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 and bovine cDNA panels and libraries showed MUC15 gene expression in adult human spleen, thymus, prostate, testis, ovary, small intestine, colon, peripheral blood leukocyte, bone marrow, lymph node, tonsil, breast, fetal liver, bovine lymph nodes and lungs of both species. Keywords: MUC15; amino-acid sequence; bovine and human cDNA; splice variant; N-glycosylation. Mucins are a heterogeneous family of high molecular mass proteins that are broadly defined by their high content of carbohydrates (50–90%), which are mainly O-linked but in some cases also N-linked. These glycoproteins are major constituents of the mucus covering the surfaces of epithelial organs and they provide selective physical barriers protect- ing the underlying epithelium. Mucins are known to be expressed in various epithelia. Nevertheless, the overall expression patterns have not been completely elucidated (reviewed in [1,2]). To date 15 human mucin genes encoding epithelial mucin type proteins have been identified: MUC1, -2, -3A, -3B, -4, -5AC, -5B, -6, -7, -8, -9, -11, -12, -13, and -16 [3–15]. In addition, two mouse mucin genes, MUC10 and MUC14, have been isolated ([16], GenBank accession number NM_016885). Mucins can be divided into at least two structurally and functionally distinct classes, the secreted (gel-forming or nongel-forming) mucins and the membrane-associated mucins. Four of the secreted mucins are encoded by a cluster of genes (MUC2, MUC5AC, MUC5B and MUC6) contained within a 400-kb genomic DNA fragment on chromo- some 11 band p15.5 [17]. The MUC7, MUC8 and MUC9 are relatively small mucins expressed in the salivary gland, 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: trige@imsb.au.dk Abbreviations: PAS, periodic acid-Schiff’s; MFGM, milk fat globule membrane; MTC, multiple tissue cDNA. Note: reported nucleotide sequences are available from the EMBL Nucleotide Sequence Database under the accession numbers AJ417816, AJ417817 and AJ417818. (Received 5 December 2001, revised 13 March 2002, accepted 22 April 2002) Eur. J. Biochem. 269, 2755–2763 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02949.x mass ranging from 95 to over 100 kDa in polyacrylamide gels. Antibody staining of sections from bovine prelactating and lactating mammary gland using monoclonal and polyclonal antibodies has shown that PAS III is largely concentrated on apical surfaces of the mammary epithelial cells [23]. The present work was initiated in order to isolate and characterize the bovine mucin-like glycoprotein PAS III. A purification method has been established, together with a determination of the complete amino-acid sequence enco- ded by the corresponding cDNA. In addition, the cDNA encoding the human homologue has been isolated and sequenced, thereby identifying a novel human transmem- brane mucin gene named MUC15 by appointment of the HUGO Gene Nomenclature Committee. Presence of O-glycosylation and sites of N-glycosylation in bovine MUC15/PAS III have been determined, and a splice variant encoding a short secreted mucin was identified. Finally, PCR on cDNA panels revealed MUC15 expression in a variety of human tissues. MATERIALS AND METHODS Purification of bovine MUC15 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 the same gradient. Additional peptides were produced treating unmodified or deglycosylated MUC15 with five different proteases independently [Staphylococcus aureus V8 protease, (Worthington Biochemical Corp.), endopeptidase LysC (Roche, Basel, Switzerland), thermolysin, chymotryp- sin or elastase (Sigma, St Louis, MO, USA)] and successively purifying generated peptides by RP-HPLC as described above. Deglycosylation of bovine MUC15 was achieved by an initial treatment with neuraminidase (Roche) in 50 m M ammonium acetate, pH 5.0 at 37 °C for 18 h. After that, N-linked oligosaccharides were removed with peptide-N 4 -(acetyl-b-glucosaminyl)-asparagine amidase (PNGase F; Roche) in 50 m M sodium phosphate, pH 7.5, 0.5% SDS, 5 m M dithioerythritol, 2% octyl-glycopyrano- side for 18 h at 37 °C. Finally, a part of