©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Zone Mapping of the Binding Domain of the Rat Low Affinity Nerve Growth Factor Receptor by the Introduction of Novel N-Glycosylation Sites (*)

(Received for publication, August 5, 1994; and in revised form, December 22, 1994)

Anne N. Baldwin (§) Eric M. Shooter

From the Department of Neurobiology, Stanford University School of Medicine, Stanford, California 94305

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The binding of NGF (nerve growth factor) to the rat low affinity nerve growth factor receptor (p75) has been studied by site-directed mutagenesis of the receptor. Introduction of non-native N-glycosylation sites within the binding domain indicates that the second of the characteristic cysteine-rich repeats may be particularly important to NGF binding. Two mutants of the second repeat, S42N and S66N, are glycosylated and bind NGF at a drastically reduced level, while still maintaining a conformation recognized by the monoclonal antibody against p75, MC192. Alanine substitution at these sites does not affect NGF binding. Two other mutations that result in local structural changes in the second repeat also greatly decrease binding. One of these altered residues, Ser, appears to play an essential structural role, since it cannot be replaced by Asn, Ala, or Thr without loss of both NGF binding and MC192 recognition on a Western. Glycosylation of selected sites in the other repeats has little effect on NGF binding or antibody recognition. The introduction of non-native N-glycosylation sites may provide a generally useful scanning technique for the study of protein-protein interactions.


INTRODUCTION

The functional role of the low affinity nerve growth factor receptor, p75, (^1)is still under intensive study. Although a signal transduction pathway activated by p75 has recently been identified(1) , most of the known biological responses to NGF do not seem to require p75, whereas trk, the other known NGF receptor, can transduce a signal without p75 participation(2, 3, 4, 5, 6, 7, 8) . Moreover, transgenic mice lacking p75 display a rather mild phenotype(9) . It would therefore seem that the role of p75 is a fairly subtle one, altering the dose response curve of neurons or neuronal precursors(10) , perhaps by influencing the kinetics of trk activation(11, 12, 13, 14, 15) . Specifically, certain of the binding data suggest that p75 may accelerate the on rate of NGF binding to trk by means of ligand transfer(14) . To understand this mechanism, it will clearly be necessary to understand binding of NGF to p75 itself.

p75 belongs to a family of cell surface proteins that share a common four-repeat cystine motif in the extracellular domain. The three-dimensional structure of the cysteine-rich domain is not known for p75, but the corresponding domain of the p55 has been crystallized in complex with its ligand, TNFbeta, and the structure determined to 1.8 Å resolution(16) . The four repeats form a curved wand that binds longitudinally along the groove between two TNF subunits.

In p75, as in p55, the ligand binding site is fully contained within this cysteine-rich domain(17, 18) . The pattern of half-cystine residues in the two proteins is almost identical. Moreover, mutation of at least some pairs of presumably bonded cysteines in p75 yields active protein, strongly suggesting that the disulfide connectivity is the same in the two proteins(19) . In addition, certain residues that have been shown to form critical hydrogen bonds between beta-strands in the p55 structure are conserved in the p75 structure. It seems highly probable that the backbone structure of the two proteins will turn out to be very similar, although the surfaces are expected to vary considerably.

All four repeats of p75 are required for binding, even though the individual repeats seem to fold independently of each other(18) . Neither half of the binding domain can be replaced by the corresponding two repeats of either TNF receptor(20) , and insertions that disrupt the tertiary structure are not tolerated(21) . Deletion of the second repeat prevents expression of the protein(18) , whereas destabilization of the first or the fourth repeat by removal of the first disulfide bridge of the repeat results in a protein that is expressed but unable to bind NGF(19) . It seems that binding is extremely sensitive to conformational changes in any of the four repeats and that the conformational stability of the whole domain requires each repeat to be intact.

It is known from mutagenesis of NGF that multiple contacts between ligand and receptor participate in binding(5, 22) . When our first attempts to disrupt binding by mutagenesis of individual receptor side chains failed, we therefore looked for a way to alter the surface of the receptor over a more extended range than the space sampled by a single side chain, while minimizing the disruption of the backbone conformation.

It occurred to us that we might sterically block specific binding by the introduction of novel N-glycosylation sites. Glycosylation has been shown to prevent aggregation of polypeptides in the unfolded state(23) . In some proteins that utilize the secretion pathway, including the epidermal growth factor and low density lipoprotein receptors(24, 25) , glycosylation is required for transport to the cell surface, a role that might also be attributed to the prevention of unwanted protein-protein interactions. In influenza hemaglutinin, new glycosylation sites have appeared and disappeared spontaneously over much of the surface, possibly as a viral strategy for masking antigenic epitopes(26, 27) . Those regions of hemaglutinin in which the consensus sequence for glycosylation never seems to occur spontaneously are those involved in protein-protein interfaces between monomers, the binding domain for the receptor, or the stem region, where a helical coiled coil undergoes a conformational change(28) . Thus the effect of glycosylation at specific sites can provide information about the surfaces involved in protein interactions.

