Isolation and characterization of MUC15, a novel
cell membrane-associated mucin
Lone T. Pallesen, Lars Berglund, Lone K. Rasmussen, Torben E. Petersen and Jan T. Rasmussen
Protein Chemistry Laboratory, Department of Molecular and Structural Biology, University of Aarhus, Denmark
The present work reports isolation and characterization of a
highly glycosylated protein from bovine milk fat globule
membranes, known as PAS III. Partial amino-acid sequen-
cing of the purified protein allowed construction of degen-
erate oligonucleotide primers, enabling isolation of a
full-length cDNA encoding a protein of 330 amino-acid
residues. N-terminal amino-acid sequencing of derived
peptides and the purified protein confirmed 76% of the
sequence and demonstrated presence of a cleavable signal
peptide of 23 residues, leaving a mature protein of 307 amino
acids. Database searches showed no homology to any other
proteins. A survey of the human genome indicated the
presence of a corresponding gene on chromosome band
11p14.3. Isolation and sequencing of the complete cDNA
sequence of the human homologue proved the existence of
the gene product (334 amino-acid residues). This novel
mucin-like protein was named MUC15 by appointment of
the HUGO Gene Nomenclature Committee. The deduced
amino-acid sequences of human and bovine MUC15 dem-
onstrated structural hallmarks characteristic for other
membrane-bound mucins, such as a serine, threonine, and
proline-rich extracellular region with several potential
glycosylation sites, a putative transmembrane domain, and a
short cytoplasmic C-terminal. We have shown the presence
of O-glycosylations, identified N-glycosylations at 11 of 15
potential sites in bovine MUC15, and a splice variant
encoding a short secreted mucin. Finally, analysis of human

respiratory tissue and fallopian tube, respectively. The
family of epithelial membrane-associated mucins includes
MUC1,-3,-4,-12,-13and-16.MUC3,MUC11,and
MUC12 have been located to chromosome 7q22 suggesting
the presence of yet another cluster of mucin genes. It should,
however, be noted that only partial sequences are known for
the MUC11 and MUC12 genes and that it is possible that
they are produced as a result of alternative splicing of a
single, large mucin gene [13]. Human MUC1 was the first
mucin to be cloned and is to date probably the best
characterized of the mucins. Generally, MUC1 is expressed
on the apical cell surface of nearly all polarized epithelial
tissues that line ducts and glands, e.g. mammary gland [18].
MUC1 is found to be a major constituent of human and
bovine milk fat globule membranes (MFGM) surrounding
the lipid droplets secreted from the mammary gland
epithelial cells [19,20].
Bovine MFGM has been shown to contain another
heavily glycosylated mucin-like glycoprotein with high
molecular mass, named PAS III. Glycoprotein C, Glyco-
protein 4, Component II and PAS3, are alternative names
that have been used for this glycoprotein as well [21]. This
poorly characterized glycoprotein has been named accord-
ing to its mobility upon separation by SDS/PAGE and
ability to stain with periodic acid-Schiff’s reagent (PAS) [22].
The protein appears heterodisperse with apparent molecular
Correspondence to J. Trige Rasmussen, Protein Chemistry
Laboratory, Gustav Wieds Vej 10C, 8000 Aarhus C, Denmark.
Fax: + 45 86136597, Tel.: + 45 89425093,
E-mail:

