Glycoprotein methods protocols - biotechnology
Separation and Identification of Mucin 777Separation and Identificationof Mucins and Their GlycoformsDavid J. Thornton, Nagma Khan, and John K. Sheehan1. IntroductionThis chapter describes a strategy for the separation and identification of the mucinspresent in mucous secretions or from cell culture, focusing primarily on those mucinsinvolved in gel formation. At present, the mucins MUC2, MUC5AC, MUC5B, andMUC6 are known to be gel-forming molecules (1–4). These mucins share commonfeatures in that they are oligomeric in nature and consist of a variable number of mono-mers (subunits) linked in an end-to-end fashion via the agency of disulfide bonds. Inaddition, their polypeptides comprise regions of dense glycosylation interspersed with“naked” cysteine-rich domains (4–7).Histological and biochemical investigations suggested that mucous-producing tis-sues and their secretions contained a complex mixture of mucin-type glycoproteins.However, until recently and with the advent of the new mucin-specific probes arisingfrom cDNA cloning studies, this theory was not definitively proven. In situ hybridiza-tion and Northern blotting studies have shown that more than one gel-forming MUCgene product can be expressed in a single mucus-producing epithelia, i.e., MUC5ACand MUC5B in the respiratory tract and MUC5AC and MUC6 in the stomach (4,8).Subsequent biochemical studies on human airway mucus have shown that these twomucin genes are not only expressed but that their glycosylated products are present inrespiratory tract secretions (2,3). A further more recent insight into the complex natureof the mucin component of mucous has been the demonstration that a mucin geneproduct from a single epithelium can have a different oligosaccharide decoration andthus exist in what are termed glycoforms. For example, the MUC5B mucin in the res-piratory tract can exist in two distinctly charged states (3). Thus, these studies demon-strate the need to have techniques available to dissect these complex mixtures toascertain mucin type, amount, and glycoform. Such investigations may lead to theidentification of novel members of this growing family of molecules.Owing to the extreme size and polydispersity (Mr= 5–50 × 106) of the gel-formingmucins in particular, there are few separation techniques available to use with these77From: Methods in Molecular Biology, Vol. 125: Glycoprotein Methods and Protocols: The MucinsEdited by: A. Corfield © Humana Press Inc., Totowa, NJ 78 Thornton et al.molecules. However, the smaller and more homogeneous reduced mucin monomersgenerated after reduction (Mr= 2–3 × 106) are amenable to conventional biochemicalseparation methods. We describe a separation protocol based on anion exchange chro-matography to fractionate the different species of mucin coupled with agarose gelelectrophoresis as a method to measure their homogeneity. Identification of mucinpolypeptides is achieved by use of MUC-specific antisera and fragmentation followedby amino acid compositional analysis and peptide purification and sequencing. Mucinglycoforms are detected using carbohydrate-specific probes, i.e., lectins or monoclonalantibodies. Figure 1 summarizes the overall procedure.2. Materials2.1. Extraction and PurificationSee Chapter 1 for details.Fig. 1. Outline of the protocol for separation and identification of mucins. Separation and Identification of Mucin 792.2. Preparation of Reduced Mucin Subunits1. Reduction buffer: 6 M guanidinium chloride, 0.1 M Tris-HCl, 5 mM EDTA, pH 8.0.2. Dithiothreitol (DTT).3. Iodoacetamide.4. Buffer A: 6 M urea, 10 mM piperazine pH 5.0, containing 0.02% (w/v) CHAPS.5. PD-10 (Amersham Pharmacia Biotech, St. Albans, UK) or equivalent desalting column.2.3. Separation of Mucin2.3.1. Anion-Exchange Chromatography1. Mono Q HR5/5 column (Pharmacia).2. Buffer A (see Subheading 2.2.).3. 