Membrane binding kinetics of protein kinase C betaII mediated by the C2 domain.

Conventional isoforms of protein kinase C (PKC) are activated when their two membrane-targeting modules, the C1 and C2 domains, bind the second messengers diacylglycerol (DG) and Ca2+, respectively. This study investigates the mechanism of Ca2+-induced binding of PKC betaII to anionic membranes mediated by the C2 domain. Stopped-flow fluorescence spectroscopy reveals that Ca2+-induced binding of the isolated C2 domain to anionic vesicles proceeds via at least two steps: (1) rapid binding of two or more Ca2+ ions to the free domain with relatively low affinity and (2) diffusion-controlled association of the Ca2+-occupied domain with vesicles. Ca2+ increases the affinity of the C2 domain for anionic membranes by both decreasing the dissociation rate constant (k(off)) and increasing the association rate constant (k(on)) for membrane binding. For binding to vesicles containing 40 mol % anionic lipid in the presence of 200 microM Ca2+, k(off) and k(on) are 8.9 s(-1) and 1.2 x 10(10) M(-1) x s(-1), respectively. The k(off) value increases to 150 s(-1) when free Ca2+ levels are rapidly reduced, decreasing the average lifetime of the membrane-bound C2 domain (tau = k(off)(-1)) from 110 ms in the presence of Ca2+ to 6.7 ms when Ca2+ is rapidly removed. Experiments addressing the role of electrostatic interactions reveal that they stabilize either the initial C2 domain-membrane encounter complex or the high-affinity membrane-bound complex. Specifically, lowering the phosphatidylserine mole fraction or including MgCl2 in the binding reaction decreases the affinity of the C2 domain for anionic vesicles by both reducing k(on) and increasing k(off) measured in the presence of 200 microM Ca2+. These species do not affect the k(off) value when Ca2+ is rapidly removed. Studies with PKC betaII reveal that Ca2+-induced binding to membranes by the full-length protein proceeds minimally via two kinetically resolvable steps: (1) a rapid bimolecular association of the enzyme with vesicles near the diffusion-controlled limit and, most likely, (2) subsequent conformational changes of the membrane-bound enzyme. As is the case for the C2 domain, k(off) for full-length PKC betaII increases when Ca2+ is rapidly removed, reducing tau from 11 s in the presence of Ca2+ to 48 ms in its absence. Thus, both the C2 domain and the slow conformational change prolong the lifetime of the PKC betaII-membrane ternary complex in the presence of Ca2+, with rapid membrane release triggered by removal of Ca2+. These results provide a molecular basis for cofactor regulation of PKC whereby the C2 domain searches three-dimensional space at the diffusion-controlled limit to target PKC to relatively common anionic phospholipids, whereupon a two-dimensional search is initiated by the C1 domain for the more rare, membrane-partitioned DG.

C2 domains from different Ca2+ signaling pathways display functional and mechanistic diversity.

The ubiquitous C2 domain is a conserved Ca2+ triggered membrane-docking module that targets numerous signaling proteins to membrane surfaces where they regulate diverse processes critical for cell signaling. In this study, we quantitatively compared the equilibrium and kinetic parameters of C2 domains isolated from three functionally distinct signaling proteins: cytosolic phospholipase A2-alpha (cPLA2-alpha), protein kinase C-beta (PKC-beta), and synaptotagmin-IA (Syt-IA). The results show that equilibrium C2 domain docking to mixed phosphatidylcholine and phosphatidylserine membranes occurs at micromolar Ca2+ concentrations for the cPLA2-alpha C2 domain, but requires 3- and 10-fold higher Ca2+ concentrations for the PKC-beta and Syt-IA C2 domains ([Ca2+](1/2) = 4.7, 16, 48 microM, respectively). The Ca2+ triggered membrane docking reaction proceeds in at least two steps: rapid Ca2+ binding followed by slow membrane association. The greater Ca2+ sensitivity of the cPLA2-alpha domain results from its higher intrinsic Ca2+ affinity in the first step compared to the other domains. Assembly and disassembly of the ternary complex in response to rapid Ca2+ addition and removal, respectively, require greater than 400 ms for the cPLA2-alpha domain, compared to 13 ms for the PKC-beta domain and only 6 ms for the Syt-IA domain. Docking of the cPLA2-alpha domain to zwitterionic lipids is triggered by the binding of two Ca2+ ions and is stabilized via hydrophobic interactions, whereas docking of either the PKC-beta or the Syt-IA domain to anionic lipids is triggered by at least three Ca2+ ions and is maintained by electrostatic interactions. Thus, despite their sequence and architectural similarity, C2 domains are functionally specialized modules exhibiting equilibrium and kinetic parameters optimized for distinct Ca2+ signaling applications. This specialization is provided by the carefully tuned structural and electrostatic parameters of their Ca2+ and membrane-binding loops, which yield distinct patterns of Ca2+ coordination and contrasting mechanisms of membrane docking.

