An Autocatalytic Cleavage in the C Terminus of the Human MUC2 Mucin Occurs at the Low pH of the Late Secretory Pathway*

Martin E. Lidell, Malin E. V. Johansson, and Gunnar C. HanssonDagger

From the Department of Medical Biochemistry, Göteborg University, Medicinaregatan 9A, S-413 90 Gothenburg, Sweden

Received for publication, October 1, 2002, and in revised form, January 14, 2003

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

During purification of a recombinant MUC2 C terminus expressed in CHO-K1 cells, the protein was partly cleaved when buffers with a pH of 6.0 were used. When buffers with higher pH values were used, less cleavage was found. Disulfide bonds held the two fragments generated together as these were only observed after reduction. Edman sequencing of the C-terminal 110-kDa fragment revealed that the cleavage had occurred at an Asp-Pro bond, a site described previously to generate the so-called "link peptide" after disulfide bond reduction. In vitro studies on the conditions for cleavage showed that it occurred in a time-dependent manner at a pH below 6.0. Furthermore, the reaction was not enzyme-mediated as it occurred in pure preparations of the MUC2 C terminus and was not inhibited by protease inhibitors. When expressed in the mucin producing cell line LS 174T, the C terminus was cleaved to a higher extent compared with the CHO-K1 cells. Neutralizing the secretory pathway with either NH4Cl or bafilomycin A1 inhibited this cleavage. Altogether, our results suggest that the cleavage is an autocatalytic reaction that occurs in the acidic environment of the late secretory pathway. Furthermore, the cleavage produced a new, reactive C terminus that has the potential to attach the mucin to itself or other molecules. Because a pH below 6 can be reached in the late secretory pathway and on mucosal surfaces, the cleavage and possible cross-linking are likely to be of biological importance.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Mucins are large glycoproteins with the carbohydrates accounting for up to 90% of its molecular mass (1, 2). These sugars are mainly O-linked to Ser and Thr residues localized to regions called mucin domains. These domains contain tandemly repeated sequences (rich in Ser, Thr, and Pro) that vary in number, length, and amino acid composition depending on the particular mucin (3). Mucins account for the major part of the mucous layers covering the epithelial surfaces throughout the body, where they are believed to have several functions including protection and lubrication of the underlying epithelia.

There are two major types of mucins, membrane-bound and secreted. In human, eight membrane-bound (MUC1, MUC3A, MUC3B, MUC4, MUC12, MUC13, MUC16, and MUC17) (4-11) and five secreted mucins (MUC2, MUC5B, MUC5AC, MUC6, and MUC7) (12-16) have been identified so far. Two additional ones cannot be classified (MUC8 and MUC11) (7, 17). The secreted mucins can be further sub-divided as being either gel-forming (MUC2, MUC5B, MUC5AC, and MUC6) or not (MUC7). The ability to form gels is dependent on the capacity of monomers to form polymeric structures. The intermolecular interactions are of disulfide-bond nature and mediated through the N- and C-terminal Cys-rich domains of mucins (reviewed in Ref. 2). Both these domains show considerable sequence homology to the human prepro-von Willebrand factor (vWf)1 (18).

The most thoroughly studied mucin is the porcine submaxillary mucin (PSM) (19). PSM has been shown to dimerize via its C-terminal domain in the endoplasmic reticulum (ER) (20, 21) and oligomerize through its N-terminal domain in the acidic compartments of the Golgi complex, presumably making up a branched structure (22, 23).

In analogy with the results from the studies of PSM, we have reported previously (24, 25) that the human MUC2 mucin forms disulfide-stabilized dimers in the ER. These studies were made on the full-length MUC2, but due to the large size and high carbohydrate content of the mucins, it was hard to do further studies on its assembly by conventional methods. We have therefore expressed the last 981 amino acids of the human MUC2 mucin (corresponding to its C-terminal Cys-rich domain) as a fusion protein with the mycTag epitope and the green fluorescent protein (GFP) in Chinese hamster ovary cells (CHO-K1) and in the human colon adenocarcinoma cell line LS 174T. With this tool we could verify that the domain was capable of forming dimers and that these dimers were secreted from the cells.2 These results were also supported by previous studies (26, 27) of the MUC2 homologue in rat (Muc2).

In this study, we have studied a cleavage in the Cys-rich C-terminal part of MUC2. The cleavage was first observed during attempts to purify and study the glycosylation pattern of the recombinant C-terminal Cys-rich domain expressed in CHO-K1 cells. We found that when buffers of pH 6.0 were used during purification or glycosidase treatments, part of the material was cleaved. The cleavage caught our attention and was further analyzed, revealing that it was formed by an autocatalytic mechanism triggered by low pH. Furthermore, the cleavage produced a new, reactive C terminus that has the potential to link the cleaved mucin to other compounds.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Antisera and Antibodies-- The polyclonal antiserum alpha -MUC2C2 has been described before (28). The alpha -mycTag monoclonal antibody (mAb) was from spent culture media of the 1-9E10.2 hybridoma (ATCC CRL-1729). Other antibodies used were alpha -GFP mAb (Clontech), goat anti-mouse immunoglobulins coupled to alkaline phosphatase (AP) (Dako), goat anti-rabbit immunoglobulins coupled to alkaline phosphatase (Dako), and streptavidin coupled to alkaline phosphatase (streptavidin-AP) (Dako).

Recombinant Expression and Tissue Culture-- An expression plasmid with the bases 12,622-15,708 of the human MUC2 sequence (12) was generated from the plasmids SMUC41 (bases 12,243-13,083) and V5 (bases 12,880-15,708) (29). These were cloned into the eukaryotic expression vector pEGFP-C1 (Clontech) where the signal sequence had been replaced with the murine immunoglobulin kappa -chain signal sequence from pSecTag A (Invitrogen) followed by the mycTag (EQKLISEEDL).2 The resulting pSMG-MUC2C plasmid encoding the kappa -chain signal sequence, the mycTag, GFP, and the MUC2 C terminus was sequenced and transfected into the human colon adenocarcinoma cell line LS 174T (ATCC CL-188) and Chinese hamster ovary cells CHO-K1 (ATCC CCL-61). Both cell lines were selected for permanent expression of the recombinant protein and named LS 174T-pSMG-MUC2C and CHO-K1-pSMG-MUC2C,2 respectively. These were cultured as described earlier (24) for LS 174T with the addition of 125 µg ml-1 G418 for LS 174T-pSMG-MUC2C and 250 µg ml-1 G418 for CHO-K1-pSMG-MUC2C, respectively.