Here we describe a set of eight mutants in which extra N-glycosylation sites have been introduced to the binding domain of p75. In choosing the sites, we looked for polar residues, likely to be solvent exposed, with a serine or threonine 2 residues downstream, so that a fairly conservative change would create a consensus sequence of N-glycosylation. We cannot assume a priori that a novel N-glycosylation site will not perturb the backbone conformation. However, we have at our disposal two monoclonal antibodies against the extracellular domain of p75, both of which are sensitive to conformational changes in the protein, and whose epitopes do not overlap with the NGF binding site(29, 30) . We have used these to assess whether each individual mutation has resulted in a global conformational change. Finally, we have made a number of control mutants, mostly with alanine replacements, to confirm our conclusions.

The mutants fall into three categories: 1) those that bind NGF normally and recognize both monoclonal antibodies even after heat denaturation and Western blotting, 2) those that bind the antibodies even after denaturation, but show greatly reduced binding of NGF, and 3) those that fail to bind well NGF and also fail to recognize one or both of the monoclonal antibodies following denaturation. The mutations that resulted in a drastic loss of NGF binding were all located in the second repeat, and no consensus sequence for N-glycosylation was introduced in this repeat without major loss of NGF binding. We suggest that this repeat plays a crucial role in NGF binding and may form direct contacts with the ligand.


MATERIALS AND METHODS

beta-NGF was purchased from Bioproducts for Science, Inc. and was iodinated by the lactoperoxidase method(31) . Sulfosuccinimidobiotin and horseradish peroxidase-conjugated streptavidin were obtained from Pierce, and endo F/N-glycosidase F was from Boehringer Mannheim.

Antibodies

MC192 is a monoclonal antibody against rat p75(29) . It is specific for the extracellular cysteine-rich domain and requires the first three but not the fourth cysteine repeat(18) . 217c is a monoclonal antibody first isolated against a cell surface antigen on C6 rat glioma cells and later found to be directed against p75(30, 32, 33) . It was a gift of Dr. J. de Vellis. Its epitope is centered close to the fourth repeat, and binding does not require the first repeat(18) . Both of these antibodies require the native conformation of the receptor, and any modification that destabilizes this structure, such as removal of even a single disulfide bond, reduces or eliminates recognition by the antibody. Immunoprecipitations were carried out using a polyclonal antibody directed against a large portion of the intracellular domain. This antibody was a gift of Dr. Stuart Decker of Parke-Davis.

Construction and Expression of Mutants

All mutants were constructed by means of the polymerase chain reaction overlap extension method (34) as described previously (18) and were subcloned into the mammalian expression vector pBJ5(34) . COS 7 cells were grown and transfected with p75 cDNA in pBJ5 as described previously(18) .

Binding of NGF

The ability of mutant proteins to bind NGF was usually assessed by cross-linking I-NGF to transfected COS 7 cells with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide as described previously (18) . Proteins were reduced and denatured before separation on a 6% Laemmli gel (35) and were detected by autoradiography. Binding of I-NGF was carried out as described by Vale and Shooter (37) . For each point, nonspecific binding, determined with a 100-fold excess of unlabeled NGF, was subtracted from total binding, and each point was the average of three separate determinations. Nonspecific binding represented approximately 10% of the total binding. Incubations were carried out at 0 °C for 2 h, with vortexing every 15 min. Equilibrium binding was achieved in this time.

