Cloning, chromosomal localization and characterization of the murine mucin gene orthologous to human MUC4 Jean-Luc Desseyn, Isabelle Clavereau and Anne Laine Unite ´ 560 INSERM, Place de Verdun, Lille, France We report here the full coding sequence of a novel mouse putative membrane-associated mucin containing three extracellular EGF-like motifs and a mucin-like domain consisting of at least 20 tandem repeats of 124–126 amino acids. Screening a cosmid and a BAC libraries allowed to isolate several genomic clones. Genomic and cDNA sequence comparisons showed that the gene consists of 25 exons and 24 introns covering a genomic region of  52 kb. The first intron is  16 kb in length and is followed by an unusually large exon ( 9.5 kb) encoding Ser/Thr-rich tandemly repeated sequences. Radiation hybrid mapping localized this new gene to a mouse region of chromo- some 16, which is the orthologous region of human chro- mosome 3q29 encompassing the large membrane-anchored mucin MUC4. Contigs analysis of the Human Genome Project did not reveal any other mucin on chromosome 3q29 and, interestingly, our analysis allowed the determination of the genomic organization of the human MUC4 and showed that its exon/intron structure is identical to that of the mouse gene we cloned. Furthermore, the human MUC4 shares considerable homologies with the mouse gene. Based on these data, we concluded that we isolated the mouse ortho- log of MUC4 we propose as Muc4. Expression studies showed that Muc4 is ubiquitous like SMC and MUC4, with highest levels of expression in trachea and intestinal tract. Keywords: MUC4; SMC; expression; large exon; tandem repeat. Epithelial mucins are high molecular mass glycoproteins synthesized by secretory epithelia. All mucins have a large domain composed of tandemly repeated sequences rich in serine and threonine residues that carry O-linked oligo- saccharides. Epithelial mucins are usually subdivided into secretory and membrane-associated classes [1] and, in humans, the latter family contains at least five members. Four of them, MUC3A, 3B, 11 and 12, are organized in a cluster of genes on chromosome 7q22 [2–4], while MUC4 andMUC1havebeenmappedtothechromosomes3and 1, respectively (reviewed in [5]). Except for the small mucin MUC1, all membrane-associated mucins are very large and seem to share four common domains: a short cytoplasmic domain, a transmembrane domain, EGF-like domains and the large O-glycosylated region with an amino-acid sequence that differs from one mucin to another and is not conserved during evolution. To date, MUC1 and the rat tumor sialomucin complex (SMC) are the two membrane-bound mucins best char- acterized. They are both expressed on the cell surface as a complex composed of two subunits coming from the same polypeptide precursor [6,7]. It has been shown by cell transfection and coimmunoprecipitation that SMC can act as a ligand for the tyrosine kinase p185 neu (homolog of ErbB2) [8] suggesting that SMC may play a role in malignancy. Furthermore, SMC seems to be implicated in the metastasis and in the resistance of SMC-expressing cells to natural killer cells [9,10]. More recently, cloning and sequencing human MUC4 cDNAs showed similarities between MUC4 and SMC at the N- and C-terminal portions of the molecules [11,12]. Although less is known about other large membrane-bound mucins, it has been suggested that MUC4 is a homolog of SMC. Cloning a complete mucin cDNA and/or a complete mucin gene is not an easy task for several reasons: (a) the RNA messenger is very large (>10 kb) and often expressed in low abundance in normal tissues; (b) the highly repetitive sequence of the central portion makes it difficult to map, subclone and sequence; (c) 5¢ and 3¢ ends are usually very similar between genes from the same class; and (d) clones may show instability presumably due to the repetitive structure of sequences and this may explain that BAC and YAC clones covering mucin genes clusters are still lacking in the Human Genome Project. In an effort to determine by genetic strategies, the specific functions of large membrane-associated mucins, we cloned and determined the complete cDNA and genomic sequences of a new large putative membrane-bound mouse mucin. This gene was assigned to mouse chromosome 16. RT-PCR experiments showed high expression of Muc4 in trachea, duodenum and intestine in contrast with a lower expression in stomach, in salivary glands, in liver and gallbladder, and in kidney. Chromosomal localization, sequence and expres- sion analyses provide strong evidence that the human MUC4 and the rat SMC are both the ortholog of the new mouse gene we characterized and proposed as Muc4.We analyzed human contigs of the evolving human draft Correspondence to A. Laine, Unite ´ 560 INSERM, Place de Verdun, 59045 Lille Cedex, France. Fax: + 33 320538 562, Tel.: + 33 320298 850, E-mail: laine@lille.inserm.fr Abbreviations:SMC,sialomucincomplex;vWD,vonWillebrand-D domain; vWF, von Willebrand Factor; RH, radiation hybrid. Note: the nucleotides sequences reported in this paper have been submitted to GenBank with accession numbers AF441785, AF441786 and AF441787. (Received 5 February 2002, revised 1 May 2002, accepted 2 May 2002) Eur. J. Biochem. 