Cathepsin E

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of cDNA for embryonic chicken pepsinogen: phylogenetic relationship with prochymosin. J. Biochem. (Tokyo) 103, 290
296. [45] Kageyama, T. (2002). Pepsinogens, progastricsins, and prochymosins: structure, function, evolution, and development CMLS. Cell. Mol. Life Sci. 59, 288
306. [46] Yasugi, S., Mizuno, T. (1981). Purification and characterization of embryonic chicken pepsinogen, a unique pepsinogen with large molecular weight. J. Biochem. (Tokyo) 89, 311
315. [47] Fukuda, K., Saiga, H., Yasugi, S. (1995). Transcription of embryonic chick pepsinogen gene is affected by mesenchymal signals through its 50 -flanking region. Adv. Exp. Med. Biol. 362, 125
129.

[48] Foltmann, B. (1992). Chymosin a short review on foetal and neonatal gastric proteases. Scand. J. Clin. Lab. Invest. 52(suppl. 210), 65
79. [49] Harboe, M., Budtz, P. (1999). The production, action and application of rennet and coagulants, in: Technology of Cheesemaking, Law, B.A., ed., Sheffield: Sheffield Academic Press, pp. 33
65. [50] Suzuki, J., Sasaki, K., Sasao, Y., Hamu, A., Kawasaki, H., Nishiyama, M., Horinouchi, S., Beppu, T. (1989). Alteration of catalytic properties of chymosin by site-directed mutagenesis. Protein Eng. 2, 563
569. [51] Chitpinityol, S., Goode, D., Crabbe, M.J.C. (1998). Site-specific mutations of calf chymosin B which influence milk clotting activity. Food Chem. 62, 133
139.

Pal Bela Szecsi Department of Laboratory Medicine, Lund, Division of Clinical Chemistry and Pharmacology, Lund University Hospital, 221 85 Lund, Sweden. Email: [email protected]

Marianne Harboe DK-2800 Kgs, Lyngby, Denmark. This article is a revision of the previous edition article by Bent Foltmann, Volume 1, pp. 29
33, r 2004, Elsevier Ltd. Handbook of Proteolytic Enzymes, 3rd Edn ISBN: 978-0-12-382219-2

© 2013 Elsevier Ltd. All rights reserved. DOI: http://dx.doi.org/10.1016/B978-0-12-382219-2.00005-3

Chapter 6

Cathepsin E DATABANKS MEROPS name: cathepsin E MEROPS classification: clan AA, family A1, subfamily A1A, peptidase A01.010 IUBMB: EC 3.4.23.34 (BRENDA) Tertiary structure: Available Species distribution: superclass Tetrapoda Reference sequence from: Homo sapiens (UniProt: P14091)

Name and History Cathepsin E (CatE), an intracellular aspartic protease of the pepsin superfamily, is homologous to the analogous aspartic protease cathepsin D (Chapter 8). The first study to report on cathepsin E was from Lapresle & Webb [1]. Several other reports implicated the presence of an aspartic protease distinct from cathepsin D in various vertebrate cells. Different research groups assigned different designations to this enzyme, such as cathepsin D-like proteinase [2,3], gastric mucosa non-pepsin acid proteinase

[4], slow moving proteinase [5] and erythrocyte membrane acid proteinase [6]. Later, it was shown that all of these activities were mediated by the same enzyme, which was termed cathepsin E.

Activity and Specificity CatE possesses two homologous domains, each containing the highly conserved tripeptide sequence DTG. These two domains are involved in the formation of the active site [7
9]. The DTG sequence is present in all species tested with the exception of rabbit CatE, in which the tripeptide sequence near the N-terminal region is replaced by DTV [10]. Liu et al. [11] synthesized active site mutants of CatE by site-directed mutagenesis to examine the significance of these residues. They found that in mouse CatE Asp98, Asp283, and Thr284 are indeed critical for catalysis [11]. CatE is synthesized as an inactive precursor and is autoactivated by proteolytic removal of Nterminal propeptide; the process is triggered by acidic pH.