Purification of Secreted MUC2 C Terminus from CHO-K1-pSMG-MUC2C Cells-- After growing CHO-K1-pSMG-MUC2C cells until 3-4 days after confluence, the cells were washed twice with phosphate-buffered saline (PBS) and Iscove's modified Dulbecco's medium (Invitrogen) was added. The spent culture media were harvested, and new media were added every 3rd day. After the addition of protease inhibitors (1 mM phenylmethylsulfonyl fluoride (PMSF) (Sigma), 100 µM leupeptin (Sigma), 100 KIE ml-1 aprotinin (Trasylol, Bayer), and 5 mM N-ethylmaleimide (NEM, Sigma)), the media was centrifuged 1,000 × g for 10 min (+4 °C), and 0.02% (w/v) NaN3 was added. 200 ml of media were filtrated in an Amicon Stirred Cell model 8400 through an Amicon Ultrafiltration Membrane YM-100 (Millipore). The volume was reduced to 20 ml, filled up to 200 ml with 50 mM Tris-HCl, pH 8.0, and reduced to 20 ml again. This procedure was repeated 3 times. The concentrate was filtered (0.22 µm Millex-GV, Millipore) and loaded on a Mono Q HR 5/5 column (Amersham Biosciences) using a flow rate of 1.0 ml min-1. After washing with 150 mM NaCl in 50 mM Tris-HCl, pH 8.0, bound components were eluted with a linear NaCl gradient (150-600 mM) in 50 mM Tris-HCl, pH 8.0, over 45 min. The eluting components were monitored at 280 nm and collected in 1-ml fractions. The fractions were individually analyzed by SDS-PAGE and silver staining. Seven column fractions, eluting between 230 and 300 mM NaCl, containing the recombinant MUC2 C terminus were pooled, concentrated to 200 µl by ultrafiltration (Vivaspin 2-ml concentrator, 30,000 molecular weight cut-off RC, 3000 × g, 4 °C), loaded directly on a Superose 6 HR 10/30 column (Amersham Biosciences) eluted with 150 mM ammonium acetate, pH 8.5, at a flow of 250 µl min-1. The eluting components were monitored at 280 nm and collected in 0.5-ml fractions. All the fractions contained in the symmetrical unimodal peak eluting at Kav = 0.095 and positive for the recombinant MUC2 C terminus, as assessed by SDS-PAGE and silver staining, were pooled and used for the subsequent studies.

Metabolic Labeling-- Confluent cells were metabolically labeled by preincubation in Met- and Cys-free minimal essential medium (Invitrogen) containing 10% (v/v) fetal bovine serum, 100 IU ml-1 penicillin, and 100 µg ml-1 streptomycin for 1-2 h followed by addition of labeling mix (Redivue Pro-mix L-35S in vitro labeling mix, Amersham Biosciences) to a final concentration of 36-107 µCi ml-1. After incubation for 6-16 h, cell lysates and media were prepared. When pulse-chase studies were performed, the labeling mix was removed after 5 or 15 min and the cells washed once and chased with normal cell culturing media with excess of Met and Cys (15 and 25 µg extra/ml, respectively).

Preparation of Cell Lysates and Media-- Media were removed and the cells washed twice with PBS. The cells were lysed in lysis buffer (50 mM Tris-HCl, pH 7.9, 150 mM NaCl, 1% (v/v) Triton X-100) containing protease inhibitors and NEM (concentrations as above) and sonicated (intensity 15) 3 times for 2 s (MSE Soniprep 100 sonifier), and the cell debris was removed by centrifugation (16,000 × g for 10 min, 4 °C). The media were supplemented with protease inhibitors and NEM (concentrations as above) and centrifuged (1000 × g for 10 min, 4 °C).

Immunoprecipitation-- Immunoprecipitations using the mAbs alpha -mycTag or alpha -GFP antibodies were performed by precoating these on goat anti-mouse IgG-coupled magnetic beads (Dynabeads, Dynal). The immunocomplexes were washed 4 times in 10 mM Tris-HCl, 2 mM EDTA, 0.1% (v/v) Triton X-100, 0.1% SDS (w/v), pH 7.4, and 1 time in lysis buffer.

SDS-PAGE-- Immunoprecipitated material was released from the magnetic beads in sample buffer (25 mM Tris-HCl, 10% (v/v) glycerol, 2.5% (w/v) SDS, pH 6.8) with or without 5% (v/v) beta -mercaptoethanol or 100 mM dithiothreitol for 5 min at 95 °C. Samples were analyzed by discontinuous SDS-PAGE using 3-10 or 3-15% gradient gels with 3% stacking gels or 5% linear gels with 3% stacking gels (30). Coomassie Blue staining was performed according to the manufacturer (BioSafe Coomassie G-250, Bio-Rad). After analysis of metabolically labeled samples, the gels were fixed for 30 min in 30% methanol, 7% acetic acid, incubated in Amplify (Amersham Biosciences) for 15 min, dried, and exposed to BioMax MS films (Eastman Kodak Co.). The molecular markers used were the High-Range Rainbow 14C-Methylated Protein Molecular Weight Markers (Amersham Biosciences) and Precision Protein Standards (Bio-Rad).