269, 3150–3159 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02988.x sequence and this allowed to determine the complete genomic organization of the human MUC4 which spans at least 70 kb and we found that its organization is very close to the one of the mouse Muc4 we described in this paper. Furthermore, domain analyses of Muc4, SMC and the human MUC4 revealed that these three large molecules have a Nido domain followed by a von Willebrand-D domain (vWD) and three EGF-like motifs. EXPERIMENTAL PROCEDURES Isolation of total RNAs Adult tissues from mice were obtained fresh, rapidly frozen andstoredat)80 °C before use. Total RNA was extracted from parotid, submaxillary gland, salivary glands, trachea, stomach, liver and gallbladder, duodenum and intestine and kidney by using guanidine hydrochloride as previously described [13]. RT-PCR amplifications Single-stranded cDNA was generated from 1 lgoftotal RNA using random hexamers or oligo(dT) primer. Several oligonucleotides (Fig. 1) were designed by comparison of the similar regions in the 3¢ regions of the gene coding for the rat SMC and of the human MUC4 gene. The two sense oligonucleotides NAU728 (5¢-TCCACTATCTGAACAA CCAACT-3¢) and NAU727 (5¢-ATGCTGATTTCTCTAG CTCCA-3¢) and the two antisense oligonucleotides NAU 726 (5¢-AACTTGTTCATGGAGCAGCCGC-3¢)and NAU729 (5¢-AGTTGGTTGTTCAGATAGTGGA-3¢) were designed from the SMC sequence (GenBank accession number M91662). The sense oligonucleotide NAU576 (5¢-CCCCACATCACCACCTTGGAT-3¢) and the anti- sense oligonucleotide NAU484 (5¢-AGAGAAACAGGGC ATAGGACC-3¢) have been chosen from the human MUC4 cDNA sequence (GenBank accession number AJ000281). The antisense oligonucleotides NAU750 (5¢-CTACATTTCTTGGAGAGGCTGAGT-3¢)and NAU762 (5¢-TGGAGCTAGAGAAATCAGCAT-3¢) were designed from our mouse cDNA sequences. NAU762 was used in RT-PCRs with the sense oligonucleo- tide NAU941 from within the repeat region (see further) to obtain the cDNA corresponding to the end of the repeat region. The sense oligonucleotide NAU972 (5¢-GAGCTGC CTGTGTTCTTGCCTCCT-3¢) was designed from the sequence coding for the signal peptide of SMC (GenBank accession number U06746) and used in RT-PCR experi- ments with the antisense oligonucleotide NAU966 from within the repeat region (see below) to obtain the 5¢ part of the cDNA 2308. PCR amplification was carried out in 50-lL reaction volumes containing 5 lL of the first strand cDNA, 0.3 m M dNTPs, 15 pmol of each primer, 2.5 U of Taq DNA polymerase (Roche) and PCR buffer (final concentration 10 m M Tris/HCl; 1.5 m M MgCl 2 ;50m M KCl). PCR parameters were 94 °C for 2 min, followed by 30 cycles at 94 °C for 45 s, 55 °Cfor1minand72°Cfor 2 min, followed by a final extension at 72 °C for 10 min. 5¢ RACE The three antisense primers used in this experiment (NAU1102, NAU1126 and NAU1123) were designed from the 5¢ part of the cDNA 2308. First-strand cDNA was synthesized using the 5¢-AmpliFINDER RACE kit (Roche) and RNA (1 lg) from trachea or duodenum and intestine with the antisense oligonucleotide NAU1102 (5¢-TGGAAC TTGGAGTATCCCTTG-3¢). The cDNA was then tailed and PCR reaction was performed using the nested antisense primer NAU1126 (5¢-ATGTTGATGAGGTCGATG CTT-3¢) and the oligo dT-anchor primer. Nested PCR involving a second round amplification using the antisense oligonucleotide NAU1123 (5¢-CTGCTGGAAAGGGACA TGGGT-3¢) and the anchor primer was carried out with 1 lL of the reaction mixture obtained from the previous round of PCR as template. A major band of 230 bp was amplified, cloned and sequenced. Screening of genomic libraries The mouse cDNA probe 1719 was generated by reverse- transcription PCR using the two oligonucleotides NAU728 and NAU726 and used to screen a mouse pWE15 cosmid library (Stratagene). Four clones were obtained and studied but their analysis showed that they did not contain the central region or the 5¢ end of the gene. The cosmid clone containing the longest part of the new gene was named CAR1 and studied. The 1719 probe and a cDNA probe (1820) obtained by RT-PCR using the two oligonucleotides NAU727 and NAU484 were then used to screen a mouse BAC (bacterial artificial chromosome) library (Incyte Genomics, Inc.). Filters were prehybridized, hybridized and washed according to the manufacturer’s instructions and one positive clone was obtained and named BAC4. Fig. 1. Muc4 cDNA (A) and genomic (B) cloning strategy, and protein domains organization (C). (A,B) Several cDNA clones and DNA fragments are indicated with their numbers. Some primers and their directions are indicated (not to scale) by horizontal arrows and their NAU (N) numbers. Restriction enzymes: N, NdeI; B, BamHI; K, KpnI. The hatched part corresponds to the repeat region. (C) Box with dashes represents the signal peptide; dense dots, Ser/Thr-rich non- repetitive sequence domain; diagonal lines, repetitive domain; square blocks, first Cys-rich region; wavy lines, domain rich both in Ser/Thr and N-glycosylation sites; black boxes, EGF-like domains; white box, unique sequence; grey box, transmembrane domain; horizontal lines, cytoplasmic domain. The star indicates the GDPH sequence. The thin vertical lines locate the 24 introns. Some exon numbers are indicated below. Ó FEBS 2002 The mouse mucin gene Muc4 (Eur. J. Biochem. 