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Mucinase Activity 38538531Mucinase ActivityRoger M. Stark, Rebecca Wiggins, Elizabeth Walley,Sally J. Hicks, Gulnaz A. Gill, Stephen D. Carrington,and Anthony P. Corfield1. IntroductionTurnover of the mucous “barrier gel” overlying mucosal surfaces is essential forhydration, mechanical protection, the physical removal of contaminants and toxins,the generation of sacrificial binding ligands that prevent microbial penetration, andthe provision of a suitable environment to renew other defensive molecules that areincorporated into mucus. Mucinase activity is crucial to this turnover process in loca-tions such as the gut and the reproductive tract. Similar activity may also be of rel-evance at other mucosal surfaces that are not normally colonised by significantmicrobial populations, such as the eye and the respiratory tract.Mucinase activity is owing to a mixture of enzymes that are expected to includeproteinases, peptidases, glycosidases, and sulphatases of prokaryotic or eukaryoticorigin. In addition, the presence and action of other hydrolases, including phosphatasesesterases and lipases should also be considered.The assay of individual enzyme activity resulting in the release of single compo-nents from mucin substrates does not give a complete analysis of the total mucin-degrading potential of the sample under study. The examination of mucinase activityfrom any source must be correlated with the nature and origin of the mucin used assubstrate. Mucins are fragmented differently as a result of structural variabilitybetween apomucin peptides and their individual patterns of glycosylation. Equallysignificant is the composition of the mucinase activity that they encounter. A carefulexamination of the fragments derived from incubations of specific mucins withmucinase activity from different sources can yield information on the succession ofdegradative steps, as well as the enzymes that carry them out.The development of methods for detecting mucinase activity and resolving it intoits component stages is in its infancy. However, with the increasing knowledge of thestructure and organization of mucin (1,2) it is now becoming possible to match frag-From:Methods in Molecular Biology, Vol. 125: Glycoprotein Methods and Protocols: The MucinsEdited by: A. Corfield © Humana Press Inc., Totowa, NJ 386 Stark et al.mentation patterns with known sequence information for both peptides and oligosac-charides. Clearly, this is best assessed using a whole mucin substrate and analyzingthe progressive degradation to small molecular weight fragments. However, the analy-sis of size-fractionated products for carbohydrate and/or amino acid compositionrequires large amounts of mucin substrate, is time-consuming, and is therefore notsuitable for the screening of large numbers of samples. Larger-scale screening formucinase activity is possible using radiolabeled mucins prepared from organ or cellcultures. Mucins can be labeled with suitable precursors; for example, cultures ofcolonic mucosa can be labeled with [35S]sulfate, [3H]glucosamine, and [3H]threonine.The purified mucins are subsequently incubated with a source of mucinase activity,and the degradation is assessed by gel filtration (3–5).An alternative to the radioactive methods is the use of biotinylated mucin sub-strates tagged through either the peptide or carbohydrate moieties of the molecule(6,7). Such assays have been described for protein substrates and adapted for use withmicrotiter plates (8). They require small amounts of pure substrate, have high sensitiv-ity, are rapid, and can be applied to large numbers of samples. Such assays are suitablefor the detection of “total” mucinase activity. Thus, their main use is for the rapidscreening of sources of mucinase activity that may be enzymatically heterogeneous.The known structure and properties of mucins, together with the ability to biotinylateeither their protein or carbohydrate domains, has important implications for the inter-pretation of microtiter plate assays. It is important to be aware that the release ofadherent mucin from a plastic surface may be owing to