Glycoprotein methods protocols - biotechnology 048-9-129-141.pdf

Glycoprotein methods protocols - biotechnology

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

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

12

Synthetic Peptides for the Analysis and Preparation

of Antimucin Antibodies

Andrea Murray, Deirdre A O’Sullivan, and Michael R Price

1 Introduction

Since the mid-1980s, the family of high molecular weight glycoproteins known as mucins have evoked considerable interest among those in the field of cancer research Mucins, which are constituents of mucus, have a lubricating and protective function in

normal epithelial tissue (1) However, expression of mucin by the cancer cell is often

highly disorganized and upregulated, sometimes to the extent that mucin can be detected in the circulation of the cancer patient These changes in expression of mucin observed in neoplasia have led to the exploitation of some members of the mucin

family as circulating tumor markers (2,3) or targets for diagnostic imaging (4–6) and

therapy of cancer.

The first mucin to have its primary amino acid sequence determined, MUC1, is also the most extensively studied This molecule is highly immunogenic, and a consider-able number of anti-MUC1 monoclonal antibodies (mAbs) and fragments have been produced by various methods Some of these have found applications for radio-immunoscintigraphy and targeted therapy of cancer, and others have been used to detect circulating MUC1 Although such studies have yielded promising results, their present application is somewhat restricted In this age of genetic and protein engineer-ing, we have, at our disposal, the technology to design antibodies with ideal character-istics of size, affinity, and specificity for any desired application However, before considering such ambitions, we must first gain an understanding of the molecular interactions between epitope and paratope when an antibody binds to its antigen It is essential that key residues involved in the interaction are identified so that a model of how the interaction takes place on a three-dimensional level can be constructed This identification will enhance our ability to design antibodies with the correct character-istics for our chosen application.

1.1 Immunoassays

Both enzyme-linked immunosorbant assays (ELISAs) and radioimmunoassays have been used in various formats to test antibody binding to synthetic peptides The indi-rect ELISA has the advantages of being easy to perform, having no requirement for radioactive tracers, and producing results that are simple to interpret The disadvan-tage of the indirect ELISA is that the procedure requires that the antigen, in this case a synthetic peptide, be immobilized on to the surface of a microtiter plate well Classi-cally this would be achieved by dispensing a solution of antigen into the wells of a microtiter plate to allow adsorption, leaving the plate coated with antigen However, short synthetic peptides adsorbed on to plates in this way provide unpredictable and inconsistent results This problem may be owing to the fact that the orientation of the peptide on the plate cannot be controlled or simply that short peptides do not adhere well to polystyrene plates Several methods of peptide modification have been utilized

to overcome these problems One such procedure involves preparing branched-chain polypeptides in which MUC1 immunodominant peptides ware conjugated to a

polyl-ysine backbone (7) These polylpolyl-ysine conjugates provide very potent MUC1-related

antigens for the interrogation of antibody specificity; however, the methodology for their preparation is beyond the scope of this chapter By far the most widely used method for modifying short peptides so that they can be used as antigens in indirect ELISA procedures is to conjugate the peptides to a large carrier protein such as bovine

serum albumin (BSA) (see Subheading 3.1 and Notes 1–3).

1.2 Tethered Peptide Libraries for Exploring Antibody Specificity

The peptide synthesis techniques developed by Geysen and colleagues (8)

repre-sent a significant development in the study of epitopes defined by antibodies reactive with antigens of known primary structure Unlike most other methods of simultaneous peptide synthesis, this technique allows the concurrent synthesis of hundreds to thou-sands of peptides so that libraries can be produced and simultaneously used as targets for antibody binding The peptides are synthesized on derivatized polyethylene or

polypropylene gears that are held on stems (Fig 1) arranged in a microtitre plate

for-mat so that a simple ELISA procedure can be used to measure antibody binding Pep-tides are tethered via the carboxyl terminus.

