Cathepsin E: a mini review

Cathepsin E is a major intracellular aspartic protease which is predominantly present in the cells of immune system and is frequently implicated in antigen processing via the MHC class II pathway. In the present review some of the known features of cathepsin E such as tissue distribution, subcellular localization, enzymatic properties, intracellular trafficking, gene regulation and associated physiological conditions are highlighted.     

Cathepsin E: a novel target for regulation by class II transactivator

The aspartic proteinase cathepsin E (CatE) has been implicated in Ag processing. In this study we report that CatE expression is negatively regulated by the MHC class II transactivator (CIITA). CIITA-deficient murine and human B cells expressed greater CatE than wild-type B cells, whereas overexpression of CIITA in a human gastric carcinoma cell line, AGS, resulted in decreased CatE mRNA and protein. AGS cells expressing CIITA also exhibited decreased processing of OVA Ag. Inhibition of CatE expression is specific to the type III CIITA isoform and maps to the acidic and proline/serine/threonine-rich (PST) protein domains of CIITA. We found that CatE expression is inducible by PU.1 and p300, and that this induction can be reversed by CIITA. These findings demonstrate a novel phenomenon: regulation of CatE Ag processing by CIITA in an isoform-dependent manner.     

Crystal structure of an activation intermediate of cathepsin E

Cathepsin E is an intracellular, non-lysosomal aspartic protease expressed in a variety of cells and tissues. The protease has proposed physiological roles in antigen presentation by the MHC class II system, in the biogenesis of the vasoconstrictor peptide endothelin, and in neurodegeneration associated with brain ischemia and aging. Cathepsin E is the only A1 aspartic protease that exists as a homodimer with a disulfide bridge linking the two monomers. Like many other aspartic proteases, it is synthesized as a zymogen which is catalytically inactive towards its natural substrates at neutral pH and which auto-activates in an acidic environment. Here we report the crystal structure of an activation intermediate of human cathepsin E at 2.35A resolution. The overall structure follows the general fold of aspartic proteases of the A1 family, and the intermediate shares many features with the intermediate 2 on the proposed activation pathway of aspartic proteases like pepsin C and cathepsin D. The pro-sequence is cleaved from the protease and remains stably associated with the mature enzyme by forming the outermost sixth strand of the interdomain beta-sheet. However, different from these other aspartic proteases the pro-sequence of cathepsin E remains intact after cleavage from the mature enzyme. In addition, the active site of cathepsin E in the crystal is occupied by N-terminal amino acid residues of the mature protease in the non-primed binding site and by an artificial N-terminal extension of the pro-sequence from a neighboring molecule in the primed site. The crystal structure of the cathepsin E/pro-sequence complex, therefore, provides further insight into the activation mechanism of aspartic proteases.     

Cathepsin E (EC 3.4.23.34)--a review

Cathepsin E belongs to the third class of enzymes - hydrolases, a subclass of peptide bond hydrolases and a sub-subclass of endopeptidases with aspartic catalytic sites. Cathepsin E is an endopeptidase with substrate specificity similar to that of cathepsin D. In a human organism, cathepsin E occurs in: erythrocytes, thymus, dendritic cells, epithelial M cells, microglia cells, Langerhans cells, lymphocytes, epithelium of gastrointestinal tract, urinary bladder, lungs, osteoclasts, spleen and lymphatic nodes. In human cells, loci of the gene of pre-procathepsin E are located on chromosome 1 in the region 1231-32. The catalytic site of cathepsin E is two residues of aspartic acid - Asp96 and Asn281, occurring in amino acid triads with sequences DTG96-98 and DTG281-283. To date, no particular role of cathepsin E in the metabolism of proteins in normal tissues has been found. However, it is known that there are many documented pathological conditions in which overexpression of cathepsin E occurs.     

