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Department of Molecular Biology, University of Aarhus, 8000 Aarhus C, Denmark
1 Corresponding author: jatr{at}mb.au.dk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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130 kDa) in ovine, caprine, porcine, and buffalo milk samples. Purification and N-terminal amino acid sequencing confirmed the presence of ovine and caprine MUC15 orthologs in milk fat globule membranes. Expression of MUC15 in human milk was demonstrated by immunostaining (150 kDa) as well as by mass spectrometry. Screening of a human multiple tissue expression array showed abundant MUC15 gene expression in placenta, salivary gland, thyroid gland, trachea, esophagus, kidney, testis, and the leukemia K-562 cell line. Furthermore, moderate expression was seen in the pancreas, adult and fetal lung, fetal kidney, lymph node, adult and fetal thymus, and parietal lobe. Structural motifs for interactions (epidermal growth factor receptor and Src homology 2 domains) are identified in the intracellular region. Implication of the mucin in signal transduction and the potential physiological function of MUC15 are discussed.
Key Words: MUC15 • ortholog • milk fat globule membrane • mucin expression
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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Expression of a human MUC15 ortholog has been demonstrated in the human mammary epithelium by reverse transcriptase (RT)-PCR, and the corresponding cDNA encodes an unprocessed protein of 334 AA showing 67% sequence similarity with the bovine counterpart (Pallesen et al., 2002). This suggests that MUC15 might be associated with human MFGM along with the 2 other membrane-bound mucins MUC1 and MUC4 (Shimizu and Yamauchi, 1982; Zhang et al., 2005). Additional MUC15 orthologs have been identified, including mouse, rat, and chimpanzee MUC15 (NCBI nucleotide accession numbers AK052840, BC083925, and XM_508334, respectively).
Little is known about the physiological function of MUC15. Mucins, in general, are known to play a central role in maintaining homeostasis because they act as selective, lubricating, and protecting cell barriers. Membrane-associated mucins also mediate adhesion and signal transduction. A broad expression pattern has emerged for these glycoproteins with expression in nonepithelial cell types as well (for a review, see Hollingsworth and Swanson, 2004). Accordingly, RT-PCR studies have shown MUC15 mRNA expression in various human and bovine tissues: ocular surface epithelium (Corrales et al., 2003), 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 (Pallesen et al., 2002). Recently, human MUC15 has been shown to be highly expressed in placenta, where it may be a key molecule in the regulation of trophoblast invasion (Shyu et al., 2007).
The present work describes the isolation and identification of caprine, ovine, and human MUC15 orthologs from milk. Cloning of a human splice variant, MUC15/S, encoding a short, potentially secreted isoform has been accomplished, together with supplementary semiquantitative expression studies on human MUC15 in a variety of tissues and cell types. Finally, aspects in relation to the function of MUC15 are discussed.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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Immunodetection of MUC15 Orthologs
Milk samples from different species including bovine, ovine, caprine, and porcine whole milk, cream from the buffalo, and MFGM from human milk (prepared as described below) were separated by SDS-PAGE (18%) using standard procedures. Western blotting was performed as described (Benfeldt et al., 1995) using peptide antibodies against MUC15 (5 µg/mL) or polyclonal bovine MUC15 antibodies (0.75 µg/mL) both with alkaline phosphatase-conjugated swine anti-rabbit secondary antibodies (1:3,000, DakoCytomation Norden A/S, Glostrup, Denmark).
Preparation of Human, Ovine, and Caprine MFGM
Preparation of human, caprine and ovine MFGM was performed by a method modified from Hvarregaard et al. (1996). Milk fat globule membranes were prepared from fresh unprocessed caprine and ovine milk, whereas the human milk sample had been frozen. Briefly, the cream fraction was isolated by centrifugation (25 min at 4,700 x g, 4°C), resuspended once in cold PBS (0.14 M NaCl, 3 mM KCl, 8 mM Na2HPO4, 2 mM KH2PO4, pH 7.2, 1 mM EDTA) and centrifuged again (25 min, 4,700 x g, 4°C). Cream fractions were then blended in PBS. The resulting buttermilk was filtered through cheese cloth, and MFGM were then precipitated by centrifugation (130 min, 33,000 x g, 4°C). Precipitated membranes were resuspended in 50 mM NH4HCO3, pH 7.8, and homogenized. The MFGM samples were diluted to 3 mg of protein/mL in 20 mM Tris-HCl, pH 7.4, 0.15 M NaCl, and 1 mM ethylene glycol tetraacetic acid (EGTA). The MFGM-associated proteins were extracted from the membranes with the nonionic detergent Triton X-100 to 1% (vol/vol). Extracted MFGM proteins were finally isolated by centrifugation (1 h, 33,000 x g).