MFGM was prepared as described by Hvarregaard et al.
[24] using the cream fraction of freshly collected unpasteur-
ized bovine milk samples. Bovine MUC15 was purified
from MFGM using a method essentially as the one used for
isolation of bovine MUC1 [20]. Briefly, MFGM proteins
were extracted from the membranes using the nonionic
detergent Triton X-100. Extracted proteins were subjected
to cation- and anion-exchange chromatography on CM-
Sepharose and DEAE-Sepharose columns, respectively
(Amersham Pharmacia Biotech, Uppsala, Sweden).
MUC15 containing fractions were dialyzed, freeze-dried,
and finally subjected to further purification by reverse-phase
chromatography using a 1-mL Resource RPC column
(Amersham Pharmacia Biotech) with a gradient of
2-propanol in 20% formic acid. MUC15 containing sam-
ples appearing at 48% 2-propanol were collected and freeze-
dried. Standard procedures were employed analysing pro-
tein samples by SDS/PAGE using 18% polyacrylamide
gels, and for the staining of proteins using Coomassie
Brilliant Blue R-250 and PAS reagent.
Peptide mapping of bovine MUC15
Bovine MUC15 peptides were generated by enzymatic
cleavage of the purified protein with trypsin (Worthington
Biochemical Corp., Lakewood, NJ, USA) for 4 h at 37 °C.
Resulting peptide mixtures were separated by RP-HPLC on
aVydacC18column(4· 250 mm, Vydac, Hesperia, CA)
using a linear gradient of acetonitrile (0–80%) in 0.1%
trifluoroacetic acid. Selected peptide fractions were further
purified by reverse-phase chromatography on a Sephacil C8
SC 2.1/10 column (Amersham Pharmacia Biotech) using

Protein Sequencer (Applied Biosystems, Foster City, CA,
USA) with online identification of the phenylthiohydantoin
derivatives. N-glycosylation sites were assigned to aspara-
gine residues lacking an identifiable phenylthiohydantoin
derivative during amino-acid sequencing of glycosylated
samplesorshowingupasasparticacidinPNGaseFtreated
MUC15 peptides.
Cloning of the bovine MUC15 cDNA by PCR
with degenerate primers
Isolation of total RNA from the mammary gland of a
lactating Danish Holstein cow was performed by means of
an RNeasy kit (Qiagen, Hilden, Germany). Synthesis of
cDNA was performed by oligo(dT) primed reverse tran-
scription of the isolated total RNA using M-MLV Reverse
Transcriptase (Life Technologies, Inc., Gaithersburg, MD,
USA) in accordance with the manufacturer’s instructions.
Six degenerate oligonucleotides were synthesized corres-
ponding to partial bovine MUC15 amino-acid sequences
obtained by peptide mapping and N-terminal sequencing of
the mature protein (DNA Technology, Aarhus, Denmark):
P1, 5¢-GARGARGGICARAARAC-3¢ (forward), corres-
ponding to the amino-acid sequence E(24)EGQKT(29)
(residues underlined in Fig. 1B); P2, 5¢-AARACNATGGA
RAAYCA-3¢ (forward), K(40)TMENQ(45); P3, 5¢-TCYT
TRTCISWIGTIARRTT-3¢ (reverse), N(54)LTSDKE(60);
P4, 5¢-GGYTCRTTICKRTCRTCRTA-3¢ (reverse), Y(271)
DDRNEP(277); P5, 5¢-CATRTCRTAIGGYTCIGGNG
C-3¢ (reverse), A(284)PEPYDM(290); P6, 5¢-GCNGTIGG
RTTRTARTA-3¢ (reverse), Y(297)YNPTA(302); where
R ¼ AorG,Y¼ CorT,K¼ GorT,S¼ Cor

M
of the specific P7 primer and degenerate primers
(P4-P6), respectively. The cDNA sequence of the bovine
MUC15wasextendedinboth5¢ and 3¢ directions by PCR
screening of an oligo(dT) primed mammary gland Uni-ZAP
cDNA library, derived from a lactating Holstein cow
(Stratagene, La Jolla, CA, USA), using MUC15-specific
and library vector primers. The full-length cDNA was
obtained sequencing overlapping clones and PCR products
derived by RT-PCR on the isolated RNA from the Danish
Holstein cow. The bovine MUC15 cDNA was sequenced
on both strands using a BigDye Sequencing kit and an ABI
PRISM 310 Genetic Analyser (Applied Biosystems).
Identification of the human MUC15 cDNA
The bovine MUC15 nucleotide sequence was employed in a
BLASTn search of the human genome database at NCBI,
and a match was found on a Ôchromosome 11 working draft
sequence segmentÕ (GenBank accession number
NT_008952). Identified partial sequences of the putative
human homologue were examined and specific PCR
primers were designed enclosing the coding sequence of
the bovine protein. To investigate the presence of MUC15
expression in epithelial cells of the human mammary gland,
we proceeded to isolate the cellular fraction of human milk
samples obtained from four lactating women at different
stages in the lactation. Samples were collected immediately
after milking and stored on ice. Milk cells were harvested by
centrifugation at 3200 g for 20 min at 4 °C, and the cellular
fraction was washed in NaCl/P
i