A novel cell penetrating aspartic protease inhibitor blocks processing and presentation of tetanus toxoid more efficiently than pepstatin A

Selective inhibition of enzymes involved in antigen processing such as cathepsin E and cathepsin D is a valuable tool for investigating the roles of these enzymes in the processing pathway. However, the aspartic protease inhibitors, including the highly potent pepstatin A (PepA), are inefficiently transported across the cell membrane and thus have limited access to antigen processing compartments. Previously described mannose-pepstatin conjugates were efficiently taken up by the cells via receptor mediated uptake. However, cells without mannose receptors are unable to take up these conjugates efficiently. The aim of the present study was to synthesize new cell-permeable aspartic protease inhibitors by conjugating pepstatin A with well-known cell penetrating peptides (CPPs). To achieve this, the most commonly used CPPs namely pAntp_(43-58) (penetratin), Tat_(49-60), and 9-mer of l-arginine (R9), were synthesized and coupled to pepstatin. The enzyme inhibitory properties of these bioconjugates and their cellular uptake into MCF7 (human breast cancer cell line), Boleths (EBV-transformed B-cell line) and dendritic cells (DC) were the focus of our study. We found that the bioconjugate PepA-penetratin (PepA-P) was the most efficient cell-permeable aspartic protease inhibitor tested, and was more efficient than unconjugated PepA. Additionally, we found that PepA-P efficiently inhibited the tetanus toxoid C-fragment processing in peripheral blood mononuclear cells (PBMC), primary DC and in primary B cells. Therefore, PepA-P can be used in studying the role of intracellular aspartic proteases in the MHC class II antigen processing pathway. Moreover, inhibition of tetanus toxoid C-fragment processing by PepA-P clearly implicates the role of aspartic proteinases in antigen processing.     

MHC class II-restricted presentation of native protein antigen by B cells is inhibitable by cycloheximide and brefeldin A

The presentation of protein Ag with MHC class II proteins involves the uptake of the protein Ag by endocytosis followed by processing, probably proteolysis, in an intracellular acidic compartment. However, there remains considerable controversy as to the precise route taken by the antigen and the MHC class II protein during this process. The unusual stability of Ag-MHC class II protein complexes has led to speculation that antigen can only associate with newly synthesized MHC class II molecules. An alternate possibility is that the MHC class II binding site can be regenerated within the cell during internalization and recycling of MHC class II proteins. To address these possibilities, three different murine B lymphoma lines were tested for their ability to process and present native protein Ag in the presence of the protein synthesis inhibitor cycloheximide or the protein synthesis inhibitor cycloheximide or the protein export inhibitor, Brefeldin A. Both agents blocked the presentation of native OVA or native hen egg lysozyme to Ag-specific T cell hybridomas. No effect was seen on peptide presentation or on presentation to allo- or autoreactive T cells. Inasmuch as Brefeldin A has been previously shown to block protein export without affecting protein internalization or protein degradation in the endocytic pathway, the simplest interpretation of these data is that antigenic fragments generated in the APC after uptake by the endocytic pathway, preferentially associate with newly synthesized rather than mature MHC class II proteins.     

The crystal structure of human cathepsin F and its implications for the development of novel immunomodulators

Cathepsin F is a lysosomal cysteine protease of the papain family, and likely plays a regulatory role in processing the invariant chain that is associated with the major histocompatibility complex (MHC) class II. Evidence suggests that inhibiting cathepsin F activity will block MHC class II processing in macrophages. Consequently, inhibitors of this enzyme may be useful in treating certain diseases that involve an inappropriate or excessive immune response. We have determined the 1.7A structure of the mature domain of human cathepsin F associated with an irreversible vinyl sulfone inhibitor. This structure provides a basis for understanding cathepsin F's substrate specificity, and suggests ways of identifying potent and selective inhibitors of this enzyme.     

