JOURNAL OF PATHOLOGY, VOL.

180: 8-17 (1996)

REVIEW ARTICLE

MUCUS GLYCOPROTEINS AND THEIR ROLE IN

COLORECTAL DISEASE
A. P . CORFIELD AND B. F. WARREN

Depurtment qf Medicine Laboratories, Bristol Royal Infirmary, Bristol, BS2 8H W, U.K. and
Department of Cellular Pathology, John Radclife Hospital, Headington, Oxford, OX3 SOU, U K.

SUMMARY
In this review, the nature and impact of progress in the study of mucins is outlined, emphasizing the current understanding of the
structure and physiological function of these molecules in the colorectum. The use of new methods for preparation and separation has
led to improvements in the analysis of mucins; these are detailed, as are their difficulties and pitfalls. Results obtained with these methods
are correlated with long-established histochemical techniques and the use of chemical, lectin, and antibody reagents for general and
specific detection of mucins in all procedures is described. Improvements in the detection and analysis of mucins in biopsy-size tissue
samples and in larger numbers of individual clinical cases have now permitted a much wider approach to the pathological evaluation of
mucin biology and progress with these techniques is outlined. The significance of the discovery of a family of much genes is presented
and new concepts of mucin structure resulting from these studies are described. Bacterial degradation of the mucus layer at the surface
of the colorectal mucosa is considered in line with the homeostatic relationship with mucosal mucin synthesis. Finally, the implications
of abnormal mucins in colorectal disease are considered in the light of recent methodological advances.
KEY woRDs-mucin; blood group antigens; ulcerative colitis; Crohn's disease; colorectal cancer; metabolic labelling; MUC genes;
glycosyltransferases

INTRODUCTION tandem repeat sequences and which carry the 0-linked

Mucus glycoproteins-or mucins-are present at all
mucosal surfaces. Recent methodological advances have
led to a greater understanding of their importance and
biological roles in mucosal defence, cellular signalling
mechanisms, and cell-cell interactions. They are discrete
molecular entities which occur as both secreted and
cell surface forms, each having distinct functional and
structural characteristics. In secreted ('classical') form,
they are part of the total mucosal secretion termed
'mucus', giving it the viscoelastic properties associated
with the mechanical aspects of mucosal defence.'-' The
cell surface, membrane-located mucins are smaller, are
not present as oligomers, and do not form extracellular
gels,*.9 but form part of the glycocalyx. Many of the
functions of these cell surface glycoproteins are associ-
ated with cellular signalling mechanisms and cell-cell
interactions.'O l 5
Mucins are composed of 'subunits' or monomers,
containing around 20 per cent protein and 80 per cent
carbohydrate (dry weight). The polypeptide component
is characteristically rich in serine and threonine, the
amino acid partners in the 0-glycosidic linkage to
N-acetylgalactosamine in the many and varied oligosac-
c h a r i d e ~ . ' . ~ . ~The
. ' ~ .peptide
'~ structure is coded for by a
number of genes, the MUC Two types of
protein domain are found. One contains the majority of
the serine and threonine residues, which usually occur as
Addressee for correspondence. Dr B. F Warren, Department of
Cellular Pathology, John Radcliffe Hospital, Headington, Oxford,
OX3 9DU, U.K.
CCC 0022-3417/96/090008-10
01996 by John Wiley & Sons, Ltd.
oligosaccharide chains. In contrast, the second domain
is relatively carbohydrate-free and contains cysteine
residues which are involved in the formation of inter-
and intra-molecular disulphide bridges. Only one of the
mucin genes discovered so far codes for a membrane
mucin (MUC1).19
Oligosaccharide chains form the main part of the dry
weight of the mucins. Most are 0-linked, although a
small proportion of N-linked chains have been identified
and are implicated in subcellular compartmentalization
during the early stages of biosynthesis. The 0-linked
chains are made up of a linkage or core region, an
extension or backbone section (which may or may not
be present), and peripheral structures.20These terminal
additions include blood group and other antigens
with fucose, sialic acids, and sulphate residues, and-a
feature largely confined to the large intestine-0-
acetylation of sialic acids with two or more 0-acetyl
ester^.^'-^^ Variation in the oligosaccharide structures
is related to the nature of the tissue, its stage of
development, and the presence of disease.
HOW ARE MUCINS STUDIED?
Preparation of mucins
The development of methods for isolating mucins free
of contaminants has been vital in revealing the differ-
ences between intact and degraded mucins.1,6,16,24,26
Mucins can be separated on the basis of their very large
molecular weight, buoyant density, and overall negative
charge.
Received 7 February 1995
Accepted 8 February 1996
 MUCUS GLYCOPROTEINS AND THEIR ROLE IN COLORECTAL DISEASE
The secreted mucins appear at mucosal surfaces in
solution (sol) or as viscoelastic gels. Conversion from gel
to sol, as a result of mechanical, chemical, and enzym-
atic actions, is a dynamic and continuous p r o ~ e s s . ~ , ~ are rendered inactive on dissociation. 16,38,39 As a result,
However, some secreted mucins occur in sol form alone,
as in salivary mucins. Demonstration of a sol form has
been difficult to study in the case of mucosae with an
adherent gel. Extracellular mucus contains many other
proteins and lipids in normal physiological complexes;
preparation of mucins from mucus or mucosal tissue