Solution structure and membrane interactions of the C2 domain of cytosolic phospholipase A2.

The amino-terminal, 138 amino acid C2 domain of cytosolic phospholipase A2 (cPLA2-C2) mediates an initial step in the production of lipid mediators of inflammation: the Ca2+-dependent translocation of the enzyme to intracellular membranes with subsequent liberation of arachidonic acid. The high resolution solution structure of this Ca2+-dependent, lipid-binding domain (CaLB) has been determined using heteronuclear three-dimensional NMR spectroscopy. Secondary structure analysis, derived from several sets of spectroscopic data, shows that the domain is composed of eight antiparallel beta-strands with six interconnecting loops that fits the "type II" topology for C2 domains. Using a total of 2370 distance and torsional restraints, the structure was found to be a beta-sandwich in the "Greek key" motif. The solution structure of cPLA2-C2 domain is very similar to the X-ray crystal structure of the C2 domain of phospholipase-C-delta and phylogenetic analysis clarifies the structural role of highly conserved residues. Calorimetric studies further demonstrate that cPLA2-C2 binds two Ca2+ with observed Kds of approximately 2 microM in an entropically assisted process. Moreover, regions on cPLA2-C2 interacting with membranes were identified by 15N-HSQC-spectroscopy of cPLA2-C2 in the presence of low molecular weight lipid micelles. An extended binding site was identified that binds the phosphocholine headgroup in a Ca2+-dependent manner and also interacts with proximal regions of the membrane surface. Based upon these results, a structural model is presented for the mechanism of association of cPLA2 with its membrane substrate.

Independent folding and ligand specificity of the C2 calcium-dependent lipid binding domain of cytosolic phospholipase A2.

The Ca(2+)-dependent lipid binding domain of the 85-kDa cytosolic phospholipase A2 (cPLA2) is a homolog of C2 domains present in protein kinase C, synaptotagmin, and numerous other proteins involved in signal transduction. NH2-terminal fragments of cPLA2 spanning the C2 domain were expressed as inclusion bodies in Escherichia coli, extracted with solvent to remove phospholipids, and refolded to yield a domain capable of binding phospholipid vesicles in a Ca(2+)-dependent manner. Unlike other C2 domains characterized to date, the cPLA2 C2 domain bound preferentially to vesicles comprised of phosphatidylcholine in response to physiological concentrations of Ca2+. Binding of the cPLA2 C2 domain to vesicles in the presence of excess Ca2+ chelator was induced by high concentrations of salts that promote hydrophobic interactions. Despite the selective hydrolysis of arachidonyl-containing phospholipid vesicles by cPLA2, the cPLA2 C2 domain did not discriminate among phospholipid vesicles containing saturated or unsaturated sn-2 fatty acyl chains. Moreover, the cPLA2 C2 domain bound to phospholipid vesicles containing sn-1 and -2 ether linkages and sphingomyelin at Ca2+ concentrations that caused binding to vesicles containing ester linkages, demonstrating that the carbonyl oxygens of the sn-1 and -2 ester linkage are not critical for binding. These results suggest that the cPLA2 C2 domain interacts primarily with the headgroup of the phospholipid. The cPLA2 C2 domain displayed selectivity among group IIA cations, preferring Ca2+ approximately 50-fold over Sr2+ and nearly 10,000-fold over Ba2+ for vesicle binding. No binding to vesicles was observed in the presence of greater than 10 mM Mg2+. Such strong selectivity for Ca2+ over Mg2+ reinforces the view that C2 domains link second messenger Ca2+ to signal transduction events at the membrane.

