Intracellular Processing of Mucin Precursors 249
249
21
Mucin Precursors
Identification and Analysis of Their Intracellular Processing
Alexandra W. C. Einerhand, B. Jan-Willem Van Klinken,
Hans A. Büller, and Jan Dekker
1. Introduction
MUC-type mucins are generally very large glycoproteins. They are encoded by
very large mRNAs, and possess polypeptides between 200 and more than 900 kDa (1).
The only notable exception is MUC7, which is considerably smaller, i.e. the polypep-
tide is only 39 kDa (1). Without exception however, mucins are very heavily O-
glycosylated: Up to 50-80% of their molecular mass is due to O-glycosylation (1,2).
Moreover, potential N-glycosylation sites are found in virtually all mucin sequences,
and in several MUCs N-glycosylation is actually demonstrated (1,2). Human MUC2
for instance contains 30 potential N-glycosylation sites, and if these are all used, the
N-glycans together would constitute a molecular mass of about 60 kDa. It is only the
very large size of the mature mucins, that makes the amount of N-glycosylation seem
insignificant (3). Generally, the sizes of the mature mucins are difficult to estimate;
The approximations run from 1 to 20 MDa for single mucin molecules, which ham-
pers many forms of biochemical analysis (3). Also, the extensive glycosylation of
mucins results in an intrinsically very heterogeneous population of mature mucins.
The detection of mucin precursors forms an attractive alternative to assess the
expression of specific mucins and to quantify mucin synthesis. Each precursor of the
MUC-type mucins can be identified by immunoprecipitation using specific anti-mucin
polypeptide antibodies (see Chapter 20). Very importantly, each of these precursors
can be identified on reducing SDS-PAGE by its distinct molecular mass (3–5). Thus,
immunoprecipitation in combination with sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) can be used to detect expression of individual MUC-
type mucins with high specificity in homogenates of tissue or cell lines. The mucin
precursor bands, recognizable on SDS-PAGE, can be quantified as sensitive measures
of mucin biosynthesis (see Chapter 6).
From:
Methods in Molecular Biology, Vol. 125: Glycoprotein Methods and Protocols: The Mucins
Edited by: A. Corfield © Humana Press Inc., Totowa, NJ
250 Einerhand et al.
Biochemically and cell biologically, MUC-type mucin precursors can be recog-
nized by a number of characteristics, which will help in their identification (2,3). Like
any glycoprotein, the MUC polypeptide is synthesized at the rough endoplasmic reticu-
lum (RER) and cotranslationally N-glycosylated. The product of this initial stage of
biosynthesis will be referred to as the mucin precursor. Then, the precursors will
oligomerize through formation of disulfide bonds, and be transported to the Golgi
apparatus, where they will be fully O-glycosylated and sulfated, as many of the O-
glycans of mucins contain terminal sulfate (see Chapter 17). Mucins that have com-
pleted synthesis are referred to as mature mucins.
In this Chapter, we focus on the identification of each of the known MUC-type
mucin precursors by immunoprecipitation using antipeptide antibodies. Moreover, a
number of biochemical and cell biological assays will be described which establish
the presence in the RER of each alleged MUC-type mucin precursor. These assays are
based on the following characteristics of the mucin precursors (1–3): (1) The pre-
cursors contain only high mannose N-glycans, (2) Most precursors form, over time,
disulfide-linked dimers within the RER, (3) O-glycosylation of the precursors, and
conversion of the N-linked glycans to complex N-glycans, occurs only after their trans-
port to the Golgi apparatus, and (4) A clear precursor/product relationship exists, as a
result of the conversion over time of the precursors into their cognate mature mucins.
The described methods will help researchers in the field to recognize and quantify the
precursors of the known MUC-type mucins, and we will provide appropriate control
experiments to verify the specificity of each of these procedures. Moreover, these
methods will help to allocate previously unidentified mucin precursors.
2. Materials
1. Source of mucin-producing cells, such as biopsies, tissue explants, or cell lines, which are
cultured as described in Chapter 18.
2. Radioactively labeled essential amino acids (Amersham, Little Chalfont, Bucking-
hamshire, UK), described in detail in Chapter 19:
a.
L
-(
35
S)methionine/(
35
S)cysteine (Pro-Mix™).
b.
L
-(
3
H)threonine.
