Glycoprotein methods protocols - biotechnology 048-9-143-155.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

13

Mucin Domains to Explore Disulfide-Dependent

Dimer Formation

Sherilyn L Bell and Janet F Forstner

1 Introduction

The viscoelastic properties needed for the protective functions of secretory mucins are in part conditional on the capacity of mucin macromolecules to form linear poly-mers stabilized by disulfide bonds The individual mucin monopoly-mers have a distinctive structure, consisting of a long central peptide region of tandem repeat sequences, flanked by cysteine-rich regions at each end, which are presumed to mediate

polymer-ization Secretory mucins contain approx 60–80% carbohydrate, with extensive O-glycosylation in the central tandem repeat regions, and N-linked oligosaccharides in

the peripheral regions (1).

The ability of mucin peptides to form large polymers, combined with their exten-sive posttranslational glycosylation and sulfation, results in complexes that reach

molecular masses in excess of 10,000 kDa (2) This leads to difficulties in resolving

mucins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)

or agarose gel electrophoresis, because both unreduced and reduced mucin samples are capable of only limited movement A related difficulty inherent in analyzing

mucins lies in their strong negative charge owing to sialic acid and sulfate content (3).

Migration through polyacrylamide gels becomes more influenced by charge than by mass The result is that interpretations of the size of mucin from electrophoretic mobility are not as straightforward as with other proteins.

Hypotheses concerning the regions of secretory mucins that could be involved in the initial dimer formation have centered on the terminal cysteine-rich, poorly glycosylated domains Indirect evidence that these domains are involved has been shown by treating mucins with proteolytic enzymes and reducing agents that act on these regions, and noting a resultant decrease in the size of mucin and gel formation

(4–6) Intriguingly, more indirect evidence was found when database searches

identi-fied a functionally unrelated protein, von Willebrand factor (vWF) (which also forms S-S–dependent polymers), to exhibit a mucinlike pattern of cysteine residue

align-ments in its N- and C-terminal domains (7,8) The function of vWF to cause

aggrega-tion of platelets is also dependent on its ability to polymerize via disulfide bonds More important for the present report, the DNA encoding vWF was expressed suc-cessfully in heterologous cells and shown to undergo S-S–dependent dimer and

multimer formation (9,10) The minimum component necessary for initial dimeriza-tion is a region of approx 150 amino acids at the C-terminal end (11) A similar vWF

“motif” has now been recognized near the C-terminus of several secretory mucins,

including frog integumentary mucin FIM-B.1 (7), bovine submaxillary mucin (12), porcine submaxillary mucin (13), human and rat intestinal mucin MUC2 (8,14), human tracheobronchial mucin MUC5AC (15), human gall bladder mucin MUC5B (16,17), and human gastric mucin MUC6 (18) Since these mucins are known to form

oligo-mers in vivo, their shared C-terminal motif may also be involved in forming mucin dimers Dimerization of mucin molecules represents the crucial first step in the trans-formation of individual mucin molecules into gel structures.

In this chapter, we describe a domain construct and expression approach used to examine directly whether the C-terminal domain of rat intestinal Muc2 is capable of dimerization through its cysteine residues This method avoids, to a large extent, the numerous difficulties of dealing with full-length mucins, since the domain peptide is expressed as a relatively small, less glycosylated monomer or dimer The principle is that DNA encoding the domain of interest is ligated to a known epitope sequence (for detection by a suitable antibody), and to a signal peptide sequence (to ensure secretion), by recombinant polymerase chain reaction (PCR) strategies The resulting construct is ligated

to an expression vector for transfection into heterologous cells Once the expected peptide has been translated and processed, dimerization by disulfide bond formation is shown by comparing the sizes of immunoreactive, thiol-reduced and nonreduced products in cells and media by SDS-PAGE and Western blotting The use of specific antibodies to various regions of the domain can provide assurance that the domain is expressed in an intact form

or, alternatively, has been proteolytically processed during dimerization After establish-ing the dimerization capability of the isolated C-terminal domain of rat Muc2, we describe methods for examining the role of glycosylation in dimerization by manipulating the sys-tem with inhibitors of glycosylation and/or deglycosylating enzymes.