Western Blotting and Detection-- Transfer of the proteins to PVDF membranes (Immobilon-P, 0.45 µm, Millipore) was done in a Transfer-Blot SD-Dry Transfer Cell (Bio-Rad) at 2.5 mA/cm2. The transfer buffer used contained 48 mM Tris, 39 mM glycine, 1.3 mM SDS, and 10% (v/v) methanol. After blotting, the membranes were placed in blocking solution (PBS containing 5% (w/v) milk powder, 0.1% (v/v) Tween 20, and 0.05% (w/v) NaN3) overnight at 4 °C and incubated with either streptavidin-AP (diluted 1:1000) or the primary antibody alpha -mycTag mAb (diluted 1:10) or alpha -MUC2C2 (diluted 1:100) for 1.5 h at room temperature. The membranes were washed 3 times for 5 min with PBS-T (PBS containing 0.1% (v/v) Tween 20) and incubated with secondary antibodies (either goat anti-mouse immunoglobulins coupled to AP or goat anti-rabbit immunoglobulins coupled to AP 1:1000 in blocking solution) for 1.5 h at room temperature. After another PBS-T wash (3 times for 5 min) the membranes were developed with nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate. In the cases where biotin ethylenediamine hydrobromide were used (see below), another blocking solution was used (10 mM Tris-HCl, pH 7.5, containing 2% (w/v) bovine serum albumin, 100 mM NaCl, 0.1% (v/v) Tween 20, and 0.05% (w/v) NaN3).

Characterization of the Cleavage Reaction-- Nonlabeled material were immunoprecipitated with the alpha -mycTag mAb from CHO-K1-pSMG-MUC2C cell lysates and media, incubated in citric acid-Na2HPO4, pH 6.0, buffer (McIlvaine buffer) (2.5 h, 37 °C) while still attached to the beads, analyzed by SDS-PAGE, Western-blotted, and detected by immunostaining. The citric acid-Na2HPO4 buffer system used here, as well as below, is the one described by McIlvaine (31). Radiolabeled and immunoprecipitated (alpha -mycTag mAb) material from CHO-K1-pSMG-MUC2C cell lysates or media was treated as described below while still attached to the magnetic beads. The pH dependence of the cleavage reaction was studied by incubating the radiolabeled material from cell lysates or media in citric acid-Na2HPO4 buffers (McIlvaine buffers) with pH from 4.8 to 7.8 for 20 min at 37 °C. Time dependence of the cleavage reaction was studied by incubating aliquots of the lysate material in a citric acid-Na2HPO4, pH 6.0 buffer (McIlvaine buffer), at 37 °C for 0-300 min. The influence of different protease inhibitors on the cleavage reaction was studied by incubating material from cell lysates for 1 h at 37 °C in 50 mM BisTris, pH 6.2 (pH 5.9 at 37 °C), containing one of the following inhibitors: PMSF (1 mM), bestatin (10 mM)(Sigma), leupeptin (100 µM), pepstatin (1 µM) (Roche Molecular Biochemicals), aprotinin (2.15 µM), NEM (5 mM), EDTA (10 mM), or a mixture of protease inhibitors (Complete mini-EDTA-free, Roche Molecular Biochemicals). All incubations above were terminated by removal of the incubation buffers and addition of SDS-PAGE sample buffer before analysis by SDS-PAGE.

Neutralization of the Secretory Pathway-- Confluent LS 174T-pSMG-MUC2C cells were used in these studies. When NH4Cl was used, the cells were preincubated with media with or without 25 mM NH4Cl for 12 h before the 1-h starvation and 8-h labeling period (both performed with 25 mM NH4Cl present). When bafilomycin A1 (a kind gift from AstraZeneca, Mölndal, Sweden) was used, it was present at 300 mM during the starvation (1 h) and labeling (6 h). The cell lysates and media were immunoprecipitated using the alpha -GFP antibody and analyzed by SDS-PAGE.

Linking of a Biotinylating Agent to the Cleavage Product-- Aliquots (114 ng) of purified recombinant MUC2 C terminus secreted from CHO-K1 cells were incubated for 1.5 h at room temperature in either a citric acid-Na2HPO4, pH 7.4 buffer, a citric acid-Na2HPO4, pH 5.4 buffer, or in the latter containing 1 µmol of biotin ethylenediamine hydrobromide (Sigma). The incubations above were terminated by addition of SDS-PAGE sample buffer before analysis by SDS-PAGE and Western blotting.

Edman Sequencing-- Purified C terminus was dried (using a SpeedVac concentrator), dissolved in 50 mM pyridine/acetic acid, pH 5.5 buffer, and incubated overnight at 37 °C. The sample was dried, dissolved in reducing SDS-PAGE sample buffer, separated by SDS-PAGE, blotted to a PVDF membrane, and stained with Coomassie Blue (BioSafe Coomassie G-250, Bio-Rad) for 1 h. The membrane was destained in 50% (v/v) methanol, 10% (v/v) acetic acid, and the band corresponding to the C-terminal cleavage fragment was cut out and sequenced on a Procise 492 Protein Sequencer (Applied Biosystems).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Purification of Secreted MUC2 C Terminus from CHO-K1-pSMG-MUC2C Cells-- The cell line CHO-K1-pSMG-MUC2C permanently expressed a construct encoding the mycTag epitope at the N-terminal end followed by GFP and the last 981 amino acids of the human MUC2 mucin. The murine immunoglobulin kappa -chain signal sequence was used to direct the fusion protein to the secretory pathway. The recombinant MUC2 C terminus (SMG-MUC2C) formed dimers in the ER (with a molecular mass of about 330 kDa) and was later secreted as dimers of higher molecular mass (about 470 kDa), a difference partly due to glycosylation.2 These cells were grown in the presence of 10% (v/v) fetal bovine serum, and the secreted fusion protein was purified by ultrafiltration, gel filtration, and ion exchange chromatography using a 50 mM BisTris, pH 6.0 buffer. The purified SMG-MUC2C was analyzed by SDS-PAGE under nonreducing conditions revealing a single band with an approximate molecular mass of 470 kDa, corresponding to the size of a dimer (Fig. 1). When the same material was analyzed under reducing conditions, three bands were observed (Fig. 1). The largest band corresponded to the expected monomer with an approximated molecular mass of about 250 kDa. The two additional bands migrated at about 130 and 110 kDa, suggesting that these could be due to a cleavage of the full-length SMG-MUC2C. Western blot analysis of the nonreduced band revealed that this was immunoreactive with both the alpha -mycTag mAb and the alpha -MUC2C2 antiserum (Fig. 2A). This was also true for the 250-kDa monomer found after reduction. The 130-kDa band only reacted with the mycTag mAb, whereas the 110-kDa band reacted with the alpha -MUC2C2 antiserum raised against a peptide sequence in the C-terminal end of MUC2. The results indicated that some of the SMG-MUC2C had been cleaved, producing a 130-kDa N-terminal and a 110-kDa C-terminal fragment, and that these fragments were still held together by disulfide bonds.


