The stoichiometry of trimeric SIV glycoprotein interaction with CD4 differs from that of anti-envelope antibody Fab fragments.

Human and simian immunodeficiency viruses infect host lymphoid cells by binding CD4 molecules via their gp160 envelope glycoproteins. Biochemical studies on recombinant SIVmac32H (pJ5) envelope ectodomain gp140 precursor protein show that the envelope is a trimer. Using size exclusion chromatography, quantitative amino acid analysis, analytical ultracentrifugation, and CD4-based competition assay, we demonstrate that the stoichiometry of CD4 receptor-oligomeric envelope interaction is 1:1. By contrast, Fab fragments of both neutralizing and non-neutralizing monoclonal antibodies bind at a 3:1 ratio. Thus, despite displaying equivalent CD4 binding sites on each of the three gp140 protomers within an uncleaved trimer, only one site binds the soluble 4-domain human CD4 extracellular segment. The anti-cooperativity and the faster k(off) of gp140 trimer:CD4 versus gp120 monomer:CD4 interaction suggest that CD4-induced conformational change is impeded in the intact envelope. The implications of these findings for immunity against human immunodeficiency virus and simian immunodeficiency virus are discussed.

Expression, purification, and characterization of recombinant HIV gp140. The gp41 ectodomain of HIV or simian immunodeficiency virus is sufficient to maintain the retroviral envelope glycoprotein as a trimer.

Efforts to understand the molecular basis of human immunodeficiency virus (HIV) envelope glycoprotein function have been hampered by the inability to generate sufficient quantities of homogeneous material. We now report on the high level expression, purification, and characterization of soluble HIV gp140 ectodomain proteins in Chinese hamster ovary-Lec3.2.8.1 cells. Gel filtration and analytical ultracentrifugation show that the uncleaved ADA strain-derived gp140 proteins are trimeric without further modification required to maintain oligomers. These spike proteins are native as judged by soluble CD4 (sCD4) (K(D) = 1-2 nm) and monoclonal antibody binding studies using surface plasmon resonance. CD4 ligation induces conformational change in the trimer, exposing the chemokine receptor binding site as assessed by 17b monoclonal antibody reactivity. Lack of anti-cooperativity in sCD4-ADA trimer interaction distinct from that observed with sCD4-SIV mac32H implies quaternary structural differences in ground states of their respective spike proteins.

A critical role for p59(fyn) in CD2-based signal transduction.

TCR- but not CD2-triggered IL-2 production is p56(lck) dependent. To test the hypothesis that p59(fyn), a second src-family protein tyrosine kinase (PTK) expressed in T lymphocytes, might be an essential upstream component of the CD2 signaling pathway, we generated human (h) CD2 transgenic (tg) fyn(+/+) and fyn(-/-) mice. Clustering of hCD2 molecules on resting peripheral T lymphocytes results in Ca(2+) mobilization, activation of MAPK and cellular proliferation. In contrast, in the absence of p59(fyn), these CD2-initiated activities are markedly reduced, while TCR-triggered proliferation is unaffected. Several CD2 pathway components regulated by p59(fyn) have been identified including phospholipase C-gamma1 (PLC-gamma1), Vav, protein kinase C-theta isoform (PKC-theta), docking protein (Dok), focal adhesion kinase (FAK) and Pyk2. Decreased inducible PKC-theta catalytic activity and Vav phosphorylation likely account for diminished p38 and JNK activation in hCD2tg fyn(-/-) mice. Moreover, deficiency in fyn-dependent PLC-gamma1 catalytic activity may contribute to reduced PKC-alpha-dependent ERK activation. Of note, CD2-dependent Dok but not linker from activated T cells (LAT) tyrosine phosphorylation requires p59(fyn). Furthermore, that FAK and Pyk2 are target substrates implies that p59(fyn) may be an important regulator of T cell adhesion as well. Collectively, these data identify p59(fyn) as a key PTK in CD2-mediated activation of mature T lymphocytes.

Expression, purification, and characterization of gp160e, the soluble, trimeric ectodomain of the simian immunodeficiency virus envelope glycoprotein, gp160.

