Characterization of the oligosaccharide structures associated with the cystic fibrosis transmembrane conductance regulator

Catherine R. O’Riordan1, Amy L. Lachapelle, John Marshall, Elizabeth A. Higgins and Seng H. Cheng

Genzyme Corporation, 31 New York Avenue, Framingham, MA 01701–9322, USA

Received on April 26, 2000; revised on June 26, 2000; accepted on June 26, 2000.


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Material and methods
 Acknowledgments
 Abbreviations
 References
 
The cystic fibrosis transmembrane conductance regulator (CFTR) is a plasma membrane-associated glycoprotein. The protein can exist in three different molecular weight forms of approximately 127, 131, and 160 kDa, representing either nonglycosylated, core glycosylated, or fully mature, complex glycosylated CFTR, respectively. The most common mutation in cystic fibrosis (CF) results in the synthesis of a variant ({Delta}F508-CFTR) that is incompletely glycosylated and defective in its trafficking to the cell surface. In this study, we have analyzed the oligosaccharide structures associated with the different forms of recombinant CFTR, by expressing and purifying the channel protein from either mammalian Chinese hamster ovary (CHO) or insect Sf9 cells. Using glycosidases and FACE analysis (fluorophore-assisted carbohydrate electrophoresis) we determined that purified CHO-CFTR contained polylactosaminoglycan (PL) sequences, while Sf9-CFTR had only oligomannosidic saccharides with fucosylation on the innermost GlcNAc. The presence of PL sequences on the recombinant CHO-CFTR is consistent with a normal feature of mammalian processing, since endogenous CFTR isolated from T84 cells displayed a similar pattern of glycosylation. The present study also reports on the use of FACE for the qualitative analysis of small amounts of glycoprotein oligosaccharides released enzymatically.

Key words: CFTR/FACE analysis/ polylactosaminoglycans/oligomannosidic saccharides


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Material and methods
 Acknowledgments
 Abbreviations
 References
 
Cystic fibrosis (CF) is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR; Boat et al., 1989Go). Patients with CF are marked by dysfunctional epithelia due to the absence of functional CFTR chloride (Cl) channels at the apical plasma membranes of the affected cells. Studies have shown that CFTR is a membrane-associated glycoprotein whose Clchannel activity is regulated by protein kinase A-mediated phosphorylation (Cheng et al., 1990Go; Gregory et al., 1990Go). Electrophoretic analysis has indicated that CFTR can exist as three different molecular weight forms of approximately 127 kDa, 131 kDa, and 160 kDa, referred to as bands A, B, and C, respectively (Gregory et al., 1990Go). Bands A, B, and C represent different glycoforms of CFTR with band A representing nonglycosylated CFTR, band B representing core glycosylated CFTR, and band C representing the mature CFTR with complex glycosylation (Cheng et al., 1990Go). These posttranslational modifications of CFTR may represent an important marker of protein processing (Cheng et al., 1990Go). The presence of core glycosylated band B CFTR, for example, indicates processing of the protein in the endoplasmic reticulum (ER), while mature band C CFTR with complex glycosylation is representative of processing in the Golgi (Cheng et al., 1990Go).

The significance of CFTR glycosylation is understood best, however, by studying the mutant {Delta}F508-CFTR. The deletion of the phenylalanine residue at position 508 ({Delta}F508) in CFTR is the most common cause of cystic fibrosis (Boat et al., 1989Go). At 37°C, mutant {Delta}F508-CFTR is synthesized predominantly as bands A and B, suggesting that the variant protein is unable to traffic normally to the Golgi apparatus (Cheng et al., 1990Go; Gregory et al., 1991Go). For this reason no CFTR chloride channels are detected. However, the processing of recombinant {Delta}F508-CFTR can revert towards that of wild-type CFTR when the incubation temperature for the cells is reduced to 26°C (Denning et al., 1992Go). At this lower temperature, some CFTR becomes fully processed and is presented at the plasma membrane, as evidenced by the appearance of very small amounts of mature band C CFTR and the detection of cAMP-regulated Cl channel activity at the cell surface (Denning et al., 1992Go).

