Complete sequence of the human mucin MUC4: a putative cell membrane-associated mucin.
The MUC4 gene, which encodes a human epithelial mucin, is expressed in various epithelial tissues, just as well in adult as in poorly differentiated cells in the embryo and fetus. Its N-terminus and central sequences have previously been reported as comprising a 27-residue peptide signal, followed by a large domain varying in length from 3285 to 7285 amino acid residues. The present study establishes the whole coding sequence of MUC4 in which the C-terminus is 1156 amino acid residues long and shares a high degree of similarity with the rat sialomucin complex (SMC). SMC is a heterodimeric glycoprotein complex composed of mucin (ascites sialoglycoprotein 1, ASGP-1) and transmembrane (ASGP-2) subunits. The same organization is found in MUC4, where the presence of a GlyAspProHis proteolytic site may cleave the large precursor into two subunits, MUC4alpha and MUC4beta. Like ASGP-2, which binds the receptor tyrosine kinase p185(neu), MUC4beta possesses two epidermal growth factor-like domains, a transmembrane sequence and a potential phosphorylated site. MUC4, the human homologue of rat SMC, may be a heterodimeric bifunctional cell-surface glycoprotein of 2.12 micrometers. These results confer a new biological role for MUC4 as a ligand for ErbB2 in cell signalling. (+info)
An intramembrane modulator of the ErbB2 receptor tyrosine kinase that potentiates neuregulin signaling.
The ErbB2 receptor tyrosine kinase plays a critical role in a variety of developmental processes, and its aberrant activation may contribute to the progression of some breast and ovarian tumors. ASGP2, a transmembrane glycoprotein found on the surface of the highly metastatic ascites 13762 rat mammary adenocarcinoma cell line, is constitutively associated with ErbB2 in these cells and in mammary tissue from pregnant rats. Expression studies indicate that ASGP2 interacts directly and specifically with ErbB2 through one of its epidermal growth factor-like domains and that the co-expression of the two proteins in the same cell dramatically facilitates their direct stable interaction. Ectopic expression of ASGP2 in human melanoma tumor cells potentiates the response of endogenous ErbB2 to the neuregulin-1 growth factor. These observations point to a novel intramembrane mechanism for the modulation of receptor tyrosine kinase activity. (+info)
Overexpression of sialomucin complex, a rat homologue of MUC4, inhibits tumor killing by lymphokine-activated killer cells.
Sialomucin complex (SMC) is a large heterodimeric glycoprotein complex composed of a mucin subunit ascites sialoglycoprotein-1 and a transmembrane subunit ascites sialoglycoprotein-2. It is a rat homologue of human mucin gene MUC4 and is abundantly expressed on the cell surface of highly metastatic ascites 13762 rat mammary adenocarcinoma cells. Because of their extended and rigid structures, mucin-type glycoproteins are suggested to have suppressing effects on cell-cell and cell-matrix interactions. During the metastatic process, these effects presumably cause tumor cell detachment from the primary tumor mass and facilitate escape of the tumor cells from immunosurveillance. Analyses of human breast cancer cells in solid tumors and tumor effusions showed that the more aggressive cells in effusions are stained with polyclonal antibodies against SMC more frequently than cells in solid tumors, suggesting a role for MUC4/SMC in tumor progression and metastasis. Previously, we generated recombinant cDNAs for SMC that vary in the number of mucin repeats to study the putative functions of SMC in tumor metastasis. These cDNAs were transfected into human cancer cell lines and tested for the effect of the expression of this gene. Here, using a tetracycline-responsive inducible expression system, we demonstrate that overexpression of SMC masks the surface antigens on target tumor cells and effectively suppresses tumor cell killing by cytotoxic lymphocytes. This effect results from the ability of SMC to block killer cell binding to the tumor cells and is dependent on both overexpression of the mucin and the number of mucin repeats in the expressed SMC. These results provide an explanation for the proposed role of SMC/MUC4 in tumor progression. (+info)
Developmental expression of mucin genes ASGP (rMuc4) and rMuc5ac by the rat ocular surface epithelium.
