Functional glycosylation sites of the rat luteinizing hormone receptor required for ligand binding.

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 1 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.
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 highaffinity hormone-binding splice variant, form B, which lacks the transmembrane and cytoplasmic domains (8). These receptors and their mutated forms were expressed in Baculovirusinfected 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 (Man 3 GlcNAc 2 ) (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.

Design of LHR Form A and B Wild-type and Mutant DNA Constructs
The full-length rat ovarian LH/CG receptor (LHR form A) cDNA and the alternate splice variant form B cDNA (lacking the first 266-bp of exon 11) were subcloned into the human cytomegalovirus promoterdriven expression vector, pCMV4 (a gift from Dr. D. Russell, University of Texas Southern Medical Center) as described previously (8). LHR mutants were constructed using the recombinant circle polymerase chain reaction (10). Synthetic oligonucleotides (Midland Certified Reagent Co., Midland, Texas) were designed to replace Asn-77, Asn-152, or Asn-173 with Gln and Ser-79, Ser-154, or Thr-175 with Ala. WT and mutant form A cDNAs were subcloned into the SmaI site of baculovirus transfer vector pVL1392, and form B was subcloned into the EcoRI site of pVL1393 (Pharmingen, San Diego, CA), for infection of insect cells. The orientation of the WT and mutant form A and form B constructs was determined by restriction enzyme analysis, and mutations were verified by DNA sequencing.

Expression of WT and Mutant LHR in Mammalian COS1 and Insect Cells
WT and mutant LHR cDNAs in pCMV4 expression vectors were transfected in mammalian COS1 cells by the lipofectAMINE (Life Tech-* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ‡ To whom correspondence should be addressed. Tel.: 301-496-2021; Fax: 301-480-8010. 1 The abbreviations used are: LHR, luteinizing hormone receptor; CHO, carbohydrate; WT, wild-type; hCG, human chorionic gonadotropin; PAGE, polyacrylamide gel electrophoresis; Endo H, endo-␤-Nacetylglucosaminidase H; N-glycanase, peptide-N-glycosidase F; Endo F, endo-␤-N-acetylglucosaminidase F; LH/CG, luteinizing hormone/chorionic gonadotropin. nologies, Inc.) method according to manufacturer's protocol. The expressed membrane-bound receptor activity was determined by 125 I-hCG binding 48 h after transfection (5). 125 I-hCG (specific activity, 34.2 Ci/g, 37% maximal bindability) was prepared by a modification of a lactoperoxidase method as described previously (11). Spodoptera frugipera (Sf9) cells (Pharmingen) were co-transfected with 4 g of WT or mutant LHR form A (pVL1392LHRA) or form B (pVL1393LHRB) and 0.5 g of BaculoGold DNA (Pharmingen) according to manufacturer's protocol. The recombinant virus titers were amplified by reinfection of Sf9 cells, and titers were monitored by plaque assay. The overexpression of WT and mutant LHRs was achieved by infecting Sf9 cells (7 ϫ 10 6 cells/75 cm 2 flask) with recombinant virus (10 8 plaque-forming units/ml) (multiplicity of infection ϭ 10) at 27°C for 2 h. At 48 h postinfection, the cells were harvested, washed, and homogenized in phosphate-buffered saline containing 1 mM phenylmethylsulfonyl fluoride, 5 g/ml leupeptin, and 24 trypsin inhibitor units of Trasylol (aprotinin, Sigma). The cell homogenates in 20% glycerol were stored at Ϫ70°C for further studies. When indicated, tunicamycin B2 (Boehringer-Mannheim) 100 ng/ml was added to the cell cultures at the time of infection with recombinant wild-type LHR form B in Baculovirus and incubated for 48 h. Cells were harvested as described above.

Preparation of Detergent-solubilized Extracts of Transfected COS1 Cells and Sf9 Cells
COS1 Cells-48 h after transfection with WT or mutant LHR cDNAs, COS1 cells were recovered, homogenized, and solubilized as described previously (8). Protein concentration was determined by BCA protein assay (Pierce).
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.

I-hCG binding to LHR
Intact COS1 cells or solubilized extracts from transfected COS1 or insect Sf9 cells were used for 125 I-hCG binding assay as described previously (8). Binding was performed using displacement assays by incubating intact cells or solubilized extracts with 125 I-hCG and increasing concentration of unlabeled hCG. Nonspecific binding was determined in samples containing an excess of the unlabeled hCG (1 g). The binding parameters (K d and binding capacities [Q]) were analyzed from Scatchard plots and saturation curves using a nonlinear model curve fitting program (12). All experiments were performed at least 3 times in triplicate. Statistical analysis for all studies were performed using analysis of variance (Supernova, Berkeley, CA) and Post Hoc Fisher LSD tests at the 99% significance level.