Studies on the relationship of glycosylation to the function of CFTR suggest that the addition of carbohydrate to CFTR is not a necessary prerequisite for the protein to target to the plasma membrane or to function as a cAMP-stimulated Cl channel (Gregory et al., 1990Go; Morris et al., 1993Go); however, determining the oligosaccharide structures associated with CFTR should contribute to a better understanding of the structure of this complex transmembrane protein and the influence that {Delta}F508-CFTR has on other cell membrane glycoproteins. In this study we have analyzed the pattern of glycosylation of the different forms of CFTR (O'Riordan et al., 1995Go) by expressing the channel protein in either mammalian or insect cells. We show that the pattern of glycosylation of recombinant CFTR expressed in mammalian cell systems is representative of that which occurs on endogenous CFTR, while CFTR expressed in insect cells is not fully processed.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Material and methods
 Acknowledgments
 Abbreviations
 References
 
Different glycosylation forms of CFTR are expressed in mammalian and insect cells
Two types of mammalian cells, CHO (Chinese hamster ovary) and C127 (mouse mammary adenocarcinoma), and an insect cell line (Sf9) were used for the overexpression of human CFTR (Marshall et al., 1994Go; O'Riordan et al., 1995Go). The different glycosylation forms of CFTR (bands A, B, and C) were expressed at varying levels depending on the cell type used. CFTR prepared from lysates of recombinant C127 and CHO cells were predominantly of the fully mature band C form (160 kDa) with a minor component of band B (Figure 1A, lane 2, and Figure 1B, lane 3). However, CFTR prepared from recombinant Sf9 insect cells appeared predominantly as an ~131 kDa polypeptide (Figure 1B, lane 2), which is similar in size to the band B form observed in mammalian cells (Figure 1A, lane 2). This is expected, since proteins expressed in Sf9 cells have been reported to harbor solely oligomannosidic glycosylation (Vialard et al., 1990Go; Altmann et al., 1993Go). Further studies on the oligosaccharide structures of wild type CFTR were performed using CFTR purified from both these sources.



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Fig. 1. Expression of CFTR in different cell types. Autoradiographs of immunoprecipitated CFTR from transfected (A) C127 cells and (B) CHO and Sf9 cells. CFTR was labeled by phosphorylation with [{gamma}-32P]-ATP and the catalytic subunit of cAMP-dependent PKA, and then electrophoresed on 6% SDS–polyacrylamide gels. Bands B and C represent core-glycosylated and fully glycosylated CFTR respectively. (A) Lane 1, mock-transfected C127 cells; lane 2, C127 cells transfected with wild type CFTR; lane 3, C127 cells transfected with {Delta}F508-CFTR. (B) Lane 1, uninfected Sf9 cells; lane 2, Sf9 cells infected with wild type CFTR baculovirus; lane 3, CHO cells transfected with wild type CFTR, lane 4, parental CHO cells.

 
Fractionation of the bands B and C forms of CFTR by lectin affinity chromatography
Since the bands B and C forms of CFTR differed in carbohydrate content, they could be further fractionated and purified by lectin affinity chromatography. The lectin wheat germ-agglutinin specifically recognizes N-acetylglucosamine residues (Nagata and Burger, 1974Go) that are present on complex carbohydrate glycoproteins and therefore could be used to separate the band C CFTR (which contains complex carbohydrate structures) from band B CFTR (which contains only oligomannose structures). As shown in Figure 2, chromatography of T84 cell lysates on wheat-germ agglutinin Sepharose resulted in retention of the band C, but not the band B, form of CFTR. The bound band C form of CFTR could be recovered from the resin in a highly purified form by elution with buffer containing 0.5 M N-acetylglucosamine (Figure 2 lanes 3 and 4). Similarly, insect Sf9-derived CFTR which was synthesized predominantly as the band B species of CFTR failed to bind to wheat germ-agglutinin Sepharose (data not shown). These results are consistent with band B CFTR containing only core glycosylation and band C CFTR containing carbohydrates of the complex type (Cheng et al., 1990Go).



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Fig. 2. Affinity chromatography of CHO-CFTR using wheat germ agglutinin. T84 cells expressing CFTR were lysed with 1% CHAPSO and applied onto wheat germ lectin Sepharose. Bound proteins were eluted from the resin with 0.5 M N-acetylglucosamine. Column fractions were immunoprecipitated with MAb 13–1 and phosphorylated in the presence of [{gamma}-32P] ATP and the catalytic subunit of protein kinase A. Lane 1 represents the load, lane 2, the flow through, and lanes 3 and 4 represent band C-CFTR specifically eluted with 0.5 M N-acetylglucosamine.

