The pathology of cystic fibrosis (CF) is characterized by organ damage resulting from failure to clear mucous secretions from epithelial surfaces primarily in the airways, the pancreas, and the male genital ducts. The cause of the mucus clearance problems remains poorly understood. Several hypotheses to explain the mucus abnormalities have been put forward, including mucin hypersecretion, dehydration of mucins due to ion transport defects that result from mutations in the CFTR cAMP-activated chloride ion channel, and biochemical abnormalities in the glycosylation of mucins in CF epithelial cells including increased sulfation and fucosylation and reduced sialylation.
Analyses of mucin from CF patients have been based on the evaluation of bulk mucus purified directly from CF patients and controls (Roussel et al., 1975; Boat et al., 1976; Wesley et al., 1983; Lo-Guidice et al., 1994) or on the investigation of mucins secreted by epithelial tissues or cell lines derived from CF patients (Frates et al., 1983; Scanlin et al., 1985; Cheng et al., 1989; Wang et al., 1990; Mohapatra et al., 1995; Mergey et al., 1995; Zhang et al., 1995; Hill et al., 1997; Jiang et al., 1997). Data derived from analysis of bulk mucus have suggested increased glycosylation and sulfation in CF intestinal mucins and abnormal glycosylation in CF airway mucins. However, bulk mucin preparations from patients are complicated by secondary modifications that result from bacterial infection or disease pathology. It has been difficult to determine whether mucins are altered as a secondary effect of mutant CFTR protein. Analysis of mucins secreted by epithelial tissues or cell cultures from CF patients offers advantages over patient samples (Frates et al., 1983; Scanlin et al., 1985; Cheng et al., 1989; Wang et al., 1990; Mohapatra et al., 1995; Zhang et al., 1995), though these experiments also have their limitations: to date it has only been possible to analyze mixtures of mucous glyco-proteins produced by these cells because of a lack of reagents that discriminate among different mucin core proteins. Moreover, it is difficult to control for normal variation in the glycosylation pathways between different individuals and in different culture conditions (Emery et al., 1997). Recently, the availability of epithelial cells carrying defined mutations in the CFTR gene has enabled the generation of matched pairs of mutant cell lines and 'corrected" cells following expression of the wild-type CFTR cDNA. These provide model systems to evaluate the effect of mutant CFTR on mucin gene transcription, glycoprotein processing and secretion (Hill et al., 1997; Jiang et al., 1997). A confounding factor is that some of these matched pairs of mutant and normal cells lines exhibit spontaneous downregulation of CFTR transcription.
Since the biochemical and biophysical properties of a mucin are dependent on O-glycosylation, our aim was to evaluate the O-glycosylation of a single mucin gene product in matched pairs of cells that differed only with respect to CFTR expression. However, it is probable that loss of CFTR expression may be accompanied by other intracellular changes. To achieve this an epitope-tagged MUC1 mucin cDNA (MUC1F; Burdick et al.,1997) was stably expressed in cell lines and an antibody specific for the epitope was used to purify this mucin. This approach detected variation in mucin glycosylation (expression of blood group antigens) in different cell lines (Burdick et al., 1997), including the colon carcinoma cell lines HT29 and Caco2. We examined the expression of blood group antigens on MUC1F mucin in matched pairs of Caco2 cell lines that either express wild-type CFTR or have spontaneously switched off CFTR expression and so may be considered equivalent to CFTR null mutants. Glycosylation of MUC1F was evaluated by reactivity with a series of monoclonal antibodies against known blood group and tumor-associated carbohydrate antigens. Metabolic labeling experiments were then carried out to estimate the gross levels of glycosylation and sulfation of MUC1F mucin in these matched pairs of cell lines. Expression of CFTR in this experimental system did not affect the gross levels of glycosylation or sulfation of the MUC1F mucin nor the antigenicity of the carbohydrates structures attached to the MUC1F protein.
Generation of HT29 and Caco2 cell lines carrying an epitope tagged MUC1 gene (MUC1F)
Eight HT29 clones transfected with MUC1F and five HT29 clones transfected with pH[beta]-APr1-neo were isolated. Genomic DNA was extracted from each clone, digested with EcoRI, and Southern blotted. The blots were probed with a MUC1 cDNA (Chambers et al.,1994) to confirm that each cell line had stably integrated one or more copies of the transfected plasmid.