3. Media (Gibco/BRL, Gaitersburg MD) for metabolic pulse-labeling and chase incubations,
as described in detail in Chapter 19.
4. Homogenization buffer for immunoprecipitation, as described in Chapter 20.
5. Glass/Teflon tissue homogenizer, 5 mL model (Potter/Elvehjem homogenizer).
6. Anti-mucin antisera directed against the mucin-polypeptide of interest (see Chapter 20,
Table 1).
7. Protein A-containing carrier to precipitate immunocomplexes, as described in Chapter 20.
8. ImmunoMix, as described in Chapter 20.
9. PBS: 10-fold diluted.
10. SDS-PAGE gels: 4% polyacrylamide running gels with 3% polyacrylamide stacking gel,
as described in Chapter 20.
11. SDS-PAGE sample buffer containing 1% SDS and 5% (v/v) 2-mercaptoethanol.
12. SDS-PAGE sample buffer containing 1% SDS, without reducing agent.
13. 10% (v/v) acetic acid/10% (v/v) methanol in water.
14. Schiff’s reagent for PAS staining (Sigma, St. Louis MO).
Intracellular Processing of Mucin Precursors 251
15. Amplify™ (Amersham).
16. X-ray film (Biomax-MR, Kodak, Rochester, NY).
17. Brefeldin A (BFA), stock solution, 1 mg/mL in water.
18. Tunicamycin (Calbiochem, La Jolla CA), stock solution, 1 mg/mL in 10 mM NaOH in water.
19. Carbonyl cyanide M-chlorophenylhydrazone (CCCP, Sigma), stock solution, 1 mM in
ethanol.
20. Endoglycosidase H (Endo H, New England Biolabs, Beverly MA), 500,000 U/mL.
21. 10-times concentrated Endo H-buffer (New England Biolabs), containing 0.5 M sodium
citrate (pH 5.5).
22. Peptide:N-glycosidase F (PNGase F, New England Biolabs), 1,000,000 U/mL.
23. 10-times concentrated PNGase F-buffer (New England Biolabs), containing 0.5 M sodium
phosphate (pH 7.5).
24. Nonidet-40 (New England Biolabs), 10% in water.
25. 10-times concentrated denaturing buffer (New England Biolabs), containing 5% SDS and
10% 2-mercaptoethanol.
26. Dolichos biflorus-agglutinin (DBA) Sepharose CL-4B beads (Sigma).
27. DBA column buffer: PBS (pH 7.2), supplemented with 1% (v/v) Triton X-100, 1 mM
phenylmethylsulfonyl fluoride, 50 µg/mL pepstatin A, 25 µg/mL leupeptin, 1% (w/v)
BSA, 10 mM iodoacetamide, and 0.1% NaN
3
.
28. N-acetyl-Galactosamine (GalNAc), 100 mM solution in the above mentioned DBA col-
umn buffer.
29. Freunds complete adjuvant (Difco, Detroit MI,).
3. Methods (Note 1)
3.1. Identification of the Precursors of MUC-Type Mucins
by Their Distinct Molecular Masses Through Metabolic Labeling
and Immunoprecipitation (Note 1)
1. Metabolically pulse-label the mucin-producing tissue or cells with radiolabeled essential
amino acids (see Chapter 19).
2. Homogenize the samples and isolate the radiolabeled mucin precursor of interest by im-
munoprecipitation using specific antipolypeptide antibodies (see Chapter 20).
3. Analyze the immunoprecipitated mucin precursors on 4% SDS-PAGE using reducing
sample buffer.
4. Identify the mucin precursor according to its apparent molecular mass, using the appro-
priate molecular mass markers and/or control samples (see Notes 2–6).
3.2. Relation of the Mucin Precursor to its Mature Form Revealed
by Pulse/Chase Experiments (Notes 1, 7, and 8)
1. Metabolically pulse-label seven samples of mucin-producing tissue or cells using radio-
labeled essential amino acids, as described in Chapter 19. Immediately homogenize one
sample after pulse-labeling. The pulse-medium is discarded.
2. Chase-incubate the remaining six tissue or cell samples, homogenize one sample after 1,
2, 3, 4, 5, and 6 h, respectively, of chase incubation, and isolate the media of each respec-
tive chase sample.