The envelope glycoprotein, gp160, of simian immunodeficiency virus (SIV) shares approximately 25% sequence identity with gp160 from the human immunodeficiency virus, type I, indicating a close structural similarity. As a result of binding to cell surface CD4 and co-receptor (e.g. CCR5 and CXCR4), both SIV and human immunodeficiency virus gp160 mediate viral entry by membrane fusion. We report here the characterization of gp160e, the soluble ectodomain of SIV gp160. The ectodomain has been expressed in both insect cells and Chinese hamster ovary (CHO)-Lec3.2.8.1 cells, deficient in enzymes necessary for synthesizing complex oligosaccharides. Both the primary and a secondary proteolytic cleavage sites between the gp120 and gp41 subunits of gp160 were mutated to prevent cleavage and shedding of gp120. The purified, soluble glycoprotein is shown to be trimeric by chemical cross-linking, gel filtration chromatography, and analytical ultracentrifugation. It forms soluble, tight complexes with soluble CD4 and a number of Fab fragments from neutralizing monoclonal antibodies. Soluble complexes were also produced of enzymatically deglycosylated gp160e and of gp160e variants with deletions in the variable segments.

Human CD4 residue Phe 43 is critical for repertoire development and maturation of MHC class II restricted CD4 single-positive T lineage cells in vivo.

To determine the functional significance of structural alteration of CD4-MHC class II interaction in vivo, two human (h)CD4-transgenic (tg) mice were established on a murine (m)CD4(-/-) H-2(b) background. The MHC class II binding-competent hCD4 (R240AhCD4) rescues the number and helper activity of hCD4(+)CD8(-) single-positive (SP) mature T cells in mCD4(-/-) mice. In contrast, the MHC class II binding-deficient F43I hCD4 mutant cannot facilitate normal differentiation of double-positive thymocytes to CD4(+)CD8(-) SP thymocytes. Hence, only 20 - 25% of CD4(+)CD8(-) SP T cells found in wild-type or R240A hCD4tg mice are generated, with resultant diminished helper responses. Differentiation of F43I hCD4 SP T cells is MHC class II but not class I dependent as demonstrated by crossing F43I hCD4tg mice onto MHC-deficient mice. These cells show a different pattern of TCR Valpha and Vbeta gene usage relative to comparable R240A hCD4 SP T cells from R240 AhCD4tg animals. Expression of activation markers including CD25 and CD69 on F43I hCD4 SP T cells suggests that autoreactive specificites may not have been eliminated intrathymically. Collectively, the results show that CD4-MHC class II interaction significantly influences intrathymic repertoire selection.

The crystal structure of a T cell receptor in complex with peptide and MHC class II.

The crystal structure of a complex involving the D10 T cell receptor (TCR), 16-residue foreign peptide antigen, and the I-Ak self major histocompatibility complex (MHC) class II molecule is reported at 3.2 angstrom resolution. The D10 TCR is oriented in an orthogonal mode relative to its peptide-MHC (pMHC) ligand, necessitated by the amino-terminal extension of peptide residues projecting from the MHC class II antigen-binding groove as part of a mini beta sheet. Consequently, the disposition of D10 complementarity-determining region loops is altered relative to that of most pMHCI-specific TCRs; the latter TCRs assume a diagonal orientation, although with substantial variability. Peptide recognition, which involves P-1 to P8 residues, is dominated by the Valpha domain, which also binds to the class II MHC beta1 helix. That docking is limited to one segment of MHC-bound peptide offers an explanation for epitope recognition and altered peptide ligand effects, suggests a structural basis for alloreactivity, and illustrates how bacterial superantigens can span the TCR-pMHCII surface.

Expression, purification, and functional analysis of murine ectodomain fragments of CD8alphaalpha and CD8alphabeta dimers.

Soluble mouse CD8alphaalpha and CD8alphabeta dimers corresponding to the paired ectodomains (CD8(f)) or their respective component Ig-like domains (CD8) were expressed in Chinese hamster ovary cells or the glycosylation variant Lec3.2.8.1 cells as secreted proteins using a leucine zipper strategy. The affinity of CD8alphaalpha(f) for H-2K(b) as measured by BIAcore revealed a approximately 65 microM K(d), similar to that of CD8alphabeta(f). Consistent with this result, CD8alphaalpha(f) as well as CD8alphabeta(f) blocked the effector function of N15 T cell receptor transgenic cytolytic T cells in a comparable, dose-dependent fashion. Furthermore, both Lec3.2.8.1-produced and Chinese hamster ovary-produced CD8 homodimers and heterodimers were active in the inhibition assay. These results suggest that the Ig-like domains of CD8 molecules are themselves sufficient to block the requisite transmembrane CD8-pMHC interaction between cytolytic T lymphocytes and target cells. Moreover, given the similarities in co-receptor affinities for pMHC, the findings suggest that the greater efficiency of CD8alphabeta versus CD8alphaalpha co-receptor function on T cells is linked to differences within their membrane-bound stalk regions and/or intracellular segments. As recently shown for sCD8alphaalpha, the yield, purity and homogeneity of the deglycosylated protein resulting from this expression system is sufficient for crystallization and x-ray diffraction at atomic resolution.