 
Characterization of CHO-CFTR using the glycosidases N-glycanase and endo–ß-galactosidase
Digestion of the purified band C CFTR (160 kDa) from CHO cells with N-glycanase, which cleaves N-linked oligosaccharides between the oligosaccharide structure and an asparagine residue, resulted in a reduction of the size of the protein to that observed for band A CFTR (127 kDa), indicating that the mature band C CFTR was modified by N-linked glycosylation (Figure 3, lane 2). As CFTR is a membrane glycoprotein, we also determined whether it harbored polylactosamine carbohydrate structures that are known to be associated with this class of glycoproteins (Gahmberg et al., 1976Go). Indeed, digestion of purified band C CFTR with endo-ß-galactosidase (which hydrolyzes polylactosamine-containing structures) reduced its size to ~131 kDa (Figure 3, lane 3), arguing for the presence of such structures on CFTR. Additional digestion of the endo-ß-galactosidase treated band C with N-glycanase resulted in a further reduction in size to that of band A (Figure 3, lane 4).



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Fig. 3. Endo-ß-galactosidase and N-glycanase digestion of CHO-CFTR. Purified band C-CFTR from CHO cells (lane 1) was digested with N-glycanase (lane 2), endo-ß-galactosidase alone (lane 3), or endo-ß-galactosidase followed by N-glycanase (lane 4). Samples were separated on 6% SDS–polyacrylamide gels followed by Coomassie blue staining.

 
Characterization of CHO-CFTR using lectin blots
Further characterization of the structure of the N-linked oligosaccharides present on band C CFTR purified from CHO cells was performed using lectin blots. Two lectins were used, DSA (Datura stramonium agglutinin) and MAA (Maackia amurensis agglutinin). DSA is known to bind unbranched (Yamashita et al., 1987Go) and branched polylactosamine sequences (Fukuda and Fukuda, 1984Go) and oligosaccharides containing an outer {alpha}-mannose residue substituted with Gal ß (1,4) GlcNAc at both the C-2 and C-6 positions (Cummings et al., 1982Go; Cummings and Kornfeld, 1984Go; Merkle and Cummings, 1987Go). The lectin MAA recognizes sialic acid that is linked either {alpha}(2–3) or {alpha}(2–6) to galactose (Haseley et al., 1999Go). Since CHO cells are unable to effect {alpha} (2–6) sialylation (Carr et al., 1989Go; Spellman et al., 1989Go), it is predicted that CHO-CFTR, if sialylated, would be of the {alpha} (2–3) type. This was indeed confirmed when band C CFTR isolated from CHO cells failed to react with SNA (Sambucus nigra agglutinin), a lectin that recognizes specifically only {alpha} (2–6) sialic acids (Haseley et al., 1999Go; data not shown). Figure 4 shows that band C CFTR reacted strongly with the lectin DSA but weakly with MAA. However, following treatment with endo-ß-galactosidase, there was a marked decrease in reactivity of the digested CFTR with both of these lectins (Figure 4). The observation that treatment of CFTR with endo-ß-galactosidase greatly reduced its ability to bind DSA, coupled with the demonstration above that band C CFTR was sensitive to endo-ß-galactosidase digestion, indicate that mature CHO-CFTR is comprised predominantly of polylactosamine structures. That CFTR also reacted, albeit weakly, with MAA suggests the presence of sialic acid residues on the polylactosamine chains.



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Fig. 4. Endo-ß-galactosidase treatment of CFTR followed by lectin blotting. Immunoaffinity purified band C-CFTR from CHO cells was treated with endo-ß-galactosidase as described in Materials and methods, transferred to nitrocellulose and probed using either the lectin DSA (Datura stramonium agglutinin) or MAA (Mackia amurensis agglutinin). The position of CFTR is indicated. (+) refers to CFTR which had been treated with endo-ß-galactosidase and (-) to untreated samples.