Several different strategies have been described for peptide sequence design that all provide different information on epitope structure and the fine specificity of an antibody-peptide interaction The Pepscan approach has been the most widely used and involves the synthesis of a set of overlapping peptides that span the length of the

antigenic sequence (Subheading 3.2.) In a short peptide sequence, such as that of the

MUC1 variable number of tandem repeat (VNTR), each peptide may overlap the next

by all but one amino acid, giving rise to a set of 21 heptapeptides that spans the VNTR

sequence (Fig 2) For larger proteins, it is more appropriate to produce longer

sequences that overlap each other by less residues, thereby spanning the length of the

antigenic sequence with a feasible number of peptides (see Note 4) In the Pepscan

approach, peptides are assayed for antibody-binding capacity by ELISA (Subheading

3.3.), and residues that are common to all the antibody-binding pins represent the

mini-Fig 1 The Multipin Peptide Synthesis System contains detachable polyethylene gears that fit on to the end of stems The stems are held in a block in an 8 x 12 microtiter plate format The surface of the gear is derivatized to give a solvent-compatible polymer matrix on which the peptides are coupled during synthesis The matrix also provides a two amino acid spacer group

Fig 2 Schematic representation of the overlapping peptides corresponding to the MUC1 VNTR sequence synthesized according to the Pepscan approach to epitope mapping Antibod-ies are allowed to react with each peptide, and those containing the epitope or minimum bind-ing unit produce positive results In this example, the epitope can be deduced as consistbind-ing of

the amino acids that are common to all positive pins (7–10) Hence, the epitope is PDTR.

mum binding unit or epitope for that antibody (Fig 2) Having identified the epitope

defined by an antibody using Pepscan, it may be useful to prepare a number of analogs

of that sequence in order to investigate the role of individual amino acids in the epitope and to identify critical contact residues Such peptide design stategies include

ommission analysis, alanine substitution and replacement net (RNET) analysis (see

Notes 5–9).

Libraries of peptides on pins can be obtained that comprise 400 different dipeptides prepared with all possible combinations of the 20 natural amino acids This approach provides qualitative information on antibody specificity and permits identification of significant features of an epitope that may contribute to antibody recognition and

bind-ing (see Notes 10 and 11).

1.3 Purification of Antibodies Using Peptide Affinity Chromatography

The identification of a linear peptide epitope within a protein sequence facilitates the design of peptide affinity matrices that can be used to purify antibodies from bio-logical feedstocks Such an epitope affinity matrix has been produced by covalently linking a synthetic peptide corresponding to the MUC1-immunodominant domain to

cyanogen bromide-activated Sepharose (Pharmacia, Uppsala, Sweden) (9) The

resulting matrix was remarkably efficient for the purification of a range of anti-MUC1 mAbs from biological feedstocks containing high levels of contaminating proteins

such as ascitic fluid and hybridoma supernatant (see Note 12).

Epitope affinity chromatography matrices have an advantage over other affinity adsorbents in that the antibody is bound to the matrix specifically via the paratope Thus, eluted antibody is fully immunoreactive and of only the desired specificity Sepharose-peptide conjugates are simple to prepare and affinity chromatography is more robust than other conventional chromatographic techniques in terms of column

packing and operation (see Subheading 3.4., Notes 13–15, and Fig 3).

1.4 General Comments

The techniques described for the analysis of antimucin antibodies using synthetic peptides can provide a great deal of information on epitope topography and structure The identification of critical binding residues within an epitope can provide clues to the forces and residues involved in the antibody-antigen interaction However, bear in mind that the use of linear synthetic peptides can only provide a one-dimensional solution to what is essentially a three-dimensional problem Further structural studies such as X-ray crystallography, nuclear magnetic resonance spectroscopy, and compu-tational molecular modeling are essential if the knowledge gained is to be confirmed and translated into a useful model on which to base antibody design strategies The structural information provided by studies such as those previously described may be of use in peptide vaccine design However, the analyses performed so far have been mainly concerned with the interaction of murine antibodies, and it may be naive

to assume that the human immune system will process mucin-related antigens in the same way Preliminary epitope-mapping studies on human serum would suggest that the immune response to MUC1 may differ considerably from that observed in the

mouse (10).