Surface Biotinylation of Transfected Cells

COS 7 cells were transfected at 30-50% confluence on 100-mm plates. At 64 h after transfection, they were washed and treated with 2 ml of cold Krebs-Ringer solution containing 300 µg/ml sulfosuccinimidobiotin (sulfo-NHS-biotin), freshly diluted from a stock solution in dimethyl sulfoxide (50 mg in 250 µl). After 30 min on a rocker at 4 °C, the cells were washed three times with phosphate-buffered saline containing 20 mM Tris-Cl, pH 7.6, to remove excess biotinylating reagent and were lysed directly on the plate with 1 ml of radioimmune precipitation buffer (10 mM Tris-Cl, pH 8.0, 150 mM NaCl, 10 mM KCl, 1 mM EDTA, 1% Nonidet P-40, 0.1% SDS, 1% sodium deoxycholate) containing 1 mM phenylmethysulfonyl fluoride, leupeptin, benzamidine, and phenanthroline. After 30 min on the rocker at 4 °C, the lysate was collected and centrifuged to remove the DNA. Receptor protein was immunoprecipitated with the Parke-Davis polyclonal antibody, added to give a 1:500 dilution. The samples were tumbled overnight at 4 °C with primary antibody, then another 2 h with goat anti-rabbit IgG-agarose beads (Sigma). The beads were collected by centrifugation at 3000 rpm and were washed eight times with radioimmune precipitation buffer. Samples were eluted by incubating the washed beads with 100 µl of 2 times gel loading buffer (0.125 M Tris-Cl, pH 6.8, 4% SDS, 20% glycerol) for 30 min at 37 °C. Aliquots of the eluted samples were reduced by boiling for 5 min with 5% 2-mercaptoethanol prior to loading onto a 7.5% Laemmli gel. Protein was transferred electrophoretically in Towbin buffer (38) to Millipore Immobilon-P(TM) membrane. The blots were blocked with 10 mM Tris-Cl, pH 8.0, 150 mM NaCl, 0.2% Tween 20/5% non-fat milk for 1.5 h and were then stained with horseradish peroxidase-conjugated streptavidin (Pierce). The protein was visualized by enhanced chemiluminescence, using reagents from Amersham Corp., according to their instructions.

Deglycosylation of Receptor Protein

Receptor proteins were deglycosylated with endo F/N-glycosidase F, purchased from Boehringer Mannheim. This enzyme cleaves at the asparagine residue and does not discriminate between different sugar residues(39) . COS 7 cells were transfected and biotinylated as described above. The p75 protein was immunoprecipitated and eluted from the beads by boiling for 5 min with 2 times gel loading buffer containing 5% 2-mercaptoethanol. Half of this sample was used directly for Western analysis; the other half was treated with glycosidase as follows. To 50 µl of eluted protein we added 50 µl of glycosidase buffer, containing 50 mM potassium phosphate, pH 7.4, 10 mM EDTA, and 1% 2-mercaptoethanol. The samples were boiled for 3 min. Then Nonidet P-40 was added to a final concentration of 1%. Enzyme was added (0.15 units/sample), and the samples were incubated overnight at 37 °C. They were boiled again for 5 min after the addition of an equal volume of 2 times loading buffer and 5% 2-mercaptoethanol prior to electrophoresis and transfer. The blots shown in Fig. 3were stained with peroxidase-conjugated streptavidin.


Figure 3: Deglycosylation of mutants with novel N-glycosylation sites. Cell surface proteins were biotinylated as in Fig. 3, and the receptor protein was immunoprecipitated with the Parke-Davis polyclonal antibody. A, an aliquot of each sample was reduced and analyzed on a Western blot as in Fig. 3; B, another portion was reduced, boiled briefly with SDS, and then treated with endo F/N-glycosidase F as described under ``Materials and Methods,'' prior to Western analysis as in A.



Immunostaining of Transfected COS 7 Cells

Cells were grown nearly to confluence on coverslips that had been coated with polylysine (1 mg/ml in phosphate-buffered saline, 37 °C overnight) and were transfected as described previously(19) . Although the efficiency of transfection was lower when the cells were fairly dense, they adhered better to the coverslips during subsequent fixation and washing procedures. After about 64 h, the cells were fixed with 4% paraformaldehyde, washed, and stained with MC192 or 217c, followed by rhodamine-conjugated donkey anti-mouse IgG (Jackson Laboratories) as described previously(19) . They were photographed on Ilford 400 35-mm black and white fine grain film at 25-fold magnification, using a Zeiss M35W camera attached to a Zeiss Axioscope. After determining the correct exposure for a brightly stained cell, all cells were exposed for the same number of seconds. Prints were likewise made from the negatives using identical exposures and the same contrast paper.


RESULTS

The p75 mutants considered in this paper are summarized in Table 1. These mutants include the eight mutants with novel N-glycosylation sites and also control mutants with Ala or Thr replacements at certain of these sites. In one case, we made a double mutation, changing Asp to Asn and Ala to Ser. (One other construct, E89N/V35M, has an unintentional second mutation due to a polymerase chain reaction error, but since this mutant binds NGF at an apparently wild-type level, we have included it in the study.) A few other mutants, listed as ``Miscellaneous,'' are included because they shed further light on individual glycosylation mutants. A mutant lacking the naturally occurring N-glycosylation site and one lacking both this site and the O-glycosylated stack region of the receptor are also listed. For the mutants with novel glycosylation sites, the consensus sequence created is given, along with the original triad of amino acids from which it was created.