(human MTC Panel II, Cat. # K1421-1 and human
Immune System MTC Panel, Cat. # K1426-1, Clontech,
Palo Alto, CA, USA). The panels contained normalized,
first-strand cDNA preparations generated from each of the
following human tissues and cell types: spleen, thymus,
prostate, testis, ovary, small intestine, colon, peripheral
Fig. 1. Purification of bovine MUC15 and obtained tryptic peptidemap.
(A) RP-HPLC chromatography of bovine milk fat globule membrane
proteins eluted from the DEAE column. Separation was performed on
a 1-mL Resource RPC column with a linear gradient of 0–80%
2-propanol in 20% formic acid at 40 °C (dotted line). Proteins were
monitored at 278 nm (solid line). The peaks containing MUC1 and
MUC15 are indicated. (B) RP-HPLC separation of peptides generated
by trypsin digestion of bovine MUC15. Peptides were eluted from a
Vydac C18 column using a linear gradient from 0 to 80% acetonitrile
in 0.1% trifluoroacetic acid (dotted line), and monitored at 226 nm
(solid line). Amino-acid sequences of labelled peaks are shown.
Underlining indicates amino-acid residues used for design of degen-
erate oligonucleotide primers.
Ó FEBS 2002 MUC15, a novel membrane-associated mucin (Eur. J. Biochem. 269) 2757
blood leukocyte, bone marrow, fetal liver, lymph node, and
tonsil. Further tissue specific studies were performed by
PCR screening of oligo(dT) primed cDNA libraries of
bovine lymph node, bovine lung, human lung (Stratagene),
and human breast tissue (Clontech). Specific bovine and
human MUC15 primer sets were employed in the PCR
screening reactions. PCR products were separated by
electrophoresis on 1% agarose gels, visualized with ethi-
dium bromide and finally sequenced.
RESULTS

was constructed. By additional use of degenerate and
specific MUC15 primers, a full-length cDNA sequence was
obtained. Reported nucleotide sequence data are available
from the EMBL Nucleotide Sequence Database under the
accession number AJ417816.
Analysis of the obtained full-length cDNA sequence
(3125 nucleotides in total) showed the presence of an open
reading frame encoding a protein of 330 amino-acid
residues (Fig. 3). Approximately 76% of the cDNA-enco-
ded amino-acid sequence was confirmed by N-terminal
sequencing of the mature protein and enzymatic generated
peptides (Fig. 3, underlined residues). The proposed trans-
lational start codon (ATG) follows a 5¢ untranslated
sequence of 120 nucleotides. The translational stop codon
(TAA), positioned at residues 1111–1113, is followed by a
3¢ untranslated sequence of 1994 nucleotides, including a
polyadenylation signal (AATAAA) (position 3085–3090)
Fig. 2. SDS/PAGE analysis of purified bovine MUC15. Analysis was
performed on 18% Tris/glycine polyacrylamide gels. Positions of
molecular mass standards are indicated to the left. Gels were stained
with periodic acid-Schiff’s reagent (PAS). Lane 1, bovine milk fat
globule membrane proteins (MFGM); lane 2, fraction from the
Resource RPC column containing purified bovine MUC15; lane 3,
neuraminidase and O-glycosidase treated bovine MUC15; lane 4,
PNGase F treated bovine MUC15; lane 5, neuraminidase treated
bovine MUC15.
Fig. 3. Alignment of the deduced amino-acid sequences of bovine and human MUC15. Fully conserved residues are indicated with black boxes.
Amino-acid sequence obtained by peptide mapping and Edman degradation of the bovine protein is underlined. Identified bovine N-glycosylation
sites are marked with asterisks and arrows indicate the signal peptide and transmembrane region. The alignment was performed using the
BIOLOGY