may also result in the formation of similar complexes
in addition to those with nucleic acids. Separation of
mucins from other molecules in such mixtures requires
the use of dissociating agents such as guanidine hydro-
chloride or urea.lJ6 Insoluble mucins resistant to the
action of these solvents have been found in the small and
large intestine; reduction and alkylation are required
before these become soluble.27
Separation of mucins
Fractionation on the basis of buoyant density is
achieved by density gradient centrifugation in caesium
salts; this remains the most valuable procedure for
isolation and identification. Gel filtration, separating on
the basis of molecular size, is used to obtain mucin-rich
fractions, as few other molecules have molecular weights
in the same high range. A combination of density
gradient centrifugation and gel filtration is best used to
obtain purified native mucins and to characterize much
fragments obtained after reduction and alkylation or
proteolytic digestion.1,6,7,16
SDS-polyacrylamide gel electrophoresis systems have
also been widely used to analyse mucins. Problems,
however, are encountered due to the inability of many
mucins to penetrate the gels. Studies on the first step
in mucin biosynthesis-polypeptide formation-have
shown that even at this early stage, mucin precursors
have molecular weights above 200 kD.8,19,28Fraction-
ation of mature or partially completed much molecules
with 70-80 per cent carbohydrate means that in many
experiments it is impossible to judge whether all or only
part of the mucin sample loaded has entered the gels.
The recent application of agarose gel electrophoresis has
offered a dramatic improvement (Fig. 1); this is the
method of choice for the immediate f ~ t u r e . ~ ~ . ~ ~ useful information on the mucin oligosaccharide
Neutral and charged mucins have been successfully
fractionated using ion-exchange chromatography31p34
and the continued refinement of this technique through
more sensitive detection methods makes it an import-
ant part of current analytical procedure for the
identification of multiple mucin glycoforms.
Electron microscopy has shown the linear nature of
the majority of these molecules and has provided evi-
dence for the presence of ‘naked’ and carbohydrate
‘protected’ peptide domain^.^^-^^ It has also directly
confirmed the consequent decrease in size after
reduction to subunits or glycopolypeptides.
During the development of these separation methods,
it became clear that degradation of the intact mucins
occurs. The extraction ‘cocktails’ which finally evolved
include proteinase inhibitors to prevent attack of the
9
peptide region forming inter-subunit disulphide bridges
and dissociating salts, such as guanidine, which also
have a protective effect, as many degradative enzymes
a pattern of ‘native’ (often insoluble) mucins, mucin sub-
units, and proteolytically degraded ‘glycopolypeptides’
has been identified. In spite of the reductions in size due
to disulphide bond and proteolytic cleavage, the result-
ing subunits and glycopolypeptides are still very large,
with molecular weights typically in the range of 500 kD
and above.
Histologicat detection of mucins-histochemistry
A variety of histochemical reagents have been
employed, as described below, and these have often
given leads for the biochemical study of m u c i n ~ . ~ ~ . ~
The negative charge in mucins is due to sialic acids
and sulphate, which determine the physicochemical
properties such as viscosity, elasticity, and gel forma-
tion. Neutral mucins tend to contain low levels of these
two components, with a higher content of neutral sugars
such as galactose, fucose, or the N-acetylhexosamines.
Mucin carbohydrate has been widely detected using
the periodic acid/Schiff (PAS) stain, often in combi-
nation with Alcian blue. The mild PAS stain, where the
concentration of periodate is reduced from approxi-
mately 45 mM to 1 mM (hence mild periodate), has been
used in combination with saponification for the detec-
tion of 0-acetylated sialic acids, a major feature of
colonic mucins.24,4144Studies of 0-acetylation detected
by the mPAS method have identified three patterns of
phenotypic expression consistent with a single poly-
morphic autosomal gene for the 0-acetyl transferase.&
These are uniform positive staining for the homozygous
recessive genotype (OAT - /OAT - ), uniform negative
staining for the homozygous dominant genotype
(OAT+/OATi), and a negative mucosa scattered with
positive discordant crypts representing the heterozygotes
(OAT+/OAT - ). Saponification to remove 0-acetyl
esters has not been employed in the majority of studies
on colonic mucosal tissues. It has been used tradition-
ally with the mild PAS methods but has not been
properly studied in relation to staining with many anti-
bodies currently available. This may provide further
side-chains.
Sulphated and non-sulphated sialomucins are
detected histochemically using high iron diamine/Alcian
b 1 ~ e . 2 4 , ~ ~ ,This
~ 5 , ~technique
6 has fallen into disrepute,
mainly due to poor control. Success with this method
depends on the use of fresh reagents prepared at the
correct pH, as incorrect preparation leads to a complete
absence of positivity, rendering interpretation without
control tissue impossible. It is essential to use a com-
posite block containing, for example, jejunum, gastric
body, and right colon to show both positive and
negative staining.47 If controlled properly, it is still a
valuable, if somewhat toxic, technique.48New, non-toxic
techniques may offer some competition as the prime
histochemical methods for these substances and deserve
full evaluation.
10 A. P. CORFlELD AND B. F. WARREN

