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Biochemical and Biophysical Research Communications 367 (2008) 517–522

Mini Review

Cathepsin E: A mini review

Nousheen Zaidi, Hubert Kalbacher *

Medical and Natural Sciences Research Centre, University of Tubingen, Ob dem Himmelreich 7, 72074 Tuebingen, Germany

Received 21 December 2007Available online 4 January 2008

Abstract

Cathepsin E is a major intracellular aspartic protease which is predominantly present in the cells of immune system and is frequentlyimplicated in antigen processing via the MHC class II pathway. In the present review some of the known features of cathepsin E such astissue distribution, subcellular localization, enzymatic properties, intracellular trafficking, gene regulation and associated physiologicalconditions are highlighted.� 2007 Elsevier Inc. All rights reserved.

Keywords: Cathepsin E; Aspartic proteases

Cathepsin E (CatE) is an intracellular aspartic proteaseof the pepsin superfamily. CatE is highly homologous tothe analogous aspartic protease cathepsin D. Early reportsimplicated the presence of an aspartic protease distinctfrom cathepsin D in vertebrate cells [1]. Different designa-tions for this enzyme have been suggested, such as cathep-sin D-like proteinase [2], gastric mucosa non-pepsin acidproteinase [3], slow moving proteinase [4] and erythrocytemembrane acid proteinase [5]. Later, it was shown that allof these activities were mediated by the same enzyme,which was termed cathepsin E (reviewed in [1]). In the pres-ent review, the findings regarding the expression, biochem-ical properties and functions of cathepsin E aresummarized and the discrepancies and similarities in theconclusions drawn by different groups are highlighted.

Tissue distribution and subcellular localization

Cathepsin E is mainly present in cells of the immune sys-tem, including antigen-presenting cells (APC) such as lym-phocytes [6], microglia [7], dendritic cells [8], Langerhanscells [9], interdigitating reticulum cells [9] and human Mcells [10]. Cathepsin E is not present in resting B-lympho-

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doi:10.1016/j.bbrc.2007.12.163

* Corresponding author. Fax: +49 7071 294507.E-mail address: kalbacher@uni-tuebingen.de (H. Kalbacher).

cytes but is up-regulated late in human B cell activationat both the mRNA and protein level [11]. It has also beendetected in gastric epithelial cells [12] and osteoclasts [13].Furthermore, tissue-specific distribution of CatE is notthe same in different mammalian species, e.g. it has beendetected in red blood cells from humans and rats but notguinea pigs, cattle, goats or pigs [14].

The intracellular localization of cathepsin E alsoappears to vary according to cell type. In APC such asmicroglia [15], dendritic cells [8] and macrophages CatEis mainly present in endosomal compartments. In contrast;in erythrocytes [5], gastric cells [2], renal proximal tubulecells [16] and osteoclasts [13], it is found in the plasmamembrane. CatE is also detected in ER and Golgi complexin different cell types, such as gastric epithelial cells [4] Lan-gerhans cells, interdigitating reticulum cells [9] and humanM cells [10].

Activity and specificity

Active sites

CatE possesses two homologous domains, each contain-ing the highly conserved tripeptide sequence DTG. Thesetwo domains are involved in the formation of the active site[17]. The DTG sequence is present in all species with the

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exception of rabbit CatE, in which the tripeptide sequencenear the N-terminal region is replaced by DTV [17]. Liuet al. synthesized active site mutants of CatE by site-direc-ted mutagenesis to examine the significance of these resi-dues [18]. They found that in mouse CatE Asp98,Asp283, and Thr284 are indeed critical for catalysis [18].

Specificity

Cathepsin E, like cathepsin D, prefers hydrophobicamino acids at the P1 and P10 positions [1]. b-Branched res-idues, e.g. Val and Ile are not allowed at P1 [19]. PositionP20 accepts a broad range of amino acids, includingcharged and polar ones [19]. For CatE, the basic residue,e.g. Lys is acceptable at position P2, which is not the casefor CatD [20]. The presence of proline at P4 might beimportant and may facilitate the presentation of the scissilepeptide bond in extended b strands for cleavage by theenzyme [21].

According to some reports, CatE also retains activity atneutral pH and shows a distinct cleavage specificity [22].However, in all studies reporting proteolytic activity ofCatE at pH 7.4, the enzyme was isolated either fromhuman gastric mucosa [23] or human red blood cells [23].In our recent study, we investigated proteolytic potentialof recombinant cathepsin at neutral pH [24]. Our resultsfail to reveal any proteolytic activity of recombinant CatEat neutral pH even in the presence of ATP, which is knownto stabilize this enzyme.