Clan AA (A1) | 6. Cathepsin E

Tsukuba et al. [12] published that active site mutants of CatE, in which one or both aspartic acid residues were substituted with alanine, not only lacked catalytic activity but were also unable to mature, thus remaining as a 46 kDa precursor [12]. This indicates that catalytic activity is essential for the processing and maturation of this enzyme. Cathepsin E, like cathepsin D (CatD), prefers hydrophobic amino acids at the P1 and P10 positions [13]. β-Branched residues, e.g. Val and Ile, are not allowed at P1 [14]. Position P20 accepts a broad range of amino acids, including charged and polar ones [14]. For CatE, a basic residue, e.g. Lys, is acceptable at position P2, which is not the case for CatD [15]. The presence of proline at P4 might be important and may facilitate scissile peptide bonding in extended β-strands for cleavage by the enzyme [16]. According to some reports, CatE also retains activity at neutral pH and shows a distinct cleavage specificity [17,18]. In one of these reports [17], proteolytic activity and cleavage specificity of CatE towards the B chain of oxidized insulin was examined. It was reported that the cleavage specificity changed significantly, with more specific cleavage at pH 7.4 and above, as compared to pH 5.5 and 3.0. At acidic pH, several peptide bonds, especially Phe-X, Tyr-X and Leu-X were cleaved, whereas at pH 7.4 the Glu13-Ala14 bond was selectively cleaved. In a more recent study [18], preferential cleavage of Arg-X and GluX bonds at pH 7.4 was reported, with the Arg-Arg bond the preferred cleavage site. However, in all studies reporting proteolytic activity of CatE at pH 7.4, the enzyme was isolated either from human gastric mucosa [17
19] or human red blood cells [19]. In a more recent report, it was observed that recombinant CatE is proteolytically inactive at neutral pH even in the presence of ATP, which is known to stabilize this enzyme [20]. The reason for this discrepancy could be that the purified CatE used in previous studies might have been contaminated with some other factor. Alternatively, these discrepant results may reflect conformational differences between recombinant CatE and the native enzyme. These differences may also arise from posttranslational modifications of the native enzyme in vivo. The most common assay for measuring aspartic protease activity in biological samples employs acid-denatured bovine hemoglobin as the substrate [6,21,22]. Enzymatic reaction liberates trichloroacetic acid-soluble products from hemoglobin which are detected by their absorbance at 280 nm and by the Folin reaction. This method is timeconsuming and has the additional disadvantage that it cannot discriminate CatE activity from other aspartic proteases. Several synthetic chromogenic or fluorogenic substrates have been developed to measure CatE activity [23
26]. These methods are simple and fast but the described substrates are restricted in their selectivity. In a recent study, a highly selective substrate for cathepsin E has been reported [27].

43

The most widely used and potent inhibitor of CatE is pepstatin A (PepA). Additionally, a wide variety of synthetic peptidomimetic inhibitors has been described that mediate potent CatE inhibition [28,29]. However, none of these small molecule inhibitors discriminates clearly between cathepsin D and E. A specific inhibitor for CatE, the Ascaris pepsin inhibitor, which has no activity against cathepsin D, has been described [30], but is not readily available in quantities sufficient for functional studies because of difficulties in purification. Furthermore, the recombinant inhibitor may have a slightly different inhibitory profile [31]. Although pepstatin A is not specific for CatE or CatD, it is widely used in cell-based studies aimed at understanding the function of these enzymes; using cells from CatD-deficient mice allows pepstatin specifically to target CatE. However, another limitation of PepA for cell-based studies is that it is inefficiently transported across the cell membrane [32]. To address this problem mannosepepstatin conjugates were developed as targeted inhibitors of antigen processing [32,33]. These mannosylated conjugates were efficiently incorporated into cells via receptormediated uptake. This approach is obviously limited to cells carrying mannose receptors. More recently, Grassystatins A-C from marine cyanobacteria, were reported to be potent cathepsin E inhibitors that also reduce antigen presentation [34]. Kitamura et al. [35] have developed cathepsin E inhibitory peptides that inhibit cathepsin E (CE) specifically at a submicromolar IC (50).

