Tài liệu Báo cáo khoa học: The role of the SEA (sea urchin sperm protein, enterokinase and agrin) module in cleavage of membrane-tethered mucins pdf

The role of the SEA (sea urchin sperm protein,
enterokinase and agrin) module in cleavage
of membrane-tethered mucins
Timea Palmai-Pallag
1
, Naila Khodabukus
1
, Leo Kinarsky
2
, Shih-Hsing Leir
1
, Simon Sherman
2
,
Michael A. Hollingsworth
2
and Ann Harris
1
1 Paediatric Molecular Genetics, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, UK
2 Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE, USA
SEA (sea urchin sperm protein, enterokinase and
agrin) modules, minimally comprised of 120 amino
acids of which 60% show strong conservation between
proteins, are usually found in extracellular domains of
dimeric or multimeric membrane-tethered proteins
[1,2]. SEA modules contain proteolytic cleavage sites
and amino-acid sequences that are important in the
noncovalent association of protein subunits [2]. A
heavily O-glycosylated domain is located N-terminal to
the SEA module in the membrane-tethered mucins
MUC1, MUC3, MUC12, MUC13, MUC16 and

MUC17 [3]. Membrane-tethered mucins establish
selective molecular barriers at epithelial cell surfaces
and are implicated in diverse functions, including
cell-adhesion, signalling, immune regulation and meta-
stasis (reviewed in [3]). MUC1, the best-characterized
membrane-tethered mucin, is transcribed as a single
precursor protein that undergoes cleavage early in its
processing to produce a heterodimer, which is further
modified post-translationally and expressed in the cell
Keywords
MUC3, MUC12, proteolytic cleavage, SEA
Correspondence
A. Harris, Paediatric Molecular Genetics,
Weatherall Institute of Molecular Medicine,
John Radcliffe Hospital,
Oxford OX3 9DS, UK
E-mail:
(Received 25 January 2005, revised
11 March 2005, accepted 7 April 2005)
doi:10.1111/j.1742-4658.2005.04711.x
The membrane-tethered mucins are cell surface-associated dimeric or multi-
meric molecules with extracellular, transmembrane and cytoplasmic por-
tions, that arise from cleavage of the primary polypeptide chain. Following
the first cleavage, which may be cotranslational, the subunits remain closely
associated through undefined noncovalent interactions. These mucins all
share a common structural motif, the SEA module that is found in many
other membrane-associated proteins that are released from the cell surface
and has been implicated in both the cleavage events and association of the
subunits. Here we examine the SEA modules of three membrane-tethered
mucins, MUC1, MUC3 and MUC12, which have significant sequence

homology within the SEA domain. We previously identified the primary
cleavage site within the MUC1 SEA domain as FRPG ⁄ SVVV a sequence
that is highly conserved in MUC3 and MUC12. We now show by site-
directed mutagenesis that the F, G and S residues are important for the
efficiency of the cleavage reaction but not indispensable and that amino
acids outside this motif are probably important. These data are consistent
with a new model of the MUC1 SEA domain that is based on the solution
structure of the MUC16 SEA module, derived by NMR spectroscopy. Fur-
ther, we demonstrate that cleavage of human MUC3 and MUC12 occurs
within the SEA domain. However, the SEA domains of MUC1, MUC3
and MUC12 are not interchangeable, suggesting that either these modules
alone are insufficient to mediate efficient cleavage or that the 3D structure
of the hybrid molecules does not adequately recreate an accessible cleavage
site.
Abbreviations
SEA, sea urchin sperm protein, enterokinase and agrin.
FEBS Journal 272 (2005) 2901–2911 ª 2005 FEBS 2901
surface membrane [4]. MUC1 subunits remain associ-
ated through a poorly understood non–covalent associ-
ation, but can be released from the cell surface by
additional proteolytic cleavage events [5,6], or other
unknown mechanisms. The cleavage and release of
membrane-tethered mucins is relevant to mucus accu-
mulation in cystic fibrosis, and these molecules are
implicated in the pathogenesis of many cancers inclu-
ding pancreatic and breast carcinomas, where there is
evidence for aberrant expression and processing of a
number of mucin molecules [3]. Further, there is accu-
mulating evidence that the cytoplasmic tail of MUC1
is involved in signal transduction and control of gene

expression (reviewed in [3]), which would be affected
by proteolytic cleavage events and the status of the
extracellular domains [7].
We previously identified the early cleavage site of
MUC1 in the SEA module at the Gly-Ser peptide bond
located 58 amino acids upstream of the transmembrane
domain (as defined in [8]), by N-terminal sequencing [9].
This cleavage site lies within a FRPG ⁄ SVVV motif that
is highly conserved among the SEA modules of MUC1
glycoproteins from different species, and is found in
other membrane-tethered mucins including MUC3,
MUC12, MUC16 and MUC17 and other proteins with
cleaved SEA domains (Table 1). The SEA module of
rat MUC3 is cleaved soon after translation in vivo and
in vitro at the Gly-Ser peptide bond of the LSKG ⁄
SIVVV motif [10], though additional amino acids
C-terminal to this site are also required for efficient
cleavage [11].
The solution structure of the SEA domain of the
mouse homologue of MUC16 (which in the human
encodes the core protein of the ovarian cancer antigen
CA125) was recently solved by multidimensional NMR
spectroscopy [12]. Similarity in sequence among the
SEA modules of MUC1 and MUC16 enabled the gen-
eration of a 3D model of MUC1 based on the MUC16
data. This model predicts that the FRPG ⁄ SVVV motif
is contained in a surface-exposed loop. To further
investigate the role of the SEA domain in cleavage of
membrane-tethered mucins we carried out site-directed
mutagenesis of the FRPG ⁄ SVVV site and evaluated

hybrid mucins in which the SEA module of MUC1
was replaced by that from MUC3 and MUC12. Our
aim was to determine whether cotranslational cleavage
of membrane-tethered mucins was solely dependent on
the FRPG ⁄ SVVV motif or whether additional sequen-
ces, within or outside the SEA module, were required.
We show that mutagenesis of the FRPG ⁄ SVVV
motif does not completely prevent cleavage of MUC1.
Though mutation of the Phe, Gly and Ser residues has
a significant effect on the efficiency of the cleavage
reaction, it is not completely abolished, suggesting that
the precise sequence of this motif is not critical for clea-
vage. In addition we determined that the SEA domains
of human MUC3 and MUC12 are not interchangeable
with that of MUC1, as substitution of these yielded
cleaved products inefficiently. This suggests that motifs
outside the SEA module may contribute to the cotrans-
lational cleavage event in vivo or that the structures of
the hybrid molecules do not enable efficient cleavage.
Results
Mutation of the FRPG ⁄ SVVV motif does not
completely inhibit cleavage of MUC1FDTR
MUC1FDTR undergoes early proteolytic cleavage at
the FRPG ⁄ SVVV site (Fig. 1), which generates two
Table 1. Conservation of the G ⁄ S cleavage site sequence in MUC1 from several species, other membrane-tethered mucins and SEA domain
containing proteins.
Protein Amino acid sequence Accession number
MUC1 (human) GLSNIKFRPGS VVVQLTLAFREGTIN J05582
Muc1 (mouse) GISSIKFRSGS VVVESTVVFREGTFS U16175
Muc1 (cow) GLSEIKFRPGS VVVELTLAFREGTTA L41543

