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Muc1 mucins on the cell surface are adhesion sites for Pseudomonas aeruginosa

E. P. Lillehoj, S. W. Hyun, B. T. Kim, X. G. Zhang, D. I. Lee, S. Rowland, and K. C. Kim

Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, Maryland 21201


    ABSTRACT
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ABSTRACT
INTRODUCTION
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RESULTS AND DISCUSSION
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Recently, we cloned and characterized a full-length cDNA of the hamster Muc1 gene, the expression of which appears to be associated with secretory cell differentiation (Park HR, Hyun SW, and Kim KC. Am J Respir Cell Mol Biol 15: 237-244, 1996). The role of Muc1 mucins in the airway, however, is unknown. In this study, we investigated whether cell surface mucins are adhesion sites for Pseudomonas aeruginosa. Chinese hamster ovary (CHO) cells not normally expressing Muc1 mucin were stably transfected with the hamster Muc1 cDNA, and binding to P. aeruginosa was examined. Our results showed that 1) stably transfected CHO cells expressed both Muc1 mRNA and Muc1 mucins based on Northern and Western blot analyses, 2) Muc1 mucins present on the cell surface were degraded by neutrophil elastase, and 3) expression of Muc1 mucins on the cell surface resulted in a significant increase in adhesion of P. aeruginosa that was completely abolished by either proteolytic cleavage with neutrophil elastase or deletion of the extracellular domain by mutation. We conclude that Muc1 mucins expressed on the surface of CHO cells serve as adhesion sites for P. aeruginosa, suggesting a possible role for these glycoproteins in the early stage of airway infection and providing a model system for studying epithelial cell responses to bacterial adhesion that leads to airway inflammation in general and cystic fibrosis in particular.

glycoprotein; binding; cystic fibrosis; airway; infection


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES

MUCINS ARE HIGH MOLECULAR MASS glycoproteins constituting the major protein component of mucus and are thought to be encoded by 12 separate genes (15, 32, 39). Although the majority of mucin genes encode secretory glycoproteins, four of them, namely, MUC1 (4, 14), MUC3 (40), MUC4 (21), and MUC12 (39), contain transmembrane domains indicating that they most likely encode cell surface mucins. MUC1 (Muc1 in nonhuman species) mucins are abundantly expressed on most carcinoma cells and have been shown to reduce intercellular (16, 36) as well as matrix adhesion (38) through their extended and rigid structure (9). These cell surface mucins are also expressed by normal epithelial cells in various glandular tissues of the respiratory, gastrointestinal, and female reproductive organs (7, 10, 25) and have been shown to be associated with cell differentiation in mammary (23), uterine epithelial (7), and airway epithelial (22) cells. Similar to cancer cells, Muc1 mucins in uterine epithelial cells have been shown to exhibit an antiadhesive property, suggesting that they play an important role in maintaining the prereceptive phase in the uterus (8). The role of MUC1 mucin in the airway, however, is unknown.

Previously, Kim et al. (12) reported that mucins released by neutrophil elastase via its proteolytic action were found on the surface of primary hamster tracheal surface epithelial (HTSE) cells. These cell surface mucins were composed of two major types: one similar to secreted mucins both in size and charge and the other resembling human MUC1 mucins based on its immunoreactivity (24). Expression of Muc1 mRNA in HTSE cells was confirmed through cDNA cloning of its gene and shown to be closely associated with goblet cell differentiation (22). Pseudomonas aeruginosa, a gram-negative bacterium almost invariably associated with airway mucus in cystic fibrosis patients, is known to bind to secreted mucins purified from airway mucus (29). Because Muc1 mucins constitute only a minor, if any, component of airway secreted mucins, it was of interest to determine whether P. aeruginosa could also adhere to these glycoproteins present on the cell surface. We approached this question by stably transfecting the hamster Muc1 gene into a hamster epithelial cell line that normally does not produce Muc1 mucins followed by monitoring P. aeruginosa adhesion to the transfected cells.


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Materials. All materials used in this study were purchased from Sigma (St. Louis, MO) unless otherwise stated.