2 Materials

2.1 Synthesis of Domain Constructs Using Recombinant PCR

1 PCR reagents: buffer, dNTPs, MgCl2, Taq DNA polymerase obtained from Perkin Elmer

(Foster City, CA)

2 Primer 1 (see Fig 1) This is a sense primer containing an XbaI site to facilitate cloning,

and also encoding part of the rat Muc2 signal peptide:

a 5'-CGTCTAGAATGGGGCTGCCACTAGCTCGCCTGGTGGCT-3'

3 Primer 2 (see Fig 1) The antisense primer containing signal peptide and “linker”

sequence to be paired with primer 1:

a 5'-CACAGTTAGATTCCAGCCCTTGGCTAAGGCCAGGACTAGGCACACAG-3'

4 Primer 3 (see Fig 1) This primer specifies the “linker” sequence and primes the 5' end of

the target domain of rat Muc2:

a 5'-GGCTTGGAATCTAACTGTGAAGTTGCTGC-3'

5 Primer 4 (see Fig 1) The antisense primer encoding the 3' end of rat Muc2 with an

additional SacI site sequence for cloning:

a 5'-CGAGCTCCTATCACTTCCTTCCTAGAAGCCG-3'

6 Clone MLP-3500, which encodes the C-terminal 1121 amino acids of rat Muc2, was used

as the DNA template (8).

7 Outer primers 5:

a 5'-CGTCTAGAATGGGGCTGC-3'

8 Antisense primer 6 (see Fig 1):

a 5'-CGAGCTCCTATCACTTCC-3'

9 Thermal cycler such as Perkin Elmer DNA Thermal Cycler 480

2.2 Ligation of Construct to Transfection Vector

1 Invitrogen TA cloning kit (Invitrogen, Carlsbad, CA)

2 Transfection vector pSVL available from Pharmacia (Uppsala, Sweden)

Fig 1 Schematic showing the synthesis of construct pRMC and its expression in COS cells

The protocol is described in Subheadings 3.1.–3.3 Oligonucleotide primers are designed that

facilitate the synthesis and joining of DNA sequence coding for the signal peptide and car-boxyl-terminal 534 amino acids of rat Muc2 via recombinant PCR The resulting construct is then subcloned into the expression vector pSVL for expression in COS cells

3 Restriction enzymes SacI, XbaI with supplied incubation buffer(s).

4 T4 DNA ligase and ligase buffer (Boehringer Mannheim, Mannheim, Germany)

5 Subcloning efficiency competent DH5α Escherichia coli in 50-µL aliquots.

6 Convection incubator maintained at 37˚C

7 DNA maxiprep columns (Qiagen, Chatsworth, CA)

8 Luria broth (LB) plates containing 50 µg/mL of ampicillin (19).

9 Agarose gels containing 0.5 µg/mL of ethidium bromide

10 HindIII-digested DNA λ markers for size and quantity estimation

11 1X TAE: 0.04 M Tris base, 1 mM EDTA, 1.14 mL/L glacial acetic acid.

2.3 Transfection

1 COS-1 or COS-7 cell line obtained from American Type Culture Collection, (Rockville, MD)

2 Dulbecco’s modified Eagle’s medium (Gibco-BRL, Gaithersburg, MD) supplemented with 10% fetal bovine serum (FBS) (CanSera, Etobicoke, ON, Canada) and with 100 U/mL

of penicillin and 100 µL of streptomycin (Gibco-BRL)

3 Hemocytometer

4 Lipofectamine (Gibco-BRL)

5 Cell culture incubator to maintain an atmosphere of 37˚C and 5% CO2

6 Transfection efficiency reporter such as pCMVßGAL or luciferase systems

2.4 Harvesting of Transfected Cells

1 2X Laemmli SDS sample buffer: 125 mM Tris-HCl, pH 6.8, 20% glycerol, 4% SDS,

0.005% bromophenol blue, plus or minus 1,4-Dithiothreitol (DTT) to give a final

concen-tration of 10 mM.