Structural Chemistry Most of the other known aspartic proteases from vertebrates exist as single polypeptide chains in their mature form, whereas cathepsin E is a dimer and has a molecular mass of B84 kDa. It is known that the amino terminal region of mature cathepsin E contains a Cys residue at position 43, which is responsible for disulfide bond formation between the two identical subunits [36]. The homodimeric form is easily converted into a monomeric form exhibiting full catalytic activity under reducing conditions [8,37]. As described previously, CatE possesses two homologous domains, both containing the highly conserved tripeptide sequence DTG. These two domains are involved in the formation of the active site [7
9]. Moreover, CatE is known to be N-glycosylated with high-mannose and/or complex oligosaccharides in the native state [38
40]. Although the crystal structure of CatE has not been resolved so far, based on available information on CatE and the crystal structures of other aspartic proteases, KuoChen Chou [41] has predicted a three-dimensional structure for CatE. Ostermann et al. [42] have crystallized and solved the structure of an activation intermediate of CatE. They reported that the overall structure resembles intermediate 2 in the proposed activation pathway of

44

aspartic proteases like gastricsin and cathepsin D [42]. The pro-sequence is cleaved from the protease and remains stably attached to the mature enzyme by forming the outermost sixth strand of the interdomain β-sheet. The pro-sequence remains attached to the mature enzyme and the primed binding site is in a closed conformation [42].

Preparation Mature cathepsin E has been purified from a variety of vertebrate cells and tissues such as human gastric mucosa [5], human erythrocytes [6], rat spleen [21], rat neutrophils [43] and rat epidermis [44]. Briefly, the cell homogenate from 20
30 ml cell pellets is applied to Con-A-Sepharose. Glyoproteins are eluted by 6% methyl α-D-mannopyranoside. Elute is resolved on a MonoQ 5/5 column using an FPLC system. Cathepsin E elutes in the range 0.20
0.25 M NaCl in 5 mM sodium phosphate pH 7.0 buffer. Alternatively, cathepsin E can be isolated by FPLC on a Mono-Q column and affinity chromatography on pepstatin-agarose or on an immobilized antibody. Procathepsin E can also be purified from different cell types but the process is comparatively difficult [39]. Recombinant procathepsin E has been produced in Escherichia coli [45] which autoactivates in an acidic environment to generate the mature enzyme. Recombinant human cathepsin E has also been produced and purified from Chinese hamster ovary cells [46], Pichia pastoris [47] and the human kidney cell line 293 [48]. Different rat cathepsin E mutants have been successfully expressed in embryonic kidney 293T cells [12].

Biological Aspects Tissue Distribution and Subcellular Localization Cathepsin E is mainly present in cells of the immune system including antigen-presenting cells (APC) [49] such as lymphocytes [50], microglia [38], dendritic cells [33], Langerhans cells [51], interdigitating reticulum cells [51] and human M cells [52]. Cathepsin E is not present in resting B-lymphocytes but is up-regulated late in human B-cell activation at both the mRNA and protein level [53]. It has also been detected in osteoclasts [54]. Furthermore, tissue-specific distribution of CatE is not the same in different mammalian species, e.g. it has been detected in red blood cells from humans and rats but not guinea pigs, cattle, goats or pigs [55]. The intracellular localization of cathepsin E also appears to vary according to cell type. In APC such as microglia [56], dendritic cells [33] and macrophages CatE is mainly present in endosomal compartments. In contrast; in erythrocytes [6,39,57], gastric cells [2,3,5,58], renal proximal tubule cells [58] and osteoclasts [54], it is

Clan AA (A1) | 6. Cathepsin E

exclusively confined to the plasma membrane. CatE is also detected in the endoplasmic reticulum and Golgi complex in different cell types, such as gastric epithelial cells [5,58], Langerhans cells, interdigitating reticulum cells [51] and human M cells [52].

Regulation of Gene Expression The limited tissue distribution of cathepsin E necessitates a mechanism that regulates the transcription of the gene encoding CatE so that expression is facilitated only in certain types of cells. The promoter region flanking the procathepsin E gene from human [59] and mouse [9] does not contain a TATA box. Instead, both sequences contain an initiator element [9,59] which offers an alternative binding site for TFIID (transcription factor IID) to initiate gene transcription [60]. Cook et al. [61] have reported that regulation of human and mouse procathepsin E gene expression is not influenced by CpG methylation. Furthermore, transcription of the CatE gene is dependent on the balance between the effects produced by positive-acting tissuespecific transcription factors such as GATA1 and PU1 and the negative influence of the ubiquitous factor YY1 [61]. Additionally, it is also known that CatE transcription is negatively regulated by CIITA (class II transactivator), a non-DNA-binding transcription factor [62].

