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J. Biol. Chem., Vol. 275, Issue 36, 28100-28109, September 8, 2000

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Signaling Lymphocytic Activation Molecule (CDw150) Is Homophilic but Self-associates with Very Low Affinity^*

Nasim Mavaddat^§, Don W. Mason, Paul D. Atkinson^¶, Edward J. Evans^§¶, Robert J. C. Gilbert^**, David I. Stuart^**, Janet A. Fennelly^¶, A. Neil Barclay, Simm J. Davis^¶§§, and Marion H. Brown

From the  Sir William Dunn School of Pathology, The University of Oxford, Oxford OX1 3RE, United Kingdom, ^¶ Molecular Sciences Division, Nuffield Department of Clinical Medicine, The University of Oxford, Oxford OX3 9DU, United Kingdom, ^** Division of Structural Biology, Wellcome Trust Centre for Human Genetics, The University of Oxford, Roosevelt Drive, Oxford OX3 7BN, United Kingdom, and the  Oxford Centre for Molecular Sciences, The University of Oxford, The Rex Richards Building, South Parks Road, Oxford OX1 3QU, United Kingdom

Received for publication, May 15, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Signaling lymphocytic activating molecule ((SLAM) CDw150) is a glycoprotein that belongs to the CD2 subset of the immunoglobulin superfamily and is expressed on the surface of activated T- and B-cells. It has been proposed that SLAM is homophilic and required for bidirectional signaling during T- and B-cell activation. Previous work has suggested that the affinity of SLAM self-association might be unusually high, undermining the concept that protein interactions mediating transient cell-cell contacts, such as those involving leukocytes, have to be weak in order that such contacts are readily reversible. Using surface plasmon resonance-based methods and analytical ultracentrifugation (AUC), we confirm that SLAM is homophilic. However, we also establish a new theoretical treatment of surface plasmon resonance-derived homophilic binding data, which indicates that SLAM-SLAM interactions (solution K_d ~200 µM) are in fact considerably weaker than most other well characterized protein-protein interactions at the cell surface (solution K_d ~0.4-20 µM), a conclusion that is supported by the AUC analysis. Whereas further analysis of the AUC data imply that SLAM could form "head to head" dimers spanning adjacent cells, the very low affinity raises important questions regarding the physiological role and/or properties of such interactions.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Transient, low affinity interactions between the proteins present on the surfaces of leukocytes or other cell types are critical for the normal functioning of the immune system (1). Among the best characterized interactions of this type are those mediated by the CD2 subset of the immunoglobulin superfamily (IgSF)¹ (2, 3). Proteins in this subset are characterized by the presence of paired membrane-distal V set and membrane-proximal C2 set IgSF domains with distinctive disulphide bond arrangements (4), and the subset now includes CD2, CD58, CD48, 2B4, SLAM (CDw150), CD84, and Ly-9.

It is postulated that the CD2 subset arose via successive duplications of a common ancestral gene originally encoding a homophilic cell adhesion molecule (5). In addition to sequence and structural relationships, this proposal is supported by the fact that many of the genes encoding these molecules are clustered at two duplicated loci in humans and mice and that most, if not all, of the interactions of these proteins are restricted to one or more members of the CD2 subset (6, 7). Substantial overlap in the ligand binding specificities and signaling properties of these molecules further underscores their close evolutionary history. For example, murine CD2 and 2B4 both bind CD48 (6), the cytoplasmic domains of 2B4 and SLAM each bind an adaptor signaling protein known as SLAM-associated protein (8, 9), and ligand or antibody-mediated cross-linking of human CD2 (reviewed in Ref. 10) or SLAM (11) induces T-cell proliferation. Finally, rat and human soluble (s) CD2 crystals are dominated by homophilic head to head crystal lattice contacts proposed (12, 13), and subsequently shown (14), to mimic the topology of natural ligand interactions. Previously, however, there has been no clear evidence that any homophilic interactions of potential physiological significance occur among the existing members of this subset of the IgSF.

SLAM was initially identified with a monoclonal antibody (mAb) that activated T-cells and bound a previously uncharacterized activation antigen (11). It is expressed by CD45RO+ T-cells, immature thymocytes, and a proportion of B-cells and is rapidly up-regulated upon the activation of T-cells, B-cells, and dendritic cells (11, 15, 16) but not NK cells (17). It has been shown in mice that highly polarized Th1 but not Th2 cells express high levels of SLAM (17). Cross-linking studies with mAbs have suggested that SLAM is a receptor that influences T-cell (18, 19) and B-cell responses to antigen (16, 20). Activated human T-cells and B-cells express mRNA species encoding three forms of SLAM, a membrane-bound protein encoded by the longest open reading frame and containing three consensus cytoplasmic Src homology 2 domain binding sequences, a variant membrane-anchored form with a truncated cytoplasmic domain, and a soluble, secreted form of SLAM (11, 15). One or more of the Src homology 2 domain binding (TIYXX(V/I)) sequences, which are also present in the cytoplasmic domains of 2B4, CD84, and Ly-9 (21), bind the Src homology 2 domain of SLAM-associated protein (8, 21, 22). It is proposed that signal transduction by SLAM is regulated via a novel mechanism in which SLAM-associated protein competes with an Src homology 2 domain-containing phosphatase (8, 20) for the TIYXX(V/I) sequences (8). Defects in the SLAM-associated protein gene are responsible for an X-linked, lymphoproliferative syndrome characterized by enhanced susceptibility to primary Epstein-Barr virus infections in humans (8, 23, 24).

