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Proc Natl Acad Sci U S A. Jun 13, 2006; 103(24): 9298–9303.
Published online Jun 5, 2006. doi:  10.1073/pnas.0600623103
PMCID: PMC1482604
Microbiology

Entamoeba histolytica cysteine proteases cleave the MUC2 mucin in its C-terminal domain and dissolve the protective colonic mucus gel

Abstract

In order for the protozoan parasite Entamoeba histolytica (E.h.) to cause invasive intestinal and extraintestinal infection, which leads to significant morbidity and mortality, it must disrupt the protective mucus layer by a previously unknown mechanism. We hypothesized that cysteine proteases secreted from the amoeba disrupt the mucin polymeric network, thereby overcoming the protective mucus barrier. The MUC2 mucin is the major structural component of the colonic mucus gel. Heavily O-glycosylated and protease-resistant mucin domains characterize gel-forming mucins. Their N- and C-terminal cysteine-rich domains are involved in mucin polymerization, and these domains are likely to be targeted by proteases because they are less glycosylated, thereby exposing their peptide chains. By treating recombinant cysteine-rich domains of MUC2 with proteases from E.h. trophozoites, we showed that the C-terminal domain was specifically targeted at two sites by cysteine proteases, whereas the N-terminal domain was resistant to proteolysis. The major cleavage site is predicted to depolymerize the MUC2 polymers, thereby disrupting the protective mucus gel. The ability of the cysteine proteases to dissolve mucus gels was confirmed by treating mucins from a MUC2-producing cell line with amoeba proteases. These findings suggest a major role for E.h. cysteine proteases in overcoming the protective mucus barrier in the pathogenesis of invasive amoebiasis. In this report, we identify a specific cleavage mechanism used by an enteric pathogen to disrupt the polymeric nature of the mucin gel.

Keywords: colon, parasite

The mucus that covers the intestinal tract forms a physical barrier between the intestinal contents and the underlying epithelia. It acts both as a sieve and as a trap for microorganisms, preventing their access to the epithelia. The mucus is especially thick in the colon, where it has been measured to be 800 μm in rats (1). MUC2 is the major gel-forming mucin secreted by goblet cells of the small and large intestines and is the main structural component of the mucus gel (2, 3). MUC2 is assembled into large polymers by disulfide bonds between mucin subunits with a mass of ≈2.5 MDa. The assembly process is initiated in the endoplasmic reticulum, where C-terminal end-to-end dimers are formed between monomers of the MUC2 apomucin (5,179 aa) (4, 5). After extensive O-glycosylation in the Golgi apparatus, multimerization is triggered in the later and more acidic part of the secretory pathway (6). These multimers are formed by disulfide bond-mediated trimerization of the N termini of MUC2 (7). An additional, still uncharacterized nonreducible bond renders the MUC2 polymer insoluble (3, 6).

The protective role of the MUC2 mucin in the intestine is maintained despite the abundant pancreatic digestive proteases. This protection is possible because its two large mucin domains are highly O-glycosylated and thus resistant to proteolytic cleavage because the glycans prevent access of the proteases to the protein core. In contrast, the N- and C-terminal cysteine-rich domains are less glycosylated, and the protein core is probably more exposed. However, studies on the recombinant N-terminal cysteine-rich domain of MUC2 have revealed that the N-terminal trimers are held together within a core that is resistant to proteolytic enzymes. The protease resistance is likely due to the high number of intramolecular disulfide bonds that shield potential cleavage sites from the pancreatic enzymes (7). Despite tryptic cleavages in the MUC2 C terminus, the covalent disulfide bonds maintain its polymeric nature (M.E.L. and G.C.H., unpublished data). However, reduction of the MUC2 polymer not only dissolves the mucus gel but also renders the protein core outside of the mucin domains highly sensitive to proteolytic cleavages (2, 3, 8).