Muc1 (hamster) GISTIEFRSGS VVVDSTVIFREGAVN U36918
Muc1 (guinea pig) GLLNIKFRPGS VAVESTVIFRKNAVN L41546
MUC3 (human) GVEILSLRNGS IVVDYLVLLEMPFSP AF143371
Muc3 (rat) GVIIKNLSKGS IVVDYDVILKAQYTP U76551
MUC12 (human) GVNIRRLLNGS IVVKNDVILEADYTL AF147790
MUC16 (human) DCQVSTFRSVPNRHHTGVDSLCNFS PL AF361486
Muc16 (mouse) DCQVLAFRSVSNNNNHTGVDSLCNFS-PL [12]
MUC17 (human) GVNITKLRLGS VVVEHDVLLRTKYTP AF430017
Ig-Hepta SVTVTQFTKGS VVVDYIVEVASAPLP AB019120
SPACR QLEILNFRNGS VIVNSKMKFAKSVPY AF017776
SPACRCAN QNLEILFRNGS¼¼¼IVVNSRMKFANSVPP AF157624
SEA modules and mucin cleavage T. Palmai-Pallag et al.
2902 FEBS Journal 272 (2005) 2901–2911 ª 2005 FEBS
subunits: an extracellular component and a cell surface
associated subunit containing the transmembrane
region and the cytoplasmic tail. These two parts of
the protein remain associated by noncovalent interac-
tion, probably involving sequences within the SEA
domain. The contribution of five different amino
acids at the proteolytic cleavage site (shown in
bold) FRPG ⁄ SVVV to the efficiency of cleavage of
MUC1FDTR was investigated by mutating these resi-
dues individually to alanine (A). The glycine (G) and
serine (S) residues at the proteolytic cleavage site (resi-
due numbers 185 and 186 in MUC1FDTR) were
mutated first as these residues are conserved in MUC1
from several species, and in MUC3, MUC12, MUC17
and IgHepta (Table 1). Three additional residues close
to the cleavage site, phenylalanine (F) 182, arginine
(R) 183 and valine (V) 189, that are highly conserved

amongst different mucins, were also mutated and
evaluated.
Caco2 colon adenocarcinoma cells were stably trans-
fected with the wild-type (WT) and mutant constructs
(G ⁄ A, S ⁄ A, F ⁄ A, R ⁄ A and V ⁄ A) and clones expres-
sing high levels of the individual mutant MUC1FDTR
constructs were identified. In Fig. 2 each lane contains
mucin immunoprecipitated with an excess of M2 or
CT-2 from 300 lg of total cell lysate. When inter-
preting these data comparison should be made of the
ratio of cleaved to noncleaved protein within each
clone as the total amount of M2-reactive material var-
ies between clones due to different expression levels.
Figure 2A shows a western blot of MUC1FDTR pro-
tein immunoprecipitated with M2 (anti-FLAG) from
each clone, separated by 6% SDS ⁄ PAGE, and probed
with M2. The major M2- reactive species in each clone
migrates at around 75 kDa though resolution of this
gel does not enable discrimination between cleaved
and noncleaved protein. (Additional M2-reactive spe-
cies, such as those at about 50 kDa are probably
incompletely processed or differentially glycosylated
forms of the epitope-tagged mucin as described previ-
ously [9,13]). However, in Fig. 2B a western blot of a
10% SDS ⁄ PAGE gel of the same material probed with
the CT-2 anti-cytoplasmic tail revealed both cleaved
and noncleaved mutant MUC1FDTR. Wild-type
MUC1FDTR generates CT-2 reactive species migrating
between 15 and 30 kDa corresponding to the cytoplas-
mic tail following cleavage at the G ⁄ S site. Multiple

cytoplasmic tail species are routinely observed with the
CT-2 antibody [13], which may be due to additional
cleavage events and ⁄ or other modifications of the pro-
tein that affect migration in the SDS ⁄ PAGE gel. CT-2
reactive species between 15 and 30 kDa are seen for all
the mutants hence they must all undergo at least par-
tial cleavage at the G ⁄ S motif. However, the G ⁄ A,
MUC1
MUC1F∆
∆
TR
MUC1F∆TR /MUC3-CL
MUC1F∆TR /MUC3-GA
MUC1F∆TR /MUC12-CL
MUC1F∆TR /MUC12SEA
MUC1F∆TR /MUC3SEA
MUC1F∆TR /MUC12FREG
TR
926 aa
TM CTSEAS
SSEATMCT
FRPGSVVV
** ** *
STMCT
LRNGSIVV
STMCT
LRNGSIVV
*
TM CTS
LLNGSIVV

STMCT
LLNGSIVV
CTTMS
LRNGSIVV
TM CTS
LLNGSIVV
FREG
Fig. 1. Schematic representation of the dif-
ferent MUC1FDTR constructs.Full length
MUC1 is at the top of the figure and
MUC1FDTR, derived from it but lacking the
tandem repeat (TR) domain and containing a
FLAG epitope (black box with flag) is shown
below. All constructs are based on
MUC1FDTR and contain the MUC1 signal
sequence (S), transmembrane domain (TM)
and cytoplasmic tail (CT) with the C-terminal
black box representing the epitope for the
CT2 antibody. The SEA domain (SEA) in
each construct is as follows: MUC1, blank
box, MUC3 diagonal stripe box, MUC12
diamond hatched box. The closed
arrowheads denote the FRPG ⁄ SVVV
cleavage site in MUC1 and predicted
cleavage sites in MUC3 (LRNG ⁄ SIVV) and
MUC12 (LLNG ⁄ SIVV); stars denote the sites
of mutations. The open arrowhead denotes
the LEAD to FREG change in MUC12.
T. Palmai-Pallag et al. SEA modules and mucin cleavage
FEBS Journal 272 (2005) 2901–2911 ª 2005 FEBS 2903