PURPOSE: To determine site and time of initiation of expression of the membrane-spanning mucin ASGP (rMuc4) and the goblet cell-specific, gel-forming mucin rMuc5AC by the developing rat ocular surface epithelium. METHODS: Newborn Sprague-Dawley rat pups were killed at 1, 7, and 14 days after birth. Adult rats (weight, 200 g) were used as controls. Reverse transcription-polymerase chain reaction (RT-PCR) was performed to detect ASGP mRNA using beta-actin as an internal control. Competitive RT-PCR was performed to quantitate rMuc5AC mRNA using an rMuc5AC-competitive reference standard (CRS) as an internal control. In situ hybridization was performed to localize ASGP and rMuc5AC mRNA. Goblet cells were detected by staining with periodic acid-Schiff (PAS) reagent. RESULTS: ASGP mRNA was detected by RT-PCR at 1 day after birth. Compared with beta-actin, the amount of ASGP mRNA showed a progressive increase from 1 to 14 days of postnatal development. By in situ hybridization, the expression of ASGP was first clearly detected at 14 days after birth at the lid margin, where the most stratification of epithelium was seen, and along the adjacent palpebral conjunctiva. This pattern was seen in rat eyelids that were not yet open but appeared about to open. In rat eyelids already open at 14 days after birth, ASGP mRNA was diffusely spread in the apical cell layer of both conjunctival and corneal epithelia. The expression of rMuc5AC was detected by RT-PCR in ocular surface epithelium in rat pups 1 day after birth. Quantitative RT-PCR showed a low level of rMuc5AC RNA expression in conjunctiva of 1-, 7-, and 14-day-old rats followed by a large increase in expression between 14 days and adulthood. The expression of rMuc5AC was first detected by in situ hybridization in a few goblet cells at 7 days after birth. One or two labeled cells were present in the fornical area; some were on the palpebral side of the fornix; others were present on the bulbar side. The distribution and time of appearance of rMuc5AC correlated with that of PAS staining of goblet cells. CONCLUSIONS: The developmental expression of the membrane-spanning mucin ASGP (rMuc4) and the gel-forming mucin rMuc5AC are regionally and temporally separated. Expression of the gel-forming mucin begins at the fornix at 7 days after birth and is correlated with the appearance of goblet cells, whereas, expression of the membrane-spanning mucin begins later at the lid margin at day 14. Expression of the membrane-spanning mucin correlates to eyelid opening. (+info)
Sialomucin complex (Muc4) expression in the rat female reproductive tract.
Previous studies in our laboratory demonstrated the presence of sialomucin complex (SMC)/Muc4 covering the rat uterine luminal epithelium. SMC/Muc4 expression in the uterus is regulated by estrogen and progesterone and lost at the time of receptivity. In contrast to this hormonal regulation at the uterine luminal surface, SMC/Muc4 in the uterine glandular epithelium, oviduct, cervix, and vagina was constitutively expressed at all stages of the estrous cycle. Furthermore, SMC was expressed in the cervix and vagina of the ovariectomized rat, even though it is not found in the uterine luminal epithelium. Both soluble and membrane-bound forms of SMC were present in these tissues. Immunohistochemical analyses showed distinctive localization patterns of SMC in the various tissues during the estrous cycle. Moreover, the previously unreported expression of SMC/Muc4 in the isthmus, ampulla, and infundibulum of the oviduct suggests potential functions in gamete development. These results indicate that SMC/Muc4 is expressed in most tissues of the female reproductive tract, in which it may have multiple functions. However, hormonal regulation appears to be restricted to the uterine luminal epithelium. (+info)
Vitamin A deficiency alters the expression of mucin genes by the rat ocular surface epithelium.
PURPOSE: To study effects of depletion of retinoic acid on expression of the mucins ASGP (rMuc4), rMuc5AC, and rMuc1, by the corneal and conjunctival epithelia of the rat. METHODS: Nineteen-day-old Sprague-Dawley male rats were fed a casein-based vitamin A- deficient diet or casein-based diet with vitamin A as control. Rats from both groups were killed at 1, 3, 5, 13, 15, 18, and 20 weeks after initiation of feeding. Expression of the three mucin genes by the ocular surface epithelium was assayed by reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization. RESULTS: In vitamin A-deficient rats, ASGP mRNA was not detected by RT-PCR after 15 weeks of feeding. rMuc5AC mRNA was detected by RT-PCR at 15 weeks, but by 18 and 20 weeks was no longer detectable. By in situ hybridization, ASGP mRNA was localized in the entire ocular surface epithelium after 1 week of feeding, was diminished but detectable above background by 13 weeks, and was not detectable at 20 weeks. rMuc5AC mRNA was detected in the goblet cells of vitamin A- deficient rats by in situ hybridization at 13 weeks, but was lost by 20 weeks, as were identifiable goblet cells. rMuc1 mRNA were detected by RT-PCR through all time points of 1 to 20 weeks in both vitamin A-deficient and control rats, indicating no significant change in rMuc1 mRNA expression with vitamin A deficiency. CONCLUSIONS: Both the membrane-spanning mucin ASGP (rMuc4) and the secretory mucin rMuc5AC are directly or indirectly regulated by vitamin A in the ocular surface epithelium, whereas the membrane-spanning mucin rMuc1 is not. (+info)
Membrane-associated mucins in normal human conjunctiva.
