The Role of N-Glycosylation for Functional Expression of the Human Platelet-activating Factor Receptor

doi:10.1074/jbc.270.42.25178

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Volume 270, Number 42, Issue of October 20, 1995 pp. 25178-25184
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.

The Role of N-Glycosylation for Functional Expression of the Human Platelet-activating Factor Receptor
GLYCOSYLATION IS REQUIRED FOR EFFICIENT MEMBRANE TRAFFICKING (*)

(Received for publication, July 10, 1995; and in revised form, August 14, 1995)

Carmen García Rodríguez (1), Diana R. Cundell (5), Elaine I. Tuomanen (5), Lee F. Kolakowski , Jr. (1), Craig Gerard (1), (2), (3), (4), Norma P. Gerard (1) (2) (3) (4)(§)

From the (1)Ina Sue Perlmutter Laboratory and Department of Pediatrics, Children's Hospital, the (2)Department of Medicine, Beth Israel and Brigham and Women's Hospitals, the (3)Center for Blood Research, and the (4)Thorndike Laboratory of Harvard Medical School, Boston, Massachusetts 02215 and the (5)Laboratory of Molecular Infectious Diseases, Rockefeller University, New York, New York 10021-6399

ABSTRACT

INTRODUCTION

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION

FOOTNOTES

ACKNOWLEDGEMENTS

REFERENCES

ABSTRACT

Streptococcus pneumoniae has been shown to utilize the platelet activating factor receptor for binding and invasion of host cells (Cundell, D. R., Gerard, N. P., Gerard, C., Idanpaan-Heikkila, I., and Tuomanen, E. I.(1995) Nature, in press). Because bacterial binding is in part carbohydrate dependent, and the human platelet-activating factor (PAF) receptor bears a single N-linked glycosylation sequence in the second extracellular loop, we undertook studies to determine the role of this epitope in PAF receptor function. Binding of pneumococci to COS cells transfected with the human PAF receptor is greatly reduced for a receptor mutant that bears no N-linked glycosylation site. Immunohistochemical and binding analyses show decreased expression of the non-glycosylated molecule on the cell membrane relative to the wild type receptor; however, metabolic labeling and immunopurification indicate it is synthesized intracellularly at a level similar to the native molecule. A mutant receptor encoding a functional glycosylation site at the NH (2) terminus is better expressed at the cell surface compared with the non-glycosylated form, indicating that trafficking to the cell surface is facilitated by glycosylation, but its location is relatively unimportant. The binding affinity for PAF is not significantly effected by the presence or location of the carbohydrate, and variations in cell surface expression have little influence on signal transduction, as the non-glycosylated PAF receptor is equally effective for activation of phospholipase C as the native molecule. These data are supportive of pneumococcal binding on protein moiety(ies) of the PAF receptor and indicate that N-glycosylation facilitates expression of the protein on the cell membrane.

INTRODUCTION

Platelet-activating factor (PAF) ()is a proinflammatory lipid involved in multiple patophysiological processes (1, 2, 3, 4) . The PAF receptor, a member of the rhodopsin family of seven-transmembrane segment receptors linked to heterotrimeric GTP-binding proteins(5, 6, 7, 8) , activates multiple intracellular signaling mechanisms, including phospholipid turnover via phospholipases A (2) , C, and D(9) . The human PAF receptor(10, 11, 12) , and several orphan receptors are members of a small subset of G-protein-coupled receptors that lack consensus N-linked glycosylation sequences in the amino-terminal extracellular domain. The human PAF receptor contains a single N-linked consensus glycosylation sequence in the putative second extracellular loop; PAF receptors cloned from other species, including guinea pig (13) and rat (14) , have an additional NH (2) -terminal consensus sequence for N-glycosylation as well.

The carbohydrate moieties of glycoproteins in general are believed important for intracellular trafficking, stability, secretion, and/or cell surface expression. They may also be important for protein folding, enzymatic activity, and additional structural functions (15, 16, 17) . Among G-protein-coupled receptors, however, the role of carbohydrate adducts is somewhat less clear, with unpredictable and non-uniform effects on ligand binding, signal transduction, and/or cell surface expression(18, 19, 20) . The role of the oligosaccharide moiety(ies) in functional expression of the PAF receptor has not previously been addressed.