0.4 M lithium perchlorate.2.3.2. Agarose Gel Electrophoresis1. Horizontal electrophoresis apparatus, e.g., Bio-Rad DNA subcell Model 96 (25 × 15 cmgels) and the Bio-Rad subcell (15 × 15 cm gels).2. Agarose (ultrapure).3. Electrophoresis buffer: 40 mM Tris-acetate, 1 mM EDTA, pH 8.0, containing 0.1% (w/v)sodium dodecyl sulfate.4. Loading buffer: electrophoresis buffer containing 30% (v/v) glycerol and 0.002% (w/v)bromophenol blue.5. Transfer buffer: 0.6 M NaCl, 60 mM sodium citrate.2.4. Identification of Mucin2.4.1. Tryptic Digestion1. Digestion buffer: 0.1 M ammonium hydrogen carbonate, pH 8.0.2. Modified trypsin or other proteinase.2.4.2. Trypsic Peptide Analysis1. Digestion buffer: 0.1 M ammonium hydrogen carbonate, pH 8.0.2. Superose 12 (Pharmacia) or equivalent column.3. µRPC C2/C18 PC 3.2/3 column (Pharmacia) or other C2/C18 column.4. 0.1% (v/v) trifluoroacetic acid (TFA).5. Acetonitrile.2.4.3. Amino Acid Analysis1. 6 M HCl.2. Redrying buffer: ethanol:triethylamine:water (2:2:1 [v/v/v]).3. Coupling buffer: ethanol:triethylamine:water:phenylisothiocyanate (PITC) (70:10:19:1[v/v/v/v]).4. 3µ ODS2 column (4.6 × 150 mm) (Phase Separations, Clwyd, UK).5. Buffer A: 12 mM sodium phosphate, pH 6.4.6. Buffer B: 24 mM sodium phosphate, pH 6.4, containing 60% (v/v) acetonitrile. 80 Thornton et al.3. Methods3.1. Extraction and PurificationFor a detailed description of extraction and purification, see Chapter 1. In brief,mucins are extracted from samples at 4°C with 6 M guanidinium chloride containingproteinase inhibitors and are purified by isopycnic centrifugation in CsCl density gra-dient first in 4 M guanidinium chloride (removal of proteins) and then in 0.2 Mguanidinium chloride (removal of nucleic acids). Finally, the preparation of mucin isdialyzed into 4 M guanidinium chloride for storage.3.2. Preparation of Reduced Mucin SubunitsMucin subunits (monomers) are prepared by reduction and alkylation of purifiedmucins as follows:1. Transfer (by dialysis or dilution) the mucins into reduction buffer.2. Reduce the mucins by the addition of DTT to a final concentration of 10 mM for 5 h at37°C.3. Alkylate the free-thiol groups generated by reduction with the addition of iodoacetamideto a final concentration of 25 mM. This step can be performed overnight in the dark atroom temperature.4. Transfer the reduced mucin subunits into buffer A by chromatography on a PharmaciaPD-10 column (follow manufacturer’s instructions) or by dialysis.3.3. Separation of MucinSeparation of reduced mucin subunits is achieved by using anion-exchange chro-matography on a Mono Q HR 5/5 column (Fig. 2A). Assessment of the effectivenessof the separation is monitored with a periodic acid-Schiff (PAS) assay and with avariety of lectins and mucin- or carbohydrate-specific antibodies (for a discussion ofthe relative merits and drawbacks of these analytical tools, see Chapter 4). The homo-geneity of the fractions is monitored by agarose gel electrophoresis and Western blotsof the gels can be probed with lectins and antibodies. Using this methodology, wehave shown that with pooling and rechromatography it is possible to prepare mucinsubunit samples enriched in specific MUC gene products and also to isolate differentglycosylated forms (glycoforms) of a single MUC gene product (see, e.g., refs.2 and 3).An example of the data obtained, using this methodology with a salivary mucinreduced subunit preparation, is shown in Fig. 2. The range of charge density of themolecules (Fig. 2a) is typical of that observed for the reduced subunits prepared fromthe gel-forming mucins isolated from other epithelial secretions (respiratory and cer-vical) and from intestinal cell lines (HT-29 and PC/AA). In this example the electro-phoretic mobility of the reduced subunits (Fig. 2b) is dependent on their charge densitysince the molecular weight and size (radius of gyration) of the molecules (determinedby light scattering) across the charge distribution are essentially the same (8a). Anal-ysis of Western blots with MUC-specific antisera suggest that the major mucin in thissample is the MUC5B gene product (8a). Thus, the Mono-Q column appears to beseparating differently charged forms of this mucin. The continuum in the electro- Separation and Identification of Mucin 81phoretic