The prevailing view of the function of SLAM is that it is homophilic and that through bidirectional signaling it regulates T- and B-cell responses (8). Preliminary data implying that the affinity might be as high as 10¹⁰ M¹ (15) argue against a role in T-cell-B-cell interactions, however, since protein contacts in this context generally have much lower affinities, presumably to ensure that such interactions are readily reversible (1). To resolve this issue, we have characterized the self-association of soluble (s) forms of SLAM using SPR-based methods and analytical ultracentrifugation. This analysis, for which a new theoretical framework for characterizing homophilic protein-protein interactions using SPR-based methods had to be established, indicates that SLAM self-associates with an affinity much lower than was previously anticipated.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Monoclonal Antibodies

mAbs used were: SLAM mAbs IPO-3 (16) (Kamiya Biomedical Company, Seattle, WA) and A12 (11) (kindly provided by DNAX, Palo Alto, CA); rat CD4 mAb, OX68; and rat CD2 mAb, OX34. The OX mAbs are referenced in the European Collection of Animal Cell Cultures. IgG FITC goat anti-mouse was supplied by Serotec (Kidlington, UK).

Expression of Recombinant Proteins

Chimeric Protein-- Vectors for expressing a chimeric form of SLAM (sSLAMCD4) consisting of the entire extracellular region fused with domains 3 and 4 of rat CD4 and containing a COOH-terminal biotinylation sequence were constructed as described (6, 25) from human SLAM cDNA ((11) kindly provided by DNAX, Palo Alto, CA). The join at the SalI (g tcg acc) junction of SLAM with CD4 was SLAM: DPSST (the residues containing the SalI site are underlined).

sSLAM-- The extracellular region of SLAM (sSLAM) was amplified by polymerase chain reaction from cDNA prepared from MT-2 cells (human T-cell lymphotrophic virus-1-transformed human T-cells). The 5'-primer was complementary to the SLAM leader sequence (MDPKGLLS (11)), added an XbaI site, and inserted, immediately upstream of the initiation codon, the 25 bases that precede the rat CD4 initiation codon (26). The 3'-primer was complementary to the membrane proximal SPWPGCRTDPSETK-encoding sequence and added nucleotides encoding a short linker (amino acids GGG), the BirA biotinylation signal sequence (amino acids LNDIFEAQKIEW (27)), a tag consisting of six histidines, a stop codon, and another XbaI site. The polymerase chain reaction fragment was subcloned into the glutamine synthetase-based gene expression vector, pEE14 (28). All constructs were checked by dideoxy sequencing.

Chimeric proteins were expressed by transiently transfecting 293T-cells with 40 µg of plasmid DNA/5 × 10⁶ cells/175-cm² flask using calcium phosphate as described (6). Chinese hamster ovary-K1 cells were transfected with the pEE14 construct (28, 29), and methionine sulfoximine-resistant clones were screened for expression by Western blotting of the tissue culture supernatant with an anti-penta-His antibody (Qiagen GmbH). The best clone was grown to confluence in bulk culture before switching to medium supplemented with 2 mM sodium butyrate, as described (29).

Protein Purification, His Tag Removal, and Biotinylation

sSLAM was purified from spent tissue culture supernatant by affinity chromatography using nickel-nitrilotriacetic acid resin (Qiagen GmbH) followed by size exclusion chromatography on a Superdex 200 HR 10/30 column. When required, the histidine tag at the carboxyl terminus was removed by incubating 2.5 mg of sSLAM in 1.5 ml of Hepes-saline buffer (10 mM Hepes, pH 7.4, 150 mM NaCl) with 1.2 units of carboxypeptidase A conjugated to agarose beads (Sigma) for 16 h at 30 °C with agitation. Removal of the carboxyl-terminal histidines was at least 80% efficient according to amino acid analysis. The extinction coefficient of the detagged protein was determined by amino acid analysis to be 1.03 cm²/mg. Prior to BIAcore^TM analysis, sSLAM was passed through a Superdex 75 gel filtration column to remove aggregated protein (Amersham Pharmacia Biotech). For immobilization in BIAcore^TM experiments, purified sSLAM or sSLAMCD4 in concentrated tissue culture supernatant were biotinylated by incubation with recombinant BirA enzyme (obtained from Avidity, Denver, CO or as a kind gift from Dr C. A. O'Callaghan) in 10 mM Tris-HCl, pH 8, 7.5 mM MgCl₂, 5 mM NaCl, 5 mM ATP, and 1 mM biotin, overnight (6, 30). The protein was then buffer-exchanged into Hepes-buffered saline to remove free biotin prior to use.

General SPR Methods

All binding experiments were carried out on a BIAcore 2000 instrument (BIAcore AB, St. Albans, UK) using Hepes-buffered saline buffer, 25 mM Hepes, pH 7.4, 150 mM NaCl, 3.4 mM EDTA, and 0.005% surfactant P20 supplied by the manufacturer. Streptavidin (Pierce) was coupled at 0.2 mg/ml in 10 mM sodium acetate, pH 5, to research grade CM5 chips (BIAcore AB) using amine coupling kits (BIAcore AB) and an activation time of 5 min, resulting in immobilization levels of ~3000-4000 response units (RU). Equilibrium binding analysis was undertaken as described (7); increasing and decreasing concentrations of sSLAM (5-µl injections at a flow rate of 20 µl/min) were passed over proteins immobilized at high, intermediate, and low levels. For kinetic analysis, dissociation rates were measured as described (7). Experiments were performed at 25 °C unless indicated. Affinity and kinetic data were analyzed using the curve fitting tools of Origin v.5.0 (MicroCal Software Inc, Northampton, MA).