A) SDS-POLYACRYLAMIDE B) AGAROSE GEL ELECTROPHORESIS

GEL ELECROPHORESIS
ulcerative colitis mntml/anti-muanantibody

Fig. 1-Electrophoresis of human colorectal mucins purified as the excluded (Vo) fraction after gel filtration on Sepharose CL 2B.
(A) SDS-polyacrylamide gel electrophoresis on 3 per cent separating gel, electroblotting, and detection with wheat germ agglutinin-horseradish
peroxidase conjugate/diaminobenzidine staining. (B) Agarose gel electrophoresis on 1 per cent gels, vacuum blotting, and staining with the same
detection system for an ulcerative colitis mucin and staining with monoclonal antibody PR3A5” to normal colonic mucin fractions of density
1.41 g/ml (1) and 1.52 dml(2) from a CsCl density gradient. The migration of markers on these gels is indicated for IgM (I), myosin (M), and
phosphorylase B (P). L is the position of loading slots on the agarose gels

Histological detection of mucins-lectin histochemistry
Lectin histochemistry has been used for many years to
subdivide mucins expressing specific sugar side-chains.
Lectins are protein agglutinins which bind to particular
carbohydrate structures and many examples have been
studied. Care must be taken in using lectins, as they may
bind to a number of related structures-this is the case
for the peanut lectin, which has been claimed to be
specific for the T-antigen (Galpl-3GalNAc). The lectin
binds to pGal with highest affinity for Gal/l1-3GalNAc,
but will also bind to Galpl-4GlcNAc, especially if this
is present in high concentration or in clusters.49 In
addition, individual lectins may be sensitive to different
configurations of the same sugar, as has been shown for
the binding of Ulex europeus lectin 1 (UEA-1) to alpha-
linked fucose in blood group substance H or Lewis
variants. There are important regional variations in
lectin binding throughout the normal colon. Examples
relating to blood group antigens are given below where
antibodies have also been used to probe this phenom-
enon. Furthermore, lectin binding, as well as staining
with mPAS and mucin antibodies, may depend on sialic
acid residues. The presence of sialic acids alone may
block the binding of some lectins, but in addition the
0-acetylation normally present in the colon of 0-acetyl
transferase-positive (OAT + /OAT +) individuals must be
removed by mild saponification before neuraminidase
can be effectively used to eliminate the sialic acids
themselves. The presence of 0-acetylation itself
may block the binding of some lectins. In colorectal
carcinoma and in OAT - /OAT - individuals, no
0-acetylated form of sialomucin is present.
Histological detection of mucins-immunohistochemistry
Immunohistochemistry has been widely used with
antibodies specific for blood group and other related
carbohydrate antigens, mucin peptide core antigens, and
a variety of other mucin-specific epitopes. Reactivity in
the proximal colon for the blood group antigens is
influenced by both blood group and secretor status. In
the adult large bowel, ABH and Leb are found in the
 MUCUS GLYCOPROTEINS AND THEIR ROLE IN COLORECTAL DISEASE
proximal colon up to the level of the transverse colon.
The goblet cell mucin in the distal colon and rectum
does not normally express blood group antigens, while
Lea (secretor gene-independent) occurs throughout the
colon. In neoplasia, aberrant ABH expression occurs,
with loss in proximal tumours and re-expression in distal
cancers.50
Several shortened or truncated carbohydrate epitopes
have been proposed as cancer markers; these result from
incomplete or aberrant glyc~sylation~' and include the
Tn (GalNAc), sialosyl-Tn, and T (Galp-3GalNAc) anti-
gens. The T-antigen was detected initially using peanut
lectin, but did not bind anti-T antibodies, and was later
shown to be due to another related epitope which also
binds the l e ~ t i n .Tn
~ ~antigen
. ~ ~ is not present in normal
colorectal mucosa but appears in adenomas and carci-
nomas; its specificity is limited, however, as it is also
present in benign metaplastic polyps and in the transi-
tional mucosa adjacent to most colorectal masses.54
Sialosyl-Tn is a natural end-product of glycosylation,
as the disaccharide cannot be elongated. It was also
thought to be a cancer marker. However, the specificity
of some anti-sialosyl-Tn antibodies remains doubtful,
and the confirmed anti-sialosyl-Tn specific antibody
TKH2 reveals expression in normal mucosa after
saponification, showing a similar distribution to the
mPAS stain,55revealing a block by the normal sialic acid
0-acetylation.
Other antibodies including MMMl 7,56PR3A5,57and
3NM,58which detect sialic acids largely in 0-acetylated
form, have been used to demonstrate the changes in
these sialic acids in normal and neoplastic colorectal
mucosa. The small intestinal mucin antibody (SIMA),
binding to sialidase-resistant sialic acids (not yet
defined), has also been employed in the same way.59
These are reagents of great value in parallel histo-
chemical and biochemical studies.
Detection of sulphated epitopes in colonic mucins has
been reported with a novel antibody, 91.9H.60This has
proved useful in the demonstration of sulphate loss in
the adenoma-carcinoma sequence.61The epitope recog-
nized by this antibody has not been defined in detail and
probably includes sialic acid. In addition, some discrep-
ancies remain regarding the cellular location of antibody
staining compared with the HID stain and quantitative
binding of the antibody relative to metabolic labelling of
mucin with 35S-sulphate.
Many antibodies recognizing mucin peptide backbone
structures have been described and used to demonstrate
abnormal glycosylation patterns in disease. These
include the membrane-bound MUCl mucin peptides62
and the MUC genes for secreted m ~ c i n sThe. ~ ~majority
of these are directed against the tandem repeat sequence
in the various MUC genes and would therefore normally
be blocked by 0-glycosylation. However, binding to
mature mucins has been described with some anti-
bodies and the structurai relationship of these epitopes
to glycosylated and 'naked' peptides is not fully
explained. A series of antibodies have been reported
which recognize conformational, non-tandem repeat
peptide epitopes, the MI antigens, in human gastric
mucins. These antigens are also expressed in fetal and
I1
precancerous colon, but not in the normal No
MUC gene identity has been reported for these epitopes
to date. Further characterization of these antibody-
peptide interactions will be of particular use with regard
to peptide-oligosaccharide structure in mucins and
for confirmation of MUC gene peptide expression in
conjunction with in situ hybridization experiments.
Histological detection of mucins-direct measurement
Direct measurement of the adherent gel layer at the
surface of the gastrointestinal mucosa is a valuable,
physiologically significant means of assessing the status
of the mucosal defensive barrier. Using fresh, non-
treated mucosal tissue, the thickness of the layer can be
measured reliably by phase contrast rnicro~copy.~~ The
same report demonstrated the variable loss of the layer
in other techniques designed to preserve the mucus
layer before fixation, confirming the value of direct
measurement with no treatment of the section.
Can these methods be used for multiple samples and
with very small mucin quantities?
Previous work has focused on individual samples
where larger amounts of mucin could be prepared for
analysis, such as whole colons rather than mucosal
biopsies. This situation has dramatically improved in
recent years, due to the introduction of sensitive tracer
techniques in conjunction with the improved protective
preparation methods described above (Fig. 2). Radio-
labelling of mucins in culture with either monosac-
charides, amino acids, or sulphate has allowed tracer
levels of much to be identified and analysed, and dual
labelling techniques have proved especially useful in the
rapid assessment of mucin q ~ a l i t y . The ~ ~ less
~ ~ ~ ~ ~
sensitive histochemical stains used to detect mucins in
histological sections and more recently in biochemical
techniques are being supplemented by more specific and
sensitive reagents.71 Combination of the established
separation techniques with membrane blotting and sub-
sequent probing with anti-mucin, anti-carbohydrate
antibodies, and lectins has become commonplace. When
this is combined with highly sensitive fluorometric detec-
tion systems, pg and ng amounts can be routinely
detected, thus reducing the amounts required for analy-
sis. The same reagents are used in histological analysis of
tissue sections and provide comparative information on
the tissue distribution of purified glycoproteins.
Although these methods have great sensitivity, each
has required careful consideration of limitations apply-
ing to the study of mucins, both at histological and at
biochemical levels. Radiolabelling experiments, for
example, must be considered within the background of
labelling and secretion conditions during the culture
experiment. Short-term cultures may only label basal
turnover of mucins in a restinglconstitutive condition.
Stored mucins may be present in mucosal cells, but
will not be labelled if they are not secreted and the
stores replenished during the labelling period of the
experiment. Experiments addressing the problems of
secretion and storage must therefore take into account
12 A. P. CORFIELD AND B. F. WARREN

ORGAN CULTURE WITH METABOLIC LABELLING

TISSUE RADIOLABEL
colonic mucosa, 2-4mm2 D-[3H]-glucosamine
1-6 biopsies per experiment [35SI-sulphate
on steel grid \ /
ATMOSPHERE
95% air or oxygen
CULTURE MEDIUM(2ml) 5% carbon dioxide
Dulbeccos Minimal humidified
Eagles Medium, 10%
foetal calf serum, 20mM
HEPES, lOmM INHIBlTOR COCKTAIL
bicarbonate, 2mM Phosphate buffered saline,
glutamine, streptomycin, PMSF, EmA, N-ethylmaleimide,
penicillin, gentamycin 24 h at 3 7 0 ~ benzamidine, soybean trypsin
inhibitor, sodium azide

Remove medium and adherent mucus

Wash with lml PBS inhibitor cocktail
SECRETED FRACTION

Homogenise tissue in PBS/inhibitor cocktail

Centrifuge to sediment cell debris and membranes
Collect supernatant
CELLULAR FRACTION

Gel filtration of secreted and cellular samples on Sepharose CL 2B

Collection of fractions eluting at Vo
MUCIN ENRICHED FRACTION

MUCIN ENRICHED SAMPLES

measurement of [35S1:[3HI ratio
measurement of turnover dpm/ug DNA or protein
measurement of total mucin content (lectin, antibody assay)
Fig. 2-Flow diagram of the method for organ culture with metabolic labelling used for colonic biopsy tissue

