©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Both Extracellular Immunoglobin-like Domains of CD80 Contain Residues Critical for Binding T Cell Surface Receptors CTLA-4 and CD28 (*)

(Received for publication, April 14, 1995; and in revised form, June 12, 1995)

Robert J. Peach (§) Jürgen Bajorath Joseph Naemura Gina Leytze JoAnne Greene Alejandro Aruffo Peter S. Linsley

From the Bristol-Myers Squibb Pharmaceutical Research Institute, Seattle, Washington 98121

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
CONCLUSION
FOOTNOTES
REFERENCES

ABSTRACT

The B7-related molecules CD80 and CD86 are expressed on antigen-presenting cells, bind the homologous T cell receptors CD28 and CTLA-4, and trigger costimulatory signals important for optimal T cell activation. All four molecules are immunoglobulin superfamily members, each comprising an extracellular Ig variable-like (IgV) domain, with CD80 and CD86 containing an additional Ig constant-like (IgC) domain. Despite limited sequence identity, CD80 and CD86 share similar overall receptor binding properties and effector functions. We have identified, by site-directed mutagenesis of soluble forms of CD80 and CD86, residues in both the IgV and IgC domains that are important for CTLA4Ig and CD28Ig binding. Mutagenesis in the IgV domain of CD80 identified 11 amino acids that support receptor binding. Many of these residues are conserved in the B7 family, are hydrophobic, and approximately map to the GFCC`C`` beta-sheet face of an IgV fold. Mutagenesis of corresponding residues in CD86 established that some, but not all, of these residues also played a role in CD86 receptor binding. In general, mutations had a similar effect on CTLA4Ig and CD28Ig binding, thereby indicating that both receptors bind to overlapping sites on CD80 and CD86. Further, mutagenesis of several conserved residues in the ABED beta-sheet face of the IgC domain of CD80 completely ablated receptor binding. Point mutagenesis had a more pronounced effect than complete truncation of the IgC domain. Thus, full CTLA4Ig and CD28Ig binding to B7 molecules is dependent upon residues in the GFC`C`` face of the IgV domain and the ABED face of the IgC domain.


INTRODUCTION

Distinct activation signals from antigen-presenting cells are required for T lymphocyte-dependent immune responses (1, 2, 3) . Antigen-specific activation of T cells is mediated by peptide/major histocompatibility complexes on antigen-presenting cells interacting with specific T cell antigen receptors. Binding of CD28 and/or CTLA-4 on T cells to B7-related receptors on antigen-presenting cells provides important antigen-nonspecific costimulatory signals essential for optimum immune responses. Blocking the delivery of these costimulatory signals in vitro can lead to T cell unresponsiveness or anergy(4, 5, 6) . Two B7-related molecules, each having different patterns of expression, have been identified: CD80 (B7-1) (7, 8) and CD86 (B7-2/B70)(9, 10, 11) .

CD80 and CD86 are members of the immunoglobulin superfamily (IgSF), (^1)with their extracellular regions consisting of one amino-terminal Ig variable-like (IgV) and one membrane proximal Ig constant-like (IgC) domain. The B7 IgV domains may include some structural features that depart from currently known Ig folds (12) and share sequence similarity with three major histocompatibility complex-encoded members of the IgSF(13) . In contrast, the IgC domains display significant sequence-structure compatibility with beta(2)-microglobulin(12) . B7 molecules are not disulfide-linked, unlike their coreceptors CTLA-4 and CD28, which are also IgSF members(14, 15, 16) .

Despite having only 25% sequence homology, CD80 and CD86 have similar equilibrium receptor binding properties. Both molecules bind CTLA-4 with high avidity and CD28 with low avidity(17, 18) . CD80 and CD86 also share T cell costimulatory properties(9, 10, 11) . However, some kinetic and molecular properties distinguishing the B7 molecules were observed, implying that these molecules have distinct interactions with CD28 and CTLA-4(18) . Little is known of the molecular details of the interaction between CD80 and CD86 and their counter-receptors. B7 residues critical for interactions with CTLA-4 and CD28 are currently unknown. We report the results of site-directed mutagenesis of the IgV- and IgC-like domains in CD80Ig and CD86Ig and the effects that deletion of the IgC domain of a recombinant CD80Ig has on CTLA4Ig and CD28Ig binding. We demonstrate that residues in both Ig-like domains are critical for binding CTLA4Ig and CD28Ig.