Finally, it may be owing to the very nature of the mucins that such a wealth of informa-tion has been provided by the techniques described The VNTR provides a convenient short sequence on which to base peptide synthesis strategies In addition, most murine antimucin antibodies analyzed to date have been shown to define short linear determi-nants It is unlikely that all other proteins and antibodies will be so accommodating. Fig 3 Schematic representation of the apparatus and reagents needed for the purification of antibodies by peptide epitope affinity chromatography

2 Materials

2.1 Preparation of BSA-Peptide Conjugates

1 Conjugation buffer: sodium hydrogen carbonate buffer (0.1 M, pH 8.4).

2 BSA: crystalline, greater than 96% pure

3 Glutaraldehyde: when used as a crosslinker must be freshly distilled or high commercial grade (Sigma, Poole, UK)

4 Dialysis buffer: sodium chloride 1% (w/v)

2.2 Solid-Phase Peptide Synthesis on Pins

All reagents used in solid-phase peptide synthesis should be of the highest avail-able purity (analytical reagent grade or better) unless stated otherwise.

1 Mulitpin Peptide Synthesis Kit (Chiron Mimotopes, Clayton, Victoria, Australia)

2 Amino acids: All amino acids recommended for use with the Multipin Peptide Synthesis Kit have their α-amino group protected with the 9-fluorenylmethyloxycarbonyl (Fmoc)

group Table 1 appropriate side chain protecting groups Alternatively, protected amino

acid esters may be used These have the advantage of requiring no prior activation How-ever, they are prone to decomposition with prolonged storage and are best stored at –20°C

3 Activators: The activation of protected amino acids with diisopropylcarbodiimide (DIC)/

1-hydroxybenzotriazole (HOBt) is recommended, but other coupling reagents can be used

4 N,N-Dimethylformamide (DMF): DMF used in peptide coupling procedures must be pure

and free from amines Several methods may be used to purify DMF (see Note 16).

5 Piperidine 20% v/v: used for Fmoc deprotection Piperidine should be redistilled before use and made up to a 20% (v/v) solution in DMF

6 Bromophenol blue: used as an indicator of coupling efficiency Stock reagent is prepared

by dissolving 33.5 mg of bromophenol blue in 5 mL of DMF This should be diluted 1:200 for working concentration

7 Acetylation mixture: DMF, acetic anhydride and triethylamine in a 50:5:1 (v/v/v) ratio

8 Side chain deprotection mixture: trifluoroacetic acid, ethanedithiol, and anisol in a 38:1:1 (v/v/v) ratio

9 Final wash solution: acetic acid 0.5% (v/v) in methanol/water (1:1, v/v).

10 Other reagents: methanol (MeOH), purified water

2.3 ELISA Testing Procedure

1 Phosphate buffered saline (PBS), 0.01 M, pH 7.2 (1.34 g of Na2HPO4·2H2O, 0.39 g of NaH2PO4·2H2O, and 8.5 g of NaCl made up to 1 L with distilled water) is used as the buffer base for most of the following buffer reagents

Table 1

Suitable Amino Acid Side Chain Protecting Groups

for Solid Phase Peptide Synthesis on Pins

2 Blocking buffer: 2% (w/v) BSA, 0.1% (v/v) Tween-20, and 0.1% sodium azide in 0.01 M PBS.

3 Conjugate diluent: 1% (v/v) sheep serum, 0.1% (v/v) Tween-20, and 0.1% sodium

casein-ate (USB, Bioscience, Cambridge, UK) in 0.01 M PBS.