 
FACE analysis of oligosaccharides released following digestion of band C CFTR with glycosidases
The structure of the oligosaccharides released by N-glycanase or endo-ß-galactosidase digestion of band C CFTR isolated from CHO cells was assessed with a relatively simple and rapid method called FACE (fluorophore-assisted carbohydrate electrophoresis; Friedman and Higgins, 1994Go). In FACE, 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS) is covalently linked with high efficiency by reductive amination to the reducing end of oligosaccharides (Jackson, 1990Go). The resulting ANTS-oligosaccharide conjugates have fluorescence emission properties, such that conjugates that have been electrophoretically resolved on high-density polyacrylamide gels are detectable at very low (picomole) levels. In the case of neutral oligosaccharides, ANTS labeling also confers charge on the labeled conjugate. Figure 5 shows fluorescent band patterns of ANTS-labeled oligosaccharides released from band C-CFTR following digestion with either N-glycanase (Figure 5A) or endo-ß-galactosidase (Figure 5B). The oligosaccharides released from CFTR after treatment with N-glycanase alone co-migrated between the trisialo triantennary and asialo tetraantennary oligosaccharide standards. (Figure 5A and Table IA). The N-glycanase–released oligosaccharides from neuraminidase-treated band C CFTR had slower mobility and migrated between the asialo biantennary and asialo tetraantennary oligosaccharide standards (data not shown). This slower mobility was due to removal of sialic acid by neuraminidase, which affects the charge on the released oligosaccharides. This data suggests that the oligosaccharide structures present on mature CFTR are no larger than that of the tetra antennary type. To further analyze the nature of these oligosaccharide chains, FACE analysis was also performed on oligosaccharides released by endo-ß-galactosidase treatment of band C CFTR. As predicted, the molecular size of the oligosaccharides released by endo-ß-galactosidase digestion were of very low molecular weight (Figure 5B), which is consistent with release of sialylated lactosamines (Figure 5B).



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Fig. 5. FACE analysis of oligosaccharides released from band C CHO-CFTR. Immunoaffinity-purified band C-CFTR from CHO cells was separated on a (4–20%) gradient polyacrylamide gel, transferred onto Immobilon and then stained using Amido black. The CFTR protein bands were then excised from the Immobilon and treated with either N-glycanase (A) or endo-ß-galactosidase (B). The oligosaccharides released were labeled using ANTS and analyzed on a high density polyacrylamide gel. Lanes: 1, standards; 2, control (enzyme alone); and 3, oligosaccharides released after glycosidase digestion.

 

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Table I. Structural representations of oligosaccharide standards
 
Glycosylation pattern of CFTR produced in insect Sf9 cells
It has been shown that insect glycoproteins, like those in other cell systems, are first synthesized and attached to a high-mannose oligosaccharide, [Asn]-GlcNAc2-Man9-Glc3 (Hsieh and Robbins, 1984Go). These oligosaccharide structures are sensitive to endoglycosidase-H, which removes sugars from the high-mannose glycoprotein, leaving one molecule of N-acetylglucosamine attached to asparagine. Analyses of N-glycans isolated from endogenous or recombinant glycoproteins expressed in insect cells revealed that this innermost N-acetylglucosamine can be subject to fucosylation (Jarvis and Summers, 1989Go; Kuroda et al., 1990Go; Chen et al., 1991Go; Wathen et al., 1991Go; Williams et al., 1991Go; Stadacher et al., 1992Go). In the case of N-linked glycans, further processing can lead to the formation of proteins with a trimannosyl core ([Asn]-GlcNAc2-Man3). There are conflicting reports as to whether insect cells have the necessary enzymes for the processing of high-mannose carbohydrates into complex type oligosaccharides. In the case of CFTR, for example, expression in insect Sf9 cells results in the expression of the endoglycosidase-H sensitive band B only (data not shown). To characterize this glycosylation further we analyzed the oligosaccharides released by both endoglycosidase-H and N-glycanase digestion of Sf9-CFTR using FACE. Figure 6 (lane 2) shows the pattern of ANTS labeled oligosaccharides released upon digestion of Sf9-CFTR with endoglycosidase-H. The oligosaccharides released co-migrated with the oligomannose standards (Figure 6, Table IB), confirming that Sf9-CFTR contained endoglycosidase-H sensitive oligomannose glycosylation. Oligosaccharides released by Endo-H will migrate faster than the indicated standards, since Endo-H leaves the GlcNAc residue attached to the asparagine of the glycoprotein. Interestingly, when insect CFTR was digested with N-glycanase, which releases both complex and high-mannose oligosaccharide structures, the main oligosaccharide released had an electrophoretic mobility slightly slower than Man3 consistent with the presence of fucosylated Man3 (Figure 6, lane 1). This structure is most likely fucosylated in {alpha}1–6 linkage, since {alpha}1–3 fucosylation inhibits N-glycanase (Tretter et al., 1991Go). Endo-H cannot release either {alpha}1–3 or {alpha}1–6 fucosylated structures (Trimble and Maley, 1984Go), which explains why several bands are visible in the Endo-H lane while one band predominates in the N-glycanase lane. This pattern of glycosylation is very similar to what was reported by Kuroda et al. (1990)Go, for influenza virus hemagglutinin expressed in insect cells. This result is also consistent with the fact that fucosylation of insect oligosaccharides occurs on the innermost N-acetylglucosamine, which is only released with N-glycanase treatment, but remains attached to the peptide with endoglycosidase-H digestion.