Isolation of Caprine and Ovine MUC15
Caprine MUC15 was isolated essentially as described for isolation of bovine MUC15 (Pallesen et al., 2002). In the final step, MUC15 was separated by reversed-phase chromatography using a 1-mL Resource RPC column (GE Healthcare) with a gradient of 2-propanol in 20% formic acid. Ovine MUC15 was isolated from Triton X-100–extracted MFGM proteins that were precipitated with acetone (1:10, vol/vol) at –20°C (overnight). The precipitate was dissolved in 10% formic acid, centrifuged, and subjected to chromatography on a 1-mL Resource RPC column. Samples containing ovine MUC15 were collected, freeze-dried, and repurified on the Resource column with a modified gradient. Resulting samples were analyzed and subjected to N-terminal amino acid sequencing.
Identification of MUC15 in Human Milk
Human MFGM Triton X-100–extracted proteins were separated as described for ovine MUC15. Obtained fractions were analyzed by SDS-PAGE and Western blotting as described above. Human MUC15 was quantified semiquantitatively in MFGM samples by densitometric scanning of Western blots using anti-peptide polyclonal antibodies (5 µg/mL) as described previously (Pallesen et al., 2007). Bovine MUC15 (Pallesen et al., 2002) was used as a standard for quantification in a range from 15 to 50 ng/lane. Blots were analyzed using a flat-bed scanner and QuantiScan 2.1 software (Biosoft, Cambridge, UK). The anti-peptide antibodies were expected to demonstrate equivalent specificity and affinity toward bovine and human MUC15 because the antibody epitopes are 100% identical.
In-Gel Digestion and Analysis of Generated Peptides
Peptides from purified protein material were identified from dissected 1-dimensional SDS-PAGE gel bands essentially as described in Devold et al. (2006). Resulting tryptic peptides were desalted and concentrated on a Zip-tip column (Millipore, Billerica, MA) and successively allowed to dry on the MS target in presence of acidified 
-cyano-4-hydroxycinnamic acid and trifluoroacetic acid. Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectra of the peptide mixtures were obtained in positive reflector-ion mode on a Voyager DE Pro mass spectrometer (Applied Biosystems, Boston, MA). Monoisotopic peptide masses (m/z) were assigned using the GPMAW program (Lighthouse Data, Odense, Denmark).
Screening of a Human Multiple Tissue Expression Array
Screening of MUC15 expression was performed on the human multiple tissue expression (MTE) Array 2 (Clontech, Mountain View, CA) containing normalized loadings of poly A+ RNA from 58 different human tissues, 8 cancer cell lines, and 8 different control RNA and DNA sequences. The cDNA probes used were made by RT-PCR on total human milk cell RNA prepared as described in Pallesen et al. (2002). Specific PCR primers were synthesized (DNA Technology, Aarhus, Denmark) enclosing the entire coding region of the human MUC15 (1,538 bp in total): 5'-ACACTTAACCCATCTGTTTTCTC-3' (forward) and 5'-CAGGGCTGTAATGGTGAAATCT-3' (reverse). Obtained PCR products were cloned into pCR 2.1-TOPO cloning vectors using the TOPO TA Cloning Kit (Invitrogen, Carlsbad, CA) and confirmed by DNA sequencing. The hybridization cDNA probe was radioactively labeled with [-32P] dATP using the Random Primed DNA labeling system (Roche Diagnostics, Mannheim, Germany). Removal of unincorporated nucleotides from the labeled probe was achieved using a Jetquick PCR Purification Spin Kit (Genomed, Bad Oeynhausen, Germany). Prehybridization (65°C, 45 min) of the MTE array was performed in ExpressHyb (Clontech) with sheared salmon testes DNA (0.1 mg/mL). The radiolabeled probe was mixed with 30 µg of Cot-1 DNA (Roche Diagnostics), 150 µg of sheared salmon testes DNA, and 50 µL of 20x saline sodium citrate (SSC; 3 M NaCl, 0.3 M Na3Citrate, pH 7.0) in a total volume of 200 µL. Upon heat denaturation the probe was mixed into the prehybridization solution and incubated with the array overnight at 65°C. Following hybridization, the array was washed 4 times at 65°C for 20 min in 2x SSC with 1% SDS, and once in 0.2x SSC with 0.5% SDS (5 min, 25°C). Blots were analyzed by using a phosphorimager. Human MUC15/S was cloned using total human milk cell RNA and following the strategy of Pallesen et al. (2002). Obtained PCR products were ligated into pCR 2.1-TOPO cloning vectors and sequenced on both strands.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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). Furthermore, a band was observed at 150 kDa in human MFGM.