Location of the membrane-docking face on the Ca2+-activated C2 domain of cytosolic phospholipase A2.

Docking of C2 domains to target membranes is initiated by the binding of multiple Ca2+ ions to a conserved array of residues imbedded within three otherwise variable Ca2+-binding loops. We have located the membrane-docking surface on the Ca2+-activated C2 domain of cPLA2 by engineering a single cysteine substitution at 16 different locations widely distributed across the domain surface, in each case generating a unique attachment site for a fluorescein probe. The environmental sensitivity of the fluorescein-labeled cysteines enabled identification of a localized region that is perturbed by Ca2+ binding and membrane docking. Ca2+ binding to the domain altered the emission intensity of six fluoresceins in the region containing the Ca2+-binding loops, indicating that Ca2+-triggered environmental changes are localized to this region. Similarly, membrane docking increased the protonation of six fluoresceins within the Ca2+-binding loop region, indicating that these three loops also are directly involved in membrane docking. Furthermore, iodide quenching measurements revealed that membrane docking sequesters three fluorescein labeling positions, Phe35, Asn64, and Tyr96, from collisions with aqueous iodide ion. These sequestered residues are located within the identified membrane-docking region, one in each of the three Ca2+-binding loops. Finally, cysteine substitution alone was sufficient to dramatically reduce membrane affinity only at positions Phe35 and Tyr96, highlighting the importance of these two loop residues in membrane docking. Together, the results indicate that the membrane-docking surface of the C2 domain is localized to the same surface that cooperatively binds a pair of Ca2+ ions, and that the three Ca2+-binding loops themselves provide most or all of the membrane contacts. These and other results further support a general model for the membrane specificity of the C2 domain in which the variable Ca2+-binding loops provide headgroup recognition at a protein-membrane interface stabilized by multiple Ca2+ ions.

Ca2+-signaling cycle of a membrane-docking C2 domain.

The C2 domain is a Ca2+-dependent, membrane-targeting motif originally discovered in protein kinase C and recently identified in numerous eukaryotic signal-transducing proteins, including cytosolic phospholipase A2 (cPLA2) of the vertebrate inflammation pathway. Intracellular Ca2+ signals recruit the C2 domain of cPLA2 to cellular membranes where the enzymatic domain hydrolyzes specific lipids to release arachidonic acid, thereby initiating the inflammatory response. Equilibrium binding and stopped-flow kinetic experiments reveal that the C2 domain of human cPLA2 binds two Ca2+ ions with positive cooperativity, yielding a conformational change and membrane docking. When Ca2+ is removed, the two Ca2+ ions dissociate rapidly and virtually simultaneously from the isolated domain in solution. In contrast, the Ca2+-binding sites become occluded in the membrane-bound complex such that Ca2+ binding and dissociation are slowed. Dissociation of the two Ca2+ ions from the membrane-bound domain is an ordered sequential process, and release of the domain from the membrane is simultaneous with dissociation of the second ion. Thus, the Ca2+-signaling cycle of the C2 domain passes through an active, membrane-bound state possessing two occluded Ca2+ ions, one of which is essential for maintenance of the protein-membrane complex.

The C2 domain calcium-binding motif: structural and functional diversity.