3. Isolate the radiolabeled mucin of interest from the seven homogenates and the six media,
respectively, by immunoprecipitation using antipolypeptide antibodies (see Note 8).
4. Analyze the immunoprecipitated mucin precursors on 4% SDS-PAGE using reducing
sample buffer and the appropriate molecular mass markers (see Notes 2–5).
252 Einerhand et al.
5. PAS-stain the gels to reveal the position of the mature mucins. Prepare fluorographs of
the gels using Amplify and X-ray film.
6. Analyze the kinetics of disappearance of the precursor and the appearance of the mature
mucin, and the appearance of the mature mucin in the medium (see Note 9).
3.3. Identification of the Mucin Precursors
as RER-Localized Proteins (
see
Note 1)
3.3.1. Inhibition of Vesicular RER-to-Golgi Transport (
see
Note 10)
3.3.1.1. I
NHIBITION OF
V
ESICULAR
RER-
TO
-G
OLGI
T
RANSPORT
BY
B
REFELDIN
A (BFA) (
SEE
N
OTE
11)
1. Treat seven samples of mucin-producing tissue or cells with BFA for 30 min under nor-
mal culturing conditions; 10 µg/mL for tissue, 0.1–2 µg/mL for cell lines (see Note 12).
2. Metabolically pulse-label the tissue or cells by radiolabeled essential amino acids, as described
in Chapter 19 (see Note 12). Homogenize one sample immediately after the pulse-labeling.
3. Chase-incubate the six remaining samples of the tissue or cells in continued presence of
BFA (identical concentrations as above), chase the samples for 1, 2, 3, 4, 5, and 6 h,
respectively. Homogenize each sample immediately after its respective chase incubation.
Also isolate and homogenize the media of the chase incubations.
4. Isolate the radiolabeled mucin precursor of interest from the homogenates and media by
immunoprecipitation using anti-polypeptide antibodies (see Chapter 20).
5. Analyze the immunoprecipitated mucin precursors on 4% SDS-PAGE using reducing
sample buffer. Compare the mobility of the mucin precursor bands in the BFA-treated
samples to the precursor bands in a pulse/chase experiment under normal conditions,
described in Subheading 3.2. (see Note 13). Perform DBA affinity chromatography to
study initial O-glycosylation (see Subheading 3.3.1.2.).
3.3.1.2. DBA A
FFINITY
C
HROMATOGRAPHY
TO
D
ETECT
I
NITIAL
O
-G
LYCOSYLATION
(
SEE
N
OTE
14)
1. Perform this entire procedure at 4°C. Prepare a DBA-Sepharose column, and wash exten-
sively with DBA column buffer.
2. Prepare a homogenate of [
35
S]amino acids-labeled tissue or cells in DBA column buffer
(Avoid the use of Tris). Apply this homogenate to the column, and elute with DBA col-
umn buffer. Collect the flow-through and store on ice.
3. Elute the terminal GalNAc-containing proteins from the column by 100 mM GalNAc in
DBA column buffer. Collect the eluate and keep on ice.
4. Immunoprecipitate the mucin precursor from the flow-through (containing the nonbound pro-
teins), and from the eluate (the GalNAc-containing proteins), as described in Chapter 20.
5. Analyze the presence of mucin precursor in both column fractions by reducing SDS-
PAGE (see Note 14).
3.3.1.3. I
NHIBITION OF
V
ESICULAR
RER-
TO
-G
OLGI
T
RANSPORT BY
CCCP (
SEE
N
OTE
15)
1. Metabolically pulse-label seven samples of mucin-producing tissue or cells by radio-
labeled essential amino acids, as described in Chapter 19. Homogenize one sample im-
mediately after the pulse-labeling. Discard the pulse-medium.
2. Chase-incubate the six remaining samples of the tissue or cells in the presence of CCCP
(tissue; 10 µg/mL, cells; 0.1–1 µM), and chase the samples for 1, 2, 3, 4, 5, and 6 h,
respectively. Homogenize each sample immediately after its respective chase incubation.
Also isolate and homogenize the media of the chase incubations.
Intracellular Processing of Mucin Precursors 253
3. Isolate the radiolabeled mucin precursor of interest from the homogenates and media by
immunoprecipitation using anti-polypeptide antibodies (see Chapter 20).