PURPOSE: To examine the presence of specific membrane-associated mucins in normal human conjunctiva. METHODS: Glycoconjugates were extracted from membranes with two detergents: octylglucoside and Triton X114. Mucins were separated by cesium chloride density gradient centrifugation. Size was assessed by gel filtration on Sepharose CL2B and charge by ion-exchange chromatography on MonoQ. Cross reaction with antibodies against mucin gene products was assessed in blots of electrophoresis gels. RESULTS: Extraction of total tissue membranes yielded material with a buoyant density typical of mucins. Gel filtration showed material reacting with antimucin antibodies in a range of molecular sizes. Agarose electrophoresis confirmed the presence of MUC1 and MUC4 and the absence of MUC2 or MUC5AC. Isolation of membrane mucins by sequential, exhaustive extraction with octylglucoside followed by Triton X114 suggested the existence of mucins in different membrane environments. Reagents to carbohydrate epitopes revealed high mobility material, comigrating with MUC1 and MUC4. Low mobility membrane-bound mucins did not cross-react with any antibodies to mucin genes known to be expressed in human conjunctiva. CONCLUSIONS: Membrane-associated mucins are distinct from secreted mucins in normal human conjunctiva. MUC1 and MUC4 mature products decorate the membranes of conjunctival epithelial cells. Their segregation between octyl glucoside and the detergent and aqueous phases of Triton X114 suggests a variety of membrane anchoring modes. (+info)
Detection of sialomucin complex (MUC4) in human ocular surface epithelium and tear fluid.
PURPOSE: To evaluate human ocular surface epithelium and tear fluid for the presence of sialomucin complex (MUC4), a high-molecular-weight heterodimeric glycoprotein composed of mucin (ASGP-1) and transmembrane (ASGP-2) subunits. METHODS: Reverse transcription-polymerase chain reaction (RT-PCR) and Northern blot analysis assays were used to identify sialomucin complex RNA in ocular surface epithelia. Immunoprecipitation and immunoblot analysis were used to identify immunoreactive species in human tears and in the corneal and conjunctival epithelia using antibodies specific for carbohydrate and peptide epitopes on the sialomucin complex subunits. Immunofluorescence staining was used to detect sialomucin complex in frozen sections and impression cytology specimens of human cornea and conjunctival epithelia. RESULTS: ASGP-1- and ASGP-2-specific sequences were amplified from RNA extracted from both conjunctival and corneal epithelial biopsies by RT-PCR. Sialomucin complex transcripts were also detected in these tissues by Northern blot analysis, with a greater level of RNA detected in the peripheral than the central corneal epithelium. Sialomucin complex was immunoprecipitated from tear fluid samples and both corneal and conjunctival epithelia and detected by immunoblot analysis with specific anti-ASGP-1 and anti-ASGP-2 antibodies. The ASGP-1 peptide antibody HA-1 stained the full thickness of the corneal and conjunctival epithelia. In contrast, antibody 15H10, which reacts against a carbohydrate epitope on ASGP-1, stained only the superficial epithelial layers of these tissues. No staining was observed in the conjunctival goblet cells. CONCLUSIONS: Sialomucin complex was originally identified in rat mammary adenocarcinoma cells and has recently been shown to be produced by the ocular surface epithelia of rats. Furthermore, it has been identified as the rat homologue of human MUC4 mucin. The present studies show that it is expressed in the stratified epithelium covering the surface of the human eye and is present in human tear fluid. Expression of a carbohydrate-dependent epitope on the mucin subunit (ASGP-1) of sialomucin complex occurs in a differentiation-dependent fashion. Sialomucin complex joins MUC1 as another membrane mucin produced by the human ocular surface epithelia but is also found in the tear fluid, presumably in a soluble form, as found on the rat ocular surface. (+info)