Thirteen Caco2 clones transfected with MUC1F, and seven Caco2 clones transfected with the pH[beta]-APr1-neo were isolated. The presence of one or more stably integrated copies of MUC1F was confirmed as for the HT29 clones. To assess the level of expression of MUC1 and CFTR mRNA in these Caco2 clones, RNA was extracted, separated on denaturing agarose gels, transferred to Hybond N+, and Northern blotted. The blots were probed with a MUC1 cDNA probe (Chambers et al.,1994) or with a CFTR cDNA probe (CF10-1). The levels of expression of MUC1 and CFTR in each clone were determined by comparing the intensity of the MUC1 or CFTR-derived signal on an autoradiograph with that obtained from a GAPDH probe subsequently hybridized to the same filter (not shown). On the basis of these data, six cell clones were chosen for further analysis. The clones either expressed CFTR mRNA levels equivalent to those of the Caco2 parental population (wild type) or had CFTR mRNA levels that were not detectable on Northern blot analysis. In addition they expressed high or low levels of MUC1F mRNA. Three clones expressed wild-type levels of CFTR mRNA and low (M12) or high (M36 and M51) levels of MUC1F mRNA. These clones were designated MUC1F+/CFTR+ (see Table III). Three clones lacked CFTR mRNA and expressed low (M24) or high (M17 and M50) levels of MUC1F mRNA. These were designated MUC1F+/CFTR-. These cell clones were used to study the effect of the presence or absence of CFTR protein on the O-glycosylation of the MUC1F glycoprotein.
The Caco2 cell line, in common with some other colon adenocarcinoma cell lines, contains populations of cells with a number of different morphologies. These may correspond to cells with distinct characteristics of differentiation. In order to ensure that the clones chosen for further analysis on the basis of their CFTR expression status were of common differentiated types, the morphology of each clone was analyzed (Figure
Figure 1. Morphology of MUC1F transfected Caco2 clones. (A) M12, (B) M24, (C) M17, (D) M36, (E) M50, (F) M51, (G) N30 (pH[beta]-APr1-neo control transfected). Magnification, 215×. Analysis of MUC1F in HT29 cells
Cell lysates were prepared for eight clones carrying MUC1F (H1M, H2M, H4M, H5M, H7M, H8M, H9M, and H10M) and five control clones carrying the pH[beta]-APr1-neo vector (H3K, H7K, H8K, H9K, and H10K). Data are presented from only one vector control clone as these clones showed identical results. Cell lysates were analyzed by Western blotting using a panel of antibodies against carbohydrate structures commonly found on O-glycosylated proteins (tumor- associated and blood group antigens; Table I). On each Western blot the M2 antibody, which reacts with the FLAG epitope, was used to identify the MUC1F glycoforms. Some of the antibodies to carbohydrate structures react with endogenous glycoproteins in the HT29 clones. Hence, MUC1F glycoproteins were scored as positive for a carbohydrate epitope only if an antibody specific for that epitope reacted with a glycoprotein that was not present in the pH[beta]-APr1-neo vector control clones and colocalized with the M2 reactive glycoprotein in Western blots. Though this interpretation is probable, it cannot be excluded that other glycoproteins have the same mobility. However, in the case of the Caco2 clones described below, M2-immunoprecipitated MUC1F gave the same results as total cell lysates, suggesting that our interpretation is correct.
Table I.
Antigen
mAbs
N1, vector pH[beta]
HT29 clone (vector MUC1F)
HIM
H2M
H4M
H5M
H7M
H8M
H9M
H10M
Flag
M2
+
+
+
+
+
+
+
+
Ca2
+
+
+
+
+
+
+
+
Lea
CO5143
Sialyl Lea ([alpha]2-3)
CA19-93
+
+
+
+
+
+
+
+
Sialyl Lea ([alpha]2-3)
B67.43
+
+
+
+
+
+
+
+
Le2
CO431
sialyl Lec (LSTa)
Dupan2
Lex
B93.1
+
+
+
sialyl Lex
CSLEX12
+
+
+
+
+
+
Ley
B32.4
sialyl Tn
CC492
+
+
+
+
+
+
+
+
Table II. Glycosylation of MUC1F in Caco2 cell lines Expression and glycosylation of MUC1F glycoprotein in HT29 cell lines.
