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Glycoprotein methods protocols - biotechnology
Proteinase Activity 39339332Proteinase ActivityDavid A. Hutton, Adrian Allen, and Jeffrey P. Pearson1. IntroductionEarly studies concerning proteolytic degradation of mucins demonstrated that theprotein core of mucins consisted of two distinct regions, glycosylated regions: pro-tected from degradation by the densely packed carbohydrate side chains and non-glycosylated regions susceptible to proteases (1,2). Since the late 1980s, sequencingof mucin genes has underlined these studies and provided a firm molecular basis forthese concepts (3). Gene-cloning studies have shown that the protein backbone of thesubunits of secreted polymeric mucins can be up to 5000 amino acids in length (approx20% by weight of the molecule) and consists of two major types of domain that alter-nate throughout the sequence (3). One type of domain, situated centrally accounts forabout 50% of the protein core and is characterized by tandem repeat (TR) sequences,rich in threonine, serine, and proline, and the hydroxyl amino acids form the sites ofattachment of the oligosaccharide chains (approx 80% by weight of the molecule).The other major type of domain, situated at the N- and C-terminals and between regionsof TR sequences, is relatively poor in these three amino acids and relatively rich incysteine (3). Some of these cysteine residues can form disulphide bridges with othermucin monomers (Mr2–3 × 106) to form large polymeric mucins linked end to end(Mr~ 107) and capable of forming gels (4–6). The tandem repeat domains are pro-tected from proteolysis by the carbohydrate side chains that sterically inhibit protein-ases from gaining access to the protein core, however, proteinases can hydrolyze thecysteine-rich regions of accessible nonglycosylated protein, thereby fragmenting thepolymeric mucins (6). The soluble glycopeptides resistant to proteolysis remain ofrelatively high Mr (200–700 kD) and recent studies have suggested that individualmucin gene products may contain different types and lengths of glycosylated domains.For instance, analysis of high Mr glycopeptides produced by trypsin digestion of theMUC5B subunit indicated that it contained different types and lengths of glycosylateddomains; one domain of Mr 7.3 × 105, two domains of 5.2 × 105and a third domain of2 × 105(7). Similarly rat small intestinal Muc2 mucin subunit contains two glycopep-From:Methods in Molecular Biology, Vol. 125: Glycoprotein Methods and Protocols: The MucinsEdited by: A. Corfield © Humana Press Inc., Totowa, NJ 394 Hutton et al.tides with an estimated mass of 650 and 335 kDa (8). The significance of the differ-ences in size of these domains is unclear.Protective essentially insoluble mucus gels adherent to mucosal surfaces aresolubilised by proteinases, e.g., in the lumen of the gastrointestinal tract (6). Themechanism of solubilisation is cleavage of the susceptible regions of the mucin pro-tein core which are responsible for the gel-forming properties of mucus gels. Thismucolysis results in degradation of the polymeric mucins (high viscosity, gel-form-ing) into relatively low Mr glycopeptides (low viscosity, soluble). A dynamic balanceexists between secretion of gel-forming mucins and their degradation by proteinases,if this relationship is tilted in favor of proteolytic degradation the protective propertiesof the mucus gel will be compromized and disease may result. The proportion of poly-meric mucins present in mucus gels has been shown to be an indicator of gel strength(9) and evidence exists for enhanced mucolysis by proteinases and/or inferior mucinpolymerisation and consequent impairment of the mucus barrier in peptic ulcerationand inflammatory bowel disease (10,11). Techniques are therefore required for mea-surement of the integrity of mucin polymeric structure in large numbers of samplesisolated from mucus gels and the mucosa