A recent investigation demonstrated that Streptococcus pneumoniae utilizes the PAF receptor for bacterial adherence and invasion in host cells(21) . A phosphoryl choline-containing teichoic acid in the pneumococcal cell wall is essential for the interaction (22, 23) , and binding is blocked in the presence of PAF or PAF receptor antagonists. As binding of pneumococcus to target cells is also mediated in part by interactions with carbohydrate residues(24) , we questioned whether specificity for the PAF receptor is conferred by the presence and/or position of the carbohydrate group. Since preliminary experiments indicated complex results we undertook a more extensive investigation into the role of N-glycosylation in the functional expression of the human PAF receptor. Our approach involved mutagenesis of the PAF receptor cDNA to delete the glycosylation sequence and/or incorporate a new glycosylation site in the amino-terminal sequence, testing resulting molecules for interaction with ligand and signal transduction in transfection systems.

EXPERIMENTAL PROCEDURES

Materials

[

H]PAF (36 Ci/mmol), [

H]WEB 2086 (10.5 Ci/mmol), [

S]methionine + cysteine (1175 Ci/mmol), [

H]mannose (21 Ci/mmol), myo-[2-

H]inositol (20 Ci/mmol), and EnHance were purchased from DuPont NEN. pCRScript was supplied by Stratagene (La Jolla, CA). DMEM and DMEM without inositol, glucose, or methionine were obtained from Life Technologies, Inc.. Sheep blood was from Micropure Medical Inc. (Stillwater, MI). Trypticae soy agar was purchased from Difco (Detroit, MI). Protein G-Sepharose was supplied by Pharmacia Biotech Inc. (Upsala, Sweden). PAF, protein A-Sepharose, and FITC were from Sigma. m2 anti-Flag was purchased from Eastman Kodak. Anti-horseradish peroxidase was from Zymed (South San Francisco, CA). Biotinylated anti-mouse IgG was from Vector Labs (Burlingame, CA). WEB 2086 was a generous gift of Boeringher Ingelheim (Ridgefield, CT). Glass slide chambers were supplied by Nunc Inc. (Naperville, IL). Glass fiber filters (GF/C) were from Whatman International Ltd. (Maidstone, United Kingdom). Dowex 1 resin and protein standards were from Bio-Rad.

Plasmid Construction

The human myeloid PAF receptor cDNA was cloned and expressed with the Flag epitope at the NH (2)

terminus in the mammalian expression vector pCDM8, as described previously(12) . The Flag-PAF receptor cDNA was modified by PCR to eliminate the single N-linked glycosylation site by mutating Asn

Ala (the dCHO mutant), taking advantage of the unique NarI restriction site (GG/CGCC) introduced by the alanine codon. Sense and antisense oligonucleotide primers corresponding to nucleotides 496-516 of the coding sequence (sense 5`-GGC TCA GGC GCC GTC ACT GCG-3`; antisense 5`-GCG AGT GAC G GC GCC TGA GCC-3`, mutated nucleotides are underlined) were paired with antisense and sense primers corresponding to the 3` and 5` ends of the PAF receptor coding sequence, respectively, and amplified through 25 cycles of denaturation at 94 °C for 1 min, annealing at 55 °C for 2 min, and extension for 3 min at 72 °C. PCR products were subcloned individually into pCRScript, ligated to join the 5` and 3` fragments, and the entire coding sequence was ligated to the pCDM8-Flag construct(12) .

Receptors containing consensus N-linked glycosylation sites in the NH (2) -terminal extracellular sequence were generated for both the wild type and dCHO receptor cDNAs by PCR. Primers were designed to mutate His Asn in the human Flag-PAF receptor, yielding the sequence, LEPNDSS (sense: Nt1 5`GCGAATTC CTG GAG CCA AA C GAC TCC TCC CAC ATG-3`, mutations underlined, EcoRI site in italics). Alternatively, the amino-terminal sequence was altered to generate the sequence corresponding to the first 7 amino acids of the guinea pig PAF receptor, mutating Pro Leu, His Asn, and Asp Ser (sense: Nt 5`-GCGAATTC CTG GAG C TA AA C AGC TCC TCC CAC ATG GAC-3`). These primers were paired with antisense primers corresponding to the 3` end of the coding sequence using the PCR conditions described above, except that annealing was carried out at 60 °C, and products were ligated to pCDM8-Flag following digestion with EcoRI and XbaI. This resulted in a total of five PAF receptor mutants, as shown schematically in Fig. 1, bearing no (dCHO), one (Nt1/dCHO and Nt/dCHO), or two consensus N-linked glycosylation sites (Nt1/WT and Nt/WT) in addition to the wild type molecule (WT). All constructs were confirmed by DNA sequencing.

Figure 1: Mutations in the N-linked glycosylation site of the human PAF receptor. Schematic representation of the extracellular domain of the human PAF receptor and sequence alignment with the guinea pig PAF receptor and the mutants constructed. The mutant dCHO encodes Asn Ala, deleting the single N-linked glycosylation site in the second extracellular loop of the wild type receptor. Alteration of His Asn introduces a potential new glycosylation site into the wild type receptor, Nt1/WT, or the dCHO mutant, Nt1/dCHO. A second set of mutants introduced the guinea pig consensus sequence for N-linked glycosylation, making Nt/dCHO, with a single glycosylation site at the position 4, and Nt/WT with two glycosylation sites at positions 4 and 169. The consensus sequences for N-linked glycosylation are in bold type.