MUC1F from all HT29 clones reacted with the anti-FLAG M2 and also with antibody CA-2 which reacts with the MUC1 core protein (Briggs et al., 1993). All eight clones showed the same pattern of expression of MUC1F glycoforms: a protein with a MW greater than 220 kDa showed the strongest reactivity with M2 and CA-2 (Figure
MUC1F glycoprotein from all HT29 clones carried the structures sialyl Lea (CA19-9 and B67.4) and sialyl Tn (CC49), while 6 out of 8 clones carried sialyl Lex CSLEX1 (Figure
Figure 2. Analysis of MUC1F expressed in HT29 cells. Each panel shows a Western blot of cell lysate separated on SDS-PAGE gels probed with M2 (lanes 1-3); CA19-9 (lanes 4-6); B67.4 (lanes7-9); CSLEX (lanes 10-12). Each lanes contains 200 µg of cell lysate: lanes 1, 5, 7, clone H7; lanes 2, 6, 8, clone H8; lanes 3, 4, 9, 10, clone N3; lane 11, clone H4; lane 12, clone H5. Analysis of MUC1F in Caco2 cell lines
Thirteen MUC1F transfected clones (M1, M3, M4, M7, M12, M14, M17, M20, M24, M27, M36, M50, and M51) and seven pH[beta]-APr1-neo controls (N22, N23, N25, N26, N29, N30 and N34) were chosen for analysis of their glycosylation patterns (Table III). Data are presented for only one vector control as all showed identical results. Cell lysates were prepared for each clone and analyzed by Western blotting. MUC1F glycoproteins were scored as positive for a carbohydrate epitope as described above for the HT29 cell clones (Table III). Expression and glycosylation of MUC1F glycoprotein in Caco2 cell lines
All the MUC1F transfected clones reacted with anti-FLAG antibody (M2) and with CA-2, which is specific for the MUC1 core peptide. The majority of clones showed the same pattern on Western blots: with one glycoform at greater than 250 kDa, which reacted most strongly with M2 and Ca2; and two lower MW forms at about 200 kDa and 150 kDa, which reacted less strongly (Figure
Figure 3. Analysis of MUC1F expressed in Caco2 clone M51 (lanes 2 and 3) and clone M50 (lanes 4 and 5). A Western blot probed with M2 of total cell lysates (200 µg) in lanes 1, 2, and 4, and immunoprecipitated material (from 200 µg of total cell lysate) in lanes 3 and 5 separated on SDS-PAGE gels. Lane 1 contains the N30 control clone.
Glycosylation data for MUC1F in Caco2 clones are summarized in Figure
Figure 4. Analysis of MUC1F expressed in Caco2 clones. Each panel shows a Western blot of cell lysate separated on SDS-PAGE gels probed with M2 (lanes 1-3), CA-2 (lanes 4-6), and CC49 (lanes7-9). Each lanes contains 200 µg of cell lysate. Lanes 1, 4, and 7, clone N30 (vector only); lanes 2, 5, and 8 clone M50; lanes 3, 6, and 9 clone M51. Analysis of MUC1F glycosylation in CFTR± Caco2 cell lines
MUC1F glycoprotein from three CFTR+/MUC1F+ cell lines (M12, M36, and M51) and three CFTR-/MUC1F+ cell lines (M24, M17, and M50) was immunoprecipitated from cell lysates with the M2 antibody. Total cell lysate (200 µg) and immunopurified material from 200 µg of total lysate were loaded in adjacent lanes and analyzed by Western blotting (Figure Glycosylation and sulfation of MUC1F in Caco2 cells in the presence or absence of CFTR
The relative levels of glycosylation and sulfation of MUC1F glycoprotein from the same three CFTR+/MUC1F+ cell lines (M12, M36, and M51) and three CFTR-/MUC1F+ cell lines (M24, M17, and M50) were evaluated. The cell lines were grown to ~30% confluence, washed in PBS, and labeled with 14C amino acids and 3H glucosamine for glycosylation experiments and with 35S sulfate and 3H glucosamine for sulfation assays. Cell lysates were prepared at confluence, and the MUC1F glycoprotein purified by immunoprecipitation with the M2 antibody. Immunoprecipitated material was separated on SDS-PAGE gels and visualized by autoradiography (Figure
Table III.