4. Analyze the immunoprecipitated mucin precursors on 4% SDS-PAGE using reducing
sample buffer. Compare the presence of the mucin precursor band in the homogenates to
the pulse/chase experiment under normal conditions, described in Subheading 3.2. (see
Note 15).
3.3.2. Analysis of Disulfide Bond Formation
of Mucin Precursors (
see
Notes 1 and 16)
1. Perform a pulse/chase experiment on mucin-producing tissue or cells, using [
35
S]amino
acids, as described in Subheading 3.2.
2. Immunoprecipitate the mucins, as described in Chapter 20, until the second of the two
wash steps in 10-fold diluted PBS.
3. Add the second aliquot (i.e. the last wash step) of 1 mL of 10-fold diluted PBS. Divide the
resuspended pellet into two equal aliquots of 500 µL in separate vials. Centrifuge these
two suspensions, and remove the buffer thoroughly.
4. Boil one pellet in sample buffer containing 5% 2-mercaptoethanol, and the duplicate pel-
let in sample buffer without reducing agent, and analyze these samples on SDS-PAGE
(see Notes 16–18).
3.3.3. Identification of Mucin Precursors
as High Mannose N-Glycan Containing Glycoproteins (
see
Note 1)
3.3.3.1. C
HARACTERIZATION OF
N
-G
LYCANS
BY
E
NDO
H
AND
PNG
ASE
D
IGESTION
(
SEE
N
OTE
19)
1. Metabolically pulse-label a sample of mucin-producing tissue or cells using [
35
S]amino
acids, as described in Chapter 19. Immediately homogenize the sample after pulse-labeling.
2. Isolate the radiolabeled mucin precursor of interest from the homogenate by immunopre-
cipitation using antipolypeptide antibodies (see Note 8).
3. Endo H digestion: Add 10 µL denaturing buffer to the S. aureus or protein A Sepharose
pellet, denature the sample for 5 min at 100°C. Cool to room temperature, add 1.2 µL
Endo H-buffer and 500 U Endo H to the sample, and incubate 1 h at 37°C.
4. PNGase F digestion: Add 10 µL denaturing buffer to the S. aureus or protein A Sepharose
pellet, denature the sample for 5 min at 100°C. Cool to room temperature, add 1.2 µL
PNGase F-buffer and 1000 U PNGase F to the sample, and incubate 1 h at 37°C.
5. Add reducing Lemmli sample buffer to the digestion mixtures, and analyze the mucin
precursors on 4% SDS-PAGE, using the appropriate molecular mass markers (see Notes
2–5, and 19).
3.3.3.2. I
NHIBITION OF
N
-G
LYCOSYLATION BY
T
UNICAMYCIN
(
SEE
N
OTES
20
AND
21)
1. Incubate one sample of mucin-producing tissue (50 µg/mL) or cells (5–20 µg/mL) for 3 h
with tunicamycin. Perform a control incubation under identical conditions.
2. Metabolically pulse-label both samples of mucin-producing tissue or cells using [
35
S]amino
acids, as described in Chapter 19. Immediately homogenize the samples after pulse-labeling.
3. Isolate the radiolabeled mucin precursor of interest from the homogenate by immunopre-
cipitation using antipolypeptide antibodies (see Note 8).
4. Analyze the mucin precursors on 4% SDS-PAGE using reducing sample buffer, using the
appropriate molecular mass markers (see Notes 2–5, and 20).
254 Einerhand et al.
3.4. Identification of Previously Unidentified Mucins Through
Detection of Their Precursors (
see
Notes 1, 22, and 23)
1. Isolate mucins using density centrifugation on CsCl/guanidinium·HCl gradients (see
Chapter 1). Thoroughly dialyze the isolated mucins against water.
2. Prepare a polyclonal antiserum in rabbits against the isolated mucins, using Freunds com-
plete adjuvant (8).
3. Metabolically pulse-label a sample of the mucin-producing tissue or cells from which the
mucin was isolated using [
35
S]amino acids, as described in Chapter 19. Immediately
homogenize the sample after pulse-labeling.
4. Isolate the radiolabeled mucin precursors from the homogenate by immunoprecipitation
using the polyclonal antiserum raised against the isolated mucins from this particular
source.
5. Analyze the mucin precursors on 4% SDS-PAGE using reducing sample buffer, using the
appropriate molecular mass markers (see Notes 2–5, 22, and 23).