The C2 domain is a Ca(2+)-binding motif of approximately 130 residues in length originally identified in the Ca(2+)-dependent isoforms of protein kinase C. Single and multiple copies of C2 domains have been identified in a growing number of eukaryotic signalling proteins that interact with cellular membranes and mediate a broad array of critical intracellular processes, including membrane trafficking, the generation of lipid-second messengers, activation of GTPases, and the control of protein phosphorylation. As a group, C2 domains display the remarkable property of binding a variety of different ligands and substrates, including Ca2+, phospholipids, inositol polyphosphates, and intracellular proteins. Expanding this functional diversity is the fact that not all proteins containing C2 domains are regulated by Ca2+, suggesting that some C2 domains may play a purely structural role or may have lost the ability to bind Ca2+. The present review summarizes the information currently available regarding the structure and function of the C2 domain and provides a novel sequence alignment of 65 C2 domain primary structures. This alignment predicts that C2 domains form two distinct topological folds, illustrated by the recent crystal structures of C2 domains from synaptotagmin 1 and phosphoinositide-specific phospholipase C-delta 1, respectively. The alignment highlights residues that may be critical to the C2 domain fold or required for Ca2+ binding and regulation.

Cytosolic phospholipase A2.

To summarize the regulation of cPLA2, we have proposed a model for the activation of cPLA2 based both on our previous studies (Clark et al., 1991; Lin et al., 1993) and the work of many others (Fig. 5). In this model, cPLA2 is tightly regulated by multiple pathways, including those that control Ca2+ concentration, phosphorylation states and cPLA2 protein levels, to exert both rapid and prolonged effects on cellular processes, such as inflammation. cPLA2 is rapidly activated by increased intracellular Ca2+ concentration and phosphorylation by MAP kinase. When cells are stimulated with a ligand for a receptor, such as ATP or PDGF, PLC is activated via either a G protein-dependent or -independent process, leading to the production of diacylglycerol (DAG) and inositol triphosphate (IP3). The rise in these intracellular messengers cause the activation of PKC and mobilization of intracellular Ca2+. Alternatively, the increase in intracellular Ca2+ can result from a Ca2+ influx. Increased Ca2+ acts through the CaLB domain to cause translocation of cPLA2 from the cytosol to the membrane where its substrate, phospholipid, is localized. This step is essential for the activation of cPLA2 and may account for the partial activation of cPLA2 in the absence of phosphorylation. MAP kinase activation can occur through both PKC-dependent and -independent mechanisms (Cobb et al., 1991; Posada and Cooper, 1992; Qiu and Leslie, 1994). In many cases, this pathway is also G protein-dependent. Activated MAP kinase phosphorylates cPLA2 at Ser-505, causing increased enzymatic activity of cPLA2, which is realized only upon translocation of cPLA2 to the membrane. Therefore, full activation of cPLA2 requires both increased cytosolic Ca2+ and cPLA2 phosphorylation at Ser-505. In a more delayed response, cPLA2 activity in the cells can be controlled by changes in its expression levels, such as in response to inflammatory cytokines and certain growth factors. Thus the expression level of cPLA2 is regulated by both transcriptional and post-transcriptional mechanisms.

An ion pair in class II major histocompatibility complex heterodimers critical for surface expression and peptide presentation.

In this report we demonstrate that the ion pair Arg-80 alpha and Asp-57 beta, located in the peptide-binding site of nearly all class II major histocompatibility complex (MHC) proteins, is important for surface expression and function of the murine class II heterodimer I-Ad. Charge reversal at either of these two residues by site-directed mutagenesis generated mutant class II molecules that failed to appear at the cell surface. This defect in surface expression was partially reversed when the invariant chain was present or when the mutants were paired with the corresponding charge-reversed variant of the opposite chain. Surprisingly, surface expression was restored when cells expressing the single-site mutants were cultured at reduced temperature. In addition, the substitution of Asp-57 beta with residues found in alleles of class II molecules associated with diabetes resulted in heterodimers that were inefficiently expressed at the cell surface and presented foreign peptide poorly. Together, these results demonstrate that the formation of a salt-bridge between Arg-80 alpha and Asp-57 beta is required for efficient surface expression of class II MHC molecules, therefore representing an important step in the assembly and transport of functional class II heterodimers to the cell surface.

Delineation of two functionally distinct domains of cytosolic phospholipase A2, a regulatory Ca(2+)-dependent lipid-binding domain and a Ca(2+)-independent catalytic domain.