269) 3151 Restriction mapping of the BAC clone and Southern blot analyses TheBACwasdigestedtocompletionwiththerestriction enzyme NotI. For each of the other restriction enzymes used, one part of this NotI-digested BAC was digested to completion and a second part partially digested with the enzyme in order to generate a set of fragments that begin at the T7 or SP6 promoters and end at the site of cleavage of the chosen enzyme. These digestion products were fraction- ated on an agarose gel (0.6%) and blotted overnight to Hybond TM -N + membrane (Amersham Corp.). The frag- ments were then mapped relative to the T7 or SP6 promoters by hybridizing the membrane with end-labeled oligonucleotide-sequencing primers specific for these prim- ers. To determine the fragments to study we used various end-labeled oligonucleotides designed from the cDNA sequences. Sequence determination and analyses Fragments of the cosmid clone CAR1 were obtained after restriction enzyme digestion and subcloned into pBlue- scriptII KS(+) vector (Stratagene). Genomic fragments of interest obtained by PCR on cosmid CAR1 DNA and on the clone BAC4 DNA using oligonucleotides designed from cDNA sequences were cloned in pCR2-1 vector. Large fragments of interest were cut from BAC4, electrophoresed on a 0.8% agarose gel, electroeluted and cloned to be sequenced and further digested and sub- cloned into pBluescriptII KS(+) vector (Stratagene). To determine the sequences of the 5¢ and 3¢ ends of intron 1, we performed PCR experiments using BAC4 DNA as template, the sense oligonucleotide NAU972 (located in exon 1) and NAU1126 (located in exon 2), respectively, and a mixture of hexamers used as second primer. PCR products were subcloned into pCR4-TOPO vector (Invi- trogen Ltd). Plasmid inserts were sequenced on both strands several times on LI-COR 4000 and computer analyses were performed using PC / GENE Software. The mouse cDNA and genomic sequences reported in this paper have been deposited in the GenBank with accession numbers AF441785 and AF441786, respectively. BLAST searches of the human draft sequence using the MUC4 cDNA (GenBank accession numbers AJ000281 and AJ010901) and the Ensembl Genome Server (http://www.ensem- bl.org/) revealed two clones spanning MUC4: RP11- 423B7 and RP11-171N2, respectively. These two sequenc- es were aligned with the human cDNA, the mouse cDNA and genomic sequences we determined. The full genomic organization of the human MUC4 has been deposited in the GenBank with accession number AF441787. Zoo blot An interspecies Zoo blot containing EcoRI-digested DNA from human, monkey, rat, mouse (Balb/c), dog, cow, rabbit, chicken and yeast from Clontech was prehybridized, hybridized and washed according to the manufacturer’s instructions with the probe 2155 (594 bp) containing one- and-a-half repeats. Expression of the mouse gene In order to determine the expression of the new gene, RNAs isolated from various tissues were reverse-transcribed using random hexamers as primers. cDNAs were subjected to PCR amplification using the oligonucleotides NAU764 and NAU726 located within the 3¢ end and designed to amplify a 324-bp fragment. After electrophoresis on a 1% agarose gel (FMC, Rockland, ME, USA), the amplified products were stained with ethidium bromide and transferred for analysis by Southern blot using a specific internal antisense oligonucleotide NAU1432 (5¢-GCATTGGGGCCCATCT GGCAGG-3¢) as a probe. The efficiency of the cDNA synthesis was estimated by PCR using two mouse b-actin specific primers: sense 5¢-GTGGGCCGCTCTAGGCAC CA-3¢ and antisense 5¢-TGGCCTTAGGGTTCAGGG GG-3¢ for an expected band of 240 bp. Radiation hybrid (RH) mapping The chromosomal localization of the new gene was performed by PCR analysis using the T31 mouse/hamster RH panel (Research Genetics) [14]. Primers NAU726 (antisense, see above) and NAU764 (sense 5¢-AAGTATGC TGGAGGAGTACTT-3¢) located within the 3¢ end of the mouse gene were tested on mouse and hamster DNA. These oligonucleotides allow the amplification of a mouse DNA fragment of 1182 bp without amplification from hamster genomic template. The PCR reaction (50 lL) consists of 25 ng of DNA template, 15 pmol of each primer, 1.5 m M MgCl 2 ,25l M of each dNTP and 2 U of Taq DNA polymerase (Roche). The cycling conditions were: 2 min at 94 °C, 38 cycles of 15 s at 95 °C, 40 s at 56 °Cand90sat 72 °C followed by a final extension at 72 °Cfor7min.PCR products were electrophoresed through a 1% agarose gel, transferred overnight to membranes and hybridized with an internal 32 P-labeled oligonucleotide NAU914 (5¢-GCTGCC TAAGAATGGATACCCT-3¢). Logarithm of odds (lod) scores were analyzed using the Jackson Laboratory mouse RH data base (http://www.jax.org). RESULTS Isolation and sequencing of cDNAs Oligonucleotides and cDNA fragments are located on Fig. 1A. Using the sense oligonucleotide NAU728 and the antisense oligonucleotide NAU726, a first cDNA fragment of 481 bp (named 1719) was obtained by RT-PCR on mouse trachea RNA. Using the sense oligonucleotide NAU727 and the antisense NAU484 or the antisense oligonucleotide NAU750, which were upstream of NAU728 and NAU726 in the aligned sequences of the human MUC4 and rat SMC, we obtained by RT-PCR the two fragments of 258 bp (named 1820) and 1755 bp (named 1751), respectively. Using the sense NAU576 and the antisense NAU729, we obtained a cDNA fragment of 1615 bp (named 1729) that overlaps the fragment 1751 by 900 bp. All the fragments were subcloned and sequenced. The cDNA compiled sequence of the 3¢ region is  3 kb. Using NAU762 and NAU941 which was designed from the repeated sequence we found within the sequence of a fragment from the BAC4 clone (see Characterization of the 3152 J L. Desseyn et al. (Eur. J. Biochem. 269) Ó FEBS 2002 BAC genomic clone and sequencing strategy section), we obtained two fragments of 1.6 and 2 kb named 2350 and 2355, respectively. They both overlap the 3¢ end of the repeat region. To sequence the 5¢ region, we performed RT-PCR using NAU972 (designed from SMC) and the antisense oligo- nucleotide NAU966 chosen within the repeated sequence. We obtained a cDNA of 523 bp (named 2308). Using an internal sense oligonucleotide (NAU1109, 5¢-CAAGTAAA ACAGAACAAACAT-3¢) and NAU966 we obtained the cDNA named 2367 (1474 bp) that contains two repeats of 363 and 366 bp surrounding a unique sequence. 5¢ RACE PCR was then performed and we cloned and sequenced a major 230-bp band (named 2359). Within this unique sequence is an ATG with a Kozak consensus sequence [15] suggesting that it represents the codon for initiation of translation and, therefore, codes for the N-terminus of the protein. This fragment contains a 105-bp fragment of 5¢ untranslated sequence upstream of the putative start site ATG. Characterization of the cosmid clones and sequencing strategy Four positive cosmid clones were obtained by screening a mouse cosmid library using the cDNA probe 1719. The clone that contains the longest part of the new gene, CAR1, was studied. Two adjacent KpnI–KpnI fragments of 5 and 4 kb (named 1941 and 1956, respectively, Fig. 1B) overlap- ping the 3¢ end of the cDNA were subcloned. We also performed PCR using the CAR1 DNA as template and the oligonucleotides we used previously to clone the cDNAs. All PCR products were subcloned into pCR2.1 vector and sequenced on both strands. We then obtained the complete genomic sequence of the 3¢ part of the gene encompassing 17 kb. Three poly(A) signals were found after the stop codon. Analysis of the four cosmid clones by PCR revealed that they do not contain any sequence upstream of the oligonucleotide NAU484. Characterization of the BAC genomic clone and sequencing strategy We screened a BAC library using the cDNA probe 1820 and one clone, BAC4, was obtained and studied. PCR amplifi- cations and hybridization experiments revealed that this clone included the entire gene (data not shown). A PCR amplification product (1743) of 2320 bp was obtained using the two oligonucleotides NAU727 and NAU484 and the BAC4 DNA as template. This fragment was completely sequenced and was shown to contain one NdeIrestriction site. BAC4 DNA, digested by various restriction enzymes, was blotted and hybridized with the end-labeled oligo- nucleotide NAU856, designed from the sequence of the fragment 1743. One NdeI–NdeI genomic fragment of 7 kb was identified, purified and subcloned (named 1850). It contains two repeats of 372 bp followed by one repeat of 378 bp at its 5¢ end. We then chose to synthesize the two following oligonucleotides from the repeat sequence: the sense oligonucleotide NAU941 (5¢-GAGACAGAAACAA GTTCCCAA-3¢) and the antisense oligonucleotide NAU966 (5¢-CTGGGATGAAGGTGTCAATGA-3¢). RT-PCR experiments on trachea RNA using this pair of primers allowed the amplification of several fragments containing various numbers of repeats. PCR performed on BAC4 DNA allowed determination of the exon–intron junctions. Genomic organization and BLAST searches Exon–intron boundaries were defined by alignment of the cDNA and the genomic sequences and this revealed a total of 25 exons (Fig. 1C) ranging in size from 71 bp to  9.5 kb (Table 1). The size of the largest exon, containing the repeat sequence (exon 2), was estimated from restriction mapping experiments. The last exon is composed of a coding sequence of 193 bp and of an untranslated region of at least 131 bp. The size of the 24 introns ranges from 79 bp to about 16 kb. The size of the largest intron was determined by restriction mapping. Each intron begins with a GT and ends with an AG. The gene spans  52 kb from the initiation ATG