©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Glycosylation of Human Truncated FcRI Chain Is Necessary for Efficient Folding in the Endoplasmic Reticulum (*)

(Received for publication, December 12, 1994; and in revised form, February 1, 1995)

Odile Letourneur (1)(§) Salvatore Sechi (1) Jami Willette-Brown (1) Michael W. Robertson (2) Jean-Pierre Kinet (1)(¶)

From the  (1)Molecular Allergy and Immunology Section, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, Maryland 20852 and the (2)Centre for Protein Engineering, Medical Research Council, Cambridge CB2 2QH, United Kingdom

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The high affinity immunoglobulin E (IgE) receptor is an alphabeta(2) tetrameric complex. The truncated extracellular segment (alphat) of the heavily glycosylated alpha chain is sufficient for high affinity binding of IgE. Here we have expressed various alphat mutants in eukaryotic and prokaryotic cells to analyze the role of glycosylation in the folding, stability, and secretion of alphat. All seven N-linked glycosylation sites in alphat are glycosylated and their mutations have an additive effect on the folding and secretion of alphat. Mutation of the seven N-glycosylation sites (Delta1-7 alphat) induces misfolding and retention of alphat in the endoplasmic reticulum. Similarly, tunicamycin treatment reduces substantially the folding efficiency of wild-type alphat. In contrast, no difference in folding efficiency is detected between wild-type alphat and Delta1-7 alphat expressed in Escherichia coli. In addition, maturation of N-linked oligosaccharides and addition of O-linked carbohydrates are not required for either the transport or the IgE-binding function of alphat. Furthermore, complete enzymatic deglycosylation does not affect the stability and the IgE-binding capacity of alphat. Therefore, glycosylation is not intrinsically necessary for proper folding of alphat but is required for folding in the endoplasmic reticulum. Our data are compatible with the concept that specific interactions between N-linked oligosaccharides and the folding machinery of the endoplasmic reticulum are necessary for efficient folding of alphat in eukaryotic cells.


INTRODUCTION

The high affinity IgE receptor (FcRI), which is required for the initiation of IgE-mediated allergic reactions (Dombrowicz et al., 1993), is expressed constitutively on mast cells and basophils (Metzger et al., 1986; Kinet, 1990). It is also expressed on Langerhans cells (Bieber et al., 1992; Wang et al., 1992), on monocytes of allergic individuals (Maurer et al., 1994), and on eosinophils of some patients with hypereosinophilia (Gounni et al., 1994). FcRI is a multimeric complex consisting of an IgE-binding alpha chain, a beta chain, and a dimer of disulfide-linked chains which associate through noncovalent interactions (Kinet, 1990; Ravetch and Kinet, 1991). The alpha chain is a type I integral membrane protein which is homologous to Fc receptors for IgG (FcRII, FcRIII) (Hogarth et al., 1992). The extracellular domain of human alpha contains 181 amino acid residues, and is organized into two immunoglobulin-like domains defined by two pairs of cysteine residues forming two disulfide bridges (Kochan et al., 1988; Shimizu et al., 1988). Previous studies have shown that expression on the plasma membrane of the extracellular domain of alpha reconstitutes IgE binding (Blank et al., 1989; Hakimi et al., 1990). Furthermore, truncation of the transmembrane and cytoplasmic domains of human FcRI alpha results in a protein (alphat) efficiently secreted by CHO (^1)cells and yet able to mediate high-affinity binding of IgE (Blank et al., 1991). The same alphat construct has also been expressed in a secreted and active form in Escherichia coli (Robertson, 1993). Recently, the second Ig domain has been shown to contain at least a portion of the receptor binding site (Mallamaci et al., 1993; Robertson, 1993). However, this second domain binds IgE only with low affinity. Therefore, it is likely that the first domain is somehow also involved in providing the receptor with a high-affinity binding site.

The extracellular domain of the alpha chain is heavily glycosylated. The core protein of alpha has a theoretical molecular weight (M(r)) of 19,275, but the apparent M(r) estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is 50,000 (Blank et al., 1991). This increase in M(r) is primarily due to N-linked glycosylations, which represent 38-42% of the total M(r) of the full alpha chain, and secondarily to O-linked carbohydrates which represent 4% of the total M(r) (La Croix and Froese, 1993). Sequence analysis reveals seven potential N-glycosylation sites in the extracellular part of the human alpha chain (Kochan et al., 1988; Shimizu et al., 1988). The mouse and rat FcRI alpha chains display 6 and 7 N-glycosylation sequons, respectively (Kinet et al., 1987; Ra et al., 1989), but only one sequon position is conserved among murine, rat, and human alpha chains.

While a role for N-linked oligosaccharides in the folding of glycoproteins is widely recognized, the mechanisms underlying this function are still unclear. Current understanding does not allow accurate predictions of the role of glycosylation for a particular protein. For some glycoproteins, N-linked oligosaccharides are necessary for their overall stability, while for others the presence of the sugars is only needed during the folding process. Some proteins, such as IgD, fold more efficiently without sugars, while the trimming of the N-linked oligosaccharides is essential for others (reviewed in Helenius(1994)). In the present study, we clarify the role of glycosylation in the maturation of alphat. We show that the main function of glycosylation is to facilitate the folding process of alphat in the endoplasmic reticulum of eukaryotic cells.