2 Filter concentrators such as Centricon with a molecular weight cutoff of 30 kDa

3 Phenylmethylsulfonylfluoride (PMSF)

4 Beckman J2-21 centrifuge, JA-20.1 rotor (Beckman Instruments, Palo Alto, CA)

2.5 SDS-PAGE and Western Blot Analysis

1 Tris-glycine polyacrylamide gels (precast) (Novex, San Diego, CA)

2 Gel electrophoresis apparatus (Novex Xcell II Mini Cell)

3 Gel running buffer: 25 mM Tris, 192 mM glycine, 0.1% SDS, pH 8.3.

4 Prestained protein standards

5 Transfer buffer: 25 mM Tris, 192 mM glycine, 20% methanol, pH 8.3, store at 4˚C.

6 Transfer apparatus such as Mini Trans-Blot (Bio-Rad, Hercules, CA)

7 Nitrocellulose membrane, cut to the size of the gel

8 Blotting paper (0.33 mm)

9 Tris-buffered saline (TBS): 20 mM Tris base, 137 mM NaCl, final pH 7.6, and TBS with

0.1% Tween-20 added (TBST)

10 Blocking solution: 3% bovine serum albumin (BSA) in TBS

11 Primary incubation solution: 1:1000 dilution (v/v) of rabbit polyclonal antibody raised

against the deglycosylated C-terminal “link” glycopeptide (20) or against synthetic

pep-tides D4553 corresponding to a 14 amino acid segment in the mucin domain, and E20-14,

corresponding to the C-terminal 14 amino acids of the mucin domain (21) (see Fig 1) in

TBS and 0.1% BSA

12 Secondary incubation solution: 1:10,000 dilution of goat antirabbit IgG alkaline phos-phatase conjugate in TBS and 0.1% BSA

13 Alkaline phosphatase detection system: equilibration solution (100 mM Tris-HCl, 100 mM NaCl, 50 mM MgCl , pH 9.5; store at 4˚C), reaction solution (same as equilibration but

with the addition of 6.6 mg of 4-nitro blue tetrazolium chloride and 1.65 mg of

5-bromo-4-chloro-3-indolyl-phosphate), and stop solution (10 mM Tris-HCl, 1 mM EDTA, pH 8.0).

Nitro blue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolyl-phosphate (BCIP) are avail-able from Boehringer Mannheim at 100 and 50 µg/µL concentrations, respectively

2.6 The Role of Glycosylation

1 Tunicamycin (Sigma, St Louis, MO) in DMEM, filter sterilized

2 20 mM benzyl 2-acetamido-2-deoxy-α-D-galactopyranoside (benzyl-α-GalNAc) (Sigma), filter sterilized

3 Peptide-N4-(acetyl-ß-glycosaminyl) asparagine amidase (N-glycosidase F, EC 3.5.1.52)

(Boehringer Mannheim)

4 Nonidet P-40

5 N-acetylneuraminidase from Vibrio cholerae (EC 3.2.1.18), (Boehringer Mannheim).

6 Lysis buffer: 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, and 1X

Com-plete™ protease inhibitor cocktail (Boehringer Mannheim)

7 Protein A-Sepharose (Boehringer Mannheim)

8 Immunoprecipitation buffer: 20 mM Tris-HCl, pH 8.0, containing 0.1 M NaCl, 1 mM

Na2EDTA, 0.1 mM PMSF, with and without 0.5% Nonidet P-40.

9 Neuraminidase incubation buffer: 40 mM Tris-HCl, 4 mM CaCl2, pH 7.8

3 Methods

3.1 Synthesis of Rat Muc2 Domain Construct (pRMC)

Using Recombinant PCR

Figure 1 shows the general scheme of the synthesis of construct pRMC Two DNA

constructs are synthesized encoding the entire rat Muc2 signal peptide and the C-ter-minal 534 amino acids, respectively Each construct is synthesized to contain an

iden-tical region (Fig 1, shaded area) at which a single strand of one construct is able to

complement the other during the annealing cycle of the second PCR step (22),

result-ing in a recombinant product (pRMC) that can be cloned, usresult-ing the incorporated restriction sites, into a suitable vector such as pSVL The detailed procedure is as follows:

1 Synthesize DNA fragments to be joined via recombinant PCR The 5' fragment will

encode the 534 amino acids of the C-terminal end of rat Muc2 and a 3' SacI restriction site

for subcloning This domain of Muc2 can be detected by the antibodies anti-d-link, anti-D4553,

and anti-E20-14 (8,20), which span the length of the pRMC peptide product (Fig 1).