MATERIALS AND METHODS

Monoclonal Antibodies (mAbs) and Fusion Proteins

Murine anti-CD86 mAb IT2.2 was purchased from Pharmingen (San Diego, CA). Anti-CD80 mAbs BB1 (IgM) and 104 were kind gifts from Dr. E. Clark, University of Washington, Seattle, WA, and Dr. J. Banchereau, Schering Plough Corp., Dardilly, France, respectively. BB1 (IgG1, anti-CD80) was purchased from Becton Dickinson (San Jose, CA). Another anti-CD80 mAb, CD80.3, was prepared in our laboratory. BALB/c mice were immunized subcutaneously and intraperitonealy with human CD80 mIg (18) (25 µg/site) in complete adjuvant. Secondary and tertiary booster immunizations were administered intraperitonealy in incomplete adjuvant. A final immunization in phosphate-buffered saline was performed 3 days before fusion with murine myeloma Ag8.653. The specificity of CD80.3 was established by binding to immobilized CD80Ig fusion protein and CD80-CHO cells. In competition experiments using flow cytometry and enzyme immunoassays CD80.3 did not block CTLA4Ig or CD28Ig binding to CD80.

CTLA4 mIg and CD28 mIg were prepared by PCR-amplification of the extracellular domains of human CTLA-4 and CD28 with primers that allowed fragments to be digested with HindIII and BclI and ligated in frame to a mouse Fc IgG2a cDNA fragment (18) encoded in a HindIII/BglII-cut LN expression vector. CD80Ig and CD86Ig were prepared in transiently transfected COS cells(17, 18) . CD80VIg, a fusion protein lacking the IgC-like domain of CD80, was prepared using the PCR. cDNA encoding the IgV-like domain was amplified using full-length CD80 as template, a forward primer from the expression vector, and a reverse primer, CTCACCCTCGGGATCCTTGACTGATAACGTCAC, encoding a BamHI restriction site and the final six amino acids of exon 3, corresponding to the carboxyl terminus of the CD80 V domain(19) . This PCR fragment was digested with HindIII and BamHI and ligated in frame to a human IgG1 genomic DNA fragment (E1, (20) ) in the CDM8 expression vector. A control CD80Ig fusion protein having the same IgG1 Fc region (CD80E1) was prepared by a similar method, except the reverse primer used ensured that cDNA encoding the complete extracellular region of CD80 was PCR-amplified. In control experiments, CD80E1 fusion protein had identical receptor binding properties to CD80Ig. CD80VIg and CD80E1 were prepared in transiently transfected COS cells, which were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Media from transfected COS cells were used as a source for Ig fusion proteins.

Site-directed Mutagenesis

CD80Ig and CD86Ig site-directed mutants were prepared by encoding the desired mutation in overlapping oligonucleotide primers and, using appropriate plasmid cDNA as template, generating the mutants by PCR(21) . Other flanking PCR primers used for these reactions were designed to allow the final PCR products to be digested with HindIII and XbaI and ligated into HindIII/XbaI-digested CDM8 vector. PCR conditions were as described previously(22) . Each mutant was sequenced by the dideoxy chain termination/extension reaction to confirm that the desired mutation had been inserted and that secondary mutations had not been introduced. Sequenase reagents were used according to the manufacturer's recommendations (U. S. Biochemical Corp.).

Enzyme Immunoassays

Quantitation of Ig fusion proteins in culture media was performed as described previously(22) . Triplicate determinations were made, and replicates differed from the mean by <15%. COS cell culture media containing CD80Ig, CD86Ig, or CD80VIg mutants were assayed for their ability to bind CTLA4 mIg coated on microtiter plates at 0.25 µg/ml for 16 h at 4 °C, using assay conditions described previously(22) . Binding of mutants was compared with wild-type CD80Ig or CD86Ig. Experiments were repeated at least twice. A similar immunoassay was used to measure binding of CD80Ig, CD86Ig, and CD80VIg mutants to CD28 mIg coated on microtiter plates at 10 µg/ml. mAb binding to mutant CD80Ig and CD86Ig was assayed by initially coating microtiter plates with 0.5 µg/ml goat anti-human IgG (Jackson ImmunoResearch Labs., Inc., West Chester, PA) for 16 h at 4 °C. After blocking, mutant Ig fusion proteins were added at 0.2 µg/ml for 1 h at 22 °C. Wells were washed with phosphate-buffered saline/Tween 20 and increasing amounts of mAb added and incubated at 22 °C for 1 h. After washing, horseradish peroxidase-conjugated goat anti-mouse IgG was added, and then horseradish peroxidase substrate was added and absorbances were measured(22) .