Assays

The most common assay for measuring aspartic prote-ase activity in biological samples employs acid-denaturedbovine hemoglobin as the substrate [5]. This method istime-consuming and has the additional disadvantage thatit cannot discriminate CatE activity from other asparticproteases. Several synthetic chromogenic or fluorogenicsubstrates have been developed to measure CatE activity[25]. These methods are simple and fast but the describedsubstrates are restricted in their selectivity. In a recentreport a new selective substrate for cathepsin E based onthe cleavage site sequence of a2-macroglobulin has beendescribed [26]. However, this substrate was also not exclu-sive for CatE because it had low level of activity for CatDand pepsin as well.

Recently, we have developed a new approach for distin-guishing cathepsin E and D activity in subcellular fractions[27]. In this method we made use of our new monospecificCatE antibody and substrate Mca-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Leu-Lys(Dnp)-D-Arg-NH2 [where Mca is(7-methoxycoumarin-4-yl)acetyl and Dnp is dinitrophenyl].This substrate is digested by both cathepsin E and cathep-sin D and therefore can be used to detect the total asparticproteinase activity (TAPA) in biological samples [25]. CatEis depleted from the samples by immunoprecipitation usingthis new highly-specific antibody and the remaining activity

is therefore due to CatD. The decrease of activity can thusbe assigned to CatE. For a schematic representation of theassay, see Fig. 1.

Inhibitors

The most widely used and potent inhibitor of CatE ispepstatin A (PepA). Additionally, a wide variety of syn-thetic peptidomimetic inhibitors has been described thatmediate potent CatE inhibition [28]. However, none ofthese small molecule inhibitors discriminates clearlybetween cathepsin D and E. A specific inhibitor for CatE,the Ascaris pepsin inhibitor, which has no activity againstcathepsin D, has been described [29], but is not readilyavailable in quantities sufficient for functional studiesbecause of difficulties in purification.

Although pepstatin A is not specific toward CatE orCatD, it is widely used in cell-based studies aimed at under-standing the function of these enzymes; using cells fromCatD-deficient mice allows pepstatin to specifically targetCatE. However, another limitation of PepA for cell-basedstudies is that it is inefficiently transported across the cellmembrane [30]. To address this problem mannose–pepsta-tin conjugates were recently developed as targeted inhibi-tors of antigen processing [8]. These mannosylatedconjugates showed higher solubility than pepstatin in waterand were efficiently incorporated into the cells via receptor-mediated uptake. This approach is obviously limited tocells carrying mannose receptors.

Our group has synthesized new cell-permeable asparticprotease inhibitors that are not specific for any particularcell type. To achieve this, the most frequently employedCell-penetrating peptides, namely pAntp(43–58) (penetratin),Tat(49–60), and 9-mer of L-arginine (R9), were synthesized.Pepstatin A was then coupled to these peptides. We foundthat the bioconjugate PepA-penetratin (PepA-P) was themost efficient cell-permeable aspartic protease inhibitor,and may serve as a useful new tool to investigate the roleof aspartic proteinases in different types of cells [31].

Regulation of gene expression

Cathepsin E has a limited tissue distribution. It is appar-ent that there must be a mechanism that regulates the tran-scription of the gene encoding CatE so that expression isfacilitated only in certain types of cells. The promoterregion flanking the procathepsin E gene from human [32]and mouse [33] do not contain a TATA box. Instead, bothsequences contain an initiator element [32,33] which offersan alternative binding site for TFIID (transcription factorIID) to initiate gene transcription [34]. Cook et al. havereported that regulation of human and mouse procathepsinE gene expression is not influenced by CpG methylation[35]. Furthermore, transcription of the CatE gene is depen-dent on the balance between the effects produced by posi-tive-acting tissue-specific transcription factors such as

Fig. 1. (A) TAPA (total aspartic proteinase activity) was measured by hydrolysis of fluorogenic substrate. (B) The observed activity was pepstain A(PepA) sensitive hence was only due to aspartic proteases. (C) Samples were subjected to immunoprecipitation (IP) using our monospecific antibody beforeabove described assay. Now increase in fluorescence intensity is exclusively caused by CatD. The difference between total aspartic proteinase and CatDactivity can be assigned to CatE activity.

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GATA1 and PU1 and the negative influence of the ubiqui-tous factor YY1 [35].

Additionally, it is also known that CatE transcription isnegatively regulated by CIITA (Class II transactivator), anon-DNA-binding transcription factor [36]. The signifi-cance of regulation of CatE gene expression by this tran-scription factor will be discussed later in this review.