S ⁄ A and F ⁄ A mutants also generate a CT-2 reactive
noncleaved species that migrates close to 75 kDa
(Fig. 2B). Thus mutating G185, S186 or F182 to alan-
ine partially inhibits the cleavage at the FRPG ⁄ SVVV
motif whereas mutating R183 or V189 to alanine does
not impair cleavage. Comparison of the relative inten-
sity of the noncleaved and cleaved forms of the S ⁄ A,
G ⁄ A and F ⁄ A mutants suggests that the S ⁄ A mutation
has the most dramatic effect on the cleavage event.
This observation was confirmed when mutants were
transfected transiently into COS-7 cells as the results
were similar to those observed in Caco2 cells with the
exception of the S ⁄ A mutant, which showed almost
complete inhibition of cleavage (data not shown). We
have previously shown that low levels of MUC1F are
released from Caco2 cells into the culture supernatant
[9,13]. To evaluate the effect of the cleavage site muta-
tions on release of MUC1FDTR, media conditioned by
these clones was immunoprecipitated with M2 (with
M2-beads in excess) (Fig. 2C,D). These data are not
quantitative as the individual clones express different
levels of the MUC1FDTR glycoprotein and some
material enters the media via routes other than by
membrane-tethered release (as demonstrated by CT-2
binding to cleaved MUC1FDTR cytoplasmic tail in the
media, Fig. 2D). However, it is clear that despite the
inefficient cleavage of the F ⁄ A, G ⁄ A and S⁄ A mutants,
MUC1FDTR carrying these mutations is released from
the cell (Fig. 2C).
The cleavage site of MUC1 is predicted to lie in a

surface loop on the SEA domain
The recent publication of the solution structure of the
SEA domain from murine MUC16, derived by NMR
spectroscopy [12] enabled computer modelling of the
MUC1 SEA domain (Fig. 3). The model has the same
a ⁄ b sandwich fold as the template SEA domain of mu-
rine MUC16 [12] and consists of three a-helices and
six b-strands. This model predicts that the
FRPG ⁄ SVVV cleavage site of MUC1 is located in a
surface loop (residues marked) between b2 and b3
strands of the SEA module. Interestingly, the glycine
(G185) and serine (S186) residues, which when
mutated to alanine have the greatest impact on MUC1
cleavage, lie in the most exposed part of this loop. The
phenylalanine (F182), which also affects MUC1 clea-
vage when mutated to alanine, is located at the base of
the loop and may interact with other residues in the
adjacent b2 and b3 strands, affecting conformation
and tightness of the loop. Next to this phenylalanine,
the arginine residue (R183), which is located on the
WT
F/A
R/A
U
G/A
V/A
S/A
50
75
105

160
250
U
WT
F/A
R/A
G/A
S/A
V/A
250
50
75
105
35
30
25
WT
F/A
R/A
G/A
S/A
V/A
WT
F/A
R/A
G/A
S/A
V/A
75
105

30
25
A
B
C
D
Fig. 2. Expression and release of wild-type and cleavage site
mutants of MUC1FDTR in Caco2 cells. Western blots of
M2-immunopurified material from Caco2 clone cell lysates (A, B)
or cell culture supernatant (C, D) from clones expressing wild-
type (WT) or mutant (R ⁄ A, F ⁄ A, G ⁄ A, S ⁄ A, V ⁄ A) MUC1FDTR as
shown. Samples were run on a 6% (A) or 10% (B, C, D)
SDS ⁄ PAGE gel. The western blots was probed with M2 (A, C)
or CT-2 (B, D). U, untransfected Caco2 cells. Mucins immunopre-
cipitated with an excess of M2 beads or CT-2 from 300 lgof
total cell lysate were loaded in each lane (A, B). Volumes of cell
culture supernatant-derived M2 immunoprecipitated material loa-
ded on gels in panels C and D were based on the intensity of
the M2-reactive species in the corresponding whole cell lysate
(not shown).
SEA modules and mucin cleavage T. Palmai-Pallag et al.
2904 FEBS Journal 272 (2005) 2901–2911 ª 2005 FEBS
N-terminal side of the loop, faces solvent and its muta-
tion to alanine has little if any effect on the cleavage
event. On the other side of the loop, the valine residue
(V189) that interacts only with the C-terminal residues
of the b1 strand, also has little impact on a loop con-
formation and can be mutated to alanine without sig-
nificant effect on the cleavage. It should be noted that
relatively low overall sequence homology ( 25%)

between the SEA modules of MUC1 and MUC16 and
the shortening of the predicted loop in MUC1
(FRPGSV_ _ _VV in MUC1 compared to FRS-
VSNNNNHT in MUC16) makes detailed structural
analysis of specific interactions within this loop some-
what speculative.
Substitution of the MUC1 FRPG ⁄ SVVV motif
with LRNG ⁄ SIVV from MUC3 or LLNG ⁄ SIVVV
from MUC12 largely abolishes cleavage
The conservation of the known proteolytic cleavage
sites of MUC1 (FRPG ⁄ SVVV) and human MUC3
(LRNG ⁄ SIVV) and the predicted cleavage site of
MUC12 (LLNG ⁄ SIVV) raises the possibility that a
common protease and cleavage mechanism might be
involved in the processing of these three membrane-
associated mucins (Tables 1,2). To evaluate this hypo-
thesis, 33 amino acids encompassing the FRPG ⁄ SVVV
site (12 amino acids N-terminal and 13 amino acids
C-terminal) of MUC1FDTR were replaced with equiv-
alent sequences from MUC3 and MUC12 (Table 2).
These hybrid constructs (MUC1FDTR ⁄ MUC3-CL and
MUC1FDTR ⁄ MUC12-CL) were stably transfected
into Caco2 colon adenocarcinoma cells and clones
expressing high levels of the hybrid glycoproteins
identified. Figure 4A and B show western blots of
M2-immunoprecipitated MUC1FDTR ⁄ MUC3-CL and
MUC1FDTR ⁄ MUC12-CL glycoproteins separated on
10% SDS ⁄ PAGE and probed with M2 antibody
(Fig. 4A) or CT-2 (Fig. 4B). As shown in Fig. 2, the
MUC1FDTR glycoprotein migrates at about 75 kDa