Metabolic Labeling and Autoradiography of SDS-polyacrylamide gel electrophoresis

COS cells were transfected with cDNA encoding mutant B7Ig fusion proteins, and 48 h later they were incubated in Cys/Met-free Dulbecco's modified Eagle's medium for 30 min. Cells were then incubated with TranS-label (100 µCi, ICN Biomedicals, Inc., Costa Mesa, CA) for 16 h. Fusion proteins were harvested from culture media using protein A-Sepharose as described previously (23) and then subjected to SDS-polyacrylamide gel electrophoresis and visualized by autoradiography.

CD80 IgC-like Domain Molecular Model

A three-dimensional model of the CD80 IgC-like domain was generated by homology modeling (24) on the basis of the crystal structure of the class 1 beta(2)-microglobulin domain using a previously reported sequence alignment(12) .


RESULTS AND DISCUSSION

Construction of CD80Ig and CD86IgV Domain Mutants

CD80 and CD86 IgV domain residues were selected for mutagenesis based on sequence alignments, mapping of residues to potentially surface accessible positions, and the premise that sequence conservation between molecules, outside IgSF consensus residues, is of functional significance. Due to the lack of sequence-structure compatibility of regions of CD80 and CD86 to currently known three-dimensional IgSF V domain structures, structural homology criteria could not be applied in the selection of all IgV-like domain residues. Fig. 1shows the alignment of B7 family IgV-like and IgC-like domain sequences. Conserved non-IgSF consensus residues of the IgC domain (except Phe-108), selected here for mutagenesis (described in a later section), were predicted to be surface accessible. In addition to sequence conservation, Fig. 1illustrates a tentative assignment of residues to core beta-strands of Ig folds. Assignment of framework beta-strands was done on the basis of consensus residues of the IgSF V-set(25) , I-set(26) , and C1-set(25) . Putative assignment of IgV beta-strands do not necessarily imply close structural similarity (12) since the structural core common to known IgSF folds only includes the four central beta-strands B, C, E, and F(27) . A total of 24 CD80 V domain and 10 CD86 V domain residues were selected for mutagenesis. CD80 and CD86 mutants are numbered from the mature NH(2) terminus of the respective proteins.


Figure 1: Sequence alignment of the extracellular regions of CD80/CD86 family members. Sequences are aligned from the NH(2) terminus of mature human CD80 (8) and that predicted for human CD86(10) . Residues at the end of each row are numbered from their respective NH(2) terminus. Tentative beta-strand assignment of residues in IgV/I-set (amino-terminal) and IgC1-set (carboxyl-terminal) domains are indicated; G-strand assignment in the IgV domain was possible using only I-set consensus residues. The relative position of the exon boundary between the two domains is indicated with an arrowhead. Areas with a dashedunderline highlight positions that could not be assigned to an IgV domain (structurally ambiguous) based on the absence of IgSF consensus residues(13) . Darkboxedareas highlight residues common to other IgV/I-like or IgC-like domains; open and shadedboxedareas highlight additional residues completely conserved and conservatively substituted within the B7 family, respectively. Asterisks highlight human CD80 residues selected for mutagenesis. Accession numbers of sequences are described elsewhere(13) .



Site-directed mutants of soluble Ig fusions of CD80 and CD86 were prepared using PCR oligonucleotide primer-directed mutagenesis. The concentration of each mutant Ig fusion protein in serum-free COS cell culture media was determined by an Ig quantitation assay, and the mutant was tested for its ability to bind CTLA4Ig and CD28Ig in enzyme immunoassays. The structural integrity of mutant fusion proteins was assessed by testing their ability to bind mAbs specific for CD80 (BB-1, IgG; BB-1, IgM; 104; CD80.3), or CD86 (IT2.2) in indirect immunoassays. Failure of mAbs to bind suggested that either the mutations directly disrupted the mAb epitope, or that the mutation had compromised the structural integrity of the molecule. All CD80 IgV domain mutants except V39R, V39A, Y53A, and D60A, bound at least one of the anti-CD80 mAbs at levels 50-100% of wild-type CD80Ig. Asp-60 is charged and is predicted to map to a loop region between putative beta-strands D and E (Fig. 1). Thus, global conformational defects caused by mutagenesis of this residue are unlikely. Since Val-39 and Tyr-53 map to a structurally ambiguous region (Fig. 1), it is less clear whether mutation of these residues would affect conformation of the molecule. All CD86 IgV domain mutants except Y59A bound mAb IT2.2. Tyr-59, like the equivalent Tyr-53 in CD80, maps to a structurally ambiguous region, so the effect of its mutation on conformation of the molecule is difficult to assess. All CD80 and CD86 mutants bound anti-human Fc antibodies indicating the presence of this domain. S metabolic labeling of transfected COS cells followed by protein A purification from conditioned media and autoradiography of SDS-polyacrylamide gel electrophoresis, showed that all mutant proteins migrated as M(r) 70,000 proteins (data not shown).