In order for enteric pathogens to invade and make contact with the colonic epithelium, they must overcome the thick protective mucus barrier (1). Microbes accomplish this task by a variety of mechanisms, including degradation of mucin oligosaccharides, proteolytic degradation of the mucin polymer, and induction of mucus hypersecretion (912). The protozoan parasite Entamoeba histolytica (E.h.) colonizes the mucus layer of the colon by adhering to mucin oligosaccharides by means of a 170-kDa Gal/GalNAc-lectin (13). In most cases, E.h. causes little harm to the host and remains in or on the mucus layer, feeding on bacteria and cellular debris. However, in a small percent of the infected patients, the parasite is able to overcome the mucus barrier and invade the underlying epithelium. Previous work has shown that the parasite secretes cysteine proteases, which degrade colonic mucins (8). Moreover, it was demonstrated that the degraded mucins were less effective in inhibiting adherence of amoebae to target epithelial cells, indicating that the mucin polymer must be intact to maintain its protective function. Similar observations have been reported for Candida albicans, where the mucin was degraded by the secreted aspartyl protease Sap2p (14). E.h. trophozoites expressing the antisense message to EhCP5 (E.h. cysteine protease 5) (15) have an impaired ability to disrupt an intact colonic mucus barrier and invade epithelial cell monolayers (16). These observations suggest that the cysteine proteases from E.h. facilitate invasion of the colon by disrupting the mucus gel. However, a more detailed molecular mechanism of how the parasite disassembles the mucin polymer has yet to be determined. In this study, we examined the hypothesis that cysteine proteases secreted from E.h. target the cysteine-rich domains of MUC2. Whereas the N-terminal cysteine-rich domain was resistant to proteolysis, the C-terminal domain was cleaved at two distinct sites, and the cleavage at the major site resulted in depolymerization of the MUC2 polymer. This finding marks the identification of a specific proteolytic cleavage used by an enteric pathogen to disrupt the polymeric nature of a mucin, thereby overcoming the protective mucus barrier.

Results

The Effect of E.h. Cysteine Proteases on MUC2 Cysteine-Rich Domains.

To determine the mechanism by which E.h. overcomes the protective mucus barrier, we investigated the effects of secreted cysteine proteases on the recombinant cysteine-rich domains of MUC2 (Fig. 1A). These domains are the most likely targets for proteases because the heavily O-glycosylated mucin domains are resistant to proteolytic enzymes (2). The N- and C-terminal cysteine-rich domains are responsible for the formation of the mucin polymers by linking monomers together by disulfide bonds. When expressed in CHO-K1 cells, the recombinant N-terminal cysteine-rich domains (1,376 aa) were secreted from the cells as disulfide-linked trimers (7), whereas the C-terminal domains (981 aa) were secreted as dimers (5). The recombinant N and C termini of MUC2 produced in CHO-K1 cells were treated with secretions from E.h., and the digests were analyzed by SDS/PAGE and silver staining. The digests were initially separated under reducing conditions to visualize the monomeric forms of the proteins. As shown in Fig. 1B, the MUC2 N terminus was resistant to proteolytic degradation. In contrast, the recombinant MUC2 C terminus was sensitive to the secretions, and two cleavage fragments were generated, the first with a molecular mass of ≈170 kDa (α-fragment) and the second, seen as a weaker band, with a molecular mass of ≈75 kDa (β-fragment; Fig. 1C). These cleavages were eliminated by pretreatment of the proteases with the specific inhibitor of cysteine proteases, E-64, suggesting that E.h. cysteine proteases are responsible for the degradation. Because the cysteine-rich domains contain many cysteine residues, it is important to determine whether these cleavages cause the disulfide-stabilized protein to fall apart or whether the cleavage fragments are still held together by disulfide bonds. Analysis of the digests by SDS/PAGE under nonreducing conditions showed that the N terminus remained intact, as expected (not shown). In contrast, the nonreduced C terminus appeared smaller and migrated at ≈300 kDa after protease treatment, compared with the intact dimer, which migrated at 470 kDa (Fig. 1D). This product was larger than expected for a monomer and suggested that the 300-kDa band was a dimer. To elucidate the identity of the cleavage products, the digests were separated by SDS/PAGE under both reducing and nonreducing conditions, blotted to membranes, and probed with either the α-MUC2C2 polyclonal antiserum (Fig. 1E) or α-GFP mAb (Fig. 1F). The α-MUC2C2 polyclonal antiserum detected the 300-kDa component in the nonreduced samples, whereas the α-GFP mAb did not, suggesting that the 300-kDa band lacks the N terminus of the C-terminal domain. When these samples were analyzed under reducing conditions, the α-MUC2C2 polyclonal antiserum detected both the α- and β-fragments at 170 and 75 kDa, respectively. If the digested sample was separated under nonreducing conditions and stained with the α-GFP mAb, the blot revealed two released bands, one at 70 kDa and one at ≈30 kDa (Fig. 1F). These bands were also stained by an α-myc mAb (not shown). The 70-kDa band has the expected size for the γ-fragment (Fig. 1G). The 30-kDa band, on the other hand, can only contain the far N terminus, including the GFP, and is likely due to a cleavage in the linker region between GFP and the MUC2 C terminus. These results together suggest that the 300-kDa band lacks the γ-fragment and that this fragment is released also when the disulfide bonds are intact. The nonreduced MUC2 C terminus is known to migrate relatively quickly compared with its calculated molecular mass (4), a property that is probably even more pronounced for the 300-kDa band. The 300-kDa band is therefore suggested to be a dimer of two α-fragments, a conclusion that is also supported by the fact that the C-terminal domain of MUC2 is held together by disulfide bonds located in the far C terminus (4, 17).