Table IV.
The six clones each showed different levels of glycosylation (Table IV) with ratios of carbohydrate to protein ranging from4.4 ± 0.5:1 for M12 (~4.4 mol of 3H glucosamine to 1 mol of 14C amino acids) to 24.0 ± 1.71:1 for M50 (~24 mol of 3H glucosamine to 1 mol of 14C amino acids). The presence or absence of CFTR had no effect on the level of glycosylation of MUC1F in these cell lines. Statistical analyses of these data by Mann-Whitney and Kruskal-Wallis tests did not reveal any statistically significant differences between levels of glycosylation of MUC1F in CFTR+ and CFTR- clones (not shown). The ratio of 3H glucosamine to 14C amino acids was lower in clones M12 (CFTR+) and M24 (CFTR-) than in the other clones. This correlates with low levels of MUC1F mRNA and protein expression in these two lines (as assessed by Northern and Western blot analysis) in comparison to clones M36, M51, M17, and M15.
Figure 5. Immunoprecipitation of 14C, 3H, and 35S-labeled MUC1F from Caco2 clone M51. Material was separated on SDS-PAGE and visualized by autoradiography. Each lane represents the MUC1F isolated from an individual T75 cell culture flask. The bars indicate the gel regions excised for scintillation counting.
The level of sulfation in the 6 clones varied (Table V) ranging from 0.220 ± 0.036:1 for M12 (~0.22 mol of 35S sulfate to 1 mol of 3H glucosamine) to 0.044 ± 0.016:1 for M17. There was no correlation between the presence or absence of CFTR and the level of sulfation of MUC1F in these Caco2 clones.
The relatively large clonal variation in gross glycosylation and sulfation makes the interpretation of these data difficult. The clones that express very low levels of MUC1F (clone M12 [CFTR+] and clone M24 [CFTR-]) give rise to this large variation. If clones expressing high levels of MUC1F are compared (clone M36 and M51 [CFTR+], M17 and M50 [CFTR-]), the clonal variations are reduced to ratios of 1:2. This renders the comparisons more informative and confirms that there is no correlation between the presence or absence of CFTR and the gross levels of glycosylation and sulfation of MUC1F.
Table V.
Antigen
mAbs
pH[beta]
CFTR+
CFTR-
M12
M36
M51
M17
M24
M50
Flag
M2
+
+
+
+
+
+
Muc1 Core
Ca2
+
+
+
+
+
+
Lea
CO514
Sialyl Lea ([alpha]2-3)
CA19-9
Sialyl Lea ([alpha]2-3)
B67.4
+
+
+
+
+
+
Leb
CO431
Sialyl Lec (LSTa)
Dupan2
Lex
B93.1
Sialyl Lex
CSLEX1
+
+
+
Ley
B32.21
Sialyl Tn
B72.3
Sialyl Tn
CC49
+
+
+
+
+
+
Tri -Tn
B230.1
Caco2 clone
Muc1 expressiona
CFTR expressiona
Ratio of 3H glucosamine to 14C amino acids
M12
Low
+
4.4 ± 0.5:1
M36
High
+
22.0 ± 2.8:1
M51
High
+
14.5 ± 1.3:1
M24
Low
8.7 ± 0.32:1
M17
High
12.4 ± 1.26:1
M50
High
24.0 ± 2.0:1
Caco2 clone
Muc1 expressiona
CFTR expressiona
Ratio of 35S sulfate to 3H glucosamine
M12
Low
+
0.220 ± 0.036:1
M36
High
+
0.061 ± 0.009:1
M51
High
+
0.108 ± 0.017:1
M24
Low
0.146 ± 0.012:1
M17
High
0.044 ± 0.016:1
M50
High
0.062 ± 0.003:1
The aim of these experiments was to evaluate the effects of CFTR expression on glycosylation and sulfation of MUC1F. The published data on glycosylation and sulfation of mucins in CF are inconsistent, and many experiments have used inadequate model systems. Reports of increased glycosylation and sulfation of mucins from CF airway tissue (Boat et al., 1976; Frates et al., 1983; Lo-Guidice et al., 1994) and intestine (Wesley et al., 1983) are complicated by the potential for secondary modifications in mucins secreted from diseased epithelia. Increased levels of fucosylation observed in skin fibroblasts from CF patients (Scanlin et al., 1985; Wang et al., 1990) are difficult to interpret as the CFTR gene is not expressed in fibroblasts, so it is unclear how mutations in CFTR would affect glycoprotein processing. Experiments showing increased sulfation of glycoconjugates secreted by CF nasal epithelial cells in culture (Cheng et al., 1989) or CF bronchial epithelial cells in a xenograft model (Zhang et al., 1995) are more relevant. Elevated sulfation of glycoconjugates has also been reported in transformed airway epithelial cell lines established from CF patients though only in cell surface glycoconjugates rather than secreted forms (Mohapatra et al.,1995).