4. Notes
1. Mucin precursors, because of their low abundance, can only be detected through meta-
bolic labeling. All methods described in this chapter are based on the methods to culture
tissue and cell lines (see Chapter 18), methods for metabolic labeling of the mucin pre-
cursors (see Chapter 19), and methods to specifically immunoprecipitate the mucin pre-
cursors (see Chapter 20).
2. Each precursor of the known human, rat or mouse MUC-type mucins can be distinguished
by its unique apparent molecular mass by SDS-PAGE. These data are summarized in
Table 1, which serves as a reference table to identify each known mucin precursor by
SDS-PAGE (see also Chapter 20 for listed molecular mass markers).
3. The distribution of MUC2-MUC6 based on detection by immunoprecipitation of their
respective precursors in gastrointestinal tissue and in cell lines are summarized in Table
2, which serves as reference table for mucin precursor synthesis in these organs and cells.
MUC1 is not included, as it is expressed in virtually all epithelia at low levels, i.e., its
expression is not tissue specific. Thus far, no data are available for other MUC-type mu-
cins, like MUC7 and MUC8.
4. The information on the molecular masses of the mucin precursors of the rat and mouse is
incomplete. However, the analogy to their human counterparts suggests that also in these
species a clear distinction can be made between the various mucin precursors based on
their molecular masses (Table 1).
5. Three cell lines are included for reference, which collectively produce the precursors of
MUC1 through MUC6 (Table 2). These cell lines are available at low costs through the
American Type Culture Collection (ATCC), and can be cultured as described in Chapter
19. The mucin precursors immunoprecipitated from these cell lines serve as excellent
markers to detect these respective mucin precursors in other human mucin-producing
sources. Moreover, immunoprecipitation of a particular mucin precursor from one of these
cell lines can provide the proper positive control for the immunoprecipitation procedure
of this particular mucin precursor from other sources.
6. MUCs often display genetic polymorphisms, which affect the number of tandemly
repeated amino acid sequences (1,2). Therefore, different individuals or cell lines may
biosynthesize precursors of a particular MUC gene of slightly variable lengths. When
immunoprecipitating precursors of a particular MUC, we sometimes observe distinct
interindividual differences in the molecular masses of these MUC precursors (Table 1).
Intracellular Processing of Mucin Precursors 255
This phenomenon is best documented for MUC1 in which the variation in molecular
mass of the precursors, produced from these different alleles, can be quite high: approx
160–310 kDa (6,18). However, for the other mucins the interindividual variations in the
molecular masses of the mucin precursors are quite small. That is, there is variation in the
Table 1
Apparent Molecular Masses of MUC-Type Mucin Precursors as
Determined by Immunoprecipitation and Reducing SDS-PAGE
Mucin Species Molecular massa References
MUC1 Human 160-400b 6
MUC2 Human 600b 4,5,7–9
Muc2 Mouse 600 10
rMuc2 Rat 600b 11
MUC3 Human 550b 4,5
MUC4 Human >900 4,5
MUC5AC Human 500 4,5,12
rMuc5AC Rat 300b 13–15
MUC5B Human 470 16,17
MUC6 Human 400 4,5
a The apparent molecular masses were estimated (expressed as kDa) after
immunoprecipitation by reducing SDS-PAGE.
bThese mucin precursors were shown to display interindividual heterogeneity,
leading to small variations in the apparent molecular masses on reducing SDS-
PAGE (see also Note 6).
Table 2
Distribution of Mucin Precursors in Human Gastrointestinal Tissues and
in Cell Lines as Determined by Metabolic Labeling and Immunoprecipitation
Tissue MUC2 MUC3 MUC4 MUC5AC MUC5B MUC6 Refs.
Stomach –a ––+++ – + 5,12
Duodenum ++ ++ – – – – 5
Jejunum ++ ++ – – – – 5
Ileum + ++ – – – – Unpub.b
Proximal colon +++ – + – + – 5
Distal colon +++ – + – + – 5,7
Gallbladder – + – – +++ – 16
LS174T +++ – – + + ++ 4
Caco–2 + ++ – – – – 4
A431 – NDc ++ ND ND ND Unpub.b
aPer organ or cell line we have indicated, in a semi-quantitative manner, the relative amounts of
mucin precursors: –, no expression ; +, detectable; ++, moderate expression; +++, strong expression.
bData on human ileum and A431 cells; Van Klinken, B. J. W., Büller, H. A., Dekker, J., and
Einerhand, A. W. C., unpublished.
cND, not determined.
