Disulfide-Bound Proteolytic Fragments of Gastric Mucin Are 100- and 140-kDa Proteins

Biochemical and Biophysical Research Communications 270, 722–727 (2000) doi:10.1006/bbrc.2000.2497, available online at http://www.idealibrary.com on

Disulfide-Bound Proteolytic Fragments of Gastric Mucin Are 100- and 140-kDa Proteins Iwona Minkiewicz-Radziejewska,* Andrzej Gindzien´ski,* and Krzysztof Zwierz† *Department of General and Organic Chemistry and †Department of Pharmaceutical Biochemistry, Institute of Chemistry, Medical Academy of Bialystok, Bialystok 8, Poland

Received February 24, 2000

Pig gastric mucus was tested for its autodegradative proteolytic degradation at pH 7.0, in the presence or absence of proteinase inhibitors and SDS. Samples of crude mucus were incubated at room temperature for 48 and 96 h in sodium azide stabilized buffer, pH 7.0, and urea-extracted mucin was purified. Electrophoretically homogenic mucin preparation was reduced and alkylated with iodo[ 14C]acetamide, and analyzed for labeled products. On 7.5% SDS/PAGE protein bands at 80 and 120 kDa were noted, but radioactivity was incorporated into 100- and 140-kDa bands, with increasing intensity from T 0 to T 96, and into high molecular mass mucin subunits. The results confirmed the autodegradative properties of gastric mucin and demonstrated that the 100- and 140-kDa fragments are the main proteolytical products of pig gastric mucin and are disulfide bound with the rest of the molecule. © 2000 Academic Press Key Words: disulfide depolymerization; gastric mucin; proteolysis.

Native secretory mucins, isolated from different sources, have a similar structure. They consist of high molecular mass heterogenous monomers containing densely O-glycosylated tandem repeats protected from proteolytic degradation, flanked by sparsely N-glycosylated and cysteine reach N and C terminals sensitive to proteolytic degradation. The cysteins of N and C ends link mucin monomers by disulfide bridges forming linear mucin oligomers of 2– 40 mDa molecular mass (1– 6) (Fig. 1). During preparation of mucin without elimination the activity of proteolytic enzymes there are released 60 – 120 kDa fragments of mucins as demonstrated by SDSPAGE (2, 4 –7). The 70 kDa fragments of gastric mucins were first described by Allen et al. who suggested their key role in maintaining polymeric structure of mucin (8). Recently was described that the 118 kDa link glycopeptide of C terminal part of intestinal mucin 0006-291X/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.

probably resulted from cleaving of Asp-Pro bond located approximately 690 residues from C terminus (9). There are the possibility of appearance of 60 –120 kDa fragments of gastric mucin caused by the action of proteolytic enzymes, which are linked to the rest of mucin by disulfide bridges. The main goal of the experiments presented here was to check time-dependent release of proteins and determination molecular mass of disulfide-bound proteins. MATERIALS AND METHODS The mucus from pig stomachs was obtained by the method elaborated in our laboratory (10). Briefly, pig stomachs (1 h after sacrificing the animals) were washed with water and incubated overnight at 4°C in 50 mM boric buffer pH 7.0 containing 10 mM EDTA-Na 2, 0.02% NaN 3 at 4°C overnight. The mucus appearing at the surface of gastric epithelium was gently scraped and homogenized in 6 M urea containing inhibitors of proteinases (1 mM PMSF phenylmethylsulfonyl fluoride), 5 mM EDTA-Na 2, 2 mM NEM (N-etylmaleimide), 11 mM iodoacetamide). The homogenate was additionally gently stirred for 20 h at 4°C to solubilize maximal amount of mucin. Material insoluble in 6 M urea was removed by centrifugation at 15000g for 30 min. Supernatant was dialysed and resulted mucin precipitate (crude mucin) was resolubilized in 1% of SDS. Resolubilized crude mucin was concentrated, the proteinase inhibitors were added and the sample was applied on a 2.3 ⫻ 56 cm Sepharose CL 2B column. Mucin was eluted with 50 mM boric buffer pH 7.0 with 10 mM EDTA-Na 2, 0.02% NaN 3, 1% SDS. The eluted fractions were assayed for carbohydrate and protein (by absorbance at 280 nm). The high molecular weight fractions were pooled, concentrated and assigned to rechromatography on another Sepharose C1 2B column (2.3 ⫻ 135 cm) and then on Sephacryl S-500 column (2.4 ⫻ 56 cm) eluted with the above mentioned buffer. Material received after chromatography on Sephacryl S-500 column was designed purified mucin. Purified mucin was concentrated by dialysis against polyethyleneglycol 35 000 and then SDS was removed by acetone extraction (11). Resulted precipitate of purified mucin was suspended in the 50 mM boric buffer pH 7.0 by gentle homogenization in a Potter homogenizer and submitted to further analysis, or dissolved in the 50 mM boric buffer with 1% SDS. In order to establish conditions which minimalize proteolytic activity in the crude mucin we incubated it for 48 and 96 h at 4°C and at room temperature in: (1) 50 mM boric buffer pH 7.0, (2) boric buffer with proteinase inhibitors (1 mM PMSF, 2 mM NEM, 5 mM EDTA-Na 2 mixture), (3) boric buffer with 1% SDS, and (4) boric buffer with SDS and proteinase inhibitors. Proteolytic activity was