the metabolic characteristics of the tissue under study. In
goblet cells, knowledge of the constitutive production
and turnover of mucins must be balanced against the
time taken for mucins to be synthesized and ‘matured’
within vesicles prior to secretion. In the same cells, the
formation, maturation, and mode of secretion as a result
of specific mechanical or chemical stimuli represents a
different pathway and is likely to involve different
mucins in non-goblet cells, where vesicle storage is not
part of the mechanism of synthesis and secretion.
Specific reagents recognizing epitopes on individual
mucins are valuable provided that the complete battery
 MUCUS GLYCOPROTEINS AND THEIR ROLE IN COLORECTAL DISEASE
Fig. 3--In situ hybridization analysis of human colorectal tissue for expression of MUC2 in normal (N) and ulcerative colitis (UC) individuals.
A 48-mer oligonucleotide to the tandem repeat sequence for MUC2 was prepared and end-labelled with [35S]-deoxy-(a-thio)adenine triphosphate
and used for in situ hybridization
of mucin molecules for a given tissue under study is
documented. Many carbohydrate-specificreagents react
with a wide range of glycoconjugates and are not limited
to mucins.24,40,72-76 The most reliable approach to
mucin metabolic studies, therefore, is to combine mucin
separation with measurement of both total and radio-
labelled molecules. This requires the use of ‘general’
reagents to give an indication of the total mucin comp-
lement. The expression of any more limited, ‘specific’
epitopes can then be assessed in relation to the complete
mucin spectrum.
MUCIN MOLECULAR BIOLOGY-WHAT
THE GENES TELL US?
The application of molecular biological techniques
has thus far resulted in the identification of eight mucin
genes.** This approach is based on the detection of
unique peptide sequences for individual mucins. Expres-
sion of these sequences has been detected with anti-
bodies recognizing the peptides, or with nucleic acid
probes for the detection of RNA or DNA sequences. As
a result, the molecular organization of secreted and
membrane mucins has become better understood. The
first mucin to be successfully cloned and sequenced
was a molecule known by several names--episialin,
polymorphic epithelial rnucin, epithelial membrane
antigen-and shown to possess the same polypeptide,
coded for by a gene now termed MUC1.8,77This is
still the only example of a membrane-bound mucin
gene in the MUC series, as all remaining genes
identified code for secreted mucins. Two main types
of peptide domain have been identified.I8 The first
consists of tandem repeat sequences distinctive for
13
individual mucins and rich in serine, threonine, and
proline. As noted earlier, these repetitive motifs carry
the majority of the 0-linked oligosaccharides. The
second domain is found in secreted mucins and con-
tains the cysteine residues present in inter- and intra-
molecular disulphide bonds, but only small amounts
of carbohydrate. In MUC2, these regions have hom-
ology to the von Willebrand factor D domain.78
Such domains have been shown to play a role in the
linking of von Willebrand factor subunits to form
oligomeric proteins and also to be important in the
storage of the molecules prior to their secretion. Their
appearance in MUC2 strongly suggests that they are
DO responsible for similar roles in mucin oligomerization
and packaging.
Preparation of suitable nucleic acid probes has opened
the say for in v i t u hybridization studies to assess the
tissue and cellular location of MUC gene expres-
sion.63,79,80
This type of work has shown a pattern of
tissue and cell-specific expression for MUC1-6 gene
products in a variety of mucosae (Fig. 3) and cell lines.
However, analysis of the full spectrum of mucin-
secreting mucosal cells is not complete and furthermore,
the total complement of MUC genes remains undeter-
mined. The ratio of serine and threonine derived from
individual MUC gene sequence has been matched with
the same ratio in mucins purified from different tissues.
In human colon, a lack of correlation has been reported
between serinekhreonine ratios in the major mucin
product and any predicted MUC gene ratio (Fogg and
Allen, University of Newcastle upon Tyne, U.K.,
personal communication). This illustrates the need for
continued surveillance of data to assess new,
unidentified MUC genes and their relationship to total
mucin expression in tissues.
14 A. P. CORFIELD AND B. F. WARREN
The mucosae in the gastrointestinal tract have differ-
ent embryological origins and structures; mucin prod-
ucts from different cell types and not only from goblet
cells need therefore to be evaluated.24 Results obtained
with anti-MUC2 and MUC3 anti-peptide antibodies
in the colon have shown the synthesis of mucins by
non-goblet cells.8' Biochemical assessment of mucins
from these sources in the colon is still outstanding, but
differences in the protein and carbohydrate structures of
isolated mucins from, for example, the stomach and
colon, may reflect differences in mucosal cell lineages,
MUC gene expression, and glycosyltransferase
complements.
Tissue-specific variations in the glycosylation of
individual MUC gene peptides, such as the different
carbohydrate substitution of MUC2 at various sites
in the gastrointestinal tract, are still not understood.
Studies are currently focusing on the initial glycosylation
steps, namely N-acetylgalactosamine to serine or threo-
nine residues in various MUC peptides.20,61It appears
likely that individual cells have a programme of glyco-
sylation regulated by the specificity of glycosyltrans-
ferase complexes which interact with given MUC
peptides. This biochemical work has required knowledge
of the MUC sequences in order to synthesize peptide
acceptors for the initial glycosylation reactions. The use
of this technique has shown that the initial steps in
glycosylation are regulated by the amino acid sequence
itself and by the nature of sugars already added.82The
majority of the glycosyltransferases which catalyse the
formation of the many mucin 0-linked oligosaccharide
sequences have been identified.20 Knowledge of the
substrate specificities and expression of these enzymes
can be used to construct normal pathways for oligo-
saccharide synthesis and to predict the products when
mutations or deletions occur. This approach has been
successfully used to demonstrate significant alterations
taking place in the adenomaxarcinoma sequence in
human colorectal cancer.61 The assay for the glycosyl-
transferases is dependent on unambiguous identification
of both substrate(s) and product(s) and is thus labour-
intensive. Combination of enzyme assays with the detec-
tion of defined carbohydrate epitopes in glycoconjugate
products has also proved to be helpful in demonstrating
the normal or modified biosynthetic pathways. A final
adjunct to this biochemical strategy is the determination
of the chemical structure of the oligosaccharide c h a i n ~ . ~ 3