this material was treated with endo-a-N-acetylgalactosaminidase (O-glycosi- dase; Sigma) in 50 m M sodium citrate, pH 6.0 at 37 °Cfor 20 h. Purified MUC15 and resolved peptide fragments were subjected to N-terminal amino-acid sequencing by automa- ted Edman degradation by means of an ABI 477 A/120 A 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 G, W ¼ AorT,N¼ A, G, C or T, and I ¼ deoxyin- deoxyinosine. The degenerate primers were employed in PCR amplifications of cDNA performed in a total volume of 25 lL containing 0.4 m M dNTPs, 2.5 lL10· PCR buffer, 2.5 U of HotStarTaq polymerase (HotStarTaq Master Mix Kit, Qiagen), 5 lL first-strand cDNA and 4 l M each of the forward and reverse degenerate primers. After a 15-min, 95 °C activation step of the HotStarTaq DNA polymerase, amplification was performed as follows: five cycles of denaturation at 94 °C for 45 s, annealing at 46 °C for 45 s and extension at 72 °C for 120 s, followed by 35 amplification cycles with an annealing temperature of 50 °C. Obtained PCR products were cloned into pCR 2.1- TOPO cloning vectors using the TOPO TA Cloning Kit (Invitrogen, Groningen, the Netherlands). Sequencing inserts from 50 positive clones a single was found to contain a MUC15 fragment of 62 nucleotides generated with the P2 2756 L. T. Pallesen et al. (Eur. J. Biochem. 269) Ó FEBS 2002 and P3 primers. From the obtained MUC15 nucleotide sequence a single specific oligonucleotide primer was designed: P7, 5¢-CAATCTGTCCCTTTAGA-3¢ (forward). The major part of the coding sequence was then cloned and sequenced using PCR as described above with 0.4 l M and 4 l 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 buffer and processed for total RNA purification using a RNeasy Blood kit (Qiagen). Synthesis of cDNA was performed by oligo(dT) primed reverse transcription of the mRNA isolated from milk cells using M-MLV Reverse Transcriptase. Using the specific primers in RT-PCR a 1501 base pair cDNA composite of the human gene was obtained. RT-PCR products were purified using a Jetquick PCR Purification Spin Kit (Genomed, Bad Oeynhausen, Germany) and sequenced as described for the bovine counterpart. Detection of an alternatively spliced MUC15 variant First-strand cDNA was prepared from the mammary gland RNA of a Danish Holstein cow using M-MLV Reverse Transcriptase as described above. Specific forward and reverse primers were designed to produce a PCR product of 513 bp containing the transmembrane domain: 5¢-CATCC ATAGCAGATAACAGTC-3¢ (forward) and 5¢-TCCCA AAGCTCATGTCATAAG-3¢ (reverse) corresponding to amino-acid residues S(123)SIADNSL(130) and P(287)YD MSFGN(294), respectively (see below). The PCR products were subjected to DdeI restriction enzyme treatment (Roche) following standard procedures, and a second round of PCR was performed using the same primers. Obtained PCR products were ligated into pCR 2.1-TOPO cloning vectors and sequenced on both strands. MUC15 expression analysis MUC15 mRNA expression was examined in a variety of tissues and cell types by PCR screening. The screening analysis was performed using commercial multiple tissue cDNA (MTC) panels of fetal and adult human tissues (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 Purification of bovine MUC15 Bovine MUC15 copurifies with MUC1 during the initial steps of the protocol designed for isolation of the latter mucin from MFGM [20]. Complete separation was achieved by RP-HPLC on a Resource RPC column with a gradient of 2-propanol in 20% formic acid (Fig. 1A). The bovine MUC15 eluted at approximately 48% 2-propanol, and the purity of this fraction was confirmed by SDS/ PAGE (Fig. 2). N-terminal amino-acid sequencing of the isolated mature bovine MUC15 was performed and revealed a segment of 30 residues (EEGQKTXTTESTAED LKTMENQSVPLESKA), which did not show similarity to any known sequences as revealed by BLASTP and FASTA 3 homology searching of databases accessed through the NCBI and EBI, respectively. Sequence description of bovine MUC15 To obtain sequence information from peptide mapping, purified bovine MUC15 was subjected to enzymatic diges- tion