Dimerization of Domains of Mucin 143
143
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 monomers 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-
144 Bell and Forstner
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, MgCl
2
, 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'.
Dimerization of Domains of Mucin 145
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.
146 Bell and Forstner
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% CO
2

.
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

2
, pH 9.5; store at 4˚C), reaction solution (same as equilibration but
Dimerization of Domains of Mucin 147
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-N
4
-(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
Na
2
EDTA, 0.1 mM PMSF, with and without 0.5% Nonidet P-40.
9. Neuraminidase incubation buffer: 40 mM Tris-HCl, 4 mM CaCl
2
, 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, using 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
MgCl
2
, 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 MgCl
2
, 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.