Cell Cultures and Transfection

COS cells were maintained in DMEM (high glucose), containing 6 mML-glutamine, 10% fetal calf serum, 100 units/ml penicillin G, and 100 µg/ml streptomycin, and transfections were performed using DEAE-dextran as described previously(12) . Cells were used for subsequent studies 48-96 h later. Parallel transfections using a plasmid encoding bacterial beta

-galactosidase (pRSV beta

Gal) followed by X-gal staining indicate transfection efficiencies of

30%.

Adherence of Pneumococci to Transfected Cells

S. pneumoniae of the unencapsulated strain R6 was grown on trypticase soy agar containing 3% sheep blood for 18 h at 37 °C. Bacteria were harvested from the plate into 1 ml of Dulbecco's phosphate-buffered saline, heat-killed, and labeled with FITC as described previously(24, 25) . The bacteria were washed twice by centrifugation (13,000 times

g, 3 min), resuspended in 1 ml of albumin buffer(25) , and diluted to 10

-10

colony-forming units/ml. For some experiments, R6 pneumococci were grown in defined medium containing ethanolamine in place of choline as the amino alcohol(22, 24) . Monolayers of COS cells were washed twice with Medium 199 and incubated with bacteria for 30 min at 37 °C. Nonadherent bacteria were removed by washing the monolayers three to five times with Medium 199. Cells were fixed in 2.5% glutaraldehyde and adherent bacteria counted visually with an inverted microscope equipped for fluorescence with an IF DM-510 filter (Diaphot-TMD; Nikon Inc., Melville, NY). Adherence was expressed as the number of attached bacteria/100 cells counted in a times

40 field(24, 25) . Values for two wells were averaged, and each experiment was performed on at least six separate occasions. To control for possible effects on adherence due to FITC labeling of bacteria, direct comparison was made between counts using FITC-labeled bacteria and unlabeled bacteria detected by Gram stain. For experiments to determine the ability of carbohydrates to inhibit adherence, FITC-labeled pneumococci (2 times

colony forming units/ml) were preincubated 15 min at room temperature with monosaccharides or glycoconjugates, centrifuged to remove unbound sugar, resuspended to 10

or 10

colony-forming units/ml in albumin buffer and added to the adherence assay.

Ligand Binding and Uptake

Ligand binding to receptor transfected COS cell membranes was performed essentially as described (11) . Membranes were prepared by scraping cells into 25 mM HEPES, pH 7.5, 10 mM MgCl (2)

, and Dounce homogenized on ice. Homogenates were centrifuged at 800 times

g for 10 min at 4 °C to remove nuclei and unbroken cells, and membranes were harvested at 100,000 times

g for 20 min. Membrane protein was quantitated by Coomassie Blue staining calibrated with BSA (Pierce Protein Assay), and 30 µg were incubated in 25 mM HEPES, pH 7.5, 10 mM MgCl (2)

, 0.1% BSA, at 22 °C with 2 nM [

H]WEB 2086 or 0.5 nM [

H]PAF and increasing concentrations of unlabeled antagonist or ligand, respectively, for 90 min. Mixtures were filtered on 1% BSA-soaked glass fiber filters (GF/C), washed, and subjected to liquid scintillation counting. Nonspecific binding was assessed in the presence of 10 µM WEB 2086 or PAF, and data were analyzed using the Ligand program. All points were measured in duplicate and experiments repeated at least three times.

Receptor-dependent uptake of [H]PAF on transfected COS cells was performed as described previously(26) . Cells in 6-well culture plates were washed with 150 mM choline chloride, containing 10 mM Tris-HCl, pH 7.4, 10 mM MgCl (2) , and 0.25% BSA, and incubated in the same buffer with 2 nM [H]PAF for 45 min at 37 °C. Cell layers were washed three times with buffer containing 2% BSA to remove extracellular ligand. Cell-associated ligand was quantitated by trypsinizing the cell layers and liquid scintillation counting. All experiments were performed at least three times in duplicate or triplicate. Data are corrected for nonspecific binding in the presence of 10 µM unlabeled PAF and expressed as the mean ± S.E.