secreting them and methods are needed toassess the mucolytic potential of host and pathogen secretions.The digestion of mucin polymers and their reduced subunits by proteases has beenfollowed by measuring molecular size using analytical ultracentrifugation or light scat-tering (2,5). These techniques, whilst providing the most empirically quantitativedescriptions of size, rely on highly purified samples, expensive equipment, a highlevel of expertise and can require relatively large quantities of sample (e.g., measure-ment of sedimentation coefficient). Therefore, they are generally unsuitable for theassessment of the polymeric integrity of large numbers of mucin samples or for thescreening of the mucolytic potential of protease containing samples.For most laboratories gel-filtration chromatography, commonly on Sepharose CL-2B or Sephacryl S-500, has provided the most convenient method of assaying mucindegradation in terms of both integrity of mucins and mucolytic activity (5,10,12,13).Polymeric mucins are excluded in the void volume of the column, and proteolyticallydegraded subunits are partially included (Kav~ 0.5), eluted mucins being assayed insolution or after blotting onto nitrocellulose membranes (see Note 1). However, thistechnique also requires relatively large quantities of sample and is time consuming inprocessing large numbers of samples. The use of radiolabelled mucin samplesimproves the sensitivity of the technique, however it remains laborious (14).Mucin polymers and their reduced subunits do not penetrate polyacrylamide gels(>3%), however, proteolytically digested subunits will enter gels of >4%. The adventof readily commercially available precast polyacrylamide gradient gels (4–15%)requiring small amounts of sample and with rapid running and staining times has vastlyimproved the analysis of the in vivo polymeric integrity of mucins isolated fromadherent mucus gels and mucosa and is described below.Assessment of the mucolytic potential of endogenous secretions or extracts frominvasive pathogens is initially underpinned by measurement of nonspecific proteinaseactivity. General protease activity has often been assayed by methods distinguishing Proteinase Activity 395hydrolyzed protein from nonhydrolyzed protein by precipitation of the latter, e.g., withtrichloroacetic acid. These methods are reliant on qualitative size and conformationalsize changes preventing protein precipitation (15). A sensitive and more accuratemethod for estimating proteolytic activity involves measuring trinitrophenylatedderivatives of new N-terminal groups which form on peptide bond hydrolysis (16).The sensitivity of the assay is improved by blocking existing amino groups on theprotein substrate, e.g., by succinylation. The preparation of succinyl albumin isdescribed subsequently. The assay can also be used with mucin substrates (12) and thelarge size of mucin protein cores means that fewer N-terminals are present on a weightfor weight basis and therefore blocking is unnecessary to achieve low backgroundabsorbances. Large quantities of highly purified mucin are, however, required. Theassay has also been adapted for use after separation of proteinase isoenzymes on aga-rose gels (16).Attempts to measure specific mucolytic activity have been dogged by similar prob-lems to those of general proteinase activity. Cetyl trimethylammoniumbromide(CTAB) or protamine sulfate precipitation has been used to precipitate only undi-gested mucins, but again depends upon qualitative assessment of precipitability ofundegraded macromolecules (17,18). Other methods that depend on the appearance ofhydrolysis zones on petri dishes containing mucin/agarose gels often rely on crudemucin preparations (contaminated with hydrolyzable protein) to provide sufficient ma-terial or require large amounts of purified mucin (19,20).Dilute solution viscosity studies measuring mucolysis by following the fall in vis-cosity of mucin