A cdc15-like adaptor protein (CD2BP1) interacts with the CD2 cytoplasmic domain and regulates CD2-triggered adhesion.

A human CD2 cytoplasmic tail-binding protein, termed CD2BP1, was identified by an interaction trap cloning method. Expression of CD2BP1 is restricted to hematopoietic tissue, being prominent in T and natural killer (NK) cells, with long (CD2BP1L) and short (CD2BP1S) variants arising by alternative RNA splicing. Both CD2BP1 molecules are homologous to Schizosaccharomyces pombe cdc15, and include a helical domain, variable length intervening PEST sequence and C-terminal SH3 domain. Although the CD2BP1 SH3 domain binds directly to the CD2 sequence, KGPPLPRPRV (amino acids 300-309), its association is augmented markedly by the CD2BP1 N-terminal segment. Upon ligand-induced clustering of surface CD2 molecules, CD2BP1 redistributes from a cytosolic to a surface membrane compartment, co-localizing with CD2. In turn, CD2-stimulated adhesion is downregulated by CD2BP1, apparently through coupling of the protein tyrosine phosphatase (PTP)-PEST to CD2. These findings offer the first molecular view into the control processes for T cell adhesion.

Structural basis of CD8 coreceptor function revealed by crystallographic analysis of a murine CD8alphaalpha ectodomain fragment in complex with H-2Kb.

The crystal structure of the two immunoglobulin variable-like domains of the murine CD8alphaalpha homodimer complexed to the class I MHC H-2Kb molecule at 2.8 A resolution shows that CD8alphaalpha binds to the protruding MHC alpha3 domain loop in an antibody-like manner. Comparison of mouse CD8alphaalpha/H-2Kb and human CD8alphaalpha/HLA-A2 complexes reveals shared as well as species-specific recognition features. In both species, coreceptor function apparently involves the participation of CD8 dimer in a bidentate attachment to an MHC class I molecule in conjunction with a T cell receptor without discernable conformational alteration of the peptide or MHC antigen-presenting platform.

Identification of a common docking topology with substantial variation among different TCR-peptide-MHC complexes.

Whether T-cell receptors (TCRs) recognize antigenic peptides bound to major histocompatability complex (MHC) molecules through common or distinct docking modes is currently uncertain. We report the crystal structure of a complex between the murine N15 TCR [1-4] and its peptide-MHC ligand, an octapeptide fragment representing amino acids 52-59 of the vesicular stomatitis virus nuclear capsid protein (VSV8) bound to the murine H-2Kb class I MHC molecule. Comparison of the structure of the N15 TCR-VSV8-H-2Kb complex with the murine 2C TCR-dEV8-H-2Kb [5] and the human A6 TCR-Tax-HLA-A2 [6] complexes revealed a common docking mode, regardless of TCR specificity or species origin, in which the TCR variable Valpha domain overlies the MHC alpha2 helix and the Vbeta domain overlies the MHC alpha1 helix. As a consequence, the complementary determining regions CDR1 and CDR3 of the TCR Valpha and Vbeta domains make the major contacts with the peptide, while the CDR2 loops interact primarily with the MHC. Nonetheless, in terms of the details of the relative orientation and disposition of binding, there is substantial variation in TCR parameters, which we term twist, tilt and shift, and which define the variation of the V module of the TCR relative to the MHC antigen-binding groove.

Atomic structure of an alphabeta T cell receptor (TCR) heterodimer in complex with an anti-TCR fab fragment derived from a mitogenic antibody.

Each T cell receptor (TCR) recognizes a peptide antigen bound to a major histocompatibility complex (MHC) molecule via a clonotypic alphabeta heterodimeric structure (Ti) non-covalently associated with the monomorphic CD3 signaling components. A crystal structure of an alphabeta TCR-anti-TCR Fab complex shows an Fab fragment derived from the H57 monoclonal antibody (mAb), interacting with the elongated FG loop of the Cbeta domain, situated beneath the Vbeta domain. This loop, along with the partially exposed ABED beta sheet of Cbeta, and glycans attached to both Cbeta and Calpha domains, forms a cavity of sufficient size to accommodate a single non-glycosylated Ig domain such as the CD3epsilon ectodomain. That this asymmetrically localized site is embedded within the rigid constant domain module has implications for the mechanism of signal transduction in both TCR and pre-TCR complexes. Furthermore, quaternary structures of TCRs vary significantly even when they bind the same MHC molecule, as manifested by a unique twisting of the V module relative to the C module.

