The CD of ligand-DNA systems. 2. Poly(dA-dT) B-DNA.

A systematic theoretical study of the CD of [poly(dA-dT)]2 and its complexes with achiral small molecules is presented. The CD spectra of [poly(dA-dT)]2 and of poly(dA):poly(dT) are calculated for various DNA structures using the matrix method. The calculated and experimental spectra agree reasonably well for [poly(dA-dT)]2 but less well for poly(dA):poly(dT). The calculated CD spectrum of [poly(dA-dT)]2 fails to reproduce the wavelength region of 205-245 nm of the experimental spectrum. This discrepancy can be explained by a magnetic dipole allowed transition contributing significantly to the CD spectrum in this region. The induced CD of a transition moment of a molecule bound to [poly(dA-dT)]2 is also calculated. As was the case for [poly(dG-dC)]2, the induced CD of a groove bound molecule is one order of magnitude stronger than that of an intercalated molecule. The calculations also show considerable differences between pyrimidine-purine sites and purine-pyrimidine sites. Both signs and magnitudes of the CD induced into ligands bound in the minor groove agree with experimental observations.     

Characterization of the anion transport channel protein in human erythrocytes. Induced circular dichroism of inhibitors bound to the anion transport channel

The induced circular dichroism (CD) of erythrocyte ghosts with anion-transport inhibitors has been studied. A ghost-EITC (eosin 5-isothiocyanate) system shows an induced CD spectrum at the wavelength region corresponding to the absorption bands of EITC. Also a ghost-EMI (eosin 5-maleimide) system shows induced CD, but has bands of opposite sign to the EITC system. From the change of the CD intensity, the number of EITC molecules bound to one erythrocyte was estimated to be about 1.4 X 10(6), being close to the number of band 3 copies per ghost. The CD spectra of EITC and EMI systems show that a configurational structure of the moiety anchoring the EMI molecule is the reverse to that of EITC. The preferred conformation of bound EITC may be twisted in a right-handed sense. From the signs of the induced CD bands in ghost-stilbene disulfonate systems, the chirality of twisted stilbene derivatives seems to be a left-handed sense, as is the case for the EMI derivative. The CD spectra of EITC in the presence of DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonate) shows that the binding site of EITC may not be identical with that of DIDS. The results observed in this study reflect the ternary arrangement of the functional amino groups in anion recognition sites.     

The origin of the DNA induced circular dichroism of CC-1065 and analogs

The calf thymus DNA (CT-DNA) and poly(dI-dC).poly(dI-dC) binding properties of the natural antitumor antibiotic CC-1065 and selected analogs of CC-1065 were studied by circular dichroism (CD) and absorbance methods. The results indicate that the intense long wavelength DNA-induced CD band of these molecules originates from a chiral electronic transition which is delocalized over the whole molecule. Both the covalently bound species (N-3 adenine adduct) and the reversibly bound species exhibit the characteristic spectral behavior of an inherently dissymmetric chromophore when these agents bind within the minor groove of B-form DNA. This mechanism of optical activity accounts for why CC-1065 shows a weak CD in buffer but a very intense induced CD at long wavelength when bound to DNA, why the intensity of the induced CD of CC-1065 analogs depends upon how many fused ring systems the analog contains, and why covalently bound analogs having the mirror image configuration of the natural configuration also exhibit an intense positive induced CD band at long wavelength.     

Circular dichroism and circular polarization of luminescence of reduced nicotinamide adenine dinucleotide in solution and bound to dehydrogenases.

The CD (circular dichroism) and CPL (circular polarization of luminescence) spectra of NADPH in aqueous solution were studied and found to be markedly different. The spectra were not affected by cleavage of the coenzyme molecule with phosphodiesterase. The differences are thus not due to the existence of extended and folded conformations of NADPH and it is concluded that they originate in excited state conformational changes of the nicotinamide--ribose fragment. Opposite signs of both the CD and CPL spectra were observed for NADH bound to horse liver alcohol dehydrogenase and to beef heart lactate dehydrogenase indicating structural differences between the nicotinamide binding sites. The binding of substrate analogues to enzyme--coenzyme complexes did not affect the CD spectra and hence no significant conformational changes are induced upon formation of the ternary complexes. No changes in the CPL spectrum of NADH bound to lactate dehydrogenase were observed upon adding oxalate to form the ternary complex. Marked differences were found between the CPL spectra of binary and ternary complexes with liver alcohol dehydrogenase, while the CD spectra of these complexes were identical. It is concluded that a conformational change of the excited NADH molecule occurs in the binary but not in the ternary complex involving LADH, thus indicating an increased rigidity of the latter complex.     