solutions (10,12) have a number of advantages over other methods.They provide information on the kinetics of digestion and the size (molecular spaceoccupancy) of the degraded species, and crude mucus samples can be studied as wellas purified mucins. The technique is readily compatible with other techniques, e.g., ifmucolysis is inhibited at particular time points, aliquots of sample can be further stud-ied by gel filtration or N-terminal analysis (12).The measurement of mucolysis by quantitating digoxigenin-labeled mucin boundto and released from microtitre plates requires little mucin substrate and may thereforebe useful for screening large numbers of potentially mucolytic samples (20). Differ-ences in binding to the plates between undegraded and degraded mucins may, how-ever, make interpretation of the results difficult (see Note 1).1.1. Proteinases in the Gastrointestinal TractProteinases from all regions of the gastrointestinal tract degrade mucus gel andmucins in vivo and in vitro to produce soluble glycopeptide fragments which are fur-ther degraded by glycosidases in the colon (1).In the stomach, the secreted mucins in the adherent mucus gel layer are faced withpepsins (maximum concentration ~0.7 mg/ml in humans). Of the seven pepsin isoen-zymes in human gastric juice, pepsin 3 is the most abundant with some pepsin 5.Pepsin 1 is normally a minor component (<5%) but can account for up to 25% of theproteolytic activity in peptic ulcer patients. Pepsin 1 has increased collagenolytic andmucolytic activities compared with other pepsins (15). In peptic ulcer patients, there is 396 Hutton et al.disruption of the mucus layer and an increase in the percentage of nongel forming lowermolecular mass mucin in the adherent gel (21) and this is associated with increasedmucolytic activity of the gastric juice owing to raised levels of pepsin 1 (10).The human pancreas produces 1–3 g of chymotrypsin, 1–3 g of trypsin and approx0.5 g of elastase per day, however total fecal levels of pancreatic enzymes are ~ 1 mg/d(22). This fall in levels is due partly to bacterial degradation and partly to autode-gradation (23). Human fecal extracts contain proteinases of pancreatic (23) and bacte-rial (24) origin whereas enzymes associated with inflammation, e.g., white cell elastaseare present only in trace amounts (25). The bacterial microflora can produce copiousamounts of luminal proteinases (24). Fecal proteinases have been demonstrated tohave mucus degrading activity (12,14). Human fecal proteinase activity is raised inulcerative colitis patients compared to nonsymptomatic control subjects (26,27) andthis raised luminal proteinase activity probably equates with elevated mucolytic activ-ity. These observations could explain in part the thinner colonic mucus in ulcerativecolitis (28) by allowing mucin degradation to outweigh mucus secretion.1.2. Specific Proteolysis of MucinsSpecific sites for proteolytic cleavage are also known to exist in the amino acidsequences of mucin protein cores. cDNA studies on human and rat MUC2 have shownthat these mucins share a proteolytic cleavage site sequence approx 700 amino acidsfrom their C-terminus (TGWGD PH(Y/F*)VTFDGLYY) and cleavage of the aspartylproline bond generates a C-terminal glycopeptide fragment of approximately 120 kDa(29). This site is homologous with a site cleaved during the synthesis of rat mammarysialomucin at an early stage of intracellular transport. The timing of cleavage (duringor after biosynthesis) of intestinal mucins has not been established. A heparin bindingsite sequence, SRRARRSPRHLGSG, is also cleaved from the protein core of MUC2at an early stage of biosynthesis (30).2. Materials1. Bovine serum albumin (BSA), Fraction V, (Sigma, Poole, Dorset, UK).2. Succinic anhydride (Sigma ).3. Trinitrobenzenesulphonic acid (TNBS; 5% [w/v] aqueous solution) (Sigma).4. Enzymes: porcine pepsin (pepsin A; EC 3.4.23.1), porcine pancreatic trypsin (E.C2.4.21.4) and porcine pancreatic elastase (EC 3.4.21.36) (Sigma).5. Proteinase inhibitor cocktail. 