P. aeruginosa Two P. aeruginosa strains were used: CF3, a mucoid isolated from a cystic fibrosis patient, and PAK, a nonmucoid laboratory strain (11) (a generous gift from Dr. Alice Prince, Department of Pediatrics, College of Physicians and Surgeons, Columbia University, New York, NY). Both strains were grown in Luria-Bertani broth for 7 h at 37°C and metabolically radiolabeled in sulfate-free M9 medium containing 10 µCi/ml of Na235SO4 (1,175 Ci/mmol, 100 mCi/ml, carrier-free; American Radiolabeled Chemicals, St. Louis, MO) for 16 h at 37°C. The radioactive bacteria were washed twice and resuspended to the appropriate concentrations in PBS containing 2 mg/ml of glucose for use in the binding assay.

Establishment of Chinese hamster ovary-Muc1 and Chinese hamster ovary-X cell lines. The hamster Muc1 expression plasmid pcDNA/Muc1 was constructed by inserting a full-length hamster Muc1 cDNA (22) into the pcDNA3 vector (Invitrogen, San Diego, CA). The pcDNA/Muc1 plasmid and pcDNA3 vector alone were separately transfected into Chinese hamster ovary (CHO) cells (American Type Culture Collection, Manassas, VA) with a cationic liposome, LIPOFECTIN (Life Technologies, Manassas, VA), according to the manufacturer's protocol. Clones were selected and passaged 10 times to identify stable cell lines. On the basis of Northern blot analysis, a stable clone expressing hamster Muc1 mRNA is referred to as CHO-Muc1 and a clone derived from the vector alone as CHO-X. The latter was used as a negative control in the adhesion assay. Both cell lines were maintained in Ham's F-12 medium-Dulbecco's modified Eagle's medium containing 200 µg/ml of G-418, 5% (vol/vol) fetal bovine serum, 100 U/ml of penicillin, and 100 µg/ml of streptomycin (all from Life Technologies).

Construction and expression of Muc1 mucin deletion mutants. Two Muc1 mucin deletion mutants were constructed: pcDNA/Muc1-NTR, lacking the tandem repeat region (NTR), and pcDNA/Muc1-NEC, lacking the entire extracellular (EC) domain (NEC). For both, a polymerase chain reaction (PCR) was used to amplify pcDNA3/Muc1 outside of the region to be deleted, and the amplicon was gel purified, digested with restriction endonucleases, ligated, and cloned into pcDNA3. PCR primer pairs were as follows, with the underlined nucleotides corresponding to the restriction sites used for joining amplicons and plasmid insertion: pcDNA/Muc1-NTR NH2-terminal region: MucTR-1, 5'-GCATGGATCCGGCACGAGCACAGCCACAGC-3', and MucTR-2, 5'-CGTACCCGGGGTTCAAGGTTGAGGAACTGC-3'; pcDNA/Muc1-NTR COOH-terminal region: MucTR-3, 5'-GCATCCCGGGTCCTCCATGCAAACCACCGA-3', and MucTR-4, 5'-CGATCTCGAGGCGCGTGTCAGGAAGGCTGG-3'; pcDNA/Muc1-NEC NH2-terminal region: MucTR-1, 5'-GCATGGATCCGGCACGAGCACAGCCACAGC-3', and MucTR-5, 5'-CGATGTCGACACTGTTTGGATCTGTTAC-3'; and pcDNA/Muc1-NEC COOH-terminal region: MucTR-6, 5'-GATCCTCGAGATGCAGTTTCCTTCCTCTG-3', and MucTR-7, 5'-GATCTCTAGAACTCTGGCTCACCAGCCC-3'. PCR was performed for 1 cycle at 94°C for 3 min followed by 35 cycles at 94°C for 1 min, 55°C for 1 min, 72°C for 1 min, and final extension at 72°C for 10 min. Stably transfected CHO-NTR cells were isolated as described in Establishment of Chinese hamster ovary-Muc1 and Chinese hamster ovary-X cell lines. Transiently transfected CHO-NEC cells were prepared by incubating 2 × 105 CHO cells/well in 24-well culture plates for 24 h at 37°C in 5% CO2, adding 0.2 µg of pcDNA/Muc1-NEC in serum-free Ham's F-12 medium-Dulbecco's modified Eagle's medium containing 1 µl of SuperFect reagent (QIAGEN, Valencia, CA), incubating for 2.5 h, replacing the medium with Ham's F-12 medium-Dulbecco's modified Eagle's medium containing 200 µg/ml of G-418 and 5% fetal bovine serum and incubating the cells for 48 h at 37°C.