This too has proved to be an immense task and has been
limited by the amounts of purified much available and
the need for high-resolution equipment to obtain struc-
tural information. An alternative to these methods is to
clone the glycosyltransferases and assess their expression
by molecular biological m e a r ~ s . ~This ~ , * ~is an area of
current interest and development, but awaits the prep-
aration of probes for many of the glycosyltransferases
involved in 0-linked oligosaccharide formation.
0-Acetylation of sialic acids is of additional genetic
interest as it is absent in 6-9 per cent of normal
Europeans and up to 40 per cent of some Far East
populations, indicating homozygous 0-acetyl
transferase-negative i n d i v i d ~ a l s .The
~ ~ advantages or
disadvantages of 0-acetylation are not yet clear.
HOW DO ENTERIC BACTERIA AFFECT MUCIN
DEGRADATION?
The interaction of enteric bacteria with mucus in the
gastrointestinal tract is part of a dynamic equilibrium
which includes the degradation of mucins. The balance
of degradation and synthesis is required to maintain a
functional defensive barrier at the mucosal surface. The
enteric bacterial population includes a small proportion
(approximately 1 per cent) of mucin degrader strains
which possess all of the enzymes necessary for the
complete degradation of rnuciqs6 but mucin in the colon
must retain resistance to resident bacterial enzymes in
order to maintain a functional mucus barrier. This is
achieved by the unique structure of colonic mucin
carbohydrate. The presence of 0-acetylated sialic acid
and sulphate residues limits the rate of oligosaccharide
degradation and controls the overall rate of mucin
d e g r a d a t i ~ n . * ~The
. ~ ~bacterial
-~~ enzymes which hydro-
lyse the oligosaccharide chains act in a sequential man-
ner from the peripheral non-reducing end. This means
that their action is blocked as long as 'non-substrate'
sugars are present in terminal positions. Both sialic acid
and sulphate act in this way. The sialic acids are
removed by sialidases, but the natural presence of
0-acetyl esters on the C7-C9 tail of these monosac-
charides slows down or blocks the action of the
enzyme.90 Release from this block is achieved through
the action of a specific sialic acid 0-acetyl esterase
produced and secreted by a number of enteric bacterial
strains.88 The actions of the glyco-degrading enzymes
together with bacterial proteases are responsible for the
rate at which mucins at the surface of the mucosa will be
degraded. Changes in the level of mucin 0-acetylation
and sulphation occur in gastrointestinal disease, as do
levels of 0-acetylesterase and sulphatase activity, once
again implicating these residues in the normal function
of colonic mucus.87~y1,92 Proteinases are able to act on
intact mucins, destroying viscoelastic properties and
creating g l y c o p ~ l y p e p t i d e s . ~
Their
~ ~ ~ ~activities are
found to be elevated in disease, but specificities have not
yet been evaluated with respect to MUC peptide
sequence or partially glycosylated substrates.
MUCIN CHANGES IN INFLAMMATORY
BOWEL DISEASE
Although most mucin-related theories of inflamma-
tory bowel disease have been received with a degree of
scepticism, it is accepted that there are differences
in mucin composition between normal and inflamed
intestine and between ulcerative colitis and Crohn's
disease; these have been detected largely at the histo-
chemical The first biochemical changes were
identified by Teague et al. in 19739sand further analysis
indicated a reduction in the size of the oligosaccharide
chains in ulcerative colitis.y6 More recent work has
supported the identification of carbohydrate changes
associated in particular with ulcerative ~ o l i t i s . ~ ~ , ~
Podolsky has identified a specific mucin subtype IV,
which is deficient or absent in both human ulcerative
 MUCUS GLYCOPROTEINS AND THEIR ROLE IN COLORECTAL DISEASE
colitis patient^^^,^^ and in the cotton top tamarin, a New
World monkey affected by a disease closely resembling
human ulcerative colitis.100--102The existence and
identification of this subtype remain c o n t r ~ v e r s i a lIt
has been known for some time that ulcerative colitis and
other forms of colitis are associated with a loss of
colonic sialomucin sulphation.47-97~103~104 This modifi-
cation would probably lead to alteration in the visco-
elastic properties of the secreted mucins and hence a
reduction in the efficiency of the defensive adherent
mucus layer. Recently, we have found that this is not
always true.47 In Asian populations with ulcerative
colitis, there is no reduction in colonic sialomucin sul-
phation, irrespective of the level of disease activity. An
additional epidemiological factor of interest in this
population is the lack of progression to colorectal car-
cinoma in ulcerative colitis. There has been a strong
epidemiological suggestion that carcinoma in ulcerative
colitis in Europeans may be genetically determined,lo5
but it is not known whether there is any genetic link
between failure to lose sulphation and the lack of
progression to colorectal carcinoma. A fundamental
difference between Crohn's disease and ulcerative colitis
has been described recently; the mucus layer over a
Crohn's ulcer is much thicker than the almost non-
existent mucus layer overlying an ulcerative colitis
ulcer.65
There are few biochemical data on mucins in inflam-
matory bowel disease other than ulcerative colitis and
Crohn's disease. However, those mucin changes which
have been identified in inflammatory bowel disease are
significant and occur in different forms. It remains
unclear whether they are primary or secondary events
and their significance will only become apparent after
careful monitoring with time, as in the case of the
normal mucin sulphation in Asian colitics and its poss-
ible predictive value for the development of carcinoma
in ulcerative colitis.
WHAT HAPPENS TO GASTROINTESTINAL
MUCINS IN CANCER?
Cancer-related changes in the nature of mucins
detected histologically at mucosae throughout the gas-
trointestinal tract are well known. The search for mucin-
related epitopes in cancer has produced a bewildering
array, detected with a wide range of reagent^.^^^^^,'^^
Cancer-related changes have been detected by mucin
histochemistry, lectin histochemistry, and immunohisto-
chemistry for blood group and Lewis antigens and for
the truncated oligosaccharide epitopes Tn (GalNAc),
sialyl Tn, and T (Galpl-3GalNAc) antigen.'7%24,75,107-109 8. New
In addition, antibodies recognizing sialylated epitopes
and sulphomucin (9 1.9H) have been used to demon-
strate the mucin epitopes appearing during the
progression to malignancy.61
A variety of mucin structures account for these
results. Formation of truncated oligosaccharides can
result from defects leading to the inability to complete
the chains, or the abnormal termination of oligosac-