1.0 mM phenylmethylsulfonyl fluoride (PMSF), 50 mMiodoacetamide, 100 mM aminohexanoic acid, 5 mM benzamidine HCl, 1 mMN-ethylmaleimide, 1 mg/Lsoybean trypsin inhibitor (all from Sigma Chemical Company),10 mM EDTA (B.D.H. Poole, Dorset, UK) in 0.67 M phosphate buffer, pH 7.5.6. Sodium dodecyl sulfate (SDS)-polyacrylamide gels for electrophoresis: 4–15% Phast gelswere purchased from Pharmacia, L.K.B. Biotechnology, Uppsala, Sweden.7. Schiffs reagent, commercial solution (Sigma).8. Sepharose CL-2B (Pharmacia) use in 1.5 ID × 150 cm glass columns equilibrated with0.2 M NaCl/0.02%(w/v) NaN3 (B.D.H., Poole, UK).9. Pharmacia Phast-gel system (Pharmacia).10. Contraves low shear 30 viscometer (Contraves A.G. Zurich, Switzerland). Proteinase Activity 3973. Methods3.1. Preparation of Substrate, Blocking of N-Terminals1. Dissolve 10g of BSA in 100ml 0.1M phosphate buffer pH 7.5.2. Add 1.4 g of succinic anhydride , stir to dissolve and maintain the pH at 7.5 with 2 MNaOH using a pH stat as the succinic anhydride dissolves (see Note 2).3. Dialyze the solution exhaustively against distilled water at 4°C, freeze-dry and store theprotein substrate with blocked N-terminals at –20°C (see Note 3).3.1.1.N-Terminal Assay1. Mix proteinase (see Note 4) or extract containing proteinase activity (200 µL, 0.1–0.5 µg)with 0.5 mL buffer of choice (see Note 5). Prepare control samples by heating the pro-teinase preparation at 100°C for 10 min to destroy activity. Alternatively add the sub-strate immediately prior to step 4 below. (This will give a background value for any freeN-terminals in the proteinase sample). Prepare a reagent blank by replacing enzyme orextract with buffer.2. Add substrate (0.5 mL 8mg/mL succinyl albumin) to start the reaction, mix and incubatesamples for 30 min at 37°C in a waterbath.3. Add 0.5 mL of sodium bicarbonate (to increase the pH of the solution to pH 8.0) and add0.5 mL 0.05% (w/v) TNBS in water (to trinitrophenylate any free amino groups formed).Incubate at 50°C for 10 min in a water bath to develop color.4. Add 0.5 mL 10% (w/v) SDS to prevent protein precipitation. Then add 0.25 mL 1 M HClto complete the reaction.5. Read the absorbance at 340 nm in a spectrophotometer.6. Calculations: Proteinase activity in millimoles new N-terminals/min/g extract is calcu-lated using the following equation:Vt × dilution × A340× 103E × t × Vs × gwhere Vt = final tube volume (mL); E = molar extinction coefficient of trinitrophenylamino acids (1.3 × 104 cm2/mole); t = time (min); Vs = volume of enzyme; A340= absor-bance at 340 nm; g = wet weight of extract/mL (see Note 6).The example for g is for proteinase activity in fecal extracts, and g refers to the weight ofmaterial present in 1 mL of a fecal homogenate.3.1.2. Collection, Biopsies, and Brushings: Purification of MucinSamples of human mucin can be obtained from samples obtained at surgery, e.g.,during routine endoscopy or colonoscopy. Adherent mucus gel is obtained by brush-ing the mucosa with a cytology brush. Mucosal biopsies provide both intracellularmucin and adherent mucus gel.1. Place samples immediately into a cocktail of proteinase inhibitors, i.e., 1.0 mMphenylmethylsulphonyl fluoride (PMSF), 50 mM iodoacetamide, 100 mM aminohexanoicacid, 5 mM benzamidine HCl, 1 mMN-ethylmaleimide, 1 mg/L soybean trypsin inhibitor,10 mM EDTA in 0.67 M phosphate buffer, pH 7.5, at 4°C to minimize proteolytic degra-dation (see Note 5) and store at –20°C until processed.2. Solubilize mucins by brief homogenization (30 s, low-speed, hand-held homogenizer)and centrifuge (8000g, 1 h, 4°C) to remove cell debris. 