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). Caprine MUC15 co-eluted with another periodic acid–Schiff (PAS)-staining 80-kDa glycoprotein (Figure 2d, lane 2), which was recognized by the anti-bovine MUC15 polyclonal antibodies (Figure 2d, lane 3) and by the anti-peptide polyclonal antibodies (results not shown). N-terminal sequencing of the isolated caprine MUC15 did not reveal any alternative N-terminals. In addition, mass spectrometry analysis of in-gel trypsin digests of the caprine 80- and 130-kDa bands revealed only the presence of peptides found in the cytoplasmic area close to the transmembrane region (residues 261 to 280, bovine numbering). Accordingly, it is not this cytoplasmic region that is missing, because the peptide antibody epitope is also located within this region.
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) was recognized by the anti-peptide polyclonal antibody (Figure 2e, lane 3). N-terminal AA sequencing of the isolated caprine and ovine proteins demonstrated that they were orthologous to bovine MUC15, and that 11 of the first 18 AA residues were fully conserved between these 3 species (Figure 2g). Analogous to caprine milk, a minor immunoreactive band was seen at about 80 kDa in ovine milk. The nature of this band has not been investigated further.
Identification of MUC15 in Human Milk
Expression of human MUC15 in the mammary gland was demonstrated by Western blotting, as a single band of 150 kDa was recognized in isolated MFGM (Figure 1) and also in buttermilk (results not shown). Isolation from human MFGM was performed and MUC15-containing samples were found to elute late from the column (Figure 2c), as documented by Western blotting (Figure 2f, lane 2). Upon SDS-PAGE analysis and staining with PAS and Coomassie Brilliant Blue R-250, only faint bands were seen around 150 kDa (Figure 2f, lane 1), implying that MUC15 only constitute minute amounts of the total protein in the obtained fractions. In agreement with that, a semiquantitative approach using Western blotting and the anti-peptide antibodies demonstrated that MUC15 constitutes approximately 0.025% (wt/wt) of human MFGM proteins. In-gel trypsin digests of human MUC15-containing HPLC fractions were performed and mass spectrometry was carried out on extracted tryptic peptides. By this method 2 membrane proximal peptides were identified, the extracellular 208TTLQPTLKFTNNSKLFPNTSDPQK231 and the intracellular 274LYDDRNEPVLR284. The latter peptide locates to the same region in which tryptic peptides of bovine and caprine MUC15 could be identified by mass spectrometry (see above).
Screening of Human MTE Array
A human MTE array was adopted to examine the expression profile and relative abundance of human MUC15. The MTE array is normalized to 8 housekeeping genes and contains a range of tissue-specific poly-adenylated RNA sequences (73 in total); for example, parts of the central nervous system, cardiovascular, digestive, and glandular tissues and several cancer cell lines. Human MUC15 mRNA was abundantly detected in placenta, salivary gland, thyroid gland, trachea, esophagus, kidney, the leukemia K-562 cell line, and testis. Moderate expression was seen in the pancreas, adult and fetal lung, fetal kidney, lymph node, adult and fetal thymus, and parietal lobe (Figure 3). During preparation of the full-length MUC15 cDNA probe for the MTE screening experiment, an additional smaller and weaker PCR product was amplified. Investigation by cloning and sequencing showed that the short variant was an alternative splice variant of human MUC15. The spliced-out region of 150 nucleotides corresponds to the entire exon 4 (Pallesen et al., 2002), which encodes a region of 50 AA covering the transmembrane domain. This variant was named MUC15/S (accession number AJ507429).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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Expression of human MUC15 mRNA has been shown in the mammary gland (Pallesen et al., 2002). The present data demonstrate expression at the protein level as well. The molecular weight of the human protein is approximately 20 kDa greater than the bovine, caprine, and ovine proteins. The calculated molecular weight of unsubstituted human MUC15 is 33,875 Da, which is only 558 Da more than the bovine ortholog. A sequence comparison shows that the extracellular domains of human and bovine MUC15 are only 59% similar, whereas the transmembrane and cytoplasmic regions are more conserved (87% similarity). Consequently, the extracellular domains may well have very different glycosylation patterns or larger glycans, as was previously shown for human milk protein (Patton, 2001). Despite several purification attempts it was not possible to obtain sufficient human MUC15 to document its presence by Edman-based N-terminal sequencing. This seems reasonable because human MUC15 was estimated to constitute only 0.025% (wt/wt) of the MFGM protein, which is 62 times less than in the bovine system (Pallesen et al., 2007).