Enzymatic Deglycosylation of WT and Mutant LH Receptor
Using
Nonreducing, Nondenaturing Conditions-Reaction mixture and incubation were same as above without SDS or ␤-mercaptoethanol in the buffer, and samples were not boiled prior to digestion.

Western Blot Analysis of Wild-type and Mutant LH/CG Receptor
25 g of protein from detergent-solubilized Sf9 cells transfected with WT and mutant LHR form B cDNAs or from enzyme deglycosylation reactions were boiled for 5 min in sample buffer (final concentration, 10% glycerol, 2% SDS, 2% ␤-mercaptoethanol, 62.5 mM Tris-HCl, pH 6.8, 0.02% bromphenol blue) and subjected to 10% SDS-PAGE electrophoresis analysis under reducing conditions. Following transfer to nitrocellulose membrane (Bio-Rad), the LH/CG receptor was probed with 1:800 dilution of immunoaffinity purified rabbit polyclonal antibody raised against an peptide antigen (amino acid 36 -51) of the rat LH/CG receptor for 1 h at 22°C and washed 3 times with phosphate-buffered saline, 0.1% Tween 20. Subsequently, the membranes were incubated with goat anti-rabbit antiserum conjugated to horseradish peroxidase (Life Technologies, Inc.) (1:1000 dilution) for 1 h at 22°C. Membranes was washed and developed with the enhanced chemiluminescence detection kit (ECL, Amersham Corp.). Negative controls are Sf9 cells transfected with pVL1393 vector without LH/CG receptor form B cDNA.
Ligand Blot 100 g of protein from cell extracts or enzymatic digestions were added to sample buffer, electrophoresed on 10% SDS-PAGE, and transferred electrophoretically to nitrocellulose. Binding of hCG to WT and mutant receptor was performed by incubation of the blots with 125 I-hCG (10 6 cpm/ml) for 16 h at 4°C in the absence or presence of excess unlabeled hCG (1 g/ml). The blots were processed and autoradiographed as described previously (13).

Radioimmunoassay
Detergent solubilized extracts of Sf9 cells transfected with wild-type or mutant LH receptor form B cDNAs were quantitated by radioimmunoassay using affinity purified polyclonal rabbit antisera raised against rat LH receptor peptide antigen 36 -51. ϳ1-25 g of crude extracts from Sf9 cell were suspended in 100 l of radioimmunoassay buffer (50 mM sodium phosphate, pH 7.4, 12 mM EDTA, 0.02% NaN 3 , 0.1% Nonidet P-40, 0.1% bovine serum albumin (fraction V, Sigma) and 24 trypsin inhibitor units of Trasylol (aprotinin, Sigma)) and incubated with 100 l of LH receptor-(36 -51) antibody (final dilution 1:3000), 200 l of radioimmunoassay buffer and 50,000 disintegrations/min of 125 I-labeled peptide-(36 -51) (specific activity of 234 Ci/mmol) for 24 h at 4°C. Separation of antibody-bound from free peptide was performed as described previously (14). Sample values were derived from reference standard curves (ID 50 of displacement of 0.20 pmol/assay tube). Assays were performed in triplicate with at least three dilutions. The intraassay coefficient of variation was 5%, and the interassay coefficient of variation was 8%. Expressed wild-type and mutant LHR concentrations from COS1 and insect cells displayed parallelism to the standard curve, while samples from cells transfected with the expression vector only showed no displacement (Fig. 2). Wild-type form A LHR protein was expressed in the COS1 cell at levels of 0.46 Ϯ 0.01 pmol/mg of protein, whereas wild-type form A LHR was expressed at 60.3 Ϯ 1.0 pmol/mg of protein in the insect cell. Wild-type form B was expressed in the insect cell at levels of 107 Ϯ 3 pmol/mg of protein.

