The MUC3 Gene Encodes a Transmembrane Mucin and Is Alternatively Spliced

doi:10.1006/bbrc.1999.1001

Biochemical and Biophysical Research Communications

Volume 261, Issue 1, 22 July 1999, Pages 83-89

https://doi.org/10.1006/bbrc.1999.1001 Get rights and content

Abstract

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.

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Rainbow trout chemokine 2 (CK-2) is currently the only known CC chemokine to have a mucin stalk. Further analysis of the mucin stalk region revealed a second, related CC chemokine sequence, denoted here as CK-2.1. This second sequence was determined to be an allele of CK-2 following genomic PCR analysis on several outbred individuals. Furthermore, in both in vivo and in vitro trials, CK-2 and CK-2.1 were both present, but appeared to have differential tissue expression in both control and PHA stimulated samples. Upon the development of a polyclonal antibody to rCK-2, CK-2 was only observed in the brain, liver and head kidney of PHA stimulated rainbow trout tissues. In comparison, when using the rainbow trout monocyte/macrophage-like cell line, RTS-11, CK-2 protein was observed in both control and PHA stimulated conditions. When studying the function of CK-2, a chemotaxis assay revealed that both peripheral blood leukocytes and RTS-11 cells migrated towards rCK-2 significantly at all concentrations studied when compared to truncated β₂m. Interestingly, this migration was lowest at both the highest concentration and the lowest concentrations of CK-2. Thus, teleostean chemokine receptors may become desensitized when overstimulated as has been observed in mammalian models. The observed chemotactic function was indeed due to rCK-2 as cell migration was inhibited through pre-treatment of both the cells and the polyclonal antibody with rCK-2. As has been observed thus far with all other chemokines, CK-2 does appear to function through binding to a G-coupled protein receptor as chemotaxis could be inhibited through pre-treatment with pertussis toxin. Overall, the results of this study indicate that CK-2 is a functional chemokine that is encoded by two differentially expressed alleles in rainbow trout, CK-2 and CK-2.1.
Global Gene Expression Patterns and Somatic Mutations in Sporadic Intracranial Aneurysms
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MUC3B may be associated with the development of IAs. MUC3B is a member of epithelial mucins involved in epithelial cell adhesion modulation.40 Later studies showed that MUCs were also expressed in vascular endothelium.41
High-throughput sequencing technologies can expand our understanding of the pathologic basis of intracranial aneurysms (IAs). Our study was aimed to decipher the gene expression signature and genetic factors associated with IAs.
We determined the gene expression levels of 3 cases of IAs by RNA sequencing. Bioinformatics analysis was conducted to identify the differentially expressed genes (DEGs) and uncover their biological function. In addition, whole genome sequencing was performed on an additional 6 cases of IAs to detect the potential somatic alterations in DEGs.
Compared with the normal arterial tissue, 1709 genes were differentially expressed in IAs arterial tissue. The most significantly up-regulated gene and down-regulated gene, H19 and HIST1H3J, may be essential for tumorigenesis of IAs. Hub protein of IKBKG in protein–protein interaction network was probably involved in the inflammation process in aneurysms. Another 2 hub proteins, ACTB and MKI67IP, as well as up-regulated genes, might be abnormally activated in aneurysms and involved in the pathogenesis of IAs. Further whole genome sequencing and filtering yielded 4 candidate somatic single nucleotide variants including MUC3B, and BLM may be involved in the pathogenesis of IAs. Even though, our results do not support the hypothesis of somatic mutations occurred in the DEGs.
Two-dimensional genomic data from transcriptome and whole genome sequencing indicated that no somatic mutations occurred in DEGs. In addition, 3 DEGs (IKBKG, ACTB, and MKI67IP) and 2 mutant genes (MUC3B and BLM) were essential in IAs.
Membrane-bound mucin modular domains: From structure to function
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Transcription variants of MUC3 encode truncated proteins, suggesting the possibility of expression of soluble forms [71]. Williams et al. confirmed the existence of two secreted isoforms of MUC3 that lack the TM domain [72]. In a similar manner, an alternative MUC17 splice event occurs and lacks the second EGF domain, the TM domain, and the cytoplasmic tail and leads to a secreted form MUC17/SEC [73].
Mucins belong to a heterogeneous family of large O-glycoproteins composed of a long peptidic chain called apomucin on which are linked hundreds of oligosaccharidic chains. Among mucins, membrane-bound mucins are modular proteins and have a structural organization usually containing Pro/Thr/Ser-rich O-glycosylated domains (PTS), EGF-like and SEA domains. Via these modular domains, the membrane-bound mucins participate in cell signalling and cell interaction with their environment in normal and pathological conditions. Moreover, the recent knowledge of these domains and their biological activities led to the development of new therapeutic approaches involving mucins. In this review, we show 3D structures of EGF and SEA domains. We also describe the functional features of the evolutionary conserved domains of membrane-bound mucins and discuss consequences of splice events.