Double-positive T cell receptor(high) thymocytes are resistant to peptide/major histocompatibility complex ligand-induced negative selection.

To investigate negative selection events during intrathymic ontogeny, we established T cell receptor (TCR)-transgenic mice [N15tg/RAG-2-/- (H-2b)] expressing a single TCR specific for vesicular stomatitis virus nuclear octapeptide N52-59 (VSV8) in the context of the major histocompatibility complex (MHC) class I molecule, K(b). Administration of VSV8 in vivo induced apoptosis in less than 4 h, deleting the majority of immature double-positive (DP) thymocytes by 24 h. In contrast, DP TCRhigh as well as single-positive (SP) thymocytes were refractory to this death process. Moreover, DP TCRhigh cells differentiated into SP thymocytes in vitro and in vivo, maturing into functional cytotoxic T lymphocytes upon intrathymic transfer to beta RAG 2-/- recipients. Hence, negative selection processes involving MHC-bound peptide ligands are operative only prior to the late DP thymocyte stage in this MHC class I-restricted TCR transgene system.

Crystallization of a deglycosylated T cell receptor (TCR) complexed with an anti-TCR Fab fragment.

A strategy to overexpress T cell receptors (TCRs) in Lec3.2.8.1 cells has been developed using the "Velcro" leucine zipper sequence to facilitate alpha-beta pairing. Upon secretion in culture media, the VSV-8-specific/H2-Kb-restricted N15 TCR could be readily immunopurified using the anti-leucine zipper monoclonal antibody 2H11, with a yield of 5-10 mg/liter. Mass spectrometry analysis revealed that all attached glycans were GlcNAc2-Man5. Following Superdex 200 gel filtration to remove aggregates, wild-type N15 or N15(s), a C183S variant lacking the unpaired cysteine at amino acid residue 183 in the Cbeta domain, was thrombin-cleaved and endoglycosidase H-digested, and the two derivatives were termed iN15DeltaH and N15(s)DeltaH, respectively, and sized by Superdex 75 chromatography to high purity. N-terminal and C-terminal microsequencing analysis showed the expected unique termini of N15 alpha and beta subunits. Nevertheless, neither protein crystallized under a wide range of conditions. Subsequently, we produced a Fab fragment of the murine TCR Cbeta-specific hamster monoclonal antibody H57 and complexed the Fab fragment with iN15DeltaH and N15(s)DeltaH. Both N15(s)DeltaH-Fab[H57] and iN15DeltaH-Fab[H57] complexes crystallize, with the former diffracting to 2.8-A resolution. These findings show that neither intact glycans nor the conserved and partially exposed Cys-183 is required for protein stability. Furthermore, our results suggest that the H57 Fab fragment aids in the crystallization of TCRs by altering their molecular surface and/or stabilizing inherent conformational mobility.

Delineation of a T-cell activation motif required for binding of protein tyrosine kinases containing tandem SH2 domains.

To define the T-cell receptor signal transduction motif, we have transfected human and murine T-cell lines with a chimeric receptor consisting of the extracellular and transmembrane domains of human CD8 alpha and the membrane-proximal portion of CD3 zeta containing at its C terminus either an 18-amino acid segment (NQLYNELNLGRREEYDVL) or alanine-scanning point mutant derivatives. Crosslinking of the extracellular domain of the chimera is sufficient to initiate Ca2+ flux, interleukin 2 production, and tyrosine phosphorylation of cellular proteins including the chimera. Subsequently, the chimera becomes associated with several tyrosine-phosphorylated proteins, among them the 70-kDa protein tyrosine kinase ZAP70. Mutational data identify the T-cell activation motif as Y(X)2L(X)7Y(X)2L and show that each of the four designated residues is necessary for the above activation events. Recombinant protein containing the two tandem SH2 domains derived from ZAP70 binds to a synthetic peptide corresponding to the above 18-amino acid motif but only when both tyrosines are phosphorylated; in contrast, little or no binding is observed to monophosphorylated or nonphosphorylated analogues. These results imply that after receptor crosslinking in T cells, and by inference also in B cells and mast cells, the motif is phosphorylated on both tyrosine residues, thereafter serving as a docking site for protein tyrosine kinases containing tandem SH2 domains.