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Fig. 6. FACE analysis of oligosaccharides released from Sf9-CFTR. Oligosaccharides released by either endoglycosidase-H or N-glycanase digestion of Sf9-CFTR were ANTS labeled and analyzed by polyacrylamide gel electrophoresis. Lane 1 contains ANTS-labeled oligosaccharides released by N-glycanase digestion of Sf9-CFTR; lane 2, ANTS labeled oligosaccharides released by endoglycosidase-H digestion of Sf9-CFTR; lane 3, glucose polymer standards. Migration of oligosaccharide standards of Man3–Man9 and Man3F is also indicated.

 
Comparison of recombinant and nonrecombinant forms of CFTR
It would therefore appear that there are distinct differences in the pattern of glycosylation of CFTR, depending on the cell system used to express the protein. Mature CFTR from CHO cells, for example, showed unusual polylactosamine structures, in contrast to the oligomannose glycosylation observed on Sf9-CFTR. To examine whether the presence of polylactosamine glycosylation is a feature only of recombinant CFTR expressed in CHO cells, but does not truly represent the type of glycosylation that occurs on endogenous CFTR, non-recombinant CFTR expressed in T84 cells was characterized. To address this issue, CFTR from T84, CHO and Sf9 cells was digested with N-glycanase (Figure 7). Digestion of band C-CFTR isolated from T84 cells with N-glycanase generated a protein with an apparent molecular weight of 127 kDa (band A). Thus, mature CFTR expressed in T84 cells and digested with N-glycanase comigrated with the product of N-glycanase digestion of mature CFTR from CHO cells (Figure 7). The large reduction in molecular weight after N-glycanase digestion of T84-CFTR suggests that T84-CFTR also likely contained polylactosamine glycosylation. Furthermore, T84-CFTR showed the same molecular weight shift as did CHO-CFTR upon digestion with endo-ß-galactosidase (Figure 8). This suggests that the type of glycosylation observed on recombinant CHO-CFTR was probably representative of the type of glycosylation that occurs on nonrecombinant forms of CFTR.



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Fig. 7. Comparison of CFTR expressed in Sf9, CHO, and T84 cells. Cell lysates were prepared from CHO, T84 and Sf9 cells and then immunoprecipitated with MAb 24–1 (anti-CFTR carboxy terminus antibody). Immunoprecipitates of CFTR were digested with the glycosidases N-glycanase or endo-ß-galactosidase and then phosphorylated using [{gamma}-32P]-ATP and the catalytic subunit of protein kinase A. The labeled, digested immunoprecipitates of CFTR were then subsequently analyzed on a 6% SDS–polyacrylamide gel.

 


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Fig. 8. Endo-ß-galactosidase digestion of CFTR from T84 and CHO cells. Band C-CFTR from CHO or T84 cells were immunoprecipitated with MAb 24–1 (anti-CFTR carboxy terminus antibody), digested with endo-ß-galactosidase and phosphorylated using [{gamma}-32P]-ATP and the catalytic subunit of protein kinase A. The labeled, digested immunoprecipitates of CFTR were then resolved on a 6% SDS–polyacrylamide gel.