398 Hutton et al.3. Purify mucins by equilibrium density gradient centrifugation in CsCl (100,000g, 48 h,4°C, starting density 1.42 g/mL) (see Note 7).4. Dialyze the pooled mucins exhaustively against distilled water, freeze-dry and store at –20°C.3.2. SDS Polyacrylamide Gel Electrophoresis3.2.1. Phast-Gel ElectrophoresisThis method refers to SDS polyacrylamide gel electrophoresis using 4–15% gelson the Pharmacia Phast-gel system (Pharmacia).1. Solubilize freeze-dried mucin purified from human gastrointestinal mucosal brushings orbiopsies in, e.g., 100 µL of 0.0125 M Tris-HCl, 0.4% (w/v) SDS, 2% (v/v) glycerol, and0.0002% bromophenol blue, pH 6.8, at a concentration of 3 mg/mL.2. Heat the samples for 2 min at 100°C.3. Load 1-µL aliquots onto the gel (3 µg).4. Electrophorese for 45 min, 15°C, 250 V, 10 mA.3.2.2. Staining of SDS 4–15% Gels1. Following electrophoresis incubate gels in 25 mL 7% (v/v) acetic acid for 1h. at roomtemperature.2. Incubate in 25 mL of periodic acid solution (7% acetic acid containing 0.04% [v/v] peri-odic acid) for 1 h at room temperature.3. At the same time as step 2 above incubate a 24-mL solution of Schiffs reagent containing0.4 g sodium metabisulphite at 37°C for 1 h.4. Pour the periodic acid solution from the gel and incubate for 30 min at 37°C in Schiff’sreagent.5. Wash the gel several times (approx 15 min each wash) with 25 mL of 7.5% (v/v) aceticacid until the washing solution becomes colorless.6. Store the gel overnight at 4°C in 7% acetic acid and scan at 555 nm with a gel scanner.3.3. Solution Viscosity as an Assay for Mucolytic ActivityMucolytic activity by proteinases can be monitored by measuring the fall in viscos-ity of mucin solutions on incubation with enzyme or extract. Viscosity can be mea-sured using a Couette rotating cup viscometer (e.g., Contraves’ Low Shear 30). Anelectronic speed programmer (Rheoscan 20) allows the speed of rotation of the cup tobe increased under controlled conditions. The cup is filled with the solution underinvestigation (~2 mL), and a bob is gently lowered into the solution without introduc-ing any air bubbles. The bob is attached to a torsion wire, and when the cup is rotated,the resistance offered by the solution is transferred to the bob and the torsion wire.This resistance is converted into an electrical signal recorded on a chart recorder. Thetemperature of the cup and bob can be maintained at 37°C using a water bath thatcirculates water through the viscometer block. The shear rate is increased linearlyfrom 1.7 to 128.5/s.1. Solubilize freeze-dried mucin (either crude or purified) at 2–5 mg/mL by stirring over-night at 4°C in buffer (e.g., 67 mM sodium phosphate buffer, pH 7.5) containing 0.08 MNaCl (see Note 8).2. Equilibrate the mucin, the buffer and the enzyme preparation or extract at 37°C. Proteinase Activity 3993. Add the enzyme to the mucin and measure the viscosity at various time intervals over a24-h period. Prepare a control solution containing mucin and buffer and measure its vis-cosity over the same time period (see Note 9). Measure the viscosity of the buffer beingused.4. Calculations: The solution viscosity of the sample is directly proportional to the gradientof the curve plotted on the chart recorder, the Y-axis being the percentage deflection andthe X-axis speed. The ratio of this gradient and the gradient produced for the solvent(buffer) alone gives the relative viscosity (ηrel).ηrel=gradient of test samplegradient of solventSpecific viscosity may then be calculated as:ηsp= ηrel–1Specific viscosity can then be plotted against time to follow the course of digestion.Reduced specific viscosity ηredcan be calculated as ηsp/C, (where C = concentration ofmucin), and intrinsic viscosity calculated by plotting ηredagainst mucin concentrationand extrapolating to zero concentration (Huggins Plot). Alternatively, plotting ln ηrelas afunction of concentration and extrapolating to zero also gives values of intrinsic viscosity(Kraemer Plot).4. Notes1. The high Mrglycopeptides produced by proteinase digestion bind poorly to nitrocellulosemembranes, e.g., in the PAS slot blot assay (31) after gel filtration on Sepharose CL-2B.A method to improve binding by previously