Genzyme Corporation, 31 New York Avenue, Framingham, MA 017019322, USA
Received on April 26, 2000; revised on June 26, 2000; accepted on June 26, 2000.
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Abstract |
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Key words: CFTR/FACE analysis/ polylactosaminoglycans/oligomannosidic saccharides
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Introduction |
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The significance of CFTR glycosylation is understood best, however, by studying the mutant F508-CFTR. The deletion of the phenylalanine residue at position 508 (
F508) in CFTR is the most common cause of cystic fibrosis (Boat et al., 1989
). At 37°C, mutant
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., 1990
; Gregory et al., 1991
). For this reason no CFTR chloride channels are detected. However, the processing of recombinant
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., 1992
). 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., 1992
).
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., 1990; Morris et al., 1993
); 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
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., 1995
) 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.
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Results |
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Discussion |
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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, 1985; Feizi, 1985
). It was speculated (Beppu, 1992
) 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, 1992
). 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., 1991). Interestingly, the effect of lowering the temperature on two important enzymes responsible for the synthesis of PL sequences, namely ß13-N-acetylglucosaminyltransferase and ß14-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., 1991
). This was attributed to a longer association of LAMPS with Golgi compartments where the ß(13)-N-acetylglucosaminyltransferase and ß(14)-galactosyltransferase enzymes are normally resident. Perhaps the observation that lowering the temperature of incubation of
F508-CFTR expressing cells facilitates processing of the mutant protein to band C (Denning et al., 1992
) 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., 1987; Greenfield, 1988
; Luckow and Summers, 1988
) 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)
, 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, 1993). 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., 1991
). Similarly, Sf9-CFTR that lacks complex oligosaccharide structures also exhibited functional CFTR channel activity (Bear et al., 1992
; Kartner et al., 1992
; O'Riordan et al., 1995
). However, other studies have suggested that the state of glycosylation of CFTR may affect its stability at the plasma membrane (Luckas et al., 1993
; Wei et al., 1996
). 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.
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Material and methods |
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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., 1994; O'Riordan et al., 1995
). The growth and infection of Sf9 cells with recombinant CFTR-expressing viruses have also been described previously (O'Riordan et al., 1995
).
Purification of recombinant CFTR from CHO cells for carbohydrate analysis
CHO-CFTR was immunoaffinity purified using the monoclonal antibody MAb 131 cross-linked to a hydrazide resin, essentially as described by O'Riordan et al. (1995). Briefly, CFTR was solubilized out of the membranes using 1.5%
-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 TrisHCl, 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 SDSPAGE sample buffer followed by electrophoresis on a 420% 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, 1994). The carbohydrate moiety of CHO-CFTR was prepared for FACE analysis by first running 100 to 200 µg of immunoaffinity purified material on a 420% 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)
. 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 100200 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 SDSPAGE analysis
Immunoaffinity purified CFTR was electrophoresed under standard conditions and then transferred to nitrocellulose paper as described before (O'Riordan et al., 1995). 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 TrisHCl, 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 131 (Gregory et al., 1991) and the immunoprecipitates then incubated with protein kinase A (20 ng) and [
-32P]ATP (10 mCi) in a final volume of 50 µl in kinase buffer (50 mM TrisHCl 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 131. The immunoprecipitates were incubated with protein kinase A and [-32P]ATP (10 mCi) as described above and labeled proteins were analyzed by SDSPAGE.
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Acknowledgments |
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Abbreviations |
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Footnotes |
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References |
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