Human MUC4 mucin cDNA and its variants in pancreatic carcinoma

The human MUC4 gene is not expressed in normal pancreas; however, its dysregulation results in high levels of expression in pancreatic tumors. To investigate the tumor-associated expression, MUC4 cDNA was cloned from a human pancreatic tumor cell line cDNA expression library using a polyclonal antibody raised against human deglycosylated mucin and RT-PCR. Pancreatic MUC4 cDNA shows differences in 12 amino acid residues in the non-tandem repeat coding region with no structural rearrangement as compared with tracheal MUC4. The full-length MUC4 cDNA includes a leader sequence, a serine and threonine rich non-tandem repeat region, a central large tandem repeat domain containing 48 bp repetitive units, regions rich in potential N-glycosylation sites, two cysteine-rich domains, EGF-like domains, and a transmembrane domain. We also report the presence of a new EGF-like domain in MUC4 cDNA, located in the cysteine-rich region upstream from the first EGF-like domain. Four distinct splice events were identified in the region downstream of the central tandem repeat domain that generate three new MUC4 cDNA sequences (sv4, sv9, and sv10). The deduced amino acid sequences of two of these variants lack the transmembrane domain. Furthermore, two unique forms of MUC4 (MUC4/Y and MUC4/X) generated as a result of alternative splicing lack the salient feature of mucins, the tandem repeat domain. A high degree of polymorphism in the central tandem repeat region of MUC4 was observed in various pancreatic adenocarcinoma cell lines, with allele sizes ranging from 23.5 to 10.0 kb. MUC4 mRNA expression was higher in differentiated cell lines, with no detectable expression in poorly differentiated pancreatic tumor cell lines.     

Cloning, chromosomal localization and characterization of the murine mucin gene orthologous to human MUC4.

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 approximately 52 kb. The first intron is approximately 16 kb in length and is followed by an unusually large exon (approximately 9.5 kb) encoding Ser/Thr-rich tandemly repeated sequences. Radiation hybrid mapping localized this new gene to a mouse region of chromosome 16, which is the orthologous region of human chromosome 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 ortholog 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.     

Cysteine-rich regions of pig gastric mucin contain von willebrand factor and cystine knot domains at the carboxyl terminal(1).

In order to sequence the cysteine-rich regions of pig gastric mucin (PGM), we used our previously identified pig gastric mucin clone PGM-2A to screen a pig stomach cDNA library and perform rapid amplification of cDNA ends to obtain two cysteine-rich clones, PGM-2X and PGM-Z13. PGM-2X has 1071 base pairs (bp) encoding 357 amino acids containing five serine-threonine-rich 16 amino acid tandem repeats, downstream from a cysteine-rich region similar to human and mouse MUC5AC. PGM-Z13 encodes the complete 3'-terminus of PGM and is composed of 3336 bp with a 2964 bp open reading frame encoding 988 amino acids with four serine-threonine-rich tandem repeats upstream from a cysteine-rich region similar to the carboxyl terminal regions of human and rat MUC5AC and human MUC5B. This region is homologous to von Willebrand factor C and D domains involved in acid induced polymerization, and to the carboxyl terminal cystine-knot domain of various mucins, TGF-beta, vWF and norrin, which is involved in dimerization. These newly sequenced cysteine-rich regions of pig gastric mucin may be critical for its gelation and for its observed increased viscosity induced by low pH.     

Molecular cloning of cDNAs derived from a novel human intestinal mucin gene

A human small intestinal lambda gt11 cDNA library was screened with antibodies to deglycosylated small intestinal mucin. Four partial cDNA clones were isolated that define a novel human mucin gene. These include two partial cDNA clones, SIB 124 and SIB 139, that contain 51 nucleotide tandem repeats which encode a seventeen amino acid repetitive peptide with a consensus sequence of HSTPSFTSSITTTETTS. SIB 139 hybridized to messages produced by small intestine, colon, colonic tumors and also by high mucin variant LS174T colon cancer cells. The gene from which cDNAs SIB 124 and SIB 139 are derived (proposed name MUC 3) maps to chromosome 7, distinct from other known human mucin genes.     