several factors. First, the deg-radation of mucin substrates and their release from the plates may be entirely the resultof mucinase action over the whole domain structure of the mucin. Second, it mayresult from the cleavage of regions of the mucin molecules which specifically bind tomicrotiter plates. A third caveat is the possibility that paradoxical results may beobtained when digesting mucins labeled through their peptide vs their carbohydrateresidues. In extreme cases, e.g., where adhesion of mucins to the plastic of microtiterplates is mainly related to hydrophobic binding to peptide sequences, mucins labeledthrough their carbohydrate may appear to be fully digested when exposed only topeptidases. In such cases, however, any released glycopeptides would be protectedfrom digestion by their carbohydrate chains. Where adhesion is mainly related to ionicinteractions between charged carbohydrates and the microtiter plate, sialidase or sul-fatase activity alone may release the majority of bound mucin while causing only lim-ited degradation of the whole molecule. Therefore, the microtitre plate assay may notappear to be specific for enzyme type with respect to the nature of the mucin biotinlabel. In reality, however, binding is probably the result of multiple binding interac-tions, which tends to reduce the significance of such extremes of binding interaction.Nevertheless, the composition of mixed enzyme preparations cannot be deduced di-rectly from the plate assays. To obtain this information, an examination of fragmentationprofiles by size fractionation and electrophoresis would define the pattern and sequenceof degradation of mucin for both methods of biotinylation. Further information can beobtained by comparing such profiles with those obtained using commercially availablepeptidases and glycosides, and by subsequent enzyme inhibition experiments. Mucinase Activity 3872. Materials2.1. Enzyme SourcesThe nature of possible samples to be screened for mucinase activity is diverse andthe sources given here serve only as examples.1. Commercially prepared enzymes: proteases, e.g., trypsin (Sigma, Poole, UK), pronase E(Boehringer Mannheim, Lewes, UK), pepsin (Calbiochem-Novabiochem, Nottingham,UK); glycosidases, e.g., α-sialidase, α- and β-galactosidase (Oxford Glycosciences,Abingdon, UK), α- and β-N-acetylhexosaminidase (Boehringer Mannehim), α-fucosidase(Sigma), O-glycanase (Calbiochem -Novabiochem).2. Bacterial culture supernatants and cell suspensions or cell extracts.3. Animal/human secretions and excretions that contain enzymes can also be used, e.g.,plasma, urine, tears, fecal extracts, sputum, mucosal washings.4. Animal and insect cell culture supernatants, cell suspensions, or cell extracts.2.2. Preparation of Radiolabeled Mucin Substrates1. Radiolabeled mucin singly or dual labeled with [3H]glucosamine or [3H]threonine com-bined with either [35S]sulfate or [14C]threonine. This mucin must be purified and shouldelute as a single high molecular weight peak at the Voof a Sepharose CL-2B column. Thepreparation of these substrates is detailed in Chapter 19 (see Note 1).2. Gel filtration buffer: 10 mM Tris-HCl, pH 8.0.3. Sepharose CL-2B (Pharmacia, Uppsala, Sweden).2.3. Preparation of Biotinylated Mucin Substrates:Biotinylation of Mucins1. Prepare mucin using density gradient centrifugation and gel filtration and characterize forthe presence of noncovalently associated contaminants as described in Chapters 1, 7, and8(see Note 1).2. Sephadex G25 (Pharmacia). Sephadex column preparation: hydrate Sephadex G25 inphosphate-buffered saline (PBS) (see Subheading 2.2.1., item 8) and pack in an all-glasscolumn approx 1 × 15 cm. Thoroughly equilibrated the packed column in PBS.3. Protein biotinylation reagent: δ-biotinyl-ε-aminocaproic-N-hydroxysuccinimide ester(BNHS) (Sigma).4. Carbohydrate biotinylation reagent: δ-biotinyl-ε-aminocaproic-acid hydrazide (BACH)(Boehringer Mannheim).5. Dimethylformamide (DMF) (Sigma).6. Dimethylsulfoxide (DMSO) (Sigma).7. Sodium periodate (Sigma).8. PBS: 0.375 g of sodium dihydrogen phosphate dihydrate, 1.155 g of disodium hydrogenphosphate, and 8.765 g of sodium chloride in 1000 mL water.2.4. Mucinase Assay with Biotinylated Mucin2.4.1. Coating of Plates with Biotinylated Mucin (seeNotes 4–6)1. Microtiter plates, 96-well, Nunc-Immuno™ plates, MaxiSorp Surface™ (Nalge Nunc,Life Technologies, Glasgow, Scotland) (see Note 2).2. Coating buffers: 0.1 M sodium acetate buffer, pH 5.0, for carbohydrate labelled mucinand 0.1 M sodium phosphate buffer, pH 7.0, for protein label (see Notes 3 and 4). 