with trypsin. Generated tryptic peptides were separated by RP-HPLC, and subjected to N-terminal amino-acid sequencing (Fig. 1B). To enable deduction of the complete amino-acid sequence of bovine MUC15 by cDNA cloning, six degenerate oligonucleotide primers were designed from the acquired partial amino-acid sequences. After RT-PCR on mammary gland mRNA of a Danish Holstein cow a single MUC15 fragment was cloned and a specific primer 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 WORKBENCH 3.2, San Diego Supercomputer Center, University of California, San Diego. EMBL Accession Numbers: bovine (AJ417816), human (AJ417818). 2758 L. T. Pallesen et al. (Eur. J. Biochem. 269) Ó FEBS 2002 and a poly(A) tail of 18 nucleotides. Two alternative poly(A) signals [A(1259)TAAA and A(1430)ATTAAA] giving rise to poly(A) tails were observed by PCR-screening of the bovine mammary gland cDNA library. The N-terminal amino-acid sequencing of purified bovine MUC15 revealed Glu24 as the initial residue of the mature protein, showing that the preceding 23 residues comprise a cleavable signal peptide. Computer analysis of the transla- ted protein sequence suggested presence of a single mem- brane-spanning domain (residues 234–256, Fig. 3), giving rise to a type 1 integral membrane protein spanning the plasma membrane once. The protein appears to be oriented with an intracellular C-terminal region of 74 residues (residues 257–330) and an extracellular N-terminal part (amino acids 24–233, Fig. 4). The N-terminal region of MUC15, rich in serine, threonine and proline residues, contains 15 consensus motifs for N-glycosylation and numerous potential O-glycosylation sites. N- and O-glycosylation of bovine MUC15 The calculated average molecular mass of the mature MUC15 at 33 317 Da is quite distant from the approxi- mately 100 kDa extrapolated from the electrophoretic mobility (Fig. 2). The heavy glycosylation, suggested by the staining behaviour of the protein, might explain at least a part of this discrepancy. The carbohydrate might thereby constitute up to 67% of the relative molecular mass, 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 cellular fractions of milk obtained from four lactating women, and expression of a human MUC15 mRNA transcript was shown in all samples. Examination of the obtained cDNA sequence (1501 nucleotides in total, EMBL accession number AJ417818) showed the presence of an open reading frame encoding a protein of 334 amino-acid residues (Fig. 3). Analysis of the coding sequence of human MUC15 suggested that it contains a signal peptide (amino acids 1– 23), an extracellular Ser, Thr, Pro, Leu and Asn rich area (residues 24–237) containing 10 N-glycosylation motifs and numerous possible O-glycosylation sites, a transmembrane domain (residues 238–260), and a short cytoplasmic C-terminal (residues 261–334). Thus, the mature human Fig. 4. Schematic representation of MUC15. (A) Schematic representation of the human MUC15 gene. Nucleotide positions (in AJ417818) are indicated by numbers. Exons and introns are indicated by E and I, respectively. Intron sizes are given in parentheses. Shaded boxes represent the coding regions whereas white boxes indicate the noncoding regions. (B) Schematic representation showing the organization of the bovine MUC15 protein: The 23 amino-acid signal peptide (SP), the extracellular Ser, Thr, and Pro rich region, the transmembrane domain (TM), and the cytoplasmic C-terminal (CYT). Positions of the domains are indicated with amino-acid numbers. Identified N-glycosylation sites are marked with hexagons. The protein is oriented with an exoplasmic N-terminal and a cytoplasmic C-terminal tail. The 50-amino-acid region skipped in the MUC15/S splice variant is shown. Ó FEBS 2002 MUC15, a novel membrane-associated mucin (Eur. J. Biochem. 269) 2759 MUC15 is proposed to comprise 311 amino acids with a calculated average mass of 33 875 Da. Alignment of the bovine and human MUC15 sequences showed 67% similarity (Fig. 3). The majority of the differ- ences exist in the extracellular part where similarity with the bovine mucin is only 59%. The similarity rises to 87% in the transmembrane domain and cytoplasmic area, suggesting that these regions may be of functional importance. 