Cell Surface Expression of the PAF Receptors

The NH

-terminal Flag epitope was used to detect receptors expressed on the cell surface as described previously(12) . COS cells were plated on fibronectin-coated glass slide chambers, transfected as described above, and immunostained 3 days later. Cells were blocked with phosphate-buffered saline (PBS) containing 3% BSA, incubated with the primary antibody, m2 anti-Flag at 10 µg/ml for 30 min at 22 °C, washed three times with PBS, and incubated with biotinylated anti-mouse IgG, followed by peroxidase-labeled avidin biotin complex as described by the supplier. Staining was accomplished with 0.05% diaminobenzidine and 0.01% H (2)

in PBS, and the cells were examined by light microscopy (Olympus BH2 microscope). Cells transfected with the pCDM8 vector without insert were used as controls.

Labeling of PAF Receptors

For metabolic labeling with [

S]methionine + cysteine, cells transfected 2 days previously were washed and incubated in methionine-free DMEM containing 5% dialyzed fetal bovine serum for 1 h at 37 °C. [

S]Methionine + cysteine (200 µCi/ml) was then added to the medium and incubation continued for 3 h at 37 °C.

For analysis of carbohydrate incorporation, transfected COS cells were incubated in glucose-free DMEM containing 10% fetal calf serum for 1 h at 37 °C. D-[2-H]Mannose (100 µCi/ml) was then added to the medium and incubated at 37 °C for an additional 2 h.

Immunopurification

Cells labeled with [

S]amino acids or [

H] mannose were washed twice with PBS and lysed in 1% Triton X-100 in 10 mM Tris-HCl, pH 7.4, 300 mM NaCl, 1 mM CaCl (2)

, containing 10 mg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride, by incubating at 4 °C for 45 min. Immunoprecipitations were performed as modified from previously described methods(27) . Nuclear debris was removed by centrifugation at 12,000 times

g for 10 min. Lysates were centrifuged at 100,000 times

g for 1 h and precleared with Staphylococcus aureus followed by a mixture of protein A- and G-Sepharose. Solubilized samples were incubated with 10 µg/ml m2 anti-Flag antibody for 90 min at 4 °C. Protein G-Sepharose was added and incubation continued for 45 min. Immune complexes were recovered by centrifugation at 12,000 times

g for 30 s at 4 °C, washed four times with lysis buffer without protease inhibitors and once with 10 mM Tris-HCl, pH 7.4, 140 mM NaCl, 1 mM CaCl (2)

. Immunoprecipitates were dissociated by incubating in SDS-sample buffer containing 2-mercaptoethanol for 20 min at 55 °C. Protein G-Sepharose beads were pelleted and the supernatant fractions applied to 10% SDS-PAGE gels. Following electrophoresis, gels were dried and subjected to fluorography (Enhance). Controls included PAF receptor-transfected cells incubated with irrelevant antibody (anti-horseradish peroxidase) in place of anti-Flag, or mock transfected COS cells incubated with anti-Flag antibody in the same manner as transfected cells.

Inositol Phosphate Production

Ligand-stimulated activation of phosphatidylinositol-specific PLC was assessed as described previously(28) . Two days after transfection, cells were washed with inositol-free DMEM and incubated for 18-24 h in inositol-free DMEM, containing 10% fetal calf serum and 10 µCi/ml myo-[2-

H]inositol. Labeling medium was removed and cells were incubated in inositol-free DMEM containing 10 mM LiCl, 0.25% BSA, and increasing concentrations of PAF at 37 °C for 30 min. Reactions were terminated by addition of 10% HClO (4)

containing 4 mg/ml IP (6)

and inositol phosphates purified by chromatography on Dowex 1 as described previously(29) . All points were determined in triplicate, and experiments were repeated at least three times. Data are expressed as the mean ± S.E., percent above unstimulated controls.

RESULTS

Pneumococcal Binding to PAF Receptors

Previous investigations have demonstrated specificity of S. pneumoniae for binding to the PAF receptor and invasion of cells on which it is expressed (21) . Pneumococci are also appreciated to interact with carbohydrate moieties, particularly those containing GlcNAc(24) . The human PAF receptor contains a single N-linked glycosylation consensus sequence in the putative second extracellular loop, on Asn

, as depicted in Fig. 1(12) . A mutant PAF receptor (dCHO), in which Asn

was changed to Ala, binds only

30% as many pneumococci as the native molecule, compared with 3-6% on untransfected controls (Fig. 2, Table 1). As previously demonstrated for lung and endothelial cells in culture, pneumococcus binding to native PAF receptor transfected COS cells is inhibited by

50% in the presence of 100 mM GlcNAc or 100 µM alpha

1 acid glycoprotein, containing the GlcNAc determinant (Table 1). Asialo-GM2 and globoside at 100 µM had no inhibitory effect (data not shown). Pneumococcal binding is also inhibited by PAF or PAF receptor antagonists(21) . Further, bacteria grown in ethanolamine instead of choline do not contain phosphoryl choline in their cell wall and are unable to adhere to PAF receptor-transfected cells (Fig. 2). These data suggest that while pneumococcal adherence involves a multiplicity of targets, some specificity may reside in the PAF receptor carbohydrate. Alternatively, specificity may be determined by the PAF receptor protein, and the non-glycosylated mutant is not expressed at the cell surface with the efficiency of the native molecule.