A Theoretical Framework for SPR-based Analysis of Homophilic Interactions

Assuming that all of the molecules of immobilized biotinylated sSLAM (sSLAMb) are equally available to take part in homophilic interactions, the relevant equations at equilibrium are as follows for interactions between molecules in solution.

(Eq. 1)

The following is for interactions between immobilized molecules.

(Eq. 2)

The interactions between soluble and immobilized molecules is as follows,

(Eq. 3)

where [A_s] and [A_f] are the concentrations of monomeric soluble and monomeric immobilized molecules, respectively, and [A_s · A_s], [A_f · A_f], and [A_s · A_f] are the concentrations of the three species of dimeric complexes. The corresponding dissociation constants are designated K_d1, K_d2, and K_d3. In keeping with the general interpretation of BIAcore^TM data, in what follows K_d3 will be assumed to be equal to K_d1. The relationship between K_d1 and K_d2 will be considered when the experimental data are discussed.

In calculating [A_s] and [A_s · A_s] from Equation 1, use is made of the fact that, at equilibrium, the concentration of these molecular species is the same in the chip as it is in the solution passing through it. Putting [A_s] = [A_so]  2[A_s · A_s], where [A_so] is the total concentration of molecules in the soluble phase, Equation 1 can be solved to give the following.

(Eq. 4)

Similarly, Equations 2 and 3 yield the following, after some rearrangement,

(Eq. 5)

where [A_fo] is the total concentration of immobilized molecules on the chip, and [A_s] is given by Equation 4.

Equations 4 and 5, which give [A_s] and [A_f] in terms of the two concentrations [A_so], [A_fo], and the two dissociation constants, K_d1 and K_d2, are used, in Equation 6 below, to give theoretical values for the response units obtained from the BIAcore^TM. Equation 6 is

(Eq. 6)

where P is the number of response units/M of dimeric molecules formed by association between molecules in solution and those immobilized on the chip. For a molecule with an M_r of 30,000 the value of P is taken to be 3 RU/µM. Titration of the sSLAM signal in the reference flow cell (see "Results"; Fig. 2B) allowed accurate determination of P and the level of sSLAMb immobilized (i.e. [A_fo]).

With these substitutions, the full binding equation is shown below.

(Eq. 7)

To derive K_d1 and K_d2 from the experimental data involves choosing values of these parameters that best fit the observations. This choice is facilitated by the fact that at low values of [A_so] the soluble molecule is virtually all in monomeric form, that is [A_s] approximately equals [A_so]. Further, the value of [A_f] is given by the following.

(Eq. 8)

It follows that, at values of [A_so] much lower than the K_d, the fraction [A_so]_bound/[A_so]_free (defined as g), is given by the following.

(Eq. 9)

Equation 9 may be rearranged to give the following.

(Eq. 10)

This expression may be used to express K_d1 in terms of g and K_d2 in Equation 7. The result is as follows.

(Eq. 11)

If the value of g, for a given value of [A_fo], is determined experimentally, Equation 11 may be used to calculate response units for various values of [A_so], for any chosen value of K_d1. In practice a curve-fitting computer program is used to determine the value of K_d1 that best fits the observed dependence of RU on [A_so].

Alternatively, if g is measured for two or more different values of [A_fo], Equation 10 may be used to calculate K_d1 and K_d2. Thus, if g_a and g_b are two values of g, obtained by using two different concentrations, [A_fo]_a and [A_fo]_b of immobilized molecules on the chip, then the equation shown below follows from Equation 10.

(Eq. 12)

Alternatively, if K_d1 is assumed to be equal to K_d2, then again the following is from Equation 10.

(Eq. 13)

Analysis of the experimental results for sSLAM indicates that they are compatible with the two dissociation constants being equal. Consequently, Equation 13 can be used to calculate K_d1 from the RU values obtained at low concentrations of sSLAM passed through the BIAcore^TM chip. More generally, if K_d1 and K_d2 are not assumed to be equal, Equation 11 must be used to evaluate K_d1.

Calculation of the Effective Concentration of the Immobilized Phase

To set up the immobilized phase (A_f), sSLAMb was generated by biotinylating the COOH terminus of sSLAM to anchor it to streptavidin covalently attached to the sensor surface. Streptavidin has four biotin binding sites, but considerations of steric hindrance strongly suggest that at most three of these remain accessible to biotin after immobilizing the streptavidin to the chip. Consequently, the immobilized sSLAMb probably exists in monomeric, dimeric, and trimeric forms. The presence of the bivalent and trivalent forms introduces a complication in calculating equilibrium constants for the immobilized sSLAMb molecules in that these multimers may be expected to make more stable homophilic complexes between themselves than does the monomeric form of sSLAMb. To take this complexity into account, we assume that only the monomeric form of immobilized sSLAMb is available to react with the soluble sSLAM passing through the chip.

If [B] is the total molar concentration of sSLAMb on the chip and [Av] is the concentration of streptavidin, then, assuming that three biotin binding sites remain accessible on each streptavidin molecule, the proportion of the total immobilized sSLAMb that exists as a monomer is given by the following.