although the massive glycosylation most likely affects the
electrophoretic migration of the protein. Removal of sialic
acid by neuraminidase resulted in a slight decrease in the
mobility of bovine MUC15 in SDS/PAGE (Fig. 2). Pres-
ence of O-linked glycans was shown by incubating neura-
minidase treated protein with O-glycosidase, which reduced
the relative molecular mass (Fig. 2). This indicates the
presence of core-1 O-linked glycans, as O-glycosidase
specifically liberates Galb1–3GalNAc from serine and
threonine residues. Upon PNGase F treatment, the appar-
ent molecular mass of MUC15 shifted from 100 kDa to
approximately 80 kDa (Fig. 2), demonstrating ample pres-
ence of N-linked glycans. Hydrolysis of the Asn-oligosac-
charide linkage by PNGase F leads to deamination of
asparagine to aspartic acid [25]. This facilitates identification
of N-glycosylation sites during amino-acid sequencing, as
an Asp-phenylthiohydantoin derivative is seen instead of
the unidentifiable glycosylated asparagine derivative. Fol-
lowing sequence analysis of the generated peptides, 11 of the
15 possible sites in bovine MUC15 showed to contain
N-linked glycosylations (marked with asterisks in Fig. 3).
Identification and cloning of the human MUC15 cDNA
In order to investigate the existence of a human MUC15
homologue, the bovine MUC15 nucleotide sequence was
employed in a search of the human genome database, and a
similar sequence was located. The milk cell fraction of
lactating tissue contains bud-off epithelial cells, enabling
performance of an indirect assay for expression of this
possible human homologue in mammary epithelium.
RT-PCR was performed on the RNA isolated from the

By comparison of the human MUC15 cDNA sequence
with the working draft sequence version of the human
genome, available from the NCBI, homologous sequences
were located on chromosome 11 (p14.3 region). With two
minor exceptions, the derived and genomic sequences were
identical. These differences correspond to nucleotide vari-
ations observed at positions 495 (a–g polymorphism) and
827 (t–c polymorphism), the latter causing an amino-acid
change from Ile to Thr (residue 202 in Fig. 3). Comparing
the obtained human MUC15 cDNA and the genomic
sequence revealed the boundaries of five exons and four
introns (Fig. 4A). The signal peptide and the major part of
the extracellular part are encoded by a single exon (exon 3),
which is followed by a 150-bp exon encoding the trans-
membrane domain (exon 4). Nucleotides encoding the
cytoplasmic domain span exons 4 and 5, which also contain
the stop codon as well as a 274-bp 3¢ untranslated region.
Alternatively splicing and expression pattern of MUC15
Database searches showed that MUC15 is widely expressed,
as numerous human EST clones have been isolated from
fetal liver and spleen, fetal ear, placenta, lung, pancreas and
kidney (e.g. accession numbers; H53268, BI491080,
BG434403, BG485125, AA386131, BG425830). By PCR
screening of human MTC panels using MUC15-specific
primers we have also demonstrated human MUC15 mRNA
expression in a wide range of tissues; adult human spleen,
thymus, prostate, testis, ovary, small intestine, colon,
peripheral blood leukocyte, bone marrow, lymph node,
tonsil, and fetal liver. Furthermore, PCR screening of
bovine and human cDNA libraries showed the presence of

Ovary
a
–++
Peripheral blood leukocyte
a
–++
Prostate
a
–++
Small intestine
a
–++
Spleen
a
–++
Testis
a
–++
Thymus
a
–++
Bone marrow
a
–+ND
Fetal liver
a
–++
Lymph node
a
–+ND

Lung
c
–+
d
ND
Lymph node
c
–+
d
ND
a
Primer pair: 5¢-AATACCAAAGAAGCCTACAATG-3¢ and 5¢-GTACGAAGTGGAGGTATGTCATC-3¢.
b
Primer pair: 5¢-GCCATTT
TAGGTGCTATTCTGG-3¢ and 5¢-TATTTTCTTTATCTGAGTTTA-3¢.
c
Primer pair: 5¢-CATCCATAGCAGATAACAGTC-3¢ and 5¢-T
CCCAAAGCTCATGTCATAAG-3¢.
d
Generated PCR products have been additionally verified by nucleotide sequencing.
2760 L. T. Pallesen et al. (Eur. J. Biochem. 269) Ó FEBS 2002
showed deletion of a segment of 150 nucleotides, corres-
ponding to the entire exon 4 of the human homologue
encoding the transmembrane domain. This variant was
called MUC15/S in analogy with the secreted short variant
of human MUC1 (GenBank accession number AF348143).
Thus, bovine MUC15/S encodes a potential secreted mucin
of 257 amino acids with a calculated molecular mass of
27 842 Da.
DISCUSSION