Targeted disruption within the CD3 zeta/eta/phi/Oct-1 locus in mouse.

To elucidate the role of the CD3 eta subunit of the T cell receptor (TCR) in thymic development, a CD3 eta -/- mouse was generated by gene targeting. Insertion of a neomycin resistance gene into exon 9 of the CD3 zeta/eta/phi locus disrupted expression of CD3 eta and CD3 phi without affecting the expression of CD3 zeta. Little difference was observed between wild type and CD3 eta -/- mice with regard to cellularity or subset composition in thymus and peripheral lymphoid organs. Furthermore, neither alloproliferative responses nor cytotoxic T lymphocyte generation and effector function was affected by the mutation. The effect of the CD3 eta -/- mutation on thymic selection was examined by crossing the CD3 eta knockout animals with anti-HY TCR transgenic animals: the absence of the CD3 eta subunit altered neither positive nor negative selection. Thus, CD3 eta is not required for thymic selection. Of note, the birth rate of the CD3 eta -/- animals was significantly lower than that of wild type or heterozygous animals (P = 0.041-0.002). This unexpected result is probably the consequence of an alteration in mRNA expression of the Oct-1 nuclear transcription factor in CD3 eta -/- animals. The CD3 zeta/eta/phi locus partially overlaps the gene encoding Oct-1 whose transcription is dysregulated by the CD3 eta -/- mutation. Our results clearly underscore the value of characterizing all products of a genetic locus disrupted by gene targeting.

Overexpression of CD3 eta during thymic development does not alter the negative selection process.

To elucidate the role of CD3 eta in thymic development and to determine whether CD3 eta is involved in the negative selection process, CD3 eta was overexpressed > 100 fold in transgenic (tg) mice using a Thy-1 promoter and regulatory elements. CD3 eta was readily observed in the majority of cortical thymocytes and in a fraction of medullary thymocytes in tg mice by immunohistochemical staining with an anti-CD3 eta-specific mAb. In contrast, endogenous CD3 eta levels were too low to detect in normal littermates. Flow cytometric analysis demonstrated an increased level of TCR on thymocytes with intermediate TCR density in tg animals and parallel biochemical studies showed a marked increased in TCR-associated CD3 zeta-eta heterodimers and CD3 eta-eta homodimers relative to controls. Despite this change in surface TCR phenotype, there was no significant alteration in the total numbers or proportion of CD4+CD8+ double-positive or CD4+CD8- or CD4-CD8+ single-positive thymocytes or peripheral T cells. Percentages of SP V beta 5, V beta 6, and V beta 8 thymocytes in tg animals were unaltered compared to normal littermates when backcrossed either to C57BL/6 (H-2b) or DBA/2 (H-2d) backgrounds. Furthermore, induction of DNA fragmentation with anti-CD3 epsilon mAb treatment in vivo was not significantly different for tg and normal littermates. Collectively, these data imply that CD3 eta is not a limiting component of the negative selection process.

T-cell-receptor isoforms.

Early work on T-cell hybridomas lacking the T-cell-receptor (TCR) sub-unit CD3 eta had suggested a correlation between the presence of CD3 zeta-eta heterodimers and signalling leading to phosphatidyl-inositol (PI) turnover as well as activation-induced cell death. The cloning of CD3 eta has now allowed thorough and direct analysis of the signal transduction properties of CD3 zeta-zeta-, CD3 zeta-eta- and CD3 eta-eta-containing TCRs. We have found that all forms of the TCR are capable of transducing signals leading to PI turnover, Ca2+ mobilization, IL-2 production and cell-cycle arrest. CD3 zeta and CD3 eta utilize the same promoter which yields coordinate expression of both products, so that restricted CD3 eta expression in a sub-population of thymocytes is unlikely. Immunohistochemical methods employing an anti-CD3 eta-specific monoclonal antibody (MAb) show no detectable staining of thymic sections from adult mice, implying at best a low level of constitutive CD3 eta expression. In contrast, CD3 eta expression is readily detected in the majority of cortical thymocytes of CD3 eta transgenic mice using a Thy-1 promoter construct. However, over-expression of CD3 eta in mice transgenic for this polypeptide does not result in increased negative selection in vivo, consistent with the in vitro findings that induction of cell death is not strictly dependent on CD3 eta. Despite earlier reports of the detection of human CD3 eta protein, we find no CD3 eta message in human thymus or T cells. Cloning of the human CD zeta-eta genomic locus has demonstrated approximately 70% homology between the mouse and human genomic sequence, corresponding to the mouse CD3 eta-specific exon. However, translation of the DNA sequence does not result in a homologous amino acid sequence. Thus, there does not appear to be a CD3 eta protein in humans.