Cathepsin E in follicle associated epithelium of intestine and tonsils: localization to M cells and possible role in antigen processing

A specific rabbit anti-human serum was used selectively to localize the aspartic proteinase cathepsin E to follicle associated epithelium (FAE) of human and rat intestine, including jejunum, ileum, appendix, colon and rectum, as well as of human palatine, pharyngeal and lingual tonsils. Coexpression of class II histocompatibility antigen HLA-DR antigen has been observed in some of the cathepsin E-positive epithelial cells. In addition, cathepsin E has been detected in a few mononuclear cells of intestinal lymphoid structures and tonsils resembling interdigitating reticulum cells of lymph nodes. Another aspartic proteinase, cathepsin D, has been found to be poorly represented in FAE and intensely expressed by macrophages. Electron immunocytochemistry localized cathepsin E to endosomal vesicles and endoplasmic reticulum of M cells in rat and human ileum as well as of M-like cells in human palatine tonsil. The results suggest a possible role of endosomal cathepsin E in the processing of macromolecules and microorganisms transported by M cells and related epithelial cells to mucosal associated lymphoid tissue (MALT).     

Cathepsin B cleavage of Ii from class II MHC alpha- and beta-chains

Class II MHC-associated invariant chain (Ii) might regulate binding of digested peptides to the Ag binding site (desetope) of class II MHC proteins by directly or allosterically blocking that site until cleavage and release of Ii from MHC alpha- and beta-chains at the time of peptide charging. We examined the cleavage and release of Ii from class II MHC alpha/beta Ii trimers by cathepsin B, which has been shown by others to colocalize with class II MHC molecules in intracellular compartments and to generate antigenic peptide fragments. Cathepsin B at pH 5.0 cleaved and released Ii from class II MHC alpha- and beta-chains. Cathepsin B digested Ii from alpha- and beta-chains in a dose-dependent fashion, yielding 23-, 21-, and 10-kDa fragments. Blockage of cathepsin B activity with leupeptin restored the 2D(nonequilibrium pH gradient gel electrophoresis/SDS) PAGE patterns of Ii and sialic acid-derivatized forms of Ii seen without the protease. The fragmentation pattern of cathepsin D treatment was different from that of cathepsin B, yielding 25-kDa intermediates.     

Action of brain cathepsin B,cathepsin D,and high-molecular-weight aspartic proteinase on angiotensins I and II

The action of three previously isolated electrophoretically homogeneous brain proteinases—cathepsin B (EC 3.4.22.1), cathepsin D (EC 3.4.23.5), and high-molecular-weight aspartic proteinase (M_r=90K; EC 3.4.23.−)—on human angiotensins I and II has been investigated. The products of enzymatic hydrolysis have been identified by thin-layer chromatography on Silufol plates using authentic standards and by N-terminal amino acid residue analysis using a dansyl chloride method. Cathepsin D and high-molecular-weight aspartic proteinase did not split angiotensin I or angiotensin II. Cathepsin B hydrolyzed angiotensin I via a dipeptidyl carboxypeptidase mechanism removing His-Leu to form angiotensin II, and it degraded angiotensin II as an endopeptidase at the Val³-Tyr⁴ bond. Cathepsin B did not split off His-Leu from Z-Phe-His-Leu. Brain cathepsin B may have a role in the generation and degradation of angiotensin II in physiological conditions. Special Issue dedicated to Dr. Eugene Kreps.     

MHC molecules protect T cell epitopes against proteolytic destruction.

There is a subtle duality in the role of proteolytic enzymes in Ag processing. They are required to fragment protein Ag ingested by APC. However, prolonged exposure to proteolytic enzymes may lead to a complete degradation of the Ag, leaving nothing for the T cell system to recognize. What ensures that some of the Ag is salvaged? Using a cell-free system we demonstrate that an Ag fragment, once bound to a MHC class II molecule, is effectively protected against proteolytic destruction by cathepsin B and pronase E. The bound fragment, however, can be modified by aminopeptidase N. We suggest that MHC class II molecules play an important regulatory role in the physiologic processing of Ag.     