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FIG. 1. The structure of mucin. Monomers containing densely O-glycosylated tandem repeats are linked together via disulfide bonds located on sparsley glycosylated N- and C-terminus. Arrows indicate possible Asp-Pro bonds located approximately 690 residues from the C-terminus, which cleavage can release the 118 kDa component (9).

determined by measuring the amounts of released free-NH 2 groups according to (12). To release every 60 –120 kDa fragments of mucin linked to the rest of the purified mucin, oligomers of the purified mucin (500 ␮g) were reduced in 0.2 M 2-mercaptoethanol for 3 h at 37°C. Alkylation of free -SH groups was performed at pH 8.0, by adding iodoacetamide to 0.4 M and incubated overnight at 4°C in the dark. Alkylated proteins were dialysed against Mili Q water for 24 h at 4°C (4 changes of water). To check if 60 –120 kDa fragments released from oligomeric mucin are produced by the action of proteolytic enzymes, mucosal scrapings were incubated for 48 and 96 h at room temperature in boric buffer with sodium azide but without proteinase inhibitors. Then samples were purified with the presence of proteinase inhibitors and resulted mucin (1 mg of each sample) was reduced and alkylated with iodo[ 14C]acetamide. At first we alkylated free -SH groups by incubation in “cold” 0.1 M iodoacetamide under nitrogen at 37°C for 4 h in the dark. Iodoacetamide was thoroughly removed by dialysis against water for 12 h (2 changes) and 0.05 M boric buffer pH 8.0 for 12 h (2 changes) at 4°C. After that easily accessible -S-S- groups were reduced by adding 0.14 ␮moles of dithiothreitol and incubated under nitrogen at 37°C for 4 h. Then free -SH groups were alkylated by adding 0.28 ␮moles of iodo[ 14C]acetamide (60 mCi/mmol) and incubated at 4°C in darkness overnight. For complete breaking of the disulfide bonds, samples were reduced in 10 mM DTT and alkylated by 20 mM iodoacetamide. The reduced and alkylated samples were dialysed against water (for 48 h) and boric buffer (for 12 h). The dialysed, reduced and alkylated mucin was subjected to SDS/PAGE. Then the SDS/PAGE gels were protein stained, dried and submitted to autoradiography. The gels were exposed to close contact with X-ray film (Hyperfilm-␤max, Amersham) for 1–2 weeks at ⫺70°C. Aliquots of the reduced and alkylated mucin were also chromatographed on a column with Sephacryl S-500 in buffer supplemented with 0.02 M of 2-mercaptoethanol. Neutral sugars were measured by the phenol–sulfuric acid method (13) and proteins by ultraviolet absorbance measurements at 260 and 280 nm (14). The polyacrylamide gel electrophoresis was performed as described by Laemli (15) and proteins in gels were stained by the silver method (16).

FIG. 2. Gel filtration of crude mucin on a Sepharose C1 2B column. The precipitate of crude mucin was dissolved in 1% SDS and concentrated, the proteinase inhibitors were added, and the sample was applied to a 2.3 ⫻ 56 cm Sepharose CL 2B column. The column was eluted with 50 mM boric buffer, pH 7.0, containing 10 mM EDTA-Na 2, 0.02% NaN 3, and 1% SDS. 5-ml fractions were collected and the eluate was assayed for carbohydrate (F) by the phenol– sulfuric acid method and by absorbance at 280 nm (E). The highmolecular-mass fractions under the bar were pooled, concentrated and assigned to rechromatography on another Sepharose CL 2B column (2.3 ⫻ 135 cm).