Northern blot analysis. Total RNA was extracted from CHO-X, CHO-Muc1, and HTSE cells as previously described (22). Details of the primary HTSE cell culture were reported elsewhere (37). RNA was electrophoresed in 1% agarose-formaldehyde gels at 5 V/cm, transferred to nitrocellulose membrane (Bio-Rad, Richmond, CA), prehybridized for 2 h, and hybridized overnight at 42°C in a solution containing the 1.5-kb fragment of the hamster Muc1 cDNA (22), 50% formamide, and 6× saline-sodium citrate (SSC). The probe was labeled with a random-primer DNA labeling kit (Boehringer Mannheim, Indianapolis, IN). The membrane was washed in 0.1× SSC containing 0.1% sodium dodecyl sulfate (SDS) at room temperature and exposed to X-ray film with intensifying screens at -80°C for 2 days.

Metabolic radiolabeling, immunoprecipitation, and SDS-PAGE. In an experiment designed to radiolabel the EC domain of Muc1 mucins, CHO-X and CHO-Muc1 cells were incubated in Ham's F-12 medium-Dulbecco's modified Eagle's medium containing a "low" level of glucose (0.4 mg/ml) and D-[6-3H]-glucosamine (100 µCi/ml, 20 Ci/mmol; American Radiolabeled Chemicals) for 24 h and lysed as previously described (24). The lysis buffer contained 40 mM sodium phosphate, pH 7.2, 250 mM NaCl, 50 mM NaF, 5 mM EDTA, 1% Triton X-100, 1% deoxycholate, 10 mM benzamidine, 25 µg/ml of leupeptin, 1 mM phenylmethylsulfonyl fluoride, and 10 µg/ml of aprotinin. Lysates were immunoprecipitated overnight at 4°C as described by Boshell et al. (1) using a polyclonal antibody (CT-1) raised against a synthetic peptide (SSLSYTNPAVAATSANL) corresponding to the COOH-terminal region of the human MUC1 cytoplasmic domain (25). Both the CT-1 antibody and its blocking peptide (the synthetic peptide used to prepare it) were generous gifts from Dr. Sandra Gendler (Mayo Clinic, Scottsdale, AZ). Although produced against the human sequence, this antibody cross-reacts with hamster Muc1 mucin (24, 25). Immune complexes were precipitated with protein A agarose, and isolated proteins were resolved by SDS-PAGE on a 5% acrylamide gel and visualized by fluorography.

Immunoblot analysis. CHO-X and CHO-Muc1 cell lysates were immunoprecipitated with CT-1 antibody, resolved on a 15% SDS-acrylamide gel, and transblotted to nitrocellulose membrane. The membrane was blocked with 5% nonfat dry milk in Tris · HCl, pH 7.2, containing 0.15 M NaCl and 0.1% Tween 20 (TBS-T), reacted overnight with the CT-1 antibody (1:2,000 dilution), and washed with TBS-T, and the bound antibody was reacted with horseradish peroxidase-conjugated goat anti-rabbit antibody (1:5,000 dilution; Pierce, Rockford, IL). After the membrane was washed with TBS-T, immunoreactive bands were visualized with enhanced chemiluminescence reagent (Amersham Pharmacia, Piscataway, NJ).

Bacterial adherence assay. Adhesion of P. aeruginosa to transfected CHO cells was performed according to the method described by Rostand and Esko (33) with slight modification. Briefly, the cells were plated in 24-well dishes at 2 × 105 cells/well and grown for 24 h to confluence. The monolayer was washed twice with PBS and fixed for 10 min with 2.5% (vol/vol) glutaraldehyde in PBS at room temperature. The fixed monolayer was washed three times with PBS and incubated with 35S-radiolabeled bacteria [2 × 107 colony-forming units (cfu) · 0.5 ml-1 · well-1] for 40 min at 37°C. After incubation, the monolayer was extensively washed three times with PBS to remove the unbound bacteria. Adhered cells were lysed with 1.0 ml of 2% SDS, and radioactivity was measured with a liquid scintillation counter. The degree of adhesion is expressed as number of bacteria bound per cell.