Alternative splicing generates a family of putative secreted and membrane-associated MUC4 mucins.

The MUC4 mucin gene encodes a putative membrane-anchored mucin with predicted size of 930 kDa, for its 26.5-kb allele. It is composed of two regions, the 850-kDa mucin-type subunit MUC4alpha and the 80-kDa membrane-associated subunit MUC4beta. In this study, we cloned and characterized unique MUC4 cDNA sequences that differ from the originally published sequence. Eight alternative splice events located downstream of the central large tandem repeat array generated eight new, distinct cDNAs. The deduced sequences of these MUC4 cDNAs (sv1-MUC4 to sv8-MUC4, the full length cDNA being called sv0-MUC4) provided seven distinct variants, five secreted forms and two membrane-associated forms. Furthermore, two other alternative splicing events located on both sides of the tandem repeat array created two variants, MUC4/Y and MUC4/X, both lacking the central tandem repeat. Therefore, MUC4 can be expressed in three distinct forms, one membrane-bound, one secreted, and one lacking the hallmark feature of mucin, the tandem repeat array. Although no specific function has yet been discovered for the family of proteins putatively produced from the unique MUC4 gene, we suspect that the MUC4 proteins may be implicated in the integrity and renewal of the epithelium.     

Isolation and characterization of MUC15, a novel cell membrane-associated mucin.

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 sequencing of the purified protein allowed construction of degenerate 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 demonstrated 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.     

Molecular cloning and chromosomal localization of a novel human tracheo-bronchial mucin cDNA containing tandemly repeated sequences of 48 base pairs.

A lambda gt11 cDNA library constructed from human tracheo-bronchial mucosa was screened with a polyclonal antiserum raised to chemically deglycosylated pronase glycopeptides from human bronchial mucins. Out of 20 positives clones, one partial cDNA clone was isolated and allowed to map a novel human tracheo-bronchial mucin gene. It contains 48 nucleotide tandem repeats quite perfectly identical which encodes a protein containing about 50% of hydroxy amino-acids. This clone hybridized to polydisperse messages produced by human tracheo-bronchial and human colonic mucosae. The gene (proposed name MUC 4) from which cDNA is derived maps to chromosome 3.     

The MUC3 gene encodes a transmembrane mucin and is alternatively spliced.

Epithelial mucins are a family of secreted and cell surface glycoproteins expressed by epithelial tissues and implicated in epithelial cell protection, adhesion modulation and signaling. The gene encoding human MUC3 (hMUC3), localised to chromosome 7q22, is most highly expressed in the small intestine. It has previously been reported to be a non-transmembrane mucin with minimal homology to its suggested orthologues from rat (rMuc3) and mouse (mMuc3). RT-PCR was performed to investigate the carboxyl terminus of the published sequence of hMUC3 from normal colon and small intestine tissues and also from a series of 10 colorectal cancer cell lines. Two distinct PCR products were identified. In contrast to the previously published hMUC3 sequence, which terminates shortly after a single cysteine-rich EGF-like domain, conceptual protein translation of the dominant and largest PCR product identified two extracellular cysteine-rich EGF-like domains separated by an N-glycosylation-rich domain and a potential coiled-coil region, followed by a putative transmembrane region and a 75 amino acid cytoplasmic tail. The smaller of the two PCR products was found to be an alternative splice variant of MUC3 including the first EGF-like domain but lacking part of the second EGF-like domain and the transmembrane region. Nine out of 10 colorectal cancer cell lines were found to express MUC3. Interestingly, one of the cell lines, LoVo, expressed predominantly the alternative splice form lacking a transmembrane domain. Structural homology of the new protein sequence of hMUC3 with rMuc3 and mMuc3 indicates it is closely related to the rodent proteins and is likely to be involved in ligand-binding and intracellular signaling. The new finding that MUC3 encodes a transmembrane molecule presents a new paradigm for the structure of this mucin and the manner in which it may function.     