256 Einerhand et al.
exact position of the precursor band on reducing SDS-PAGE, and sometimes double bands
can be observed in particular individuals. However, it is very important to note that these
variations in apparent molecular mass are relatively small, and that they will not lead to
any confusion regarding the identity of the immunoprecipitated mucin precursor.
7. For gastrointestinal tissues, over a period of up to 6 h, at 37°C under normal culture
conditions, all precursor will be processed to mature mucin. For cell lines, like LS174T,
this conversion may take longer (up to 24 h). In these experiments, the mature mucin can
be recognized on SDS-PAGE by its molecular weight, by PAS-staining, and often by its
heterogeneous appearance (smear). Also the position of the mature mucin on SDS-PAGE
can be revealed by metabolic labeling of duplicate tissue or cell samples with [
3
H ]galac-
tose or [
35
S]sulfate (see Chapters 19 and 20).
8. Pulse/chase experiments will only reveal the precursor/product relationship of the mucin
precursor and its cognate mature mucin if antibodies are used, which are able to recog-
nize both the precursor as well as the mature mucin. Therefore, the antibodies used in
these experiments must be able to recognize the mucin polypeptide in a manner indepen-
dent of O-glycosylation (extensively described in Chapter 20).
9. Precursors are never present in the medium. If however, a known precursor is found in the
medium, this can be taken as evidence of cell lysis during the experiment.
10. Inhibition of vesicular transport from the RER to the Golgi complex will lead to the accu-
mulation of mucin precursors in the RER. This accumulation is generally accepted as
evidence of RER localization (2).
11. BFA is a fungal metabolite, which inhibits the anterograde vesicular transport from the
RER to the Golgi complex, but not the retrograde transport of vesicles from the Golgi
complex to the RER. This results in accumulation of RER-localized protein in the RER,
but also in an enrichment within the RER with enzymes (like glycosyltransferases), which
are normally present in the cis-Golgi cisternae (2,22).
12. BFA is added to the medium during the 30 min period, which is used to deplete the
compound to be used as label. During the metabolic pulse-labeling the medium is not
changed, i.e., BFA remains present in the medium.
13. BFA will retain the mucin precursors in the RER. However, some enzymes involved in
initial O-glycosylation are redistributed to the RER in the presence of BFA, resulting in
initial O-glycosylation of these precursors. As a result, the precursor band will gradually
transform over time into a smear, slightly above the normal precursor position on reduc-
ing SDS-PAGE (14,20). As BFA is a potent inhibitor of secretion, none of these partly O-
glycosylated precursors will appear in the medium as secreted product (14,20,22).
14. DBA has a high affinity for terminal GalNac residues. Therefore, the binding of mucin
precursors to this lectin is taken as evidence that initial O-glycosidic α(1–0) GalNac ad-
dition to serine and threonine residues has occurred (14). This initial O-glycosylation will
occur in the presence of BFA, but not in the presence of CCCP (14,20).
15. CCCP inhibits the oxidative phosphorylation in the mitochondria, resulting in a sharp
drop in ATP levels in the cells. As the RER-to-Golgi transport is highly energy depen-
dent, the addition of CCCP will almost instantaneously inhibit this transport. The pres-
ence of CCCP will lead to accumulation of all mucin precursors, formed in the
pulse-labeling, in the RER (14,20). Never, add CCCP prior to or during the pulse-label-
ing, as this will inhibit nearly all protein synthesis (20).
16. Most mucin precursors form disulfide-bound dimers in the RER (14,20). When we per-
form a pulse/chase experiment on tissue or cells with radiolabeled amino acids, and ana-
lyze the immunoprecipitated mucin precursors on nonreducing SDS-PAGE, we are able
Intracellular Processing of Mucin Precursors 257
to demonstrate, next to the monomeric precursor band, a band with a much higher appar-
ent molecular mass than the monomeric mucin precursor. Reduction of parallel samples
will show that radioactivity in this high molecular weight band can be retrieved as the
monomeric mucin precursor on reducing SDS-PAGE, thus proving the dimerization of
the mucin precursor. The pulse sample usually only contains only monomeric precursors,
when analyzed on nonreducing SDS-PAGE. The precursor dimer appears during the
chase-period (typically within 30–60 min), and shows clear precursor/product relation-
ship with the monomeric precursor (14,20). It is advisable, to perform electrophoresis for
extended time to ensure that all putative dimers enter the running gel (20).