LHR Expression from Mammalian COS1 and Insect Cell
Lines-Transfection of the membrane-bound LH holoreceptor (form A) into either the mammalian COS1 or the baculovirusinfected insect Sf9 cell line results in the expression of high affinity receptors. Scatchard analysis of the wild-type receptors expressed in COS1 cells (Ref. 8 and Table I) and in insect cells revealed that their gonadotropin binding affinity (K a ) were equivalent in both cell lines but somewhat lower than that observed in particulate ovarian membrane fractions (Table I) (15). Hormone binding activity was detectable on the surface of the intact COS1 cell, while in the insect cell, LHR holoreceptor binding activity was only recovered following detergent solubilization (Table I). Overexpression of the form A LHR was achieved in the insect cell where wild-type form A LHR was expressed in the insect cell at levels of 129-fold greater than that observed in the transfected COS1 cell and 30-fold greater than that found in ovarian membranes (Fig. 2).
The wild-type LHR soluble splice variant form B (Ref. 8, Fig.  1) that is overexpressed in insect cells at levels of 107 Ϯ 3 pmol/mg of protein, shows similar high hCG hormone binding affinities when expressed from insect or COS1 (8) cells (Table  I). 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.
Tunicamycin Treatment of Wild-type LHR form B-To investigate the role of N-linked glycosylation in the synthesis of the high affinity LHR, form B-infected insect cells expressing the spliced variant were treated with tunicamycin, which prevents synthesis of dolichyl N-acetylglucosamine pyrophosphate, the initial step in N-linked glycosylation in the endoplasmic reticulum. In western immunoblots, anti-LHR-(36 -51) antibodyspecifically recognized the nonglycosylated LHR expressed from tunicamycin-treated cells (Fig. 3, left, lanes a and b). The nonglycosylated form B LHR exhibits a reduction in molecular weight of approximately 6 kDa in comparison with the wildtype LHR of 37.8 Ϯ 0.9 kDa (n ϭ 10) (Fig. 3, left, lanes b and c), corresponding to the absence of N-linked carbohydrate chains. Ligand blots showing the binding of labeled hCG to the form B LHR reveal that the tunicamycin-treated receptor does not bind labeled hCG (Fig. 3, right, lane b), whereas the wild-type form B LHR does bind the labeled hormone (Fig. 3, right, lane  a), and this binding is abolished in the presence of excess cold hCG (Fig. 3, right, lane d). Thus, ligand and Western blots show that tunicamycin treatment abolishes LHR hormone binding, without reducing LHR protein levels (Fig. 3, left, lanes  b and c). Neither tunicamycin treatment, nor the absence of N-linked carbohydrates affected steady state levels of the receptor molecule in comparison with untreated controls. This experiment demonstrates that synthesis of the high affinity hormone binding domain in the LH receptor requires the posttranslational addition of N-linked carbohydrate chains.
Identification of Glycosylation Sites on the LHR-Initial studies indicated that three of the six potential glycosylation sites on the LHR are of functional importance to hormone binding activity (Asn-77, Asn-152, and Asn-173) ( Fig. 1 and Ref. 5). The relevance of these reductions in binding activity to the elimination of carbohydrate chains at these glycosylation positions was investigated by mutation of the Asn or Ser/Thr of the consensus site Asn-Xaa-Ser/Thr, and SDS-gel electrophoresis. Mutation of either the Asn or Ser/Thr position should result in similar changes in hormone binding activity if the CHO chain is the only factor in binding activity (see below). Substitution of either Asn-173 to Gln or Thr-175 to Ala in the form A holoreceptor abolished hormone binding activity when measured from the surface of intact COS1 cells (Fig. 4, left). This loss of activity could not be attributed to deviations in transport to the cell surface since the same effect was noted in soluble fractions prepared from COS1 cells (Fig. 4, right). Similarly, in the insect cell, the detergent extracted form A and form B LHR mutants Asn-173 3 Gln and Thr-175 3 Ala exhibited no detectable hormone binding activity (Fig. 5). To assess whether the loss of hormone binding activity of Asn-173 3 Gln and Thr-175 3 Ala was related to defects in protein synthesis, radioimmunoassay analysis of expressed wild-type and mutant LHR protein was performed, and protein levels of the Asn-173 3 Gln and Thr-175 3 Ala mutants were not significantly different from wild-type. Thus, loss of binding activity of the mutant LHRs was solely attributed to defects in hormone binding rather than reduced protein levels (Table II).
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 3 Gln or Thr-175 3 Ala show equivalent reductions in apparent molecular mass of about 2 kDa from the wild-type molecular mass (37.8 Ϯ 0.9 kDa (n ϭ 10) to 35.5 Ϯ 0.3 kDa (n ϭ 7)), indicating that position 173 is glycosylated (Fig.  6, left, lanes 1, 4, and 5). Ligand blots of the Asn-173 3 Gln and Thr-175 3 Ala mutant LHRs confirm that these receptors cannot bind hCG (Fig. 6, right (A), lanes 2 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. 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 3 Gln and Ser-154 3 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, lanes 1, 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 II), but mutation of Ser-154 to Ala exhibited only a 60% reduction in hormone binding activity (Table II). These results were verified in ligand blots where the Asn-152 3 Gln mutant exhibited no hCG binding (Fig. 6B, lane 2), whereas, the Ser-154 3 Ala mutant exhibited reduced but visible binding (Fig. 6B, lane 3). 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 recep-