Antigenic sites on cytochrome c2 from Rhodospirillum rubrum.

The antigenic determinants for three monoclonal antibodies against cytochrome c2 from Rhodospirillum rubrum were partially characterized by differential chemical modification of free and antibody-bound cytochrome c2 and by cross-reactivity analysis with different antigens. Circular dichroism spectroscopy was used to probe the effect of antibody binding on the conformation of cytochrome c2. The binding of two antibodies was strongly dependent on the native folding of the antigen. The first antibody bound to a determinant around the exposed heme edge on the 'front side' of the molecule which is not antigenic in mitochondrial cytochrome c2. Binding of this antibody to cytochrome c increased the induced CD of the ferric heme in a manner similar to that observed previously when mitochondrial cytochrome-c oxidase bound to the front side of cytochrome c. This observation points to a subtle conformational adaptation of the antigen induced by the antibody. The determinant for the second antibody, which also affected the heme CD spectrum of the antigen, was on a polypeptide loop where cytochrome c2 differs from mitochondrial cytochrome c by an eight-residue insertion. The third antibody, which did not induce a change in CD, bound to a sequential determinant near the amino end of cytochrome c2. Only this antibody cross-reacted with isolated cytochrome-c-derived peptides and with apo-cytochrome c2. A preliminary analysis of the polyclonal immune response of five rats against cytochrome c2 indicates that, unlike in eukaryotic cytochrome c, antigenic determinants are distributed over the whole polypeptide chain of the prokaryotic immunogen.     

Gangliosides induce selective modulation of CD4 from helper T lymphocytes

The cluster designation (CD)4 molecule is one of several nonpolymorphic T lymphocyte surface proteins that have been implicated in T cell-target cell interactions, and is thought to play an important role in regulating T helper cell function. Previously, we found that gangliosides inhibited the function of rat T helper cell lines, and simultaneously inhibited the expression of the rat CD4 molecule identified by the W3/25 antibody. We have now evaluated the generality and mechanism(s) of ganglioside-induced modulation of CD4 expressed by mouse, rat, and human T helper lymphocytes. Ganglioside pretreatment induced rapid and selective disappearance of the CD4 molecule from T helper cells of all three species. The ganglioside effect was temperature- and dose-dependent, reversible within 24 hr of ganglioside removal, azide-insensitive, and was neutralized completely by 10% serum. CD4 modulation appeared to be a general property of gangliosides since the effect could be induced similarly by highly purified individual gangliosides with varying amounts of sialic acid, or by mixed gangliosides. The activity of gangliosides appeared to require both the lipid and sialated oligosaccharide moieties. Gangliosides did not inactivate antibody function, but prevented binding at the cell surface by 12 different monoclonal antibodies specific for a variety of different CD4 epitopes. Preclearance of CD4 by antibody-mediated capping reduced binding of 3H-GM1 to T helper cells. Labeled GM1 bound to several detergent-extracted and transblotted lymphocyte-associated proteins, but apparently did not bind directly to the CD4 molecule under these conditions. These results indicate that gangliosides induce a profound change in the molecular orientation of CD4 within the T helper cell membrane which renders epitopes on the CD4 molecule inaccessible to antibody. This ganglioside effect represents a novel pathway which may contribute to the understanding of the role of CD4 as a regulatory molecule and as a specific receptor for the acquired immune deficiency syndrome virus.     

Monomeric class I molecules mediate TCR/CD3 epsilon/CD8 interaction on the surface of T cells.

