Role of the cystine-knot motif at the C-terminus of rat mucin protein Muc2 in dimer formation and secretion

Bell, Sherilyn L.; Xu, Guangzhi; Forstner, Janet F.

doi:10.1042/bj3570203

Cited by 26 publications

(21 citation statements)

References 34 publications

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“…Studies on the pig submaxillary mucin (PSM) and the rat Muc2 mucin suggest that the C-terminal parts of these proteins are responsible for dimer formation [10][11][12]. These findings support the hypothesis that dimerization of the human MUC2 takes place via its C-terminus.…”

Section: Introductionsupporting

confidence: 78%

“…This proposes that this cysteine might be involved also in the dimerization of mucins. Studies on rat Muc2 [11,12] point out that this cysteine residue is essential for dimer formation, whereas studies on vWF [15], PSM [16] and Norrin [17] indicate that additional cysteine residues may be of importance. The mucin genes MUC2, MUC5AC, MUC5B and MUC6 are clustered on chromosome 11p15.5 [18], all of which contain the cystine-knot motif and show large similarities in the positions of other cysteine residues also outside of the cystine-knot motif, indicating that these mucin genes are part of a family having a common ancestral gene.…”

Section: Introductionmentioning

confidence: 99%

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The recombinant C-terminus of the human MUC2 mucin forms dimers in Chinese-hamster ovary cells and heterodimers with full-length MUC2 in LS 174T cells

Lidell

Johansson

Mörgelin

et al. 2003

Biochemical Journal

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The entire cDNA corresponding to the C-terminal cysteine-rich domain of the human MUC2 apomucin, after the serine-and threonine-rich tandem repeat, was expressed in Chinese-hamster ovary-K1 cells and in the human colon carcinoma cell line, LS 174T. The C-terminus was expressed as a fusion protein with the green fluorescent protein and mycTag sequences and the murine immunoglobulin κ-chain signal sequence to direct the protein to the secretory pathway. Pulse-chase studies showed a rapid conversion of the C-terminal monomer into a dimer in both Chinese-hamster ovary-K1 and LS 174T cells. Disulphide-bondstabilized dimers secreted into the media of both cell lines had a higher apparent molecular mass compared with the intracellular forms. The MUC2 C-terminus was purified from the spent culture medium and visualized by molecular electron microscopy. The dimer nature of the molecule was visible clearly and revealed that each monomer was attached to the other by a large globular domain. Gold-labelled antibodies against the mycTag or green fluorescent protein revealed that these were localized to the ends opposite to the parts responsible for the dimerization. The Cterminus expressed in LS 174T cells formed heterodimers with the full-length wild-type MUC2, but not with the MUC5AC mucin, normally expressed in LS 174T cells. The homodimers of the MUC2 C-termini were secreted continuously from the LS 174T cells, but no wild-type MUC2 secretion has been observed from these cells. This suggests that the information for sorting the MUC2 mucin into the regulated secretory pathway in cells having this ability is present in parts other than the C-terminus of MUC2.

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Section: Introductionsupporting

confidence: 78%

Section: Introductionmentioning

confidence: 99%

The recombinant C-terminus of the human MUC2 mucin forms dimers in Chinese-hamster ovary cells and heterodimers with full-length MUC2 in LS 174T cells

Lidell

Johansson

Mörgelin

et al. 2003

Biochemical Journal

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“…8). The mucin polypeptide is made (within 20 min), folded, and dimerized in the endoplasmic reticulum (within 1 h), and we infer on the basis of other studies that the dimerization occurs by disulfide linkage between the C termini (7,23,24). A small proportion of the polypeptide (approximately 1-5%) remains monomeric and may undergo further modification and/or represent material that is bound for degradation.…”

Section: Discussionmentioning

confidence: 99%

“…The model is based on findings presented here and those previously presented by other workers. In the endoplasmic reticulum (ER) the mucin polypeptide forms disulfide-linked dimers via their C termini (filled circles) (7,23,24). GalNAc substitution (late endoplasmic reticulum/Golgi) then precedes glycan elaboration to yield fully glycosylated mucin dimers, which multimerize via disulfide linkage between their N termini (open circles) (7,8).…”

Section: Discussionmentioning

confidence: 99%

Identification of Molecular Intermediates in the Assembly Pathway of the MUC5AC Mucin