The approximate location of each mutation site relative to the cysteine-rich domain is shown in Fig. 1, a cartoon based roughly on the crystal structure of p55(16) . This drawing is meant only to help in orienting the reader and does not imply precise knowledge of the molecular geometry. The results described below are summarized in Table 2.


Figure 1: Cartoon showing approximate positions of the sites mutated in this study, based roughly on the crystal structure of p55 by Banner et al.(15) .





All of the Glycosylation Mutants Are Expressed in COS 7 Cells and Are Transported to the Cell Surface

Transfected cells were biotinylated, then lysed, and the p75 immunoprecipitated with a polyclonal antibody against the intracellular domain. The biotinylated protein was detected on a Western blot with peroxidase-conjugated streptavidin, followed by enhanced chemiluminescence development. Fig. 2shows the surface expression of a number of these mutant proteins. For other mutants, cross-linking to whole cells demonstrates surface expression, as discussed below.


Figure 2: Expression of mutant protein at the cell surface. Transfected COS 7 cells were biotinylated as described under ``Materials and Methods.'' The cells were then lysed, and the p75 protein was immunoprecipitated with the Parke-Davis polyclonal antibody. Fractions were reduced and separated on a Laemmli gel prior to transfer to Immobilon-P(TM) membrane. The blot was stained with peroxidase-conjugated streptavidin and developed by enhanced chemiluminescence.



Six of the Eight Mutants with Extra Glycosylation Sites Are Glycosylated at the Novel Positions

From Fig. 2, it can be seen that most of the mutants with novel N-glycosylation consensus sequences migrated on a gel more slowly than wild-type protein, although not all at exactly the same rate. There were two exceptions. One was the double mutant of the second repeat, D75N/A77S. The other exception was T10N. When a Western blot of the crude extract was stained with the polyclonal antibody, there appeared to be some p75-T10N protein migrating more slowly than wild-type protein, but not as slowly as the mutant protein S42N, for example (data not shown). This component did not show up by surface biotinylation (Fig. 2). Thus there may be a partially glycosylated component of T10N that is not transported to the cell surface.

All other mutants glycosylated at novel sites were transported to the cell surface in the fully glycosylated form (Fig. 3A). When the mutant proteins were immunoprecipitated with the polyclonal antibody, and then treated with endo F/N-glycosidase F, all comigrated on a Laemmli gel with the mutant protein, N32D, which lacks the single naturally occurring N-glycosylation site (Fig. 3B).

The Native N-Glycosylation Present in the Wild-type Protein Is Not Required for Surface Expression or for NGF Binding

Mutant N32D, which lacks the natural N-glycosylation site, showed wild-type levels of binding as assessed by cross-linking (Fig. 4A & 5) and by Scatchard analysis (Fig. 4B). This demonstrates that N-glycosylation is not strictly required for surface expression, or for formation of the active conformation of the binding domain, consistent with the earlier finding by Grob and Bothwell (40) that tunicamycin did not inhibit binding of NGF to PC12 cells.


Figure 4: A, cross-linking of [I]NGF to COS 7 cells transfected with mutants N32D and N32D/DeltaBS. N32D lacks the naturally occurring N-glycosylation site, and mutant N32D/DeltaBS lacks both N-glycosylation and also the stalk region between the binding domain and the transmembrane domain. The latter region appears to be heavily O-glycosylated. B, Scatchard analysis of I-NGF binding to COS cells transfected with wild-type p75 and with p75-N32D, as described under ``Materials and Methods.'' The points determined for binding to wild-type cells were based on 2 times 10^4 cells/sample and the binding to N32D cells on 5 times 10^4 cells/sample.



We had previously shown that the stalk region between the cysteine-rich domain and the membrane-spanning domain, which appears to be heavily O-glycosylated, was likewise not essential for NGF binding(18) . Here we show further that when the N-glycosylation site and the stalk region were simultaneously deleted cross-linking still appeared to be normal (Fig. 4A).

Binding of NGF Is Strongly Dependent on the Location of the Extra Glycosylation Site

Fig. 5A shows the cross-linking of I-NGF to COS 7 cells transfected with cDNA for each of the mutants with altered glycosylation sites. When extraN-glycosylation sites were incorporated (always in addition to the normal glycosylation site at Asn), the effect on binding depended critically on the position of the site. Strikingly, each new glycosylation site we created in the second repeat greatly inhibited binding. In contrast, extra glycosylation at several positions in other repeats was introduced without such loss of function. These mutations are described in the order of their position in the protein.