charides, as a result of changes in glycosyltransferase
15
a ~ t i v i t i e sIn
.~~many cases, characterization of the mucin
epitopes has not been confirmed biochemically with
lectin or antibody binding. The normal high level of
.~~ colonic mucin sialic acid 0-acetylation is known to
interfere with the binding of some antibodies, such as
the anti-sialyl-Tn reagent TKH2,5s and it also blocks
sialidase treatment, which is used to expose crypt anti-
gens for detection. Removal of these 0-acetyl esters by
mild saponification is a necessary control in the histo-
logical analysis of mucins but has remained unexploited.
This feature is compounded by the common loss of sialic
acid 0-acetylation in cancer.61,110 Sulphation may pose
similar problems, but sufficient biochemical data are
lacking for prediction of reagent binding properties. In
addition, the absence of specific methods for sulphate
removal at biochemical or histological levels makes it
impossible to test this hypothesis at present. As noted
before, changes in cancer have been detected at the
glycosyltransferase level and these remain predictive of
certain structural epitopes which require direct chemical
demonstration.20,6'These data, together with lectin and
antibody studies, will provide a better indication of the
significant changes in mucin structure occurring as a
result of malignant transformation.
CONCLUSIONS
The recent advances in mucin science stress the
importance of combined biochemical, molecular bio-
logical, and histological study. Each of these areas is
providing information which cannot be interpreted fully
without input from the others, and it is only by this
combined approach that both the role of mucins in the
development of the gut and their importance in diseases
of the gastrointestinal tract will be revealed.
REFERENCES
1. Allen A, Pearson JP. Mucus glycoproteins of the normal gastrointestinal
tract. Eur J Gastroenterol Hepatol 1993; 5 193-199.
2. Allen A, Hutton DA, Pearson JP, Sellers LA. The colonic mucus gel barrier:
structure, gel formation and degradation. In: Peters TJ, ed. The Cell Biology
of Inflammation in the Gastrointestinal Tract. Hull: Corners Publications,
1990; 113-125.
3. Corfield A. The role of mucins in the gastrointestinal tract: an overview with
emphasis on nutrition. In: Renner B, Sawatzki G, eds. New Perspectives in
Infant Nutrition. Stuttgart: Georg Thieme, 1993; 5 7 4 3 .
4. Lamont JT. Mucus: the front line of intestinal mucosal defense. Ann N Y
Acad Sci 1982; 364: 190-201.
5 . Smith AC, Podolsky DK. Colonic mucin glycoproteins in health and
disease. Clin Gastroenterol 1986; 1 5 815-837.
6. Strous GJ, Dekker J. Mucin-type glycoproteins. Crit Rev Biochem Mol B i d
1992; 27: 57-92.
I. Neutra MR, Forstner JF. Gastrointestinal mucus: synthesis, secretion and
function. In: Johnson LR, ed. Physiology of the Gastrointestinal Tract. 2.
York: Raven Press, 1957; 975-1009.
Hilkens J, Ligtenberg MJL, Vos HL, Litvinov SV. Cell-membrane associ-
ated mucins and their adhesion-modulating property. TIES 1992; 17:
359-363.
9. Carraway KL, Hull SR. Cell surface mucin-type glycoproteins and mucin-
like domains. Glycobiology 1991; 1: 131-138.
10. Briskin MJ, McEvoy LM, Butcher EC. MAdCAM-I has homology to
immunoglobulin and mucin-like adhesion receptors and to IgAl. Nature
1993; 363 461464.
11. Fukuda M. Leukosialin, a major 0-glycan-containing sialoglycoprotein
defining leukocyte differentiation and malignancy. Glycobiology 1991; 1:
347-356.
12 Lasky LA, Singer MS, Dowbenko D, er al. An endothelial ligand for
L-selectin is a novel niucin-like molecule. Cell 1992; 69 927-938.
16 A. P. CORFlELD AND B. F. WARREN
13. Phillips TE, Wilson J . Signal transduction pathways mediating much
secretion from intestinal goblet cells. Dig Diu Sci 1993; 3 8 10461054.
14. Sgroi D, Varki A, Braesch-Anderson S. Stamenkovic I. CD22, a B
cell-specific immunoglobulin superfamily member, is a sialir, acid-binding
Iwtin. J Biol Chem 1993; 268 7011-7018.
15. Shiniizu Y , Shltw S. Mucins in the mainstream. Nature 1993; 366: 63C631.
16. Carlstedt I, Sheehan JK. Corfield AP, Gallagher JT. Mucous glycoproteins:
a gel of a problem. Es.suys Biochem 1985; 2 0 4&76.
17. Kim YS. Mu& glycoproteins in gastrointestinal malignancies and metas-
tasis. Eur J Gustroznrerol Hrpurol 1993; 5: 219-225.
18. Gum JR. Mucin genes and the proteins they encode: structure diversity and
regulation. A m J Respir Cdl Mol Bid 1992; 7: 557-564.
19. Gendler SJ, Spicer AP. Lalani E-N, et ul. Structure and biology of a
carcinoma associated mucin MUCI. Am Rev Re@ Dis 1991; 1 4 4 S42--S47.
20. SChdchter H, Brockhausen 1. The hiosynthesis of serine (threonine)-N-
acetylgalactosainine-1inlit.dcarbohydrate moieties. In: Allen HJ, Kisailus
EC, eds. Glyeoconjugatea. New York: Marcel Dekker, 1992; 263-332.
21. Varki A. Diversity in the sialic acids. G/ycohio/og.v 1992; 2: 25-40.
22. Schauer R. Biosynthesis and function of N- and 0-substituted sialic acids.
Clycchiotogy 1991; 1: 449152.
23. Reid PE, Culling CFA, Dunn WL, Ramey CW, Magi1 AB, Clay MG.
Ditferences between the 0-acetylated sialic acids of the epithelial mucins of
human colonic tumours and nornial controls: a correlative chemical and
histochemical study. J Hir~ochrmCytochenr 1980; 28: 217-222.
24. Jass JR. Roberton AM. Colorectal mucin histochemistry in health and
disease: a critical review. Pathol Int 1994; 44: 487-504.
25. Campbell F. Appleton MAC, Fuller CE, Williams GT, Williams ED. Racial
variation in 0-acetylation of human colonic sialomucins. J Puthol 1993;
169 54A.
26. Corfield AP. Paraskeva C. Secreted mucus glycoproteins in cell and organ
culture. In: Hounsell EF, ed. Glycoprotein Analysis in Biomedicine. 14.
Totowa: Humana Press, 1993: 21 1-232.
27. Carlstedt I, Herrnldnn A. Karlsson H, Sheehan J, Fransson L-A. Hansaon
GC. Characterization of two different glycosylated domains from the
insoluble mucin complex of rat small intestine. J Biol Chenr 1993; 268
I877 1-1 8781.
28. Dekker J, Van Beurden-Lammers WMO, Strous GJ. Riosynthesis of gastric
mucus glycoprotein of the rat. J B i d Chenr 1989; 264: 10431-10437.
29. Thornion DJ. Devine PL. Hanski C, Howard M, Sheehan JK. Identification
of two major populations of mucins in respiratory secretions. A m J Rrspllir
Crir Cure Metf 1994; 150: 823-832.
30. Thornton DJ, Howard M. Devine PL. Sheehan JK. Methods for separation
and deglycosylation of mucin subunits. A n d Biochenr 1995; 227: 162-167.
Thornton DJ, Sheehan JK, Carlstedt I. Heterogeneity of mucus glycopro-
31.
teins from cystic fibrotic sputum. Are there different i'amilies of mucins?
Biochrm J 1991; 276: 677-682.
32. Shamamoto C, Deshmukh GD, Rigot WL, Boband CR. Analysis of
cancer-associated colonic mucin by ion-exchange chromatography: evidence
for a muciii species of lower molecular charge and weight in cancer. Biochini
Biophys Actu 1989: WI: 284~295.
33. Raouf A. Parker N. lddon D, et al. lon-exchange chromatography of