For the long lasting procedure of gel exclusion chromatography, we tried to find out conditions which would minimize proteolytic degradation of mucin. The results of the measurements of proteolytic activity of the crude mucus extract incubated in different conditions are presented in Table 1. Data in the table show that in the samples of crude mucin incubated without proteinase inhibitors or SDS, proteolysis took place as a time dependent increase of free-NH 2 groups. This proteolysis was partially inhibited in the presence of proteinase inhibitors or SDS.

RESULTS In our study crude oligomeric mucin was partially purified by chromatography on a Sepharose CL 2B column in buffers with 1% SDS and proteinase inhibitors (Fig. 2). High molecular fraction was rechromatographed on another Sepharose CL 2B column and on a Sephacryl S-500 column (Fig. 3). Mucin isolated by this procedure was free of non-mucin proteins as determined by SDS gel electrophoresis (Fig. 4, lane a).

FIG. 3. Rechromatography of the mucin on a Sephacryl S-500 column. Partially purified mucin obtained after chromatography on two Sepharose CL 2B columns was concentrated and applied to a 2.4 ⫻ 56-cm Sephacryl S-500 column eluted with buffer with SDS. The fractions (3.8 ml) were eluted with the 0.05 mM boric buffer, pH 7.0, with SDS and assayed for carbohydrates (F) and by absorbance at 280 nm (E). The high-molecular-mass fractions under the bar were pooled, concentrated, and submitted to further analysis.

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FIG. 4. Electrophoresis of reduced and alkylated mucin. Purified mucin was tested for its purity (lane a) and then reduced and alkylated as described under Materials and Methods. 50 ␮g (lane a) and 10 –15 ␮g (lane b) of proteins were applied on 7.5% of separating gel. After electrophoresis gels were silver stained. Lane b, mucin after reduction and alkylation. Lane c, molecular mass markers.

Purified oligomeric mucin was intensively reduced, alkylated and subjected to SDS/PAGE. We could observe high molecular mucin on the top of the gel and a distinct band of released proteins at approx. 70 – 80 kDa (Fig. 4, lane b). In a few cases, another weakly stained 120 kDa protein band was also noted. To evaluate endopeptidase activity in the crude mucin, homogenate of mucus scrapings was divided into three pools. One was frozen and the two others were incubated for 48 and 96 h respectively, at room temperature, in the boric buffer pH 7.0 containing sodium azide. After incubation, mucin from each sample was purified in the typical way, as described (see Materials and Methods) in the presence of both proteinase inhibitors and SDS. Each preparation of pure mucin was reduced and alkylated with iodo[ 14C]acetamide (see

FIG. 5. Electrophoresis (A) and autoradiography (B) of purified mucin reduced and alkylated with iodo[ 14C]acetamide. Mucus scrapings were incubated for 48 and 96 h at room temperature, then the samples were purified and high molecular mass mucin peak material was reduced and alkylated with iodo[ 14C]acetamide (see Materials and Methods). 15–20 ␮g of protein were applied to the 7.5% separating gel. After electrophoresis the gel was silver stained. Autoradiogram was developed after two weeks exposition of the gel with X-ray film at ⫺70°C. Lanes a, b, and c, samples after 0, 48, and 96 h incubation time, respectively. Lane d, molecular mass markers.

Materials and Methods) and divided into two portions. One portion was subjected to SDS/PAGE and the other to Sephacryl S-500 gel filtration in reducing conditions. SDS/PAGE gels were silver stained, dried and subjected to autoradiography. On the protein silver staining, we observed three diffuse bands of 80, 120, and approximately 200 kDa of apparent molecular mass, weak in the control sample and stepwise increasing in intensity in the samples incubated for 48 and 96 h (Fig. 5A). However, autoradiography revealed the radioactivity incorporation into oligomeric non-penetrating gel subunit fraction and, for the sample incubated for 96 h, into two distinct diffuse bands of approx. 100 and 140 kDa of apparent molecular mass. In the control and 48 h incubated samples, weakly saturated bands were

TABLE 1

Proteolytic Activity in the Crude Mucin at pH 7.0 Free-NH 2 groups [␮mol/mg of protein] Temperature 22°C