Cytosolic phospholipase A2 (cPLA2) associates with natural membranes in response to physiological increases in Ca2+, resulting in the selective hydrolysis of arachidonyl phospholipids. The isolation and sequence analysis of cPLA2 cDNA clones from four different species revealed several highly conserved regions. The NH2-terminal conserved region is homologous to several other Ca(2+)-dependent lipid-binding proteins. Here we report that the first 178 residues of cPLA2, containing the homologous Ca(2+)-dependent lipid-binding (CaLB) motif, and another recombinant protein containing the cPLA2(1-178) fragment placed at the COOH terminus of the maltose-binding protein (MBP-CaLB) associate with membranes in a Ca(2+)-dependent manner. cPLA2 and MBP-CaLB also bind to synthetic liposomes at physiological Ca2+ concentrations, demonstrating that accessory proteins are not required. In contrast, delta C2, a truncated cPLA2 lacking the CaLB domain, fails to associate with membranes and fails to hydrolyze liposomal substrates. However, both delta C2 and cPLA2 hydrolyze monomeric 1-palmitoyl-2-lysophosphatidylcholine at identical rates in a Ca(2+)-independent fashion. These results delineate two functionally distinct domains of cPLA2, the Ca(2+)-independent catalytic domain, and the regulatory CaLB domain that presents the catalytic domain to the membrane in response to elevated Ca2+.

Normal peripheral T-cell function in c-Fos-deficient mice.

The ubiquitous transcription factors Fos and Jun are rapidly induced in T cells stimulated through the T-cell antigen receptor and regulate transcription of cytokines, including interleukin 2, in activated T cells. Since positive and negative selection of thymocytes during T-cell development also depends on activation through the T-cell receptor, Fos and Jun may play a role in thymocyte development as well. Fos and Jun act at several regulatory elements in the interleukin 2 promoter, including the AP-1 and NFAT sites. Using antisera specific to individual Fos and Jun family members, we show that c-Fos as well as other Fos family members are present in the inducible AP-1 and NFAT complexes of activated murine T cells. Nevertheless, c-Fos is not absolutely required for the development or function of peripheral T cells, as shown by using mice in which both copies of the c-fos gene were disrupted by targeted mutagenesis. c-Fos-deficient mice were comparable to wild-type mice in their patterns of thymocyte development and in the ability of their peripheral T cells to proliferate and produce several cytokines in response to T-cell receptor stimulation. Our results suggest that other Fos family members may be capable of substituting functionally for c-Fos during T-cell development and cytokine gene transcription in activated T cells.

Simultaneous involvement of all six predicted antigen binding loops of the T cell receptor in recognition of the MHC/antigenic peptide complex.

The TCR is predicted to resemble the Fab fragment of an Ig molecule and by analogy to possess six Ag binding loops that contact MHC proteins bound with antigenic peptides. We have identified residues in the predicted Ag binding loops (beta 1, beta 2, and beta 3) on a TCR beta-chain that are important in the recognition of the MHC/antigenic peptide complex. Using site-directed mutagenesis, we altered the residues forming the predicted Ag binding site on the beta-chain expressed by the T lymphocyte clone D5, which specifically recognizes p-azobenzenearsonate-conjugated peptides presented by the class II MHC molecule I-Ad. Amino acid substitution of individual residues in each loop affected Ag recognition, demonstrating that all three putative Ag binding loops of the D5 TCR beta-chain are important in interaction with I-Ad/arsonate-conjugated Ag. Taken together with our previous work on the D5 TCR alpha-chain (Nalefski et al., J. Exp. Med. 175:1553), these results suggest that all six Ag binding loops of the D5 TCR alpha- and beta-chains interact simultaneously with the MHC/peptide complex. Consequently, the area of interaction between the TCR and the MHC/antigenic peptide complex is predicted to be extensive.

Nature of the ligand recognized by a hapten- and carrier-specific, MHC-restricted T cell receptor.