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 MUC15 mRNA in human breast, bovine mammary gland, bovine lymph nodes and lungs of both species (Table 1). Of the identified ESTs a single clone, isolated from the human lung (GenBank accession number BG485125), appeared to have been derived from an alternative splicing event. In agreement with this, 11 of the 19 PCR screening experi- ments revealed a smaller and weaker band in addition to the expected product (Table 1). Therefore, to investigate the possible existence of an alternatively spliced mRNA variant of MUC15, RT-PCR experiments were performed on total RNA extracted from the mammary gland of a Holstein cow. Using MUC15-specific primers flanking the region containing the potential splice site, a major band of 513 bp was amplified by RT-PCR, along with a second shorter and weaker band. To specifically amplify the shorter variant in a second round of PCR, the products were subjected to specific enzymatic cleavage with the DdeIenzyme,which should only cut generated products comprising the trans- membrane region. Isolation and sequencing of a clone corresponding to the short variant confirmed the presence of an alternatively spliced form of bovine MUC15. The isolated variant (EMBL accession number AJ417817) Table 1. Expression of MUC15 in human and bovine tissues and cell types. MUC15 mRNA expression was examined by PCR screening of commercial multiple tissue cDNA panels and oligo(dT) primed cDNA libraries and by RT-PCR on RNA isolated from the mammary gland of a Holstein cow and the cellular fraction of human milk samples. ND, not detected; NI, not investigated. Tissue Template MUC15 mRNA MUC15/S mRNA Human Colon a cDNA Panel + + 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 Tonsil a –+ND Breast b cDNA library + d NI Lung b –+ d NI Milk cells a cDNA + d ND Bovine Mammary gland c cDNA + d + d Mammary gland c cDNA library + d + 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 The present paper describes the purification and character- ization of a hitherto unknown bovine membrane-associated mucin-like glycoprotein, MUC15, and cloning of a human homologue. The mature protein contains a single trans- membrane domain, and is proposed to be oriented with a small intracellular C-terminal part and an extracellular N-terminal comprising numerous N- and O-glycosylation sites (Fig. 4). Furthermore, database searches performed to look for other proteins with significant sequence similarity turned out fruitless. Several features of the isolated bovine glycoprotein suggest that it is a mucin-type molecule; a high molecular mass, a high content of carbohydrate, and third expression at the apical surface in epithelial cells of the mammary gland. Likewise, the deduced amino-acid sequences of both bovine and human MUC15 resemble the mucins in having serine, threonine and proline as the predominant amino acids, however, their high contents of leucine and aspara- gine is a characteristic shared only with the MUC8, MUC9, MUC13, and MUC16. Like the membrane-associated members of the mucin family, MUC15 appears to be derived from a precursor sequence including a signal peptide, a serine/threonine/proline-rich extracellular region, a hydrophobic transmembrane domain and a cytoplasmic tail. Although most structural elements of the membrane- associated mucins turned out to be present in MUC15, it is unique in its short extracellular domain and lack of repetitive segments with the typical mucin tandem repeats. However, lack of tandem repeats is also seen in the mucin- like glycoproteins mouse MUC14, endomucin-1, and 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. Localization of the human MUC15 to chromosome 11p14.3 on the human genome, showed the structure of the gene (Fig. 4A). A cluster of four secreted gel-forming mucin genes (MUC2, MUC5AC, MUC5B, and MUC6) has been localized within a 400-kb genomic DNA fragment on chromosome 11 band p15.5, and appears to