairy Board) and the Danish Government.
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