The hapten- and carrier-specific T lymphocyte clone D5 and a T hybridoma (D5h) derived from D5 cells recognize several different protein Ag conjugated with p-azobenzenearsonate (arsonate) presented by the class II MHC protein I-Ad. We show here that the ligand recognized by the D5 TCR is a complex of a haptenated peptide bound to I-Ad. We have identified a peptide fragment generated by enzymatic cleavage of arsonate-conjugated OVA (Ars-OVA), which stimulates D5 cells when presented by I-Ad-bearing APC. A synthetic peptide corresponding to this fragment, OVA(36-50), forms a ligand for D5h cells when it is conjugated with arsonate and presented by cells bearing I-Ad. Paraformaldehyde-fixed, I-Ad-bearing cells present Ars-OVA(36-50), or the longer stimulatory peptide Ars-OVA(33-49), to D5h cells, demonstrating that haptenated synthetic peptides can substitute for naturally processed antigenic peptides. The peptide Ars-OVA(33-49) binds to the major peptide-binding site of I-Ad because it competitively inhibited presentation of the peptide OVA(323-339), previously demonstrated to bind to I-Ad directly in vitro, to the OVA/I-Ad-specific T cell hybridoma 3DO-54.8. The unconjugated OVA(33-49) peptide failed to inhibit the presentation of OVA(323-339), demonstrating that the hapten facilities binding of the peptide to I-Ad. Conversely, the peptide OVA(323-339) competitively inhibited the presentation of Ars-OVA(33-49) to D5h cells, indicating that the two peptides Ars-OVA(33-49) and OVA(323-339) bind to overlapping sites on I-Ad. Amino acid substitutions introduced into the beta 1 domain of I-Ad that affected recognition of OVA(323-339) by 3DO-54.8 cells also affected recognition of Ars-OVA(33-50) by D5h cells, demonstrating that similar regions on I-Ad are required for TCR recognition of conventional as well as haptenated peptides. These results represent the first demonstration that the ligand recognized by a hapten- and carrier-specific T cell clone restricted to an MHC class II protein is a haptenated peptide Ag bound to the MHC molecule.

CD3- T cells with cis- or trans-acting mutations affecting expression of T cell receptor beta-chain mRNA.

Mutants of an untransformed T cell clone that no longer respond to TCR/CD3 stimulation have been derived using a selection procedure based on the loss of functional response to Ag. This functional selection gives rise to clones of several different phenotypes. We have previously described mutants with a TCR/CD3+ cell surface phenotype whose TCR are uncoupled from cellular responses. We describe six additional mutants that do not express TCR/CD3 at the cell surface. One of the CD3- clones contains a deletion in the successfully rearranged TCR-alpha gene, whereas another carries a deletion in the successfully rearranged TCR-beta gene. TCR/CD3 expression in these deletion mutants can be restored by transfection of TCR-alpha or TCR-beta DNA. Four other clones do not express TCR-beta mRNA, yet contain no obvious deletions or rearrangements in the TCR-beta genes. One of these clones does not transcribe TCR-beta chain mRNA. The mutation in this clone does not reside in the TCR-beta gene itself, but may instead reside in a trans-acting regulatory element affecting TCR-beta gene expression, because the TCR-beta mRNA-phenotype is complemented by fusion with a TCR-alpha-beta- cell line. TCR-beta chain regulatory mutants will be valuable in contributing to our understanding of how TCR expression is regulated.

Functional analysis of the antigen binding site on the T cell receptor alpha chain.