388 Stark et al.2.4.2. Detection of Biotin-Labeled Mucin1. PBS (see Subheading 2.3.1., item 8).2. PBST: Add Tween-20 (Sigma) to PBS to give a final concentration of 0.2%.3. Blocking buffer: 1% bovine serum albumin in PBST. Use enough to fill the well, 200–300 µL.4. Streptavidin-horseradish peroxidase (HRP) solution: Streptavidin-HRP conjugate (Vec-tor, Peterborough, UK) at 1 mg/mL is diluted to 1:1500 in blocking solution (75 µL/well)5. OPD Solution: 1,2-phenylenediamine dihydrochloride (Dako, High Wycombe, Bucks,UK). Dissolve four (2 mg) tablets in 12 mL of distilled water and add 5 µL of 30% hydro-gen peroxide immediately prior to use.6. Stop solution: 0.5 M sulfuric acid (28 mL of 95–97% acid in 1000 mL of distilled water).3. Methods3.1. Biotin Labeling of Mucins in the Protein Moiety1. Dissolve BNHS in DMF to give a final concentration of 20 mg/mL.2. Dissolve 1 mg of mucin in 0.9 mL of PBS (larger batches can be prepared at the samemucin buffer ratio).3. Add 0.1 mL of BNHS solution and incubate at 4°C overnight or at room temperature for4 h.4. Load the biotin/mucin incubation (1 mL) onto a Sephadex G25 column and run in PBSbuffer, collecting 1-mL fractions up to 30 mL total volume. The Sephadex column isdiscarded (see Note 2).5. Test fractions from the column for mucin using the slot blot assay with the periodic acidSchiff’s stain (see Chapter 4).6. Test fractions for their biotin labeling by adherence to 96-well microtiter plate assay (seeSubheading 3.4).7. Pool the labeled fractions (approx 5 × 1 mL) to give a concentration of 20 µg/mL mucin,and aliquot as 0.2-mL samples. Store at 4°C until used.3.2. Biotin Labeling of Mucins in the Carbohydrate MoietyCarbohydrate labeling requires the periodate oxidation of the carbohydrate moi-eties of the mucin before biotinylation of these oxidized residues. In the case of co-lonic mucins, the presence of O-acetyl esters will block this oxidation and asaponification step is needed first.1. Dissolve 1 mg of mucin in 0.5 mL of 0.1 M sodium hydroxide and incubate for 45 min atroom temperature. Neutralize to approx pH 7.0 with 0.05 mL of 1 M HCl. Check the pH.2. Adjust to pH 5.5 with 0.1 M acetate buffer. Add sodium periodate so that the final con-centration is 1 mM.3. Incubate for 20–60 min at room temperature4. Apply the oxidised mucin to a Sephadex G25 column as for the biotinylation of protein-labeled mucin (see Note 6) and collect fractions.5. Detect mucin containing fractions mucin using the slot-blot assay with the periodic acidSchiff’s stain (see Chapter 4), and pool these fractions (approx 5 × 1 mL).6. Add BACH in DMSO to a final concentration of 1 mM.7. Incubate at room temperature for 2 hours or overnight at 4°C.8. Separate the biotinylated mucin on a Sephadex G25 column as for protein-labeled mucin. Mucinase Activity 3899. Collect fractions and detect mucin using the slot blot assay with the periodic acid Schiff’sstain and with the 96-well microtiter plate assay (see Subheading 3.4.).10. Pool the labeled fractions (approx 8 × 1 mL) to give a concentration of 12.5 µg/mL mucinand aliquot as 0.2-mL samples. Store at 4°C until used.3.3. Mucinase Assay with Radioactive Substrates (see Notes 7 and 8)1. Mix 20–500 µL of enzyme extract or commercially available enzyme (see Subheading2.1., item 1) with 5000–50,000 cpm of radiolabeled mucin in incubation buffer in a finalvolume of 1 mL, maximum (see Notes 7 and 8).2. Incubate at 37°C for periods up to 24 h (usually 6, 12, or 24 h, but start with 24 h if theactivity is unknown).3. Prepare a blank and incubate under the same conditions as step 2 above (see Note 9).4. After incubation, either load onto Sepharose CL 2B column immediately, or freeze at–20°C until ready to start gel filtration.5. Load as a 