codon to the stop codon. Three poly(A) signals are present located 122, 191 and 361 bp downstream of the stop codon. BLAST searches showed homologies with SMC and the human MUC4 mucin. Sequence similarity searches using the MUC4 cDNA identified two clones on human chromosome 3 from the evolving working draft sequence. The clone RP11-423B7 is  173 kb and consists of 16 ordered pieces and the clone RP11-171N2 is  164 kb and consists of 29 unordered pieces. Multiple alignments of genomic sequence pieces with the MUC4 cDNA allow for the first time determination of the complete exon–intron structure of this large membrane-bound mucin gene. Because several differences exist between the human cDNA and the two human genomic sequences, exons sizes given in the Table 1 for the human MUC4 gene correspond to the sizes deduced from the cDNA sequence. All introns have the same classes and positions in MUC4 and in the mouse gene we describe here. Chromosomal localization Using the T31 mouse/hamster RH panel that consists of 100 hybrid cell lines [14], the new gene was mapped by PCR screening on the chromosome 16 with highest lod score of linkage (13.7) to the marker D16Mit60. This region exhibits synteny with human chromosome 3q where the human MUC4 gene is located [16]. This and sequence similarities suggest that the new gene we cloned is the mouse ortholog of both MUC4 and SMC; we have named this gene Muc4. Analysis of the nucleotide and deduced amino-acid sequences and domains organization A schematic representation of the deduced amino acids sequence of Muc4 is depicted in Fig. 1C. The first 29 amino acids, predicted as the signal sequence, are followed by a Ser/Thr-rich region coded by exons 2–8, followed by a cysteine-rich region of 139 amino acids (Cys ¼ 7.2%) coded by exons 9–11, a second Ser/Thr-rich region of 374 amino acids coded by exons 12–18 and a C-terminal cysteine-rich region of 357 amino acids (Cys ¼ 7.6%) coded by the last seven exons. The first Ser/Thr-rich region is made of a 63-amino-acid peptide followed by 20 or 21 tandem repeats of 124–126 amino acids and this region ends with a 422-amino-acid peptide that is Ser/Thr-rich Ó FEBS 2002 The mouse mucin gene Muc4 (Eur. J. Biochem. 269) 3153 Table 1. Comparison of nucleotide sequences of intron–exon junctions between the mouse Muc4 and human MUC4 showing that exon sizes and positions are conserved between the two genes. The mouse exons (except exon 2) and introns (except intron 1) have been entirely sequenced. Intron positions have been determined by alignment between genomic and cDNA sequences. Uppercase and lowercase letters are for exon and intron sequences, respectively. Exons Introns No. Size (bp) 5¢ end 3¢ end No. Size (bp) Class Mouse 1 >178 a ACCTGgtaagacaag 1  16000 b 1 Human 1 >110 a CCCAGgtaagtgatg 1 >2008 1 Mouse 2  9500 b tttcaagaagATGCT CTCAGgtgagtcagc 2 223 1 Human 2 >15 432 c ttcactccagGAACC ATCAGgtagctgcca 2 334 1 Mouse 3 129 ccatgtccagGATTG TCAGGgtaagtgata 3 1669 1 Human 3 153 acgtgtccagGAATG GAGAGgtgaggccat 3 >2579 c 1 Mouse 4 134 ccttgtctagGCATT TCTACgtgagtctct 4 761 0 Human 4 134 cctggcccagGAGTT TCTACgtgagtccgg 4 >2147 c 0 Mouse 5 165 ttgtgctcagGTTAC ACCAGgtgagtcatt 5 945 0 Human 5 165 atgtgctcagTTCAC ATCAGgtgagccttt 5 1629 0 Mouse 6 156 cctttcctagGAATA TTGGGgtgagtggat 6 1102 0 Human 6 156 cctttcctagGAATA TCGGGgtgagtagac 6 1063 0 Mouse 7 131 caacttccagACCAA TCCAGgtaagatcgg 7 1437 2 Human 7 131 ccacccccagAGCAA TCTAGgtaggatggg 7 >1759 c 2 Mouse 8 89 ttgcctgcagTGGAG ATTAGgtaaaagtgc 8 1680 1 Human 8 90/89 d tttcctgcagTGGAG CTCAGgtaaaagtgc 8 1213 1 Mouse 9 180 tgttcctcagGCATC CCCAGgtgatacctc 9 202 1 Human 9 180 ccgacctcagGCCTC CATAGgtgacacctc 9 145 1 Mouse 10 117 ctctttgcagGTTGG GTTTGgtaagtatct 10 681 1 Human 10 114/126 d cttgtttcagGTCGC GTTGGgtgatctcaa 10 801 1 Mouse 11 120 ttcttcacagATGAG GCCCGgtgagcatca 11 303 1 Human 11 120 ttctccgcagCCCAG GCCCGgtgagcgaca 11 396 1 Mouse 12 212 tttcttttagCTTGG TCACGgtaagtgagg 12 1174 0 Human 12 209 ttccttccagCCTGG TCACGgtgagtgagg 12 487 0 Mouse 13 85 tctttcccagGTTCA TGAAGgtaggctccg 13 363 1 Human 13 91 ctccttccagGTCCA CGGAGgtaggttggg 13 820 1 Mouse 14 168 tgtcttccagTCTTA TCTGGgtaagatgca 14 445 1 Human 14 168 gatgctccagGCCAG CCTGGgtgagggcgg 14 501 1 Mouse 15 102 tgtctcacagGAGTG GACATgtgagtctgg 15 351 1 Human 15 102 tgtccctcagGGGTC GACCTgtgagtctgg 15 366 1 Mouse 16 234 tgtgttacagGGCAC CCTCAgtaagtgaca 16 1133 1 Human 16 234 tgtgttacagGGCAG CCTCAgtaagtggcc 16 2059 1 Mouse 17 138 tctgtttcagATCAG CTTTGgtatgaatct 17 3067 1 Human 17 138 tgtgtttcagATCAG CTTTGgtaggactat 17 1806 1 Mouse 18 182 ctctggacagAAAAC TTGAGgtgagtagtg 18 838 0 Human 18 182 cccggggcagAGAAT TGGAGgtgagtgttg 18 >2683 c 0 Mouse 19 160 tgtcattcagGTGAC TGCAGgtgagtgtgg 19 862 1 Human 19 160 cctcctccagGTGGC TGCGGgtgagccggg 19 982 1 Mouse 20 174 ccctttacagCTCTG TACGGgtatggctaa 20 695 1 Human 20 180 cactctgcagCTCTG CCTAGgtaccgccag 20 1659 1 Mouse 21 74 ttatctccagAGCTT CCTCGgtcagtgctg 21 1020 0 Human 21 74 ccatctccagAACTT CCTCGgtcagtgctg 21 1101 0 Mouse 22 71 tccattgtagGTGGC CGAACgtaagtagag 22 79 2 Human 22 65 tctaacctagGTGGC CGAATgtaagtggga 22 94 2 Mouse 23 236 ttccatacagCTCTC GGCTCgtgagtcact 23 1613 1 Human 23 224 tcccacacagCGATT AGCCCgtgagtccgt 23 1819 1 Mouse 24 163 ctgcctacagTGAAC TGCAGgtgggtaggg 24 858 2 Human 24 163 ttcccgacagTGAAC TGCAGgtgcataggg 24 1522 2 Mouse 25 >411 a ctgtcttcagCTGCG Human 25 >323 a tggtcaccagCTGTG a the precise sizes of UTRs have not been determined. b Size estimated by restriction mapping. c Size estimated from the two contigs analysis (see text). d Two different sizes depending on the contig considered. 