Immortalized airway epithelial cells have also been used to demonstrate that cAMP-mediated glycoconjugate secretion may be defective in CF tracheal cells and that this can be corrected by transfer of a normal CFTR transgene to these cells (Mergey et al., 1995). However, in other experiments using equivalent cell lines, the differences in glycosylation seen between CF lines and lines with CFTR function restored by a normal transgene were shown to be independent of CFTR and rather due to clonal differences in carbohydrate processing between cell lines (Jiang et al.,1997).
All the experiments in which CF mucins have been analyzed to date, regardless of the experimental model, have analyzed a mixture of glycoproteins secreted from epithelial cells, rather than evaluating the biochemistry of individual mucins. Our aim was to look at an individual mucin processed by matched pairs of cells that express normal CFTR or mutant CFTR. Though the MUC1 mucin is not a classical gel-forming mucin, we chose to examine the processing of this molecule due to the availability of a well characterized set of reagents, not currently available for any of the gel-forming mucins. We analyzed an epitope-tagged MUC1 (MUC1F) that enabled the purification of this mucin from all other cellular glycoproteins prior to analysis of glycosylation and sulfation of the molecule.
Evaluation of the glycosylation of MUC1F in HT29 and Caco2 cell lines showed that the profile of carbohydrate epitopes added to MUC1F was different in these two lines though individual clones of each cell line showed little variation. Hence MUC1F synthesized in HT29 cells carries sialyl Lea , Lex , Lea, sialyl Lex, and sialyl Tn while MUC1F synthesized in Caco2 clones only carries sialyl Lea, sialyl Tn, and in some clones sialyl Lex. These data extended our previous observations that the epitope-tagged MUC1F allowed identification of O-linked oligosaccharides found on MUC1 expressed in pancreatic and colon tumor lines (Burdick et al., 1997). It was of interest that both the M2 (anti-FLAG) antibody and the CA-2 (anti MUC1 tandem repeat) antibody detected three MUC1F glycoforms on Western blots of Caco2 clones. The antibodies directed against carbohydrate structures interacted most strongly with the slowest mobility glycoform, while the two faster migrating forms reacted either less strongly or not at all. It is possible that the slowest mobility glycoforms are mature MUC1 which has been more highly processed and probably undergone several rounds of recycling at the cell membrane while the faster mobility forms are not fully processed. Alternatively, other modifications that significantly affect the charge of the MUC1F glycoprotein, such as sialylation and sulfation, are known to affect the mobility of the molecule (Litvinov and Hilkens, 1993; Burdick et al., unpublished observations). These glycoforms of MUC1F are probably equivalent to those described previously for endogenous MUC1 biosynthesis (Hilkens and Buijis, 1988; Linsley et al., 1988; Litvinov and Hilkens, 1993).
Next we analyzed the O-glycosylation of MUC1F in matched pairs of Caco2 cells that either expressed wild type CFTR or had lost CFTR expression in culture and were equivalent to CFTR null mutants. Morphological characterization of these clones suggested that we had not merely selected differentiated cell types with distinct functions that might express divergent glycosyltransferases. Alterations in the glycosylation of mucins produced in CF cells might be reflected in the precise carbohydrate structures that are attached to the mucin peptide backbone or in the gross levels of glycosylation.