2 Make up 100 µL of PCR samples containing 10 µL of 10X polymerase buffer, 2.5 mM

MgCl2, 400 µM dNTPs, 200 ng of primers 1 and 2 or 3 and 4, 1 ng of template DNA (with only primers 3 and 4), and 5 U of Taq polymerase Perform a PCR program in a suitable

thermal cycler at 94˚C for 5 min, followed by 30 cycles at 94˚C for 1 min, 60˚C for 1 min, and 72˚C for 1 min, ending with an extension period of 72˚C for 7 min

3 Couple the signal peptide and RMuc2 PCR products Make up 100-µL reaction mixtures containing 10 µL of 10X polymerase buffer, 2.5 mM MgCl2, 400 µM dNTPs, and approx

50 ng of each PCR product as templates Denature the DNA in a thermal cycler for 1 min

at 94˚C, followed by a 5-min incubation at 55˚C to allow both templates to anneal Add 5

U of Taq polymerase, and incubate the reaction at 72˚C to allow extension of the linked

templates Add 2 µg each of primers 5 and 6, and continue the PCR with 30 cycles at 94˚C for 30 s, 50˚C for 1 min and 72˚C for 2 min, ending with a 72˚C extension for 7 min

4 Check the PCR product for size and purity by electrophoresis on a 1% agarose gel The construct may be verified by sequencing at this point or after insertion into a vector

3.2 Ligation of Construct to the Transfection Vector

TA cloning may be necessary if direct ligation is unsuccessful In cases where subcloning is impeded by incomplete restriction enzyme digestion, a specialized vec-tor containing 3' deoxythymidine (T) overhangs can be utilized to ligate the PCR prod-uct generated by Taq polymerase (which leaves complementary deoxyadenosine or A, overhangs) The ligated TA overhangs can then be digested without difficulty Alter-natively, it can be assumed from the beginning that TA cloning will be used, which would result in a small change in primer design Specifically, it would not be

neces-sary to incorporate SacI and XbaI sites into the beginning primers, because these sites

already exist in the TA cloning vector.

1 Using fresh recombinant PCR product (less than 1 d old), perform TA cloning as outlined

in the protocol of the supplier (Invitrogen) Pick isolated white colonies and check for the

correct insert by restriction digest of DNA minipreps with SacI and XbaI.

2 Add 5 µL of recombinant PCR product or positive TA clone DNA to 3 µL of H2O, 1 µL of

incubation buffer, and at least 1 U of SacI to a microfuge tube Incubate for 1 h in a 37˚ C water bath Add at least 1 U of XbaI to the reaction and incubate for a further hour at

37˚C Inactivate the enzymes according to the manufacturer’s instructions

3 Purify the SacI/XbaI restriction digest product We used electroelution from the agarose

fragment (19) in TAE buffer for 1 h followed by precipitation in 0.1 vol of 3 M sodium

acetate and 2 vol of ethanol Electrophorese on a 1% agarose gel and quantitate the sample Use sufficient quantities of pSVL and insert DNA to result in an approx 1:3 molar ratio, respectively Bring the ligation reaction to 8 µL with H2O, and then add 1 µL of the 10X ligation buffer supplied with the enzyme and 1 µL (1 U) of T4 DNA ligase Incubate the ligation reaction at 16˚C for 16 h

4 Thaw competent DH5α cells on ice, and to each 50-µL aliquot add 2 µL of the ligation reaction mixture (or H2O for mock-transformation control), stir gently with a pipet tip, and incubate the cells on ice for 30 min

5 Heat shock the reactions in a 37˚C water bath for 20 s, and then incubate them on ice for 2 min

6 Add 950 µL of LB to each reaction tube and incubate on a shaker at 37˚C for 1 h

7 Add 50 and 200 µL from each tube of transformed cells to warmed LB plus ampicillin plates Distribute the cells over the plate evenly with a sterile glass spreader, allow plates

to dry, and leave in a 37˚C convection incubator for 16 hr

8 After confirming that no growth has occurred on control plates of mock-transformed DH5α, pick at least five positive, isolated colonies Check DNA minipreps of these positive

colo-nies by restriction digestion with SacI and XbaI The positive clones may be sequenced at

this point to confirm that the correct sequence is contained within the plasmid

9 Prepare large-scale amounts of transfection-quality construct pRMC and control vector pSVL by resin purification such as with the Qiagen Maxiprep kit

3.3 Transfection

The transfection system uses the expression vector pSVL, which is ideally suited for transfection into COS cells owing to the production of T-antigen, which increases

pSVL expression (23).