Treatment with human neutrophil elastase. Cells were incubated with human neutrophil elastase (HNE; 5 µg/ml) (12) for 30 min at 37°C in serum-free Ham's F-12 medium-Dulbecco's modified Eagle's medium and washed twice with PBS before being fixed with glutaraldehyde for the bacterial adherence assay.

Statistical analyses. The number of bacteria adhered to epithelial cells was calculated in the following order: 1) determination of the number of bacteria adhered to each well by converting disintegrations per minute to actual number of bacteria, 2) determination of the number of epithelial cells at the end of the assay using a separate parallel group not exposed to bacteria, and 3) assessment of the significance of the difference between groups with Student's t-test for unpaired samples or analysis of variance.


    RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
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To study the functional role of Muc1 mucins, we sought to identify a nonexpressing hamster epithelial cell line to serve as a suitable host for transfection of our previously cloned hamster Muc1 cDNA. During initial screening of various cell lines, we observed that CHO cells did not express Muc1 mRNA (Fig. 1). Furthermore, a recent report by Rostand and Esko (33) showed that P. aeruginosa adhesion to CHO cells was fully accounted for by the presence of cholesterol and cholesterol esters on the epithelial cell surface, whereas glycoconjugates such as glycoproteins, glycolipids, or proteoglycans were not involved. Therefore, CHO cells turned out to be an ideal system for transfecting the hamster Muc1 gene in the present study.


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Fig. 1.   Northern blot analysis of Muc1 mRNA expression. A: total RNA was isolated from Chinese hamster ovary (CHO; lane 1) cells, CHO cells transfected with the pcDNA3 vector alone (CHO-X; lane 2), CHO cells transfected with the pcDNA/Muc1 plasmid (CHO-Muc1; lane 3), and hamster tracheal surface epithelial (HTSE; lane 4) cells. RNA samples (15 µg) were electrophoresed, transferred, probed, and analyzed by autoradiography as described in METHODS. Muc1, hamster Muc1 mRNA at 2.8 kb. B: blots were stripped and reprobed with a beta -actin probe to normalize the amount of RNA.

After two stable clones (CHO-Muc1 and CHO-X cells) were obtained (Fig. 1) as described in METHODS, we examined whether Muc1-encoded proteins are expressed on the cell surface based on the following structural information available for human MUC1 mucin: 1) MUC1 mucins are made up of two subunits noncovalently bound via a hydrophobic interaction: a larger subunit containing the EC domain where all the O-linked glycosylation sites are present and a smaller subunit containing the transmembrane region and cytoplasmic domain (9, 17); 2) the COOH-terminal amino acid sequence of hamster Muc1 mucin is virtually identical to human and mouse sequences, rendering it immunoreactive with rabbit CT-1 antibody against the 17 COOH-terminal residues (22); and 3) hydrophobic binding between the two subunits can be disrupted by high concentrations of SDS (17). We also exploited the previous observation that HNE released Muc1 mucins from the surface of airway goblet cells (37). In the present experiment, cells were metabolically radiolabeled with [3H]glucosamine to identify the glycosylated EC domain and immediately treated with HNE to cleave the EC domain, the cells were then lysed in detergent solution, and the lysates were immunoprecipitated with CT-1 antibody (25). After immunoprecipitation, the cell lysates were subjected to SDS-PAGE, and the larger subunit containing most of the radiolabeled sugar moieties was identified by 3H fluorography, whereas the smaller subunit containing the cytoplasmic tail was identified by immunoblotting with the CT-1 antibody.