4 Citrate phosphate buffer: 17.8 g of Na2HPO4·2H2O and 16.8 g of citric acid monohydrate made up to 1 L with distilled water, pH 4.0

5 2,2'-azino-bis[3-ethylbenz-thiazoline-6-sulfonic acid] (ABTS) substrate solution (Sigma): 0.5 mg/mL in citrate phosphate buffer with hydrogen peroxide (35% w/w) added to give

a final concentration of 0.01% (w/v)

6 Disruption buffer: Sodium dihydrogen orthophosphate (0.1 M) pH 7.2, containing sodium

dodecyl sulfate (SDS) (0.1% w/v) β-Mercaptoethanol (5 mL) is added immediately prior to use

2.4 Purification of Antibodies Using Peptide Affinity Chromatography

1 Affinity support: CNBr-activated Sepharose 4B (Pharmacia, Uppsala, Sweden)

2 Equilibration buffer: 0.01 M PBS with azide (PBSA), pH 7.2 (1.34 g of Na2HPO4·2H2O, 0.39 g of NaH2PO4·2H2O, and 8.5 g of NaCl made up to 1 L with distilled water) with sodium azide 0.02% (w/v) added as a preservative

3 Wash buffer: 0.5 M NaCl, pH 7.2, in distilled water.

4 Elution buffer: 3 M NaSCN, pH 7.2, in distilled water.

5 Desalting column: Sephadex G25 (Pharmacia)

3 Methods

3.1 Preparation of BSA-Peptide Conjugates ( see Notes 1–3)

1 Dissolve BSA (10 mg) in 3 mL conjugation buffer in a clean glass vial

2 Dissolve peptide (10 mg) in 1 mL conjugation buffer

3 To the BSA solution, add 1 mL of peptide solution and 10 µL of glutaraldehyde Then seal and agitate on a roller for 4 h at room temperature

4 The conjugate is finally dialysed against sodium chloride (1%) for 48 h at 4°C

3.2 Solid-Phase Peptide Synthesis on Pins ( see Notes 4–11, and 17)

The Multipin Peptide Synthesis Kit (Chiron Mimotopes) contains derivatized gears, stems, 8 × 12 format pin holders, and reaction trays In addition, it contains all the software needed for creating a synthesis schedule, running dispensing aids, and read-ing and plottread-ing assay results.

3.2.1 Creating a Synthesis Schedule

The method described by Geysen et al (8) for linear epitope scanning requires that

many different peptides be synthesized simultaneously To plan and execute a manual synthesis schedule for creating hundreds of peptides simultaneously is extremely time-consuming and fraught with the possibility of errors Fortunately, computer software is available to generate a synthesis schedule based on any given protein sequence with any of the manipulations described (Chiron Mimotopes) These schedules calculate the weights and volumes of the various reagents required on each day of the synthesis and then instruct

the operator where in the 96-well reaction tray each amino acid should go (see Notes 5–8).

3.2.2 Peptide Synthesis

The peptide synthesis procedure consists of cycles of N-terminal deprotection, washing and coupling until the desired peptides have been assembled, followed by

side chain deprotection The synthesis schedule provides details of the amounts of amino acids and activators required It is advisable to weigh out all these reagents before beginning a synthesis since this is the most time-consuming step of the proce-dure All steps are carried out at room temperature unless stated otherwise.

1 The appropriate number of gears required for that synthesis on that day should be removed from storage and assembled on to the block according to the synthesis schedule It is impor-tant that only gears requiring deprotection in the next cycle of synthesis be added to the block

2 Achieve deprotection of the amino terminus by immersing the pins in a bath containing 20% piperidine for 20 min The piperidine solution should cover the gears The pins are then washed as follows:

a DMF to cover gears for 2 min

b MeOH (complete submersion) for 2 min

c MeOH to cover gears for 2 min (three times)

The pins are then allowed to air-dry in an acid-free fume hood for a minimum of 30 min

3 Prepare HOBt and DIC solutions by dissolving in the appropriate amount of DMF (see

Note 13) The addition of bromophenol blue to the HOBt to give a final concentration of

0.05 mM as an indicator of coupling efficiency is optional The volume of HOBt solution

specified on the synthesis schedule must be added to each amino acid to dissolve it fully before adding the specified amount of DIC

4 Dispense amino acid solutions into a 96-well reaction tray according to the synthesis schedule The recommended order of activating and dispensing amino acids is as follows:

A D E F G I L M P S T V Y W Q N K C H R Care should be taken to ensure that the amino acids are dispensed into the correct wells Dispensing aids are now available that consist of a bank of LED lights set out in a microtiter plate format Lights are lit beneath the reaction tray to indicate which wells should contain which amino aid The dispensing aid is driven by the synthesis schedule software

5 Place the block of Fmoc-deprotected pins into the reaction tray in the correct orientation Place the tray into a polystyrene box to reduce evaporative losses and avoid contamina-tion and leave to incubate for at least 4 h

6 When the coupling reaction is complete, the blue colouration of bromophenol blue should have disappeared The pins are then washed as follows:

a MeOH to half the pin height for 5 min

b air-dry for 2 min

c DMF to half the pin height for 5 min

The next cycle of peptide synthesis can begin immediately with Fmoc deprotection

7 When the required peptides have been synthesized, deprotect and wash the free amino

termini as described in step 2 The amino terminus may then be acetylated to remove the

charge associated with a free amino terminus (if required) by incubating the pins in a reaction tray containing acetylation mixture at 150 µL/well for 90 min in an enclosed environment Then wash the pins in MeOH for 15 min and then air-dry

8 To achieve side chain deprotection, incubate the pins in a bath of side chain deprotection mixture for 2.5 h Next, immerse in a final wash solution for 1 h, rinse twice in MeOH for

2 min each, and air-dry overnight The pins are now ready for ELISA testing

3.3 ELISA Testing Procedure ( see Notes 7 and 12)

Antibody binding to peptides on pins is measured using an indirect ELISA proce-dure in which the solid phase on which the test antibody is captured is the

peptide-coated gear, and the presence of the test antibody is reported using an enzyme-labeled secondary antibody The enzyme catalyzes the reaction of ABTS substrate to its col-ored product, which can be measured using a spectrophotometer The degree of color change is proportional to the amount of test antibody bound to the peptide on the gear Before antibody testing begins, the newly synthesized pins should be tested for non-specific binding to the enzyme-labeled secondary antibody of choice This is achieved

by carrying out steps 1 and 4–6 Antibodies may now be tested as follows, with all

incubations and washing steps performed at room temperature unless otherwise stated.

1 First precoat the pins in blocking buffer in order to minimize nonspecific binding to the gear To achieve this, immerse the pins in a microtiter plate containing blocking buffer at

200µL/well and incubate for 1 h with agitation

2 Dilute the primary antibody to an appropriate concentration in blocking buffer and dispense into the wells of a microtiter plate at 200 µL/well After removing from blocking buffer and flicking to remove excess buffer, incubate the pins in primary antibody at 4°C overnight

3 Remove the pins from the microtiter plate and wash four times in a bath of PBS contain-ing Tween-20 (0.1% v/v) for 10 min Use fresh buffer for each wash

4 Dilute an appropriate horseradish peroxidase-labeled secondary antibody conjugate (e.g., horse-radish peroxidase-conjugated rabbit antimouse Ig is suitable for detecting murine primary anti-bodies) in conjugate diluent and dispense into the wells of a microtiter plate at 200 µL/well Then incubate the washed pins in the secondary antibody solution for 1 h with agitation

5 Wash the pins four times as in step 3 Prepare ABTS substrate solution immediately

before use and dispense into the wells of a microtiter plate at 200 µL/well Immerse the pins in the substrate solution in the correct orientation and allow to incubate for 45 min with agitation The reaction can be stopped before the time is elapsed, if it appears that the reaction will give an optical density (OD) of 2 or greater, by removing the pins from the wells and then allowing the microtiter plate to shake for a further 15 min to allow full color dispersion The OD of each well is determined spectrophotometrically at a wave-length of 405 nm

6 Bound antibodies can be removed from the pins by sonication in disruption buffer at

60°C for 2 h, followed by repeated rinses in distilled water at 60°C and methanol (two

times) The efficiency of the cleaning procedure should be tested by repeating steps 4 and

5 Absorbance levels above background indicate that antibody remains bound to the pins

and further cleaning is required Once the pins are clean, they should be sonicated for 30

min followed by rinsing as detailed just above (see Note 17).