(Eq. 14)

The application of Equation 10 to the experimental data given in "Results" indicates that, at [sSLAMb_total] values of 82.7, 186, and 438 µM, the fraction of the total immobilized sSLAMb that exists as a monomer is 0.91, 0.80, and 0.56, respectively. The corresponding values for the effective concentrations of sSLAMb are 75, 148, and 246 µM. A correction of this magnitude is clearly essential because without it the application of Equation 12 to the experimental data obtained with 82.7 µM and 438 µM sSLAMb immobilized predicts a negative value for K_d1. In contrast, the application of Equation 12 to the 82.7 and 186 µM results gives a positive value for the same dissociation constant. Similarly, uncorrected values of [A_fo] substituted in Equation 9 give very disparate values for the ratio of K_d1/K_d2 when the observed values of g are used to calculate it.

An Alternative Binding Model

In the preceding analysis it was assumed that those molecules of immobilized sSLAMb, which were not involved in the multimeric homophilic interactions described above, were available to react both with each other and equally with sSLAM passing through the chip. This assumption would be invalid if the multimeric interactions imposed some conformational restraint on the gel matrix that impeded homophilic interactions between molecules of immobilized sSLAMb but not between immobilized and soluble forms of the molecule. To analyze this model, K_d2 was taken to be essentially infinite. In this case the binding equation reduces to the following.

(Eq. 15)

The term [A_fo] is equal to g × K_d1, where g is, as before, the observed ratio of bound to free sSLAM at low concentrations of sSLAM. With this equality substituted into Equation 15, a comparison can be made between the experimental data and the prediction of this alternative model, without an explicit value of [A_fo] being required. Application of this model to the experimental data demonstrated that it was invalid because the values of K_d1 that it predicted yielded corresponding values of [A_fo] that were actually larger than those used in the experiments (see "Results").

Analytical Ultracentrifugation

Data Collection-- Samples to be analyzed by sedimentation velocity were contained in double sector centerpieces (pathlength of 3 or 12 mm depending on protein concentration) and spun at 40,000 rpm in a Beckman Optima XL-A analytical ultracentrifuge at 37 °C. The sedimentation of the protein boundary was observed using a variety of incident wavelengths of light, chosen to give an interpretable series of boundary traces for the experimental protein concentration. At high concentrations a schlieren signal was observed in the absorbance scans. This has been reported once before when it was shown that it is a function of the precise configuration of the AUC monochromator (31), which varies between instruments. This was controlled for by collecting data at different wavelengths and resulted in an additional peak in the g(s*) profile at low s* at wavelengths where a schlieren signal was elicited. The schlieren contribution was allowed for at this stage.

Data Analysis-- Data were analyzed using the time derivative (g(s*)_t) method calculated with the program dc/dt (32) wherein,

(Eq. 16)

and g(s*)_t (in arbitrary units) is the distribution of apparent sedimentation coefficients (s*) at the boundary calculated from the time derivative of a series of boundary traces, c is concentration (in absorbance units), r is radial distance from the center of the rotor (in cm), t is the time since the start of the experiment (in seconds), c₀ is the concentration at time 0,  is the rotor velocity (in radians/second), and r_m is the radius at the meniscus. The s* values determined by fitting the g(s*)_t profiles were plotted against protein concentration, extrapolated to zero concentration, and corrected for the effects of buffer density and viscosity and the temperature at which the experiment was performed to yield values of s_20,w⁰, the sedimentation coefficient in water at 293 K at infinite dilution. This value was used to calculate the frictional ratio (f/f₀) displayed by the two species using the equation (33),

(Eq. 17)

wherein M is the mass of the species (in g/mol), its partial specific volume (in ml/g), ₀ is the density of water, N_A is Avogadro's number, and ₀ is the viscosity of water (in poise). The frictional ratio is the ratio between the experimental frictional coefficient and that of a sphere with the same mass. This was corrected for a range of hydration factors to obtain the experimental Perrin function describing the axial ratio of the species (34),

(Eq. 18)

wherein P_exp is the Perrin function and _app is the apparent hydration (in g of H₂O/g of protein). Experimental values for the sedimentation coefficient in water at 293 K (see "Results") were also corrected for hydration using a similar relationship,

(Eq. 19)

where s₀ is the anhydrous sedimentation coefficient. All curve fitting was carried out using the nonlinear least-squares curve-fitting package, ProFit (Quantum Software, CA).

sSLAM Modeling-- A model for sSLAM was based on the structure of rat sCD2 (12) with eight octaglycyl chains modeled in random orientations at the positions that align with the glycosylation sequons seen in the SLAM protein sequence. The octasaccharides modeled at each position represent the average N-linked carbohydrate structure added by Chinese hamster ovary cells (35). This model was converted to hydrodynamic beads using the program AtoB (36), and hydrodynamic parameters were calculated using the program SOLPRO (37). A dimeric model was based on the complex between CD2 and CD58 (14) by aligning domain 1 of the monomeric model with the CD2 and CD58 domains present in the complex structure using the program SHP (38). The hydrodynamic parameters of this model were calculated in the same way as for the monomer.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

