Glycoprotein methods protocols - biotechnology 048-9-075-085.pdf

Glycoprotein methods protocols - biotechnology

Separation and Identification

of Mucins and Their Glycoforms

David J Thornton, Nagma Khan, and John K Sheehan

1 Introduction

This chapter describes a strategy for the separation and identification of the mucins present in mucous secretions or from cell culture, focusing primarily on those mucins involved in gel formation At present, the mucins MUC2, MUC5AC, MUC5B, and

MUC6 are known to be gel-forming molecules (1–4) These mucins share common

features 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 In addition, 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 arising

from 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 MUC gene product can be expressed in a single mucus-producing epithelia, i.e., MUC5AC

and 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 two mucin genes are not only expressed but that their glycosylated products are present in

respiratory tract secretions (2,3) A further more recent insight into the complex nature

of the mucin component of mucous has been the demonstration that a mucin gene product from a single epithelium can have a different oligosaccharide decoration and

thus 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 to ascertain mucin type, amount, and glycoform Such investigations may lead to the identification of novel members of this growing family of molecules.

Owing to the extreme size and polydispersity (Mr = 5–50 × 106) of the gel-forming mucins in particular, there are few separation techniques available to use with these

77 From: Methods in Molecular Biology, Vol 125: Glycoprotein Methods and Protocols: The Mucins

Edited by: A Corfield © Humana Press Inc., Totowa, NJ

molecules However, the smaller and more homogeneous reduced mucin monomers

generated after reduction (Mr = 2–3 × 106) are amenable to conventional biochemical separation methods We describe a separation protocol based on anion exchange chro-matography to fractionate the different species of mucin coupled with agarose gel electrophoresis as a method to measure their homogeneity Identification of mucin polypeptides is achieved by use of MUC-specific antisera and fragmentation followed

by amino acid compositional analysis and peptide purification and sequencing Mucin glycoforms are detected using carbohydrate-specific probes, i.e., lectins or monoclonal

antibodies Figure 1 summarizes the overall procedure.

2 Materials

2.1 Extraction and Purification

See Chapter 1 for details.

Fig 1 Outline of the protocol for separation and identification of mucins

2.2 Preparation of Reduced Mucin Subunits

1 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 Mucin

2.3.1 Anion-Exchange Chromatography

1 Mono Q HR5/5 column (Pharmacia)

2 Buffer A (see Subheading 2.2.).

3 0.4 M lithium perchlorate.

2.3.2 Agarose Gel Electrophoresis

1 Horizontal electrophoresis apparatus, e.g., Bio-Rad DNA subcell Model 96 (25× 15 cm gels) 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 Mucin

2.4.1 Tryptic Digestion

1 Digestion buffer: 0.1 M ammonium hydrogen carbonate, pH 8.0.

2 Modified trypsin or other proteinase

2.4.2 Trypsic Peptide Analysis

1 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 Analysis

1 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.

3 Methods

3.1 Extraction and Purification

For 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 containing

proteinase 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 M

guanidinium chloride (removal of nucleic acids) Finally, the preparation of mucin is

dialyzed into 4 M guanidinium chloride for storage.

3.2 Preparation of Reduced Mucin Subunits

Mucin subunits (monomers) are prepared by reduction and alkylation of purified mucins 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 at

37°C

3 Alkylate the free-thiol groups generated by reduction with the addition of iodoacetamide

to a final concentration of 25 mM This step can be performed overnight in the dark at

room temperature

4 Transfer the reduced mucin subunits into buffer A by chromatography on a Pharmacia PD-10 column (follow manufacturer’s instructions) or by dialysis

3.3 Separation of Mucin

Separation 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 effectiveness

of the separation is monitored with a periodic acid-Schiff (PAS) assay and with a variety of lectins and mucin- or carbohydrate-specific antibodies (for a discussion of

the 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 blots

of the gels can be probed with lectins and antibodies Using this methodology, we have shown that with pooling and rechromatography it is possible to prepare mucin subunit samples enriched in specific MUC gene products and also to isolate different

glycosylated 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 mucin

reduced subunit preparation, is shown in Fig 2 The range of charge density of the molecules (Fig 2a) is typical of that observed for the reduced subunits prepared from

the 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 density

since the molecular weight and size (radius of gyration) of the molecules (determined

by 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 this

sample is the MUC5B gene product (8a) Thus, the Mono-Q column appears to be

separating differently charged forms of this mucin The continuum in the

electro-phoretic mobility observed with the salivary mucin sample is not normally seen in respiratory mucin subunit preparations In respiratory samples there are typically two gel-forming mucins, namely MUC5AC and MUC5B The MUC5B mucin appears by Mono-Q chromatography to be in two differently charged states, and like the salivary mucin subunits, the electrophoretic mobility of the MUC5B subunits is consistent with their charge density However, the MUC5AC mucin-reduced subunits are of lower charge density than the most highly charged MUC5B molecules, but they migrate

farther 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 shows the distribution of the reduced subunits as monitored with the PAS-reagent (B) Aliquots from each 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 against reduced mucin subunits

3.3.1 Anion-Exchange Chromatography

The 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 min after application

2 Elute the sample with a linear gradient from 0 to 0.4 M lithium perchlorate in buffer A

over 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 Electrophoresis

Electrophoresis is performed in 1% (w/v) agarose gels using a standard horizontal gel electrophoresis apparatus.