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Fig. 1.   Purification of secreted MUC2 C terminus from CHO-K1-pSMG-MUC2C cells. Secreted MUC2 C terminus from CHO-K1-pSMG-MUC2C cells was purified using ultrafiltration, ion exchange chromatography, and gel filtration. The purified material was separated on a 3-15% SDS-PAGE gel and visualized by Coomassie Blue staining. NonPur, nonpurified material (used culture media); Pur pH >=  8.0, material purified using buffers with a pH >=  8.0 (as described under "Experimental Procedures"); Pur pH 6.0, material purified using a 50 mM BisTris, pH 6.0 buffer; NonRed, nonreduced samples; Red, reduced samples. Positions of molecular mass standards are indicated to the left. A schematic picture of the recombinant C terminus of MUC2 is given in the upper part of the figure. The arrows indicate the cleavage site, and the underlined sequence indicates the sequence obtained after Edman sequencing of the C-terminal cleavage product (C2). The N-terminal cleavage product is indicated by MG-C1 and MUC2-C1 and MUC2-C2 corresponds to amino acids 4199-4486 and 4487-5179 of the MUC2 sequence (12), respectively. alpha -MUC2C2 indicates the localization of the epitope detected by the antiserum with the same name.


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Fig. 2.   Identification of the cleavage products of the MUC2 C terminus. A, MUC2 C terminus, secreted from CHO-K1-pSMG-MUC2C cells and purified using a pH 6.0 buffer, was separated on a 3-10% SDS-PAGE gel, blotted, and detected with either alpha -MUC2C2 antiserum or alpha -mycTag mAb. B, cell lysates and media from CHO-K1-pSMG-MUC2C cells were immunoprecipitated (using the alpha -mycTag mAb), incubated for 2.5 h in a citric acid-Na2HPO4, pH 6.0 buffer, separated by SDS-PAGE (3-10%, reducing conditions), blotted, and detected by either the alpha -MUC2C2 antiserum or the alpha -mycTag mAb. L, material from cell lysates; M, material from the cell culturing media; Di, position of the dimer of the MUC2 C terminus; Mono, position of the monomer of the MUC2 C terminus; MG-C1, the N-terminal cleaved fragment; C2, the C-terminal cleaved fragment; NonRed, nonreduced samples; Red, reduced samples. IB indicates the antibody used for detection. Positions of molecular mass standards are indicated to the left.

When the glycosylation of the recombinant MUC2 C terminus was studied, using different exoglycosidases, two buffers with different pH values were used (pH 6.0 and 7.0, respectively). When the samples were treated in the pH 7.0 buffer, only the intact protein band was observed. However, when the samples were treated in the pH 6.0 buffer, the two cleavage products appeared (data not shown). These observations of a cleavage occurring only when the sample had been treated at pH 6.0 led to the hypothesis that this reaction was induced at low pH. Purification of secreted MUC2 C terminus from CHO-K1-pSMG-MUC2C cells grown under serum-free conditions, using ultrafiltration, ion exchange chromatography, and gel filtration with buffers having a pH >=  8.0, gave rise to a much lower extent of cleavage (Fig. 1), further supporting this hypothesis.

Localization of the Cleavage Site by Edman Sequencing-- Purified SMG-MUC2C, from spent culture media, was incubated at pH 5.5 overnight. After separation by SDS-PAGE and transfer to a PVDF membrane, the 110-kDa band corresponding to the C-terminal cleavage fragment was excised and subjected to N-terminal amino acid sequencing. The sequence obtained, PHYVTF, is located at a position compatible with the sizes of the two cleaved fragments (see Fig. 1). The localization of this cleavage corresponds to the N-terminal end of the previously found 118-kDa "link peptide" (32).

Characterization of the Cleavage Reaction-- To decipher the identity of the cleavage products produced in the in vitro studies, immunoprecipitated material from CHO-K1-pSMG-MUC2C cell lysates and media was incubated at pH 6.0 and further analyzed by SDS-PAGE, Western blotting, and immunodetection (Fig. 2B). The results showed that the N-terminal cleavage product migrated as ~50 kDa heavier after its secretion (130 relative to 80 kDa, left panel), whereas the C-terminal fragment only acquired around 10 kDa after its secretion (110 relative to 100 kDa, right panel). This caused the two bands to switch position relative to the intact SMG-C monomer, when comparing cell lysate and media. This implies that the modifications, leading to a secreted SMG-MUC2C with lower mobility compared with the intracellular form, mainly are present in the N-terminal part of the protein.

To test the hypothesis of a pH-dependent cleavage, radiolabeled material from CHO-K1-pSMG-MUC2C cell lysates and media was immunoprecipitated using alpha -mycTag antibodies coupled to magnetic beads. While still attached to the beads, aliquots of the material were treated under different conditions, and the labeled SMG-MUC2C was analyzed by SDS-PAGE and autoradiography.