mobility observed with the salivary mucin sample is not normally seen inrespiratory mucin subunit preparations. In respiratory samples there are typically twogel-forming mucins, namely MUC5AC and MUC5B. The MUC5B mucin appears byMono-Q chromatography to be in two differently charged states, and like the salivarymucin subunits, the electrophoretic mobility of the MUC5B subunits is consistent withtheir charge density. However, the MUC5AC mucin-reduced subunits are of lowercharge density than the most highly charged MUC5B molecules, but they migratefarther on a 1% (w/v) agarose gel (2).Figure 2. Separation of reduced mucin subunits: (A) Mucins were isolated from saliva,reduced and alkylated, and chromatographed on a Mono-Q HR 5/5 column. The diagram showsthe distribution of the reduced subunits as monitored with the PAS-reagent. (B) Aliquots fromeach fraction across the charge distribution were subjected to 1% (w/v) agarose gel electro-phoresis. A Western blot of the gel was probed with a polyclonal antiserum raised againstreduced mucin subunits. 82 Thornton et al.3.3.1. Anion-Exchange ChromatographyThe flow rate for anion-exchange is 0.5 mL/min throughout the chromatography,and typically 0.5-mL fractions are collected.1. Apply the sample (up to 10 mg) to the Mono Q column in buffer A and wash for 10 minafter application.2. Elute the sample with a linear gradient from 0 to 0.4 M lithium perchlorate in buffer Aover a period of 60 min.3. Analyze samples with A280nmmeasurements, an assay for carbohydrate (e.g., PAS reagent)and for lectin and antibody reactivity (see ref. 9 for detailed procedures).3.3.2. Agarose Gel ElectrophoresisElectrophoresis is performed in 1% (w/v) agarose gels using a standard horizontalgel electrophoresis apparatus.3.3.2.1. SAMPLE PREPARATION1. Dilute or dialyze reduced mucin subunits from the Mono Q separation into electrophore-sis buffer (see Note 1).2. Add 1/10 of a volume of 30% (v/v) glycerol in electrophoresis buffer containing 0.002%(w/v) bromophenol blue.3.3.2.2. GEL PREPARATIONWe typically perform electrophoresis in 15 × 15 cm or 25 × 15 cm gels of approx3 to 4 mm thickness.1. Dissolve the agarose in electrophoresis buffer in a microwave; for a small gel, use 1.6 g ofagarose and 160 mL of buffer, and for a large gel, use 2.8 g of agarose and 280 mL ofbuffer.2. Leave to cool before pouring (hand hot) and insert well-forming comb (see Note 2).3. Leave to set for at least 1 h prior to use.3.3.2.3. ELECTROPHORESIS1. Electrophorese sample in electrophoresis buffer for 16 h at 30 V at room temperature.Ensure that the buffer is at least 0.5 cm above the gel surface and always use the samebuffer volume.3.3.2.4. WESTERN BLOTTINGAfter electrophoresis, transfer the gel to nitrocellulose or poly(vinylidine difluoride)(PVDF) (see Note 3) as follows:1. Wash the gel in transfer buffer for 5 min.2. Transfer subunits to nitrocellulose or PVDF membrane by vacuum blotting in transferbuffer at a suction pressure of 45 mBar for 1.5 h. We use a Pharmacia Vacu-Gene XL forthis procedure.3. Probe the membrane for lectin and antibody reactivity (see ref. 9 for detailed procedures). Separation and Identification of Mucin 833.4. Identification of MucinThe easiest route to identification of mucin is by the use of MUC-specific antisera.However, because of sequence similarities among already known mucins, these anti-sera may not provide an unequivocal answer. Therefore, we also use N-terminalsequencing of proteolytically derived peptides and amino acid compositional analysisof mucin glycopeptides for a more definitive identification. These procedures willalso provide a route to obtain information on novel mucins. The starting point forthese analyses is a proteolytic fragmentation of reduced mucin subunits. For example,trypsin digestion of reduced mucin subunits liberates high Mrmucin glycopeptides,which correspond to the heavily O-glycosylated tandem repeat regions of the mol-ecule, and lower Mrpeptides and glycosylated peptides, which arise from the “naked”cysteine-rich regions of the molecule.3.4.1. Trypsin