(weakly evident in Fig. 4A) and a CT-2-reactive
15–30 kDa cleavage product identifies the cytoplas-
mic tail after cleavage (Fig. 4B). Three forms of
MUC1FDTR ⁄ MUC3-CL glycoprotein are seen bet-
ween about 30 and 50 kDa (apparent MW) (Fig. 4A,
MUC3-CL) in addition to a diffuse form at around
75 kDa that is seen more clearly in Fig. 4B. The fas-
ter migrating glycoforms may include incompletely
processed and differentially glycosylated forms. Similar
migration profiles are seen for the MUC1FDTR ⁄
MUC3GA glycoprotein (Fig. 4A, MUC3-GA), which
contains a Gly ⁄ Ala mutation in the cleavage site motif,
Fig. 3. Model of SEA domain of MUC1. Ribbon representation of
the modelled SEA domain of human MUC1. The model consists of
three a-helices and six b-strands forming an a ⁄ b sandwich fold.
The FRPG ⁄ SVVV residues of the MUC1 cleavage site, which is
located in a loop between b2andb3 strands, are illustrated.
Table 2. The SEA domains of MUC1, MUC3 and MUC12. The predicted cleavage site of each SEA domain is bold underlined. The FREG
sequence that is conserved in MUC1 SEA domains from many different species and corresponding sequences from MUC3 and MUC12 are
underlined. The numbered amino acids denote the limits of the SEA domain substitutions in MUC1 (J05582) by MUC3 (AF143371) and
MUC12 (AF147790) sequences.
Protein Sequence
MUC12SEA
E
273
KLN ATLGMTVKVTYRNFTEKMNDASSQEYQNFSTLFKNRMDVVL
MUC3 SEA
D
61
VVE TEVGMEVSVD.QQFSPDLNDNTSQAYRDFNKTFWNQMQKIF

MUC1SEA PQLS TG
V
128
SFFFLSFHISNLQFNSSLEDPSTDYYQELQRDISEMFLQIY
MUC12SEA KGDNLPQYRGVNIRR
LLNGSIVVKNDVILEADYT LEYEELFENLAEIVKA
MUC3 SEA ADMQGFTFKGVEILS
LRNGSIVVDYLVLLEMPFS PQLESEYEQVKTTLKE
MUC1SEA KQG GFLGLSNIK
FRPGSVVVQLTLAFREGTINVHDVETQFNQYKTEAAS
MUC12SEA KIMNETRTTLLDPDSCR.KAILCY
S
391

MUC3 SEA GLQNAS QDVNSCQDSQTLCF KPD
S
178
MUC1SEA RYNLTIS DVSVSDVPFPFSA
238
Q
T. Palmai-Pallag et al. SEA modules and mucin cleavage
FEBS Journal 272 (2005) 2901–2911 ª 2005 FEBS 2905
and for the MUC1FDTR ⁄ MUC12-CL glycoprotein
(Fig. 4A, MUC12-CL). When the same M2 immuno-
precipitated proteins were western blotted and probed
with CT-2 the major species detected for each of the
hybrid proteins was noncleaved protein migrating
close to 75 kDa (Fig. 4B, MUC1FDTR ⁄ MUC3-CL
[MUC3-CL], MUC1FD TR ⁄ MUC12-CL [MUC12-CL],
MUC1FDTR ⁄ MUC3GA [MUC3-GA]). However, a

minor component migrated close to the cleaved
cytoplasmic tail of MUC1FDTR (15–30 kDa) for
MUC1FDTR ⁄ MUC3GA (Fig. 4B, MUC3-GA) and
MUC1FDTR ⁄ MUC12-CL (Fig. 4B, MUC12-CL) gly-
coproteins (15–30 kDa, marked by a bracket) but
no cytoplasmic tail peptides were detected in
MUC1FDTR ⁄ MUC3-CL (Fig. 4B, MUC3-CL). These
data suggest that the replacement of the MUC1FDTR
cleavage site by that from MUC12 enables an ineffi-
cient cleavage event to occur but the MUC3 replace-
ment largely abolishes cleavage. Next, the CT-2
antibody was used to immunopurify the glycopro-
teins prior to western blotting (Fig. 4C,D). For the
MUC1FDTR ⁄ MUC3-CL, MUC1FDTR ⁄ MUC12-CL
and MUC1FDTR ⁄ MUC3GA constructs both M2 and
CT-2 antibodies reacted with the 75 kDa species, sug-
gesting that the majority of the glycoprotein from
each construct is not cleaved (Fig. 4C). However, for
all constructs, minor species were detected between 15
and 30 kDa (marked by the bracket) that reacted
with the CT-2 antibody (Fig. 4D, MUC3-CL,
MUC12-CL and MUC3-GA), but were not reactive
with M2 (data not shown). More than one cleaved
form of the cytoplasmic tail peptide is seen for both
MUC3 and MUC12-containing hybrid molecules.
These data show that the replacement of 33 amino
acids flanking the MUC1FDTR cleavage site by
equivalent sequences from MUC3 and MUC12
enables a cleavage event to occur within these regions
but does not provide an efficient substrate for prote-

ase-mediated cleavage at the G ⁄ SIVV motif. It may
be relevant that the G ⁄ A mutant of the MUC3 clea-
vage site is apparently more efficiently cleaved than
the wild-type sequence in these hybrid molecules
implicating structural constraints on the cleavage
motif.
U
MUC1F∆TR
MUC3-GA
MUC3-CL
MUC12-CL
MUC3SEA
MUC12SEA
MUC12FREG
105 kDa
75 kDa
50 kDa
35 kDa
30 kDa
25 kDa
15 kDa
A
U
MUC1F∆TR
MUC3-GA
MUC3-CL
MUC12-CL
MUC3SEA
MUC12SEA
MUC12FREG

105 kDa
75 kDa
50 kDa
35 kDa
30 kDa
25 kDa
15 kDa
B
105 kDa
75 kDa
50 kDa
35 kDa
30 kDa
25 kDa
15 kDa
U
MUC1F∆TR
MUC3-GA
MUC3-CL
MUC12-CL
MUC3SEA
MUC12SEA
MUC12FREG
C
U
MUC1F∆TR
MUC3-GA
MUC3-CL
MUC12-CL
MUC3SEA

MUC12SEA
MUC12FREG
105 kDa
75 kDa
50 kDa
35 kDa
30 kDa
25 kDa
15 kDa
D
}
}
Fig. 4. Expression and cleavage of the MUC1FDTR hybrid SEA domain mucins in Caco2 cells. Western blots of hybrid mucin, immunopuri-
fied from stably transfected Caco2 clones with M2 anti-FLAG antibody (A, B) or with CT-2 anti-cytoplasmic tail antibody (C, D). Mucin was
separated on 10% SDS ⁄ PAGE gels and probed with M2 (A, C) or with CT-2 (B, D). The brackets in B and D denote C-terminal cleavage
products of the hybrid mucins. Amounts of mucin per lane are as described in the legend for Fig. 2. U denotes material immunopurified with
the relevant antibody from untransfected Caco2 cells. The clones carry the constructs described in Fig. 1.
SEA modules and mucin cleavage T. Palmai-Pallag et al.
2906 FEBS Journal 272 (2005) 2901–2911 ª 2005 FEBS
In a further attempt to improve the efficiency of
cleavage of MUC1FDTR ⁄ MUC12-CL, we next intro-
duced the highly conserved MUC1 FREG sequence
into the partial MUC12 SEA module of the hybrid
protein to replace the native LEAD sequence
(Table 2). Evaluation of the hybrid mucin immunopre-
cipitated with M2 or CT-2 from stably transfected
Caco2 cells showed that substitution of the FREG
sequence did not markedly improve the efficiency of
cleavage of this glycoprotein (Fig. 4A–D, MUC12-
FREG).