To investigate if the cleavage was induced at a low pH, aliquots of immunoprecipitated SMG-MUC2C from cell lysate and media were treated in citric acid-Na2HPO4 buffers with pH from 4.8 to 7.8 for 20 min at 37 °C (Fig. 3, A and B). The cleavage was observed at a pH around 6.0 and lower for both the intracellular and extracellular forms of SMG-MUC2C. The cleavage products could be observed as weak bands also at pH >6.0 in the secreted but not the intracellular form of SMG-MUC2C. This indicated that the cleavage could occur prior to the incubation and probably inside the cell. The intracellular SMG-MUC2C has been localized to the ER,2 and thus this form has not passed through the more acidic parts of the late secretory pathway. The presence of some cleavage of the secreted SMG-MUC2C suggested that this process occurred late in the secretory pathway.


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Fig. 3.   pH requirement and time dependence of the cleavage reaction of the MUC2 C terminus. Metabolically labeled and immunoprecipitated (using the alpha -mycTag mAb) material from CHO-K1-pSMG-MUC2C cell lysates (A) or cell culture media (B) was treated in citric acid-Na2HPO4 buffers with pH from 4.8 to 7.8 for 20 min at 37 °C. Material from cell lysate was prepared in the same way as above and treated in a citric acid-Na2HPO4, pH 6.0 buffer at 37 °C for different times (0-300 min) (C). All incubations above were terminated by removal of the incubation buffers and addition of SDS-PAGE sample buffer before analysis on a 5% SDS-PAGE gel under reducing conditions. Mono, position of the MUC2 C terminus monomer; MG-C1, the N-terminal cleaved fragment; C2, the C-terminal cleaved fragment. Positions of molecular mass standards are indicated to the left.

To investigate if the cleavage reaction was time-dependent, aliquots of immunoprecipitated SMG-MUC2C from cell lysate were incubated in a pH 6.0 buffer for different times (0-300 min) (Fig. 3C). The result from this experiment showed that cleavage was time-dependent as an increased amount of the cleavage products was observed with longer incubation times. However, cleavage was not complete even after 5 h.

To explore if the cleavage was mediated by a protease, aliquots of immunoprecipitated SMG-MUC2C from cell lysate were treated in a BisTris buffer, pH 6.2 (gives pH 5.9 at 37 °C), with the addition of protease inhibitors (Fig. 4). A wide range of protease inhibitors including Ser, Cys, aspartic, and metalloprotease inhibitors as well as an aminopeptidase inhibitor were used. None of these inhibited the cleavage reaction to a significant extent. This argues for the cleavage not being mediated by an enzyme tightly attached to or being an integral part of the MUC2 C terminus itself.


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Fig. 4.   Influence of different protease inhibitors on the cleavage of the MUC2 C terminus. Metabolically labeled and immunoprecipitated (using the alpha -mycTag mAb) material from CHO-K1-pSMG-MUC2C cell lysates was treated in a 50 mM BisTris, pH 6.2 (gives pH, 5.9 at 37 °C) buffer for 1 h at 37 °C in the absence (Treated) or presence of different protease inhibitors (PMSF (1 mM), bestatin (10 mM), leupeptin (100 µM), pepstatin (1 µM), aprotinin (2.15 µM), NEM (5 mM), EDTA (10 mM), and a mixture of protease inhibitors (complete mini-EDTA-free)). All incubations above were terminated by removal of the incubation buffers and addition of SDS-PAGE sample buffer before analysis on a 5% SDS-PAGE gel under reducing conditions. Mono, position of the MUC2 C terminus monomer; MG-C1, the N-terminal cleaved fragment; C2, the C-terminal cleaved fragment. Positions of molecular mass standards are indicated to the left.

Neutralization of the Secretory Pathway-- The level of cleavage in the secreted material from the CHO-K1-pSMG-MUC2C cells was very low. This together with the fact that CHO-K1 cells are very different from mucin-producing cells led us to test a more physiological relevant cell line, LS 174T. This human colon adenocarcinoma cell line expresses endogenous MUC2 and contains mucin granules and is thus a better model for a goblet cell in studies of mucin biosynthesis. A stable cell line (LS 174T-pSMG-MUC2C) permanently expressing the same construct as the CHO-K1-pSMG-MUC2C cells was generated and shown to secrete dimers of SMG-MUC2C.2 After immunoprecipitations (using either the alpha -mycTag or alpha -GFP antibodies), SDS-PAGE, and Western blot analysis, a 130-kDa band immunoreactive with the alpha -MUC2C2 antiserum and a 210-kDa band immunoreactive with the alpha -mycTag mAb were revealed (Fig. 5A). These cleavage products are slightly larger than the ones of the CHO-K1-pSMG-MUC2C cells, in line with the observation that the secreted SMG-MUC2C is larger in this cell line. The LS 174T cell line is derived from the intestine and normally makes longer and more complex oligosaccharides compared with CHO-K1 cells, suggesting that the differences are due to glycosylation. One also observes that bands migrating in the same positions as the secreted material as well as the cleavage products could be found also in the LS 174T-pSMG-MUC2C cell lysates, something not observed in the CHO-K1-pSMG-MUC2C cells.


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Fig. 5.   Neutralization of the secretory pathway inhibits the cleavage of the MUC2 C terminus. A, cell lysates and media from LS 174T-pSMG-MUC2C cells were immunoprecipitated (using either the alpha -GFP or alpha -mycTag antibodies), separated by SDS-PAGE (3-10%, reducing conditions), blotted, and detected by either the alpha -MUC2C2 antiserum (when immunoprecipitation with alpha -GFP) or alpha -mycTag mAb (when immunoprecipitation with alpha -mycTag mAb). L, material from cell lysates; M, material from the spent culture media. Mono, position of the monomer of the MUC2 C terminus (in B only indicated in the right part); MG-C1, the N-terminal cleaved fragment; C2, the C-terminal cleaved fragment. IB indicates the antibody used for detection. Positions of molecular mass standards are indicated to the left. B, confluent LS 174T-pSMG-MUC2C cells were preincubated with media with or without 25 mM NH4Cl for 12 h. After washing the cells with PBS they where starved (1 h) and metabolically labeled (8 h) in the presence of the same concentration of NH4Cl as during the preincubation. In the case where bafilomycin A1 was used as the neutralizing agent, 300 mM bafilomycin A1 was present during the starvation (1 h) and labeling steps (6 h). After the labeling cell lysates and media were prepared, and immunoprecipitations (using the alpha -GFP antibody) and SDS-PAGE were performed (5% separation gel, reducing conditions). The mAb alpha -GFP and alpha -mycTag gave identical results.