Conserved Residues in the CD80 V Domain Are Important for Binding to CTLA4Ig

The ability of CD80 V domain mutants to bind immobilized CTLA4Ig was determined by indirect immunoassays. Of the 24 CD80 mutants prepared, nine bound to CTLA4Ig similar to wild-type CD80Ig, while 12 disrupted binding (>10-fold loss, Table 1). Mutations that disrupted binding included fully conserved, partially conserved, and nonconserved residues. Five mutations that disrupted CTLA4Ig binding involved completely conserved residues, Q33A, V39R, V39A, Y53A, and D60A (Table 1). Mutation of another fully conserved residue, L40A, had no effect on CTLA4Ig binding. Four other mutations of residues fully conserved only in CD80 family members, R29A, Y31A, W50A, and K86A, disrupted CTLA4Ig binding (Table 1, Fig. 2A). Other mutations that disrupted CTLA4Ig binding included L25A, M38A, and I49A, none of which are fully conserved (Fig. 1). Ile-67 is a putative IgV fold consensus residue(25) , and, as to be expected, its mutagenesis also reduced CTLA4Ig binding (Table 1). Mutagenesis of other residues, for example, Asn-19, Lys-37, and Asn-55 had little effect on receptor binding (Table 1, Fig. 2A).




Figure 2: Representative CD80 IgV domain mutants binding to CTLA4Ig and CD28Ig. Site-directed mutant fusion proteins were produced in transiently transfected COS cells, quantitated, and tested for their ability to bind to CTLA4Ig (A) and CD28Ig (B) immobilized on 96-well plates. Fusion proteins were quantitated as described under ``Materials and Methods.'' Binding data (representative of at least three experiments) are expressed as the average of duplicate determinations and replicates differed from the mean by <10%.



These results led to two conclusions. First, the large number of mutations that disrupted receptor binding suggested the presence of an extensive contact area between CD80 and CTLA4Ig. Second, seven of the residues have hydrophobic side chains, suggesting that hydrophobic interactions are important determinants of CTLA-4/CD80 binding. This is consistent with the role of the hydrophobic MYPPPY region in CTLA-4/CD28 binding to CD80(22) .

Conserved Residues in the CD86 V Domain Are Important for Binding to CTLA4Ig

The finding that eight of the residues that disrupt binding of CD80 are completely conserved or conservatively substituted in the B7 family suggested that the equivalent amino acids in CD86 may also be important for receptor binding. We therefore mutated several conserved and nonconserved residues in the CD86 IgV domain and assessed the ability of the mutants to bind immobilized CTLA4Ig by indirect immunoassays. Of the 10 CD86 mutants prepared, three mutations of rigorously conserved residues, Q35A, V41A, and Y59A, and two mutations of conservatively substituted residues, F33A and H91A (Y31A and K86A in CD80, respectively), disrupted CTLA4Ig binding (>10-fold, Table 1). Mutation of L72A also disrupted receptor binding, but this was probably due to disruption of the structural integrity of the molecule since Leu-72 is an Ig fold consensus residue. These results parallel those obtained for the equivalent CD80 mutants and confirm that CD80 and CD86 utilize conserved residues in their IgV domains for CTLA4Ig binding.