Autoproteolysis of the SEA module of rMuc3 C-terminal domain modulates its functional composition
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Citation Excerpt :
To explore the biological significance of the cleavage occurring at the LSKGSIVV motif of the rMuc3 C-terminal domain, LoVo cells were selected as the target cells. LoVo cells have a main spliced MUC3 mRNA and lead to the absence of the transmembrane domain, therefore native human MUC3 was secreted and did not target the plasma membrane of the LoVo cells [22]. LoVo cells transfected by p20 (coding for the wild type Muc3 C-terminal domain), p20g/a (the cleavage motif was mutated to LSKASIVV), p20s/a (the cleavage motif was mutated to LSKGAIVV) and pSecTag2 (pSec) (the vector alone) were selected with hygromycin for stably-expressing clones (Fig. 3A), and detected by western blotting with the anti-V5 antibody (Fig. 3B).
rMuc3 is a typical transmembrane mucin and contains a 174 amino acid domain called an SEA module in its C-terminal domain which is cleaved in eukaryotic cells. However, the mechanism by which the rMuc3 SEA module is proteolyzed and its biological significance has to be elucidated. In this study, we showed that the rMuc3 C-terminal domain was cleaved at LSKGSIVV motif within SEA module in prokaryotic cells, the time-dependence of the cleavage was found in the purified rMuc3 C-terminal domain carrying a mutated LSKASIVV motif expressed in bacteria. Thus, the cleavage of rMuc3 SEA module depended on autoproteolysis. The autoproteolysis of the SEA module of rMuc3 C-terminal domain played a critical role in the migration and invasion of the LoVo human colon cancer cells with rMuc3 C-terminal domain in vitro. The rMuc3 C-terminal domain induced a significant activation of HER/ErbB2 phosphorylated form (py1248) in LoVo cells. Inhibition of the phosphorylation by gefitinib (ZD1839) did attenuate migration and invasion of LoVo cells with rMuc3 C-terminal domain. Thus, rMuc3 C-terminal domain undergoes autoproteolysis at its SEA module, which maintains its availability for the potentiation of the signaling process that is modulated by HER/ErbB2 phosphorylation to promote the migration and invasion of LoVo cells.
Activity of recombinant cysteine-rich domain proteins derived from the membrane-bound MUC17/Muc3 family mucins
2010, Biochimica et Biophysica Acta - General Subjects
The membrane-bound mucins, MUC17 (human) and Muc3 (mouse), are highly expressed on the apical surface of intestinal epithelia and have cytoprotective properties. Their extracellular regions contain two EGF-like Cys-rich domains (CRD1 and CRD2) connected by an intervening linker segment with SEA module (L), and may function to stimulate intestinal cell restitution. The purpose of this study was to determine the effect of size, recombinant host source, and external tags on mucin CRD1-L-CRD2 protein activity.
Four recombinant Muc3-CRD proteins and three MUC17-CRD proteins were generated using Escherichia coli or baculovirus-insect cell systems and tested in colonic cell cultures for activity related to cell migration and apoptosis.
N-terminal glutathione-S-transferase (GST) or C-terminal His₈ tags had no effect on either the cell migration or anti-apoptosis activity of Muc3-CRD1-L-CRD2. His-tagged Muc3-CRD1-L-CRD2 proteins with truncated linker regions, or the linker region alone, did not demonstrate biologic activity. The human recombinant MUC17-CRD1-L-CRD2-His₈ was shown to have anti-apoptotic and pro-migratory activity, but did not stimulate cell proliferation. This protein showed similar in vitro biologic activity, whether produced in E. coli or a baculovirus-insect cell system.
Recombinant mucin proteins containing a bivalent display of Cys-rich domains accelerate colon cell migration and inhibit apoptosis, require a full-length intervening Linker-SEA segment for optimal biologic activity, and are functional when synthesized in either E. coli and insect cell systems.
These results indicate that an Escherichia coli-derived full-length His₈-tagged human MUC17 CRD1-L-CRD2 recombinant protein is a biologically active candidate for further development as a therapeutic agent.

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¹: To whom correspondence and reprint requests should be addressed at Mater Medical Research Institute, Level 3, Aubigny Place, Mater Misericordiae Hospitals, Raymond Terrace, South Brisbane, Q4101 Australia. Fax: +61-7-38402550. E-mail: [email protected].

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Regular ArticleThe MUC3 Gene Encodes a Transmembrane Mucin and Is Alternatively Spliced

Abstract

Curr. Opin. Struct. Biol.

J. Biol. Chem.

Anal Biochem.

J. Mol. Biol.

FEBS Lett.

J. Biol. Chem.

Biochim. Biophys. Acta

Tumori

Am. Rev. Respir. Dis.

J. Biol. Chem.

Biochem. J.

Regular Article
The MUC3 Gene Encodes a Transmembrane Mucin and Is Alternatively Spliced