(Received for publication, May 17, 1995; and in revised form, June 28, 1995)
From the
The contribution of N-linked glycosylation to the ligand binding activity of the rat luteinizing hormone receptor (LHR) was studied in wild-type and mutant LHR expressed in mammalian (COS1) cells and overexpressed in insect (Sf9) cells. The binding affinities of the holoreceptor and its truncated splice variant (form B) lacking the transmembrane domain were equivalent in both cell lines. Tunicamycin-treated transfected Sf9 cells expressed a carbohydrate-free LH receptor that lacked hormone binding activity. Functional carbohydrate chains essential for binding activity were localized to glycosylation sites at Asn-173 and Asn-152. Glycosidase treatment of the double mutant N173Q/N152Q revealed the presence of at least one additional carbohydrate chain at Asn-269, Asn-277, or Asn-291 that does not contribute to hormone binding. Asn-77 was not glycosylated, but its mutation to Gln reduced hormone binding. LHR expressed in insect cells contained only high mannose carbohydrate chains, and those located at Asn-173 and Asn-152 were sufficient for high-affinity hormone binding. Enzymatic cleavage of glycosyl chains indicated that only the proximal N-acetylglucosamine residue, which is common to high mannose and complex carbohydrate forms, is necessary for acquisition of the high affinity conformation of the receptor. The carbohydrate chains of the LHR appear to be involved in intramolecular folding of the nascent receptor rather than in its interaction with the hormone.
The LHR ()is a glycoprotein present in the cell
membrane of gonadal cells, with six potential N-linked
glycosylation sites (Fig. 1) and N-linked carbohydrate
(CHO) chains of the complex type (1, 2, 3) .
A functional role for N-linked carbohydrates in high affinity
hormone binding has not yet been established, and is a subject of some
controversy. Conflicting reports on the effects of deglycosylation (2, 4) and site-directed mutagenesis (5, 6) of the mature holoreceptor on hormone binding
activity may be a function of the original receptor state. N-Linked carbohydrate chains of the rat ovarian LH receptor
have previously been shown to be essential for high affinity hormone
binding by a number of different methods, including site-directed
mutagenesis of the Asn of the putative glycosylation sites(5) ,
tunicamycin treatment(7) , and enzymatic deglycosylation of
solubilized receptors(2) . These procedures have not resolved
the question of whether the reported carbohydrate requirement of the
receptor is based on a direct interaction with the hormone or is
related to the conformation of the ligand binding site.
Figure 1:
The
rat LH receptor. The amino acid sequence of the rat LH receptor (3) is shown. Amino acids corresponding to the cleavable signal
peptide are within hexagons, and those of the mature peptide
are within circles. The rat LHR form B sequence diverges from
holoreceptor at 294 (I) and continues with LLHGALPATHCLS
peptide tail(8) . Putative glycosylation sites are indicated in
the extracellular domain at positions 77, 152, 173, 269, 277, and 291.
Glycosylation sites/glycosyl chains that are important for hormone
binding at amino acid positions 152 and 173 (), nonglycosylated
site at position 77 (
). Nonfunctional sites (one or more
glycosylated) are at amino acid positions 269, 277, and 291
(
).
In order to
study the importance of post-translational glycosylation on LHR binding
activity, we have evaluated both the membrane-bound LHR holoreceptor
(form A) (3) and its high-affinity hormone-binding splice
variant, form B, which lacks the transmembrane and cytoplasmic domains (8) . These receptors and their mutated forms were expressed in Baculovirus-infected insect cells and in the mammalian COS-1
cells. It is noteworthy that glycoproteins in insect cells have been
shown to carry N-linked carbohydrates of the high mannose type
that are trimmed to shorter core structures
(ManGlcNAc
)(9) . In contrast, the
mammalian cell, which has been shown to express high-affinity
LHR(8) , carries a full complement of trimming and complex
glycosidases in its post-translational machinery(2) . The
present experiments demonstrate a post-translational glycosylation
requirement that is satisfied by high mannose carbohydrate chains.