3.4 Purification of Antibodies Using Peptide Affinity Chromatography ( see Notes 12–15 and Fig 3)

1 Prepare Sepharose-peptide affinity matrices and pack columns according to the manufacturer’s

instructions (see Notes 12 and 13).

2 Equilibrate the columns with 10 column volumes of PBSA at a flow rate of 1 mL/min

3 Clarify hybridoma or bacterial culture supernatants by ultracentrifugation (40,000g, 1 h)

and ultrafiltration (0.2 µm) and then store with 0.05% (w/v) sodium azide as a preservative

4 Apply clarified supernatant to the column at a rate of 1 mL/min followed by washing with PBSA, to remove unbound material, until the trace from the ultraviolet monitor has re-turned to baseline

5 Optional: Wash the column with 0.5 M NaCl (1 mL/min) to remove material that has

bound to the column nonspecifically

6 To achieve desorption of specifically bound, pure antibody, apply three column volumes

of 3 M NaSCN to the column at a rate of 1 mL/min and finally return the column to PBSA

(see Notes 14 and 15).

7 Antibody preparations desorbed using 3 M NaSCN must be desalted soon after elution

from the affinity matrix To achieve this, connect a gel filtration column containing a

medium such as Sephadex G25 in series with the affinity column (Fig 3).

4 Notes

1 Conjugation of peptides to carrier proteins can also be performed in situ in the well of a

microtitier plate (11).

2 Synthetic peptide-carrier protein conjugates and synthetic branched-chain polypeptides provide highly characterized and reproducible sources of mucin-like antigenic material However, bear in mind that these reagents are analogs of the natural antigen and do not possess the carbohydrate side chains that are a dominant characteristic of all mucins Hence, results obtained in immunoassays, especially those involving the measurement of kinetic data, must be treated with caution

3 The influence of carbohydrates on the recognition of peptide epitopes may be evaluated,

at least to some extent, using synthetic glycopeptides rather than peptide alone

Glyco-peptides can be produced by both chemical (12) and enzymatic (13,14) methods These

reagents have been of value in assessing the contribution of O-linked

N-acetyl-galactosamine (GalNAc) residues to mucin secondary structure and also in the studies to

investigate the role of GalNAc residues in the binding of protein core antibodies (15).

However, the glycosylation of mucin molecules is complex, and the production of higher-order synthetic mucin analogs with more than a single sugar at each glycosylation site is technically demanding

4 The length of peptides synthesized seems to have no effect on the result obtained as dem-onstrated by two independent studies of anti-MUC1 protein core mAbs One group used heptamers spanning the tandem repeat domain and overlapping each other by six amino

acids (16), and the other used octamers overlapping each other by seven amino acids (17).

Twelve antibodies were analyzed in total and the three that were analyzed in both studies gave identical minimum binding units

5 In the omission analysis approach, a series of peptides are synthesized based on an epitope sequence In each consecutive peptide, a single residue is omitted from the sequence This allows the role of individual residues to be assessed For example, an omission analy-sis series covering the immunodominant region of the MUC1 protein core may be synthe-sized If an antibody were allowed to interact with this series of peptides, those sequences that produced a loss in binding compared with the parent sequence can be identified as part of a peptide in which an essential residue has been omitted Antibody binding is maintained in those peptides in which the epitope is complete

6 In the substitution analysis approach, each residue is replaced in turn with another amino acid This amino acid is normally alanine, but for cases in which an alanine already exists

at that substitution position, the residue can be replaced with glycine The information provided when an antibody is allowed to react with this set of peptides is similar to that of omission analysis; however, in this case, the spatial arrangement of the respective epitope residues more closely resembles the native sequence

7 RNET analysis offers the most critical and informative method for probing a peptide epitope in the MUC1 protein core In this approach, a set of peptides with sequences based on a short minimum binding sequence or epitope are synthesized on the heads of