purified colonic mucus glycoproteins in inflammatory bowel disease. Guf
1991; 3 2 1139-1 145.
34. Podolsky DK, lsselbacher KJ. Composition of human colonic mucin:
selective alteration in inflammatory bowel disease. J Cliri Invest 1983; 7 2
142-1 53.
35. Dekker J, van der Ende A, Aelmans PH, Strous GJ. Rat gastric mucin is
synthesized and secreted exclusively as filamentous oligomers. Biochem J
1991; 279 251 256.
36. Sheehan JK, Oates K, Carlstedt I . Electron microscopy of cervical. gastric
and bronchial mucus glycoproteins. Biochrnl J 1986; 239 147-153.
37. Thornton DJ, Sheehan JK, Lindgren H, Carlstedt I. Mucus glycoproteins
from cystic fibrotic sputum. Macromolecular properties and structural
'architecture'. Biochenr J 1991; 276: 667--675.
38. Carlstedt 1, Sheehan JK. Macromolecular properties and polymeric
structure of mucus glycoproteins. In: Mucous and Mucosa. 109. London:
Pitman, 1984; 157-172.
39. Allcn A, Hutton D, Pearson JP, Sellars LA. Mucus glycoprotein structure,
gel formation and gastrointestinal mucus function. In: Mucus and Mucosa.
109. London: Pitman, 1984; 137-156.
40. Filipe MI, Rainachandra S. The histochemistry of human intestinal mucins:
changes in disease. In: Whitehead R, ed. Gastrointestinal and Oesophageal
Pathology. Edinburgh: Churchill Livingstone, 1995; 73-95,
41. Ramcy CW, Corer E, Trueman L, Clay MG.
tion of side chain substituted 0-acetylated sialic
acids: the PAT-KOH-Bh-PAS and the PAPT-KOH-Bh-PAS procedures.
Hlrtochrm J 1984; 1 6 623 639.
42. Veh RW, Meessen D, Kuiitz D, May B. Histochemical demonstration of
side-chain substituted sialic acids. hi: Malt RA, Williamson RCN, eds.
Colon Carcinogenesis. Lancaster: MTP Press, 1982; 355-365.
43. Fuller CE, Davies RP. Williams GT, Williams ED. Crypt restricted
heterogeneity of goblet cell mucus glycoprotein in histologically normal
human colonic mucosa: a potential marker of somatic mutation. Br J
Cuncer 1990; 61: 382Z384.
44. Campbell F, Appleton MAC, Fuller CE, el trl. Racial variation in the
0-acetylation phenotype of human colonic mucosa. J Pathol 1994; 174:
169-174.
45. Spicer SS. Diamine methods for differenliating mucopolysaccharides
histochemically. J Hiutochrrn Cyrochem 1965; 13: 21 1-234.
46. Spicer SS. The use of cationic reagents in histochemical diferentiation of
mucopolysaccliarides. A m J Clin Puthol 1961; 3 6 393-407.
47. Prabert CSJ, Warren BF, Perry T, Mackay EH, Mayberry JF, Corfield AP.
South Asian and European colitics show characteristic differences in colonic
mucus glycoprotein type and turnover. Potential identification of a lower
risk group for severe disease and Cancer. Gul 1995; 3 6 696702.
48. Warren BF. Review in depth-- the histopathology of mucins. Eur J
Gustroenterol Hepatol 1993; 5: 200-204.
49. Sueyoshi S, Tsuji T. Osawa T. Carbohydrate-binding specificities of five
lectins that bind to 0-glycosyl-linked carbohydrate chains. Quantitative
analysis by frontal-affinity chromatography. Ctrrhohydr Res 1988; t 7 8
213-224.
50. Bloom EJ, ltzkowitz SH, Kim YS. Carbohydrate markers in colon cancer
and polyps. In: Moyer MP, Poste GH. eds. Colon Cancer Cells. New York:
Academic Press, 1990; 429451.
S I . Singhal A. Hakomori S-I. Molecular changes in carbohydratc antigens
associated with cancer. BioEusu):~1990; 1 2 223-230.
52. Qrntoft TF, Harving N , Langkilde NC. 0-Linked miicin-type glycoproteins
in normal and malignant colon mucosa: lack of T-antigen expression and
accumulation of Tn and siaIosyl-Tn antigens in carcinomas. Int J Cuncer
1990; 43 666-672.
53. Wolf MF, Koerner U, Schumdcher K. Specificity of reagents directed to the
Thomsen-Friedenreich antigen and their capacity to bind to the surface of
human carcinoma cell lines. Cuncer Res 1986; 46 1779-1782.
54. King M-J, Chan A, Roe R, ef ul. Two different glycosyltransferase defects
that result in GalNAc-rr-0-peptide (Tn) expression. Gl~vcobioh>gy 1994; 4:
267-279.
5s. Jass JR, AlIison LM, Edgar S. Monoclonal antibody TKH2 to the
cancer-associated epitope sialosyl-Tn shows cross reactivity with variants of
normal colorectal goblet cell mucin. Purholog). 1994: 2 6 418422.
56. Milton JD, Eccleston D. Parker N, e l al. Distribntion of 0-acetyetylated
sialomucin in the normal and diseased human gastrointestinal tract assessed
using a newly characterized monoclonal antibody. J Clin Purhol 1993; 45:
211 217.
57. Richman PI, Bodmer WF. Monoclonal antibodies to human colorectal
epithelium: markers for ditferentiation and tumour characterization Inr J
Cancer 1987; 3 9 317-328.
58. Hughes NR, Walls RS, Newland RC, Payne JE Antigen expression in
normal and neoplastic colonic mucosa; three tissue specific antigens using
monoclonal antibodies to isolated colonic glands. Cuncer Res 1986 46
2 164-21 7 I .
59. Pilbrow SJ, Hertzog PJ, Linnane AW. The adenoma-carcinoma sequence in
the colorectum-early appearance of a hierarchy of small intestinal mucin
antigen (SIMA) epitopes and correlation with malignant potential. Br J
Cancer 1992; 66: 748-757.
60. Irimura T, Wynn DM, Hager LG, Cleary KR, Ota DM. Human colonic
sulfomucin identified by a specific monoclonal antibody. Cancer Rrs 1991;
51: 5728 5735.
61. Vavasseur F, Dole K, Yang J, et a I . 0-Glycan biosynthesis in human
colonic cells during progression to cancer. Eur J Bfochem 1994; 2 2 2
4 15- 424.
62. Girling A, Bartkova J, Burchell J. Gendler S. Gillett C , Taylor-
Papadimitiou J. A core protein epitope of the polymorphic epithelial mucin
detected by the monoclonal antibody SM-3 is selectively exposed in a range
of primary carcinomas. Int J Cuncer 1989; 43: 1072-1076.
63. O g m S, Uehara H, Chen A, Itzkowitz SH. Mucin gene expression in
colonic tissues and cell lines. Cuncer Re.s 1992; 52: 5971-5978.
64. Bara J, Gautier R, Daher N, Zaghouani H, Decaens C. Monoclonal
antibodies against oncofetal mucin MI antigens associated with pre-
cancerous colonic mucosae. Cuncer Rex 1986; 46: 3983-3989.
65. Pullan RD, Thomas GAO, Rhodes M, et t i / . Thickness of adherent mucus
on colonic mumsa in humans and its relevance to colitis. Gut 1994; 35:
353-359.
66. Forstner GG, Zhang Y , McCool D, Forstner J. Mucin secretion by T84
cells: stimulation by PKC, Ca2+, and a protein kinase activated by Ca'+
ionophore. Am J Physiol 1993; 2 6 4 G1096-Gl 102.
67. Heim H-K, Oestmann A, Thiele H, Sewing K-F. lncorporation of N-acetyl-
['4C]~-glucosamineand ['H]~-leucineby isolated pig gastric mucosal cells.
Digestion 1989; 4 4 2635.
68. Irimura T, Carlson DA, Price J, et ul. Differential expression of a sialoglyco-
protein with an approximate molecular weight of 900,000 on metastatic
human colon carcinoma cells growing in culture and in tumor tissues.
Cuncer Res 1988; 4 8 2353-2360.
69. Ohara S, Byrd JC, Gum JRJ, Kim YS. Biosynthesis of two distinct types of
mucin in HM3 human colon cancer cells. Biochem J 1994; 297: 509-516.
70. Smith AC, Podolsky DK. Biosynthesis and secretion of human colonic
mucin glycoproteins. J Clin Invesl 1987; 80: 30C307.