1 Twenty-four hours before the transfection, seed COS cells into 3.5-cm tissue culture dishes at a density of 8 × 106 cells, as counted by a hemocytometer

2 Pilot experiments are necessary to establish the ideal ratio of DNA to Lipofectamine For this system, add 2 µg of pRMC or pSVL control to 100 µL of DMEM Add 10 µL of Lipofectamine to 100 µL of DMEM in a separate tube

3 Add the contents of the first tube containing DNA to the second tube containing Lipofectamine, and incubate the tubes at room temperature for 20 min While the incuba-tion is ongoing, remove the medium from all dishes of COS cells and wash with 1 mL of DMEM (containing no antibiotics or FBS) Drain the dishes thoroughly

4 Add 800 µL of DMEM to the DNA-Lipofectamine mixture, pipet to mix, and add to the dish of COS cells Leave all dishes in the tissue culture incubator at 37˚C for 5 h

5 Replace the mixture with 2 mL of DMEM plus 10% FBS (do not include antibiotics) Leave the dish in the tissue culture incubator for 43 h

6 Remove serum-containing DMEM and wash COS cells thoroughly with DMEM without serum Replace medium with 2 mL of DMEM without serum and incubate at 37˚C for another 24 h If it is critical to collect conditioned media from the entire incubation pe-riod, OptiMEM (Gibco-BRL) may be substituted for DMEM to avoid later problems as-sociated with SDS-PAGE involving FBS (e.g., distortion of lower mobility proteins during electrophoresis and antibody crossreactivity)

3.4 Harvesting of Transfected Cells

1 After establishing the success of transfection with a reporter system, remove COS cells transfected with pRMC and pSVL control vector from the incubator, collect all medium, and wash the cells with 1 mL of phosphate-buffered saline (PBS)

2 Add 60 µL of 2X Laemmli sample buffer to each dish, and quickly scrape the lysates into microfuge tubes The lysates may be stored at –70˚C at this time

3 Add PMSF to the conditioned medium to give a concentration of 0.1 mM, and

concen-trate using spin columns, such as Centricons, to attain a final volume of approx 100 µL Medium may be stored at –70˚C at this time

3.5 SDS-PAGE and Western Analysis

The transfected COS cells are harvested for separation of lysate components on polyacrylamide gels The percentage of polyacrylamide used will vary with the size of the expected product and the desired separation Most experiments in the present study were performed using 8% gels.

1 Boil the cell lysates for 3 min to reduce viscosity, divide each sample equally between two microfuge tubes, and add DTT to one of each pair of samples to give a final

concen-tration of 10 mM Boil the samples for a further 2 min, briefly spin to collect droplets, and

load each sample into a well of an 8% polyacrylamide gel

2 Add 15 µL of 2X Laemmli sample buffer to 15-µL aliquots of conditioned media (two samples for each vector transfected) and reduce one of each pair with DTT (final

concen-tration 10 mM) Boil, spin, and pipet samples into wells of an 8% polyacrylamide gel.

3 Add prestained molecular mass standards to one well and 2x Laemmli sample buffer to

any remaining empty wells (see Note 3).

4 Electrophorese at 125 V for 100 min, or until the bromophenol blue dye reaches the bot-tom of the gel

5 Transfer the separated proteins from the gel to a nitrocellulose membrane at a constant voltage of 100 V for 1 h using buffer conditions suitable for the transfer apparatus; the

Bio-Rad Mini Trans-blot requires buffer maintained at 4˚C The transfer setup is detailed

in ref 19.

6 Remove and submerge the membrane in blocking solution Incubate on a rotator at room temperature for 1 h

7 Wash the membrane five times in TBST at room temperature, 5 min per wash

8 Incubate the membrane for 16 h at 4˚C in primary incubation solution We have used the

anti-d-link, anti-D4553, and anti-E20-14 antibodies (see Subheading 2.5., step 11) in

our experiments, with equivalent results Wash the membrane as described in step 5.

9 Incubate the membrane for 1 h in secondary incubation solution Wash the membrane as

in step 5.