From the MTE array it can be seen that MUC15 is widely expressed, which is in accordance with the previously conducted RT-PCR screening (Pallesen et al., 2002). Overall, MUC15 mRNA expression is now found in 24 human-derived tissues and cell types. In relative terms, the greatest expression level of human MUC15 was observed in the placenta, which is in accordance with a recent multiple human tissue Northern blot study that included 12 of the tissues found on the present MTE array. The same Northern blot study showed MUC15 only in lung and kidney in the rest of the human tissues (Shyu et al., 2007). From the present MTE array study it is notable that expression of MUC15 is seen in several epithelial tissues, trachea, lung, esophagus, and salivary gland. Expression in kidney and the endocrine glands pancreas, thymus, ovaries, testis, and thyroid gland correlates with the fact that they all are recognized as mucin-expressing tissues (Hollingsworth and Swanson, 2004). A high expression level was also observed in the leukemia cell line K-562 (chronic myelogenous leukemia), whereas no expression of MUC15 was detected in bone marrow, peripheral blood leukocytes, or any of the other leukemia cell lines in the MTE array. Tumor-associated alterations of the expression pattern of mucins have been demonstrated showing abnormal high levels and altered glycosylation profiles (Baldus et al., 2004). It is well known that human MUC1 is highly expressed by many carcinomas, and it has been speculated that the mucin plays a role in tumor progression and metastasis (Gendler, 2001).
In analogy with the bovine system, a short human MUC15 splice variant (lacking the transmembrane domain) was found to be expressed in the mammary gland. This implies a possible presence of a secreted non-gel-forming mucin. Searching the expressed sequence tag (EST) database at NCBI revealed that clones encoding part of this splice variant have been isolated from human lung, placenta, and fetal cochlea (GenBank accession numbers BG485125, BQ025249, and CN398309). Furthermore, previous results also indicated the existence of a splice variant of human MUC15 (Pallesen et al., 2002). Thus, it appears that the alternative splicing of MUC15 is conserved across species and that the short variant is widely expressed in various human tissues.
An alignment of the full-length database AA sequences of the mouse, rat, human, and bovine, and a computer-generated chimpanzee MUC15 shows that the domain structure is overall conserved (Figure 4). The human and chimpanzee MUC15 AA sequences showed 98% similarity, whereas the remaining orthologs showed between 55 and 85% similarity. In addition to a high content of serine, threonine, and proline residues, the extracellular regions show the greatest sequence variance. Both characteristics are common among the transmembrane mucins.
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). Furthermore, all 5 species contain 3 class III PDZ domain binding motifs (-X-Asp/Glu-X-hydrophobic amino acid) in the cytoplasmic C-terminal tail of the mucin. The PDZ domains have an important role in mediating interactions for the assembly of large protein complexes at specific subcellular locations and are found in proteins involved in signaling (Hung and Sheng, 2002). The initial tyrosine residue of the 301YYNP304 binding motif (numbering of human MUC15) is predicted by the NetPhos 2.0 server (Blom et al., 1999) as an epidermal growth factor receptor (EGF-R) phosphorylation site. The same motif corresponds with the consensus sequence for binding of the Src homology 2 (SH2) domain of Grb2, pTyr-X-Asn-X (Kessels et al., 2002). It is therefore tempting to suggest that MUC15 could be involved in signal transduction through the Ras signaling pathway. Similarly, the human transmembrane MUC1 has been shown to be involved in the Sos-Ras-RAF-MEK-ERK2 signaling pathway by interactions with Grb2, Sos, and EGF-R (Li et al., 2001; Meerzaman et al., 2001). Recently, we performed a preliminary yeast 2-hybrid screening of a human mammary gland library for proteins that interact with the intracellular part of MUC15. Among the isolated positive clones, 4 have been selected for further investigation: lactoferrin, vacuolar protein sorting 37 homolog C, Ser/Thr kinase 23, and pleckstrin homology domain 2 protein (results not shown). Forthcoming research must evaluate the significance of these findings, which in turn could bring us closer to a description of the physiological function(s) of MUC15.
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ACKNOWLEDGEMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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Received for publication March 26, 2008. Accepted for publication July 29, 2008.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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