MATERIALS AND METHODS

Antibodies

The following antibodies were used: mAb 15-1, a mouse monoclonal antibody anti-human FcRIalpha (Wang et al., 1992); mouse monoclonal IgE anti-dinitrophenyl antibody; rabbit anti-mouse IgE previously described (Letourneur et al., 1991); 974, a rabbit antibody raised against the peptide VSLNPPWNRI, a sequence corresponding to the amino acids 35 to 44 on the human FcRI alpha chain; and 997, a rabbit polyclonal anti-alphat generated against purified human alphat. Preparation of peptide and immunization of rabbits were as described previously (Letourneur et al., 1991).

Site-directed Mutagenesis

The cDNA encoding human alphat was excised from the plasmid pGEM-3Z (Blank et al., 1991) and subcloned into the HindIII-EcoRI sites of the expression vector pEE14 (Celltech, Berkshire, United Kingdom). Polymerase chain reactions (PCR) (Higuchi et al., 1988) were used to mutate alphat. For each mutant, the asparagine, serine, or threonine residues in the Asn-X-Ser/Thr sequence were changed to alanine using one or two nucleotide substitutions. For all constructs, PCR was used to engineer two additional restriction sites: XbaI upstream of the ATG start codon and EcoRI downstream of the TAA stop codon. The PCR products were digested using EcoRI and XbaI, agarose gel purified, and subcloned into the EcoRI and XbaI sites of the expression vector pcDL-SRalpha296 (Takebe et al., 1988). Double mutants were generated by PCR using single mutated alphat constructs as templates (Higuchi et al., 1988). The subsequent double mutants were then used as PCR templates to construct triple and multiple mutants using the same procedure. All constructs were sequenced using the Sequenase II kit (U. S. Biochemicals, Cleveland, OH). The constructs were designated by numbers defining the N-glycosylation sites mutated, e.g. a mutant of alphat lacking glycosylation sites 1, 2, and 3 was referred as Delta1-3 alphat.

Expression of alphat in E. coli and Periplasmic Preparation

The WT alphat and Delta1-7 alphat cDNAs were fused to the pel-B leader and then ligated into the pUC119 vector (Robertson, 1993). E. coli strain HB2151 was transformed by electroporation using a Gene Pulser (Bio-Rad), and single colonies isolated on 2 times YT-agarose plates containing carbenicillin. Cultures from a single transformation colony were grown in 2 times YT broth containing 0.1% glucose and 100 µg/ml carbenicillin at 37 °C with shaking until an optical density at 600 nm (A) of 0.5 was reached. Cultures were then equilibrated to room temperature with continuous shaking until A of 0.9-1.0 was obtained and then induced by adding isopropyl-beta-D-thiogalactopyranoside to a final concentration of 0.25 mM. The cultures were harvested after 9 h of induction and centrifuged for 10 min at 3000 times g. Periplasmic extracts were prepared as follows: the cell pellet was resuspended in 8 ml of 30 mM Tris-HCl, pH 8.0, 20% sucrose, 1 mM EDTA per 0.1 g of cell pellet and incubated at room temperature for 10 min before centrifugation at 8000 times g at 4 °C for 10 min; the resulting pellet was resuspended in 4 ml of 5 mM MgSO(4) per 0.1 g of cell pellet for 10 min on ice and centrifuged as before. Both culture supernatants and periplasmic extracts were assayed for IgE binding.

Immunoprecipitation and Western Blotting of alphat from E. coli

Periplasmic preparations were incubated for 1 h at 4 °C with mAb 15-1 or 974 antibodies bound to protein A-Sepharose (Pharmacia Biotech Inc.). For the immunoprecipitation with mAb 15-1 the beads were first loaded with rabbit polyclonal anti-IgG (Jackson Immunoresearch Labs, West Grove, PA). After washing, the immunoprecipitated proteins were eluted by boiling for 10 min in Laemmli sample buffer (Laemmli, 1970). Samples were then separated by 12% SDS-PAGE under nonreducing conditions and transferred to Immobilon-P membranes (Milllipore, Bedford, MA). Membranes were blocked in 20 mM Tris, pH 7.6, 137 mM NaCl (TBS) containing 2.5% bovine serum albumin for 2 h and then incubated for 2 h at room temperature with a 1:10,000 dilution of rabbit serum 997 in TBS containing 0.1% bovine serum albumin. After washing with TBS containing 0.1% Tween 20, the membranes were incubated for 20 min at room temperature in the same buffer with a 1:20,000 dilution of goat anti-rabbit antibody conjugated with horseradish peroxidase (Bio-Rad). Membranes were developed using enhanced chemiluminescence according to the manufacturer's instructions (Amersham Corp.).

Transient Transfection in COS-7 Cells

The COS-7 cell line (American Type Culture Collection, Rockville, MD) was grown to 70-80% confluence in Dulbecco's modified Eagle's medium (Biofluids Inc., Rockville, MD) supplemented with 5% fetal calf serum, 2 mM glutamine, 100 IU/ml penicillin, 100 mg/ml streptomycin (COS medium) at 37 °C in a humidified 5% CO(2) incubator before transfection by electroporation. The cells (8 times 10^6 COS-7 cells at 10^7 cell/ml) were resuspended in COS medium, placed in a Gene Pulser cuvette in the presence of 20 µg of plasmid DNA and electroporated at 250 V, 500 microfarads. The medium was replaced with fresh COS medium 18 h after transfection and the cells incubated for an additional 24 h.