Substitution of the SEA domain of MUC1 with
those of MUC3 or MUC12 provide substrates for
mucin cleavage
Rodent MUC3 is apparently cleaved twice during its
post-translational processing, first at the G ⁄ SIVV resi-
due within the SEA domain [11] and subsequently at a
second site that is currently undefined, but is located
at least 10 amino acids C-terminal to the G ⁄ S site [10].
Based on these data and the current observations that
replacement of the small fragment of the SEA domain
(33 amino acids flanking the G ⁄ SVVV motif) resulted
in substantial inhibition of the cleavage, the entire
SEA domain (boundaries established by [1], Table 2)
of MUC1FDTR was replaced with the SEA modules
from human MUC3 and MUC12. The MUC1FDTR ⁄
MUC3SEA and MUC1FDTR ⁄ MUC12SEA constructs
were stably expressed in Caco2 clones.
Western blots of M2-purified MUC1FDTR ⁄ MUC3-
SEA and MUC1FDTR ⁄ MUC12SEA glycoproteins
showed a major M2-reactive and CT-2 reactive non-
cleaved glycoform of about 105–120 kDa (Fig. 4A,C,
MUC3SEA and MUC12SEA) and a minor population
of a CT-2 reactive, M2 nonreactive cleavage product,
at about 25 kDa for MUC3 and 30 kDa for MUC12
(Fig. 4B,D, MUC3SEA and MUC12SEA). There
appear to be 2 forms of the CT-2 reactive peptide in
the MUC3-containing construct, consistent with previ-
ous observations [10]. A slight difference in the mobi-
lity of the major glycoform of MUC1FDTR and
MUC1FDTR ⁄ MUC3SEA or MUC1FDTR ⁄ MUC12-

SEA may be accounted for by the introduction of an
additional N-glycosylation site. These data suggest that
the SEA domains are not completely interchangeable
between different mucins; however, they form a mod-
ule that can be cleaved even when substituted into
another mucin. The cleavage is apparently more effi-
cient in the hybrid mucins carrying the whole SEA
domains rather than the 33 amino acid fragments
encompassing the G ⁄ SIVV motif alone as the ratio of
cleaved to uncleaved protein is greater (Fig. 4B,D),
however, it is still highly inefficient in comparison to
MUC1FDTR (Fig. 4B,D).
Discussion
The function of the SEA domain in the cleavage and
sub–unit association of membrane-tethered glycopro-
teins is well documented but poorly understood. Our
aim was to evaluate the role of this domain in the
processing of membrane-tethered mucins. The mech-
anism by which the heavily glycosylated portion of
these molecules is released at epithelial surfaces within
the airway, the intestine and pancreatic ducts is of
considerable significance in the pathology of a number
of diseases including cystic fibrosis and cancer.
Equally important is the fate of the cytoplasmic tail
of the mucins once the extracellular domain is lost.
This peptide has been shown to associate with b-cate-
nin [7,14], Grb2 [15] and erbB family members [16]
and to be involved in signal transduction (reviewed
in [3]).
We previously determined that there is an important

early cleavage of the MUC1 mucin at an FRPG ⁄
SVVV motif within the SEA module of the protein [9].
Herein we show that mutating G185, S186 or F182 to
alanine inhibits but does not completely block cleavage
at the FRPG ⁄ SVVV motif, whereas mutating R183 or
V189 to alanine does not impair cleavage. Compari-
sons of the relative intensities of the noncleaved and
cleaved forms of the S ⁄ A, G ⁄ A and F ⁄ A mutants sug-
gests that the S ⁄ A mutation has the most dramatic
effect on the cleavage event. Interestingly, the ineffi-
cient cleavage reduces, but does not abolish, the sur-
face membrane targeting of these mucin glycoproteins
(data not shown). The FRPG ⁄ SVVV site and homo-
logous sites in other membrane-tethered mucins, have
recently been studied by several other groups. Muta-
genesis studies by Lillehoj et al. [17] on the MUC1
cleavage site FRPG ⁄ SVVV yielded data that are partly
but not completely consistent with our results. Lillehoj
et al. [17] observed that the serine to alanine mutation
at the cleavage site completely abolished cleavage in
COS7 cells and in several human airway and breast
cancer cell lines that express endogenous MUC1. We
observed the same results in COS7 cells; however, in
contrast to the published findings with human airway
and breast cancer cell lines, when the Ser to Ala
mutant was transfected into the Caco2 colon carci-
noma cell lines that expresses very low levels of endo-
genous MUC1, we observed partial cleavage of the
protein. It is currently not clear whether cleavage at
the G ⁄ SVVV site is protease-mediated or autocatalytic.

If the former is true, then the difference between our
T. Palmai-Pallag et al. SEA modules and mucin cleavage
FEBS Journal 272 (2005) 2901–2911 ª 2005 FEBS 2907
data and those of Lillehoj et al. [17] may reflect the
different spectrum of proteases in different cell types
or species. Alternatively, the failure of Lillehoj et al.
[17] to see cleavage of the Ser to Ala mutant in the
breast and airway lines may be explained by the possi-
bility that the antibodies employed in that study detect
endogenous MUC1 in addition to the transfected
mutant construct. In our experiments, the use of anti-
bodies against a FLAG epitope tag in MUC1FDTR
enabled discrimination between endogenous MUC1
and MUC1FDTR.
The rat MUC3 mucin is cleaved at a site homolog-
ous to the MUC1 site, LSKG ⁄ SIVV and mutagenesis
of the glycine residue to alanine in this sequence
reduced the efficiency of cleavage [10], whereas a serine
to alanine mutation abolished cleavage in COS 7 cells.
In addition to the LSKG ⁄ SIVV mediated cleavage, a
second proteolytic event has been reported in rat
MUC3 [11] and though the precise cleavage site has
not been mapped it is at least 10 amino acids C-ter-
minal to the first site. This is of particular interest as
we, and others, have detected multiple forms of the
cytoplasmic tail of MUC1, MUC1FDTR and its deriv-
atives [9,13]. Further, the migration of the CT2-react-
ive forms of the hybrid mucins shown in the current
work predicts more than one species derived from the
MUC3- and possibly the MUC12-SEA domain. This