To analyze if the cleavage observed in the secreted SMG-MUC2C was dependent on the acidic pH in the late secretory pathway, this was neutralized by treating the cells with either NH4Cl or bafilomycin A1. NH4Cl mediates its effect through ammonia diffusing into the cells while bafilomycin A1 is an inhibitor of H+-K+-ATPase pumps present in the secretory pathway. The cells were radiolabeled, and cell lysates and media from the treated cells were immunoprecipitated with the alpha -GFP mAb followed by SDS-PAGE (Fig. 5B). Both treatments blocked the cleavage in the secreted material more or less completely as the C2 fragment almost disappeared from the media. The intracellular SMG-MUC2C precursor found at around 210 kDa was as expected not affected. These observations support the idea that the cleavage of the SMG-MUC2C can occur in vivo and that it is triggered by the lower pH in the late secretory pathway. It also argues against the cleavage being an experimental artifact. One can also observe that the secreted SMG-MUC2C acquires a slightly higher mobility after both treatments. This is in agreement with previous studies (33) showing that a neutralization of the secretory pathway, using these compounds, leads to a redistribution of glycosyltransferases and a subsequent decrease in O-glycan chain length.

Pulse-Chase Studies of Cells Expressing the Recombinant MUC2 C Terminus-- Bands migrating in the same positions as the noncleaved and cleaved secreted material were observed in the lysates from the LS 174T-pSMG-MUC2C cell (Fig. 5), but not in the lysates from the CHO-K1-pSMG-MUC2C cells. One possible explanation could be that the SMG-MUC2C cells have a slower passage through the secretory pathway in the LS 174T cells. To test if this was the case, both CHO-K1-pSMG-MUC2C and LS 174T-pSMG-MUC2C cells were pulsed with 35S-labeled Met and Cys for 15 min and chased for 40-110 min. Media from the treated cells were immunoprecipitated with the mAb alpha -mycTag, and the precipitated material was analyzed by SDS-PAGE and autoradiography. The results show that SMG-MUC2C cells appear in the media after 60 min from both cell lines, indicating that there is no difference in the passage rate through the secretory pathway (Fig. 6A).


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Fig. 6.   Pulse-chase studies of cells expressing the recombinant MUC2 C terminus. CHO-K1 and LS 174T cells expressing the C terminus of the MUC2 mucin was metabolically labeled with Redivue Pro-mix L-35S in vitro labeling mix for 15 (A) or 5 min (B) and chased in nonradioactive medium with excess of Met and Cys for different times. Cell lysates and media were prepared and immunoprecipitated by alpha -mycTag mAb coupled to magnetic beads. Samples were analyzed by SDS-PAGE on 5% gels under reducing conditions. A, media (40-110 min) from CHO-K1 (upper panel) and LS 174T cells (lower panel) expressing the C terminus of MUC2. B, cell lysates (0-70 min) and media (70 min) from CHO-K1 (upper panel) and LS 174T cells (lower panel) expressing the C terminus of MUC2. Monoec and Monoic, positions of the monomeric MUC2 C termini in media and cell lysates, respectively; MG-C1, the N-terminal cleaved fragment; C2, the C-terminal cleaved fragment. Positions of molecular mass standards are indicated to the left.

As the total passage time was identical, another reason for a cleavage to occur in the LS 174T, but not in CHO-K1 cells, could be that SMG-MUC2C spends a longer time in the latter, and slightly acidic, parts of the secretory pathway. To test this hypothesis, both cell lines were pulsed for 5 min and chased for different times, and the lysates were immunoprecipitated and analyzed as above. The fully O-glycosylated protein was found also in the cell lysates of the LS 174T-pSMG-MUC2C cells from 30 min to the last chase time (70 min) (Fig. 6B). After long exposure of the film, the cleaved fragments also could be found in the same fractions (not shown), in analogy with those observed in Fig. 5. In contrast to the LS 174T cells, no bands corresponding to the fully glycosylated SMG-MUC2C was found in the CHO-K1 cell lysates. Only very faint bands could be observed in the lysates at chase times 40-70 min after very long exposures of the film (not shown). The results indicate that SMG-MUC2C actually spends a longer time in the latter parts of the secretory pathway in the LS 174T compared with the CHO-K1 cells, a fact that could explain why the pH-dependent cleavage was observed in only in LS 174T cells.

A Primary Amine Reacts with the Cleaved MUC2 C Terminus-- Previously, a mechanism for the cleavage at Asp-Pro bonds at low pH has been proposed and involves the formation of an internal anhydride between the two carboxylic groups of the generated C-terminal Asp (34). Such anhydrides are relatively reactive with primary amines, for example. In a first attempt to indicate the presence of such an anhydride in the cleaved MUC2 C terminus, biotin conjugated with a primary amine was used as a reagent. Purified recombinant MUC2 C terminus, secreted from CHO-K1 cells, was incubated in a citric acid-Na2HPO4 buffer at a pH 7.4 or pH 5.4 with biotin ethylenediamine. Analysis by SDS-PAGE, Western blotting, and detection with streptavidin revealed that only one band was stained when incubation was done at pH 5.4 in the presence of the reagent (Fig. 7). This band migrated at the same position as expected for the N-terminal fragment (designated MG-C1) and reacted with the alpha -mycTag mAb. That the noncleaved and the C-terminal fragment (C2) was not labeled indicated that the reaction with biotin ethylenediamine was specific. The only difference between the cleaved fragment MG-C1 and the intact protein and C1 fragment is the newly produced C terminus. This make it likely that the biotin ethylenediamine did not react with carboxyl groups, for example, and that it has reacted with the Asp C terminus as suggested from the hypothesis of an anhydride formed during the cleavage (34). This should mean that the MUC2 mucin might have a possibility to covalently cross-link with other molecules.