Fig. 1.
The C-terminal cysteine-rich domain of human MUC2 is susceptible to digestion by cysteine proteases secreted by E.h. (A) Schematic representation of the human MUC2 mucin and the recombinant MUC2 fusion proteins. The entire protein sequence for MUC2 mucin ...

To decipher the exact positions of the cleavages seen in the recombinant C-terminal cysteine-rich domain, Edman sequencing was performed on the α- and β-bands. The N-terminal sequence of the α-fragment was shown to be TPHKDXT, and that of the β-fragment was TGLRPYPXXVLI (X indicates empty cycles due to cysteines or modified amino acids). Comparisons with the MUC2 sequence showed that the cleavages had occurred at the sites indicated in Fig. 1G. E.h. cysteine proteases have been reported to cleave substrates with positively charged amino acids, such as arginine, in the P2 position (18). The cleavage sites found in MUC2 (IRT/T and GKT/T) are in good agreement with the suggested sequence specificity.

The major cleavage occurs N-terminally of the first cysteine of the C-terminal cysteine-rich domain. This finding, together with the observed release of the α-fragment, suggests that the intact MUC2 will loose its C terminus and that the polymeric nature of the MUC2 mucin will be disrupted by this cleavage. This disruption is in contrast to the minor β-cleavage; the β-fragments remain linked to the N-terminal parts by disulfide bonds after this cleavage, thus not disrupting the mucin. Taken together, these results imply that cysteine proteases secreted from E.h. cleave MUC2 at one critical site in its C-terminal domain.

Mutation of the Major Cleavage Site for the E.h. Cysteine Proteases.

The specificity of the cysteine proteases for the major cleavage site, which liberates the α-fragment, was further studied by mutating this site from IRTT to ADAA. The degraded protein now migrated at ≈220 kDa as compared with 170 kDa for the cleaved wild-type MUC2 under reducing conditions (Fig. 2). The small reduction in size is probably due to a cleavage in the linker region between GFP, and the actual MUC2 C terminus as a 30-kDa band, as discussed above, was detected with the α-GFP mAb (Fig. 1F) and the α-myc mAb (data not shown). As expected, the β-fragment was also detected at 75 kDa (most clearly seen when using 0.25 μg of secreted protease; Fig. 2 Left). A faint band migrating slightly slower than the α-fragment was also noticed in the cleaved samples, which indicates another minor cleavage site located N-terminally of the major α-cleavage site. Analysis of the MUC2 sequence reveals an RGTT sequence 36 aa N-terminal to the IRTT cleavage site that might be targeted by the E.h. cysteine proteases.

Fig. 2.
Mutation of the IRTT site in the C-terminal cysteine-rich domain of MUC2 renders the protein resistant to digestion by cysteine proteases secreted by E.h. The recombinant human C-terminal cysteine-rich domain of MUC2 with the sequence IRTT [amino acids ...

Degradation of Insoluble Mucins by E.h. Cysteine Proteases.