However, binding of CD80 and CD86 to CTLA4Ig is not completely equivalent. CD80 and CD86 bind their counter-receptors with different kinetics and bind differently to the CTLA4Ig mutant Y100A(18) . These observations suggest that CD80 and CD86 differ in their interaction with CTLA4Ig. We have identified three mutations in CD86Ig that had different effects than mutation of the equivalent residues in CD80Ig. D66A in CD86Ig resulted in 30% of wild-type binding, but the equivalent mutation D60A in CD80Ig completely prevented CTLA4Ig binding (Table 1). CD86Ig mutant H56A had wild-type CTLA4Ig binding activity, while mutagenesis of the equivalent residue in CD80Ig, W50A, resulted in >10-fold loss of binding (Table 1). Finally, the CD86Ig mutation G61N bound CTLA4Ig about one-third of wild-type levels, but substitution of the equivalent residue in CD80Ig (N55A) increased receptor binding relative to wild-type CD80Ig (1.5-fold, Table 1). These molecular differences may contribute to the different binding properties of CTLA4Ig for CD80 and CD86.

CD80 and CD86 V Domain Mutations Had Similar Effects on CTLA4Ig and CD28Ig Binding

We addressed whether residues in the IgV domain of CD80 and CD86, identified as important for CTLA4Ig binding, were also important determinants for CD28Ig binding. The ability of mutants to bind immobilized CD28Ig was assessed by indirect immunoassays. Despite some quantitative differences, mutations that affected CTLA4Ig binding generally had an equivalent effect on CD28Ig binding ( Table 1and Fig. 2B). The results suggest that an overlapping set of residues on CD80 and CD86 determine the binding to both CTLA4Ig and CD28Ig.

Mapping CD80 IgV Domain Mutants

CD80 residues whose mutagenesis affected CTLA4Ig/CD28Ig binding were mapped on a schematic representation of an IgV fold. The resolution of this schematic is limited by the lack of structural data on the CD80 IgV-like domain (12) . Mutations that disrupted binding clustered in two groups (Fig. 3). The first group maps to loops at the amino-terminal end of the domain and included Leu-25 and Asn-63, and the charged amino acids Arg-29, Asp-60, and Lys-86. The second group includes Gln-33 and the hydrophobic/aromatic residues Tyr-31, Met-38, Val-39, Met-47, Ile-49, Trp-50, and Tyr-53. These amino acids are located on beta-strand C and in the structurally ambiguous region between beta-strands C and D. The overall picture suggests that receptor interactions are most likely centered on the GFCC`C`` face of the IgV domain including amino-terminal loop residues. We did not perform extensive mutagenesis of residues on the opposite BED face as most of the conserved amino acids predicted to lie on this face are IgSF consensus residues (Fig. 1). However, mutagenesis of residues that map to the margins of the BED face, N19A, N55A, and L65A, did not destroy receptor binding. Asn-19 and Asn-55 are potential N-linked glycosylation sites as is Asn-64. Since these residues map within the margins of the BED beta-sheet face (Fig. 3), attachment of carbohydrate moieties is unlikely to influence receptor binding to the opposite GFCC`C`` face. The GFCC`C`` beta-sheet face of IgV domains mediate interactions between other IgSF members(28, 29) .


Figure 3: Mapping of CD80 site-directed mutants onto a schematic representation of an IgV-fold. Residues in the CD80 amino-terminal (or V-like) domain whose mutation affects ligand binding (red) are mapped onto approximately corresponding positions of an IgV-fold. Potential N-linked glycosylation sites are blue. The beta-strands and termini of the domain are labeled. Regions that can be assigned to an IgV-fold are shown in yellow; regions where no V-set consensus residues are present are shown in gray.



Construction and Binding of CD80VIg to CTLA-4 and CD28

A previous study identified a murine CD80 splice variant, lacking the entire membrane proximal IgC domain, that had altered receptor binding characteristics(30) . To further assess a potential role for the IgC domain in receptor binding, a human CD80Ig fusion protein lacking this region was prepared using PCR. The CD80 deletion mutant (CD80VIg), harvested from transfected COS cell culture media, migrated under reducing conditions on SDS-polyacrylamide gel electrophoresis with a M(r) 50,000 compared with the 70,000 seen for CD80Ig. CD80-specific mAbs all showed greatly reduced binding to CD80VIg, indicating that the presence of the IgC domain was essential to maintain intact mAb epitopes. However, CD80VIg bound CTLA4Ig and CD28Ig with 2 and 6% activity relative to wild-type CD80Ig, respectively (Fig. 4, A and B, and Table 1). Thus, the presence of the IgC domain of CD80 was necessary for full receptor binding.