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Fig. 7.   Biotin with a primary amine react with a MUC2 C terminus cleavage product. Aliquots of purified recombinant MUC2 C terminus secreted from CHO-K1 cells were incubated for 1.5 h at room temperature in either a citric acid-Na2HPO4, pH 7.4 buffer, a citric acid-Na2HPO4, pH 5.4 buffer, or in the latter containing 1 µmol of biotin ethylenediamine hydrobromide. After SDS-PAGE on a 3-10% gel, under reducing conditions, the proteins were blotted and detected by either the alpha -mycTag mAb or streptavidin. Mono, the monomer of the MUC2 C terminus; MG-C1, the N-terminal cleaved fragment; C2 with an arrow, the position of the C-terminal cleaved fragment (not shown here). IB indicates the antibody used for detection. Positions of molecular mass standards are indicated to the left.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

It has been observed previously (32) that a 118-kDa glycopeptide could be released from purified rat intestinal mucins by thiol reduction. The peptide was called the "link" peptide, as it was believed to be responsible for linking the mucin monomers together to a polymeric gel structure. This glycopeptide was thoroughly studied (35-37) but was questioned to be a degradation product. When the cDNA encoding the link peptide was cloned, it was first believed to be a separate protein named mucin-like peptide (38), but it was later shown to be an integral part of the rat Muc2. The N-terminal amino acids of the link glycopeptide were sequenced, and it was concluded that the peptide represented the last 689 amino acids of the mucin and was generated by a cleavage at an Asp-Pro bond near the N-terminal border of the mucins D4-domain, a cleavage site identical to the one reported in the present study. The rat Muc2 mucin is the homologue of the human MUC2 mucin and as such shows extensive sequence similarities to its N- and C-terminal Cys-rich domains (12, 38-40). It has been disputed whether the link peptide is a preparatory artifact or not. The same cleavage fragment starting with the amino acids Pro and His of GDPH have been found by Herrmann et al. (41) in the human MUC2 mucin, prepared by rigorous methods, from the large intestine. This further argues for the hypothesis that a cleavage between Asp and Pro in GDPH occurs naturally. The results presented here further suggest that this cleavage is due to an autocatalytic mechanism triggered by low pH. It also argues for it being a natural phenomenon occurring in MUC2 and suggests that the cleavage could be of biological relevance as it takes place at a pH comparable with that of the late secretory pathway. The cleavage occurred at pH 6 and below, and as the pH in the trans-Golgi network is slightly below 6.0 (42) and can approach 5.2 in the secretory granules (43), the mucin is likely to encounter a milieu in which this type of cleavage may occur. The use of agents that could neutralize the secretory pathway inhibited the cleavage found in LS 174T-pSMG-MUC2C cells, thus proving that the cleavage can actually occur in the cell. Furthermore, the reaction was not inhibited by any of the used protease inhibitors supporting the hypothesis of it being a nonenzymatic process.

The GDPH sequence is also found in other proteins, but it has only been studied in the pre-alpha -inhibitor, which has shown a similar type of cleavage as we report here. The pre-alpha -inhibitor is a serum protein secreted from rat liver and whose physiological function is still not understood. This protein consists of the two polypeptides, bikunin and heavy chain 3 (H3), covalently linked to each other by a chondroitin sulfate chain (44). H3 is synthesized as a precursor that, like the MUC2 mucin, is cleaved between an Asp and a Pro in a GDPH sequence (45). After the cleavage, H3 is esterified, via the alpha -carbon of its C-terminal Asp, to C-6 of an internal N-acetylgalactosamine of the glycosaminoglycan chain attached to bikunin (44). The cleavage has been shown to be induced by the low pH in the late Golgi and mediated through an intramolecular process, suggested to generate a reactive Asp anhydride (46), which could be the case for MUC2 as well. Mutation studies of H3 have shown that the presence of the Asp and the Pro in the GDPH sequence are absolutely essential for the cleavage to occur (47). However, other residues surrounding these two amino acids as well as residues located further C-terminal seem to be of importance for the cleavage reaction (46). The sequences flanking the GDPH sequences in MUC2 and H3 show limited sequence similarity (Fig. 6), suggesting there might be differences in how the cleavage takes place and its consequences.

It is well known that Asp-Pro bonds are acid-labile, something that has been used for chemical cleavage of proteins (48). However, the conditions necessary for this type of protein fragmentation are far harsher than the ones we used, typically including treatment in diluted acids with pH <=  2.5 at 40 °C for at least 24 h. The milder conditions used for the in vitro experiments in this study may explain why the cleavage reaction does not come to completion. The mild acidic environment that proteins experience on their way through the secretory pathway is probably also the reason for the low extent of cleavage seen in the secreted material. Furthermore, the in vitro studies show that the time the proteins spend in the acidic environment is an important factor for the extent of cleavage. The SMG-MUC2C protein analyzed here was continuously secreted from both the LS 174T-pSMG-MUC2C and CHO-K1-pSMG-MUC2C cells. Although the overall passage rate of SMG-MUC2C through the secretory pathway was similar in both cell lines, the time it spent in the latter and slightly acidic parts seemed to be longer in the LS 174T cells. This might explain the higher cleavage seen in this cell line as compared with the CHO-K1 cells. A fast passage through the latter part of the secretory pathway will only expose the protein to the acidic pH for a rather short period. However, in vivo the normal full-length MUC2 mucin is directed to the regulated secretory pathway and stored in secretory granules for extended periods (49). This means that the extent of cleavage may be more pronounced as the protein encounters a more acidic environment and for a longer time. One can thus suggest that the cleavage observed in both rat and human MUC2 is natural and that the extent of cleavage could vary.