1 mL sample onto a column 30 × 1cm of Sepharose CL 2B equilibrated in 10 mMTris-HCl, pH 8.0, and elute with the same buffer.6. Collect 30 1-mL fractions7. Mix the entire fraction (or 0.5 mL where >50,000 cpm counts are present) with scintilla-tion fluid and measure the radioactivity.8. Compare profiles of the test with the blank incubations. Identify the region of lowmolecular weight product that represents degraded mucin. Figure 1 gives an exampleprofile.9. Subtract the blank incubation (background) from the test incubation, and assess the propor-tion of low molecular weight product formed from the high molecular weight substrate.3.4. Mucinase Assay with Biotinylated Substrates3.4.1. Coating Microtiter Plates1. Dilute the biotinylated mucin in coating buffer (see Notes 6 and 7).2. Carefully place the chosen volume in the bottom of the microtitre plate well (see Note 5).Typically about 50 µL is used.3. Incubate the plates overnight at 4°C for the mucin to adsorb onto the plate.4. Empty the plates carefully (see Note 5).5. Wash once with incubation buffer 1 × 50 µL followed by 3 × 200 µl. The buffer used is thebuffer that is to be used in the assay (see Notes 7 and 8).3.4.2. Digestion of Coated Plates1. Prepare a suitable dilution of the enzyme sample in incubation buffer (see Notes 7, 8, and 10).2. Place 60–100 µL of enzyme preparation in each well (i.e., more than the volume of mucinsolution which was used to coat the plate).3. Incubate at 37°C typically for 1–2 h.4. Carefully remove the digestion media.5. Wash four times with 200 µL of PBS.3.4.3. Detection of Labeled Mucins1. Block nonspecific binding with 150–300 µL of blocking buffer for 1 h at room tempera-ture or overnight at 4°C.2. Empty the plates and wash twice with 200 µL PBS per well.3. Incubate with streptavidin-HRP solution (75 µL) for 60 min at room temperature. 390 Stark et al.4. Empty the plates and wash with PBS 1 × 100 µL and 3 × 200 µL.5. Place 100 µL of OPD solution in wells and incubate in the dark for up to 60 min.6. Stop the reaction and develop color with the addition of 100 µL of stop solution (0.5 M H2SO4).7. Measure the absorbance at 490 nm (see Note 11).4. Notes1. The choice of the source mucin for mucinase assays is of great importance. If possible itis always best to prepare a mucin substrate from the mucosa that is the target for mucinasestudy. In some cases it is not possible to obtain suitable samples, e.g., owing to ethicalconsiderations, or to the low abundance of material from minor mucosal surfaces. It maybe possible to obtain equivalent material from normal animals. If “nontypical” mucinsare chosen and used to probe the presence of mucinase activity in normal and/or diseasesituations, the interpretation of the physiological significance of the results needs carefulconsideration. Such mucins can of course be used simply to detect the presence of anymucinase activity.2. The choice of plate type and buffer is critical. The amount of mucin that binds to the plateis dependent on both the type of plastic that the plate is composed of and any treatment ithas undergone (such treatments affect the charge of the plastic). The pH affects the chargeof the mucin and therefore the adherence to the plate. Preliminary experiments are crucialto assess the effect of both the plate type and the pH of the buffer used.3. When preparing the plates, it is essential to dilute the mucin in the desired coating bufferbefore placing in the plate. When placing in the plate great care must be taken to not totouch the sides of the well where the mucin will not coat Also when transporting theplate, it is essential not to distribute the mucin outside the normal coating area. The mucinsstick rapidly and disturbance will increase the coated surface area.Fig. 1. Identification of the products of mucinase activity by Sepharose CL-2B chromatog-raphy. Intact colonic mucin labeled with [3H]-D-glucosamine (ᮀ). The products of incubationwith human fecal extract are shown (•) and the fractions pooled as low molecular weight. Mucinase Activity 3914. The chemical composition and pH of the coating buffer are critical. Avoid using aminocontaining buffers (Tris, HEPES, glycine), particularly with protein-labeled mucins.Instead, use buffers such as acetate or citrate.5. The Sephadex G25 step removes free biotin. It is necessary to discard the column becauseirreversible adsorption of biotin occurs.6. It is necessary to remove any unreacted periodate from the mucin before the biotinylationreaction is started because it interferes with the reaction. The use of a Sephadex G25column is an easy method to do this and provides a high recovery.7. Mucinase activity usually shows a broad pH range as the complete activity is a compositeof enzymes acting together. Suitable incubation buffers should be chosen to cover thepotential range of activity expected. Typically, acetate buffers at lower pHs and Tris athigher pHs. Highly purified enzymes available for commercial sources can be used tovalidate plate assays, and to generate specimen fragmentation profiles.8. The use of phosphate buffers should be avoided if possible because phosphate may inhibitsulfatase activities (see Chapter 34). and thus affect overall mucinase activity.9. Blank incubations can be prepared using enzyme extract that has been heat treated for 5min at 95°C and centrifuged. Alternatively, the enzyme can be substituted by the samevolume of incubation buffer. The suitability of either of these blanks should be tested forinterference in each assay system.10. The dilution of enzyme samples should be made in incubation buffer suitable for theenzyme itself and for the mucinase plate assay. This is easiest with commercial enzymesin which the amount of enzyme is known. In cases in which crude preparations are beingused, such as fecal extracts, urine, serum, bacterial, or cell cultures as cell suspensions, orcell supernatants, the samples should be tested directly and as dilutions in incubationbuffer. It is wise to test at high, low, and intermediate pH ranges.11. If a 490-nm filter is not available, use the nearest available wavelength. If the plate readeruses a reference filter for standardization, wavelengths between 630 and 650 nm (or near-est available) should be used.AcknowledgmentsThis work was supported by grants 046530/Z/96 and 051586/Z/97 from theWellcome Trust.References1. Roussel, P. and Lamblin, G. (1996) Human mucosal mucins in diseases, in Glycoproteinsand Disease. (Montreuil, J., Vliegenthart, J. F. G., and Schachter, H. eds.), Elsevier Sci-ence BV, Amsterdam, pp. 351–393.2. Gendler, S. J. and Spicer, A. P. (1995) Epithelial mucin genes. Annu. Rev. Physiol. 57,607–634.3. Dwarakanath, A. D., Campbell, B. J., Tsai, H. H., Sunderland, D., Hart, C. A., and Rhodes,J. M. (1995) Faecal mucinase activity assessed in inflammatory bowel disease using 14Cthreonine labelled mucin substrate. Gut 37, 58–62.4. Corfield, A. P., Wagner, S. A., O’Donnell, L. J. D., Durdey, P., Mountford, R. A., andClamp, J. R. (1993) The roles of enteric bacterial sialidase, sialate O-acetyl esterase andglycosulfatase in the degradation of human colonic mucin. Glycoconj. J. 10, 72–81.5. Roberton, A. M. and Corfield, A. P. (1999) Mucin degradation and its significance ininflammatory conditions of the gastrointestinal tract, in Medical Importance of the NormalMicroflora. (Tannock, G. W. ed.), Kluwer Academic Publishers, Dordrecht, pp. 222–261. 392 Stark et al.6. Colina, A.-R., Aumont, F., Deslauriers, N., Belhumeur, P., and de Repentigny, L. (1996)Evidence for degradation of gastrointestinal mucin by Candida albicans secretory aspar-tyl proteinase. Infect. Immun. 64, 4514–4519.7. Colina, A.-R., Aumont, F., Deslauriers, N., Belhumeur, P., and de Repentigny, L. (1996)Development of a method to detect secretory mucinolytic activity from Candida albicans.J. Med. Vet. Mycol. 34, 401–406.8. Koritsas, V. M. and Atkinson, H. J. (1995) An assay for detecting nanogram levels ofproteolytic enzymes. Anal. Biochem. 227, 22–26. . (Calbiochem-Novabiochem, Nottingham,UK); glycosidases, e.g., α-sialidase, - and β-galactosidase (Oxford Glycosciences,Abingdon, UK), - and β-N-acetylhexosaminidase. reagent: δ-biotinyl-ε-aminocaproic-N-hydroxysuccinimide ester(BNHS) (Sigma).4. Carbohydrate biotinylation reagent: δ-biotinyl-ε-aminocaproic-acid hydrazide