Monoclonal antibodies against a number of tumor-associated antigens and blood group antigens were used to investigate whether MUC1F from CFTR+ Caco2 cells carried different carbohydrate structures than MUC1F synthesized in CFTR- cells. No differences were detected in the array of carbohydrate structures decorating the MUC1F peptide in CFTR+ and CFTR- lines, both carrying sialyl Lea, sialyl Tn, and in some clones sialyl Lex.
Metabolic labeling experiments also provided no evidence for gross differences in the glycosylation or sulfation of MUC1F synthesized in CFTR+ or CFTR- Caco2 clones. Though there were differences in the total levels of glycosylation and sulfation of MUC1F in different Caco2 clones, these variations reflected clonal differences particularly with respect to the total amounts of MUC1F glycoprotein being synthesized. MUC1F synthesized in two clones (MUC12 and MUC24) that expressed very low levels of the transgene was less heavily glycosylated and more heavily sulfated than MUC1F produced by clones which expressed higher levels of MUC1F glycoprotein. The clonal variation in glycoprotein processing is an important factor to consider in interpreting this type of analysis, as has been suggested by others (Jiang et al., 1997). The precise growth condition of a cell line at the time of glycoprotein extraction is also relevant (Emery et al., 1997).
CFTR has been reported to play a role in plasma membrane recycling (Bradbury et al., 1992), and cells carrying mutant CFTR were shown to lack normal cAMP-dependent regulation of endocytosis and exocytosis. It is known that fully mature glycosylated MUC1 undergoes several rounds of membrane recycling (Hilkens and Buijis, 1988; Linsley et al., 1988; Litvinov and Hilkens, 1993). Hence, we might have expected to see a different distribution of MUC1F glycoforms in Caco2 clones lacking CFTR. Though some variations in glycoform distribution were noted, these were clonal variations. We detected no consistent differences in the distribution of MUC1F glycoforms in CFTR+ and CFTR- clones.
It has been suggested that a mutant CFTR protein may cause dysfunction of intracellular membranes as well as the apical membrane of epithelial cells. Hence, mutations in CFTR result in an altered pH within intracellular organelles due to loss of normal Golgi acidification (Barash et al., 1991; Barasch and Al-Awqati, 1993). If this were the case, then the activity of glycoprotein-processing enzymes would be altered and abnormalities in glycosylation, sialylation, and sulfation might be predicted. The data presented here would not support this hypothesis. However, the model system that we have used employs the equivalent of CFTR null mutants, and it is possible that only certain processing mutations in CFTR such as [Delta]F508, which result in mislocalization of CFTR to the endoplasmic reticulum, are associated with a raised intracellular pH. Other experiments have provided evidence that trans-Golgi and endosome acidification is normal in CF cells (Seksek et al., 1996). A further suggestion is that permeability of CFTR to adenosine 3[prime]-phosphate 5[prime]-phosphosulfate, might provide a link between mutations in CFTR and the abnormalities in glycoprotein processing characteristic of CF (Pasyk and Foskett, 1997). However, these data are derived from CFTR-transfected CHO cells, and so their relevance to endogenous glycoprotein processing in CF epithelial cells is uncertain.
The failure to detect differences in the glycosylation and sulfation of MUC1F in Caco2 cells that express wild type CFTR or are effective CFTR null mutants warrants discussion. One interpretation of our results is that there are no differences in glycosylation or sulfation of mucins as a primary intracellular result of CFTR expression or lack of it. This hypothesis predicts that previous observations of aberrant glycosylation and sulfation of CF mucins result from secondary extracellular effects of the disease such as epithelial damage or bacterial infection. Further, in the experiments reported here we have analyzed membrane-bound and intracellular MUC1F mucin rather than MUC1F released into the cell culture medium. It is possible that alterations in mucin glycosylation and sulfation in cells expressing mutant CFTR are only evident in the secreted forms.
Alternatively, though differences in carbohydrate antigenicity were not detected on MUC1F in CFTR+ and CFTR- clones, this could result from limitations of the types of structures detected by the antibodies we employed. There may be differences in carbohydrate backbone structures, side chains, or terminal structures that were not detected by the available panel of monoclonal antibodies.