Identification of cathepsin B,D and H in the larval midgut of colorado potato beetle,Leptinotarsa decemlineata say (Coleoptera: Chrysomelidae)

The larval midgut of the Colorado beetle, Leptinotarsa decemlineata contains cathepsin B, D and H activity detected by use of haemoglobin, synthetic substrates specific for each enzyme, pH at which the substrate was maximally hydrolysed and effects of potential activators and inhibitors on proteolytic activity. Cysteine proteases cathepsin B, and H were activated by thiol compounds and inhibited by iodoacetamide, TLCK and epoxysuccinyl-leucyl-amido(guanidino)butane (E-64) a cysteine specific proteinase inhibitor. Cathepsin B was distinguished from H by hydrolysis of benzoyloxycarbonyl-Ala-Arg-Arg-methoxynaphthylamide, a cathepsin B specific substrate and inhibition of substrate hydrolysis by leupeptin. Cathepsin H activity, detected using the specific substrate arginine-naphthylamide, was insensitive to leupeptin. Cathepsin D had maximal activity at pH 4.5 and was inhibited by pepstatin, an aspartic proteinase inhibitor.     

Application of a novel highly sensitive activity-based probe for detection of cathepsin G

Cathepsins are crucial in antigen processing in the major histocompatibility complex class II (MHC II) pathway. Within the proteolytic machinery, three classes of proteases (i.e., cysteine, aspartic, and serine proteases) are present in the endocytic compartments. The combined action of these proteases generates antigenic peptides from antigens, which are loaded to MHC II molecules for CD4+ T cell presentation. Detection of active serine proteases in primary human antigen-presenting cells (APCs) is restricted because of the small numbers of cells isolated from the peripheral blood. For this purpose, we developed a novel highly sensitive α-aminoalkylphosphonate diphenyl ester (DAP) activity-based probe to detect the serine protease cathepsin G (CatG) in primary APCs and after Epstein-Barr virus (EBV) exposure. Although CatG activity was not altered after short-term exposure of EBV in primary myeloid dendritic cells 1 (mDC1s), the aspartic protease cathepsin D (CatD) was reduced, suggesting that EBV is responsible for mitigating the presentation of a model antigen tetanus toxoid C-fragment (TTCF) by reduction of CatD. In addition, CatG activity was reduced to background levels in B cells during cell culture; however, these findings were independent of EBV transformation. In conclusion, our activity-based probe can be used for both Western blot and 96-well-based high-throughput CatG detection when cell numbers are limited.     

Functional requirements for the lysosomal thiol reductase GILT in MHC class II-restricted antigen processing

Ag processing and presentation via MHC class II is essential for activation of CD4(+) T lymphocytes. gamma-IFN-inducible lysosomal thiol reductase (GILT) is present in the MHC class II loading compartment and has been shown to facilitate class II Ag processing and recall responses to Ags containing disulfide bonds such as hen egg lysozyme (HEL). Reduction of proteins within the MHC class II loading compartment is hypothesized to expose residues for class II binding and protease trimming. In vitro analysis has shown that the active site of GILT involves Cys(46) and Cys(49), present in a CXXC motif that shares similarity with the thioredoxin family. To define the functional requirements for GILT in MHC class II Ag processing, a GILT-deficient murine B cell lymphoma line was generated and stably transduced with wild-type and cysteine mutants of GILT. Intracellular flow cytometric, immunoblotting, and immunofluorescence analyses demonstrated that wild-type and mutant GILT were expressed and maintained lysosomal localization. Transduction with wild-type GILT reconstituted MHC class II processing of a GILT-dependent HEL epitope. Mutation of either Cys(46) or Cys(49) abrogated MHC class II processing of a GILT-dependent HEL epitope. In addition, biochemical analysis of these mutants suggested that the active site facilitates processing of precursor GILT to the mature form. Precursor forms of GILT-bearing mutations in Cys(200) or Cys(211), previously found to display thiol reductase activity in vitro, could not mediate Ag processing. These studies demonstrate that the thiol reductase activity of GILT is its essential function in MHC class II-restricted Ag processing.     