10 After a 3-min equilibration, develop the membrane in reaction solution containing NBT and BCIP until specific bands are visible Keep all membranes protected from light dur-ing the color development

11 Submerge the membrane in stop solution and dry on blotting paper Compare control and

experimental samples (Fig 2) for the appearance of immunospecific bands that increase

in mobility (i.e., decrease in size) upon treatment with DTT (see Note 4).

3.6 The Role of Glycosylation

3.6.1 Tunicamycin

1 Repeat Subheadings 3.1.–3.5 with the addition of 0.5 µg/mL of tunicamycin to the

DMEM used in Subheading 3.3 steps 5 and 6 Harvest and analyze cells and

condi-tioned media as outlined previously (see Note 5)

3.6.2 N -Glycosidase F

1 Repeat Subheadings 3.1.–3.3 inclusive.

2 Collect and concentrate the conditioned medium as described in Subheading 3.4., step 3.

Add 1 U of N-glycosidase F (or H2O as a control) to each 15-µL aliquot and incubate 16 h at

37˚C in a water bath Analyze by SDS-PAGE as described in Subheading 3.5., steps 2–10.

3 Wash the COS cells in PBS, scrape the cells into 500 µL of PBS and microfuge the suspension

at 4˚C to form a pellet Resuspend the pellet in 50 µL of 1% SDS, and boil 1 min to denature

4 Dilute the suspension with Nonidet P-40 to give final concentrations of SDS and Nonidet

P-40 of 0.1 and 0.5 %, respectively N-glycosidase F is deactivated by higher

concentra-tions of SDS

5 Add 1 U of N-glycosidase F to each cell lysate and medium sample (H2O to controls) and incubate for 16 h at 37˚C in a water bath

6 Vacuum-dry the samples and reconstitute in 60 µL of 2X Laemmli sample buffer

7 Analyze the conditioned media and cell lysates by SDS-PAGE and Western blotting (Fig

3) as described in Subheading 3.5 (see Note 6).

3.6.3 Benzyl- α -GalNAc

1 Repeat Subheadings 3.1.–3.5 with the addition of benzyl-α-GalNAc at a final

concen-tration of 2 mM to each test dish of COS cells Harvest and analyze cell lysates and

conditioned media as outlined previously (see Note 7).

3.6.4 N -Acetylneuraminidase

1 Repeat Subheadings 3.1.–3.3 inclusive.

2 Collect and concentrate the conditioned medium as described in Subheading 3.4., step 3.

Add 10 mU of N-acetylneuraminidase from V cholerae (or H O as a control) to each 50-µL

aliquot and incubate 16 h at 37˚C in a water bath Analyze by SDS-PAGE as described in

Subheading 3.5., steps 2–10.

3 Harvest COS cells in 200 µL of lysis buffer and microfuge at 4˚C to remove cell debris

4 Incubate the remaining supernatant with 4 µL of antibody (anti-D4553 was used in this study) for 1.5 h at 4˚C followed by a further 2.5-h incubation at the same temperature with the addition of 50 µL of Protein A-Sepharose Isolate the immunoprecipitates by centrifugation at 4˚C for 5 min followed by washing the resultant pellet three times with

500µL of immunoprecipitation buffer (the third wash without Nonidet P-40)

5 Analyze the conditioned media and cell immunoprecipitates by SDS-PAGE and Western

blotting (Fig 3) as described in Subheading 3.5 (see Note 8).

4 Notes

1 The procedures outlined above for specific domain expression in heterologous cells dem-onstrate that the C-terminal region of rat Muc2 can form disulfide-dependent dimers, and

that N-glycosylation plays a significant role in dimerization A general schematic

reflect-ing the results usreflect-ing these methods is shown in Fig 4 The reader is referred to

Perez-Vilar et al (25) for an earlier application of domain expression to show dimerization of

the C-terminal domain of porcine submaxillary mucin The domain approach was neces-sitated by the virtual impossibility of studying detailed structural changes in molecules as large and as viscous as typical full-length secretory mucins As has been true for the elucidation of vWF physiology and disease-associated mutants of vWF, the expression of individual mucin domains and their secretory fate holds promise of enlarging our under-standing of structure-function relationships of mucins in vivo