Figure 2A shows the presence of a glycoprotein with an apparent molecular mass of 180 kDa in CHO-Muc1 cells (lane 2) but in not CHO-X cells (lane 1). This protein was degraded by HNE (Fig. 2A, lane 3), thus identifying it as the larger subunit of Muc1 mucins. Because the concentration of HNE used in this experiment did not cause lactate dehydrogenase release from the cells (data not shown), we concluded that the reduced signal of the [3H]glucosamine-labeled EC domain after enzyme treatment was not due to nonspecific disruption of the plasma membrane. Immunoblot analysis of these samples with the CT-1 antibody (Fig. 2B) showed the presence of a 26-kDa protein in CHO-Muc1 cells regardless of HNE treatment (lanes 2 and 3), thus identifying this protein as the smaller subunit of Muc1 mucins. When the immunoprecipitation reaction was blocked with the synthetic peptide used to prepare the CT-1 antibody, appearance of the 26-kDa band was completely abolished (data not shown). Collectively, the results presented in Fig. 2 indicate that CHO-Muc1 cells express Muc1 mucins on the cell surface, whereas CHO-X cells do not.


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Fig. 2.   Immunoprecipitation and Western blot analysis of CHO-Muc1 cell lysates after treatment with human neutrophil elastase (HNE). Confluent cell cultures were metabolically radiolabeled with [3H]glucosamine overnight and treated with HNE for 30 min before cell lysis. A: cell extracts were immunoprecipitated with CT-1 antibody, and the precipitates were separated on a 5% SDS-PAGE gel and analyzed by fluorography. B: same extracts were subjected to immunoblot analysis with CT-1 antibody as described in METHODS. Lanes 1, CHO-X cells without HNE; lanes 2, CHO-Muc1 cells without HNE; lanes 3, CHO-Muc1 cells plus HNE. Nos. in middle, prestained protein size markers. Left arrowhead, larger (180-kDa) subunit; right arrowhead, smaller (26-kDa) subunit.

Having characterized the transfected cells for Muc1 mucin expression, we next compared the ability of CHO-X and CHO-Muc1 cells to bind to P. aeruginosa. In preliminary experiments, we tried both live and fixed cells for the P. aeruginosa adhesion and found no significant differences between them, with the exception that live cells tended to give greater variation in bacterial adhesion compared with that in fixed cells (data not shown). Moreover, a recent report (26) that live human airway epithelial cells may internalize P. aeruginosa led us to use fixed cells for the P. aeruginosa adhesion experiments. Results from kinetic studies with CHO-X cells (data not shown) were practically identical to those previously reported for nontransfected CHO cells (33). P. aeruginosa adhesion to CHO-X cells reached a plateau after 40 min, with approximately five bound bacteria per CHO-X cell. Based on these results, we chose adhesion assay conditions utilizing 2 × 107 cfu of radioactive P. aeruginosa incubated with 2 × 105 cells for 40 min at 37°C and quantified the number of bound bacteria. Figure 3 shows that although P. aeruginosa adhered to both CHO-X and CHO-Muc1 cells in a concentration-dependent fashion, adhesion to CHO-Muc1 cells was consistently about twofold greater than that to CHO-X cells regardless of the number of P. aeruginosa added.


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Fig. 3.   Pseudomonas aeruginosa adhesion to CHO-X and CHO-Muc1 cells. Indicated concentrations of 35S-labeled P. aeruginosa (CF3 strain) were incubated with either confluent CHO-X (open circle ) or CHO-Muc1 () cells in 16-mm wells for 40 min, and the no. of bacteria bound to the epithelial cells was quantified as described in METHODS. Values are means ± SE from 6 wells. * Significantly different, P < 0.01.

Several previous studies found that mucoid P. aeruginosa strains adhered to epithelial cells better than nonmucoid variants (18-20), but others found no difference (2, 31) or that the mucoid forms bound less strongly (30, 41). These discrepancies could be due to differences in the bacterial isolates and/or cell types used. To investigate whether the increased adhesion of the CF3 strain of P. aeruginosa to CHO-Muc1 cells that we observed was due to its mucoid nature, we next tested the nonmucoid PAK strain that has been used extensively for studying adhesion to the airway epithelial cell (3). Figure 4 shows the results of CF3 and PAK adhesion to CHO-X and CHO-Muc1 cells. Although the former P. aeruginosa strain displayed substantially greater adhesion to both CHO cell transfectants, the degree of adhesion to CHO-Muc1 cells was significantly greater than to CHO-X cells with both bacterial strains. Thus the binding of CF3 and PAK to CHO-Muc1 cells was 194 ± 6 and 242 ± 8% greater, respectively, than to CHO-X cells. The addition of a 100-fold excess of "cold" P. aeruginosa (2 × 109 cfu) displaced >90% of 35S-labeled P. aeruginosa adhesion to CHO-Muc1 cells by both strains, suggesting the presence of specific binding to the cells (data not shown).