Figure 5: Cross-linking of I-NGF to COS 7 cells transfected with p75 mutants. A, mutants with extra potential N-glycosylation sites. B, miscellaneous mutants C4G/C15G and Y83A/Y85A. In this experiment, the cross-linked protein was immunoprecipitated with the Parke-Davis polyclonal antibody prior to electrophoresis and autoradiography.



Mutations of the First Cysteine Repeat

T10N in the first repeat bound NGF slightly less well than wild type, but this may reflect the fact that the glycosylated form did not appear to be transported to the cell surface. We therefore cannot say whether glycosylation at this site would block the approach of NGF, but the mutation itself did not have much effect on NGF binding. Thr is located in the center of the first cysteine loop of the repeat. To investigate the role of this loop, we made a double mutant, C4G/C15G. This mutant did not bind NGF (Fig. 5B).

Mutations of the Second Cysteine Repeat

All mutants with an extra glycosylation site in the second repeat showed drastically reduced levels of cross-linking to NGF (Fig. 5A). These mutations included S42N and S50N in the first cystine loop, S66N on the second predicted beta-strand, and D75N/A77S on the third predicted beta-strand. The first three of these were glycosylated, whereas the double mutant was not.

To test whether the poor binding of NGF to S42N, S50N, and S66N was directly due to the glycosylation, we constructed control mutants in which the original serine was replaced by Ala instead of by Asn. Fig. 6A shows that mutants S42A and S66A were fully able to bind NGF, but S50A was not. A further variant of this site, S50T, was also unable to bind NGF (Fig. 6B). As a partial control for the double mutant D75N/A77S, we made a single mutant, D75N. This mutant often showed some reduction in cross-linking to NGF, but considerably less reduction than D75N/A77S (data not shown).


Figure 6: Cross-linking of I-NGF to mutants of Ser, Ser, and Ser, showing the effect of Ala substitution at each of these sites (A) or Thr substitution at Ser (B).



Mutants of the Third and Fourth Cysteine Repeats

Mutants E89N and Q114N of the third repeat, and E123N of the fourth repeat, all bound NGF at roughly wild-type levels (Fig. 5A). All appeared to be glycosylated and all were expressed on the cell surface in the fully glycosylated form, as judged by surface biotinylation (Fig. 2). Mutant E89N contained a second unintended mutation, V35M, but this did not appear to interfere with NGF binding either. (This particular valine is conserved in p55, and somewhat surprisingly its side chain is oriented toward the solvent in that structure(16) .)

Western Analysis

We next asked which of the mutations that markedly affect NGF binding do so because of a global change in protein conformation and which do so through direct steric hinderance by the carbohydrate or by very local structural changes. To address this, we looked at the interaction between the receptor protein and two monoclonal antibodies, MC192 and 217c, described under ``Materials and Methods.'' Both of these antibodies recognize wild-type protein on a Western blot even when the samples have been boiled with SDS prior to electrophoresis and transfer, provided that the disulfide bonds in the binding domain have not been reduced.

Some, but not all, of the mutants with novel glycosylation sites recognized the monoclonals on a Western blot, following SDS denaturation. The reactivity of the different mutants toward MC192 under these conditions is shown in Fig. 7. The results of this and previous experiments, in which smaller groups of mutants were tested, are summarized in Table 2. Mutants S42N and S66N, which were glycosylated and which did not bind NGF well, typically did bind MC192. In contrast, neither S50N nor the other Ser mutants reacted well on a Western blot. This is consistent with the lack of NGF binding to the S50A and S50T mutant proteins; evidently a structural change, most likely the loss of a critical hydrogen bond, has occurred due to the loss of the Ser side chain. T10N and T10A in the corresponding loop of the first repeat were also somewhat sensitive to the Western blotting procedure (although less so than the Ser mutants), indicating that Thr may also contribute a hydrogen bond that stabilizes some structural feature.


Figure 7: Western analysis of glycosylation mutants. Samples were boiled for 3 min in gel loading buffer without reduction. Proteins were fractionated on a 7.5% Laemmli gel (35) and transferred electrophoretically to Immobilon-P(TM) membrane. The blot was stained with MC192, followed by horseradish peroxidase-conjugated donkey anti-mouse IgG, and the proteins were visualized by enhanced chemiluminescence. D74N/A77S showed less relative stability in this experiment than in others.



All of the glycosylation mutants except T10N and S50N recognized MC192 on a Western blot. A similar pattern was seen with 217c staining, except that the effect of altering Thr or Ser was less dramatic (data not shown). This is consistent with other data that place the 217c epitope toward the C-terminal end of the repeat(18) . The D75N/A77S mutant behaved anomolously in Fig. 7; in most experiments, this protein reacted strongly with MC192. It was detected on a blot of a nondenaturing gel (Fig. 8), and was immunoprecipitated by MC192 (Fig. 9).