Metabolic Labeling and Immunoprecipitation

Forty-two h after transfection, COS-7 cells were washed and preincubated for 30 min at 37 °C in methionine-cysteine-free Dulbecco's modified Eagle's medium supplemented with 5% dialyzed fetal calf serum and 2 mM glutamine. Cells were then metabolically labeled with 0.1 mCi/ml [S]methionine-cysteine (DuPont, Boston, MA) for 3 or 16 h at 37 °C. Cells and supernatants were collected separately. Cells were lysed in ice-cold lysis buffer containing 0.5% (w/v) Triton X-100, phosphate-buffered saline, pH 7.4, 10 mM iodoacetic acid, 1 mM phenylmethylsulfonyl fluoride, 1 mM EDTA and kept on ice for 10 min. Insoluble material was removed by centrifugation at 15,000 times g for 30 min at 4 °C. Cell lysates and culture supernatants were precleared by incubation with protein G-Sepharose (Pharmacia Biotech Inc.) for 30 min at 4 °C and alphat proteins were immunoprecipitated for 3 h at 4 °C with mAb 15-1 or the 974 antibody prebound to protein G-Sepharose. Alternatively, alphat proteins were incubated for 1 h with mouse IgE and immunoprecipitated for 2 h with rabbit anti-mouse IgE bound to protein G-Sepharose. Immunoprecipitated proteins were eluted from washed immunoprecipitates with Laemmli sample buffer and resolved by SDS-PAGE. Immunoprecipitation of unfolded alphat was as follows: after immunoprecipitation using IgE, alphat was eluted by boiling for 5 min in phosphate-buffered saline containing 2% beta-mercaptoethanol, 1% SDS, 20 mM iodoacetamide; the supernatant was recovered and diluted to a final concentration of 0.014% SDS and 0.025% beta-mercaptoethanol; the diluted sample was divided into 3 aliquots which were immunoprecipitated with IgE, mAb 15-1, or 974 using the conditions described above.

Pulse-Chase Experiments

Transfected cells were metabolically labeled with 0.5 mCi/ml [S]methionine-cysteine for 20 min at 37 °C. Pulse-labeled cells were chased for different periods of time in growth medium. At each time point cells were washed in cold phosphate-buffered saline, centrifuged, and the cell pellets were stored at -80 °C. Lysis of the cells and immunoprecipitations were performed as described above. Where indicated, immunoprecipitates were treated with endo-beta-N-acetylglucosamidase H (Endo H) (New England Biolabs) as follows: after washing of the beads, the immunoprecipitated proteins were eluted by boiling for 10 min in 40 µl of 50 mM sodium citrate buffer, pH 5.5, containing 0.5% SDS and 1% beta-mercaptoethanol, and then 20 µl of the eluate were treated with 400 units of Endo H for 2 h at 37 °C.

Stable Transfection of CHO Cells and Selection of Clones

Wild type Chinese hamster ovary (CHO) cells (CHOK1, clone Pro-5;25) were obtained from the American Type Culture Collection. The glycosylation deficient CHO cell lines (Lec.3.2.8.1 and ldlD.Lec1) were kindly provided by Dr. P. Stanley (Albert Einstein College of Medicine, New York). Before transfection and during selection, CHO cells were maintained in GEMEM-S (Life Technologies, Inc./BRL) containing 10% fetal calf serum at 37 °C in a humidified 5% CO(2) incubator. CHO cells (1 times 10^7) were electroporated (250 V, 500 microfarads) with 20 µg of SalI linearized pEE14 plasmid containing WT or mutant alphat cDNAs. Cells were plated directly into 96-well plates. The medium was aspirated 24 h after transfection and replaced by fresh media containing 25 µM methionine sulfoximine (Sigma) which inhibits endogenous glutamine synthetase. Clones were detected after 2 weeks (for CHOK1 cells) or 5-6 weeks (for Lec.3.2.8.1 and ldlD.Lec1 cells) and assayed for secretion of alphat. Positive clones were subjected to gene amplification by progressively increasing the concentration of methionine sulfoximine, up to 100 µM.

Purification and Deglycosylation of alphat Secreted from ldlD.Lec1 Transfectants

The alphat secreted from ldlD.Lec1 stable transfectants was purified using an anti-alpha immunoaffinity column prepared as follows: purified mAb 15-1 was covalently linked to Affi-Gel 10 (Bio-Rad) according to the manufacturer's instructions. Culture medium from ldlD.Lec1 was passed over the immunoaffinity column at a constant flow rate of 8.5 ml/h at 4 °C. The column was sequentially washed with phosphate-buffered saline containing 0.05% NaN(3) and then 50 mM sodium phosphate, pH 7, until the absorbance at 280 nm was reduced to base-line level. The bound alphat was eluted with a 5-7 M urea step gradient in 50 mM sodium phosphate, pH 8, 0.05% NaN(3). The alphat containing fractions were pooled, concentrated, and the buffer adjusted to 3 M urea, 50 mM sodium citrate, pH 5.5, using an Amicon ultrafiltration cell stirrer with a YM10 membrane (Amicon). The concentrate (about 1 mg/ml) was then incubated for 2 h at 37 °C with Endo H (100 units/20 µg). Following this incubation, the buffer was changed to 3 M urea, 50 mM sodium phosphate, pH 8, and N-glycanase (Genzyme Corp., Cambridge, MA) was added at 0.2 unit/20 µg and then the sample was incubated overnight at 37 °C. Samples were analyzed after each step by SDS-PAGE and Coomassie Blue staining.

Detection of IgE Binding Activity of Secreted alphat

The IgE-binding activity of alphat was determined by calculating the capacity of alphat to inhibit the binding of I-IgE to FcRI expressed on rat basophilic leukemia cells (RBL-2H3) as described previously (Blank et al., 1991).