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Fig. 4.   Comparison of 2 P. aeruginosa strains for adherence to CHO-X and CHO-Muc1 cells. CHO-X and CHO-Muc1 cells were incubated with 35S-labeled CF3 (a mucoid P. aeruginosa strain) or PAK (a nonmucoid P. aeruginosa strain), and the no. of bacteria adhered was measured as described in METHODS. Values are means ± SE from 6 wells. * Significantly different, P < 0.01.

Because the only difference between CHO-Muc1 and CHO-X cells is most likely the presence of the Muc1 mucin on the surface of the former cells, the results presented above strongly suggested that increased P. aeruginosa adhesion to CHO-Muc1 cells was mediated by Muc1 mucins. However, these results do not rule out the alternative, although unlikely, possibility that transfection with the Muc1 mucin cDNA induced expression of a nonmucin protein responsible for bacterial adhesion. To more directly address the role of this glycoprotein in P. aeruginosa adhesion, two experimental approaches were taken: proteolytic digestion of mature mucins on the cell surface with HNE and analysis of EC domain deletion mutants. HNE has previously been shown to specifically and efficiently release Muc1 mucin from primary HTSE cells (12) and CHO-Muc1 cells (Fig. 2). As shown in Fig. 5, the addition of HNE to the binding assay completely abolished the increased binding of the CF3 and PAK strains to CHO-Muc1 cells to the basal levels seen with CHO-X cells. Reduced adhesion of P. aeruginosa to HNE-treated CHO-Muc1 cells was significant (P < 0.01).


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Fig. 5.   Effect of HNE on P. aeruginosa adhesion to CHO-X and CHO-Muc1 cells. CHO-X and CHO-Muc1 cells were incubated with 35S-labeled CF3 (A) or PAK (B) strain P. aeruginosa in the presence and absence of 5 µg/ml of HNE, and the no. of bacteria adhered was measured. Open bars, binding in the absence of HNE; solid bars, binding in the presence of HNE. The no. of CF3 and PAK binding to CHO-X cells in the absence of HNE was set to 100% (control). Values are means ± SE from 6 wells. * Significantly different, P < 0.01.

Finally, CHO cells transfected with mutants of the Muc1 cDNA that were either partially (CHO-NTR) or completely (CHO-NEC) deleted of the EC domain were analyzed for P. aeruginosa adhesion. Construction of the NTR deletion mutant generated a molecule devoid of O-linked sugar units while still retaining the majority of the protein core and all N-linked glycosylation sites. The complete NEC deletion mutant, on the other hand, was constructed to produce a Muc1 mucin molecule containing only the transmembrane and cytoplasmic domains of the protein. Expression of both mutants in transfected CHO cells was confirmed by immunoblot analysis with the CT-1 antibody (data not shown). As shown in Fig. 6, CHO cells expressing these mutants displayed significantly altered binding to P. aeruginosa compared with the nonmutant Muc1 mucin. Thus although PAK binding to CHO-Muc1 cells was 280 ± 24% greater than to CHO-X cells, CHO-NTR and CHO-NEC cells showed 163 ± 7 and 80 ± 4% P. aeruginosa binding, respectively, compared with CHO-X cells. In other words, deletion of the tandem repeat region led to a 60% reduction in P. aeruginosa adhesion, whereas complete deletion of the EC domain resulted in a total loss of the increased P. aeruginosa adhesion in CHO-Muc1 cells. Collectively, these results strongly suggested that Muc1 mucin is the cellular receptor for P. aeruginosa adherence.