raises the possibility of additional cleavage events
within the SEA domain that are probably subsequent
to that at the G ⁄ SVVV. Inspection of the predicted 3D
structure of the SEA domain of MUC1 suggests that
more than one cleavage event is required to release the
extracellular domain of the protein from its cytoplas-
mic tail. Recent data on additional cleavage events for
the MUC1SEA domain in uterine epithelial cells,
mediated by defined proteases including TACE and
MT1-MMP, which are not active at the G ⁄ SVVV site,
provide evidence for this [5,6]. However, it is also poss-
ible that some of the multiple forms of CT2-reactive
peptides seen on western blots are modified by other
post-translational modifications, for example by phos-
phorylation.
When we substituted 33 amino acids flanking the
cleavage site of MUC1FDTR or the whole SEA mod-
ule with equivalent sequences from the SEA module
of MUC3 or MUC12, the efficiency of the cleavage
event was greatly reduced in comparison to WT
MUC1FDTR. Nonetheless, small amounts of cleaved
cytoplasmic tail were detected on western blots for all
hybrid molecules. These observations support the data
from Wang [10] and Khatri [11] that clearly demon-
strate the cleavage of rat MUC3 at the LSKG ⁄ SIVV
site and provide in vivo confirmation for cleavage of
human MUC3 in the SEA domain. Further, our data
provide the first evidence for the cleavage of the
MUC12 mucin SEA domain that would be predicted
to occur at the LLNG ⁄ SIVV site. The greatly reduced

efficiency of the cleavage in the hybrid mucins in com-
parison to that observed for MUC1FDTR might be
accounted for by the 3D structure of the hybrid mole-
cules, which may not allow proper folding and accessi-
bility of the cleavage site. This hypothesis is consistent
with our observations that substitution of the whole
SEA domain is marginally more effective at generating
a substrate for cleavage than are the 33 amino acid
substitutions. Another feature of the hybrid mucins is
that that they lack motifs of the native MUC3 and
MUC12 that may contribute to the generation of the
cleavage substrate, for example the flanking EGF
domains. Alternatively, the SEA modules of the differ-
ent membrane-tethered mucins may not be sufficiently
homologous to allow substitution of one for another.
It is relevant that, due to poor sequence homology, we
were not able to generate a reliable model of the SEA
domain of MUC3 or MUC12 based on the solution
structure of the MUC16 SEA domain derived by
NMR spectroscopy.
In summary, the results presented here demonstrate
that cleavage of membrane associated mucins in the
SEA domain is not entirely sequence dependent, but is
instead related to general structural features of the
SEA domain. Molecular modelling of the MUC1 SEA
domain supports the hypothesis that the cleavage site
exists as an exposed loop that protrudes outward from
an otherwise folded compact structure, which we pre-
dict provides molecular topological features enabling
its recognition and cleavage by cellular proteases.

Experimental procedures
Site directed mutagenesis of the G ⁄ SVVV site of
MUC1 and the LRNG ⁄ SIVV site of human MUC3
Site-specific mutations were carried out using the Quik-
Change site-directed mutagenesis kit (Stratagene, Cedar
Creek, TX, USA). For all constructs a FLAG-epitope-tagged
MUC1 cDNA that lacked native tandem repeat sequences
(MUC1FDTR [18], [13], Fig. 1) was used. The F182, R183,
G185, S186 and V189 of the FRPG ⁄ SVVV site in
MUC1FDTR were mutated individually to alanine using
the following prime r pairs: G ⁄ A substitution: 5¢-GTTCAGGC
CAGCATCTGTGGTGGTACAATTG-3¢ (sense), 5¢ -CAAT
TGTACCACCACAGATGCTGGCCTGAAC-3¢ (antisense),
S ⁄ A substitution: 5¢-GTTCAGGCCAGGAGCTGTGGTG
GTACAATTG-3¢ (sense), 5¢-CAATTGTACCACCACAG
CTCCTGGCCTGAAC-3¢ (antisense), R ⁄ A substitution:
SEA modules and mucin cleavage T. Palmai-Pallag et al.
2908 FEBS Journal 272 (2005) 2901–2911 ª 2005 FEBS
5¢-CTCCAATATTAAGTTCGCGCCAGCATCTGTGGT
GG-3¢ (sense), 5¢-CCACCACAGATCCTGGCGCGAACTT
AATATTGGAG-3¢ (antisense), V ⁄ A substitution: 5¢-CCAGG
ATCTGTGGTGGCACAATT GACT CTG-3 ¢ (sense), 5¢-CAG
AGTCAATTGT GCCACCACAGATCCTGG-3¢ (antisense),
F ⁄ A substitution 5¢-TCTCCAATATTAAGGCCAGGCCA
GGATCTGTGGTG-3¢ (sense), 5¢-CACCACAGATCCTG
GCCTGGCCTTAATATTGGAGA-3¢ (antisense). In the
MUC1FDTR ⁄ MUC3 chimera the glycine in the LRNG ⁄
SIVV motif was mutated to alanine using the following pri-
mer pairs 5¢-CCTGTCCCTGAGGAATGCCAGCATCGT
GGTGGAC-3¢ (sense) and 5¢-GTCCACCACGATGCTGG