Human 3-hydroxy-3-methylglutaryl coenzyme A reductase. Conserved domains responsible for catalytic activity and sterol-regulated degradation

A full length cDNA for human 3-hydroxy-3-methylglutaryl coenzyme A reductase, the membrane-bound glycoprotein that regulates cholesterol synthesis, was isolated from a human fetal adrenal cDNA library. The nucleotide sequence of this cDNA shows that the human reductase is 888 amino acids long and shares a high degree of homology with the hamster enzyme. The amino-terminal membrane-bound domain is the most conserved region between the two species (7 substitutions out of 339 amino acids). This region, which is predicted to span the endoplasmic reticulum membrane seven times, mediates accelerated degradation of reductase in the presence of sterols. The carboxyl-terminal catalytic domain is also highly conserved (22 substitutions out of 439 amino acids). However, the linker region between these two domains has diverged (32 substitutions out of 110 amino acids). Conservation of the structure of the membrane-bound domain in HMG-CoA reductase supports the hypothesis that sterol-regulated degradation is an important mechanism for suppression of reductase activity and for regulation of cholesterol metabolism in humans as well as in hamsters.     

A novel protein derived from the MUC1 gene by alternative splicing and frameshifting

Genes that have been designated the name "MUC" code for proteins comprising mucin domains. These proteins may be involved in barrier and protective functions. The first such gene to be characterized and sequenced is the MUC1 gene. Here we report a novel small protein derived from the MUC1 gene by alternative splicing that does not contain the hallmark of mucin proteins, the mucin domain. This protein termed MUC1/ZD retains the same N-terminal MUC1 sequences as all of the other known MUC1 protein isoforms. The common N-terminal sequences comprise the signal peptide and a subsequent stretch of 30 amino acids. In contrast, the MUC1/ZD C-terminal 43 amino acids are novel and result from a reading frameshift engendered by a splicing event that forms MUC1/ZD. The expression of MUC1/ZD at the protein level in human tissues is demonstrated by Western blotting, immunohistochemistry, immunoprecipitation, and an ELISA. Utilization was made of affinity-purified MUC1/ZD-specific polyclonal antibodies as well as two different monoclonal antibodies that are monospecific for the MUC1/ZD protein. The MUC1/ZD protein is expressed in tissues as an oligomeric complex composed of monomers linked by disulfide bonds contributed by MUC1/ZD cysteine residues. MUC1/ZD protein expression did not parallel that of the tandem-repeat array-containing MUC1 protein. Results presented here demonstrate for the first time the expression of a novel MUC1 protein isoform MUC1/ZD, which is generated by an alternative splicing event that both deletes the tandem-repeat array and leads to a C-terminal reading frameshift.     

Evolution of the large secreted gel-forming mucins

Mucins, the major component of mucus, contain tandemly repeated sequences that differ from one mucin to another. Considerable advances have been made in recent years in our knowledge of mucin genes. The availability of the complete genomic and cDNA sequences of MUC5B, one of the four human mucin genes clustered on chromosome 11, provides an exemplary model for studying the molecular evolution of large mucins. The emerging picture is one of expansion of mucin genes by gene duplications, followed by internal repeat expansion that strictly preserves frameshift. Computational and phylogenetic analyses have permitted the proposal of an evolutionary history of the four human mucin genes located on chromosome 11 from an ancestor gene common to the human von Willebrand factor gene and the suggestion of a model for the evolution of the repeat coding portion of the MUC5B gene from a hypothetical ancestral minigene. The characterization of MUC5B, a member of the large secreted gel-forming mucin family, offers a new model for the comparative study of the structure-function relationship within this important family.     

Construction and characterization of a chimeric receptor containing the cytoplasmic domain of MUC1 mucin

MUC1 mucin is a transmembrane glycoprotein that is highly expressed in various cancer cell lines and is also present in most of the glandular epithelial cells including the airway. Although the presence of numerous phosphorylation sites in its cytoplasmic domain suggests its potential role as a receptor, the unavailability of a ligand for MUC1 mucin has limited our understanding of its function. In this paper, we tried to circumvent this problem by constructing a chimeric receptor containing the cytoplasmic domain of MUC1 mucin, which can be phosphorylated on activation. To this end, we constructed a chimeric plasmid vector (pCD8/MUC1) by replacing the extracellular and transmembrane domains of human MUC1 mucin with those of human CD8. Transient transfection of the vector into COS-7 cells resulted in expression of the chimeric receptor on the surface of the COS-7 cells as judged by immunologic assays with various antibodies as well as by fluorescence-activated cell-sorting analysis. Treatment of the transfected COS-7 cells with an anti-CD8 antibody resulted in a significant increase in phosphorylation of tyrosine moieties of the chimeric receptor. This chimeric receptor will serve as a powerful tool in elucidating the signaling mechanism as well as the functional role of MUC1 mucin in the airway.     