RESULTS

The Seven Putative N-Linked Glycosylation Sites on alphat Are Glycosylated

We generated a panel of alphat mutants in which the seven individual sequons (Asn-X-Thr/Ser) were mutated by substituting the Asn or Thr/Ser residue with Ala (Fig. 1) while trying to preserve as much as possible the consensus amino acid sequence among mouse, rat, and human species (Ra et al., 1989). For instance, the first sequon in the human alphat sequence (Asn-Val-Thr) corresponds to the sequence (Lys-Val-Thr) in mouse and rat. Therefore, we chose to mutate Asn rather than the Thr residue which is conserved.


Figure 1: Mutations of N-linked glycosylation sites. Panel A, schematic representation of FcRI alphat chain indicating the positions of the N-linked glycosylation sites within the polypeptide sequence. The shaded bar represents the leader peptide, the open bar, the mature polypeptide. The N-linked glycosylation sites are numbered from 1 to 7 and their positions in the amino acid sequence is indicated. Panel B, amino acid changes for each N-glycosylation site mutation.



In order to assess the usage of each individual sequon, COS-7 cells were transfected with mutated or WT alphat constructs and metabolically labeled with [S]methionine-cysteine for 3 h. The WT and mutated alphat proteins, including both intracellular and secreted forms, were then immunoprecipitated with the anti-alpha antibody, mAb 15-1, and resolved on SDS-PAGE. The intracellular WT alphat is composed of polypeptides with different degrees of glycosylation. This generates a ladder of bands with the two at the top, around 40 kDa, being most prominent (Fig. 2A). All mutants of alphat show a similar pattern but with a 4-kDa increase in electrophoretic mobility. This systematic downward shift indicates that all the potential N-glycosylation sequons of WT alphat are glycosylated (see also Fig. 9). The bands corresponding to the secreted forms of WT and mutated alphat are much broader, indicating an heterogeneity most likely due to the Golgi processing of alphat (Fig. 2B). These broad bands are not detected intracellularly, probably because of a rapid secretion after Golgi processing. The increase in electrophoretic mobility observed intracellularly with the mutated forms of alphat is also seen on secreted proteins, except when glycosylation sites 1 and 5 are mutated. This could result from an influence of the local environment of the glycosylation site on the type of carbohydrate processing to which this particular site is subjected.


Figure 2: Expression of WT alphat and alphat lacking single N-glycosylation sites in COS-7 cells. COS-7 cells transfected transiently with a plasmid encoding WT alphat or single-site N-glycosylation mutants were metabolically labeled with [S]methionine-cysteine for 3 h at 37 °C. The alphat proteins were immunoprecipitated with mAb 15-1 from cell lysates (A) or cell supernatants (B) before analysis by 14% SDS-PAGE in reducing conditions. The designation of the transfected construct is indicated above each line. The numbers correspond to the N-glycosylation sequons mutated. N, untransfected cells. The position of the M(r) standards is indicated on the left.




Figure 9: Enzymatic deglycosylation of alphat secreted by ldlD.Lec1. Secreted alphat was purified on an agarose-bound mAb 15-1 column (lane 1). The affinity purified product was digested with Endo H (lane 2), followed by further deglycosylation with N-glycanase (lane 3). Proteins were resolved on SDS-PAGE under reducing conditions and stained with Coomassie Blue. The position of the M(r) standards are indicated on the left.



Mutation of Several N-Linked Glycosylation Sites Decreases the Amount of Secreted Active alphat from Eukaryotic Cells

To determine whether single or multiple mutations of the seven N-linked glycosylation sequons could affect the efficiency of secretion of active alphat, we constructed mutants of alphat in which two, three, four, five, six, or all seven N-oligosaccharide sites were inactivated. The various mutants were transfected into COS-7 cells and the IgE-binding capacity of the corresponding alphat secreted in the supernatant was determined (Table 1). There was no significant difference in the IgE-binding capacity of the single alphat mutants when compared with the WT alphat (data not shown). However, there was a cumulative effect on the IgE-binding capacity as the number of mutations increased. No activity was detected in cells transfected with the Delta1-7 alphat construct. This effect could be attributed to either the lack of N-glycosylation or the changes in the amino acid sequence.



Mutation of the Seven N-linked Glycosylation Sites Does Not Affect the Production of Active alphat from E. coli

To analyze whether the substitution of amino acids could affect the folding of alphat we compared unglycosylated WT alphat and Delta1-7 alphat expressed in E. coli. The IgE-binding activity was determined for the respective proteins recovered from the culture supernatant and from the periplasmic space. IgE binding of Delta1-7 alphat was 82 ± 15% (n = 2) of WT alphat from the culture supernatant and 91 ± 17% (n = 2) of WT alphat in samples isolated from periplasmic extracts. Periplasmic extracts from WT alphat and Delta1-7 alphat transformed bacteria were subjected to immunoprecipitation with mAb 15-1. The precipitates were analyzed by SDS-PAGE and immunoblotted with the polyclonal antibody 997. Similar amounts of alphat were immunoprecipitated from both WT alphat and Delta1-7 alphat (Fig. 3). Thus the mutation of the seven glycosylation sites does not decrease the secretion of active alphat in E. coli.


Figure 3: Expression of WT alphat and mutant Delta1-7 alphat in E. coli. Periplasmic extracts from E. coli cells transformed with either WT alphat (lane 2) or mutant Delta1-7 alphat (lane 4) DNA were immunoprecipitated with mAb 15-1. Control immunoprecipitates using an isotype matched antibody are in lanes 1 (WT alphat) and 3 (Delta1-7). The immunoprecipitates were subjected to SDS-PAGE, and immunoblotted with antibody 997. The position of the M(r) standards is indicated on the left.