endomucin-2 [26]. Nevertheless, the extracellular region of
MUC15 and traditional mucin tandem repeat domains
share the same characteristics with long extended sequences
devoid of secondary structure and great potential for
extensive glycosylation.
TreatmentofbovineMUC15withO-glycosidase dem-
onstrated presence but not extend of O-glycosylation. Until
now no specific motif for O-glycosylation has been identi-
fied, however, proline is preferentially positioned in prox-
imity to the glycosylation site and especially in the )1 and/or
+3 positions [27]. According to the NetOGlyc server,
predicting mucin-type O-glycosylations using the algorithm
of Nielsen et al. [28], the extracellular region of bovine and
human MUC15 offer 22 and 14 O-glycosylation sites,
respectively. The majority of these potential O-glycosylation
sites are positioned in the central part of the extracellular
region, which also contains 10 predicted N-glycosylation
motifs in human MUC15 and 15 in the bovine counterpart.
Interestingly, doubly glycosylated Asn-Xaa-Ser/Thr motifs
have been reported, illustrating that N-glycosylations do not
hinder O-glycosylation of the surrounding serine and
threonine residues [29]. There is limited information avail-
able regarding the actual presence of N-linked oligosaccha-
rides in mucins. So far, N-glycans have only been identified
on bovine MUC1 together with human MUC2 and
MUC5AC [20,30,31]. Moreover, N-glycosylations are likely
to be present on human MUC3, MUC4, MUC7, MUC12,
MUC13, and MUC16 [10,13–15,32,33]. The present inves-
tigation shows that bovine MUC15 is N-glycosylated in 11
out of 15 potential sites.

of membrane-associated mucins has previously been repor-
ted. Experiments have shown that the nascent RNA
transcripts of the MUC1, MUC3, and MUC4 genes, are
spliced in an alternative manner possibly forming soluble
molecules that are secreted rather than retained on the cell
surface [18,35,36]. Recently, the membrane-associated
mucin MUC16 was found to be secreted from ovarian
tumours and cell lines by an unknown mechanism, however,
obtained results indicated that an alternative spliced variant
without the transmembrane region might exist [15]. More-
over, immunohistochemistry studies have demonstrated the
MUC13 protein within goblet cell thecae, indicative of
secretion in addition to presence on the cell surface [14]. To
this point, conclusive data showing that the MUC3, MUC4,
MUC13 and MUC16 mucins exist in both membrane-
associated and nonmembrane soluble forms are still miss-
ing. Likewise, at present there is no documentation for the
existence of the splice variant of MUC15 at the protein level.
The significance of the potential coexistence of MUC15
splice variants is unclear. However, the MUC1/SEC secre-
ted form of MUC1, devoid of the transmembrane and
cytoplasmic domain, has been found to constitute a cognate
binding protein for MUC1/Y, which lacks the tandem
repeat region. MUC1/SEC interacts with the extracellular
domain of MUC1/Y, resulting in the phosphorylation of the
Ó FEBS 2002 MUC15, a novel membrane-associated mucin (Eur. J. Biochem. 269) 2761
cytoplasmic domain of MUC1/Y and a concomitant change
in cell morphology [37]. These results suggest a mechanism
whereby alternative splicing regulates the relative levels of
both the receptor and its secreted cognate binding protein,

characterized as epithelia-specific, some membrane-associ-
ated mucins are also expressed in immune and hematopoi-
etic cells.
ACKNOWLEDGEMENTS
We express our thanks to Margit Skriver Rasmussen, Parisa
Mabhout and Marian Dyrberg Andersen for technical assistance,
Arla Innovation Centre, Brabrand, Denmark, for supplying the
bovine milk samples, and Department of Pediatrics, Aarhus
University Hospital, Skejby, Denmark for establishing contact to
the human milk donors. This work is part of the FØTEK program
supported by the Danish Government and the Danish Dairy
Research Foundation.
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