3154 J L. Desseyn et al. (Eur. J. Biochem. 269) Ó FEBS 2002 (S + T ¼ 28.7%). According to the results of restriction mapping, the whole repeat region encompasses  8.3 kb, of which more than 4 kb have been sequenced. Amino-acid repeats are aligned in Fig. 2A and the comparison of the 126 amino acids consensus sequence with the consensus sequence of SMC repeats is shown (Fig. 2B). A unique sequence of 119 amino acids is inserted between the first two repeats a1 and a2. A 32 P-labelled oligonucleotide designed from this unique sequence was hybridized to a Southern blot of the BAC4 DNA digested with various restriction enzymes and revealed a single HpaI–HpaIbandof1.5kb suggesting that this sequence is unique (data not shown). The order of the repeats b1-b2, c1-c2 and d1 is unknown. There is at least one potential N-glycosylation site (Asn- X-Ser/Thr where X is any amino acid except Pro) in each repeat. The tandem repeat array ends with the e1-e8 repeats. The second Ser/Thr rich region starts at a AWTFGDPH peptide and consists of 374 amino acids (S + T ¼ 21.1%). Moreover, it contains 14 potential N-glycosylation sites. The GDPH sequence is conserved in human MUC4 [11] and in SMC [17]. It has previously been shown that SMC is cleaved early in the pathway to the cell surface [7] at this site. Comparison with SMC and MUC4 and domains analysis using SMART N-Terminal parts of Muc4, SMC and MUC4 were aligned (Fig. 3) showing that signal sequences are very similar for the three molecules but it is noticeable that mouse and rat are closer. The Ser/Thr-rich region upstream of the repeat area (amino acids 30–98 in Muc4) shows strong similarity between mouse and rat while this region differs markedly in the human sequence. This region is 63 amino acids in rodent and longer in human (951 amino acids, shortened in Fig. 3). Comparison of the predicted amino-acid sequences coded by exons 3–23 with those of MUC4 and SMC peptides (Fig. 4) shows that the three peptides are very similar although areas of high sequence homology are interspersed in SMC with four sections of low homology (see sequences in italic on Fig. 4). Nevertheless, the nucleotide sequence of Muc4 shares high homology with the cDNA sequence of SMC except for a few nucleotide insertions/deletions (data not shown). It is noticeable that 12 potential N-glycosyla- tion sites are perfectly conserved in Muc4, SMC and MUC4. Amino-acid sequence analysis using the SMART program [18] shows the presence of a Nido domain followed by a von Willebrand-D domain (Fig. 1C) and three EGF- like motifs (Figs 1C and 4). Concerning the vWD domain, the sequence (residues 397–578, Fig. 4) is more similar to the vWF-D2 domain (residues 378–540, GenBank accession number P04275). A putative transmembrane motif of 23 amino acids at the C-terminal portion of the molecule is followed by a short cytoplasmic tail (18 amino acids). Conservation of Muc4 tandem repeats A comparison of the consensus sequence of Muc4 tandem repeats with the consensus sequence of SMC tandem repeats published previously [19] demonstrated that there is a good degree of sequence identity (67%) between the two Fig. 2. Alignment of the amino-acid sequences of the Muc4 repeats (A) and comparison of the repeat consensus sequences of Muc4 and SMC (B). (A) Under the consensus sequence, the repeats are numbered in order of appearance from N- to C-terminal. The order of the repeats (b–d) is unknown. Repeats e1–e8 are the last eight repeats. Dots indicate exact sequence matches with the consensus sequence and dashes gaps in the sequence. A unique sequence inserted between repeats a1 and a2 is observed. The potential N-glycosylation sites are underlined. (B) Conserved amino acids are shaded. Dash indicates a gap. Fig. 3. Alignment of the amino-terminal sequences of mouse Muc4, rat SMC and human MUC4. The dashes indicate gaps in the sequence. Identical sequences are shaded. Ó FEBS 2002 The mouse mucin gene Muc4 (Eur. J. Biochem. 