Figure 8: Detection of mutant proteins on a blot of a nondenaturing gel. Electrophoresis was run with a multiphasic buffer system of Chrambach et al.(42) . The blots were stained with (A) MC192, and (B) 217c, and peroxidase-conjugated goat anti-mouse IgG. Protein was visualized by enhanced chemiluminescence.




Figure 9: Immunoprecipitation of p75 mutant proteins with MC192. The immunoprecipitations were carried out in radioimmune precipitation buffer extracts, as described. The receptor protein was first eluted from the agarose beads and then reduced prior to electrophoresis and Western analysis using the Parke-Davis polyclonal antibody.



Antibody Analysis without Heat Denaturation

Western analysis crudely reflects the stability of an epitope and does not necessarily reflect the properties of the native structure, in spite of the extensive disulfide bridging of the ligand binding domain. NGF binding, in contrast, is determined on the surface of cells, in nondenaturing conditions.

To assess binding of the antibodies to undenatured mutant receptors, we have done three kinds of experiment: analysis of blots of nondenaturing gels, immunoprecipitation, and fluorescent surface staining of transfected cells.

All of the mutant proteins, when fractionated on a nondenaturing gel using a multiphasic buffer system of Chrambach et al.(41) , and then blotted, stained with both monoclonal antibodies (Fig. 8). The Thr and Ser mutants stained less well than some of the others, consistent with their lesser stability.

Immunoprecipitation with MC192 followed by Western analysis with the polyclonal antibody resulted in a pattern very similar to that obtained when a Western blot was stained with MC192 (Fig. 9). The mutants that failed to precipitate with MC192 were those that showed instability on a Western blot, namely T10N, S50N, S50A, S50T, C4G/C15G, and Y83A/Y85A, although Western analysis of the original extract showed that the mutant proteins were expressed at similar levels (data not shown). Not surprisingly, those proteins that are stable to heat/SDS denaturation were also stable to immunoprecipitation.

Surface staining of transfected cells with MC192 or 217c, plus rhodamine-conjugated second antibody, likewise showed some reactivity of all of the constructs to both antibodies, although staining of the Ser mutants, as well as mutant C4G/C15G, was less bright with MC192 (Fig. 10). The cell or cells photographed in each case represented one of the brightest in the field. Since there was not much difference in surface expression (Fig. 2), and since all of the constructs stained quite evenly with 217c, the fainter staining of the Ser and C4G/C15G mutants must result from decreased affinity for MC192. This result is consistent with our conclusion that a basic structural change has occurred as a result of each of these mutations.


Figure 10: Immunostaining of COS 7 cells transfected with p75 mutants. Cells were grown and transfected directly on coverslips, as described, and stained with MC192 or 217c, followed by rhodamine-conjugated donkey anti-mouse IgG. The first and third columns show representative cells stained with MC192; the fifth and seventh columns show representative cells stained with 217c. A phase contrast photograph of approximately the same field is shown immediately to the right of each fluorescent photograph. 1, antisense; 2, wild type; 3, T10N; 4, T10A; 5, S42N; 6, S50N; 7, S50A; 8, S50T; 9, S66N; 10, D75N/A77S; 11, D75N; 12, E89N/V35M; 13, Q114N; 14, E123N; 15, C4G/C15G; 16, Y83A/Y85A.



Effect of MC192 on Binding of NGF to Mutants S42N and S66N

MC192 increases the affinity of wild-type p75 for NGF by a factor of 2-3(29) . Since NGF does not bind to MC192 itself, this implies that the antibody induces a conformational change in the receptor. We therefore asked whether this effect could still be seen in the glycosylation mutants of the second repeat that bind MC192 but show greatly reduced binding of NGF. We tested mutants S42N and S66N on transfected COS 7 cells through a standard binding assay(37) . The results are shown in Fig. 11. Wild-type p75 binding was enhanced when binding was studied at an NGF concentration range of 2 nM, but since the two mutants have a much lower affinity for NGF than wild type, it was necessary to increase the concentration of I-NGF to 10 nM in order to see the effect. Conversely, at this concentration of NGF, the wild-type receptor was essentially saturated and did not show an increase on adding MC192.


Figure 11: Effect of MC192 on I-NGF binding to p75-S42N and p75-S66N on transfected COS 7 cells. Specific binding was determined as described under ``Materials and Methods.'' When MC192 was added (Fig. 12), the final concentration of purified IgG was 8 µg/ml. &cjs2112;, no MC192; , +MC192.



