Chemical and enzymatic probing (footprinting) of the reactivity of the promoter DNA backbone is applied to characterize two binary open complexes RPo1 (-Mg2+) and RPo2 (+Mg2+), formed by Escherichia coli RNA polymerase (E sigma 70) at the lambda PR promoter. We report that HO. detects major differences in backbone reactivity between RPo1 and RPo2 in the open region from -4 to +1 relative to the transcription start site. Deoxyribose sugars at positions -4 to +1 of the t (template) strand react with HO. in RPo2 but are relatively protected in RPo1. Binding of Mg2+ to convert RPo1 to RPo2 therefore increases the reactivity of two negatively charged footprinting agents [MnO4-; Suh, W.-C., Ross, W., & Record, M. T., Jr., (1993) Science 259, 358-361; and Fe(EDTA)2-/HO.] at the start site and is required for binding of the negatively-charged initiating nucleotides to the polymerase and the t strand at the start site. We propose that these effects result from binding of two Mg2+ ions to the catalytic carboxyls in the nucleotide binding sites. Except for the key region on the t strand at the start site, the promoter DNA of both RPo1 and RPo2 is continuously protected from DNase I and hydroxyl radical (HO.) cleavage between the -12 and +25 promoter positions. Protection in the upstream region, extending from -13 to about -70, is periodic, with an 11 base pair periodicity indicative of binding of polymerase to a single face of the DNA helix.(ABSTRACT TRUNCATED AT 250 WORDS)
Severe anemia is a lethal complication of Plasmodium falciparum malaria, particularly in children. Recent studies in children with severe P. falciparum anemia have demonstrated elevated levels of E-bound Abs, reduced E-associated complement receptor 1 (CR1) and decay-accelerating factor (DAF), and pronounced splenic enlargement, suggesting a mechanism for E loss involving Abs, complement, and phagocytosis. Motivated by these reports, we have developed an in vitro model in which human E with Abs and complement bound to CR1, DAF, or glycophorin A are incubated with model human macrophages (the THP-1 cell line). Previous work has demonstrated that immune complex (IC) substrates bound to E CR1, either by an Ab or via C3b, are transferred to macrophages with loss of CR1. In this study, we report that IC bound to DAF or glycophorin A by an Ab linkage are also transferred to macrophages. DAF is lost from the E during the transfer of DAF-bound IC, but the transfer of CR1-bound IC does not lead to a significant loss of DAF. Using glycophorin A-bound IC, we observe competition between transfer of IC and phagocytosis of the E: a fraction (≤15%) of the E was phagocytosed, while the remaining E were stripped of IC. We also examined the organization of CR1 and DAF in the presence of E-bound Ab/complement. We find that CR1, but not DAF, colocalizes with IgM mAb-C3b and IC-C3b substrates attached to glycophorin A. We observe that the binding of the IgM mAb-C3b to glycophorin A induces a novel unclustering of CR1.
Objective. To develop an in vitro model for investigating the mechanism by which autoantibodies in immune complexes (ICs) that are bound to primate erythrocytes via antigen-based heteropolymers (AHPs) are cleared from the circulation and localized to the liver.Methods. IgG anti-double-stranded DNA (antidsDNA) antibodies in ICs with dsDNA were bound to human erythrocytes via complement receptor 1 (CR1) either by opsonization with normal human serum as a complement source or through the use of an AHP, which consists of an anti-CR1 monoclonal antibody (mAb) that is chemically crosslinked with dsDNA. We performed parallel investigations of the mechanism of transfer of both types of erythrocyte-bound ICs to a monocytic cell line (U937). Erythrocytes with CR1-bound ICs were incubated with U937 cells under a variety of conditions, and subsequently, the levels of IgG anti-dsDNA, CR1, AHP, or C3b on both erythrocytes and U937 cells were measured by flow cytometry with appropriate fluorescently labeled probes.Results. In the presence of U937 cells, both the AHP-anti-dsDNA and C3b-opsonized ICs were rapidly removed from the erythrocytes; at 37°C, more than half of the complexes were removed in 2 minutes. Monomeric mouse IgG2a mAb blocked the transfer of both types of complexes by 75%, suggesting that Fc␥ receptor type I (Fc␥RI) is the main phagocyte receptor responsible for the removal of ICs from erythrocytes. Levels of CR1 on the erythrocyte surface were reduced during transfer of the AHP-anti-dsDNA ICs, suggesting that transfer involves a concomitant removal of CR1, presumably by proteolysis.Conclusion. Transfer of AHP-anti-dsDNA ICs from erythrocyte CR1 to model phagocytes occurs by a mechanism that is similar to the natural mechanism of IC clearance, involving recognition by Fc␥RI and removal of erythrocyte CR1 as key steps.
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