Processing, maturation and intracellular trafficking of

cathepsin E

The processing events of pro-cathepsin E include theremoval of propeptide, glycosylation and formation of adisulphide bond between two cysteine residues at theNH2-terminal region to yield a homodimer. CatE is synthe-sized as a 46 kDa precursor and is later converted into the42 kDa mature form. In this section, we will discuss themolecular basis of processing and intracellular traffickingof CatE.

Role of propeptide in processing and maturation

The unique structural characteristics of propeptide areconserved in all species [37,38]. The propeptide is com-posed of 40 amino acid residues and is highly positivelycharged. Therefore, it is hypothesized that the propeptideis bound to active CatE mainly through electrostaticinteractions.

It has been reported that the propeptide of CatE playsan important role in the correct folding, maturation andtargeting of this protein to its final destination. Tsukubaet al. have constructed CatE mutants lacking the propep-tide (Leu23-Phe58). They reported that this mutant proteinwas neither processed not matured and was found mostlyin the ER, in comparison to the wild type which was mainlylocated in endosomes [39].

Significance of catalytic activity of CatE for its acid-

dependent auto-activation

Pro-cathepsin E is readily auto-activated upon briefincubation at an acidic pH and transforms to the matureform. The active site residues (Asp98, Asp283, andThr284) are critical for catalysis, as mentioned before inthis review [18]. Tsukuba et al. published that, active sitemutants of CatE, in which one or both aspartic acid resi-dues were substituted with alanine, not only lacked cata-lytic activity but were also unable to mature, thus remainstably as a 46 kDa precursor [39]. This indicates that cata-lytic activity is essential for the processing and maturationof this enzyme.

ER-retention sequence in maturation and intracellular

targeting of cathepsin E

Finley and Kornfeld reported that amino acids 1–48 ofmature cathepsin E are important for its retention in theER [40]. Tsukuba et al. constructed a mutant lacking mostof this putative ER-retention sequence, which was not con-verted into a mature form of the enzyme, was rapidlydegraded in the cells and could not be detected in the endo-somes [39]. This indicates that the putative ER-retentionsequence is required for correct folding, processing, matu-ration and targeting of CatE to endosomes.

Role of N-glycosylation of CatE in processing, maturation

and intracellular targeting

CatE is known to be N-glycosylated with high-mannoseand/or complex oligosaccharides in the native state. InAPC such as microglia and macrophages, the mature formof CatE is localized in endosomal compartments and is N-glycosylated mainly with complex-type oligosaccharides

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[7]. CatE from erythrocyte membranes (human and rat)[41] and thymocytes (rat) [42] is also N-glycosylated withcomplex-type oligosaccharides, while the enzyme fromthe spleen (rat) [37] and stomach (rat) [43] has high man-nose-type oligosaccharides. This is interesting in view ofthe fact that CatE shows different subcellular localizationin different cell types, as discussed previously in this review,and it has been suggested that the nature of its oligosaccha-ride chains may be cell-specific or may vary with its cellularlocalization.

Yasuda et al. constructed an N-glycosylation mutant (bychanging the N residue to Q and D at position 73 and 305in potential glycosylation sites of rat CatE) [44]. It wasfound that this mutant was less stable to temperature andpH than glycosylated CatE, although its catalytic proper-ties were equivalent to wild type. Tsukuba et al. reportedthat N-glycosylation mutants were stably retained in cellsbut were not processed to the mature form and were exclu-sively confined to the ER [39]. Hence, N-glycosylation ofCatE seems to play an important role in processing, matu-ration and trafficking of CatE to its final destination, butmight not be essential for correct folding of the enzyme.

Physiological roles of cathepsin E

Pathological conditions developing in CatE-deficient mice

CatE-deficient mice are found to develop atopic derma-titis-like skin lesions [45]. It was reported that they alsoshow an increased susceptibility to bacterial infection asso-ciated with decreased expression of multiple cell surfaceToll-like receptors [46]. According to a recent study [47],CatE deficiency induces a novel form of lysosomal storagedisorder characterized by accumulation of lysosomal mem-brane sialoglycoproteins and the elevation of lysosomal pHin macrophages.

Pathological conditions in which over-expression of CatE is

observed

CatE is expressed in pancreatic ductal adenocarcinoma,and its presence in pancreatic juice may be a diagnosticmarker for this cancer [48]. Increased levels of CatE in neu-rons and glial cells of aged rats are suggested to be relatedto neuronal degeneration and reactivation of glial cells dur-ing the normal aging process of the brain [49].