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Fig. 6.   Deletion of the tandem repeats (NTR) or extracellular domain (NEC) of Muc1 mucin reduces P. aeruginosa adherence. CHO-X cells, CHO-Muc1 cells, CHO cells transfected with the pcDNA/Muc1-NTR plasmid (CHO-NTR), or CHO cells transfected with the pcDNA/Muc1-NEC plasmid (CHO-NEC) were incubated with 35S-labeled PAK, and the no. of bacteria adhered was measured as described in METHODS. The no. of bacteria bound to CHO-X cells was set to 100% for the purpose of comparison. Values are means ± SE from 6 wells. * Significantly different, P < 0.001. ** Not significantly different, P > 0.05.

The exact glycosylation patterns of MUC1 mucins, human and nonhuman, are not known. Previous reports (28, 34, 35) on P. aeruginosa binding to epithelial cells, however, implicated sugar residues in the primary binding domains. Most of the sugar moieties of human MUC1 and hamster Muc1 mucins are O-linked, are found in the tandem repeat region, and are composed of 20-amino acid repeats rich in serine, threonine, and proline (5). However, the number of repeating units varies greatly between these two species such that the hamster molecule contains 12 repeats (22) and the human 25-125 repeats (5). Thus MUC1 mucin may possess more binding sites and higher affinity for P. aeruginosa. Interspecies differences in glycosylation between MUC1 and Muc1 mucin molecules may result in heterogeneous patterns of P. aeruginosa adhesion, potentially limiting our study. On the other hand, such alterations in glycosylation may be crucially important for P. aeruginosa adhesion to epithelial cells. It is interesting that cells expressing the deletion mutant devoid of tandem repeats (CHO-NTR) showed significantly increased (~40%) P. aeruginosa adhesion compared with CHO-NEC cells, suggesting the presence of at least two binding subdomains. Current experiments are underway to further define the molecular nature of these subdomains with regard to the involvement of N-linked sugars and/or the protein core of Muc1 mucin.

The physiological significance of P. aeruginosa adhesion to MUC1 mucins remains unknown. These molecules may serve as a secondary defense barrier in the lung against airborne bacteria that manage to escape the gel phase of airway mucus, the primary physical barrier. Bacteria "trapped" by MUC1 mucins might be cleared from the airway by some as yet unidentified process, possibly involving proteolytic cleavage and release of the mucin-bacteria complex from the cell surface for subsequent removal by the mucociliary escalator. Alternatively, although not mutually exclusive, MUC1 mucins may serve as signal transduction receptors for microorganisms that penetrate the primary mucous layer, bind to epithelial cells, and induce an inflammatory response (13). For example, it was recently shown that adhesion of P. aeruginosa to glycolipids on airway epithelial cells resulted in nuclear translocation of nuclear factor-kappa B and interleukin-8 expression (3), indicating an important role for these molecules in initiating an epithelial proinflammatory response to P. aeruginosa adhesion. Important in this regard, MUC1/Muc1 mucins from human, mouse, hamster, and rabbit have virtually identical amino acid sequences in their cytoplasmic domains, with conserved consensus motifs for tyrosine phosphorylation (4, 6, 14, 19). Recent reports by Zrihan-Licht et al. (42) and Quin and McGuckin (27) that MUC1 mucins from mammary and ovarian carcinoma cell lines can be phosphorylated on both tyrosine and serine residues in the cytoplasmic domain strongly implicate activation of intracellular signal transduction pathway(s) mediated by this cell surface mucin. We are currently investigating downstream signaling mechanisms and cellular inflammatory responses in the P. aeruginosa/CHO-Muc1 model system.


    ACKNOWLEDGEMENTS

This work was supported by National Heart, Lung, and Blood Institute Grant R01-HL-47125 and research grants from the Cystic Fibrosis Foundation.


    FOOTNOTES

Address for reprint requests and other correspondence: K. C. Kim, Dept. of Pharmaceutical Sciences, Univ. of Maryland School of Pharmacy, 20 N. Pine St., Rm. 446, Baltimore, MD 21201.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 17 January 2000; accepted in final form 16 October 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES

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