sSLAM Self-associates-- Two soluble, recombinant forms of human SLAM were prepared for this study, one consisting of the extracellular region of SLAM including a COOH-terminal histidine tag (sSLAM) and another, chimeric form consisting of the extracellular region fused to domains three and four of rat CD4 (sSLAMCD4). An additional sequence was incorporated at the COOH termini so that each protein could be biotinylated using the BirA enzyme (the biotinylated forms are called sSLAMb and sSLAMCD4b, respectively). Both sSLAMb and sSLAMCD4b immobilized on sensor surfaces via streptavidin bound strongly to two SLAM mAbs, A12 (11) and IPO-3 (16), which compete for identical or overlapping binding sites on the molecule (Fig. 1, A and B; data not shown). Assuming bivalent binding of the antibodies, the responses observed indicate that at least 70% of the immobilized SLAM was antigenically active. This, together with the high levels of expression of the two proteins, indicates that both proteins were correctly folded. On gel filtration, sSLAM eluted anomalously early at all concentrations (eluting at the same volume as a globular protein of 100 kDa; data not shown). In our experience (7), such behavior is characteristic of heavily glycosylated asymmetric proteins (sSLAM has eight potential N-glycosylation sites), and overall, the gel filtration data were consistent with sSLAM being monomeric at low protein concentrations.

View larger version (33K): [in this window] [in a new window]   Fig. 1.   sSLAM self-associates. SLAM mAbs A12 (100 µg/ml) (A) and IPO-3 (50 µg/ml) (B) were injected (solid bars) through flow cells with sSLAMCD4b immobilized at 2418 RU or 1130 RU, respectively, to confirm that the immobilized protein was correctly folded. sSLAM (76 µM) was also injected over immobilized sSLAMb (2280 RU) or a control protein, 2B4CD4b (2244 RU), before (C) and after (D) saturation binding of A12 mAb (injected at 100 µg/ml). In D the decreased response is likely to be the result of sSLAM preventing rebinding of the mAb to immobilized sSLAMb.

Changes in response observed when sSLAM protein was passed over immobilized sSLAMb, or a control protein, human 2B4CD4 (6), are shown in Fig. 1C. The control response is because of the high concentration of the protein injected into the flow cell; the difference in response between the two flow cells represents specific binding (self-association). Similar levels of binding were also observed when sSLAM was passed over immobilized sSLAMCD4b (data not shown). Binding was observed following removal of the histidine tag from sSLAM with carboxypeptidase A indicating that the responses were not because of artifactual histidine tag-mediated interactions (data not shown). Moreover, no binding was observed when an irrelevant histidine-tagged protein was injected over immobilized sSLAMb (data not shown). sSLAM also did not bind to the immobilized control proteins CD4 or human sCD84CD4 (39).² To further confirm the specificity of binding, saturating levels of SLAM mAbs were passed over immobilized sSLAMb prior to injection of sSLAM. Both SLAM mAbs completely blocked binding (see for example, Fig. 1D) indicating that binding is specific and that the antibody epitopes overlap the ligand binding site of SLAM. sSLAM injection induced the dissociation of the mAb A12 from the surface (Fig. 1D). This is most likely to be a consequence of injected sSLAM preventing the rebinding of bivalently bound mAb that had partially dissociated.

Affinity of sSLAM Self-association-- The injection of a range of concentrations of sSLAM over immobilized sSLAMb at 37 °C (Fig. 2A) clearly indicated that binding was saturable (Fig. 2, B and C). The determination of homophilic affinity constants using SPR differs from that for heterophilic interactions in that there are three, rather than just one, interactions to consider. The observed responses (measured in RU) reflect only the binding of the molecules in solution to those immobilized in the flow cell, whereas interactions between immobilized molecules and between molecules in the solution phase will also occur, both of which act to reduce the observed response at a given concentration of injected sSLAM. A theoretical model was therefore developed that takes these additional interactions into account, and the equations derived from this model were used to calculate the sSLAM-sSLAM affinity constant from the binding data.

View larger version (39K): [in this window] [in a new window]   Fig. 2.   Affinity of sSLAM self-association. A, sSLAM was injected at the indicated concentrations through flow cells with immobilized sSLAMb (1273 RU) or as a negative control, 2B4CD4b (1285 RU), at 37 °C. B, the responses at equilibrium (A) in the flow cells in which sSLAMb (circles) and/or the contol protein (squares) were immobilized, and the difference between the responses (giving specific binding, C), are each plotted against the sSLAM concentration. Removal of the oligohistidine tag with carboxypeptidase A had no effect on the affinity measurements. The linear fit used to determine P (Equation 6 in "Experimental Procedures") is shown in B.

At low concentrations of soluble reactant in the flow cell, the RU observed are directly proportional to the concentration, [A_so], of the reactant used (Fig. 3A). This is true for all three values of the concentration [A_fo] of the immobilized component on the chip. A theoretical derivation of the constant of proportionality, g (the ratio [A_so]_bound/[A_so]_free), between RU and [A_so] is derived under "Experimental Procedures" (see Equation 9). The values of g obtained from Fig. 3A are as follows g = 0.248, g = 0.388, and g = 0.547 for the low, intermediate, and high values of [A_fo] used in the experiments. If pairs of these values of g are substituted into Equation 12, we obtain three values for K_d1 and K_d2. They are K_d1 = 162, 181, and 217 µM and K_d2 = 92, 134, and 220 µM, respectively. The closer concordance for the three values of K_d1, compared with K_d2, reflects the greater sensitivity of the latter parameter to small variations in the value of K_d1. In calculating these values for the dissociation constants, the values of [A_fo] were corrected to allow for multimer formation as described (see Equation 14).