3.3.2.1 SAMPLE PREPARATION

1 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 PREPARATION

We typically perform electrophoresis in 15 × 15 cm or 25 × 15 cm gels of approx

3 to 4 mm thickness.

1 Dissolve the agarose in electrophoresis buffer in a microwave; for a small gel, use 1.6 g of agarose and 160 mL of buffer, and for a large gel, use 2.8 g of agarose and 280 mL of buffer

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 ELECTROPHORESIS

1 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 same buffer volume

3.3.2.4 WESTERN BLOTTING

After 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 transfer buffer at a suction pressure of 45 mBar for 1.5 h We use a Pharmacia Vacu-Gene XL for this procedure

3 Probe the membrane for lectin and antibody reactivity (see ref 9 for detailed procedures).

3.4 Identification of Mucin

The 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-terminal sequencing of proteolytically derived peptides and amino acid compositional analysis

of mucin glycopeptides for a more definitive identification These procedures will also provide a route to obtain information on novel mucins The starting point for these analyses is a proteolytic fragmentation of reduced mucin subunits For example,

trypsin digestion of reduced mucin subunits liberates high Mr mucin glycopeptides,

which correspond to the heavily O-glycosylated tandem repeat regions of the mol-ecule, and lower Mr peptides and glycosylated peptides, which arise from the “naked” cysteine-rich regions of the molecule.

3.4.1 Trypsin Digestion

1 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 (see

Note 5) and leave the digestion overnight at 37°C

3 Separate digestion products into high M rglycopeptides 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 (see Note 7)

Tryptic peptides can be fractionated by reverse phase chromatography and indi-vidual peptides purified and their primary sequence determined by N-terminal sequencing.

3.4.2.1 PEPTIDE PURIFICATION AND N-TERMINAL SEQUENCING

1 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-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), and purify peptides to homogeneity by rechromatography on the C2/C18 column utilizing shallower gradients centered on their elution point

4 Dry down peptides by vacuum centrifugation and determine the primary sequence of peptides by automated N-terminal sequencing

5 Use sequence to search sequence databases

3.4.3 Amino Acid Analysis (see Note 9)

3.4.3.1 ACID HYDROLYSIS

1 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

3.4.3.2 DERIVATIZATION

1 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 CHROMATOGRAPHY

1 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 comparison with standard amino acid mixture

4 Notes

1 Urea can be tolerated up to a concentration of at least 6 M, but salts (particularly

guanidinium 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 between band broadness and amount of sample to be loaded Larger amounts of sample tend to yield poorer quality data; i.e., the bands become more smeared

3 Molecules transferred to PVDF can be treated with trifluoromethanesulfonic acid to

remove O-linked glycans This is often essential if using antisera that are directed against

core protein epitopes “masked” by oligosaccharides (10).

4 Treat trypsin by reductive alkylation to modify arginine and lysine residues to prevent autolysis 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 the total mass of the mucin subunit and that approx 50% of this protein is in “naked” regions that are accessible to the proteinase

6 Chromatography separates the high M rmucin glycopeptides (void volume) from the lower

M r 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 obtain

a peptide fingerprint associated with each gene product We have already performed such

an 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 four major 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 this

molecule (3) These peptides arise from cysteine-rich regions that are repeated several times 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 with 0.1% (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, an amino acid composition on the mucin glycopeptides might be a simple way to distinguish between mucins For example, the MUC2 mucin would be expected to have a much higher threonine content than any of the other identified mucins

10 Samples must be salt free; otherwise the derivatization is hampered.

11 To decrease the risk of contamination, all glassware should be prewashed in concentrated chromic 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 by

a linear gradient of 0–50% buffer B (42.34 min) Over the next minute, the concentration

of buffer B is brought to 100%

References

1 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 fully

glycosylated 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 two glycoforms 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) The carboxyl-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 both

upstream 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, human

tracheobronchial 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 alternate subdomains 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 glycosylated

populations of the MUC5B gene product Glycobiology 3, 293–302.

9 Thornton, D J., Carlstedt, I., and Sheehan, J K (1996) Identification of glycoproteins on

nitrocellulose 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 deglycosylasepara-tion of mucin subunits Anal Biochem 227, 162–167.