CATTCCTCAGGGACAGG-3¢ (antisense). Nucleotides
in bold and underlined represent the base-pair changes
required to generate the appropriate amino acid substitu-
tion. The mutations were confirmed by DNA sequencing.
Constructs were subcloned into the mammalian expression
vector pHb-APr1-neo [19] at the BamH1 site.
Generation of MUC1FDTR constructs containing
MUC3- and MUC12- cleavage sites and the MUC3
and MUC12 SEA domains
All constructs were based on the MUC1FDTR backbone
[18,13], and are illustrated in Fig. 1. Using unique PsiI
(J05582: 3351) and Dra III (J05582: 3421) restriction sites,
33 amino acids (from K170 to V202 inclusive, with
amino acids numbered according to [13]) including the
FRPG ⁄ SVVV proteolytic cleavage site of MUC1FDTR
were replaced with PCR amplified cDNA sequences
from human MUC3 (AF143371) and human MUC12
(AF147790). The primer pair used to produce the MUC3
cDNA insert was (sense) 5¢-ATTTATAAGGGCTTCACC
TTCAAG-3¢ (AF143371 324–339) and (antisense) 5¢-CTCC
ACGTCGTGCTGGGGGCTGAAGGG-3¢ (AF143371 406–
420), the MUC12 cDNA insert was (sense) 5¢-ATTTAT
AATCTTCCTCAGTATAGAGGG-3¢ (AF147790 965–983),
and (antisense) 5¢-CTCCACGTCGTGCTCTAAAGTGTA
GTC-3¢ (AF147790 1047–1061). Mucin-specific sequences
(bold) were extended by eight nucleotides carrying a
PsiI recognition site in the sense primers, or 12 nucleotides
with a DraIII site in the antisense primers. PCR parame-
ters were 30 cycles of 94 °C for 30 s, 60 °C for 30 s, 72 °C
for 1 min with a final extension at 72 °C for 5 min. The

PCR generated fragments were digested with PsiI and
DraIII and cloned into the respective sites of the
plasmid pUC18 ⁄ MUC1FDTR. The mucin constructs were
then transferred as BamHI fragments into the BamHI site
of the mammalian expression vector pHb-APr1-neo
[19]. MUC1FDTR ⁄ MUC12FREG was identical to the
MUC1FDTR ⁄ MUC12-CL construct but with the LEAD
sequence 11 amino acids C-terminal to the G ⁄ S cleavage
site replaced by the FREG sequence that is conserved in
MUC1 proteins from many different species. The following
oligonucleotides were synthesized: M12FREG-F (sense)
5¢-TAATCTTCCTCAGTATAGAGGGGTGAACATTCG
GAGATTGCTCAACGGTAGCAT CGTGGTCAA GAAC
GATGTCATCTTCCGAGA AGGTTACA CTT TAGAGCA
CGAC-3¢ and M12FREG-R (antisense) 5¢-GTGCTCTAA
AGTGTAACCTTCTCGGAAGAT GACATCGTT CTTGA
CCACGATGCTACCGT TGAGCAATCTCCGAATGTTC
ACCCCTCTATACTGAGGAAGATTA)3¢ (AF147790 965–
1061) where the PsiI and DraIII sites are in italics and
the FREG motif in bold. These oligonucleotides were
annealed and cloned into the PsiI ⁄ DraIII sites of
MUC1FDTR.
To replace the SEA domain of MUC1 with those of
MUC3 or MUC12, XcmI sites were used to remove 111
amino acids from V128 to A238 of MUC1FDTR. These
were replaced by 118 amino acids from D61 to S178 of
MUC3 (AF143371) or by 119 amino acids from E273 to
S391 of MUC12 (AF147790). Oligonucleotides were syn-
thesized to encode an XcmI-SpeI-EcoRI-XhoI-XcmI poly-
linker. SEALINK1 (sense) 5 ¢-CTACTGGACTAGTGAA

TTCCTCGAGCCAGTCTG-3¢ and SEALINK2 (antisense)
5¢- AGACTGGCT CGAGGA ATTCAC TAGTCCA GTAGA-3 ¢
were annealed and directly cloned into the XcmI site of
MUC1FDTR. The MUC3 and MUC12 SEA domains were
amplified by PCR using gene-specific primers 3-SEA-F
(sense) 5¢-GGACTAGTAGATGTAGTGGAGACCGAG-3¢
(AF143371 180–198), 3-SEA-R (antisense) 5¢-CCGCTC
GAGTCAGGCTTAAAACACAGG-3¢ (AF143371 513–530),
12-SEA-F 5¢-GGACTAGTGGAAAAACTCAAGGCCACT
TTAGG-3¢ (AF147790 818–841) and 12-SEA-R 5¢-CCGCT
CGAGTAGCACAGTATGGCCTTTC-3¢ (AF147790 1153–
1171). SpeI and XhoI sites were incorporated at the 5¢ end
of the forward and reverse primers, respectively, and
MUC3 and MUC12-specific sequences are shown in bold.
PCR conditions were as described previously. The frag-
ments were then cloned into the SpeI ⁄ XhoI sites of the
polylinker in MUC1FDTR. The remaining portions of the
polylinker introduced leucine and valine residues N-ter-
minal to the MUC3 and MUC12 specific sequences and a
serine residue C-terminal to them.
Transient expression of epitope-tagged mucin
proteins in Cos-7 cells and generation of epitope-
tagged mucin-expressing stable Caco2 cell clones
Cos7 and Caco2 cells were cultured in DMEM containing
10% (v ⁄ v) FBS, penicillin (100 UÆmL
)1
), streptomycin
(100 lgÆmL
)1
). For transient expression, Cos7 cells were

grown to 50% confluence in 90 cm dishes. For each dish,
20 lg of plasmid DNA was transformed by standard meth-
ods using Lipofectin (Invitrogen, Carlsbad, CA, USA) or
calcium phosphate. Cells were lysed 48 h post-transfection
and proteins analysed by western blot. For stable clones
Caco2 cells were transfected with Lipofectin (Invitrogen) by
standard methods and clones selected in G418 (Invitrogen)
at 600 lgÆmL
)1
.
T. Palmai-Pallag et al. SEA modules and mucin cleavage
FEBS Journal 272 (2005) 2901–2911 ª 2005 FEBS 2909
Preparation of cell lysates, immunopurification of
epitope-tagged hybrid-mucins and Western blot
analysis
Cells were lysed in NET buffer (50 mm Tris HCl pH 7.5,
5mm EDTA, 150 mm NaCl) with complete protease inhi-
bitor cocktail (Sigma, St. Louis, MO, USA) at 2–3 day post-
confluence. Epitope tagged mucins were immunopurified
with M2 anti-FLAG conjugated agarose beads (Sigma) as
described previously [13] or with CT2 antibody (directed
against the last 17 amino acids [SSLAYTNPAVAATSANL]
of the cytoplasmic tail of MUC1, kindly donated by
S. Gendler, Mayo Clinic, Scottsdale, AZ, USA [16]) in the
presence of Protein G ⁄ Sepharose (Sigma) at 4 °C. Mucins
were eluted from the beads ⁄ Sepharose with specific peptides.
Immunoprecipitates were subjected to SDS ⁄ PAGE under
reducing conditions, and transferred to Hybond ECL mem-
branes. Mucins immunoprecipitated with an excess of M2
beads or CT2 from 300 lg of total cell lysate were loaded in