Digestion1. Dissolve lyophilized reduced mucin subunits in digestion buffer.2. Add modified trypsin (Note 4) to the subunits in a weight ratio of approx- 1:1000 (seeNote 5) and leave the digestion overnight at 37°C.3. Separate digestion products into high Mrglycopeptides and tryptic peptides by chroma-tography on a Superose 12 column (or equivalent gel filtration medium) eluted with diges-tion buffer (Note 6).4. Lyophilize tryptic fragments on a freeze-drier.3.4.2. Tryptic Peptide Analysis (seeNote 7)Tryptic peptides can be fractionated by reverse phase chromatography and indi-vidual peptides purified and their primary sequence determined by N-terminalsequencing.3.4.2.1. PEPTIDE PURIFICATION AND N-TERMINAL SEQUENCING1. Solubilize lyophilized tryptic peptides in 0.1% TFA.2. Chromatograph on a C2/C18 reverse phase column (see Note 8).3. To increase the chances of obtaining unambiguous sequence data, an assessment of pep-tide homogeneity is advisable. We analyze aliquots of the peaks by using matrix-assistedlaser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), andpurify peptides to homogeneity by rechromatography on the C2/C18 column utilizingshallower gradients centered on their elution point.4. Dry down peptides by vacuum centrifugation and determine the primary sequence ofpeptides by automated N-terminal sequencing.5. Use sequence to search sequence databases.3.4.3. Amino Acid Analysis (seeNote 9)3.4.3.1. ACID HYDROLYSIS1. Dissolve the samples (1 µg to 2 mg) in 500 µL 6 M AristaR grade HCl (BDH, Poole,Dorset, UK) (see Note 10) and transfer to glass hydrolysis tubes (see Note 11).2. Flood the tubes with argon gas, seal, and hydrolyze at 110°C for 24 h. 84 Thornton et al.3.4.3.2. DERIVATIZATION1. Transfer the hydrolyzed samples to Eppendorf tubes.2. Remove all the acid by evaporation under vacuum in a centrifugal evaporator.3. Add 50 µL of redrying buffer to each sample and dry, under vacuum, for 30 min.4. Add 50 µL of freshly prepared coupling buffer to each sample and vortex to mix well.5. Leave at room temperature for 30 min.6. Remove excess PITC by centrifugal evaporation under vacuum for 1 h.7. The derivatized samples can be stored at –20°C.3.4.3.3. REVERSE PHASE CHROMATOGRAPHY1. Solubilize the derivatized amino acids in buffer A.2. Chromatograph at 38.6°C on a 3µ ODS2 column (see Note 12).3. Monitor column eluent at 254 nm, and determine amino acids in sample by comparisonwith standard amino acid mixture.4. Notes1. Urea can be tolerated up to a concentration of at least 6 M, but salts (particularlyguanidinium chloride) should be avoided.2. The comb size (width relative to thickness) is important for the quality of the data. Typi-cally we use combs that are 1.5 mm thick and 1 cm wide. There is a compromise betweenband broadness and amount of sample to be loaded. Larger amounts of sample tend toyield poorer quality data; i.e., the bands become more smeared.3. Molecules transferred to PVDF can be treated with trifluoromethanesulfonic acid toremove O-linked glycans. This is often essential if using antisera that are directed againstcore protein epitopes “masked” by oligosaccharides (10).4. Treat trypsin by reductive alkylation to modify arginine and lysine residues to preventautolysis and thus remove the problem of peptides arising from the enzyme.5. The amount of enzyme added was calculated assuming that protein constitutes 20% of thetotal mass of the mucin subunit and that approx 50% of this protein is in “naked” regionsthat are accessible to the proteinase.6. Chromatography separates the high Mrmucin glycopeptides (void volume) from the lowerMr peptides that elute near or in the total volume; for an example, see ref. 3.7. As we purify more mucins, it might become possible by using MALDI-TOF MS to obtaina peptide fingerprint associated with each gene product. We have already performed suchan analysis for the MUC5B mucin purified from respiratory, cervical, and salivary secre-tions, as well as from respiratory cells in culture, and have identified a pattern of fourmajor peptides (masses 1036, 1132, 1688, and 1980 