Sheehan¹,

Kirkham²,

Howard

et al. 2004

Journal of Biological Chemistry

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MUC5AC mucins secreted by HT-29 cells in culture are oligomeric glycoproteins with characteristics similar to the MUC5AC mucins isolated from human airway sputum (Sheehan, J. K., Brazeau, C., Kutay, S., Pigeon, H., Kirkham, S., Howard, M., and Thornton, D. J. (2000) Biochem. J. 347, 37-44). Therefore we have used this cell line as a model system to investigate the biosynthesis of this major airway mucin. Initial experiments showed that the MUC5AC mucins isolated from the cells were liable to depolymerization depending on the conditions used for their solubilization. Prevention against reduction resulted in large oligomers associated with the cells, similar to those secreted into the medium. Using a combination of density gradient centrifugation and agarose gel electrophoresis coupled with probes specific for different forms of the mucin we identified five major intracellular populations of the MUC5AC polypeptide (unglycosylated monomer and dimer, GalNAc-substituted dimer, fully glycosylated dimer, and higher order oligomers). Pulse-chase studies were performed to follow the flow of radioactivity through these various intracellular forms into the mature oligomeric mucin secreted into the medium (a process taking ϳ2-4 h). The results show that the mucin polypeptide undergoes dimerization and then becomes substituted with GalNAc residues prior to glycan elaboration to produce a mature mucin dimer, which then undergoes multimerization. These data indicate that this oligomeric mucin follows a similar assembly to the von Willebrand factor glycoprotein to yield long linear disulfide-linked chains.In vertebrates, epithelia are protected on their luminal side by mucus gels synthesized and secreted by specialized cells in the surface and/or underlying submucosa. These gels act as a physical barrier as well as provide the infrastructure to support an array of protective molecules and mechanisms for sequestering chemical toxins and pathogens. Furthermore, they contribute to the homeostasis and stability of the underlying epithelium. The molecular framework of mucus is provided by large, oligomeric, O-linked glycoproteins termed mucins. These macromolecules are polydisperse in mass (2-40 MDa) and size (0.5-10 m in length) and are assembled from a disulfide linkage of monomers (2-3 MDa) (1-5). Entanglement of the mucin chains is considered to be a major factor for gel formation, and as a consequence their size distribution is a key determinant of the physical properties of the mucus.In humans, four genes encoding oligomeric mucins have been identified: MUC5AC, MUC5B, MUC2, and MUC6, and these are found in a cluster on chromosome 11 (6). Their expression appears to be selectively controlled in a manner favoring tissue-specific expression, but the functional reasons for this are unclear. Sequence similarities of Cys-rich domains in the N and C termini of these mucins with another oligomeric, Olinked glycoprotein, von Willebrand factor (VWF), 1 suggest that mucins are assembled in a manner similar to VWF (7). Indeed, the consens...

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“…Biochemical analyses have revealed that mucins MUC5AC and MUC5B, secreted by cells lining the respiratory tract, are the major gel-forming polymer components of airway mucus (13,30,31). Cysteine domains present on these mucins contribute to polymer formation and possibly interaction with neighboring mucin chains, by disulfide bond formation (1,2). Because disulfide bonds on proteins are the preferred substrates for Trx enzymatic activity, it was hypothesized that mucin polymers were targets for reduction during the liquefaction of sputum by Trx.…”

Section: Discussionmentioning

confidence: 99%

Thioredoxin liquefies and decreases the viscoelasticity of cystic fibrosis sputum

Rancourt¹,

Tai²,

King³

et al. 2004

American Journal of Physiology-Lung Cellular and Molecular Phys

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White. Thioredoxin liquefies and decreases the viscoelasticity of cystic fibrosis sputum. Am J Physiol Lung Cell Mol Physiol 286: L931-L938, 2004. First published December 24, 2003 10.1152/ ajplung.00352.2003.-The persistent and viscous nature of airway secretions in cystic fibrosis (CF) disease leads to airway obstruction, opportunistic infection, and deterioration of lung function. Thioredoxin (Trx) is a protein disulfide reductase that catalyzes numerous thiol-dependent cellular reductive processes. To determine whether Trx can alter the rheological properties of mucus, sputum obtained from CF patients was treated with TRX and its reducing system (0.1 M thioredoxin reductase ϩ 2 mM NADPH), and liquid phase-gel phase ratio (percent liquid phase) was assessed by compaction assay. Exposure to low Trx concentrations (1 M) caused significant increases in the percentage of liquid phase of sputum. Maximal increases in percent liquid phase occurred with 30 M Trx. Additional measurements revealed that sputum liquefaction by the Trx reducing system is dependent on NADPH concentration. The relative potency of the Trx reducing system also was compared with other disulfidereducing agents. In contrast with Trx, glutathione and N-acetylcysteine were ineffective in liquefying sputum when used at concentrations Ͻ1 mM. Sputum viscoelasticity, measured by magnetic microrheometry, also was diminished significantly following 20-min treatment with 3, 10, or 30 M Trx. Similarly, this reduction in viscoelasticty also was dependent on NADPH concentration. Further investigation has indicated that Trx treatment increases the solubility of highmolecular-weight glycoproteins and causes redistribution of extracellular DNA into the liquid phase of sputum. Recognizing that mucins are the major gel-forming glycoproteins in mucus, we suggest that Trx alters sputum rheology by enzymatic reduction of glycoprotein polymers present in sputum.

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Role of the cystine-knot motif at the C-terminus of rat mucin protein Muc2 in dimer formation and secretion

Cited by 26 publications

References 34 publications

The recombinant C-terminus of the human MUC2 mucin forms dimers in Chinese-hamster ovary cells and heterodimers with full-length MUC2 in LS 174T cells

The recombinant C-terminus of the human MUC2 mucin forms dimers in Chinese-hamster ovary cells and heterodimers with full-length MUC2 in LS 174T cells

Identification of Molecular Intermediates in the Assembly Pathway of the MUC5AC Mucin

Thioredoxin liquefies and decreases the viscoelasticity of cystic fibrosis sputum

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