View larger version (36K): [in this window] [in a new window]   Fig. 3.   Estimation of the binding affinity using responses observed at different levels of sSLAMb immobilization. sSLAM was injected at 37 °C over flow cells in which sSLAMb was immobilized at 240 RU (75 µM; triangles), 540 RU (148 µM; circles), and 1273 RU (246 µM; squares), or in which the control protein 2B4CD4b had been immobilized at 1285 RU (not shown). The differences in response observed in the flow cells containing immobilized sSLAMb and 2B4CD4b are plotted against the injected sSLAM concentrations for narrow (A) and wider (B and C) ranges of injection. From A, the slope of the linear fit, g, was calculated as g = 0.248, g = 0.388, and g = 0.547 for the low, intermediate, and high values of [Afo], respectively. In B, curve-fitting is shown with various chosen values of Kd1 and calculated Kd2 for [Afo] = 148 µM using Equation 7 from the "Experimental Procedures." The actual responses obtained with sSLAMb immobilized at 148 µM are shown as triangles. In C, Kd1 was determined by curve-fitting to binding data for [Afo] = 75 µM (Kd1 = 181 µM), [Afo] = 148 µM (Kd1 = 282 µM), and [Afo] = 246 µM (Kd1 = 160 µM), using Equation 11 from "Experimental Procedures."

It is apparent from the values of K_d1 and K_d2 calculated above that, within experimental accuracy, the two equilibrium constants are very similar. If, however, the assumption is made that K_d1 = K_d2, then Equation 13 can be used to calculate K_d1 from the observed values of g given above. The results are 203, 216, and 211 µM, respectively, for 75, 148, and 246 µM effective concentrations of immobilized sSLAMb in the flow cells. A comparison of these results with those obtained in the previous section, in which no assumption was made about the equality of the two equilibrium constants, indicates that in both instances the value of K_d1 is ~200 µM.

When the concentration, [A_so], of sSLAM in the soluble phase is not negligible compared with K_d1, the variation of the observed response units with concentration is no longer linear. In this case the full equilibrium relations, described by Equations 7 and 11, can be used to analyze the experimental data. The procedure for using Equation 7 was as follows. The linear part of the response curve was used to determine g. With these values of g, Equation 10 was used to calculate the values of K_d2 for different, chosen values of K_d1. These pairs of values of K_d1 and K_d2 were then substituted into Equation 7, and the results were compared with observation. An example is illustrated in Fig. 3B, where the data obtained for [A_fo] equal to 148 µM are compared with the kinetic theory. With this value of [A_fo] the observed value of g is 0.388, and Equation 10 of the "Experimental Procedures" yields the following, after some rearrangement.

(Eq. 20)

The significance of this relationship is that, for all values of K_d1 and K_d2 that satisfy it, the value of g, the gradient, at the origin, of [A_so]_bound versus [A_so]_free, remains constant. Using this relationship, the calculated values of K_d2 in µM, for the corresponding different assumed values of K_d1 (shown in parentheses), are as follows: 0.0096 (2.16), 0.999 (21.6), 27 (100), 216 (216), 840 (300), 36,109 (383).

It is evident from Fig. 3B that the three lowest chosen values of K_d1 do not fit the data, but for values of about 200 µM and above, all values agree to within experimental error. Note that, from the above relationship between K_d1, K_d2, and g, the maximum value of K_d1 occurs when K_d2 is infinite (i.e. the immobilized sSLAMb does not self-associate). This maximum is 383 µM, and it follows that the experimental data imply that K_d1 lies in the range 200-383 µM. Fig. 3C illustrates the result of applying Equation 11 to the experimental data and using a computer-based best fit to evaluate K_d1. The values of the equilibrium constant obtained in this way are K_d1 = 181, 282, and 160 µM for, respectively, 75, 148, and 246 µM of accessible immobilized sSLAMb. These results, which are compatible with a value of K_d1 ~200 µM, suggest that the upper limit of 383 µM discussed above is likely to be an overestimate. In summary, the mean of the results obtained by the analyses described above (160, 162, 181, 181, 203, 211, 216, 217, and 282 µM) is K_d = 201 ± 37 µM. Whereas this statistical analysis is of limited value because it depends on K_d values derived from the data using three different methods of analysis, the broad level of agreement observed between them supports the conclusion that the K_d value is ~200 µM.

An alternative model was considered under "Experimental Procedures" where self-association of monomeric immobilized sSLAMb was assumed not to occur, and hence K_d2 is effectively infinite. When Equation 15 is used to simulate the experimental data where departure from linearity is observed (seven data points), the calculated values of K_d1 are 333, 534, and 532 µM, respectively, for the three concentrations of immobilized sSLAMb. As described, the values of K_d1 derived in this way can be used to calculate the corresponding values of [A_fo]. The results are 130, 207, and 182 µM. The first two of these calculated values exceed the measured raw data values of [A_fo], which were 82 and 186 µM, respectively, and the last is significantly less than the measured value of 438 µM. It is evident from these results that the alternative model does not fit the observations and that it can therefore be disregarded.

sSLAM Homodimers Dissociate Rapidly-- The low affinity of the interaction seen using the BIAcore^TM was confirmed by estimating the rate constant for dissociation of sSLAM from sSLAMb (Fig. 4), as this parameter is unaffected by interactions in the solution phase or between molecules immobilized in the flow cell. Even at 25 °C most (>80%) of the sSLAM dissociated from sSLAMb so rapidly that only lower limits (>15 s¹) could be given for the k_off, consistent with the very weak binding observed at equilibrium (Fig. 2). A minor component of the protein dissociated much more slowly (k_off <1 s¹). Whereas mass transport limitations and rebinding can lead to biphasic binding kinetics, these effects are unlikely to account for two dissociation phases with such markedly different rate constants. The slow component of binding can probably be accounting for by small amounts of aggregated protein likely to have appeared after gel filtration. The k_on, calculated from a K_d1 of 200 µM and a dissociation rate of 15 s¹, is 7.5 × 10⁴ M¹s¹.