Daltons; some of these peptides con-tain cysteine residues, which are alkylated in our analyses) that are characteristic of thismolecule (3). These peptides arise from cysteine-rich regions that are repeated severaltimes in the MUC5B polypeptide (7).8. We have used a µRPC C2/C18 PC 3.2/3 column eluted at a flow rate of 240 µL/min with0.1% (v/v) TFA (5 min) followed by a linear gradient of 0–50% (v/v) acetonitrile in 0.1%(v/v) TFA (30 min) using the Pharmacia SMART system.9. Because of unique sequences of the tandem repeat regions of mucins identified so far, anamino acid composition on the mucin glycopeptides might be a simple way to distinguishbetween mucins. For example, the MUC2 mucin would be expected to have a much higherthreonine content than any of the other identified mucins. Separation and Identification of Mucin 8510. Samples must be salt free; otherwise the derivatization is hampered.11. To decrease the risk of contamination, all glassware should be prewashed in concentratedchromic acid, rinsed in high-quality water, and dried before use.12. The column is eluted at a flow rate of 0.75 mL/min with buffer A (2.66 min) followed bya linear gradient of 0–50% buffer B (42.34 min). Over the next minute, the concentrationof buffer B is brought to 100%.References1. Sheehan, J. K., Thornton, D. J., Howard, M., Carlstedt, I., Corfield, A. P., and Para-skeva, C. (1996) Biosynthesis of the MUC2 mucin: evidence for a slow assembly of fullyglycosylated units. Biochem. J. 315, 1055–1060.2. Thornton, D. J., Carlstedt, I., Howard, M., Devine, P. L., Price, M. R., and Sheehan, J. K.(1996) Respiratory mucins: identification of core proteins and glycoforms. Biochem. J.316, 967–975.3. Thornton, D. J., Howard, M., Khan N., and Sheehan, J. K. (1997) Identification of twoglycoforms of the MUC5B mucin in human respiratory mucus: evidence for a cysteine-rich sequence repeated within the molecule. J. Biol. Chem. 272, 9561–9566.4. Toribara, N. W., Ho, S. B., Gum, E., Gum, J. R., Lau, P. and Kim, Y. S. (1997) Thecarboxyl-terminal sequence of the human secretory mucin, MUC6: analysis of the pri-mary amino acid sequence. J. Biol. Chem. 272, 16,398–16,403.5. Gum, J. R., Hicks, J. W., Toribara, N. W., Rothe, E. M., Lagace, R. E. and Kim, Y. S.(1992) The human MUC2 intestinal mucin has cysteine-rich subdomains located bothupstream and downstream of its central repetitive region. J. Biol. Chem. 267, 21,375–21,383.6. Meerzaman, D., Charles, P., Daskal, E., Polymeropoulos, M. H., Martin, B. M,. and Rose,M. C. (1994) Cloning and analysis of cDNA encoding a major airway glycoprotein, humantracheobronchial mucin (MUC5). J. Biol. Chem. 269, 12,932–12,939.7. Desseyn, J. L., Guyonnet-Duperat, V., Porchet, N., Aubert, J. P. and Laine, A. (1997)Human mucin gene MUC5B, the 10.7-kb large central exon encodes various alternatesubdomains resulting in a super-repeat: structural evidence for a 11p15.5 gene family.J. Biol. Chem. 272, 3168–3178.8. Audie, J. P., Janin, A., Porchet, N., Copin, M. C., Gosselin, B., and Aubert, J. P. (1993)Expression of human mucin genes in respiratory, digestive, and reproductive tracts ascer-tained by in situ hybridization. J. Histochem. Cytochem. 41, 1479–1485.8a. Thornton, D. J., Khan, N., Howard, M., Veerman, E., Packer, N. H. and Sheehan, J. K.(1999) Salivary mucin MG1 is comprised almost entirely of a differently glycosylatedpopulations of the MUC5B gene product. Glycobiology 3, 293–302.9. Thornton, D. J., Carlstedt, I., and Sheehan, J. K. (1996) Identification of glycoproteins onnitrocellulose membranes and gels. Mol. Biotechnol. 5, 171–176.10. Thornton, D. J., Howard, M., Devine, P. L. and Sheehan, J. K. (1995) Methods for separa-tion and deglycosylation of mucin subunits. Anal. Biochem. 227, 162–167. . assessment of pep-tide homogeneity is advisable. We analyze aliquots of the peaks by using matrix-assistedlaser desorption ionization time-of-flight mass spectrometry. contained a complex mixture of mucin-type glycoproteins.However, until recently and with the advent of the new mucin-specific probes arisingfrom cDNA cloning