FIG. 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 125 I-hCG to detect hormone binding (right). Control (lane 4A) blots are incubated in presence of 1 g/ml hCG (unlabeled). Complete displacement of bound tracer by unlabeled hormone indicates specificity of hormone binding. tors rather than a change in binding affinity (not shown) or expression (Table II). The discrepancy between the total loss of activity by Asn-152 3 Gln and the 60% reduction in hormone binding activity by Ser-154 3 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 (Figs. 6 and 7, top panel) and would be sterically blocked from hormone or receptor interaction.
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 II, Fig. 7, bottom panel). However, the change in hormone binding activity was not correlated with a reduction in the molecular mass of the mutant protein (Fig. 7, bottom panel) 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.
Carbohydrate Composition of Essential Carbohydrate Chains-Localization of the carbohydrate requirement to Asn-173, and Asn-152 in the LHR expressed from either mammalian COS1 or the insect cell suggests that a specific complex carbohydrate composition in the mammalian cell may not be essential, since the insect cell lacks the complex glycosidases of the mammalian cell (9). To determine whether the LHR expressed from the insect cell carries only high mannose chains, the receptor was treated with the glycosidase Endo H that cleaves between the two core N-acetylglucosamines of a high mannose chain, and the reduction in molecular weight was compared with LHR digestion by N-glycanase that cleaves both high mannose and complex carbohydrate chains at the amino acid Asn. The Endo H-and N-glycanase-digested LHR migrated to identical positions on an SDS gel (31.5 Ϯ 0.4 kDa n ϭ 9, 31.5 Ϯ 0.5 kDa, n ϭ 6, respectively) when visualized by antibody in an immunoblot (Fig. 7, top panel). The contribution of the proximal N-acetylglucosamine that remains after Endo H treatment was not visible on an SDS gel. The wild-type high affinity receptor expressed from the insect cell is glycosylated only with high mannose chains.
Effect of Enzymatic Deglycosylation on Hormone Binding of Form B-To determine if the high mannose carbohydrate chains of the receptor expressed from the insect cell functions exclusively in the initial folding of the nascent receptor, or also in the renaturation of the detergent solubilized processed receptor, we studied whether or not the deglycosylated soluble receptor was able to renature to an active hormone binding configuration following SDS-gel electrophoresis in ligand blots (Fig. 8). Treatment of the wild-type form B LHR with Endo H under SDS denaturing and reducing conditions (see "Materials and Methods"), followed by SDS-gel electrophoresis and 125 I-hCG ligand blotting, resulted in a decrease in the receptor molecular mass of 6 kDa and renaturation of the LHR to a high affinity binding conformation (Fig. 8). Similar results were obtained with Endo F, which cleaves at the same position as Endo H, giving a molecular mass of 31.6 Ϯ 0.2 (n ϭ 4) under reducing, denaturing, or nonreducing, nondenaturing conditions (Fig. 8).
Scheme 1 indicates 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 panel lane 3). 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. DISCUSSION 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 II). 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  (Table II). The reductions in molecular weight of the Asn-152 3 Gln and Ser-154 3 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 3 Gln LHR than the Ser-154 3 Ala LHR (Table II), 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 re-ports have described retention of hormone binding activity with the Thr-175 3 Ala mutant protein (6), and attributed the loss of activity by Asn-173 3 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 (Ref. 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 (Figs. 3, 6, and 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, Refs. 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 (Figs. 6 and 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 FIG. 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 125 I-hCG ligand blot analysis as described under "Materials and Methods." Top panel, samples were not treated with SDS or reductant prior to deglycosylation; bottom panel, samples were denatured and reduced prior to enzyme treatment and electroblotting (see "Materials and Methods"). Nontreated, control wild-type receptor nontreated with glycosidases. SCHEME 1. Glycosidase specificity of generic high mannose chain. x, y, mannose residues; z, fucose or H; ç, å, cleavage site (16). PNGase F ϭ N-glycanase. 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.