Crude mucin Crude mucin with 1 mM PMSF, 2 mM NEM, 5 mM EDTA-Na 2 Crude mucin with 1% SDS Crude mucin with proteinase inhibitors and 1% SDS

Temperature 4°C

T0

T 48

T 96

Increase during 96 h

T0

T 48

T 96

Increase during 96 h

24.0 25.0

26.6 25.8

27.4 26.0

3.4 1.0

24.0 25.0

25.0 25.6

26.2 26.0

2.2 1.0

25.2 25.0

25.4 25.6

26.0 26.0

0.8 1.0

25.2 25.0

25.4 25.6

25.8 25.8

0.6 0.8

Note. Four samples of the crude mucin were incubated at 22 and 4°C for 48 and 96 h in different conditions. After incubation, the amount of free-NH 2 groups in each sample was measured according to Habeeb (12). T, time in hours. 724

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eluted in tubes 35– 44 (fraction b from Fig. 6). Fraction a contained labeled monomeric high molecular mucin observed on the start line on the SDS/PAGE gel. The amount of radioactivity incorporated into both monomeric mucin (fraction a) and proteolytic fragments (fraction b) from the Sephacryl S-500 column increased from 48 to 96 h of incubation (Table 2). DISCUSSION

FIG. 6. Column chromatography of purified mucin, reduced and alkylated with iodo[ 14C]acetamide. Mucus scrapings were incubated for 48 and 96 h at room temperature, then the samples were purified, reduced, and alkylated with iodo[ 14C]acetamide (see Materials and Methods) and applied on the Sephacryl S-500 column. The column was eluted with SDS– boric buffer containing 1% SDS and 0.02 M of 2-mercaptoethanol. Fractions (3.8 ml) after each incubation time (T 48, E; T 96, F) were assayed for scintillation counting. Fractions under bars (for mucus sample incubated for 96 h) were pooled, concentrated, and submitted to further analysis.

The aim of our investigation was the determination of the molecular mass of proteins bound by disulphide bridges to pig gastric mucin and released on reduction and alkylation. Many authors have described the release of 118 –120 kDa proteins from the mucins (2, 6, 17) but according to our knowledge, no one has demonstrated disulfide binding of such protein(s) to the macromolecular structure of gastric mucin. Although the released proteins are observed after disulfide reduction of the mucin, the possibility of entrapment of some proteins into the mucin three dimensional network and their release on disulfide depolimerization, also can not be excluded (18). Because in the purification procedure of our mucin we used gel exclusion chromatography, performed with participation of some protease inhibitors and SDS in buffered solutions at room temperature, it was interesting to measure in the crude mucus preparation the extent of proteolytic activity at pH 7.0 and the inhibitory effect of inhibitors used and SDS. As is presented in Table 1, in the crude mucin proteolytic activity was present which was partially inhibited with both inhibitors and SDS. However, according to our experience, in some mucin preparations there was no released proteins using the same purification procedure. This

also seen at 100 and 140 kDa levels (Fig. 5B). No distinct radioactive band was found at the 70 kDa level. The elution profiles of the same reduced and alkylated with iodo[ 14]acetamide mucin samples, after chromatography on the Sephacryl S-500 column, are presented in Fig. 6. Most of the radioactivity was eluted in the wide, high molecular mass heterogenous peak (tubes from 20 to 47), and in the small one, eluted just a few tubes later (tubes 50 –57). Similarly, the amount of radioactivity incorporated also increased with the length of incubation, especially for sample incubated 96 h. To locate 100 and 140 kDa proteins of radioactive bands on SDS/PAGE electrophorograms on the elution curve from Sephacryl S-500, eluate was divided into three fractions (a, b, and c, Fig. 6). Material from each fraction was submitted to polyacrylamide gel electrophoresis with protein-staining (Fig. 7A) and autoradiography (Fig. 7B). As demonstrated on the autoradiogram, the labeled 100 and 140 kDa bands were

FIG. 7. Electrophoresis and autoradiography of the eluate from the Sephacryl S-500 column. Mucin sample after 96 h of incubation at room temperature was chromatographed as described in the legend to Fig. 6. Fractions under bars were pooled and submitted to electrophoresis, protein staining (A), and for autoradiography (B). Lane a, fractions 20 –29 (peak a from Fig. 6); lane b, fractions 35– 46 (peak b); lane c, fractions 50 –57 (peak c); lane d, molecular mass markers.