Previous studies have shown that mutation of only three of the six Asn
of the LHR putative glycosylation sites (Asn-77, Asn-152, and Asn-173)
reduced hormone binding activity, and this could not be attributed to
changes in LHR transport to the cell surface(5) . In this
study, we demonstrate by direct substitution of Asn and Ser/Thr of
these putative glycosylation sites that only Asn-152 and Asn-173 carry
carbohydrate chains that are required for hormone binding activity. In
addition, the importance of the proximal N-acetylglucosamine
of the carbohydrate chain for the high affinity conformation of the LHR
was demonstrated.
Sf9 Cells- 48 h after transfection with WT or mutant LHR cDNAs, Sf9 cells were homogenized with 50 strokes at 1800 rpm of a tissue grinder (Potter-Elvehjem with PTFE pestle, Thomas Scientific, Swedesboro, NJ); glycerol and Nonidet P-40 were added to final concentrations of 20 and 1%, respectively. Extract was rotated for 1 h at 4 °C, and diluted to a final Nonidet P-40 concentration of 0.1%. The solubilized mixture was centrifuged, and protein concentration was determined as described above.
Figure 2:
Radioimmunoassay of LHR. Dose-related
displacement of I-LHR-(36-51) peptide binding to
LHR-(36-51) peptide antibody by unlabeled LHR-(36-51)
peptide (standard curve), by extracts of ovarian membranes (OM) or COS1 and insect cells transiently transfected with
wild-type form A and B of LHR. The extracts of samples containing LHR
displayed parallel displacement to the standard curve. No displacement
was observed by extracts from cells infected or transfected with vector
in the absence of LHR insert.
The wild-type LHR soluble splice variant form B ((8) , Fig. 1) that is overexpressed in insect cells at levels of 107 ± 3 pmol/mg of protein, shows similar high hCG hormone binding affinities when expressed from insect or COS1 (8) cells (Table 1). Form B LHR expressed in insect cells was utilized in a number of subsequent experiments as a simplified prototype of the high affinity LHR form A hormone binding domain.
Figure 3:
Immuno and Ligand blots of form B LHR
expressed from insect cells in the presence or absence of tunicamycin. Left, immunoblots, form B LHR in sample buffer was reduced and
denatured and resolved on SDS-PAGE. After transfer to nitrocellulose
membrane, form B LHR protein was probed with the LHR peptide
(36-51) antisera. Lanea,negative control,
cells transfected with vector without insert. Cells transfected with
LHR form B tunicamycin treated (b) and untreated (c). Right, ligand blot of form B LHR binding to receptors
(nonreduced/nondenatured) resolved on SDS-PAGE and transferred to
nitrocellulose. The blot was incubated with I-hCG in the
presence or absence of excess unlabeled hCG. Lanes are defined by form
B LHR transfection or vector control (±), tunicamycin treatment
(±), and excess cold hCG (±).
Figure 4:
Displacement curves of I-hCG binding to wild-type and mutant LHR form A
expressed in COS1 cells. Leftpanel, hCG binding to
surface receptors on intact cells. Rightpanel, hCG
binding to total detergent solubilized receptors (detergent-solubilized
particulate fraction = total cell receptor, no binding was
observed in cytosol fraction).
Figure 5:
Wild-type and mutant receptors expressed
in insect cells. Displacement curves of I-hCG binding to
wild-type and mutant LHR form B receptors expressed in insect cells- Left, form A holoreceptor; right, form B truncated
receptor.
The equivalent changes in hormone binding activity
between the Asn-173 and Thr-175 mutant form B LHRs indicate that the
deviation in hormone binding activity could be related to the absence
of a carbohydrate chain. Immunoblots of the mutant Asn-173 Gln
or Thr-175
Ala show equivalent reductions in apparent molecular
mass of about 2 kDa from the wild-type molecular mass (37.8 ±
0.9 kDa (n = 10) to 35.5 ± 0.3 kDa (n = 7)), indicating that position 173 is glycosylated (Fig. 6, left, lanes1, 4,
and 5). Ligand blots of the Asn-173
Gln and Thr-175
Ala mutant LHRs confirm that these receptors cannot bind hCG (Fig. 6, right (A), lanes2 and 3). These studies demonstrate that the glycosylation
position at Asn-173 is of major importance to hormone binding activity
in both the mammalian and insect cell, and serves a basic hormone
binding function that does not involve transport or protein stability.
Figure 6:
Immunoblots and ligand blots of wild-type
and mutant form B LHR receptors expressed in insect cells.