Figure 2: Adherence of pneumococci to PAF receptor-transfected COS cells. Adherence of FITC-labeled pneumococci to COS cells transfected with the indicated PAF receptor construct is illustrated (approximately 70 COS cells are shown/panel). Ethanolamine-grown bacteria labeled as efficiently as wild type cells but adhered poorly to PAF receptor-bearing cells. Values for pneumococci/100 COS cells in each panel are: PAF receptor, 221; vector alone, 34; the non-glycosylated receptor (dCHO), 73; ethanolamine-grown bacteria on PAF receptor-transfected cells, 55.

Immunochemical and Pharmacological Analysis of the Non-glycosylated PAF Receptor

In order to distinguish these possibilities, we took advantage of the Flag epitope expressed at the NH (2)

terminus of the transfected receptors. Previous studies have demonstrated the utility of the Flag epitope for detection of cell surface PAF receptors independently of ligand binding(12, 26) . As shown in Fig. 3A, COS cells expressing the native human PAF receptor exhibit predominant antibody staining at the perimeter of the cell, characteristic of cell surface epitopes. In contrast, the non-glycosylated mutant, dCHO, exhibits very faint staining, consistent with greatly reduced expression on the cell surface (Fig. 3B).

Figure 3: Immunohistochemical expression of the human PAF receptor and its mutants. Cell surface expression of the Flag-PAF receptor mutants was compared immunohistochemically as described under ``Experimental Procedures'' using unfixed, unpermeabilized cells. COS cells were transfected with the wild type Flag-PAF receptor/pCDM8 (A), or the mutants dCHO (B), Nt/WT (C), or Nt/dCHO (D) and stained with m2 anti-Flag antibody as described. Antibody staining is most intense at the perimeter of the cell, characteristic of a cell surface epitope. Nontransfected cells (E) or cells transfected with the vector pCDM8 alone show no staining.

Scatchard analyses of [H]WEB 2086 binding to membranes from transfected COS cells (Fig. 4, Table 2) are consistent with pneumococcal binding data and immunochemical analysis. The non-glycosylated molecule (dCHO) exhibits only 30% as many sites/cell compared to the wild type receptor; both receptors bind antagonist with similar affinity, 14-23 nM. Comparisons based on binding of [H]PAF to these membrane preparations were not possible due to high nonspecific binding as previously reported(11) .

Figure 4: Scatchard plot of [H]WEB 2086 binding to the human PAF receptor and glycosylation mutants. Membranes were prepared from COS cells transfected with wild type human PAF or glycosylation mutant receptors and tested for binding to [H]WEB 2086 as described under ``Experimental Procedures.'' Scatchard analysis of the data obtained from a representative experiment comparing each of the mutants with wild type receptor.

Ligand uptake in intact cells mirrored antagonist and pneumococcal binding (Table 3). As previously reported, this activity is dependent on expression of the receptor in COS cells and, for the wild type molecule 8-10 times more ligand is internalized at physiological temperature compared with the amount that binds to intact cells at 4 °C(26, 30) . As indicated in Table 3, the non-glycosylated mutant (dCHO) incorporates 50% as much PAF compared with the wild type PAF receptor (WT).

Additional Glycosylation Mutants of the Human PAF Receptor

To determine whether the presence or position of the carbohydrate dictates trafficking to the cell surface, we constructed several additional mutant PAF receptor cDNAs, as depicted in Fig. 1. Glycosylation sequences were introduced in the NH (2)

-terminal extracellular sequence at positions corresponding to the guinea pig PAF receptor. The mutation His

Asn produces the sequence L (1)

EPNDSS

and was introduced in both the native receptor (Nt1/WT) and the dCHO mutant (Nt1/dCHO). Since the N-glycosylation sequences in the guinea pig and rat molecules are devoid of flanking proline or aspartic acid residues, we prepared an additional set of mutants introducing the sequence corresponding to the guinea pig PAF receptor NH (2)

terminus, L (1)

ELNSSS

(Nt/WT, Nt/dCHO).

Immunochemical analysis indicates the mutant with a single glycosylation site in the NH (2) -terminal domain Nt/dCHO is expressed on the cell surface, although staining is somewhat less pronounced than wild type (Fig. 3D). The mutant Nt/WT, with two glycosylation sites, shows relatively robust cell surface staining compared with the wild type receptor (Fig. 3C), while untransfected cells or cells transfected with the vector alone (pCDM8) show no antibody reactivity (Fig. 3E).