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The Composition of Purified Mucin and Radioactivity in Peaks a and b from Fig. 6 Composition of purified mucin, reduced and alkylated with iodo[ 14C]acetamide

T 48 T 96

Total radioactivity [dpm]

Protein [mg]

Neutral sugars [mg]

Sugars/protein ratio

Fractions 20–29 from peak a in Fig. 6

Fractions 35–46 from peak b in Fig. 6

b/a ratio

5.96 6.44

2.54 2.64

0.43 0.40

8721 61458

5173 47305

0.59 0.77

Note. Analyzed mucus samples were reduced and alkylated with iodo[ 14C]acetamide and separated on a Sephacryl S-500 column, as is presented in Fig. 6. T, times (in hours) of incubation of the mucus homogenate at room temperature.

can be explained in that mucin proteolytic destruction is an early event in mucin isolation, and, in the presence of both inhibitors and SDS, proteolysis is effectively inhibited. Similar inhibitory properties of guanidine hydrochloride, another mucus-disaggregating agent, has also been recently described (17). Although we did not perform ultracentrifugal purification in the CsCl gradient, mucin isolation by multistep gel exclusion chromatography in the presence of 1% SDS is very effective in non-covalently bound proteins dissociation and removal (19, 20). In order to prove the proteolytic origin and disulfide binding of the released proteins, we submitted the crude mucin to incubation for 48 and 96 h at room temperature in the azide protected buffer pH 7.0. The incubated mucin from each sample was purified and submitted to radioactive labeling. Firstly, free sulfhydryl groups were alkylated with an excess of iodoacetamide, and after extensive dialysis, mucin disulfide bonds were partially reduced with a low concentration of dithiothreitol. Newly formed -SH groups were alkylated with twice molar concentration of iodo[14C]acetamide. To complete mucin disulfide depolymerization, the rest of the disulfide bonds was fully reduced with an excess of dithiothreitol, alkylated with “cold” iodoacetamide and dialyzed. As can be seen in Fig. 5, on SDS/PAGE protein stained gels, as much as three protein-stained bands were detected, and the intensity of the bands is proportional to the length of time of incubation. Two radioactive bands were found on the same gels after autoradiography, however most of the radioactivity incorporated after 96 h of incubation, was found in bands of 100 and 140 kDa apparent molecular mass. These results were confirmed with Sephacryl S-500 column chromatography of the same material (Fig. 6). Released proteins of 100 and 140 kDa were eluted in the fraction b. From the data presented in Fig. 5 and Table 2 can be seen, that most of the radioactive proteins were released between 48 and 96 h of incubation. In the similar amounts of mucin in both the reduced and alkylated samples, after 96 h there is a shift of radioactivity from fraction a to fraction b (b/a ratio 0.77 vs. 0.59,

Table 2). Assuming a similar amount of disulfide bonds in both samples, it could be treated as the result of mucin protein backbone proteolysis at the C- and N-mucin subunit termini. Some authors suggest, that protein releasing with reducing agents is the result of earlier accidental proteolysis of the C-terminus fragments of mucin subunits (Fig. 1) (9, 17). Lately, the 140 and 150 kDa proteins were found to be released on reduction and carboxymethylation of rabbit intestinal mucin, both in 95 and 100% homologous with the N-terminal sequence of human MUC2 mucin (21). However, the sum of total amount of radioactivity incorporated into fractions a and b of the samples incubated for 96 h is almost 8 times greater than after 48 h. This may be the result of better accessibility of disulfide groups after proteolysis, or prolonged incubation resulted in solubilization of another population of mucin, stabilized with densely located disulfide groups. In summary, we found on autoproteolysis and disulfide reduction, the release of at least five different proteins. Several pig gastric mucin populations have been described (22, 23), and actually we cannot say if the release is common in all populations. Two proteins, 100 and 140 kDa molecular mass appeared to be cystein reach and disulfide bound to mucin subunits, but their concentration was probably to low to be protein stained. The higher concentration of the released proteins was found after 96 h, but their relative concentration increased only slightly on incubation. Timedependent concentration increase of the 100 kDa (Fig. 5) suggests a stepwise proteolytic depolymerization of the 140 kDa band. REFERENCES

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