2 Since the mucin domain forms dimers, the present approach could be extended to include larger constructs and/or other domains, e.g., constructs encoding both C- and N-terminal domains, to test whether dimers can expand into larger S-S–linked oligomers or multimers Various embellishments are also possible, including the addition to constructs

of commercially available tag epitopes at selected areas of the domain and immunopre-cipitation of translated products with specific antiepitope antibodies Crosslinking agents could also be added to examine the possibility that nonmucin proteins bind to specific mucin domains The kinetics of posttranslational modifications of the domain and its secretion could be studied by performing pulse-chase experiments using radioactive pre-cursors added to transfected cells The strategic use of inhibitors during cell incubations has the potential to reveal information about the pre- and post-Golgi movement of mucin domains along the secretory pathway Finally, truncation or site-specific mutagenesis of the initial domain constructs could be introduced to explore the role of selected amino

acids, such as cysteines required for dimerization, or N-glycosylation sites involved in

mediating the correct folding for dimerization

3 We have noted some discrepancies in mobility versus size correlations of different com-mercial batches and sources of molecular mass standards, particularly in the range above

150 kDa Thus domain product sizes should be viewed as relative rather than absolute

4 Figure 2A,B shows the results of this protocol for cell lysates and media Nonspecific

bands may be present and can be eliminated from consideration using the pSVL vector

control lanes for comparison (Fig 2A,B, lanes 1 and 3) The sizes of specific bands

reported herein are given with reference to Bio-Rad prestained molecular mass standards (Estimated dimer sizes are higher, by up to 30 kDa using Novex standards, whereas

mo-nomeric size approximations remain unchanged.) In cell lysates (Fig 2A), an

immuno-specific band is seen at approx 150 kDa (lane 2), which is replaced with an 88-kDa band

on thiol reduction (lane 4) These represent the mucin domain dimer and reduced mono-mer, respectively In nonreduced samples there is also a band at 73 kDa The identity of this species is not yet clear, but may represent misfolded or immature monomeric domain

products It converts to 88 kDa with reduction (Fig 2A, lane 4) In the cell medium (Fig.

2B), lane 2 shows a secreted immunospecific product with a mobility equivalent to about

165 kDa (dimer) that disappears on reduction, leaving a 100-kDa species (lane 4) (mer) Note that the apparent molecular masses of the secreted (medium) dimer and mono-mer are larger than the corresponding cell lysate species (i.e., 165 vs 150 for the dimono-mer,

100 vs 88 for the reduced monomer) The explanation lies in a late glycosylation step, as

noted in Subheading 3.6.

5 As seen in Fig 2C, cells treated with tunicamycin exhibit a 45-kDa product (presumably

a highly folded, nonglycosylated monomer) under nonreducing conditions (lane 1), and a 62-kDa band on reduction (lane 2) The calculated size of the expected translation prod-uct is 59 kDa (534 amino acids), which corresponds well with the observed 62-kDa band

No dimer form appears, indicating a prerequisite for N-glycosylation in domain

dimeriza-tion Because no bands were observed in the medium (not presented), it is clear that the nonglycosylated product cannot be secreted

6 Results are shown in Fig 3A, in which a mobility shift of the untreated control (lane 1)

from 165 kDa to a band at 145 kDa (lane 2) is observed after treatment with

N-glycosi-dase F

Fig 2 SDS PAGE analysis of COS-1 cell translation products after transfection with pRMC, as

outlined in Subheading 3.5 Western blots were performed using antibody to the deglycosylated

“link” glycopeptide “+” lanes refer to samples reduced with 10 mM DTT (A) Thirty

microli-ters of pRMC- (lanes 2 and 4) and pSVL control-transfected (lanes 1 and 3) cell lysates

Reduction produces a shift in mobility from a 150-kDa dimer to an 88-kDa monomer (B) A

similar shift, but of larger species, is seen using 30-µL aliquots of concentrated conditioned media from pRMC- (lanes 2 and 4) and pSVL-transfected (lanes 1 and 3) COS-1 cells DTT

reduction of samples (denoted +) causes a shift from 165 to 100 kDa (C) Culture of COS-1

cells in 0.5 µL/mL of tunicamycin followed by SDS-PAGE analysis of the cell lysates reveals

a highly mobile band, presumably tightly folded, at 45 kDa (lane 1), which yields a 62-kDa deglycosylated monomer on reduction No dimer product is detected in lysates, and no mono-mer or dimono-mer is detected in conditioned medium (not shown)