Both CD8 and the TCR bind to MHC class I molecules during physiologic T cell activation. It has been shown that for optimal T cell activation to occur, CD8 must be able to bind the same class I molecule that is bound by the TCR. However, no direct evidence for the class I-dependent association of CD8 and the TCR has been demonstrated. Using fluorescence resonance energy transfer, we show directly that a single class I molecule causes TCR/CD8 interaction by serving as a docking molecule for both CD8 and the TCR. Furthermore, we show that CD3epsilon is brought into close proximity with CD8 upon TCR/CD8 association. These interactions are not dependent on the phosphorylation events characteristic of T cell activation. Thus, MHC class I molecules, by binding to both CD8 and the TCR, mediate the reorganization of T cell membrane components to promote cellular activation.     

Fine structure of three different anti-fluorescein combining sites: induced circular dichroism of hapten bound to autologous and heterologous recombinants.

We have previously reported the sequential appearance in hyperimmunized rabbits of three distinct types of antifluorescein-combining sites that can be distinguished by the characteristic induced circular dichroism (CD) of the bound hapten, fluorescein. Such induced CD depends on the configuration of the surrounding residues, and, in the case of anti-fluorescein, can be localized to the configuration of the sub-site which binds the hydroxyxanthenone moiety of fluorescein. In the present investigation, we have studied the fine structure of both autologous and heterologous recombinant sites and of the free chains by measuring the induced CD of bound hapten. Heavy chain dimers at pH 5.4 bound fluorescein that displayed a weak negative CD band which is different from that observed in any of the native antibodies. No induced CD was observed with light chains. Thus, the hydroxyxanthenone subsite does not exist intact in any of the isolated chains. Fluorescein bound to purified autologous recombinants, when studied at pH 7.5, exhibited CD spectra very similar to those observed for the original antibodies. Most significantly, fluorescein bound to purified heterologous recombinants, prepared in all possible combinations, showed CD spectra most similar to those exhibited by sites from which the heavy chain was derived. Thus, the microenvironment of the subsite appears to be due primarily to the heavy chain.     

Multimolecular associations of the T-cell antigen receptor

T cells are activated when the T-cell receptor for antigen (TCR) interacts with an antigenic peptide bound to a major histocompatibility complex (MHC) molecule on the surface of another cell. It is often assumed that T-cell activation is induced by the crosslinking of TCRs. In this article, Albertus Beyers, Louise Spruyt and Alan Williams argue that this mechanism is not generally applicable. They hypothesize that the key event in T-cell activation is the formation of multimolecular complexes consisting of the TCR and several other polypeptides, including CD4 or CD8, CD2, CD5 and the associated tyrosine kinases p59(fyn) and p56(lck).     

Molecular interactions mediating T-B lymphocyte collaboration in human lymphoid follicles. Roles of T cell-B-cell-activating molecule (5c8 antigen) and CD40 in contact-dependent help.

In lymphoid follicles, CD4+ T lymphocytes provide contact-dependent stimuli to B cells that are critical for the generation of specific antibody responses in a process termed Th function. The CD4+ T cell-restricted surface activation protein, 5c8 Ag (T-BAM), has recently been shown to be a component of the contact-dependent helper signal to B cells. To further dissect this process, we utilized a Jurkat T cell lymphoma clone, termed D1.1, that constitutively expresses T-BAM and activates peripheral B cells to express surface CD23 in a contact-dependent mechanism that is inhibited by mAb anti-T-BAM (5c8). Similar to its effect on peripheral B cells, Jurkat D1.1 activates B cells from lymphoid organs, as well as a B cell lymphoma clone, RAMOS 266,4CN 3F10 (RAMOS 266), to up-regulate surface CD23. Interestingly, mAb to the B cell surface molecule, CD40 (mAb G28-5 and B-B20), inhibit D1.1 induced activation of RAMOS 266 and peripheral and lymphoid B cells. In contrast, mAb to CR2 or the adhesion molecules, LFA1, LFA3, or ICAM-1, have little effect. The inhibitory effect of anti-CD40 mAb on B cell activation induced by D1.1 is specific because anti-CD40 potentiates, rather than inhibits, the up-regulation of CD23 on B cells induced by rIL-4. Moreover, cross-linking CD40 molecules by anti-CD40 mAb bound to Fc gamma RII+ (CD32) L cells induces B cell CD23 expression. In vivo, T-BAM-expressing cells are CD4+ T cells that are restricted to lymphoid organs and are localized in the mantle and centrocytic zones of lymphoid follicles and the spleen periarteriolar lymphoid sheath in association with CD40+ B cells. Taken together, these data demonstrate that T-BAM on T cells and CD40 on B cells are involved in contact-dependent T-B help interactions that occur in lymphoid follicles.     