The localization of the major cleavage site and the degradation fragments suggests that the MUC2 polymer will disassemble because of E.h. cysteine proteases. Because MUC2 is the major component of the mucus gel (2, 3), it is likely that the whole mucus gel will fall apart, allowing E.h. to overcome the protective mucus barrier. To address this question, mucus gel was prepared from LS 174T cells in two different ways and incubated with E.h. proteases. As shown in Fig. 3A, the volume of the guanidinium chloride (GuHCl)-insoluble mucins was reduced by the action of the cysteine proteases present in the amoeba secretions, because the degradation was inhibited by the specific inhibitor E-64. Mucins insoluble in 1% Triton X-100 were also digested with E.h. proteases. This treatment dissolved the mucus pellet into a turbid solution with an absorption of 0.319 compared with 0.014 for the negative control and 0.004 for E-64-treated controls (Fig. 3B). Attempts to find the α- and β-fragments in the digests of the insoluble mucins were not successful. To confirm the cleavage site in wild-type MUC2, immunoprecipitations were performed by using α-MUC2-TR, an antiserum against the non-O-glycosylated tandem repeats in the mucin domain. The precipitated mucin was treated with E.h. proteases while still attached to the beads and analyzed by SDS/PAGE and Western blotting, using the α-MUC2-C2 mAb for detection. A 150-kDa band was detected in the treated samples but not in the nontreated or E-64-inhibited controls (Fig. 3C). MUC2 precipitated with α-MUC2-TR polyclonal antiserum is not O-glycosylated, and the 150-kDa band likely represents the non-O-glycosylated form of the α-fragment. The dissolution of the mucus pellets indicates that cysteine proteases secreted from E.h. can disintegrate the MUC2 mucus gel, allowing the parasite to breach the mucus barrier.

Fig. 3.
Cysteine proteases secreted from E.h. depolymerize the MUC2 network. (A and B) Mucins insoluble in 6 M GuHCl (A) or 1% (vol/vol) Triton X-100 (B) were incubated for 16 h at 37°C with 25 μg (A) or 5 μg (B) of proteins secreted from ...

Discussion

Enteric pathogens must overcome a series of innate host defenses before making contact with the intestinal epithelium. The first obstacle encountered during invasion is the protective mucus barrier. Attachment to the host mucus layer and colonization of the gastrointestinal tract are the first steps in the infection process, which determine the outcome of disease. Tissue-specific expression of mucin and mucin glycosylation patterns allow for the colonization of microbes in different regions of the gastrointestinal tract. For example, Shigella dysenteriae 1, the causative agent of shigellosis in humans, preferentially adheres to colonic mucin but not to small intestinal mucin (19). E.h. colonizes the mucus layer of the colon by binding with high affinity to Gal and GalNAc residues of colonic mucin (13, 20). The parasite also binds host epithelial cells by means of the Gal/GalNAc-lectin, and this adherence is a prerequisite for epithelial cell cytolysis and invasion (20). Mucin carbohydrates act as receptors for the commensal gut microflora as well as for invasive organisms, and binding sites on gel-forming mucins compete with those of the underlying epithelium and aim to restrict access of pathogens to the mucosa. In addition, mucus plays a protective role by being constantly renewed from the epithelial cells and expelled into the intestinal lumen. Mucus traps both commensal and pathogenic microorganisms, and this mucus flow finally removes these microorganisms during defecation (21).

After successful colonization on the mucus layer of the gastrointestinal tract, invading bacteria, viruses, or parasites must overcome the mucus barrier. Virulence factors such as proteases, glycosidases, and mucus secretagogues are produced by these organisms and are believed to be responsible for disruption and depletion of the mucus gel (912). E.h. constitutively secretes at least four cysteine proteases that are involved in tissue destruction and invasive disease (22, 23). The closely related noninvasive amoeba Entamoeba dispar produces significantly less cysteine protease activity than E.h. and does not express several homologous cysteine proteases that correlate with invasive disease (24, 25). It is possible that high levels and the specificity of cysteine protease activity in E.h. contribute to increased virulence and invasion. Previous studies have indicated that this activity plays a major role in intestinal as well as extraintestinal disease. Cysteine protease activity is necessary for the trophozoites to traverse a colonic mucus barrier before cell cytolysis (8, 16). In addition, the proteases are necessary for the parasite to disrupt the epithelial cell monolayers in vitro (15), to disseminate through host tissue (26), and to cause liver abscesses (27, 28). The mechanism by which the parasite disrupts and penetrates the mucin polymeric network was previously unknown, but recent evidence suggests that the amoeba disrupts the MUC2 polymers (8).