have originated from a common ancestral gene [34]. Despite the location of the MUC15 gene close to the cluster of mucin genes, it does not show the characteristics of this group of secreted gel- forming mucins and therefore presumably has not evolved from the same ancestral gene. Two variant forms of MUC15 cDNA were found to be expressed by the normal bovine mammary gland. The short variant of bovine MUC15 (MUC15/S) arises from an alternative splicing event in which a section of 150 nucleotides was spliced out of the mRNA transcript, leading to the synthesis of a protein lacking a 50 amino- acid residues long region covering the transmembrane domain. Hence, MUC15/S may represent a secreted nongel forming mucin-type molecule as it does not contain any cysteine-rich regions characteristic for the gel forming mucins [1]. Examination of a corresponding alternatively spliced database EST clone of the human lung showed that the missing region of this clone corresponds to exon 4 (Fig. 4A). Likewise, nucleotides absent in the bovine MUC15/S variant correspond precisely to exon 4 of the human homologue, indicating a conserved genomic struc- ture of human and bovine MUC15, and exon skipping as a possible explanation for the origin of the splice variant. Interestingly, the appearance of alternative soluble variants 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, generated from the one and same gene, and thereby also control the biological effects elicited by the interaction of these two isoforms. Alternatively, it could be speculated that the secreted isoform of MUC15 may function as a protective mucin, perhaps as a coconstituent with gel-forming mucins in mucus, or it may act at the apical cell surfaces as a ligand for other cell surface molecules. The physiological role of MUC15 is not known, however, hints might arise from gene expression profiles. PCR screening of human MTC panels and additional cDNA libraries demonstrated MUC15 and MUC15/S mRNA expression in a wide range of tissues (Table 1), but at a level lower than the housekeeping gene, glyceraldehyde-3-phos- phate dehydrogenase (results not shown). The expression of mucins is generally thought to be restricted to epithelial cells. Surprisingly, the present data indicate no restriction of the MUC15 cDNA expression to epithelial cells. In contrast, expression in hematopoietic cells and tissues with function in the immune system was seen. Thereby, it might be difficult to discriminate between expression by transiting leukocytes, penetrating vascular endothelium, and the tissue specific cells. MUC1 expression, which is associated most consistently with epithelial tissues, has also been reported at mRNA and protein level in peripheral blood lymphocytes, lymph node samples, bone marrow and in various hema- topoietic cell lines [18,38,39]. In addition, the membrane- bound MUC13, like the human MUC15, also appears to be expressed at low levels in prostate, lung, liver, spleen, peripheral blood leukocytes, lymph node, bone marrow, testis, and ovary [14]. Apparently, although historically characterized as epithelia-specific, some membrane-associ- ated mucins are also expressed in immune and hematopoi- etic cells. 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Dent, G.A., Civalier, C.J., Brecher, M.E. & Bentley, S.A. (1999) MUC1 expression in hematopoietic tissues. Am. J. Clin. Pasthol. 111, 741–747. 39. Brugger,W.,Buhring,H.J.,Grunebach,F.,Vogle,W.,Kaul, S., Muller, R., Brummendorf, T.H., Ziegler, B.L., Rappold, I., Brossart, P., Scheding, S. & Kanz, L. (1999) Expression of MUC-1 epitopes on normal bone marrow: implications for the detection of micrometastatic tumor cells. J. Clin. Oncol. 17, 1535– 1544. Ó FEBS 2002 MUC15, a novel membrane-associated mucin (Eur. J. Biochem. 269) 2763 . 5¢-AATACCAAAGAAGCCTACAATG-3¢ and 5¢-GTACGAAGTGGAGGTATGTCATC-3¢. b Primer pair: 5¢-GCCATTT TAGGTGCTATTCTGG-3¢ and 5¢-TATTTTCTTTATCTGAGTTTA-3¢. c Primer pair:. Pallesen et al. (Eur. J. Biochem. 269) Ó FEBS 2002 and a poly (A) tail of 18 nucleotides. Two alternative poly (A) signals [A( 1259)TAAA and A( 1430)ATTAAA] giving