71. Thornton DJ, Holmes DF, Sheehan JK, Carlstedt I. Quantitation of mucus
glycoproteins blotted onto nitrocellulose membranes A n d Biochem 1989;
182: 160-164.
 MUCUS GLYCOPROTElNS AND THEIR ROLE IN COLORECTAL DISEASE
72. McMahon RFT, Panesar MJR, Stoddart RW. Glycoconjugates of the
normal human colorectum. Hisrochem J 1994; 2 6 504-518.
73. Sell S. Cancer-associated carbohydrates identified by monoclonal anti-
bodies. Hum Puthol 1990; 21: 160-164.
74. Mitchell EP, Schlom J. Monoclonal antibodies in gastrointestinal cancers.
Semin Onrol 1988; 1 5 170-180.
75. Hanski C, Hanisch F-G, Rieken EO. Alteration of mucin-bound carbohy-
drate moieties in malignant transformation of colonic mucosa. Cancer J
1992; 5: 332-342.
76. Roth J. Cellular sialoglycoconjugates: a histochemical perspective.
Histocheni J 1993; 25: 687-710.
77. Gendler SJ, Lancaster CA, Taylor J. e f al. Molecular cloning and expression
of human tumor-associated polymorphic epithelial mucin. J Biol Chem
1990; 265 15286-15293.
78. Gum JR, Hicks JW, Toribara NW, Siddiki B, Kim YS. Molecular cloning
of human intestinal mucin (MUC2) cDNA. Identification of the amino acid
functions and overall sequence similarity to prepro-von Willebrand factor.
J Biol Chem 1994; 269 2440-2446.
79. Audie JP, Janin A, Porchet N, Copin MC, Goaselin 9, Aubert JP.
Expression of human mncin genes in respiratory, digestive and reproductive
tracts ascertained by in situ hybridisation. J Hisrochem Cytochem 1993; 41:
1479-1485.
80. Ho JJL, Bi N, Siddiki B, Chung Y-S, Yuan M, Kim YS. Multiple forms of
intracellular and secreted mucins in a pancreatic cancer cell line. Cancer Res
1993; 5 3 884-890.
81. Ho SB, Niehans GA, Lyftogt C, et al. Heterogeneity of mucin gene
expression in normal and neoplastic tissues. Cancer Res 1993; 5 3
64 1-65 1.
82. Granovsky M, Bielfeldt T, Peters S , et d.UDP-ga1actose:glycoprotein-N-
acetyl-u-galactosamine 3-P-~-galactosyltransferaseactivity synthesizing
0-glycan core 1 is controlled by the amino acid sequence and glycosylation
of glycopeptide substrates. Eur J Biothem 1994; 221: 1039-1046.
83. Capon C, Laboisse CL, Wieruszeski J-M, Maoret J-J. Augeron C, Fournet
B. Oligosaccharide structures of mucins secreted by the human colonic
cancer cell line CL.16E. J Bid Chem 1992; 267: 19248-19257.
84. Joziasse DH. Mammalian glycosyltransferases: genomic organization and
protein structure. Glycobiology 1992; 2 271-277.
85. Lowe JB. Molecular cloning, expression and uses of mammalian glycosyl-
transferases. Semin CeN Biol 1991; 2 289-307.
86. Hoskins LC. Much degradation in the human gastrointestinal tract and its
significance to enteric microbial ecology. Eur J Gastroenterol Hepatol 1993;
5 205-213.
87. Corfield AP, Wagner SA, O’Donnell LJD, Durdey P, Mountford RA,
Clamp JR. The roles of enteric bacterial sialidase, sialate 0-acetyl esterase
and glycosulfatase in the degradation of human colonic mucin. Glycoconj J
1993; 1 0 72-81.
88. Corfield AP, Wagner SA, Clamp JR, Kriaris MS, Hoskins LC. Mucin
degradation in the human colon: production of sialidase, sialate 0-acetyl
esterase, N-acetylneuraminate lyase, arylesterase and glycosulfatase activi-
ties by strains of fecal bacteria. Infect Immun 1992; 6 0 3971-3978.
89. Rhodes JM, Black RR, Gallimore R, Savage A. Histochemical demonstra-
tion of desialylation and desulphation of normal and inflammatory bowel
disease recal mucosa by faecal extracts. Gut 1985; 2 5 1312-1318.
90. Corfield T. Bacterial sialidases-roles in pathogenicity and nutrition.
Glycobiulogy 1992; 2 509-521.
17
91. Tsai HH, Sunderland D, Gibson GR, Hart CA, Rhodes JM. A novel mucin
sulphatase from human faeces: its identification, purification and charac-
terization. Clin Sci 1992; 8 2 447454.
92. Roberton AM, McKenzie C, Scharfe N, Stubbs L. A glycosnlphatase that
removes sulphate from mucus glycoprotein. Biochem J 1993; 293 683-689.
93. Allen A. Pre-epithelial factors in resistance to acid and pepsin. Eur J
Gastroenterol Hepatol 1990; 2: 168-171.
94.Corfield AP, Williams AJK, Clamp JR, Wagner SA, Mountford RA.
Degradation by bacterial enzymes of colonic mucus from normal subjects
and patients with inflammatory bowel disease: the role of sialic acid
metabolism and the detection of a novel 0-acetylsialic acid esterase. CIin Sci
1988; 7 4 71-78.
95. Teagne RH, Fraser D, Clamp JR. Changes in monosaccharide content of
mucus glycoproteins in ulcerative colitis. Br Med J 1973; 2 645-646.
96. Clamp JR, Fraser G, Read AE. Study of the carbohydrate content of mucus
glycoproteins from normal and diseased colons. Clin Sci 1981; 61: 229-234.
97.Raouf AH, Tsai HH, Parker N, Hoffman J, Walker RJ, Rhodes JM.
Sulphation of colonic and rectal mucin in inflammatory bowel disease:
reduced sulphation of rectal mucus in ulcerative colitis. CIin Sci 1992; 8 3
623-626.
98. Morita H, Kettlewell MGW, Jewell DP, Kent PW. Glycosylation and
sulphation of colonic mucus glycoproteins in patients with ulcerative colitis
and in healthy subjects. Gut 1993; 34: 92C932.
99. Podolsky DK, Isselbacher KJ. Glycoprotein composition of colonic
mucosa: specific alterations in ulcerative colitis. Gastroenterology 1984; 87:
99 1-998.
100. Warren BF, Watkins PE. Animal models. In: Schmolerich J, Kruis W,
Goebel H, Hohenberger W, Gross V, eds. Inflammatory Bowel Diseases.
Lancaster: Kluwer Academic Press, 1993; 3-7.
101.Boland CR, Clapp NK. Glycoconjugates in the colons of New World
monkeys with spontaneous colitis. Association between inflammation and
neoplasia. Gastroenterology 1987; 9 2 625-634.
102. Podolsky DK, Madara JL, King N, Sehgal P, Moore R, Winter H. Colonic
mucin composition in primates. Selective alterations associated with spon-
taneous colitis in the cotton-top tamarin. Gasrroenrerology 1985; 8 8 20-25.
103. Corfield AP, Warren BF, Bartolo DCC, Wagner SA, Clamp JR. Mucin
changes in ileoanal pouches monitored by metabolic labelling and histo-
chemistry. Br J Surg 1992; 7 9 1209-1212.
104. Corfield AP, do Amaral Corfield C, Wagner SA, ef al. Loss of sulphation in
human colonic mucins during ulcerative colitis. Biochem Soc Truns 1992; 2 0
95c.
105. Satsangi J, Jewell DP, Rosenberg WMC, Bell JI. Genetics of inflammatory
bowel disease. Gut 1994; 3 5 696-700.
106. Feller WF. Mucin glycoproteins as tumour markers. Immunol Ser 1990; 53:
63 1-672.
107. Irimura T, Matsushita Y, Hoff SD, el al. Ectopic expression of mucins in
colorectal cancer metastasis. Semin Cancer Biol 1991; 2: 129-139.
108. Hanski C, Bornhoeft G, Topf N, Hermann U, Stein H, Riecken OE.
Detection of a mucin marker for the adenomaarcinoma sequence in
human colonic mucosa by monoclonal antibody AM-3. J Clin Purhol 1990;
4 3 379-380.
109. Itzkowitz SH, Bloom EJ, Lau T-S, Kim YS. Mucin associated Tn and
sialosyl-Tn antigen expression in colorectal polyps. Gut 1992; 3 3 518-523.
110. Corfield AP, Wagner SA, Paraskeva C, et al. Loss of sialic acid
0-acetylation in human colorectal cancer cells. Biochem Soc Trans 1992; 2 0
94s.