In this study, we have now identified the specific cleavage sites in the MUC2 polymer that are targeted by E.h. cysteine proteases. The intact MUC2 polymeric network cannot be analyzed because the mucin forms large net-like structures that are insoluble (3, 6, 7). The only way to analyze this MUC2 gel in more detail is to reduce the inter- and intramolecular disulfide bonds. However, this form of mucin is not the form that the microorganism encounters, and it is well known that reduced mucins are very susceptible to cleavages with proteolytic enzymes. This proteolytic susceptibility is not the case for the mucin domains, which are protected against proteolytic digestion by the dense array of O-glycans shielding the protein core (2). When the mucins are nonreduced and intact, the less glycosylated cysteine-rich domains are also much more resistant to proteolytic enzymes. The large size and insolubility of the intact MUC2 mucin gel have made studies on the effects of enteric pathogens on intestinal mucins impossible. Therefore, a more practical approach was taken. Recombinant N- and C- terminal cysteine-rich domains of MUC2 were expressed and secreted from CHO-K1 cells. Previous studies using these expression systems have determined how the MUC2 polymer is assembled (5, 7). The MUC2 C terminus was shown to form homodimers, whereas the N terminus was shown to be secreted as trimers (5, 7). When these recombinant proteins were incubated with proteases secreted by E.h., the MUC2 C terminus was cleaved, but the relatively larger N terminus was uncleaved, suggesting that the E.h. cleavage must be relatively specific. Moreover, cysteine proteases were identified as the enzymes responsible for the cleavage of MUC2, as suggested by the effect of the specific cysteine protease inhibitor E-64. We identified one major and one minor specific cleavage site in the C-terminal domain of MUC2. Both sites had the sequence arginine or lysine followed by two threonines, and the cleavage occurred between the two threonines. Both sites were in agreement with the specificity suggested for cysteine proteases, with a basic amino acid in the P2 position (18). Site-directed mutagenesis of the major cleavage site further demonstrated the sequence specificity of the E.h. cysteine proteases.

A question arises: Which of the cysteine proteases secreted from the amoeba is responsible for the cleavage of MUC2? EhCP5 is a likely candidate because it is one of the major secreted cysteine proteases and, together with EhCP1 and EhCP2, it accounts for virtually all cysteine protease activity of E.h. (29). In addition, EhCP5 is one of the two cysteine proteases that seems to be absent in the noninvasive E. dispar (29). This enzyme’s role in overcoming the mucus barrier was lately implicated because antisense down-regulation of EhCP5 in trophozoites impaired its ability to disrupt an intact colonic mucus barrier and invade epithelial cell monolayers (16). In addition, digestion of the MUC2 C terminus with recombinant EhCP5 (30) suggested the generation of an α-fragment (unpublished data). However, the presence of DTT is necessary for activity of this recombinant EhCP5, and, most of the time, the reducing agent reduced the disulfide bonds of MUC2 and caused its complete degradation. Although EhCP5 is indicated as the responsible cysteine protease, the other E.h. proteases have so far not been excluded.

What is the consequence of the cleavages in the C terminus of MUC2? The minor β-cleavage occurred in a region that is surrounded by numerous cysteines, and analysis by SDS/PAGE revealed that disulfide bonds held this part together despite this cleavage. In contrast to this, the α-cleavage caused the separation of the N-terminal part from the C-terminal dimer, as revealed by analysis of the recombinant C terminus on reducing and nonreducing SDS/PAGE. This interpretation is supported by the localization of this cleavage site N-terminal of the first cysteine of the MUC2 C termini. The localization of the cleavages is illustrated in Fig. 4. Our results indicate that it is only the α-cleavage site that is responsible for breaking up the MUC2 mucin polymer. The polymeric nature of mucins is necessary for maintaining the mucus gel. The capability of E.h. cysteine proteases to disrupt MUC2-enriched mucus from LS 174T cells further indicates the importance of this α-cleavage. We have not been able to isolate the α-fragment from GuHCl-insoluble MUC2 mucin for unknown reasons, but the likely localization of the still uncharacterized nonreducible covalent linkage to the C terminus is a possible explanation (6). However, this fragment was shown to be cleaved off from an immature, not yet cross-linked MUC2 mucin.

Fig. 4.
Cysteine proteases secreted from E.h. cleave the MUC2 mucin at two specific positions in its C-terminal cysteine-rich domain and generate the α- and β-fragments. While the mucus gel is still held together by disulfide bonds after the generation ...