Scatchard analyses of antagonist binding indicate that alteration of the NH (2) -terminal sequence in the dCHO mutant to that of the guinea pig receptor increases membrane expression levels to 50% of the native molecule. Expression of both glycosylation sites increased expression to the level of the wild type human molecule (Table 2, Fig. 4).

The mutant Nt/dCHO, containing a single glycosylation site in the NH (2) terminus, internalizes 85% as much ligand as the native receptor (Table 3). Nt/WT, with two glycosylation sites, internalizes slightly more ligand than the wild type receptor. The mutant Nt1/dCHO exhibits similar behavior in uptake studies as the nonglycosylated mutant (data not shown), and untransfected cells exhibit no specific [H]PAF uptake(26) . These results support a decrease in the number of functional receptor molecules appearing on the cell surface in the absence of glycosylation. Functional expression is at least partially restored by the presence of a glycosylation site in the NH (2) -terminal sequence.

Biosynthesis of Native PAF and Mutant Receptors

To determine the relative efficiency of cellular synthesis of the mutated PAF receptors, transfected COS cells were metabolically labeled with [

S]amino acids and the receptors immunopurified using the anti-Flag antibody. SDS-PAGE analysis of the radiolabeled products indicate that all the cDNA constructs are translated with similar efficiency, and yield proteins with distinct apparent molecular weights, as anticipated based on the presence or absence of glycosylation sites (Fig. 5A). The native PAF receptor migrates with an apparent molecular mass of

42 kDa, similar to previous observations(31) . The non-glycosylated, dCHO mutant yields a major band migrating at

39 kDa, consistent with elimination of the N-glycosylation site and the predicted size based on the deduced amino acid sequence(12) . The mutant Nt/WT (containing two potential N-glycosylation sites) migrates as a relatively broad band at

44 kDa. Nt/dCHO (with an N-glycosylation site only in the NH (2)

terminus) also shows a relatively broad band at

40 kDa. The mutants encoding His

Asn, Nt1/WT, and Nt1/dCHO migrated similarly to the wild type receptor and the dCHO mutant, respectively, suggesting the single amino acid substitution in this sequence is not sufficient to produce an efficiently glycosylated sequence (data not shown). Higher molecular weight species likely represent receptor dimers, as observed for rhodopsin under similar conditions(32) ; however, unlike rhodopsin, the intensity of labeling in this position was not reduced by solubilization with octyl glucoside (not shown).

Figure 5: Immunopurification of Flag-PAF receptor and mutants. Transfected COS cells were labeled with [S]methionine + cysteine (A) or [H]mannose (B) as described under ``Experimental Procedures.'' Flag-PAF receptors were immunoprecipitated with m2 anti-Flag antibody and protein G-Sepharose followed by electrophoresis on 10% SDS-PAGE gels under reducing conditions and fluorography. Lane 1, untransfected COS cells; lane 2, cells transfected with wild type PAF receptor; lane 3, the dCHO mutant; lane 4, Nt/WT; lane 5, Nt/dCHO. Equivalent amounts of protein were applied for each sample. The apparent molecular masses were determined relative to protein standards for wild type PAF receptor as 43 kDa (A) and for the non-glycosylated mutant, dCHO, as 39 kDa (B).

Because the apparent molecular weight for Nt/dCHO, in which the glycosylation site was moved from the second extracellular loop to the NH (2) -terminal sequence, was smaller and apparently more heterogeneous than the wild type PAF receptor, incorporation of carbohydrate was confirmed by labeling transfected cells with [H]mannose and repeating the immunopurifications. As shown in Fig. 5B, the wild type PAF receptor cDNA expresses a protein that incorporates [H]mannose. The dCHO mutant exhibits no [H]mannose incorporation, consistent with expression of a non-glycosylated protein. Mutant cDNAs that introduce a consensus sequence for N-glycosylation in the NH (2) terminus (Nt/WT and Nt/dCHO) both express proteins that incorporate [H]mannose. Nt/dCHO and Nt/WT yield H-labeled bands of 39 and 44 kDa, consistent with glycosylation of one or two sites, respectively. The migration patterns of both these mutants are somewhat more diffuse than the native receptor, when labeled either with [S]amino acids or with [H]mannose, likely reflecting heterogeneity of the oligosaccharide chains. The mutant Nt/dCHO also shows somewhat less incorporation of [H]mannose compared to the wild type receptor, consistent with altered kinetics of glycosylation and/or processing of the carbohydrate at the NH (2) terminus compared with the second extracellular loop site.