Nitrocellulose blots of LHR samples resolved on SDS-gels were probed
with LHR- (36-51) peptide antibody to visualize LHR protein (left) or I-hCG to detect hormone binding (right). Control (lane4A) blots
are incubated in presence of 1 µg/ml hCG (unlabeled). Complete
displacement of bound tracer by unlabeled hormone indicates specificity
of hormone binding.
Since tunicamycin treatment reduced the molecular mass of the form B
LHR expressed from the insect cell by approximately 6 kDa, and mutation
of Asn-173 decreased the molecular weight of form B LHR by only 2 kDa,
additional glycosylation positions that carry carbohydrate chains on
the form B LHR were predicted to exist. Our previous studies showed
that mutation of the putative glycosylation position at Asn-152
significantly reduced hormone binding activity close to nonspecific
levels(5) . Immunoblots of the form B Asn-152 Gln and
Ser-154
Ala LHR mutant proteins showed similar reductions in
molecular mass of approximately 2 kDa in comparison with the wild-type
form B LHR of 38 kDa, indicating that the glycosylation position at
Asn-152 also carries a carbohydrate chain (Fig. 6, left, lanes1, 2, and 3).
Mutation of the Asn-152 in the form B LHR, expressed from insect cells,
resulted in a total loss of hormone binding activity (Table 2),
but mutation of Ser-154 to Ala exhibited only a 60% reduction in
hormone binding activity (Table 2). These results were verified
in ligand blots where the Asn-152
Gln mutant exhibited no hCG
binding (Fig. 6B, lane2), whereas,
the Ser-154
Ala mutant exhibited reduced but visible binding (Fig. 6B, lane3). Scatchard analyses
and radioimmunoassay of mutant and WT receptors indicates that the
reduction of S154A binding activity is due to an increase in the number
of inactive nonbinding receptors rather than a change in binding
affinity (not shown) or expression (Table 2). The discrepancy
between the total loss of activity by Asn-152
Gln and the 60%
reduction in hormone binding activity by Ser-154
Ala may
represent a negative effect caused by the introduction of Gln, or more
likely, an enhancement in activity caused by the substitution of
Ser-154 for Ala. In either instance, the amino acid Asn-152 should not
by itself contribute to binding activity in the wild-type receptor
since Asn-152 is linked to a carbohydrate chain in vivo ( Fig. 6and Fig. 7, toppanel) and would
be sterically blocked from hormone or receptor interaction.
Figure 7:
Top panel, Western blots of
wild-type and mutant form B LHR, and of WT LHR B after treatment with
glycosidases. Immunoblots of wild-type and mutant LHR expressed in
insect cells pretreated with mannose-specific endoglycosidase H or N-glycanase. Single mutations, N152Q or N173Q; double
mutation, N152Q/N173Q. Bottom panel, Western blot of wild-type
receptor and mutant form B LHR. Immunoblots of wild-type and mutant LHR
(N77Q, S79A, N152Q) expressed in insect cells. Mutation of amino acids
at the putative glycosylation site Asn-Xaa-Ser
showed no difference in M
from WT. The N152Q
was placed in this gel for comparison of amino acid mutation that
yields removal of an N-linked glycosyl chain with the expected
reduction of the molecular weight.
Mutation of the glycosylation position at Asn-77 of the LHR expressed from insect cells gave a 50% reduction in hormone binding activity, with no reduction in receptor protein levels (Table 2, Fig. 7, bottompanel). However, the change in hormone binding activity was not correlated with a reduction in the molecular mass of the mutant protein (Fig. 7, bottompanel) and does not appear to be associated with loss of a carbohydrate chain. Similarly, mutation of Ser-79 to Ala did not result in a reduction in LHR molecular weight (Fig. 7, bottom panel). Thus, only the glycosylation positions at Asn-152 and Asn-173 carry functional carbohydrate chains.
Immunoblot analysis of the double mutant Asn-152/Asn-173 in comparison with the deglycosylated N-glycanase-treated LHR indicates that additional carbohydrate chains at other glycosylation positions (269, 277, and 291) are present on the LHR. The double mutant exhibited an apparent molecular mass of 2 kDa greater than the deglycosylated LHR (Fig. 7, top panel). Individual mutation of each of the three remaining glycosylation positions at 269, 277, and 291 was previously shown to produce high affinity hormone binding receptor that was capable of transport to the surface of COS1 cells(5) . Clearly, putative carbohydrate chains at these positions are either not involved in hormone binding or can be substituted by carbohydrate chains at other glycosylation positions.