The Polyclonal Anti-peptide Antibody 974 and mAb 15-1 Recognize Different Forms of alphat

To further analyze why the COS-7 cell product of the Delta1-7 alphat construct does not bind IgE, we generated a polyclonal anti-peptide antibody (974) against a sequence of 10 amino acids close to the amino terminus of alphat, a sequence which does not contain any N-linked glycosylation sites. We compared the reactivity of mAb 15-1 and 974 toward WT alphat and Delta1-7 alphat expressed in E. coli. Both antibodies immunoprecipitated proteins around 20 kDa (Fig. 4A). However, the polypeptide precipitated by 974 has a slightly faster electrophoretic mobility. To test whether the two antibodies reacted with different species of alphat, we performed a depletion experiment. After a first immunoprecipitation with mAb 15-1 or 974, the resulting supernatants were subjected to a second immunoprecipitation with the same antibody to insure complete depletion of the reactive alphat species (Fig. 4B). The alphat species remaining in the supernatant after two sequential mAb 15-1 or 974 immunoprecipitations were then immunoprecipitated with 974 or mAb 15-1, respectively (Fig. 4B). Depletion with mAb 15-1 has no effect on the form and the amount of alphat that is precipitated by 974, and vice versa. Therefore the antibodies 15-1 and 974 react with different species of alphat and do not display any cross-reactivity. In addition, these antibodies immunoprecipitate similar amounts and apparently similar species from both the WT and Delta1-7 alphat constructs. Thus, mAb 15-1 and antibody 974 do not detect any difference of reactivity between the WT alphat and the Delta1-7 alphat when expressed in E. coli.


Figure 4: Reactivity of WT alphat and Delta1-7 alphat produced in E. coli with mAb 15-1 and antibody 974. Panel A, periplasmic extracts from E. coli cells transformed with either WT alphat or mutant Delta1-7 alphat DNA were immunoprecipitated with mAb 15-1, antibody 974, or control antibodies. Panel B, the resulting supernatants were subjected to a second immunoprecipitation with the same antibody, mAb 15-1 or 974. The supernatants depleted with mAb15-1 or 974 were then immunoprecipitated with 974 or mAb15-1, respectively. All immunoprecipitates were resolved on SDS-PAGE and immunoblotted with antibody 997. The position of the M(r) standards is indicated on the left.



The Polyclonal Antibody 974 Reacts Only with Denatured alphat Expressed in Eukaryotic Cells

We analyzed the reactivity of the antibody 974 toward the fully glycosylated WT alphat produced in COS-7 cells. When compared to IgE or mAb 15-1, 974 immunoprecitates WT alphat very poorly (Fig. 5, compare lane 3 with lanes 1 and 2). Therefore, unlike IgE and mAb 15-1, 974 does not recognize the native WT alphat. By contrast, after denaturation in SDS, WT alphat can be precipitated by 974 but not by IgE or mAb 15-1 (compare lane 6 with lanes 4 and 5). Thus, the antibody 974 recognizes only denatured species of alphat incapable of binding IgE or mAb 15-1.


Figure 5: Demonstration of the specifities of mAb 15-1, 974, and IgE. COS-7 cells transiently expressing WT alphat chain were metabolically labeled with [S]methionine-cysteine for 3 h at 37 °C. Cell lysates were divided into 3 portions: four-sixths was immunoprecipitated using IgE, one-sixth with mAb 15-1, and one-sixth with 974. Three-fourths of the washed IgE precipitate was then denatured and divided into 3 equal portions which were immunoprecipitated with either IgE, or mAb 15-1 or 974. Immunoprecipitates were analyzed by 12% SDS-PAGE under reducing conditions. Lanes 1-3, immunoprecipitation in native conditions; lanes 4-6, immunoprecipitation after denaturation; lanes 1 and 4, immunoprecipitation using IgE; lanes 2 and 5, using the mAb 15-1; lanes 3 and 6, using 974. The position of the M(r) standards is indicated on the left.



Mutation of Several N-Linked Glycosylation Sites Affects the Folding of alphat

The antibodies mAb 15-1 and 974 were used to study the effect of multiple mutations on the folding, stability, and secretion of alphat in eukaryotic cells. A pulse-chase metabolic labeling was conducted with COS-7 cells transfected with WT alphat (Fig. 6A). The labeled proteins from the cell lysates were immunoprecipitated with mAb 15-1 or 974, treated or not with Endo H, and resolved by SDS-PAGE. Secreted alphat was similarly analyzed except for the omission of Endo H treatment. After a 20-min pulse, nearly all of the intracellular WT alphat is Endo H sensitive (Fig. 6A), indicating a pre-Golgi localization. As expected, no material is secreted at that time. In addition, very little material is precipitated with 974 when compared with mAb 15-1. After a 2-h chase, about one-third of the labeled WT alphat is secreted in the medium; after 6 h of chase, most is secreted. The fraction remaining intracellularly is still Endo H sensitive and is not precipitated by 974. The absence of Endo H-resistant alphat species in the intracellular compartments confirms that the processed WT alphat is rapidly secreted after carbohydrate processing in the Golgi.


