The most polymorphic residue on Plasmodium falciparum apical membrane antigen 1 determines binding of an invasion-inhibitory antibody.

Apical membrane antigen 1 (AMA1) is currently one of the leading malarial vaccine candidates. Anti-AMA1 antibodies can inhibit the invasion of erythrocytes by Plasmodium merozoites and prevent the multiplication of blood-stage parasites. Here we describe an anti-AMA1 monoclonal antibody (MAb 1F9) that inhibits the invasion of Plasmodium falciparum parasites in vitro. We show that both reactivity of MAb 1F9 with AMA1 and MAb 1F9-mediated invasion inhibition were strain specific. Site-directed mutagenesis of a fragment of AMA1 displayed on M13 bacteriophage identified a single polymorphic residue in domain I of AMA1 that is critical for MAb 1F9 binding. The identities of all other polymorphic residues investigated in this domain had little effect on the binding of the antibody. Examination of the P. falciparum AMA1 crystal structure localized this residue to a surface-exposed alpha-helix at the apex of the polypeptide. This description of a polymorphic inhibitory epitope on AMA1 adds supporting evidence to the hypothesis that immune pressure is responsible for the polymorphisms seen in this molecule.

Targeted disruption of an erythrocyte binding antigen in Plasmodium falciparum is associated with a switch toward a sialic acid-independent pathway of invasion.

Erythrocyte invasion by Plasmodium requires molecules present both on the merozoite surface and within the specialized organelles of the apical complex. The Plasmodium erythrocyte binding protein family includes the Plasmodium falciparum sialic acid-binding protein, EBA-175 (erythrocyte binding antigen-175), which binds sialic acid present on glycophorin A of human erythrocytes. We address the role of the conserved 3'-cysteine rich region, the transmembrane, and cytoplasmic domains through targeted gene disruption. Truncation of EBA-175 had no measurable effect on either the level of EBA-175 protein expression or its subcellular localization. Similarly, there appears to be no impairment in the ability of soluble EBA-175 to be released into the culture supernatant after schizont rupture. Additionally, the 3'-cys rich region, transmembrane, and cytoplasmic domains of EBA-175 are apparently non-essential for merozoite invasion. In contrast, erythrocyte invasion via the EBA-175/glycophorin A route appears to have been disrupted to such a degree that the mutant lines have undergone a stable switch in invasion phenotype. As such, EBA-175 appears to have been functionally inactivated within the truncation mutants. The sialic acid-independent invasion pathway within the mutant parasites accounts for approximately 85% of invasion into normal erythrocytes. These data demonstrate the ability of P. falciparum to utilize alternate pathways for invasion of red blood cells, a property that most likely provides a substantial survival advantage in terms of overcoming host receptor heterogeneity and/or immune pressure.

Structure of a HoxB1-Pbx1 heterodimer bound to DNA: role of the hexapeptide and a fourth homeodomain helix in complex formation.

Hox homeodomain proteins are developmental regulators that determine body plan in a variety of organisms. A majority of the vertebrate Hox proteins bind DNA as heterodimers with the Pbx1 homeodomain protein. We report here the 2.35 A structure of a ternary complex containing a human HoxB1-Pbx1 heterodimer bound to DNA. Heterodimer contacts are mediated by the hexapeptide of HoxB1, which binds in a pocket in the Pbx1 protein formed in part by a three-amino acid insertion in the Pbx1 homeodomain. The Pbx1 DNA-binding domain is larger than the canonical homeodomain, containing an additional alpha helix that appears to contribute to binding of the HoxB1 hexapeptide and to stable binding of Pbx1 to DNA. The structure suggests a model for modulation of Hox DNA binding activity by Pbx1 and related proteins.

The structure of GABPalpha/beta: an ETS domain- ankyrin repeat heterodimer bound to DNA.

GA-binding protein (GABP) is a transcriptional regulator composed of two structurally dissimilar subunits. The alpha subunit contains a DNA-binding domain that is a member of the ETS family, whereas the beta subunit contains a series of ankyrin repeats. The crystal structure of a ternary complex containing a GABPalpha/beta ETS domain-ankyrin repeat heterodimer bound to DNA was determined at 2. 15 angstrom resolution. The structure shows how an ETS domain protein can recruit a partner protein using both the ETS domain and a carboxyl-terminal extension and provides a view of an extensive protein-protein interface formed by a set of ankyrin repeats. The structure also reveals how the GABPalpha ETS domain binds to its core GGA DNA-recognition motif.

Binding and repression of the latency-associated promoter of herpes simplex virus by the immediate early 175K protein.