Potential role in the MHC class II pathway

Several lines of evidence suggest that cathepsin E plays arole in antigen processing via the MHC class II pathway.CatE is detectable in APC such as Langerhans and inter-digitating dendritic cells [9] and its expression is up-regu-lated on activation of human B cells and B cell lines [11].Moreover, a peptide derived from CatE was found to beassociated with HLA-DR molecules in human EBV-trans-

formed B cells [50], suggesting that it is found at the site ofassembly of peptides and MHC class II complexes.

On the basis of studies utilizing specific inhibitors andcathepsin-deficient mice, CatE has been implicated in anti-gen processing. The first study to report involvement ofcathepsin E in antigen processing was carried out by Ben-nett et al. [51]. This study made use of a CatE-specific Asca-

ris inhibitor and demonstrated that CatE is essential forprocessing of ovalbumin by murine B cells, whereas, pre-sentation of synthetic OVA-derived peptides correspondingto the epitope was not affected by the specific CatE inhib-itor. Similar results were obtained when the role of CatE inantigen presentation by microglia [15] and dendritic cells [8]derived from CatD-deficient mice was examined. CatE wasfound to be involved in processing of ovalbumin but not inthe presentation of OVA-derived peptides.

An immunological role for CatE in microglia was fur-ther supported by the observation that its expression atthe RNA level is enhanced after treatment with IFN-cwhile expression of CatD, CatB, CatL and CatS remainsconstant [15]. Microglia also show strong pepstatin A-sen-sitive antigen-presenting ability for both native OVA andOVA-peptides only after IFN-c treatment. Because IFN-c enhances only CatE expression, it can be suggested thatCatE plays a major role in antigen processing. IFN-c-acti-vated peripheral macrophages also showed similar results[15]. The association of CatE expression and activity withIFN-c treatment in microglia indicates the involvementof CatE in the MHC class II pathway because IFN-c isknown to induce MHC class II expression as well as co-stimulatory molecules required for T-helper cell activation[52]. Moreover, in microglia and dendritic cells, CatE ispresent in the early endosomes and partially in lysosomeswhile CatD has purely lysosomal localization [8,15]. Thisindicates that localization of CatE is far more appropriatefor processing of exogenous antigens than CatD, becauseexogenous antigens are first processed in early endosomesafter endocytosis.

An interesting report by Yee et al. [36] provided the firstlink between immune-specific regulation and Ag-process-ing by CatE. They observed that CatE expression is nega-tively regulated by the MHC class II transactivator(CIITA), a non-DNA-binding transcription factor whichactivates and is required for the expression of MHC IIand other genes related to antigen presentation [53]. CIITAis known to modulate immune responses also by repressingthe transcription of other genes including IL4, collagen a2and Fas ligand [36]. It was reported that CIITA-deficientmurine and human B cells express higher amounts of CatEthan wild-type B cells, whereas cells overexpressing CIITAshowed decreased CatE mRNA, protein and proteolyticactivity [36]. This negative regulation of CatE expressionby CIITA was very specific toward CatE and did not affectCatD. This regulation of CatE by CIITA might either inhi-bit or promote Ag-processing and presentation. If the lat-ter, then production of antigenic epitopes by CatE couldbe down-regulated as a part of an activation-induced shift

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from Ag-processing to Ag-presentation [36]. Alternatively,it is possible that CatE cleaves productive antigens, such asAg-MHC complexes or chaperones and therefore it isdown-regulated by CIITA. This explanation is supportedby the fact that CatE cleaves human a2-macroglubulin, acapture protein that promotes internalization and degrada-tion of target proteins [54].

Most of the above-mentioned reports point toward theinvolvement of CatE in antigen processing and but not inthe presentation of the synthesized antigenic peptides[8,15]. Thus, the general conclusion from these findings isthat inhibition of CatE does not alter the number ofMHC molecules at the cell surface which are able to bindprocessed peptide, and that inhibition of CatE might alsohave no effect on invariant chain processing because ifthe Ii processing is blocked, loading of MHC moleculeswith processed peptides is also blocked [55].

However, there are several discrepant reports showingthat aspartic protease inhibitors effectively prevent the pro-cessing of the invariant chain [56]. CatE could be involvedin the initial cleavage of the invariant chain, in which anunidentified endosomal aspartic protease has been impli-cated [56,57]. Although CatE is absent from the classicallate endosomal class II loading compartments [8], whichcontain cathepsin S, and in which invariant chain process-ing is thought to take place, it is possible that the invariantchain traffics transiently via an early endosomal compart-ment en route to the class II-loading pathway [58].

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