Regions required for CD4 binding in the external glycoprotein gp120 of simian immunodeficiency virus.

The external domain of the envelope glycoprotein, gp120, of simian immunodeficiency virus (SIV) has been expressed as a mature secreted product using recombinant baculoviruses and the expressed protein, which has an observed molecular mass of 110 kDa, was purified by monoclonal antibody (MAb) affinity chromatography. N-terminal sequence analysis showed a signal sequence cleavage identity similar to that of the gp120s of both human immunodeficiency virus type 1 (HIV-1) and HIV type 2. The expressed molecule bound to soluble CD4 with an affinity that was approximately 10-fold lower than that of gp120 from HIV-1. A screening of the ability of SIV envelope MAbs to inhibit CD4 binding revealed two groups of inhibitory MAbs. One group is dependent on conformation, while the second group maps to a discrete epitope near the amino terminus. The particular role of the V3 loop region of the molecule in CD4 binding was investigated by the construction of an SIV-HIV hybrid in which the V3 loop of SIV was precisely replaced with the equivalent domain from HIV-1 MN. The hybrid glycoprotein bound HIV-1 V3 loop MAbs and not SIV V3 MAbs but continued to bind conformational SIV MAbs and soluble CD4 as well as the parent molecule.     

CD58 and CD59 molecules exhibit potentializing effects in T cell adhesion and activation.

We have generated stable Chinese hamster ovary (CHO) cell transfectants expressing either CD58 or CD59 or both molecules to compare their respective parts played in T cell adhesion and activation. Using a rosetting assay, we have shown the following: 1) The CD59 molecule was directly responsible for adhesive interaction between human T cells and CD59+ CHO transfectants. CD59-mediated adhesion induced 12 +/- 2% (mean +/- SEM, n = 25) of rosettes. 2) The CD58 molecule expressed on CD58+ CHO transfectants induced 29 +/- 6% (mean +/- SEM, n = 8) of rosettes. 3) Double transfected CD58+CD59+ CHO cells formed up to 80% of rosettes, largely exceeding the sum of rosettes formed by single transfectants, thus disclosing at least an additive and possibly a synergic action of both molecules in mediating adhesion to T cells. Culturing purified human T cells in the presence of fixed CHO transfectants and submitogenic doses of PHA + rIL-1 alpha showed that: 1) CD59+ CHO transfectants induced sevenfold T cell proliferation enhancement, demonstrating the direct involvement of the CD59 molecule in T cell activation; 2) CD58+ CHO transfectants induced 20-fold T cell proliferation increase; and 3) the enhancement induced by CD58+CD59+ CHO cells was more than 40-fold. These results suggest that CD58 and CD59 molecules present on the surface of accessory cells might exert synergic function in T cell adhesive interactions and in the stimulation of T cell activation.     

In vitro interactions of opioid peptides with phospholipids. II. Additional spectral evidence

The interaction of leu-enkephalin with phosphatidylserine has been studied with ultraviolet and circular dichroism spectroscopy methods as well as with fluorescence anisotropy techniques. The data reported hereunder confirm the existence of binding between the two species, and also support the hypothesis that not only the tyrosine, but also the phenylalanine residue in the leu-enkephalin molecule is involved in peptide-lipid interaction. In addition, ultraviolet and CD evidence, taken together, tend to suggest that both aromatic residues are bound, with a different degree of involvement, to the same region of the lipid molecule. The data reported are discussed in terms of the interaction model previously proposed by us.     

Activated protein kinase C directly phosphorylates the CD34 antigen on hematopoietic cells.