 

    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Material and methods
 Acknowledgments
 Abbreviations
 References
 
We have shown that the mature band C form of CFTR expressed in CHO cells has a unique pattern of glycosylation consisting of complex structures of N-acetyllactosamine repeating units. This was evidenced by its sensitivity to digestion by N-glycanase and endo-ß-galactosidase, by its binding characteristics to DSA lectin, and its electrophorectic mobility using FACE. The occurrence of such glycoforms is not necessarily a result of expression of CFTR in a recombinant cell per se, since endogenous CFTR from T84 cells was shown to have similar characteristics. These polylactosaminoglycans (PL) are heterogeneous oligosaccharides and are distinct from the more common complex type asparagine-linked oligosaccharides by having side chains of Galß1–4GlcNAcß1–3 repeats which are susceptible to endo-ß-galactosidase (Scudder et al., 1983Go).

The importance of these PL sequences on the mature, fully processed CFTR is as yet unclear. The anion exchanger Band 3, a major glycoprotein of erythrocytes, reportedly also contains similar PL structures (Fukuda, 1985Go; Feizi, 1985Go). It was speculated (Beppu, 1992Go) that IgG autoantibody against Band 3 of the human erythrocyte membrane (anti-Band 3) plays a role in removing senescent or damaged erythrocytes from circulation by recognizing the sialylated poly-N-lactosaminyl sugar chains. Other suggested functions for poly-N-lactosaminyl structures on Band 3 include shielding potential antigenic sites on external loops and protection of the protein from proteolytic degradation (Casey, 1992Go). Likewise, it could be that the presence of poly-N-lactosamine structures on CFTR may help reduce the antigenicity of the protein or maintain the channel in an active form in the membrane by improving its stability or preventing it from being degraded by proteases.

The role of the oligosaccharide chain in the activity of transport proteins has not been extensively studied. However, PL-associated structures have been reported to influence the processing, proper trafficking, and targeting of some plasma membrane proteins to their sites of action (Wang et al., 1991Go). Interestingly, the effect of lowering the temperature on two important enzymes responsible for the synthesis of PL sequences, namely ß1–3-N-acetylglucosaminyltransferase and ß1–4-galactosyltransferase, has been documented. It has been shown that LAMPS (lysosomal associated membrane glycoproteins) expressed in HL-60 cells at lower temperatures (21°C) are processed further to acquire more poly-N-acetyllactosamines than LAMPS produced at 37°C (Wang et al., 1991Go). This was attributed to a longer association of LAMPS with Golgi compartments where the ß(1–3)-N-acetylglucosaminyltransferase and ß(1–4)-galactosyltransferase enzymes are normally resident. Perhaps the observation that lowering the temperature of incubation of {Delta}F508-CFTR expressing cells facilitates processing of the mutant protein to band C (Denning et al., 1992Go) may be due, in part, to a longer association of the variant protein with the Golgi complex, thereby allowing it to acquire the extended PL structures.

Baculovirus expression of CFTR in the Sf9 insect cell system resulted in the expression predominantly of a protein band that comigrated with that for band B CFTR. The expression of a band B-like CFTR in the Sf9 cells is consistent with earlier reports indicating that only oligomannose structures are contained on recombinant proteins expressed in this insect cell line (Wojchowski et al., 1987Go; Greenfield, 1988Go; Luckow and Summers, 1988Go) and that little further processing of the original high-mannose oligosaccharide core occurs in these cells. Using FACE, we showed that Sf9-CFTR was further modified by fucosylation at the innermost N-acetylglucosamine residue. This confirms the presence of a fucosyltransferase gene in insect cells as first reported by Davidson and Castellino (1991)Go, who demonstrated the presence of fucose on recombinant human plasminogen (r-HPg) expressed in Sf21 insect cells. These investigators were also the first to demonstrate that the addition of complex carbohydrate structures to proteins expressed in insect cells could occur, but was specifically dependent on the infective process employed. In this present study CFTR expressed in the Sf9 insect cell system had only oligomannose structures; however, it may be possible to manipulate the nature of CFTR glycosylation in these cells by altering the infective process, thus generating fully processed CFTR that is more representative of endogenously expressed CFTR or CFTR expressed in recombinant mammalian cells.