applying wheat germ agglutinin to the mem-branes has been described (32).2. It is essential to maintain the pH at 7.5 to prevent denaturation of the albumin.3. Allow expansion space in the dialysis tubing to avoid bursting since it will swell duringdialysis.4. Porcine pepsin (pepsin A; EC 3.4.23.1), porcine pancreatic trypsin (EC 2.4.21.4), andporcine pancreatic elastase (EC 3.4.21.36) (Sigma) have all been successfully used asstandards in the N-terminal proteinase assay (12,16).5. Inhibition of proteinases. It is vitally important to inhibit endogenous proteinases whenattempting to isolate intact, native polymeric mucins. Unless rigorous precautions aretaken during isolation by including a mixture of wide ranging proteinase inhibitors then“nicking” of the protein core occurs to give units which, while still retaining much of theintact protein core, are of smaller size. 1 mM phenylmethylsulfonyl fluoride has beenfound to be particularly important in inhibiting proteolysis (D. A. Hutton and A. Allen,unpublished observations). PMSF should be dissolved in a small quantity (~5 mL) ofpropan-2-ol and added to the rest of the cocktail immediately before use. An alternativeinhibitor to PMSF is diisopropylphosphorofluoridate (DIFP) which while very effective(5) does require extremely careful handling6. A protein assay carried out on the proteinase sample to determine the mg/mL concentra-tion of protein will allow the activity to be expressed per milligram of protein.7. For human mucin from biopsies and brushings the mucin containing fractions are bestestimated using density, the concentration of mucin in each fraction being insufficient tomeasure even with the slot-blot method (31,32). Fractions having a density equivalent tomucins under these conditions (1.45–1.51 g/mL) should be pooled. 400 Hutton et al.8. It is essential to have isotonic conditions, i.e., 300 mosmolar to shield the negative chargeon the mucins otherwise anomalously high viscosities will result.9. It is vital to include controls without enzyme in the mucolysis experiments. Crude mucussamples may contain endogenous protease activity and some interactions in pure mucinsamples may be disrupted by repeated shearings causing an apparent loss of viscosity.References1. Allen, A. and Pearson, J. P. (1993) Mucus glycoproteins of the normal gastrointestinaltract. Eur. J. Gastroenterol. & Hepatol 5, 193–199.2. Scawen, M. and Allen, A. (1977) The action of proteolytic enzymes on the glycoproteinfrom pig gastric mucus. Biochem. J. 163, 363–368.3. Gendler, S. J. and Spicer, A. P. (1995) Epithelial mucin genes. Annu. Rev. Physiol. 57, 607–634.4. Snary, D., Allen, A., and Pain, R. H. (1970) Structural studies on gastric mucoproteins.Lowering of molecular weight after reduction with mercaptoethanol. Biochem BiophysRes Commun. 40, 844–851.5. Carlstedt, I. Sheehan, J. K., Corfield, A., and Gallagher, J. T. (1985) Mucus glycoproteins:a gel of a problem. Essays Biochem. 20, 40–76.6. Allen, A. 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R., ed.), Raven Press, New York, pp. 1255–1283.30. Xu, G., Forstner G. G., and Forstner, J. F. (1996) Interaction of heparin with syntheticpeptides corresponding to the C-terminal domain of intestinal mucins. Glycoconjugate J.13, 81–90.31. Thornton, D. J. Holmes, D. F., Sheehan, J. K., and Carlstedt, I. (1989) Quantitation of mu-cous glycoproteins blotted onto nitrocellulose membranes. Anal. Biochem. 182, 160–164.32. Ayre, D., Hutton, D. A., and Pearson, J. P. (1994) The use of wheat germ agglutinin toimprove binding of heterogeneous mucin species to nitrocellulose membranes. Anal.Biochem. 219, 373–375. . Muc2 mucin subunit contains two glycopep-From :Methods in Molecular Biology, Vol. 125: Glycoprotein Methods and Protocols: The MucinsEdited by: A. Corfield. viscosity, gel-form-ing) into relatively low Mr glycopeptides (low viscosity, soluble). A dynamic balanceexists between secretion of gel-forming mucins