The CD34 antigen is a human leukocyte membrane protein expressed specifically by lymphohematopoietic progenitor cells. We found that CD34 is a phosphoprotein and therefore examined the regulation of its phosphorylation. Activation of protein kinase C (PKC) enhanced CD34 phosphorylation. The PKC activators, 12-O-tetradecanoylphorbol-13-acetate and bryostatin-1, induced rapid, stoichiometric hyperphosphorylation of CD34 protein in cells, resulting in a 5-fold increase in CD34 phosphorylation. In vitro kinase studies revealed that purified PKC could directly phosphorylate purified CD34. Only serine phosphorylation was detected in the CD34 molecule. Two-dimensional phosphopeptide mapping experiments indicated that PKC induces the phosphorylation of identical serine residue(s) in vitro and in vivo (in KG1 cells). These are newly phosphorylated serine residue(s), which are not detectably phosphorylated in CD34 from exponentially growing KG1 cells. These data indicate that the developmental stage-specific molecule, CD34, is a phosphorylation target for activated PKC. Furthermore, these findings raise the possibility that PKC activation and phosphorylation of the CD34 molecule may play a role in signal transduction during early lymphohematopoiesis.     

Expression, regulation, and function of B cell-expressed CD154 in germinal centers.

Activated B cells and T cells express CD154/CD40 ligand in vitro. The in vivo expression and function of B cell CD154 remain unclear and therefore were examined. Tonsillar B and T cells expressed CD154 at a similar density both in situ and immediately ex vivo, whereas a significantly higher percentage of the former expressed CD154. CD154-expressing B cells were most frequent in the CD38positiveIgD+ pre-germinal center (GC)/GC founder, CD38positive GC and CD38-IgD- memory populations, and were also found in the CD38-IgD+ naive and CD38brightIgD+ plasmablast subsets, but not in the CD38brightIgD- plasma cell subset. B cell expression of CD154 was induced by engaging surface Ig or CD40 by signals that predominantly involved activation of AP-1/NF-AT and NF-kappaB, respectively. The functional importance of CD154-mediated homotypic B cell interactions in vivo was indicated by the finding that mAb to CD154 inhibited differentiation of CD38positiveIgD- GC B cells to CD38-IgD- memory cells. In addition, mAb to CD154 inhibited proliferation induced by engaging sIg or CD40, indicating the role of up-regulation of this molecule in facilitating B cell responsiveness. Of note, CD154 itself not only functioned as a ligand but also as a direct signaling molecule as anti-CD154-conjugated Sepharose beads costimulated B cell responses induced by engaging surface Ig. These results indicate that CD154 is expressed by human B cells in vivo and plays an important role in mediating B cell responses.     

Comparison of phosphorylation and internalization of the antigen receptor/CD3 complex, CD8, and class I MHC-encoded proteins on T cells. Role of intracytoplasmic domains analyzed with hybrid CD8/class I molecules

We analyzed the phosphorylation and the dynamics of TCR/CD3, CD8 and MHC class I molecules during the activation of a CD8+ cytotoxic T lymphocyte clone and of CD8- T helper hybridomas transfected with the gene coding for the native (J. Gabert, C. Langlet, R. Zamoyska, J.R. Parnes, A.M. Schmitt-Verhulst, and B. Malissen. 1987. Reconstitution of MHC class I specificity by transfer of the T cell receptor and Lyt-2 genes. Cell 50:545) or truncated CD8 alpha molecule. The CD3 components gamma and epsilon and the CD8 alpha subunit were phosphorylated after activation of the CTL clone with the protein kinase C activator PMA. Class I MHC molecules were phosphorylated irrespective of PMA activation. Constitutive phosphorylation of the MHC class I products was found to be intrinsic to the transmembrane/cytoplasmic portion of the molecules because it was transferred to the CD8 alpha hybrid molecules composed of extracellular CD8 and MHC class I transmembrane and intracytoplasmic domains (CD8-e/MHC-t-i). Measurements of the dynamics of these cell surface molecules by using radiolabeled mAb revealed distinct behaviors: TCR/CD3 complex ligand internalization was increased (around 50% after 40 to 60 min) after PMA activation, whereas the ligand of class I MHC molecules was internalized at constant rate irrespective of PMA activation. Ligand bound to native CD8 molecules was poorly internalized, irrespective of the activation of the T cells with PMA. The same ligand bound to the CD8-e/MHC-t-i hybrid molecule was internalized at the same rate as a class I MHC molecule ligand, indicating that the behavior of the hybrid molecule was characteristic of the transmembrane/cytoplasmic portion of MHC class I molecules.     