Presently, it is unclear whether these different glycoforms of CFTR exhibit different activities. Some studies have suggested that the carbohydrate moiety is dispensable for CFTR Cl channel activity (Morris, 1993Go). A mutant CFTR that was altered such that it was now unable to undergo N-glycosylation was shown to retain cAMP-stimulated Cl channel activity (Gregory et al., 1991Go). Similarly, Sf9-CFTR that lacks complex oligosaccharide structures also exhibited functional CFTR channel activity (Bear et al., 1992Go; Kartner et al., 1992Go; O'Riordan et al., 1995Go). However, other studies have suggested that the state of glycosylation of CFTR may affect its stability at the plasma membrane (Luckas et al., 1993Go; Wei et al., 1996Go). Clearly, the effect of carbohydrate addition on the structure and function of CFTR requires further investigation. This in turn may have implications for development of relevant therapies for treatment of CF patients harboring the most common form of the disease.


    Material and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Material and methods
 Acknowledgments
 Abbreviations
 References
 
Materials
FACE reagents and equipment were supplied by Glyko (Novato, CA); N-glycanase, endo-ß-galactosidase, neuraminidase, and the Lectin-Link Kit were supplied by Genzyme (Cambridge, MA); DIG Glycan Differentiation Kit was supplied by Boehringer-Mannheim; Wheat germ agglutinin Sepharose was supplied by Sigma (St. Louis, MO).

Cell cultures
The human colon carcinoma cell line T84 was obtained from the American Type Culture Collection (ATCC CCl 248). Established C127 (mouse mammary epitheloid cells) and CHO (Chinese hamster ovary) cells stably transfected with the human CFTR cDNA were generated and maintained as described previously (Marshall et al., 1994Go; O'Riordan et al., 1995Go). The growth and infection of Sf9 cells with recombinant CFTR-expressing viruses have also been described previously (O'Riordan et al., 1995Go).

Purification of recombinant CFTR from CHO cells for carbohydrate analysis
CHO-CFTR was immunoaffinity purified using the monoclonal antibody MAb 13–1 cross-linked to a hydrazide resin, essentially as described by O'Riordan et al. (1995)Go. Briefly, CFTR was solubilized out of the membranes using 1.5% {alpha}-lyso PC and the solubilized material then incubated with the resin overnight at 4°C. Following extensive washing with wash buffer (150 mM NaCl, 50 mM Tris–HCl, pH 8.0, 1 mM EDTA and 1% sodium cholate) to remove nonspecifically bound proteins, CFTR was eluted from the resin using 150 mM NaOH, 10% glycerol, 1 mM EDTA, pH 11.0 containing 0.5% sodium cholate (neutralized with 0.1M Tris pH 7.5).

Treatment with endo-ß-galactosidase and N-glycanase
Approximately 100 g of immunoaffinity purified CFTR in neutralized elution buffer (150 mM NaOH, 10% glycerol, 1 mM EDTA, pH 11.0, containing 0.5% sodium cholate buffer and neutralized with 0.1 M Tris, pH 7.5) was reacted with 0.01 units of endo-ß-galactosidase enzyme for 6 h at room temperature. Reaction was stopped by the addition of SDS–PAGE sample buffer followed by electrophoresis on a 4–20% gradient gel. Alternatively, for samples which were analyzed by FACE, the reaction was stopped by evaporating samples to dryness using a speed vacuum. For N-glycanase digestion, CFTR was first denatured by adding sodium dodecyl sulfate (SDS) to 0.1% and ß-mercaptoethanol to 50 mM and incubating at 37°C for 5 min. NP-40 was then added to 0.8% and the reaction initiated by adding 40 units of N-glycanase (Genzyme) and incubating for 2 h at 37°C. All reactions were done in neutralized elution buffer as described above. Reactions were stopped by evaporation to dryness using a speed vacuum.