Figure 6: Pulse-chase analysis of WT alphat, mutant Delta1-5 alphat, and mutant Delta1-7 alphat in transfected COS-7 cells. COS-7 cells transfected transiently with plasmids encoding WT-alphat (A), Delta1-5 alphat (B), or Delta1-7 alphat (C) constructs were pulse-labeled with [S]methionine-cysteine for 20 min at 37 °C and chased for 0, 2, or 6 h. The alphat proteins were immunoprecipitated either with mAb 15-1 (left) or antibody 974 (right). Immunoprecipitates from cell extracts were either treated with Endo H (+) or not(-), while immunoprecipitates from supernatants (S) were untreated before analysis on 14% SDS-PAGE under reducing conditions. Note that panels A and B correspond to an overnight exposure of the gels while panel C corresponds to a 4-day exposure. The position of the M(r) standards is indicated on the right.



The mutant with the first five sequons inactivated, Delta1-5 alphat, shows an increased electrophoretic mobility when compared with WT alphat (Fig. 6B). The amount of secreted Delta1-5 alphat which can be immunoprecipitated with mAb 15-1 is much less than that of secreted WT alphat and corresponds well with the drop in IgE-binding activity seen in the supernatant of the Delta1-5 alphat mutant (see above). The amount of intracellular Delta1-5 alphat immunoprecipitated by mAb 15-1 also decreases. In contrast, 974 immunoprecipitates more intracellular Delta1-5 alphat than WT alphat (compare Fig. 6, B and A). A large amount of Delta1-5 alphat remains stable and undegraded intracellularly throughout the chase. Virtually no 974 reactive Delta1-5 alphat is secreted. Therefore, the mutation of sequons 1-5 affects the intracellular folding and hence the secretion of Delta1-5 alphat, but causes little change in its stability.

The effect of multiple mutations is even clearer when all the sequons are mutated. Under these conditions, mAb 15-1 does not precipitate any material around 22 kDa from intracellular or secreted Delta1-7 alphat (Fig. 6C). However, intracellular Delta1-7 alphat is clearly seen after 974 immunoprecipitation. Thus, unlike in E. coli, mAb 15-1 and antibody 974 detect differences in reactivity between WT alphat and Delta1-7 alphat expressed in eukaryotic cells. The amount of alphat detected by mAb 15-1 decreases with the number of mutations. In striking contrast, the amount of alphat detected by antibody 974 increases with the number of mutations (compare panels A, B, and C in Fig. 6). Therefore, increasing the number of mutations and thereby decreasing the glycosylation, induces misfolding of alphat in the pre-Golgi compartment. Taken together, these data indicate that glycosylation of alphat is necessary for its efficient folding in the endoplasmic reticulum of eukaryotic cells.

Prevention of Glycosylation of alphat by Tunicamycin or Mutation Has Similar Effects on Folding and Secretion

We previously published that stable CHO transfectants treated with tunicamycin could secrete deglycosylated alphat capable of binding IgE (Blank et al., 1991). However, the amount of active alphat secreted in that experiment was very small when compared with fully glycosylated alphat. To test whether tunicamycin-induced deglycosylation could affect the folding of alphat in a manner similar to the mutations, we compared the folding of Delta1-7 alphat with the folding of unglycosylated WT alphat obtained from cells treated with tunicamycin. COS-7 cells transfected with WT alphat were treated, or not, with tunicamycin and labeled with [S]methionine-cysteine for 3 h. Both intracellular (Fig. 7A, lane 1) and secreted WT alphat (Fig. 7A, lane 2) were precipitated with mAb 15-1, but much less material was seen after treatment of the cell with tunicamycin (Fig. 7A, lanes 3 and 4). Nevertheless, after longer exposure, small amounts of WT alphat deglycosylated with tunicamycin were detected with mAb 15-1, both as intracellular and secreted material (Fig. 7B, lanes 3 and 4). Similar results to those obtained with tunicamycin-treated cells were obtained with COS-7 cells transfected with Delta1-7 alphat (Fig. 7, A and B, lanes 5 and 6). Thus deglycosylation whether by using tunicamycin or by mutations affects dramatically the amount of intracellular and secreted alphat detected by mAb 15-1. The higher amount of alphat from tunicamycin-treated WT alphat cells (Fig. 7B, lane 3) compared to that of alphat from Delta1-7 alphat cells (lane 5) probably reflects different levels of transcription/translation by the two transfectants. In agreement with the results shown in Fig. 6C, the antibody 974 immunoprecipitates more Delta1-7 alphat material than does mAb 15-1 (compare lanes 5 and 6 in Fig. 7, B and C) and only from the intracellular compartment. In addition, antibody 974 immunoprecipitates similar amounts of material from the intracellular compartment of tunicamycin-treated cells and from cells transfected with Delta1-7 alphat (compare lanes 3 and 5 in Fig. 7C). Thus misfolded forms of alphat detected by 974 are seen in equivalent amounts in tunicamycin-treated cells or in cells transfected with Delta1-7 and very little folded material is detected by mAb 15-1 in both types of cells. These data confirm that glycosylation is important for the proper folding and secretion of alphat in eukaryotic cells.


Figure 7: Comparison of WT alphat from cells grown in the presence of tunicamycin with mutant Delta1-7 alphat. COS-7 cells transiently expressing WT alphat grown without (lanes 1 and 2) or with tunicamycin (lanes 3 and 4) or expressing the mutant Delta1-7 alphat (lanes 5 and 6) were metabolically labeled with [S]methionine-cysteine for 3 h at 37 °C. Cell lysates (lanes 1, 3, and 5) or supernatants (lanes 2, 4, and 6) were divided in two portions and immunoprecipitated using the mAb 15-1 (A and B) or 974 (C) before analysis on 12% SDS-PAGE under reducing conditions. Note that Panel B is a longer exposure of the gel in Panel A. The position of the M(r) standards is indicated on the left.