A soluble, single-chain T-cell receptor fragment endowed with antigen-combining properties.

A strategy for the production of small, soluble, single-chain T-cell receptor (scTCR) fragments that carry an intact TCR antigen-combining site is presented. The rationale is based on structural similarity between TCR and antibody molecules and use of computer modeling methods to derive a model structure of a human scTCR variable (V)-domain dimer. A gene encoding the RFL3.8 TCR protein, specific for the hapten fluorescein in the context of major histocompatibility complex class II and composed of one V alpha and one V beta domain joined via a flexible peptide linker, was assembled in an Escherichia coli plasmid. Subsequently, the protein was produced in a bacterial expression system, purified, refolded, and found to be poorly soluble at neutral pH in aqueous buffers. An inspection of the computer-generated V alpha-V beta domain model showed several surface exposed hydrophobic residues. When these were replaced by water-soluble side chains via site-directed mutagenesis of the corresponding gene, a soluble protein resulted and was shown to have antigen-binding properties equivalent to those of the intact TCR of the RFL3.8 T-cell clone. These results demonstrate the feasibility of obtaining TCR fragments endowed with antigen-combining properties by protein engineering in E. coli.

CD3 eta and CD3 zeta are alternatively spliced products of a common genetic locus and are transcriptionally and/or post-transcriptionally regulated during T-cell development.

The CD3 eta subunit of the T-cell receptor is thought to subserve an important role in signal transduction and possibly T-cell development. Herein we characterize the organization of the mouse CD3 eta gene and show that it is part of one gene locus that also encodes CD3 zeta on chromosome 1. The NH2-terminal sequence of CD3 zeta and CD3 eta, which share the same leader peptide and are identical through amino acid 122 of each mature protein, is encoded by exons 1-7. However, exons 8 and 9 are differentially spliced to give rise to CD3 zeta and CD3 eta: exons 1-8 encode CD3 zeta and exons 1-7 plus 9 encode CD3 eta. RNase protection analysis with RNA from a variety of fetal, neonatal, and adult cell types indicates that expression of both gene products is T-lineage-restricted. Importantly, expression of CD3 zeta and CD3 eta mRNA appears before or on day 16 of fetal gestation. Expression is apparently coordinate since no cell types tested express CD3 zeta or CD3 eta alone. The steady-state level of CD3 zeta mRNA is greater than or equal to 40-60 times that of CD3 eta mRNA. In immature CD4+CD8+CD3low double-positive thymocytes and CD4+CD8-CD3high or CD4-CD8+CD3high single-positive thymocytes, the respective steady-state CD3 zeta and CD3 eta mRNA levels are equivalent, whereas the amount of receptor-associated CD3 zeta and CD3 eta proteins in double-positive thymocytes is approximately 10 times less than in single-positive thymocytes. Nevertheless, the CD3 zeta/CD3 eta protein ratio remains constant in all populations (40-60:1). Furthermore, discordance between mRNA and protein levels for CD3 zeta and CD3 eta is also observed in splenic T cells. Thus, posttranscriptional and/or transcriptional regulatory mechanisms control CD3 zeta and CD3 eta expression during T-cell development.

Structural and binding analysis of a two domain extracellular CD2 molecule.

The 50-kD CD2 (T11) surface glycoprotein on human T lymphocytes and thymocytes plays a critical role in T lineage cell activation and adhesion via its ligand LFA-3. To begin to define structure-function relationships in the extracellular segment of the transmembrane CD2 molecule, we have used a eukaryotic expression system and a CD2 cDNA to produce milligram amounts of recombinant soluble CD2 molecule that corresponds to the two extracellular segment exons. We show that this protein, termed T11ex2, behaves as a monomer in aqueous solution and includes a proteolytically resistant NH2-terminal fragment (domain I) encoded by the first extracellular segment exon. Circular dichroism analysis of T11ex2 demonstrates that its stabilized secondary structure is dependent on the intrachain disulfide bonds present in domain II. The T11ex2 monomer binds directly to the CD2 ligand LFA-3 with a dissociation constant of 0.4 microM. This relatively low affinity implies that cooperative binding resulting from an array of transmembrane CD2 molecules is important to facilitate physiologic T cell adhesion.