Invariant chain processing is independent of cathepsin variation between primary human B cells/dendritic cells and B-lymphoblastoid cells

As part of the endocytic antigen processing pathway, proteolytic cleavage of the invariant chain (Ii) is important for the generation of class II-associated invariant chain peptide (CLIP). CLIP remains associated with the major histocompatibility complex (MHC) class II molecule to prevent premature loading of antigenic peptides. Cysteine proteases, such as Cathepsin S (CatS), CatL, or CatV, play a pivotal role in the final stage of Ii degradation depending on the cell type studied. Less is known regarding the early stages of Ii processing. We therefore explored whether the serine protease CatG is involved in the initial step of Ii degradation in primary antigen presenting cells (APC), since the cathepsin distribution differs between primary APC and cell lines. While primary human B cells and dendritic cells (DC) do harbor CatG, this protease is absent in B-lymphoblastoid cells (BLC) or monocyte-derived DC generated in vitro. In addition, other proteases, such as CatC, CatL, and the asparagine endoprotease (AEP), are active in BLC and monocyte-derived DC. Here we demonstrate that CatG progressively degraded Ii in vitro resulting in several intermediates. However, pharmacological inhibition of CatG in primary B cells and DC did not alter Ii processing, indicating that CatG is dispensable in Ii degradation. Interestingly, stalling of cysteine proteases by inhibition in BLC vs. primary B cells and DC did not result in any differences in the generation of distinct Ii intermediates between the cells tested, suggesting that Ii processing is independent of the cathepsin variation within professional human APC.     

Emerging functional roles of cathepsin E

Cathepsin E is an intracellular aspartic protease of the endolysosomal pathway. It has been implicated in several physiological and pathological processes however, its exact functional role is yet to be elucidated. The present review gives an account of the major physiological functions that are associated to cathepsin E by various research groups and highlights the conditions developed in cathepsin E deficiency or the conditions where overexpression of cathepsin E is observed.     

Asparagine endopeptidase is not essential for class II MHC antigen presentation but is required for processing of cathepsin L in mice

Class II MHC molecules survey the endocytic compartments of APCs and present antigenic peptides to CD4 T cells. In this context, lysosomal proteases are essential not only for the generation of antigenic peptides but also for proteolysis of the invariant chain to allow the maturation of class II MHC molecules. Recent studies with protease inhibitors have implicated the asparagine endopeptidase (AEP) in class II MHC-restricted Ag presentation. We now report that AEP-deficient mice show no differences in processing of the invariant chain or maturation of class II MHC products compared with wild-type mice. In the absence of AEP, presentation to primary T cells of OVA and myelin oligodendrocyte glycoprotein, two Ags that contain asparagine residues within or in proximity to the relevant epitopes was unimpaired. Cathepsin (Cat) L, a lysosomal cysteine protease essential for the development to CD4 and NK T cells, fails to be processed into its mature two-chain form in AEP-deficient cells. Despite this, the numbers of CD4 and NK T cells are normal, showing that the single-chain form of Cat L is sufficient for its function in vivo. We conclude that AEP is essential for processing of Cat L but not for class II MHC-restricted Ag presentation.     

Cathepsin S controls MHC class II-mediated antigen presentation by epithelial cells in vivo

Epithelial cells at environmental interfaces provide protection from potentially harmful agents, including pathogens. In addition to serving as a physical barrier and producing soluble mediators of immunity, such as cytokines or antimicrobial peptides, these cells are thought to function as nonprofessional APCs. In this regard, intestinal epithelial cells are particularly prominent because they express MHC class II molecules at the site of massive antigenic exposure. However, unlike bone marrow-derived professional APC, such as dendritic cells or B cells, little is known about the mechanisms of MHC class II presentation by the nonprofessional APC in vivo. The former use the lysosomal cysteine protease cathepsin S (Cat S), whereas thymic cortical epithelial cells use cathepsin L (Cat L) for invariant chain degradation and MHC class II maturation. Unexpectedly, we found that murine Cat S plays a critical role in invariant chain degradation in intestinal epithelial cells. Furthermore, we report that nonprofessional APC present a class II-bound endogenous peptide to naive CD4 T cells in vivo in a Cat S-dependent fashion. These results suggest that in vivo, both professional and nonprofessional MHC class II-expressing APC use Cat S, but not Cat L, for MHC class II-mediated Ag presentation.