269) 3155 rodent species (Fig. 2B). We then hybridized an interspecies Zoo blot containing EcoRI-digested DNA from human, monkey, rat, mouse (Balb/c), dog, cow, rabbit, chicken and yeast with the probe 2155 corresponding to one-and-a-half repeats. This showed a 20-kb faint band with the rat DNA while a very strong signal at 18 kb is observed for the mouse DNA supporting that Muc4 and SMC are orthologs. No signal is observed for other species (Fig. 5). Tissue specific expression of Muc4 The tissue distribution of Muc4 was determined by RT-PCR using total RNA from various tissues. PCR amplifications were analyzed by ethidium bromide staining (Fig. 6) and Southern blotting using an internal primer as a probe. Strong expression of Muc4 is shown in trachea, duodenum and intestine, while a much weaker expression is shown in stomach. In each case, the quality of the cDNA was verified by amplification of the b-actin cDNA, shown to be equally expressed in all cDNAs. By Southern blot an even weaker expression in submaxillary glands, salivary glands, liver and gallbladder and in kidney (not shown) was obtained. Fig. 4. Comparison of Muc4 C-terminal protein with SMC and MUC4. Dashes indicate gaps introduced in the sequence for alignment purposes. Amino acids are numbered at the right. Conserved amino acids are shaded. Amino acids in bold italic correspond to regions with a frameshift in SMC. The GDPH sequence is underlined with triangles. The potential N-glycosylation sites conserved in the three molecules are indicated with hashes. The three EGF-like domains are underlined. The transmembrane domain is underlined with stars. Fig. 5. Zoo blot hybridized with repeated sequence (probe 2155) showing cross-hybridization between rat and mouse DNA. The Zoo-blot contains EcoRI-digested DNA from human, monkey, rat, mouse (Balb/c), dog, cow, rabbit, chicken and yeast. The size of the band is estimated to be 20 kb for rat and 18 kb for mouse. Fig. 6. Expression of Muc4 by RT-PCR. Total RNA from parotid (lane 1), submaxillary glands (lane 2), salivary glands (lane 3), trachea (lane 4), stomach (lane 5), liver and gallbladder (lane 6), duodenum and intestine (lane 7) and kidney (lane 8) was used. (A) One band of 324 bp is obtained with the couple of primers NAU764 and NAU726. (B) The efficiency of the cDNA synthesis was estimated by PCR using two mouse b-actin specific primers producing one band of 240 bp. L, ladder;C,control(H 2 O). 3156 J L. Desseyn et al. (Eur. J. Biochem. 269) Ó FEBS 2002 DISCUSSION The SMC is a large heterodimeric glycoprotein that protects tumors from immune system and may influence signaling pathways via the transmembrane subunit [8–10]. Over- expression of SMC is believed to mask antigens at the tumor cell surface. The precise function of the SMC transmem- brane subunit is poorly understood due to the large size of the molecule and the presence of several domains. In order to further investigate the biological roles of large mem- brane-bound mucins, we cloned a new mouse gene using primers designed from the cDNA sequence of SMC and human MUC4. Cloning and sequencing the human MUC4 had shown substantial similarities between MUC4 and SMC at the N- and C-terminal portions of the molecules [11,12] but such similarities may exist with rat and human mucin cDNAs that still remain to be cloned. The work we present in this paper provides strong evidence that the mouse gene we cloned, characterized and suggested as Muc4, is the ortholog of SMC and MUC4:(a)thethree molecules show high sequence similarities; (b) the region of mouse chromosome 16 on which we mapped the gene exhibits synteny with human chromosome 3q where the human MUC4 gene has been mapped; (c) Muc4 and MUC4 have a similar pattern of expression; (d) the tandem repeat sequences of Muc4 share homology with the tandem repeat sequences of SMC; and (e) BLAST searches and multiple alignments allowed to determine the complete genomic organization of MUC4 and this clearly shows that the two genes are very close. Based on sequence similarities we believe that we cloned the complete coding sequence from the ATG initiator to the stop codon of Muc4 which is followed by three poly(A) signals. Furthermore, we suggest that the ATG is embedded in a Kozak consensus sequence [15]. BLAST searches revealed one sequence deposited in the GenBank (GenBank acces- sion number AF296636) that encompasses the first exon of Muc4 and in which the initiator methionine suggested is the same as the one we suggest. Our restriction mapping and sequencing results show that the mouse Muc4 is  52 kb and codes for a transcript of a predicted size of  13 kb. Both human MUC4 and mouse Muc4 genes are virtually identical in terms of the class of introns, the exon number and size of exons. The sizes of the 24 mouse introns range from 79 to 3067 bp except the first intron, which is unusually large ( 16 kb). The genomic organization of MUC4 we determined by comparison of the published cDNA and the evolving human draft sequence [20] is very close to that of the mouse Muc4 (Table 1). The size of intron 1 of the human MUC4 has been estimated to be  15 kb by restriction mapping [12] and to be at least 20 kb by our analysis of the human draft sequence. Intronic sequences are not conserved between species but sequences surrounding splice junctions are highly similar (Table 1). Furthermore, it is noticeable that introns of the human gene are longer than in the mouse gene. Each intron of both genes begins with a GT and ends with an AG, obeying strictly the