Rodents are known to be naturally resistant to E.h. infections (31). Analyzing the mouse Muc2 sequence reveals that the cleavable human IIRT/TG sequence has been replaced by the ITSPST sequence. This sequence lacks the arginine and does not fulfill the predicted criteria for cleavage by E.h. cysteine proteases. Thus, it can be hypothesized that sequence differences in the MUC2 mucin between different species could determine the susceptibility to invasive amoebiasis. Only a minority of people infected with E.h. acquire an invasive form of the disease. One possibility for such variability could be single-nucleotide polymorphisms at this site of the MUC2 sequence. However, no SNPs were found at this site when searching SNP databases. Another possibility is that the two threonines at the cleavage site could be O-glycosylated. It is likely that glycosylation of one or both of the two threonines flanking the cleavage site will affect or perhaps block the E.h. cleavage. The human genome contains 20 peptidyl-GalNAc transferases that add GalNAc to serine or threonine. The specificities of some of these enzymes are known, but it is still not possible to predict whether any of these enzymes should glycosylate this sequence. In any case, it is known that the intestine has abundant peptidyl-GalNAc transferases and that the repertoire of these transferases can extensively modulate which glycosylation sites are used in a specific sequence. Further studies should reveal whether the two threonines at the α-cleavage site could be glycosylated and whether this glycosylation could modulate the invasiveness of E.h. Colonic mucus presents an obstacle for E.h. that must be disrupted before making contact with the epithelium, and E.h. has developed a unique strategy to overcome this barrier. In addition to degrading the mucin oligosaccharide component of the molecule (32), the parasite targets a specific cleavage site in a region of the molecule where a cleavage disrupts the mucus gel. Documentation of this event may aid in our understanding of how invading pathogens can defeat innate defenses and how the host can withstand the pathogen by its protective mucus layer.

Materials and Methods

Cell Culture and Expression and Purification of Recombinant Mucins.

The CHO-K1 and LS 174T cells stably expressing the recombinant N- and C-terminal cysteine-rich domains of the human MUC2 mucin were grown as described in refs. 5 and 7. The IRTT sequence [located at positions 4320 to 4323 in the MUC2 sequence (33)] in the recombinant C-terminal cysteine-rich domain was mutated to ADAA by site-directed mutagenesis (QuikChange, Stratagene) of the expression vector pSMG-MUC2C (5) by using the oligonucleotides 5′-CTCCACACCCAGCATCGCCGACGCCGCCGGCCTGAGGCCCTACC-3′ and 5′-GGTAGGGCCTCAGGCCGGCGGCGTCGGCGATGCTGGGTGTGGAG-3′. The obtained plasmid, pSMG-MUC2C IRTT(4320–4323)ADAA, was transfected into CHO-K1 cells by using Lipofectamine 2000 (Invitrogen), and stable clones were selected and screened as described in ref. 5. Both the mutated and the nonmutated recombinant C-terminal cysteine-rich domains of MUC2 were purified from spent cell culture media as described in ref. 34.

Preparation of Proteins Secreted from E.h.

E.h. HM1:IMSS trophozoites were serially passaged through gerbil livers to maintain high virulence and were cultured in TYI-S-33 medium as described in ref. 35. Secreted components were collected from trophozoites incubated in Hanks’ balanced salt solution (Invitrogen) for 2 h at 37°C at a final concentration of 2 × 107 amoeba per ml (36). The viability of the trophozoites was determined by using the trypan blue exclusion assay as described in ref. 36. The cysteine protease activity in the secretions was measured against the synthetic substrate z-Arg-Arg-pNA (Bachem) and a gelatin zymogram (22). The protein concentrations in the secretions were determined by the Bradford method (37).

Antibodies and SDS/PAGE.

The α-MUC2C2 and α-MUC2TR polyclonal antisera are described in refs. 4 and 6, and the α-MUC2C2–7B2 mAb was generated against the same peptide as that used for making the α-MUC2C2 polyclonal antiserum (6). Other antibodies used were α-GFP mAb (Clontech) and goat anti-mouse and goat anti-rabbit immunoglobulins coupled to alkaline phosphatase (DAKO). The samples were analyzed by discontinuous SDS/PAGE and Western blotting as described in ref. 5. The blots were developed by using nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (Promega).

Isolation of Insoluble and Soluble Mucins from LS 174T Cells.