Metabolic labeling of cells transfected with the Nt1/dCHO mutant, that introduces an asparagine at residue 4 and deletes the site located in the second extracellular loop, indicates the protein is not significantly glycosylated (data not shown). This mutation encodes the amino acid sequence L (1) EPNDSS (7) , and the absence of glycosylation may result from the proline residue just preceding the asparagine, and/or the flanking acidic amino acids.

Signal Transduction by Transfected Receptors

Previous studies have shown that the human PAF receptor couples with G alpha

q in COS cells to activate phosphatidyl inositol-specific PLC(28) . In the present study, cells transfected with each of the PAF receptor mutants were compared with wild type PAF receptor for activation of this pathway by metabolism of [

H]phosphatidyl inositol following stimulation with increasing concentrations of PAF (Fig. 6). Our data indicate that ligand-induced activation of PLC is equally efficient for the mutants as it is for the wild type receptor; transfection with each of the constructs results in essentially identical increases in IP as a function of PAF concentration. Untransfected cells do not respond to stimulation by PAF. Thus, expression of the PAF receptor is required for activation of PLC; however, the magnitude of the response is not reflected by the number of receptor sites/cell, but may be limited by another component like the cellular G-protein content or another downstream effector molecule.

Figure 6: PAF-induced PLC activation in transfected COS cells. COS cells were transfected with cDNAs encoding the human PAF receptor and glycosylation mutants. Cells labeled with [H]inositol were stimulated with increasing PAF concentrations, and IP production was determined as described in ``Experimental Procedures.'' Values shown are the percent increase in [H]inositol phosphate above background. Each point represents the mean (±S.E.) of triplicate determinations from four independent experiments.

DISCUSSION

The experiments described were undertaken as a result of the observation that S. pneumoniae appears to adhere to cells in part by binding to the PAF receptor(21) . The unusual position of the glycosylation site on the human PAF receptor, in addition to the observation that pneumococci also bind to host cells via particular carbohydrate interactions(22, 23) , prompted us to investigate the role of the PAF receptor carbohydrate for bacterial adherence as well as functional interactions with the normal ligand. Our findings indicate that a non-glycosylated human PAF receptor mutant is expressed in COS cells at 30% of the level of the native molecule based on pharmacologic and immunochemical analysis. COS cells transfected with this mutant also bind only 30% as many pneumococci as the wild type receptor, compared with only 3-5% in untransfected cells. As this mutant contains no carbohydrate determinant, pneumococci likely recognize a protein determinant on the PAF receptor. Further, pneumococci lacking cell wall phosphoryl choline are ineffective for binding PAF receptor-transfected cells. However, since carbohydrates partially block pneumococcal binding to the PAF receptor it is possible that glycosylation enhances bacterial binding but is not required for the interaction. The relatively large size of a bacterial particle could facilitate multiple binding interactions with a receptor.

Glycosylation is generally considered important for protein secretion among other functions; however, for G-protein coupled receptors, the role of carbohydrate adducts is somewhat variable(15, 16, 17) . The consensus recognition sequence for N-linked glycosylation is N-X-T/S-Y(33, 34) where X and Y are any amino acid except proline(35) , and mutagenesis of the asparagine residue prevents carbohydrate addition. The deduced amino acid sequence for the human PAF receptor predicts a single N-linked glycosylation site, Asn, located in the second extracellular loop but none in the NH (2) -terminal domain(12) . A second N-linked glycosylation sequence exists at residues 58-61, in transmembrane segment 2, although, as shown by the data of Fig. 5B, this position is not utilized, likely because of its predicted transmembrane location.

As shown in Fig. 5, the mutant Asn Ala is translated efficiently as a protein that is not glycosylated, as expected by deletion of the N-linked glycosylation site. Immunohistochemical experiments show greatly reduced expression of this mutant on the cell surface compared to the wild type receptor (Fig. 3). Ligand binding studies indicate a reduction in the number of binding sites by 70% relative to wild type, consistent with a requirement for glycosylation to effect efficient transport to the cell surface.

When the NH (2) -terminal sequence was modified to that of the guinea pig receptor, L (1) ELNSSS (7) (Nt/dCHO), the resulting protein was both glycosylated and expressed on the cell surface (Fig. 3 Fig. 4 Fig. 5). The carbohydrate moiety added at this position appears somewhat smaller and more heterogeneous than that added at the second extracellular loop site, based on SDS-PAGE (Fig. 5). Cell surface expression based on immunohistochemistry is somewhat reduced compared with the wild type molecule (Fig. 3), and analysis of binding and ligand uptake data indicate 50% and 85% as many sites/cell ( Table 2and Table 3). We have observed a similar apparent reduction in immunoreactivity of the Flag epitope with other amino terminally glycosylated receptors, potentially because of steric influences. ()The presence of functional glycosylation sites at residues 4 and 169 yields a molecule with cell surface expression that is similar to the wild type human molecule. The double mutant, Nt1/dCHO, encoding His Asn, Asn Ala, produces the amino-terminal sequence L (1) EPNDSS (7) and was neither glycosylated nor expressed on the cell surface, suggesting that the consensus sequence for N-glycosylation is adversely effected by a proline prior to the asparagine residue and/or the flanking acidic amino acids.