Alternate splicing of mRNAs encoding human mast cell growth factor and localization of the gene to chromosome 12q22-q24.

Human mast cell growth factor (MGF) complementary DNAs (cDNAs) were cloned from HeLa cells using the polymerase chain reaction with oligonucleotides corresponding to murine and human MGF sequences. Sequencing of the cloned human MGF polymerase chain reaction products revealed two types of cDNA: a full length form corresponding in size to the murine cDNA, and an alternately spliced clone with a deletion of the sixth exon of the gene. Since membrane-bound MGF is predicted to be proteolytically cleaved within the sequences encoded by exon 6 to generate a soluble protein, this alternately spliced cDNA would likely encode a noncleavable, membrane-bound form of MGF. No difference in biological activity on human bone marrow cells was observed with recombinant, soluble forms of both types of human MGF protein. Our previous localization of the murine MGF gene to the Sl locus on chromosome 10 suggested (via conserved linkage groups) that the human MGF gene would be located on human chromosome 12. Therefore, rodent-human somatic cell hybrids with or without an entire human chromosome 12 and hybrids retaining partial 12 were tested by Southern blot analysis and used to show the presence of the human Mgf locus at chromosome region 12q. Chromosomal in situ hybridization localized the gene to 12q22-q24 in the region predicted by the comparative mapping of the murine Mgf/Sl locus.     

MUC16 is produced in tracheal surface epithelium and submucosal glands and is present in secretions from normal human airway and cultured bronchial epithelial cells

The gel-forming MUC5AC and MUC5B mucins have been identified as major components of human airway mucus but it is not known whether additional mucin species, possibly with other functions, are also present. MUC16 mucin is a well-known serum marker for ovarian cancer, but the molecule has also been found on the ocular surface and in cervical secretions suggesting that it may play a role on the normal mucosal surface. In this investigation, the LUM16-2 antiserum (raised against a sequence in the N-terminal repeat domain) recognized MUC16 in goblet and submucosal gland mucous cells as well as on the epithelial surface of human tracheal tissue suggesting that the mucin originates from secretory cells. MUC16 mucin was present in 'normal' respiratory tract mucus as well as in secretions from normal human bronchial epithelial (NHBE) cells. MUC16 from NHBE cells was a high-molecular-mass, monomeric mucin which gave rise to large glycopeptides after proteolysis. N- and C-terminal fragments of the molecule were separated on gel electrophoresis showing that the MUC16 apoprotein undergoes a cleavage between these domains, possibly in the SEA domain as demonstrated for other transmembrane mucins; MUC1 and MUC3. After metabolic labeling of NHBE cells, most of the secreted monomeric, high-molecular-mass [(35)S]sulphate-labelled molecules were immunoprecipitated with the OC125 antibody indicating that MUC16 is the major [(35)S]sulphate-labelled mucin in NHBE cell secretions.     

The complete genomic organization of the human MUC6 and MUC2 mucin genes

The complete genomic organization of the two mucin genes MUC2 and MUC6 was obtained by comparison of new and published mRNA sequences with newly available human genomic sequence. The two genes are located 38.5 kb apart in a head-to-head orientation within a gene complex on chromosome 11p15.5. The N-terminal organization of MUC6 is highly similar to that of MUC2, containing the D1, D2, D', and D3 Von Willebrand factor domains followed by the large tandem repeat domains located in exons 31 and 30, respectively. MUC6 has a much smaller C-terminal domain (101 amino acids) encoded by 2 exons containing only the CK domain, compared with MUC2, which has a C-terminal domain of 859 amino acids containing the D4, C, D, and CK domains, encoded by 19 exons. The gene structures agreed partially but not completely with predictions from gene prediction programs.