17. The application of BFA or CCCP in pulse/chase experiments, as described in Subhead-
ings 3.3.1.1. and 3.3.1.3., has no effect on the kinetics of oligomerization of the mucin
precursors (14,20).
18. Care should be taken not to run samples with reducing and nonreducing sample buffer
alongside on the same gel. The reduction of disulfide bonds is a fast process and the
reducing agents (typically 2-mercaptoethanol) are highly diffusible compounds. There-
fore, the risk exists that 2-mercaptoethanol will diffuse through the gel and reduce the
disulfide bonds in nonreduced samples. If these samples are run on the same gel, at least
one lane should be left unused in between.
19. N-linked glycans are added to RER-localized proteins in a conformation known as “high
mannose” N-glycans. Upon transport through the Golgi apparatus these N-glycans are
modified to “complex” N-glycans. The high mannose N-glycans can be split from the
polypeptide by the action of Endo H. This enzyme is however not capable to release the
complex form of these glycans. PNGase F releases all N-glycans, irrespective of their
conformation. Thus, if a mucin precursor is demonstrated to contain only high mannose
N-glycans this is taken as good evidence that this molecule is present within the RER (2–
4,7,11,13,14). The sensitivity of the mucin precursors towards these enzymes is demon-
strated on SDS-PAGE by an increase in mobility.
20. Tunicamycin inhibits the N-glycosylation completely, resulting in RER-localized
polypeptides without any glycosylation. When mucin precursors are immunoprecipitated
from tunicamycin-treated tissue or cells, this will yield the “naked” mucin polypeptide.
Upon reducing SDS-PAGE this will give the most accurate indication of the molecular
mass of the mucin polypeptide. Moreover, the position of this “naked” mucin polypeptide
on reducing SDS-PAGE is identical to the position of Endo H- or PNGase F-digested
mucin precursors, which can serve as appropriate evidence that the Endo H and/or PNGase
F digestions have removed all N-glycans from mucin precursors (e.g., ref. 13).
21. The inhibition of N-glycosylation by tunicamycin slows down the process of oligomer-
ization of the mucin precursors considerably (14,17,20). Since both N-glycosylation and
oligomerization take place in the RER, this lends additional experimental evidence to the
notion that the mucin precursors are actually present in the RER. To observe this inhibi-
tory effect on oligomerization, pulse/chase experiments must be performed in the con-
tinuous presence of tunicamycin.
22. The procedures to isolate mucins from any given source and to prepare polyclonal anti-
bodies against these intact mucins are described previously (8). Polyclonal antisera raised
following this protocol are always specific for the unique, non-O-glycosylated polypep-
tide regions of the mucins, which are expressed in this particular mucin source. It has
been demonstrated for many different tissues, that these antisera will be able to recognize
the mucin precursors in the respective tissue or cells in metabolic labeling experiments
(7–17). Thus, immunoprecipitation using these antisera on pulse-labeled tissue or cells
258 Einerhand et al.
will reveal which mucins are expressed in this particular mucin source. As each mucin precur-
sor can be identified by its unique mobility on reducing SDS-PAGE (Table 1), the identity of
the immunoprecipitated mucin precursors can be established (see also Chapter 20).
23. An excellent example of the successful application of this method is the study of human
gallbladder mucin. Human gallbladder mucin was isolated using CsCl/guanidinium.HCl
density gradients, a polyclonal antiserum was raised, and the expression of mucin precur-
sors was studied by metabolic labeling experiments (17). It appeared that the antiserum
recognized only one mucin precursor with an apparent molecular mass of 470 kDa. By
comparative immunoprecipitation analysis it appeared that this mucin precursor was not
identical to the precursor of MUC1, 2, 3, 4, 5AC, 6, or 7, leading us to conclude that
gallbladder mucin was either a novel mucin or MUC5B (4,21). Finally, using specific
monoclonal antibodies to immunoprecipitate MUC5B precursor, we were able to show
that the major human gallbladder mucin was identical to MUC5B (16).
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