A third interpretation of the data presented here is that our model for CF null mutants is inadequate to reproduce the CF phenotype. We have used Caco2 cells that express very high levels of CFTR mRNA and protein under normal culture conditions and selected clonal lines that have lost CFTR expression as detected by Northern and Western blot analysis. These lines may not be equivalent to true null mutants but rather show greatly reduced CFTR protein that is still sufficient to support normal glycoprotein processing. Recent data that suggest lower levels of CFTR expression are required to effect proper glycoprotein processing than to correct the CF-associated chloride ion transport defect support this hypothesis (Zhang et al., 1998). To address this issue, we are now extending these experiments to matched pairs of cell lines that have defined mutations in both CFTR genes and in which wild-type CFTR expression has been restored by introduction of a CFTR transgene.
A fourth interpretation of our results is that processing of the MUC1 mucin is not affected by CFTR expression or lack of it while processing of other mucins might be. The rationale behind this hypothesis would be that MUC1 is not a classical gel-forming mucin and, rather than being released from epithelial cells by cAMP-mediated fusion of granules with the cell membrane, it is a membrane-bound molecule that is cleaved from the cell surface. Differences might include the specific intracellular trafficking pattern that MUC1 follows through the cellular glycosylation machinery, thereby placing it in a different compartment from the gel-forming mucins. The gel-forming mucins might then show divergent glycosylation mechanisms that could be affected by mutations in CFTR. There is currently no evidence to support this hypothesis. We are now investigating this possibility in similar experimental systems, using the goblet cell mucins MUC2, MUC5AC, and MUC5B.
HT29 (Huet et al., 1987) and Caco2 (Fogh et al., 1977) cells were grown in DMEM medium supplemented with 10% fetal calf serum. The plasmids MUC1F (Burdick et al., 1997) and pH[beta]-APr1-neo (Gunning et al., 1987) were transfected into HT29 and Caco2 cells by calcium phosphate precipitation. Stable transformants were selected with G418 at 500 µg/ml, and clonal cell lines were isolated. Total genomic DNA was prepared by standard methods, restricted with EcoRI, Southern blotted, and probed with a MUC1 cDNA (Chambers et al., 1994) to confirm the presence of an integrated copy of the transgene. RNA was extracted from the Caco2 clones (Chirgwin et al., 1979), separated on denaturing agarose gel, transferred to Hybond N+ and hybridized with a MUC1 probe (Chambers et al.,1994). Clones were defined as expressing high or low levels of MUC1 mRNA (Table I) by comparing the MUC1-derived signal with that obtained from a GAPDH probe (Benham et al., 1984) subsequently hybridized to the same filter. In addition, the MUC1F transfected clones were evaluated for CFTR mRNA by Northern blotting and probing with CF10-1 (Riordan et al.,1989) that contains exons 1-6 of the CFTR cDNA.
Preparation of cell lysates and Western blotting
Cell lysates were prepared in NET buffer (10 mM Tris pH7.5, 150 mM NaCl, 5 mM EDTA) containing PMSF (1 mM), aprotinin (10 g/ml), pepstatin A (1 µg/ml), antipain (50 µM), and leupeptin (1 µg/ml ) with 1% Triton X-114. Protein concentrations were determined using a detergent compatible DC protein assay (Bio-Rad). Protein samples (100-200 µg) were separated on SDS-PAGE gels (3% stacker, 6% resolving), transferred to Hybond C (50 mA, 4°C 12 h) and blocked in blocking buffer (1% Marvel in PBS, 145 mM NaCl, 8 mM Na2HPO4, 2.5 mM NaH2PO4). Primary antibodies were used at dilutions of 1:2000 (except the flag antibody 1:500) in blocking buffer. Blots were incubated with the primary antibodies for 1 h at room temperature followed by three 10 min washes in blocking buffer. The blots were incubated with HRP-conjugated rabbit anti-mouse Ig secondary antibody (DAKO) in blocking buffer as above followed by three 10 min washes in blocking buffer. Antibodies were visualized using ECL reagents (Amersham) applied according to the manufacturer's instructions and exposed to ECL-sensitive film. Monoclonal antibodies were obtained from the following sources: M2 (IBI); CA-2 was a gift from M Bramwell; CC49 and B72.3 from D. Colcher; CO514, CO431, B93.1, and CSLEX were donated by M. Reddish, Biomera Corp.