Processing of N-Linked Oligosaccharides and O-Linked Glycosylations Are Not Required for Transport and Secretion of an Active alphat

The trimming of the N-linked oligosaccharides is an essential step in the folding and transport of many proteins (Helenius, 1994). To study whether this is the case for alphat, we took advantage of two CHO cell lines which have defects in the glycosylation pathways. The Lec3.2.8.1 cell line produces proteins with all N-linked carbohydrates in the oligomannosyl form and O-linked carbohydrates initiating with N-acetylgalactosamine. The ldlD.Lec1 cell line gives rise to proteins with the same N-linked carbohydrates as Lec3.2.8.1 but with O-linked oligosaccharides completely absent (Stanley, 1989).

The alphat-containing pEE14 vector was transfected into the wild type CHO (CHOK1) and the two mutant Lec3.2.8.1 and ldlD.Lec1 cell lines. The alphat secreted from stable clones was metabolically labeled with [S]methionine-cysteine for 16 h, precipitated with IgE, and analyzed by SDS-PAGE (Fig. 8). As expected, the fully glycosylated alphat secreted by CHOK1 migrates as a broad heterogenous band around 50 kDa. The electrophoretic mobility of alphat secreted by the mutant ldlD.Lec1 and Lec3.2.8.1 cell lines shows a 10-15 kDa reduction. In the three cell lines, secreted alphat can be precipitated with IgE and is therefore properly folded. Furthermore, after 16 h of metabolic labeling, alphat is detected almost exclusively in the cell supernatant with virtually no intracellular alphat remaining (data not shown). This indicates an efficient secretion of alphat. Therefore the processing of high mannose-type to complex-type oligosaccharides and the addition of O-linked oligosacharides are not required for transport and secretion of efficiently folded alphat.


Figure 8: Secretion of alphat by stable CHO transfectants. CHOK1, ldlD.Lec1 (Lec1), and Lec3.2.8.1. (Lec3), either untransfected (lanes 1, 3, and 5) or stably expressing alphat (lanes 2, 4, and 6), were metabolically labeled with [S]methionine-cysteine for 16 h at 37 °C. The alphat proteins were precipitated from the cell supernatants with IgE before analysis on 11% SDS-PAGE under reducing conditions. The position of the M(r) standards is indicated on the left.



N-Linked Oligosaccharide Are Not Necessary for the Activity of alphat

We took advantage of the Endo H sensitivity of alphat secreted by ldlD.Lec1 to visualize the utilization of the various N-glycosylation sites. Truncated alpha secreted by stably transfected ldlD.Lec1 cells was purified on mAb 15-1 affinity column and then sequentially deglycosylated with Endo H and N-glycanase. After each step, the deglycosylated products were analyzed by SDS-PAGE and Coomassie Blue staining (Fig. 9). The incomplete digestion with Endo H allows to visualize a ladder of bands most likely generated by glycosylation at the seven N-linked glycosylation sites (see above). Complete digestion was achieved with a further treatment of the sample with N-glycanase. The apparent M(r) of 23,000 observed thereafter corresponds well with the M(r) of 19,275 calculated for alphat.

The IgE-binding activity of glycosylated and deglycosylated alphat was compared quantitatively. No difference was detected (data not shown). Therefore once folded properly, alphat remained active and was still capable of binding IgE.


DISCUSSION

Analyzing the role of carbohydrates in the folding and stability of glycoproteins is an area of intensive investigation. At present, our understanding of carbohydrate function is still fragmentary. Recent works indicate that glycoprotein glycans may play multiple roles and that different proteins have different requirements for carbohydrates. Glycoprotein glycans have been shown to be involved in several functions which include regulation of intracellular trafficking, modulation of enzyme and hormone activities, and participation in cell-cell interactions (Lis and Sharon, 1993). It has been proposed that N-linked oligosaccharides could play several roles during the conformational maturation of glycoproteins (Helenius, 1994). Although for many glycoproteins it has been reported that N-linked oligosaccharides are needed for folding, some, such as IgM or major histocompatibility complex molecules fold efficiently without any. Carbohydrate trimming may also be essential for the folding of many proteins (reviewed in Helenius(1994)).

Our first question was whether the core N-glycosylation which occurs in the pre-Golgi compartment has any relevance to the folding of alphat. We first generated a series of single glycosylation site mutants of alphat (Fig. 1). Transient expression of the single site mutants in COS-7 cells shows that all 7 potential N-glycosylation sites of the alphat chain are glycosylated in the mature native molecule (Fig. 2). The individual elimination of any one site has no significant effect on the secretion of active alphat (data not shown).

Once we had established that all 7 glycosylation sites were modified in vivo, and that no one site was particularly critical for the folding process, we constructed a panel of multiple mutants to test the effect of the cumulative loss of core glycosylation on the intracellular transport of alphat. As illustrated in Table 1, there is a strong cumulative effect of the number of mutated glycosylation sites on the amount of active alphat secreted, raising the question of the cause of the decreased secretion. Possible mechanisms include a primary effect of the amino acid substitutions, misfolding due to the loss of N-glycosylation, and either increased degradation or intracellular retention of unglycosylated alphat. While single replacements of serine or threonine with alanine rarely induce conformational changes (Argos, 1987), the effect of multiple substitutions could have resulted in the misfolding of alphat.