Processing, Maturation and Intracellular Trafficking Of Cathepsin E Cathepsin E is synthesized as a 46 kDa precursor and is later converted into the 42 kDa mature form. The processing events of pro-cathepsin E include the removal of propeptide, modification of oligosaccharide chains and formation of a disulfide bond between two cysteine residues at the NH2-terminal region to yield a homodimer. Like many other aspartic proteases, CatE is synthesized as a zymogen which is catalytically inactive towards its natural substrates at neutral pH and which autoactivates in an acidic environment to generate the mature enzyme by proteolytic removal of N-terminal propeptide. The unique structural characteristics of propeptide are conserved in all species [7,8,63,64]. The propeptide is composed of 40 amino acid residues and is highly positively charged. Therefore, it is hypothesized that the propeptide is bound to active CatE mainly through electrostatic interactions. It has been reported that the propeptide of CatE plays an important role in the correct folding, maturation and targeting of this protein to its final destination [65]. Tsukuba et al. [12] have constructed CatE mutants lacking the propeptide (Leu23-Phe58). This mutant protein was neither processed nor matured and was found mostly in the ER, in comparison to the wild type which was mainly located in endosomes [12]. In addition, Finley and Kornfeld [66] reported that

Clan AA (A1) | 6. Cathepsin E

amino acids 1
48 of mature cathepsin E are important for its retention in the ER. Tsukuba et al. [12] constructed a mutant lacking most of this putative ER-retention sequence which was not converted into a mature form of the enzyme, was rapidly degraded in the cells and could not be detected in the endosomes. This indicates that the putative ERretention sequence is required for correct folding, processing, maturation and targeting of CatE to endosomes. CatE is known to be N-glycosylated with high-mannose and/or complex oligosaccharides in the native state. In APC such as microglia and macrophages, the mature form of CatE is localized in endosomal compartments and is Nglycosylated mainly with complex-type oligosaccharides [38]. CatE from erythrocyte membranes (human and rat) [39] and thymocytes (rat) [40] is also N-glycosylated with complex-type oligosaccharides, while the enzyme from the spleen (rat) [7] and stomach (rat) [67] has high mannosetype oligosaccharides. This is interesting in view of the fact that CatE shows a different subcellular localization in different cell types, and it has been suggested that the nature of its oligosaccharide chains may be cell-specific or may vary with its cellular localization. Yasuda et al. [68] constructed an N-glycosylation mutant (by changing the N residue to Q and D at position 73 and 305 in potential glycosylation sites of rat CatE). It was found that this mutant was less stable to temperature and pH than glycosylated CatE, although its catalytic properties were equivalent to wild-type. Tsukuba et al. [12] reported that N-glycosylation mutants were stably retained in cells but were not processed to the mature form and were exclusively confined to the ER. Hence, N-glycosylation of CatE seems to play an important role in processing, maturation and trafficking of CatE to its final destination, but might not be essential for correct folding of the enzyme.

Physiological Roles of Cathepsin E So far the functional importance of cathepsin E is not completely understood. Recent studies with cathepsin Edeficient mice have provided several pieces of information regarding association of this protease with different physiological effects. CatE-deficient mice do not exhibit any obvious phenotypes when reared under specific pathogenfree conditions [69]. However, when kept under conventional conditions they spontaneously develop atopic dermatitis-like skin lesions. Moreover, reduced expression of CatE was observed in erythrocytes of both humans with atopic dermatitis (AD) and in the AD mouse model NC/ Nga [69]. This suggests that CatE-deficiency might be responsible for the induction of AD in humans and mice. Additionally, macrophages derived from CatE-deficient mice exhibit a novel form of lysosomal storage disorder characterized by the accumulation of the two major lysosomal membrane sialoglycoproteins LAMP-1 and LAMP-2 and the elevation of the lysosomal pH [70].