Figure 4: CD80 mutagenesis demonstrating that deletion of IgC-like domain diminishes but does not eliminate binding to CTLA4Ig and CD28Ig. Soluble CD80VIg lacking the IgC-like domain was produced and assayed for its ability to bind CTLA4Ig (A) and CD28Ig (B) as described in Fig. 2. Data are expressed as the average of duplicate determinations and differed from the mean by <10%. The data are representative of three separate experiments.



Characterization and Binding of CD80 IgC Domain Mutants to CTLA4Ig and CD28Ig

To assess a potential role for specific amino acids in the IgC domain of CD80Ig in receptor binding, nine highly conserved residues were mutated to alanine (Fig. 1). Eight of these were predicted to be surface accessible(12) . Anti-human Fc antibodies bound to each mutant fusion protein, while CD80-specific mAbs bound to six of nine mutants, the exceptions being F108A, P111A, and I113A. Lack of mAb binding to the F108A mutant probably indicates that this residue contributes to the hydrophobicity of the core of the C domain, since its side chain is predicted to be buried. Pro-111 and Ile-113 are predicted to map to surface accessible positions, suggesting that their mutagenesis may have directly affected the epitopes recognized by each of the anti-CD80 mAbs tested.

Mutagenesis of hydrophobic residues, Phe-108, Pro-111, and Ile-113 abolished CTLA4Ig binding, while Q157A, D158A, E162A, and L163 bound to CTLA4Ig at 6-35% relative to wild-type CD80Ig (Fig. 5A and Table 1). Mutagenesis of Ser-156 and Ser-167 resulted in 2-3-fold increased binding to CTLA4Ig. Perhaps increased hydrophobicity of this region because of the serine to alanine mutation enhanced receptor binding. The effects of mutagenesis on CD28Ig binding were more dramatic. All mutations, except S156A and S167A, significantly reduced or completely abolished CD28Ig binding (Fig. 5B and Table 1). While deletion of the CD80 IgC domain had a greater effect on binding to CTLA4Ig than CD28Ig, point mutagenesis of residues in the IgC domain had the opposite effect. Thus, some conserved residues in the IgC domain of CD80Ig play an important role in CTLA4Ig and CD28Ig binding.


Figure 5: Mutagenesis in CD80 IgC-like domain demonstrates the involvement of specific residues in CTLA4Ig and CD28Ig binding. Site-directed mutant fusion proteins were prepared and assayed for their ability to bind CTLA4Ig (A) and CD28Ig (B) as described in Fig. 2. Data are expressed as the average of duplicate determinations and differed from the mean by <10%. The data are representative of at least four experiments.



Mapping CD80 IgC Domain Mutants

CD80 IgC domain mutants affecting ligand binding were mapped on a three-dimensional model (Fig. 6). Predictions based on this model establish that Gln-157, Asp-158, Glu-162, and Leu-163 spatially cluster in surface accessible positions near the amino-terminal end of the domain in the loop between beta-strands D and E and at the beginning of beta-strand E. Other residues, (Phe-108, Pro-111, and Ile-113), mapped to beta-strand A. Thus, residues important for receptor binding map to the ABED face of the IgC domain, the opposite face from that shown for the IgV domain. Reversed orientation of the two domains would allow the GFCC`C`` face of the IgV domain and the ABED face of the IgC domain to align on one side of the molecule. Such an orientation would be reminiscent of that seen in crystal structures of D1D2 and D3D4 of human and rat CD4 domains(31, 32) . Five potential N-linked glycosylation sites in the IgC domain (Asn-152, Asn-173, Asn-177, Asn-192, Asn-198), mapped predominantly to the GFC face, opposite the ABED face. Thus, carbohydrate is probably not involved in the CD80-receptor interaction.


Figure 6: Mapping of CD80 site-directed mutants onto a schematic representation of the IgC domain. Residues in the C-like domain of CD80 whose mutation affect ligand binding are mapped onto a three-dimensional model of the domain (generated on the basis of sequence structure compatibility with beta(2)-microglobulin, (12) ). Mutants and potential N-linked glycosylation sites are color coded according to Fig. 3. With the exception of Phe-108, all residues are predicted to be accessible on the domain surface.