We have identified residues on a T cell receptor (TCR) alpha chain that are important for interaction with antigen/major histocompatibility complex (MHC). Using site-directed mutagenesis, we modified DNA encoding the postulated antigen/MHC binding loops on the TCR alpha chain expressed by the T cell clone D5, which recognizes p-azobenzenearsonate-conjugated antigens presented by cells bearing I-Ad. These variant TCR alpha chains were expressed in conjunction with the wild-type D5 TCR beta chain on the surface of hybridoma cells, and were tested for the ability to recognize hapten-conjugated antigens presented by I-Ad. Individual amino acid substitutions in each of the three antigen binding loops (alpha 1, alpha 2, alpha 3) of the D5 TCR alpha chain affected antigen recognition, demonstrating that all three loops are important in recognition of antigen/MHC. A subset of the single amino acid substitutions completely eliminated antigen recognition, thus identifying the residues that are particularly important in the recognition of antigenic peptide/MHC by the D5 TCR. Because the wild-type D5 TCR recognizes arsonate and certain structural analogues of arsonate conjugated to a variety of protein antigens, we were able to test whether the TCR substitutions affected the specificity of the D5 TCR for hapten or carrier antigen. One substitution introduced into antigen binding loop alpha 3 markedly altered the pattern of carrier recognition. Together, these results verify the Ig model for the TCR and are consistent with the proposition that residues forming the first and second antigen binding loops of the TCR contact the MHC, while those forming the third loop contact mainly antigenic peptides.

Amino acid substitutions in the first complementarity-determining region of a murine T-cell receptor alpha chain affect antigen-major histocompatibility complex recognition.

The T-cell antigen receptor mediates recognition of foreign antigens physically associated with major histocompatibility complex (MHC) proteins. The tertiary structure of the T-cell receptor is thought to resemble that of immunoglobulin Fab fragments and to possess corresponding complementarity-determining regions (CDRs) that contact antigen-MHC. To test such a model for the T-cell receptor, we have generated T-cell hybridomas that express a wild-type or mutant form of the T-cell receptor present on the p-azobenzenearsonate-specific T-cell clone D5. Mutation of 2 amino acids (Tyr26 to serine, Gly28 to valine) in the predicted CDR1 of the D5 T-cell receptor alpha chain caused a markedly diminished response to antigen without affecting the response to anti-CD3 and anti-T-cell receptor antibodies. These results constitute the first test of the prediction that CDR1 in the T-cell receptor alpha chain is important for antigen-MHC recognition, thus providing strong evidence for the structural model of the T-cell antigen receptor based upon immunoglobulin.

An isoelectric focusing overlay study of the humoral immune response in Theiler's virus demyelinating disease.

Oligoclonal immunoglobulin G (IgG) bands are a frequent feature of inflammatory diseases of the central nervous system (CNS). In multiple sclerosis (MS), cerebrospinal fluid (CSF) oligoclonal IgG bands are a potential clue to the pathogenesis of the disease; however, their particular antigenic target is unknown. We sought to characterize the IgG response in an experimental CNS persistent demyelinating infection by isoelectric focusing (IEF) studies of serum and CSF from mice infected with Theiler's murine encephalomyelitis virus (TMEV). Following IEF, we used a new technique in order to identify TMEV-specific antibodies; focused immunoglobulins were blotted onto nitrocellulose paper which was then overlaid with radiolabeled virus. Autoradiograms showed that most of the TMEV antibody was locally synthesized within the CNS since CSF, but not serum, TMEV antibody had an anodal distribution. CSF IEF TMEV antibody spectrotypes were very similar, presumably because the CSFs were collected from the same inbred mouse strain. CSF TMEV antibody displayed less restricted heterogeneity than the very restricted cathodal CSF oligoclonal IgG bands seen in MS. The new IEF immunoblotting antigen overlay technique will be a powerful detection system to probe for the antigenic target against which MS CSF IgG may be directed.

Oligoclonal IgA bands in multiple sclerosis and subacute sclerosing panencephalitis.

CSF oligoclonal IgG bands are often found in MS or cerebral diseases in which there is chronic antigenic stimulation. Using agarose isoelectric focusing followed by immunoblotting, we found oligoclonal IgA bands in CSF from 16 of 20 randomly selected patients with MS, 7 of 7 with subacute sclerosing panencephalitis (SSPE), and 0 of 10 with noninflammatory neurologic or psychiatric disease. IgA bands did not correlate with the course or stage of MS. Serial samples from two patients with MS and one with SSPE demonstrated only minor changes in IgA banding pattern. One MS patient without oligoclonal IgG bands had oligoclonal IgA bands, indicating that the latter test may be helpful in the diagnosis of MS.