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Measurement of the sSLAM dissociation rate. In A, sSLAM (48 µM) was injected at 20 µl·min1 through flow cells with sSLAMb (triangles) or the control protein, 2B4CD4b (squares), both immobilized on the sensor surface at 1000 RU. In B, individual points in the dissociation phase are shown with an expanded time scale. The response in the control flow cell, which represents the rate of washing of sSLAM from the control flow cell (squares), was subtracted from the response in the flow cell to which sSLAMb was immobilized (triangles). A koff = 15 s1 for sSLAM dissociation from sSLAMb, and a koff = 20 s1 for sSLAM removal from the flow cell containing 2B4CD4b, was calculated by exponential decay curve-fitting (line). To aid comparison, the data are normalized, with the response at the start of the dissociation phase set at 100%.

AUC Confirms sSLAM Self-association-- The conclusions of the SPR-based analysis were verified independently and extended using sedimentation velocity AUC methods. Fig. 5A shows a series of absorbance scans collected at regular intervals during a sedimentation velocity run. Changes in the location and shape of the boundary between the protein-depleted solvent (Hepes-buffered saline) and sample remaining in solution as the experiment proceeds can be analyzed via the time derivative of pairs of such scans to yield a function, g(s*) (Equation 16, "Experimental Procedures"), giving the distribution of sedimentation coefficients of the species present at the boundary (Fig. 5B, bottom panel (32)). A single, ideal monodisperse species will display a normal (Gaussian) distribution of s* arising from the symmetrical nature of its boundary. Systematically varying residuals (Fig. 5B, top panel) indicate that the distribution does not fit a single Gaussian peak, whereas a very good fit was observed for a two-species model (Fig. 5B, middle panel). Higher order models also gave poor fits (data not shown). The distribution midpoints represent the sedimentation coefficient of each species, which, for the data shown in Fig. 5B, were 5.48 S (Svedbergs) and 6.2 S. The effects of buffer density and viscosity because of buffer salts and the experimental temperature were corrected for using the program SEDNTERP (40). To account for the diffusion-limiting effects of the protein concentrations used, the sedimentation coefficients were measured at a series of concentrations (Fig. 5C). Extrapolation to infinite dilution yielded an s_20,w⁰ of 3.8 ± 0.1 S for the smaller species and 4.7 ± 0.2 S for the larger species.

View larger version (20K): [in this window] [in a new window]   Fig. 5.   AUC confirmation of the sSLAM self-association. In A, absorbance scans at 298 nm for sSLAM at ~3 mg/ml collected at intervals of 15 min are shown with the sample moving from left to right. Traces mark the migration of sSLAM along the centrifuge cell from the sample meniscus (A) to the cell bottom (). In B, lower panel, g(s*)t for sSLAM at ~3 mg/ml at 310 K () is fitted with two Gaussian curves (dotted line), representing species with s* of 5.48 ± 0.02 S and 6.20 ± 0.02 S (calculated from their midpoints). The residuals () for this fit are shown in the middle panel. The residuals of a single species fit (data fitted as a single Gaussian) are shown in the top panel () for comparison. In C, values for s* for species 1 (, fit (solid line)) and species 2 (, fit (dotted line)) are plotted against sSLAM concentration. The error bars represent the standard errors on the Gaussian fit midpoints. Extrapolation to zero concentration yields s*0 (s* at infinite dilution) of 5.42 ± 0.07 S and 6.73 ± 0.20 S, which were then corrected for the effects of solvent and temperature to yield s20,w0 values.

For interacting species, the ratios of complexed to uncomplexed forms at equilibrium are highly concentration-dependent close to the K_d. Because the Gaussian fits describe the population of species in the sample, the areas of the fitted peaks reflect the proportion of each species measured by their absorbance. A plot of the molar ratios (R), derived from these absorbance ratios, versus total sSLAM concentration, confirmed the concentration dependence of the formation of the two species observed at the boundary (Fig. 6). This result implies that the two species represent the two states of an interacting system, in this case monomeric and homodimeric forms of sSLAM, and eliminates factors such as oligosaccharide heterogeneity as their source, since in such cases R would be invariant. Comparison with a theoretical derivation of R in terms of total sSLAM concentration for a given range of affinities indicates that the K_d for the interaction is high, within the range of 100 µM-1 mM (Fig. 6), in good agreement with the SPR analysis.

View larger version (33K): [in this window] [in a new window]   Fig. 6.   AUC-based estimation of the affinity of sSLAM self-association. The observed ratio (R) of the areas of the two gaussian distributions seen in the g(s*)t profiles of sSLAM, determined using the following equations (Eq. 21) is plotted against total sSLAM concentration. Using the additional relationships, (Eq. 22) and (Eq. 23) (wherein ST is the total sSLAM concentration), R can be derived in terms of ST and Ka. (Eq. 24) The dotted lines illustrate the expected dependence of R on total sSLAM concentration for given values of Kd (from 10 µM to 1 mM) calculated using this equation. According to this analysis, the Kd for sSLAM self-association is between 100 µM and 1 mM.

