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

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

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

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

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

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

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%)

(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

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-

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

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-

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.

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

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

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

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

collaboration grant and NIH Grants 1R01 CA84106
to S.S and CA57326 to M.A.H.
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