Searches for GDPH sequences reveals that this is also present in other mucins. Of the gel-forming mucins, this sequence is only found in the C terminus of the MUC5AC but not in the other known gel-forming mucins MUC5B, MUC6, and PSM (Fig. 8). The absence of this sequence in the latter two is not surprising as both lack the D4-domain, the region were the GDPH sequence is located. The sequence is also absent in the vWf, which is otherwise showing high sequence similarities within the C terminus of the gel-forming mucins and contains a D4-domain. When looking at the gel-forming mucins, it is also interesting to notice that the ones that are normally expressed in goblet cells (MUC2 and MUC5AC) contain the GDPH sequence, whereas the ones normally expressed in glands (MUC5B and MUC6) do not (Fig. 8). In addition to gel-forming mucins, the GDPH sequence is also found in the transmembrane mucin MUC4 and its rat orthologue, the sialomucin complex (SMC). SMC is encoded as a single protein but is found as a heterodimeric glycoprotein complex composed of one extracellular and one transmembrane subunit (50, 51). These two subunits are formed due to an uncharacterized intracellular cleavage at an Asp-Pro bond located in a GDPH sequence (52, 53). No proteases have been identified, and we now suggest that this cleavage is also due to a pH-triggered autocatalytic process.


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Fig. 8.   Sequence comparisons of selected mucins and other proteins. Selected proteins are aligned with the sequence surrounding the GDPH sequence of the human MUC2 mucin. The residue numbers in the figure do not necessarily refer to the actual number from the true N terminus because all proteins in the figure have not been fully sequenced. The residue numbers for MUC5AC and MUC5B are from the assembly made by Perez-Vilar and Hill (2) and are marked with * in the figure. The secretory mucins that contain the GDPH sequence has this located in their early D4-domain. Both MUC5B and the vWf lack the GDPH sequence in their D4-domains, whereas MUC6 and PSM lack the whole D4-domain and are therefore not represented in the figure. The GDPH of hemolectin is located in a D-domain, corresponding to the D2-domain on the mucins. The GDPH sequences of MUC4, SMC, and H3 are not located in any D-domain, and these proteins show little sequence homology with the others. The amino acids within the GDPH sequence are printed as white on black, and the other amino acids matching the MUC2 sequence are highlighted with a gray background.

The GDPH sequence is also found in proteins other than mucins. The H3 protein has already been discussed, but another case is the recently cloned protein hemolectin from Drosophila melanogaster (52). This protein is synthesized in the hemocytes at the larvae stage and shows sequence similarities to the vWf and the gel-forming mucins. The hemolectin has been shown to be sensitive to acidic cleavage (54). The exact localization of the cleavage site has not been defined, but it has been proposed to be located in the area around its D2-domain. Interestingly, hemolectin contains a GDPH sequence as well as other similarities to MUC2 in the region surrounding this sequence (Fig. 8). It is thus likely that this protein also is cleaved at the Asp-Pro linkage and that this occurs by a similar mechanism as that for MUC2.

The consequence of the pH-dependent cleavage for the properties of the MUC2 mucin is presently not known. One possibility is that it leads to conformational changes giving the protein new properties that, for instance, could affect its viscosity. There is no experimental support for such a hypothesis, but there is for the idea that the cleavage could give the mucin an opportunity to link to other components. The present results suggest that the GDPH cleavage of MUC2 generates a reactive Asp (an anhydride) at the C-terminal end because it was possible to label this fragment with biotin substituted with a primary amine. As discussed above, this generation of an Asp anhydride has been shown for H3, where the anhydride reacts with the C-6 hydroxyl group of an internal GalNAc in the chondroitin sulfate chain attached to bikunin, generating the pre-alpha -inhibitor (44, 45). Such an ester linkage could theoretically be formed between the reactive Asp produced after cleavage of MUC2 and any of the abundant O-glycans of mucins. The previously observed covalent bond connecting MUC2 monomers even after reduction of the disulfide bonds (28, 41) could be due to such a linkage. Although it is not known to date if the cleavage of GDPH in MUC2 is accompanied by the formation of any covalent bond to another molecule or not, the potential appearance of such a linkage could be physiologically very important. An example where this might be relevant is the disease cystic fibrosis, characterized by viscous mucus and impaired clearance of the mucous layer. Recent studies (55, 56) suggest that a nonfunctional cystic fibrosis transmembrane conductance regulator, as in cystic fibrosis, causes a lowered pH value of the secretory pathway as well as a more acidic extracellular milieu. If either or both of these assumptions are correct, this lower pH value could lead to a higher degree of cleavage of MUC5AC in the lungs and MUC2 in the intestine and lungs followed by cross-linking to itself or other molecules. It could be speculated that this is one explanation for the observed, but still not explained, mucous aberration of this disease. However, these ideas have to be confirmed by biochemical proof for the presence of such cross-links and a deeper understanding of the pH-dependent autocatalytic cleavage at the C-terminal domain of the MUC2 mucins.

    ACKNOWLEDGEMENT

We thank Fredrik Olson for help with the Edman sequencing.

    FOOTNOTES

* This work was supported by Swedish Research Council Grant 7461 and by the IngaBritt and Arne Lundberg Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed. Tel.: 46-31-773-34- 88; Fax: 46-31-41-61-08; E-mail: gunnar.hansson@medkem.gu.se.

Published, JBC Papers in Press, February 11, 2003, DOI 10.1074/jbc.M210069200

2 M. E. Lidell, M. E. V. Johansson, M. Mörgelin, N. Asker, C. J. R. Gum, Jr., Y. S. Kim, and G. C. Hansson, unpublished observations.

    ABBREVIATIONS

The abbreviations used are: vWf, von Willebrand factor; PSM, porcine submaxillary mucin; ER, endoplasmic reticulum; GFP, green fluorescent protein; mAb, monoclonal antibody; AP, alkaline phosphatase; PBS, phosphate-buffered saline; PMSF, phenylmethylsulfonyl fluoride; NEM, N-ethylmaleimide; H3, heavy chain 3; SMC, sialomucin complex; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; PVDF, polyvinylidene difluoride.

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ABSTRACT
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RESULTS
DISCUSSION
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