The observed sedimenting boundary for a system in monomer-dimer equilibrium is the sum of the two partial boundaries belonging to the monomer and dimer, and the shapes of the partial boundaries will determine their relative separation. Previous simulations have suggested that a rapid equilibrium characterized by a fast off rate (k_off >10² s¹) will tend to prevent resolution of a dimeric species from the monomer (41) because of the merging of their partial boundaries. The question therefore arises as to why we observe the two species given that the SPR analysis indicates that sSLAM dissociates very rapidly (k_off >15 s¹). Boundary sharpening because of restricted diffusion has previously been shown to be an effect of both molecular elongation and solute concentration (42, 43). Sharpening the partial sedimentation boundaries of two species will tend to resolve them, whereas broadening will tend to merge the species. The elongated nature of the sSLAM dimer giving a low diffusion coefficient and high Perrin function (see below) means that, countering the fast dissociation, the dimer partial boundary will be sharpened with respect to that of the monomer (41). The boundary of the somewhat less asymmetric monomer (see below) will be similarly affected, albeit to a correspondingly smaller extent. In addition, both partial boundaries will be sharpened because of the high concentration of sSLAM present (42). We suggest that the sum of these effects explains why we observe monomeric and dimeric peaks in g(s*)_t, despite the rapid equilibrium. We were unable to fit sedimentation equilibrium data from parallel experiments because of the complex nature of the boundary shape, presumably because of nonideality at the very high sSLAM concentrations necessary to observe self-association (data not shown).

Topology of sSLAM Homodimers-- The sedimentation coefficient of a molecule is a function of its mass and shape. The experimental Perrin function, P_exp, is a size-independent parameter describing the degree of elongation of the species. The values of s_20,w⁰ calculated above were used to determine P_exp for the monomeric and homodimeric species of sSLAM at a range of possible hydration levels using Equations 17 and 18 (Table I). At each level of hydration, P_exp for the dimer is larger than that of the monomer. These values were compared with the sedimentation coefficients and calculated Perrin functions, P_calc, determined for models of the monomeric and homodimeric forms of glycosylated sSLAM, based on the crystal structures of human sCD2 (13) (Fig. 7A) and the head-to-head CD2 domain 1-CD58 domain 1 complex (14) (Fig. 7B), as described under "Experimental Procedures." The calculated s and P_calc values for these models are 4.07 S and 1.47 for the monomer, respectively, and 5.08 S and 1.60 for the dimer, in reasonable agreement with the experimental values (Table I). Our modeling is likely to be inexact in that it does not allow for any inherent flexibility such as that of the carbohydrate moieties. Nevertheless, the analysis strongly suggests that the dimer has an end-to-end rather than side-to-side geometry, because, for homodimers with side-to-side geometry, P_exp would remain essentially unchanged and the sedimentation coefficient would nearly double. The calculated s and P_calc values for the monomeric and side-to-side homodimeric forms of sB7-1 (44), for example, are 2.58 S and 1.27, and 4.08 S and 1.28, respectively.

View this table: [in this window] [in a new window]   Table I Experimental Perrin ratios (Pexp) and anhydrous sedimentation coefficients (s0) calculated for the two species observed in the g(s*)t profiles at a range of apparent hydrations (app) (see "Results").

View larger version (52K): [in this window] [in a new window]   Fig. 7.   Molecular modeling of the sSLAM structure. In A, the structure of rat soluble CD2 is shown (left) with modeled N-linked oligosaccharides (green) attached in random orientations at positions likely to correspond to SLAM glycosylation sequons according to sequence alignments (data not shown). The corresponding bead model, constructed using AtoB (36), is shown in the same orientation (right). In B, a model of the sSLAM homodimer, constructed by superimposing the monomer model (A) on the CD2 and CD58 domains solved in complex, as described under "Experimental Procedures," is shown (left). The corresponding bead model of the sSLAM homodimer is also shown. The bead models were used to calculate the theoretical hydrodynamic parameters for the sSLAM monomer and homodimer (see "Experimental Procedures"). The images were generated using BOBSCRIPT (58, 59) and RASTER3D (60).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We have shown that the extracellular region of SLAM is homophilic and that it self-associates with very low affinity. The SPR-based methods implemented in the BIAcore^TM are ideally suited to the detection of weak protein interactions, because binding can be followed in real time, and very small volumes of reactants are required for analysis. The work of Wyer et al. (44) has shown that immobilization of large amounts of highly "active" protein can facilitate the detection of very low affinity interactions by increasing the signal to noise ratio. Following this lead, we readily detected the very weak binding of sSLAM to biotinylated sSLAM immobilized via streptavidin.

Although sSLAM-sSLAM binding could be demonstrated relatively easily, the quantitative analysis of homophilic interactions using SPR methods presents special problems because of self-association that occurs within the solute fraction and between immobilized molecules. An initial difficulty involved estimating the proportion of immobilized sSLAMb available for interactions with sSLAM in solution. For injections at a single concentration, sSLAM binding does not increase with increasing levels of immobilized sSLAMb at the rate predicted by Equation 5, indicating that some of the immobilized protein is unavailable for binding. Because streptavidin was used to immobilize sSLAMb, it is likely that stable, multivalent homotypic interactions reduce the concentration of immobilized sSLAMb available to react with the soluble protein. To allow for the possibility that the immobilized sSLAMb