45

The fact that CatE-deficient mice spontaneously develop atopic dermatitis like skin lesions when reared under conventional conditions, but not under specific pathogen-free conditions, implies that development of AD in these mice is triggered by some environmental factors such as pathogenic microorganisms. Additionally, Staphylococcus aureus is postulated to be involved in development of AD [71,72] and in CatE-deficient mice, colonization by S. aureus was observed in AD-like lesions [69]. Recently, Tsukuba et al. [73] showed that the susceptibility of CatE-deficient mice to bacterial infections is increased. This was found to be associated with decreased cell surface expression of Toll-like receptor-2 (TLR-2) and TLR-4 by macrophages. As total cellular expression of these receptors was not impaired, it was hypothesized that the trafficking of these receptors was defective. It is reported that elevated lysosomal pH interferes with the maturation and fusion events of the organelles involved [74,75]. Hence, the lysosomal storage disorder in CatEdeficient macrophages that results in elevated lysosomal pH is thought to cause impaired trafficking. Elevated levels of CatE expression have also been associated with several forms of cancer, including human gastric and cervical adenocarcinomas [76
78]. Protein profiling of human lung carcinomas has shown that CatE is elevated in both adenocarcinomas and squamous cell carcinomas [79]. However, the most thoroughly studied relationship between CatE upregulation and malignancy is focused on pancreatic ductal adenocarcinomas [80
82]. With respect to cancer, CatE has been studied as a diagnostic marker [82,83] as well as an anti-tumor factor produced by the host defense system [84,85]. CatE is also known to specifically induce tumor growth arrest and apoptosis in human prostrate carcinoma cell lines without affecting normal cells by catalyzing the release of tumor necrosis factor-related-apoptosis-inducing ligand (TRAIL) from the cell surface [84]. Moreover, administration of purified CatE into human tumor xenografts in nude mice induced apoptosis in tumor cells and inhibited tumor growth. Several lines of evidence suggest that cathepsin E plays a role in antigen processing via the MHC class II pathway. CatE is detectable in APC such as Langerhans and interdigitating dendritic cells [51] and its expression is up-regulated on activation of human B cells and B-cell lines [53]. In addition, neurons and glial cells of aged rats are reported to exhibit increased levels of CatE. This overexpression of cathepsin E is suggested to be related to neuronal degeneration and reactivation of glial cells during the normal aging process of the brain [86,87]. CatE is found in cerebral cortex and brainstem of rats. Its expression, localization and conversion to the mature enzyme are strongly age-dependent. Cathepsin E is co-localized with lipofuscin and also with carboxy-terminal fragments of Alzheimer’s amyloid precursor protein (APP). The corresponding fragments might be

46

amylogenic, and therefore cathepsin E is perhaps involved in altered APP catabolism [88]. In contrast, it is observed that the expression of cathepsin E is reduced in thymocytes of aged rats. This decrease in CatE expression is restricted to thymocytes, whereas spleen cells show no significant alteration in its expression [86]. Moreover, CatE expression is also reported to vary between different rat tissues at different stages of embryonic development [89].

Distinguishing Features The dimeric/monomeric interconversion of cathepsin E can easily be detected via SDS-PAGE under nonreducing/ reducing conditions. In a recent study, a highly selective substrate (Mca-Ala-Gly-Phe-Ser-Leu-Pro-Ala-Lys(Dnp)DArg-CONH2) for cathepsin E has been reported [27]. This Cat E selectivity was established by having LeukPro-residues at the scissile peptide bond, showing a 265-fold difference in the net fluorescence between CatE and D. A specific inhibitor for CatE, the Ascaris pepsin inhibitor, which has no activity against cathepsin D, has been described [30]. Kitamura et al. [35] have also generated peptides that inhibit cathepsin E specifically at a submicromolar IC50. Several cathepsin E antibodies are commercially available, such as the anti-human Cathepsin E antibody AF1294 and the monoclonal anti-human Cathepsin E antibody MAB1294 from R & D Systems. Antibodies against cathepsin E from other species are also available from different companies.

Further Reading For research articles, see Yanagawa et al. [70], Tsukuba et al. [12,73]. For reviews, see Zaidi et al. [20,90].

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Nousheen Zaidi Department of Oncology, Johnson and Johnson, Pharmaceutical Research and Development, A Subdivision of Janssen Pharmaceutics, Beerse, Belgium. Email: [email protected]

Hubert Kalbacher Interfaculty Institute of Biochemistry, University of Tuebingen, Hoppe-Seyler-Str. 4 72076 Tuebingen, Germany. Email: [email protected] This article is a revision of the previous edition article by John Kay, Volume 1, pp. 33
38, r 2004, Elsevier Ltd. Handbook of Proteolytic Enzymes, 3rd Edn ISBN: 978-0-12-382219-2

© 2013 Elsevier Ltd. All rights reserved. DOI: http://dx.doi.org/10.1016/B978-0-12-382219-2.00006-5