To determine whether the multiple substitutions in the primary sequence could affect the folding, we expressed WT alphat and mutant Delta1-7 alphat in E. coli. As shown in Fig. 3, the mutation of the seven N-linked glycosylation sites does not influence the production and the folding of alphat. This was also confirmed with IgE-binding activity data where no significant difference was noticed between the WT alphat and the mutant Delta1-7 alphat. The above data demonstrate that the amino acid substitution per se does not interfere with the folding of alphat.

To compare the fate of the various forms of alphat, WT alphat, Delta1-5 alphat, and Delta1-7 alphat, we used a monoclonal antibody that precipitates only efficiently folded alphat molecules (mAb 15-1) and an anti-peptide antibody that precipitates unfolded alphat molecules (974) ( Fig. 4and Fig. 5). In transfected COS-7 cells, immediately after biosynthetic labeling, a mixture of properly folded and misfolded alphat is detected intracellularly in all 3 cases. The ratio of misfolded over properly folded alphat increases with the extent of loss of glycosylation (Fig. 6). The Endo H sensitivity of misfolded WT and Delta1-5 alphat indicates retention in a pre-Golgi compartment. The low levels of secretion of Delta1-5 and Delta1-7 alphat are most likely due to retention of misfolded alphat.

The above data seem to contradict our previously published work on tunicamycin-treated cells where glycosylation was demonstrated not to be required for the secretion of active alphat (Blank et al., 1991). However, in this previous study no attempt was made to determine the efficiency of secretion. To clarify this apparent contradiction we used the antibodies mAb 15-1 and 947 to compare alphat produced by the Delta1-7 multiple mutant to that produced by WT in cells treated with tunicamycin. Tunicamycin treatment of COS-7 cells transiently transfected with WT alphat results in misfolding and intracellular trapping of approximately 95% of the unglycosylated proteins, nearly identical to the results with the Delta1-7 (see Fig. 7, A and C). Despite the virtually complete absence of glycosylation in both cases, small amounts of efficiently folded alphat are formed and secreted (see Fig. 7B). This confirms the previous work demonstrating that glycosylation is not absolutely required for secretion (Blank et al., 1991), but indicates that the loss of N-glycosylation results in misfolding of alphat and a substantial decrease in secretion of alphat. Taken together, these results demonstrate that core N-glycosylation facilitates correct folding, with the individual contributions of the carbohydrate chains being additive in promoting folding and, as a consequence, transport of alphat through the secretory pathway.

Our next question was whether the maturation of core N- or O-glycosylation which occurs in the Golgi compartment is involved in the intracellular transport and in the folding of alphat. We chose to address this question by stably expressing alphat in two glycosylation deficient cell lines, Lec3.2.8.1 and ldlD.Lec1. Both cell lines produce alphat which binds IgE (Fig. 8) and mAb 15-1 (data not shown). In addition, equilibrium labeling shows that the alphat produced is completely secreted with essentially no retention in either the Golgi or the endoplasmic reticulum. Retention would be expected if the missing saccharide residues were necessary for proper folding. This result is identical to that obtained after transfection of alphat into CHOK1 cells, the parental cell line for both Lec3.2.8.1 and ldlD.Lec1. These results demonstrate that the Golgi compartment mediated maturation of N-glycosylation and the addition of O-glycosylation are dispensable for the proper folding and efficient intracellular transport of alphat.

Our data indicate that alphat belongs to a group of molecules such as human transferrin receptor, human glucuronidase, and influenza A virus hemagglutinin which are also retained in the endoplasmic reticulum as a consequence of a loss of oligosaccharides (Helenius, 1994). However, once folded, these molecules remain stable. Therefore, the oligosaccharides seem to be necessary only in the endoplasmic reticulum and pre-Golgi compartment. These striking features have been the basis for the theory of specific interactions between the carbohydrate moieties of glycoprotein and the folding machinery. These interactions would be necessary for efficient folding and would control the retention of the non-folded or partially folded forms in the endoplasmic reticulum. Our results are consistent with this concept.

Interactions of proteins with molecular chaperones such as BiP have been well documented. However, these chaperones interact directly with the polypeptide moiety (reviewed in Hartl et al.(1994)). Interactions with chaperones that are dependent on the presence of carbohydrates have been investigated only recently. One example is the folding of vesicular stomatitis virus G protein which interacts sequentially with BiP and calnexin (Hammond and Helenius, 1994). The latter interaction is necessary for proper folding and is prevented by removal of the sugars or by inhibition of the endoplasmic reticulum glucosidases. It will be interesting to investigate whether alphat could also interact with calnexin or a similar, carbohydrate-dependent, chaperone.


FOOTNOTES

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

§
Current address: CDDT Ciba-Geigy, CH-4002 Basel, Switzerland.

To whom correspondence should be addressed: National Institute of Allergy and Infectious Diseases, National Institutes of Health, Twinbrook II Bldg., 12441 Parklawn Dr., Rockville, MD 20852. Tel.: 301-402-0992; Fax: 301-402-0993.

(^1)
The abbreviations used are: CHO, Chinese hamster ovary; PAGE, polyacrylamide gel electrophoresis; mAb, monoclonal antibody; PCR, polymerase chain reaction; WT, wild-type; BiP, immunoglobulin heavy chain-binding protein.


ACKNOWLEDGEMENTS

We thank Dr. P. Stanley for his gift of the ldlD.Lec1 and Lec.3.2.8.1. cell lines and Dr. C. Beddington for the gift of expression vector pEE14. We are grateful to Dr. F. Letourneur and Dr. A. Scharenberg for helpful discussions.


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