In each experiment, FLAG antibody (M2) was used to identify the MUC1F glycoforms. MUC1F glycoproteins were scored as positive for a carbohydrate epitope if an antibody specific for that epitope reacted with a protein that was not present in the pH[beta]-APr1-neo vector control clones and colocalized with M2 reactive protein.
Immunopurification
MUC1F glycoconjugates were purified with the FLAG epitope; 20 µl of M2-conjugated agarose beads (IBI) were incubated with 200 µg of lysate at 4°C for 24 h with vigorous shaking. The agarose beads were pelleted by centrifugation and washed twice in NET buffer, and protein was eluted from the beads by incubation with 50 µl of FLAG peptide (500 µg/ml) for 24 h at 4°C with vigorous shaking.
Metabolic labeling
All metabolic labeling experiments were carried out in triplicate. Relative levels of glycosylation per unit protein were determined. Metabolic labeling was carried out using the MEM Select-Amine kit (BRL Life technologies). Cell lines were grown to about 30% confluence in T75 flasks and washed five times in PBS, and then 10 ml of DMEM selectamine media (amino acid deficient) containing 10% dialyzed fetal calf serum (BRL) was placed in each flask; 15 µg/ml methionine, 0.292 mg/ml glutamine, 0.033 MBq/ml [U-14C] protein hydrolysate (CFB25 Amersham, >1.92 GBq/milliatom Carbon), and 0.066 MBq/ml d-[6-3H] glucosamine hydrochloride (TRK398 Amersham, 962 GBq/mmol) were added to each flask. One hour later, the remaining amino acids from the Select-Amine kit (arginine, cystine, histidine, isoleucine, leucine, lysine, phenylanaline, threonine, and tryptophan) were added, and the cells were grown to confluence over 4 days. Lysates were prepared as for unlabeled cells, and the MUC1F glycoprotein was purified by immunoprecipitation with the M2 antibody. The immunoprecipitated material was separated on SDS-PAGE gels as described above and visualized by autoradiography after treatment with the fluorographic agent Amplify (Amersham), and gel areas containing the MUC1F glycoprotein were excised. The amount of 14C amino acids and 3H glucosamine present in these gel slices was determined by scintillation counting. Counts were then expressed as ratios comparing the level of glycosylation (3H glucosamine) to the level of protein (14C amino acids).
The relative level of sulfation was compared to the level of glycosylation. Cell lines were grown to about 30% confluence in T75 flasks and washed five times in PBS. Ten milliliters of sulfate deficient Basal Eagle medium (Sigma) containing 10% dialyzed fetal calf serum (BRL) and 100 µg/ml glutamine was added to each flask. After 1 h, the medium was supplemented with 6.6 MBq/ml [35S] Na2SO4 (NEN, (38.8-59.2 TBq/mmol) and 0.066 MBq/ml d-[6-3H] glucosamine hydrochloride (Amersham, 962GBq/mmol) and the cells grown to confluence over 4 days. Lysates were prepared as described for unlabeled cells, and the MUC1F glycoprotein was purified by immunoprecipitation with the M2 antibody. The immunoprecipitated material was separated on PAGE gels and visualized by autoradiography as described for the 14C amino acids/3H glucosamine experiments. The gel areas containing the MUC1F glycoprotein were excised. The amounts of 35S sulfate and 3H glucosamine were determined by scintillation counting the excised gel slices. These counts were expressed as ratios comparing the level of glycosylation (3H glucosamine) to the level of sulfation (35S sulfate).
We thank Mrs. Z.Madgwick for technical assistance; Drs. M.Reddish, D.Colcher and M.E.Bramwell for gifts of antibodies; Dr. M.Gravenor for statistical analyses and Prof. E.R.Moxon for continued support. This work was supported by the Cystic Fibrosis Trust, UK; NIH Grants CA57362, DK46589, CA69234, CA36727, and CA09476; and a Wellcome Trust Biomedical Collaboration grant.
CFTR, cystic fibrosis transmembrane conductance regulator
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