CONCLUSION

We have shown that the IgC domain of CD80 exerts considerable influence on CTLA4Ig and CD28Ig binding. Complete absence of the IgC domain in CD80VIg resulted in >10-fold reduced binding to both CTLA4Ig and CD28Ig. Thus, with human CD80, the presence of both the amino-terminal IgV and membrane proximal IgC domains was essential for full binding to CTLA4Ig/CD28Ig. However, several conserved non-IgSF consensus residues in the IgC domain were identified whose mutagenesis completely abolished CTLA4Ig/CD28Ig binding in our assays. Thus, point mutations in the IgC domain had a greater effect on receptor binding than complete removal of the domain. It is possible that mutagenesis of some residues in the IgC domain led to structural defects affecting the whole extracellular region. More likely, however, this implies that the IgC domain participates in the presentation of the receptor binding site.

Several possible interactions could occur between CD80 and CTLA-4/CD28. Since deletion of the IgC domain and mutagenesis of residues in both CD80 domains affected receptor binding, neither domain can act independently to provide full CTLA4Ig or CD28Ig binding. Instead, the results favor an interaction where both CD80 IgV and IgC domains contribute to binding. Rather than spatial separation of the IgV and IgC domains, the IgC domain may support binding by forming an extensive interface between the domains, similar to that seen with CD4 and tissue factor(31, 32, 33) . This domain interface could support CTLA-4/CD28 binding by, for example, shielding the hydrophobic binding face in the amino-terminal IgV domain from solvent. The consensus hydrophobic residues in the CD80 IgC domain, identified here as critical for receptor binding, may contribute to the formation of the domain interface. Changes in the domain-domain orientation as a consequence of interface mutations could prohibit counter-receptor binding. Alternatively, these hydrophobic residues may be directly associating with CTLA-4 and CD28, particularly via the hydrophobic MYPPPY loop. However, if these and other consensus residues in the IgC domain were directly interacting with CTLA-4 and CD28, they probably do not contribute significantly to the overall binding affinity, as the IgV domain alone can sustain some receptor binding activity.

Evidence for a close association of the IgV and IgC domains of B7 molecules comes from assignment of putative beta-strands in the two domains. Overlapping assignment of residues can be made of beta-strand G in the IgV domain with beta-strand A in the IgC domain (Fig. 1). An exon boundary, which often defines domain limits, is found after the first nucleotide of the triplet codon encoding alanine 106(19) , a residue assigned to beta-strand A in the IgC domain. However, the upstream amino acid valine 104, an IgV-fold G strand consensus residue, can also be assigned to beta-strand A of the IgC domain. This is analogous to the closely associated D1D2 and D3D4 Ig-like domains seen in the crystal structures of human and rat CD4(31, 32) .

Both CD80 and CD86 are extensively glycosylated(11) , but potential N-linked glycosylation sites in the CD80 IgV and IgC domains are localized to regions essentially opposite the site of receptor interaction, and are therefore unlikely to modulate ligand binding. Extensive glycosylation of CD80 and CD86 may instead be required to aid in solubility since their extracellular domains probably contain a number of hydrophobic residues on their surfaces. CTLA-4 and CD28 are also glycosylated. Removal of N-linked glycosylation sites by mutagenesis of CTLA4Ig does not alter its ability to bind CD80 or CD86. (^3)Thus carbohydrate moieties are not likely to play a role in B7/CTLA-4/CD28 interactions.

Reversed orientation of the CD80 IgV and IgC domains would allow residues that affect receptor binding to align on one face of the molecule. While we have not extensively mutated residues on the opposite faces of these domains, we believe they are unlikely to be involved in receptor binding for two reasons. First, the conserved residues on this face, particularly in the IgV domain, are predominantly IgSF consensus residues; these most likely function to maintain structural stability rather than to mediate receptor binding. Second, bulky carbohydrate moieties that could mask receptor binding sites, map to the opposite faces. The precise nature of the interaction of the single IgSF domain receptors, CTLA-4 and CD28, with multiple domains of B7-related molecules is yet to be determined. However, the present study provides insights into the molecular nature of these interactions and establishes the basis of future structural studies.


FOOTNOTES

*
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: Bristol-Myers Squibb Pharmaceutical Research Inst., 3005 First Ave., Seattle, WA 98121. Tel: 206-727-3565; Fax: 206-727-3501.

(^1)
The abbreviations used are: IgSF, immunoglobulin superfamily; mAb, monoclonal antibody; PCR, polymerase chain reaction.

(^3)
R. J. Peach, G. Leytze, and P. S. Linsley, unpublished results.


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