The presence or position of PAF receptor glycosylation sites has little impact on the affinity of the receptor antagonist [H]WEB 2086, as indicated in Table 2. The binding affinity is in the range of 14-23 nM, similar to a previous report(11) . Nonspecific binding of [H]PAF in membrane preparations precluded the use of the natural ligand in these studies; however, the lipid is internalized at physiological temperatures in receptor-transfected COS cells by a receptor-dependent mechanism with parameters reflecting PAF ligand-receptor interactions (26) . Data deriving from these experiments essentially mirror the binding studies; the dCHO mutant internalizes only 50% as much ligand compared with the wild type receptor and the EC values are essentially the same (Table 3). Substitution of the guinea pig amino-terminal sequence in Nt/dCHO restores essentially wild type levels of ligand internalization, and, again, the doubly glycosylated receptor exhibits as much or more ligand uptake.

While cell surface expression, pneumococcal and ligand binding, and internalization are regulated by glycosylation of the PAF receptor (i.e. dependent on the number of receptor molecules expressed on the cell surface), signal transduction appears independent. The data of Fig. 6show essentially identical increases in inositol phosphate production in response to increasing concentrations of PAF irrespective of the number or position of glycosylation sites. COS cells bearing no PAF receptor exhibit no PAF-induced increase in IP levels over base line. It seems unlikely that coupling to G-proteins is limiting, since ligand uptake data, as well as previously reported binding studies in transfected cells, provide evidence for a single high affinity class of receptor(12, 26) . Such a result might be observed if PLC or another pathway component were limiting.

Studies of the role of carbohydrate moieties in other seven transmembrane segment receptors show variable results. Rhodopsin, which contains two functional glycosylation sequences, was not effected by mutation of Asn; however, mutation of Asn adversely effected protein folding, expression, and signal transduction (36) . In contrast, the muscarinic acetylcholine receptor does not require glycosylation for synthesis, cell surface expression, or coupling to G-protein(19) . Glycosylation of the beta (2) -adrenergic receptor is also not required for high affinity binding, but non-glycosylated mutants exhibited a decrease of 50% in cell surface expression(37) . The follitropin receptor also contains several N-linked carbohydrates that are not required for high-affinity hormone binding(18) .

The majority of G-protein-coupled receptors contain glycosylation sites in their amino-terminal extracellular sequences, including PAF receptors from species other than human. The unusual position of the glycosylation site in the second extracellular loop of the human PAF receptor, in addition to preliminary studies relating to pneumococcal binding prompted the studies described here. Our data indicate a role for glycosylation in transport of the PAF receptor to the cell surface relatively independent of its position on the protein. Several other receptors of this class are reported that naturally contain no glycosylation sequences, including the dog C5a receptor(38) , several species of alpha (2) b-adrenergic receptor(39) , and a number of orphans. The relative expression levels for these molecules is not known, but, particularly in the case of the orphans, the absence of glycosylation may lead to low expression at the cell surface, making traditional ligand studies difficult.

FOOTNOTES

*: This work was supported in part by National Institues of Health Grants HL36162, HL41587 (to N. P. G.), AI23459 and AI27913 (to E. I. T.), a fellowship from Ministerio de Educación y Ciencia, Spain (to C. G. R.), a Norman and Rosita Winston Fellowship Award (to D. R. C.), and by Pfizer Central Research, Groton, CT (to N. P. G. and C. G.). 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.
§: To whom correspondence and reprint requests should be addressed: Dept. of Medicine, Beth Israel Hospital, 330 Brookline Ave., Boston, MA 02215. Tel.: 617-355-6737; Fax: 617-730-0422.
(): The abbreviations used are: PAF, platelet-activating factor; BSA, bovine serum albumin; IP, inositol phosphate; PLC, phospholipase C; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; FITC, fluorescein isothiocyanate; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis.
(): N. P. Gerard and C. Gerard, unpublished observations.

ACKNOWLEDGEMENTS

We thank Dr. J. Bischoff (Children's Hospital, Boston, MA) and Dr. Gonzalez Cabrero (Dana Farber Cancer Center, Boston, MA) for helpful discussions. Carole de Dios provided excellent technical assistance.

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