GuHCl-insoluble mucins were prepared from LS 174T cells cultured in six-well plates for 10 days with daily media changes (38). The cells were extracted with GuHCl extraction buffer (6 M GuHCl/5 mM EDTA/10 mM NaH2PO4, pH 6.5), and insoluble material was pelleted by centrifugation for 20 min at 30,000 × g, followed by six washes in GuHCl extraction buffer and six washes in Dulbecco’s PBS. The resulting insoluble material was used for degradation studies. Water-insoluble mucins were prepared from LS 174T cells cultured to confluence (in 8.5-cm Petri dishes). The cells were washed twice in cold PBS and lysed in 1.5 ml of lysis buffer [50 mM Tris·HCl, pH 7.9/150 mM NaCl/1% (vol/vol) Triton X-100] containing protease inhibitors [2× complete (Roche Diagnostics, Mannheim, Germany), 5 mM EDTA, 1 μM pepstatin (Roche), and 1 μM bestatin (Sigma)], 5 mM N-ethylmaleimide (Sigma), and 0.05% (wt/vol) NaN3. After sonication with a Soniprep 150 sonifier (intensity of 15, 3 × 2 s, MSE; Crawley, Sussex, U.K.), the insoluble material was pelleted by centrifugation (16,000 × g for 10 min at 4°C). The insoluble material was washed twice with PBS containing the same additives as during the lysis, followed by another three washes with PBS only, before it was used for degradation studies. The cleared LS 174T cell lysates saved after the preparation of water-insoluble mucins were used for isolation of soluble MUC2 by immunoprecipitation. Each cell lysate was incubated with 50 μl of protein G beads (protein G PLUS agarose; Santa Cruz Biotechnology) for 3 h at 4°C. After centrifugation (1,000 × g for 1 min), the supernatant was incubated with 50 μl of α-MUC2-TR polyclonal antiserum for 16 h at 4°C before adding 50 μl of protein G beads and incubating for another 3 h at 4°C. The immune complexes were washed four times in 10 mM Tris·HCl/2 mM EDTA/0.1% (vol/vol) Triton X-100/0.1% SDS (wt/vol), pH 7 and once in PBS before the degradation studies.

Digestions with Secretions from E.h.

Secretions from E.h. were preincubated in Dulbecco’s PBS in the presence or absence of the specific cysteine protease inhibitor E-64 (100 μM) (Roche) on ice for 30 min. Purified C-terminal cysteine-rich domain, N-terminal cysteine-rich domain from spent culture medium, or immunoprecipitated MUC2 was then added, and the mixtures were incubated at 37°C. The digestions were terminated by heating the samples at 95°C for 5 min.

When GuHCl-insoluble mucins were digested, the secretions from E.h. were pretreated as above and transferred to insoluble mucins extracted from one of the wells of the six-well plates. The mixtures were then incubated at 37°C for 16 h. The negative controls were incubated in Dulbecco’s PBS only. The incubations were stopped as above and centrifuged at 16,000 × g for 10 min, and the resulting pellets were photographed. Water-insoluble mucins were digested in PBS as the GuHCl-insoluble mucins. The incubations were stopped as above, supplemented to 50 mM Tris·HCl, pH 7.4/6 mM MgCl2/50 mM NaCl/50 mM KCl and incubated with 150 units of DNase I (Invitrogen) for 6 h at 37°C. Pictures were taken, and the absorbance of the supernatants were measured at 600 nm.

Edman Sequencing of the Major MUC2 Cleavage Products.

Recombinant MUC2 C terminus (8.6 μg) was digested with 6 μg of proteins secreted from E.h. for 4 h at 37°C, separated by SDS/PAGE, blotted to a poly(vinylidene difluoride) membrane, and stained with Coomassie blue. Bands were excised and sequenced by Edman degradation on a Procise 492 protein sequencer (Applied Biosystems).

Acknowledgments

We thank Dr. Sandra J. Gendler for comments on the manuscript. This work was supported by Swedish Research Council Grant 7461 and grants from the Ingabritt and Arne Lundberg Foundation and the Canadian Institutes of Health Research.

Abbreviations

E.h.
Entamoeba histolytica
GuHCl
guanidinium chloride
EhCP5
E.h. cysteine protease 5.

Footnotes

Conflict of interest statement: No conflicts declared.

This paper was submitted directly (Track II) to the PNAS office.

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