Figure 8:
Effect of deglycosylation on wild-type
form B LHR by N-glycanase, Endo H, and Endo F on receptor
binding. Following pretreatment of detergent extracts of form B LHR
expressed in insect cells with glycosidases, the samples were subjected
to SDS-PAGE immunoblot and I-hCG ligand blot analysis as
described under ``Materials and Methods.'' Toppanel, samples were not treated with SDS or reductant
prior to deglycosylation; bottompanel, samples were
denatured and reduced prior to enzyme treatment and electroblotting
(see ``Materials and Methods''). Nontreated, control
wild-type receptor nontreated with
glycosidases.
Fig. S1indicates that mannose residues are not required for renaturation of the mature denatured receptor and that any high mannose function occurs prior to the formation of the processed receptor. In contrast, N-glycanase treatment completely abolished ligand binding, although the receptor was visualized by antibody, and deglycosylated to 32 kDa (Fig. 8, top panel, bottom panellane3). These experiments indicate that the proximal N-acetylglucosamine linked to Asn, which is retained on the Endo H- or Endo F-treated LHR but not the N-glycanase-treated LHR, is essential for the renaturation of the high affinity hormone binding receptor following electrophoresis on an SDS-denaturing gel.
Figure S1:
Scheme 1Glycosidase specificity of
generic high mannose chain. x, y, mannose residues; z, fucose or H; ,
, cleavage site(16) . PNGase F = N-glycanase.
These studies have demonstrated that N-linked
carbohydrate chains, specifically those attached to the two
glycosylation sites Asn-173 and Asn-152, are essential for the assembly
of a high affinity hormone binding site within the extracellular domain
of the LH receptor. Mutation of either Asn-173 or Thr-175 within the
glycosylation consensus sequence Asn-Xaa-Thr in the form A or form B
LHR proteins abolished binding activity (Table 2). This loss of
activity was correlated with the loss of a carbohydrate chain in
immunoblots in which the mutant LHR proteins showed equivalent
reductions in molecular mass of approximately 2 kDa (Fig. 6).
The carbohydrate chain at Asn-173 is essential for binding activity,
but it is not the only carbohydrate chain required for production of
the high affinity hormone binding site. Significant reductions in
receptor size and hormone binding activity were also obtained with
mutations of Asn-152 and Ser-154, even in the presence of the
glycosylation position at Asn-173 (Table 2). The reductions in
molecular weight of the Asn-152 Gln and Ser-154
Ala
mutant LHRs were equivalent, indicating the disappearance of a
carbohydrate chain. However, the reduction in hormone binding activity
was much greater with the Asn-152
Gln LHR than the Ser-154
Ala LHR (Table 2), indicating a negative amino acid effect
by the substituted Gln-152 or a positive amino acid effect by the
substituted Ala-154. Since Asn-152 is glycosylated, any negative effect
by the introduction of Gln has no relevance in determining the
intrinsic importance of this Asn to hormone binding. The putative
glycosylation position at Asn-77 does not appear to be glycosylated (Fig. 7, bottom panel), and changes in binding activity
must be attributable to either the loss of the native amino acid or the
introduction of a foreign amino acid. Asn-77 is within a leucine heptad
zipper motif (Leu-75, Leu-82, Leu-89), and theoretically a putative CHO
chain at 77 would interfere with the amphiphilic helix. Therefore, it
is not unexpected that Asn-77 is not glycosylated. Single mutations of
each of the six putative glycosylation sites of the LHR established
that the putative glycosylation positions Asn-269, Asn-277, and Asn-291
did not contribute to either hormone binding activity or membrane
transport and insertion(5) . However, the present studies
indicate that at least one of them carries a carbohydrate chain (Fig. 8, top panel).