Fluorophore-assisted carbohydrate electrophoresis (FACE)
FACE analysis was applied as previously described (Friedman and Higgins, 1994Go). The carbohydrate moiety of CHO-CFTR was prepared for FACE analysis by first running 100 to 200 µg of immunoaffinity purified material on a 4–20% SDS-PAGE gel followed by transfer to a PVDF membrane in 10 mM CAPS, 10% methanol, pH 11.0. The conditions for electrophoresis are described in O'Riordan et al. (1995)Go. Proteins were stained with 0.1% amido black in 20% ethanol and the background was destained in 20% ethanol. After destaining, the membrane was kept in 50 mM NaHPO4, pH 7.7. The CFTR band was excised and cut into small pieces being careful not to let the membrane dry out. The pieces were placed in a microfuge tube and covered with approximately 100–200 µl of 50 mM NaHPO4, pH 7.7 buffer. Carbohydrates were then removed from CFTR using N-glycanase as described above. In some instances, CFTR was digested with neuraminidase prior to N-glycanase digestion. Briefly, membranes were rinsed with 50 mM NaHPO4, pH 5.5 then covered with ~200 µl of 50 mM NaHPO4, pH 5.5 buffer. Five microliters (0.1 units) of neuraminidase were added, and the reaction allowed to proceed overnight at room temperature. Fluorescence labeling of the carbohydrate residues was carried out for 18 h using reagents and protocol supplied by Glyko (Novato, CA). Electrophoresis reagents and equipment were also supplied by Glyko.

Lectin blots and SDS–PAGE analysis
Immunoaffinity purified CFTR was electrophoresed under standard conditions and then transferred to nitrocellulose paper as described before (O'Riordan et al., 1995Go). Both the DIG Glycan Differentiation Kit (Boehringer-Mannheim) and the Lectin-Link Kit (Genzyme Corp., Framingham, MA) were used. Lectin blotting was performed according to the manufacturers’ directions.

Immunoprecipitation and protein phosphorylation using protein kinase A
T84 cell lysates were prepared by treating the cells with lysis buffer (50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1% NP-40, 1 mM PMSF, and 5 mM aprotinin). After incubation on ice for 30 min, unlysed cells were removed by centrifugation at 14,000g. CFTR was immunoprecipitated with the monoclonal antibody MAb 13–1 (Gregory et al., 1991Go) and the immunoprecipitates then incubated with protein kinase A (20 ng) and [{gamma}-32P]ATP (10 mCi) in a final volume of 50 µl in kinase buffer (50 mM Tris–HCl pH 7.5, 10 mM MgCl2 and 100 mg/ml bovine serum albumin) at 30°C for 50 min. Samples were heated for 5 min at 37°C before electrophoresis. Glycosidase digestion of immunoprecipitates was performed by overnight digestion in the presence of the enzyme in 10 mM sodium phosphate buffer, pH 6.4, containing 0.75% NP-40. Digestion was performed prior to the phosphorylation assay.

Wheat germ agglutinin chromatography
Wheat germ agglutinin Sepharose was equilibrated with phosphate-buffered saline. T84 cell lysates were prepared as described above and applied to the equilibrated wheat germ agglutinin Sepharose. The flow-through fraction was collected and the column was washed with PBS before elution with 0.5M N-acetylglucosamine. Both the flow-through fraction and the N-acetylglucosamine eluate were assayed for CFTR by immunoprecipitation with the monoclonal antibody Mab 13–1. The immunoprecipitates were incubated with protein kinase A and [{gamma}-32P]ATP (10 mCi) as described above and labeled proteins were analyzed by SDS–PAGE.


    Acknowledgments
 Top
 Abstract
 Introduction
 Results
 Discussion
 Material and methods
 Acknowledgments
 Abbreviations
 References
 
We thank Drs. S.Wadsworth and Helen Romanczuk for critical review of the manuscript.


    Abbreviations
 Top
 Abstract
 Introduction
 Results
 Discussion
 Material and methods
 Acknowledgments
 Abbreviations
 References
 
ANTS, 8-aminonaphthalene-1,3,6-trisulfonic acid; CAPS, 3-(cyclohexylamino) propane sulfonic acid; CHAPSO, (3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate; CHO, Chinese hamster ovary; CFTR, cystic fibrosis transmembrane conductance regulator; CF, cystic fibrosis; FACE, fluorophore-assisted carbohydrate electrophoresis; PAGE, polyacrylamide gel electrophoresis; PC, phosphatidylcholine; PMSF, phenylmethylsulfonylfluoride; PL, polylactosaminoglycans; SA, sialic acid.


    Footnotes
 
1 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Material and methods
 Acknowledgments
 Abbreviations
 References
 
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