each lane. Rainbow marker (Amersham, Chalfont St. Giles,
Bucks, UK) was used as a molecular mass standard. Mem-
branes were incubated with M2 or CT2 and reactive protein
species were detected with the Hybond ECL western Blot
Detection Kit (Amersham).
Mucins released from the cells were collected from cell
culture medium conditioned for 4 days postconfluence. The
media was cleared by centrifugation at 300 g for 10 min
and then immunoprecipitated with 100 lL M2 agarose
beads for 30 mL of culture medium. Immunoprecipitates
were subjected to SDS ⁄ PAGE as described above and vol-
umes were loaded according to the intensity of M2-reactive
species in the corresponding whole cell lysate.
Molecular modeling of SEA domain of MUC1
The composer and biopolymer modules of the sybyl 6.91
software package (Tripos Inc., St Louis, MO, USA) were
used for comparative molecular modelling of the human
MUC1 SEA domain based on the NMR-derived structure
of the murine MUC16 SEA domain (PDB code:1IVZ). Sec-
ondary structure-based multiple sequence alignment [12]
was used to build a model. The modelled structure of the
MUC1 SEA domain was refined by energy minimizations
using the amber (Kollman all-atom) force field implemen-
ted in sybyl. The quality of the structural model was tested
with the protable module of sybyl.
Acknowledgements
We thank Dr S. Williams for the MUC3 and MUC12
partial cDNAs and Dr S. Gendler for the CT2 anti-
body. This work was supported by the Cystic Fibrosis
Trust, UK, a Wellcome Trust Biomedical Research

collaboration grant and NIH Grants 1R01 CA84106
to S.S and CA57326 to M.A.H.
References
1 Bork P & Patthy L (1995) The SEA module: a new
extracellular domain associated with O-glycosylation.
Protein Sci 4, 1421–1425.
2 Wreschner DH, McGuckin MA, Williams SJ, Baruch
A, Yoeli M, Ziv R, Okun L, Zaretsky J, Smorodinsky
N, Keydar I et al. (2002) Generation of ligand-receptor
alliances by ‘SEA’ module-mediated cleavage of mem-
brane-associated mucin proteins. Protein Sci 11, 698–
706.
3 Hollingsworth MA & Swanson B (2004) Mucins in Can-
cer: protection and control of the cell surface. Nat Rev
Cancer 4, 45–60.
4 Ligtenberg MJ, Kruijshaar L, Buijs FM, van Meijer M,
Litvinov SV & Hilkens J (1992) Cell-associated episialin
is a complex containing two proteins derived from a
common precursor. J Biol Chem 267, 6171–6177.
5 Thathiah A, Blobel CP & Carson DD (2003) Tumor
necrosis factor-alpha converting enzyme ⁄ ADAM 17
mediates MUC1 shedding. J Biol Chem 278, 3386–
3394.
6 Thathiah A & Carson DD (2004) MT1-MMP mediates
MUC1 shedding independent of TACE ⁄ ADAM17.
Biochem J 382, 363–373.
7 Wen Y, Caffrey TC, Wheelock MJ, Johnson KR &
Hollingsworth MA (2003) Nuclear association of the
cytoplasmic tail of MUC1 and beta-catenin. J Biol
Chem 278, 38029–38039.

8 Lan MS, Batra SK, Qi WN, Metzgar RS & Hollings-
worth MA (1990) Cloning and sequencing of a human
pancreatic tumor mucin cDNA. J Biol Chem 265,
15294–15299.
9 Parry S, Silverman HS, McDermott K, Willis A,
Hollingsworth MA & Harris A (2001) Identification of
MUC1 proteolytic cleavage sites in vivo. Biochem Bio-
phys Res Commun 283, 715–720.
10 Wang R, Khatri IA & Forstner JF (2002) C-terminal
domain of rodent intestinal mucin Muc3 is proteolyti-
cally cleaved in the endoplasmic reticulum to generate
extracellular and membrane components. Biochem J
366, 623–631.
11 Khatri IA, Wang R & Forstner JF (2003) SEA (sea-
urchin sperm protein, enterokinase and agrin) –module
cleavage, association of fragments and membrane tar-
geting of rat intestinal mucin Muc3. Biochem J 372,
263–270.
12 Maeda T, Inoue M, Koshiba S, Yabuki T, Aoki M,
Nunokawa E, Seki E, Matsuda T, Motoda Y, Kobaya-
shi A et al. (2004) Solution structure of the SEA
domain from the murine homologue of ovarian cancer
antigen CA125 (MUC16). J Biol Chem 279, 13174–
13182.
13 Silverman HS, Parry S, Sutton-Smith M, Burdick MD,
McDermott K, Reid CJ, Batra SK, Morris HR,
SEA modules and mucin cleavage T. Palmai-Pallag et al.
2910 FEBS Journal 272 (2005) 2901–2911 ª 2005 FEBS
Hollingsworth MA, Dell A & Harris A (2001) In vivo
glycosylation of mucin tandem repeats. Glycobiology 11,

459–471.
14 Yamamoto M, Bharti A, Li Y & Kufe D (1997) Inter-
action of the DF3 ⁄ MUC1 breast carcinoma-associated
antigen and beta-catenin in cell adhesion. J Biol Chem
272, 12492–12494.
15 Pandey P, Kharbanda S & Kufe D (1995) Association
of the DF3 ⁄ MUC1 breast cancer antigen with Grb2
and the Sos ⁄ Ras exchange protein. Cancer Res 55,
4000–4003.
16 Schroeder JA, Thompson MC, Gardner MM & Gendler
SJ (2001) Transgenic MUC1 interacts with epidermal
growth factor receptor and correlates with mitogen-acti-
vated protein kinase activation in the mouse mammary
gland. J Biol Chem 276, 13057–13064.
17 Lillehoj EP, Han F & Kim KC (2003) Mutagenesis
of a Gly-Ser cleavage site in MUC1 inhibits ecto-
domain shedding. Biochem Biophys Res Commun 307,
743–749.
18 Burdick MD, Harris A, Reid CJ, Iwamura T & Hol-
lingsworth MA (1997) Oligosaccharides expressed on
MUC1 produced by pancreatic and colon tumor cell
lines. J Biol Chem 272, 24198–24202.
19 Gunning P, Leavitt J, Muscat G, Ng SY & Kedes L
(1987) A human beta-actin expression vector system
directs high-level accumulation of antisense transcripts.
Proc Natl Acad Sci USA 84, 4831–4835.
T. Palmai-Pallag et al. SEA modules and mucin cleavage
FEBS Journal 272 (2005) 2901–2911 ª 2005 FEBS 2911