We demonstrate that the immediate early 175K protein (IE175K) of herpes simplex virus type 1 binds to the cap site of the latency-associated promoter (LAP) in an unusual manner. The complex formed on the LAP cap site was significantly larger than that formed on the IE175K cap site and the requirements for binding were qualitatively distinct with respect to both the primary sequence determinants at the site, and the regions of IE175K protein required for binding compared to those for the IE175K cap site. Although purified IE175K was sufficient for this large complex formed on the LAP cap site, the DNA-binding domain was unable to bind efficiently. This contrasted strikingly with the IE175K cap site where, using precisely analogous probes, the DNA-binding domain exhibited a strong interaction. Surprisingly, from dissociation kinetics we show that binding of the intact protein to the LAP cap site is considerably more stable than the binding of IE175K to its own cap site (half-lives of the complexes 15 min and < 1 min respectively), and this was reflected in more efficient repression of LAP-driven expression than IE175K promoter-driven expression by IE175K. Moreover, primary sequence requirements for IE175K binding to the LAP cap site region differed from previously identified IE175K recognition sequences in that in addition to a partially conserved consensus sequence, neighbouring bases were necessary for binding. Although the LAP cap site exhibits a pseudopalindromic arrangement of core consensus sites, we show that this is not the basis for the higher order, more stable binding to this region. Together these results indicate that IE175K forms an unusual complex at the LAP cap site, broadening the range of previously defined sequences recognized by IE175K.

Localization of cis-acting sequence requirements in the promoter of the latency-associated transcript of herpes simplex virus type 1 required for cell-type-specific activity.

We have previously demonstrated (A. H. Batchelor and P. O'Hare, J. Virol. 64:3269-3279, 1990) the selective activity in human neuroblastoma cells (IMR-32) of a promoter located upstream of the latency-associated transcript of herpes simplex virus type 1. In this work, we provide evidence for the basis of the selective activity of this latency-associated promoter (LAP). Recombinant constructs containing sequences up to -143 (relative to the LAP cap site) linked to the chloramphenicol acetyltransferase gene retain strong activity in HeLa cells but exhibit extremely weak activity in IMR-32 cells. Sequences mapping within the 108 bp upstream of -143 to position -251 enhance LAP activity by over 15-fold, restoring optimal levels of expression in IMR-32 cells, but have little or no effect (1.5-fold) in HeLa cells. This cell-type-specific enhancement of promoter activity took place in two major steps, with sequences between -143 and -158 conferring a four- to fivefold effect and sequences between -177 and -251 conferring a further threefold effect. Furthermore, sequences mapping from -40 to -258 could transfer the ability to be expressed in neuroblastoma cells to the normally inactive immediate-early 110K promoter (IE110K), increasing levels of expression by 35-fold. By comparison, this region had a relatively minor effect (twofold) on the activity of the IE110K promoter in HeLa cells, even though this promoter is open to activation by other mechanisms. However, neither of the overlapping subregions from -40 to -143 or -138 to -258 could confer efficient IMR-32 cell expression on the IE110K promoter, and we present alternative models for multiple element requirements or the requirement for a critical site around -140 which is not retained in either subfragment. We provide consistent evidence for a site around -140 and demonstrate the presence selectively in IMR-32 cells of a DNA-binding factor which binds a probe spanning this region. We propose that this element and the cognate factor (IC-1) may be involved in the selective activity of the LAP in neuroblastoma cells.

Regulation and cell-type-specific activity of a promoter located upstream of the latency-associated transcript of herpes simplex virus type 1.

To identify promoter regions which control expression of the latency-associated transcript (LAT) of herpes simplex virus type 1 (HSV-1), we constructed a series of recombinant vectors in which various sequences upstream of LAT were linked to the chloramphenicol acetyltransferase gene and tested for expression efficiency by transfection into tissue culture cells. In HeLa cells no activity was observed from the region (-250 to +201) immediately surrounding the nominal 5' end of LAT, but high levels of activity were observed by using different fragments within the region -1267 to -594. This promoter activity was largely contained within the 140-base-pair region from -797 to -658 and was 20- to 50-fold stronger than typical HSV delayed-early promoters and at least as strong as the activity from the simian virus 40 (SV40) enhancer-promoter region or the HSV immediate-early 110,000-Mr (IE110K) promoter region. In human neuroblastoma cells (IMR-32), there was a dramatic switch in relative activities in favor of the LAT promoter, so that it was 45- and 200-fold stronger than the IE110K and SV40 constructs, respectively. Furthermore, optimal activity in the neuroblastoma cells required sequences within the region -1267 to -797. This region had little effect on activity in HeLa cells. We also show that the LAT promoter activity was very efficiently repressed by the IE175K protein. From internal deletion analysis, the site of repression was located within a 55-base-pair region just downstream of a potential TATA box. This region exhibited a high degree of homology with the IE175K cap site and may be a binding site for the IE175K protein.

The role of CD44, CD45, CD45RO, CD46 and CD55 as potential anti-adhesion molecules involved in the binding of human tonsillar T cells to phorbol 12-myristate 13-acetate-differentiated U-937 cells.

In this study, we have shown that human tonsillar T cells adhere to phorbol 12-myristate 13-acetate(PMA)-differentiated U-937 cells. To examine the molecular mechanisms involved, the effect of a panel of monoclonal antibodies upon this adhesion was assessed in a quantitative binding assay. Antibodies against LFA-1 and ICAM-1 inhibited binding, directly implicated these molecules in T cell-PMA-induced U-937 adhesion. Furthermore, the adhesion was magnesium but not calcium dependent. Of the remaining antibodies that were tested, none of those against CD2, LFA-3, Mac-1, p150,95, CD43, CD45RA or CD56 affected binding. However, antibodies against CD44, CD45, CD45RO, CD46 and CD55 enhanced binding suggesting an anti-adhesive role for these molecules during U-937-T cell interaction.