The importance of Asn-173
glycosylation demonstrated in the present study is of particular
relevance because other reports have described retention of hormone
binding activity with the Thr-175 Ala mutant
protein(6) , and attributed the loss of activity by Asn-173
Gln LHR to the substitution of Asn-173 for Gln, rather than the
loss of a carbohydrate chain. However, in our experiments, no hormone
binding activity was observed after tunicamycin treatment, where the
expressed LHR retained the native amino acids Asn-173 and Asn-152
without the carbohydrate chains (Fig. 3), confirming the
importance of the N-linked carbohydrate chain rather than the
Asn-173 for LHR activity.
Our studies demonstrate that high mannose carbohydrate chains are sufficient for the formation of the high affinity hormone binding domain of the form B LHR expressed from insect cells. In contrast, the carbohydrate chains of the high affinity rat ovarian LHR are not high mannose but are rather of the complex type(2) . This suggests that either carbohydrate residues common to both the high mannose and complex chains are central to the formation of the hormone binding domain, or that carbohydrate function occurs in the mammalian cell prior to processing of the high mannose chains to the complex form, as has been reported with the mannose 6-phosphate receptor(17) .
Monoglucose high mannose chains have been implicated in folding of nascent proteins (18) as well as with the chaperone protein calnexin interaction (19) prior to entry into the Golgi. However, the ability of the denatured mature LHR to refold spontaneously during electrophoretic transfer to nitrocellulose in ligand blots ((2) , Fig. 6)) indicates that chaperone proteins and processing enzymes in the endoplasmic reticulum are not necessary for the formation of the hormone binding domain. The high mannose (Fig. 6) or complex carbohydrates (2) of the mature LHR are equally capable of refolding the receptor protein. Total elimination of the N-linked CHO chain by mutation of the Asn-173 glycosylation site, tunicamycin treatment, or N-glycanase treatment yields mature receptors that exhibit no hormone binding activity and are incapable of renaturation following electrophoresis (Fig. 3, Fig. 6, and Fig. 8). In the purified mature ovarian LHR, renaturation was prevented by deglycosylation with N-glycanase in one study(2) , although other studies with the membrane-bound and partially purified LHR indicate that deglycosylation of the mammalian LHR does not impair hormone binding (4, 20) . None of these studies performed the deglycosylation reaction on the SDS-denatured and reduced LH receptor, suggesting that the discrepancy may have arisen from incomplete deglycosylation. This was also suggested by reported differences in the molecular masses of the deglycosylated receptor (59 versus 62 kDa, (2) and (20) ).
In other studies, ligand blotting of nonglycosylated receptor expressed from Escherichia coli showed only hormone bound to high molecular weight aggregates, and none bound to the monomeric form of the truncated LHR-(1-294) under nonreducing conditions(21) . In the present investigation, wild-type form B LHR expressed from insect cell was capable of ligand binding to electroblotted receptor under reducing (Fig. 8) as well as nonreducing conditions ( Fig. 6and Fig. 8). N-Glycanase treatment of the SDS-denatured and reduced high mannose form B LHR yielded a nonbinding receptor (Fig. 8). Denaturation and reduction per se did not impair the refolding process, since Endo H treatment of the denatured LHR did not abolish hormone binding (Fig. 8). These experiments suggest an important role for the proximal N-acetylglucosamine residue attached to the Asn of the glycosylation site that is present in both complex and high mannose chains, in refolding of the mature receptor and perhaps folding of the nascent receptor. This was confirmed with Endo F-treated receptor even under nondenaturing/nonreducing conditions. These experiments do not preclude the possibility that mannose residues are important in the initial processing of LHR expressed from the insect or mammalian cell.
In summary, our studies indicate that putative glycosylation positions Asn-77, Asn-152, and Asn-173 are relevant to specific functions that lead to the formation of an active hormone binding domain. Of these, only Asn-152 and Asn-173 carry carbohydrate chains, and the nonglycosylated Asn-152 and Asn-173 LHR expressed from tunicamycin-treated cells exhibits no hormone binding activity (Fig. 3). The Asn-173/Thr-175 glycosyl chain is essential for acquisition of hormone binding activity. We have established that complex carbohydrate chains are not required for hormone binding in the insect cell and that any carbohydrate function may permit folding of the nascent protein to a high affinity conformation. In addition, the contribution of the proximal N-acetylglucosamine to renaturation of the high affinity binding site of the LHR was clearly established by selective cleavage of the carbohydrate chain.