GT/AG rule of splice-junction sequences [21]. Twenty-two out of the 23 internal exons are in the range 71 to 236 bp (Table 1), in good agreement with the mean length of exons [20,22]. Due to the repetitive structure, we did not succeed in cloning and sequencing the full repetitive region but we can assume by restriction mapping experi- ments that exon 2 is  9.5 kb in length and codes for two Ser/Thr-rich regions flanking 20 or 21 imperfect mucin-type repeats of 124 or 126 amino acids. An unusually large exon coding for tandem repeats rich in Ser/Thr is a common feature of mucins [23–25] and this suggests that the tandem repeat array arose through internal duplications rather than through exon shuffling. Alignment of repeat sequences (Fig. 2A) shows an insertion of a unique peptide of 119 amino acids between repeats a1 and a2. This sequence is unrelated to the three insertion sequences of 33, 43 and 94 amino acids described previously between tandem repeats of SMC [19]. It is interesting to note that all the repeats of Muc4 contain at least one potential N-glycosylation site, an uncommon feature in mucin, contained only by the mouse submandibular small mucin [26]. The consensus sequence of the Muc4 tandem repeat is very close to that of SMC. Nevertheless, the tandem repeat domain of Muc4 does not show significant identity with the human MUC4 except that they are both rich in serine and threonine. It is known that tandem repeats differ in sequence and size between the two species and different mucins [5]. Previous work on rat Muc5ac [27], and mouse Muc3 [28], together with this work, clearly shows that rat and mouse tandem repeats are conserved suggesting a high pressure of selection on the tandem repeat sequences of rodents. The derived amino-acid sequence was used to search a collection of gapped alignments of domains using the SMART program [18]. As predicted, this analysis revealed at the C-terminal portion of the molecule the two EGF-like motifs and the transmembrane helix already described for SMC and found in MUC4 [11,17] suggesting that the Muc4 is a member of the large membrane-bound mucins family. MUC4, SMC and Muc4 have a smaller cytoplasmic tail than the other members of the transmembrane epithelial mucins family [4]. It is interesting to note that SMART program revealed also a third EGF-like motif, a vW-D domain coded by exons 11–15 and a Nido domain coded by exons 5–10 (Figs 1B and 4). A third EGF-like motif has been identified previously in human MUC4 [29] encoded by a single exon (exon 19). SMART analysis shows that these three domains are also conserved in MUC4 and SMC. The vWF-D domain is a feature of the large secreted gel-forming mucins. This domain, found four times in the von Wille- brand Factor (vWF), is rich in cysteine residues and may participate in intermolecular disulfide bonds (reviewed in [30]). Nevertheless, the cysteine residues conserved between the vWF and the large secreted mucins are not conserved in Muc4. This may reflect a lost of function of this domain during evolution. The Nido domain has been found in various proteins. This domain is a part of the nidogen glycoproteins, which are expressed by mesenchymal and epithelial cells. Nidogens have a high affinity for laminin- binding protein and are believed to be important for epithelial morphogenesis [31]. The significance of this domain in membrane-bound mucins is unclear. EGF-like motifs are found in numerous growth factors and extracel- lular proteins involved in formation of extracellular matrix, cell adhesion, chemotaxis and wound healing [4]. These motifs may allow exposure of ligand-binding sites outside of the cell. To date, no ligand has been identified for MUC3 but SMC has been shown to bind the erbB2 receptor tyrosine kinase through one of the EGF-like domains of Ó FEBS 2002 The mouse mucin gene Muc4 (Eur. J. Biochem. 269) 3157 SMC and this interaction modulates the receptor tyrosine kinase activity [8]. According to these authors, SMC interacting directly with ErbB2 extracellular domain through its EGF1 domain potentiates signaling through the ErbB receptor network. Expression of epithelial mucins is tissue-specific and several mucins may be expressed in each tissue [5]. MUC4 is expressed in numerous normal tissues including ocular, salivary glands, trachea, lung, stomach, colon, ovary, uterus, prostate, and endocervix [32–34]. There is no detectable expression in normal pancreas while there is an abnormal expression of MUC4 in pancreatic tumors [35,36]. The mouse Muc4 seems to have a similar pattern of expression as MUC4. It is also expressed in numerous epithelial tissues with the highest expression in trachea, duodenum and intestine while a lower expression is observed in submaxil- lary glands, salivary glands, liver, gallbladder and in kidney. Abnormal expression of MUC4 has been reported in several human epithelial cancers [35,37,38] and it has been shown that SMC contributes to tumor progression [8]. 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