Additional Point Mutants in the Vicinity of the Second Repeat

Because basic residues of NGF have been shown to be required for binding to p75, we have looked at the effect of mutating certain acidic side chains of p75 in the second repeat. Alanine replacement of Glu Asp or Asp did not result in loss of binding function, however (data not shown).

In the p55 several aromatic residues toward the C-terminal end of the first cystine loop of the third repeat participate in TNF binding. In p75, these aromatic residues are missing, but there is an aromatic cluster at the N-terminal end of the loop. To see whether these residues might play a role in ligand binding, we made a double mutant, Y83A/Y85A. This mutant did not bind NGF appreciably (Fig. 5B). It was also not recognized by MC192 on a Western blot (Fig. 7), although it stained well on intact cells (Fig. 10). The mutation therefore appears to have affected the stability of the receptor, and we cannot be sure at this stage that the effect on NGF is direct.


DISCUSSION

The mutants described here strongly implicate the second cysteine repeat of the cysteine-rich domain in the specific binding of NGF. This conclusion is based on the markedly position-dependent effect of adding extra glycosylation sites. Extra glycosylation at two sites within the second repeat (Ser and Ser) and mutations that affect local backbone structure or stability at two other sites (Ser and Asp/Ala) greatly impede NGF binding, whereas added glycosylation at two sites in the third repeat (Glu and Gln) and one site in the fourth repeat (Glu) do not. In addition, the naturally occurring N-glycosylation, found on the last predicted beta-strand of the first repeat, does not inhibit binding.

Mutants of the Second Repeat

Within the second repeat, three of the four mutations that affect NGF binding (S42N, S66N, and D75N/A77S) do not affect the binding of MC192 even after denaturation by heating in the presence of SDS. Although 217c does not recognize D75N/A77S under these same conditions, implying that the 217c epitope has been affected in this case, any conformational changes induced by the three mutations S42N, S66N, and D75N/A77S must be confined to a local folding region, since at least one antibody recognizes each of them. In the case of S42N and S66N, it seems likely that the glycosylation itself directly blocks NGF binding through simple steric hinderance. This conclusion is strengthened by the finding that MC192 can still enhance binding of NGF to these mutant proteins, whereas any more global change would be likely to destroy a distal conformational effect by the antibody.

Mutant D75N/A77S was not extensively glycosylated. This mutant contains the amino acid sequence Asn-Asp-Ser. Asp in the middle position of this triad is suspected to prevent glycosylation, either because the local conformation is incompatible with the binding site of the glycosyl transferase (42) or because the charged side chain interferes with the putative hydrogen bonding between the amide proton and the serine oxygen(43) . Moreover, Ser does not function as well as Thr as a proton acceptor in this position, so our result is not surprising.

At another site in the second repeat (Ser), more extensive structural change undoubtedly accompanies mutation, since this residue cannot be changed to Asn (with glycosylation), or to Ala or Thr, without loss of recognition on a Western blot by MC192. Hydroxyl-containing side chains that are buried within the hydrophobic core of a protein are nearly always hydrogen-bonded. The fact that Thr cannot substitute for Ser at this position makes it likely that this side chain is buried. It might be involved in the formation of a beta-turn or some nonrepetitive structure within the cysteine loop, or it might form an H-bonded bridge to the preceding repeat.

The effect of Ser mutations on MC192 binding is more pronounced than on 217c binding, suggesting that this part of the second repeat is more critical to the MC192 epitope than to the 217c epitope. Both antibodies do detect these mutant proteins after fractionation on a nondenaturing gel, and they both stain the Ser mutants on the surface of transfected cells, although less brightly than the wild-type protein. Evidently these mutations decrease the stability of the active conformation of the domain, so the direct effect of glycosylation cannot be assessed.

Thr in the first repeat occupies a position within its cystine loop similar to that of Ser in the second repeat, and its mutants also destabilize the MC192 epitope. They do bind NGF, indicating that any local backbone distortion does not perturb the binding conformation. Opening of this loop by removal of the first disulfide bridge did, however, prevent binding of either NGF or MC192, just as did the opening of the corresponding bridge of the fourth repeat(19) . This reinforces our conclusion that each repeat serves a role in maintaining the active conformation of the overall domain. At the same time, it validates Thr mutants as position controls for the Ser mutants.

Glycosylation Mutants of the Third and Fourth Repeats

These mutants, which all bind NGF, serve as important controls for the effect of glycosylation within the second repeat.

