(Received for publication, July 10, 1995; and in revised form, August 14, 1995)
From the
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 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.
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
, 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
-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.
Receptors containing consensus N-linked glycosylation sites in the NH-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.
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
, 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.
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.
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.
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).
Immunochemical analysis indicates the mutant with a single
glycosylation site in the NH-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-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 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
-terminal
sequence.
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-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
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
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 LEPNDSS
, and the absence of
glycosylation may result from the proline residue just preceding the
asparagine, and/or the flanking acidic amino acids.
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.
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
-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-terminal sequence was modified to that of the guinea pig
receptor, L
ELNSSS
(Nt/dCHO), the resulting
protein was both glycosylated and expressed on the cell surface (Fig. 3Fig. 4Fig. 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
EPNDSS
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
-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 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.