Sialoadhesin, a macrophage sialic acid binding receptor for haemopoietic cells with 17 immunoglobulin-like domains.

Sialoadhesin is a macrophage-restricted adhesion molecule of 185 kDa that mediates sialic acid-dependent binding to cells. It is expressed strongly by macrophages in lymphoid and haemopoietic tissues where it is likely to mediate cell-cell interactions. Here we report the molecular cloning of murine sialoadhesin and show that it is a new member of the immunoglobulin (Ig) superfamily with 17 Ig-like domains. COS cells transfected with a cDNA encoding full-length sialoadhesin bound mouse bone marrow cells in a sialic acid-dependent manner. Alternatively spliced cDNAs, predicting soluble forms of sialoadhesin containing the first three or 16 Ig-like domains of sialoadhesin, were expressed in COS cells and the respective proteins purified. When immobilized on plastic, the 16-domain form bound cells in a sialic acid-dependent manner, suggesting that sialoadhesin can function in both secreted and membrane-bound forms. The most similar proteins in the database were CD22, myelin-associated glycoprotein, Schwann cell myelin protein and CD33. Like sialoadhesin, CD22 mediates sialic acid-dependent cell adhesion. The sequence similarity of sialoadhesin to CD22 and related members of the Ig superfamily indicates the existence of a novel family of sialic acid binding proteins involved in cell-cell interactions.     

Molecular recognition of human CD1b antigen complexes: evidence for a common pattern of interaction with alpha beta TCRs

Ag-specific T cell recognition is mediated through direct interaction of clonotypic TCRs with complexes formed between Ag-presenting molecules and their bound ligands. Although characterized in substantial detail for class I and class II MHC encoded molecules, the molecular interactions responsible for TCR recognition of the CD1 lipid and glycolipid Ag-presenting molecules are not yet well understood. Using a panel of epitope-specific Abs and site-specific mutants of the CD1b molecule, we showed that TCR interactions occur on the membrane distal aspects of the CD1b molecule over the alpha1 and alpha2 domain helices. The location of residues on CD1b important for this interaction suggested that TCRs bind in a diagonal orientation relative to the longitudinal axes of the alpha helices. The data point to a model in which TCR interaction extends over the opening of the putative Ag-binding groove, making multiple direct contacts with both alpha helices and bound Ag. Although reminiscent of TCR interaction with MHC class I, our data also pointed to significant differences between the TCR interactions with CD1 and MHC encoded Ag-presenting molecules, indicating that Ag receptor binding must be modified to accommodate the unique molecular structure of the CD1b molecule and the unusual Ags it presents.     

Non-cooperative Ca(II) removal and terbium(III) substitution in carp muscle calcium binding parvalbumin.

Close coorelation of atomic absorption measurements for Ca(II) contents indicates that from pH 5.8-7.4 a twentyfold excess of EGTA1 removes but one of two Ca(II) from carp parvalbumin. Thus binding of the two Ca(II) appears to be noncooperative. The maximum in emission intensity observed at a nonintegral 1.4-1.7 equivs of added Tb(III) is shown to be due to quenching by excess Tb(III). The emission intensity at the maximum increased 40% upon dialysis to remove Tb(III) not bound in the CD or EF sites. Atomic absorption results show that both Ca(CD) and Ca(EF) of native parvalbumin are easily replaced by Tb(III). Emission of Tb(EF) is not quenched by Tb(CD), but by solution Tb(III) bound at a third site, perhaps the single water molecule bound to Tb(EF). Labeling of the single sulfhydryl group with a trifluoroacetonyl gorup yields a protein with ultraviolet circular dichroism, emission, and circularly polarized emission spectra closely similar to those of native parvalbumin.