E89N in the third repeat, and E123N in the fourth repeat, is located in the first cystine loop, which, as we have said, must play a critical structural role in the repeat. The glutamic acid side chains may well extend into the solvent, however, whereas Thr of the first repeat and Ser of the second repeat may be hydrogen-bonded within the loop. Glu is found toward the C-terminal end of its cystine loop, whereas Glu is near the N-terminal end of its loop.

Gln is found in a putative beta-strand of the third repeat with a local sequence almost identical to that where the naturally occurring glycosylation site in the first repeat is found (QNTV in the third repeat compared to NQTV in the first repeat). The Q114N change therefore might not be expected to disrupt the backbone structure very much, although glycosylation might break a side chain to main chain hydrogen bond.

The fact that these mutants bind NGF does not rule out the possibility that the third and fourth repeats make contacts with NGF, nor does it preclude the possibility that these repeats are somewhat misfolded but do not normally contact NGF in the binding reaction. Nonetheless it does indicate that certain surface regions exist where bulky carbohydrate side chains do not interfere with ligand binding.

Glycosylation in Other Proteins

A major unknown in this work is how the carbohydrate moieties interact with the protein surface or backbone. The structural information available for naturally occurring N-linked sugars is limited, but so far, perturbation of the polypeptide seems to be slight. The resolved sugar moieties of elastase (44) and of human chorionic gonadotropin (45) show only limited contacts with the protein surface; O-glycosylation in the hinge region of an immunoglobulin does alter the backbone conformation, but only locally(46) . The N-linked sugars of alpha(1)-antitrypsin do not affect the thermodynamic stability or the folding transitions of the protein(47) . Naturally occurring glycosylation sites might, however, have been selected for their lack of interference with the process of protein folding.

The consequences of adding extra glycosylation sites have previously been studied in detail in several viral systems in which the secretion of a glycoprotein is dependent on naturally occurring glycan attachment, and it has been shown that some added groups block transport, very likely because they prevent correct folding of the protein, whereas others do not(48, 49, 50, 51) . Our p75 mutants were all expressed in COS 7 cells, and all were transported to the cell surface, except for the glycosylated version of T10N, and thus presumably are not grossly misfolded.

Comparison with p55

Contacts made by TNF to the p55 binding domain are concentrated in the second repeat and the first loop of the third repeat, although limited contacts to the other repeats occur too. The binding surface on p55 is complex, with asymmetric contacts to two subunits of the trimeric ligand. In p75, the novel glycosylation sites that interfere with NGF binding (without disrupting the structure) are not on the putative surface that should correspond to the p55 binding site, but we do not know how wide a surface area might be affected by the presence of a bulky carbohydrate side chain or how much solvent space it might displace. There is also no a priori reason to assume that the binding of TNF to its receptor would mimic the binding of NGF to p75 at all, since the two ligands are structurally unrelated, and the two receptors show virtually no surface homology.

Nonetheless, the simplest interpretation of our results is that the second repeat plays a direct and critical role in the binding of NGF to its p75 receptor. Our failure to identify any critical salt bridge through mutagenesis of acidic residues in this repeat may result from the small contribution of any individual hydrophilic bond to the overall binding energy.

The aromatic residues at the very beginning of the third repeat may contribute to the binding site. Aromatic side chains are often found in protein-protein interfaces, particularly at the edge (52) . Of the 3 tyrosines in the p75 cluster, the third, by analogy with conserved aromatic residues in all repeats of p55, most likely stabilizes the repeat by packing against the next disulfide bond (16) . The other two, which we have mutated together, could interact with either NGF or with the preceding repeat, thus fixing the relative orientation of the two halves of the domain. The instability of this protein to Western analysis does indicate a structural role for one or both of these aromatic side chains.

Finally, the introduction of novel glycosylation sites may have a general application in the study of specific protein-protein interactions, provided each mutant can be adequately characterized. It provides an alternative to homolog scanning (53) in targeting a binding surface, when the tertiary structure of the protein domain in question is as intricate as that of p75 or appropriate homologs are unavailable. It might also be used to identify interactions that are more elusive. In the NGF receptor system, for example, it is still not clear whether p75 forms a physical complex with trk. Some of the glycosylation mutants described here might be useful in testing the effect of p75 on trk activation.


FOOTNOTES

*
This work was supported in part by Grant NS04270 from the National Institutes of Health, Grant IIRG-92-138 from the Alzheimer's Association, and by the State of California, Department of Health Services, under Contracts 92-15942 and 93-18647. 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 should be addressed. Tel.: 415-723-5194; Fax: 415-723-0388.

(^1)
The abbreviations used are: p75, or p75, the low affinity nerve growth factor receptor; NGF, nerve growth factor; TNF, tumor necrosis factor; p55, tumor necrosis factor receptor I.


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