RESUMO
The V3 loop of the HIV-1 envelope (Env) protein elicits a vigorous, but largely non-neutralizing antibody response directed to the V3-crown, whereas rare broadly neutralizing antibodies (bnAbs) target the V3-base. Challenging this view, we present V3-crown directed broadly neutralizing Designed Ankyrin Repeat Proteins (bnDs) matching the breadth of V3-base bnAbs. While most bnAbs target prefusion Env, V3-crown bnDs bind open Env conformations triggered by CD4 engagement. BnDs achieve breadth by focusing on highly conserved residues that are accessible in two distinct V3 conformations, one of which resembles CCR5-bound V3. We further show that these V3-crown conformations can, in principle, be attacked by antibodies. Supporting this conclusion, analysis of antibody binding activity in the Swiss 4.5 K HIV-1 cohort (n = 4,281) revealed a co-evolution of V3-crown reactivities and neutralization breadth. Our results indicate a role of V3-crown responses and its conformational preferences in bnAb development to be considered in preventive and therapeutic approaches.
Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Anti-HIV/imunologia , HIV-1/imunologia , Conformação Proteica , Produtos do Gene env do Vírus da Imunodeficiência Humana/química , Anticorpos Neutralizantes/metabolismo , Linhagem Celular Tumoral , Epitopos/genética , Epitopos/imunologia , Epitopos/metabolismo , Células HEK293 , Anticorpos Anti-HIV/metabolismo , HIV-1/genética , HIV-1/metabolismo , Humanos , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Mutação , Ligação Proteica , Produtos do Gene env do Vírus da Imunodeficiência Humana/genética , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologiaRESUMO
Numerous intracellular pathogens exploit cell surface glycoconjugates for host cell recognition and entry. Unlike bacteria and viruses, Toxoplasma gondii and other parasites of the phylum Apicomplexa actively invade host cells, and this process critically depends on adhesins (microneme proteins) released onto the parasite surface from intracellular organelles called micronemes (MIC). The microneme adhesive repeat (MAR) domain of T. gondii MIC1 (TgMIC1) recognizes sialic acid (Sia), a key determinant on the host cell surface for invasion by this pathogen. By complementation and invasion assays, we demonstrate that TgMIC1 is one important player in Sia-dependent invasion and that another novel Sia-binding lectin, designated TgMIC13, is also involved. Using BLAST searches, we identify a family of MAR-containing proteins in enteroparasitic coccidians, a subclass of apicomplexans, including T. gondii, suggesting that all these parasites exploit sialylated glycoconjugates on host cells as determinants for enteric invasion. Furthermore, this protein family might provide a basis for the broad host cell range observed for coccidians that form tissue cysts during chronic infection. Carbohydrate microarray analyses, corroborated by structural considerations, show that TgMIC13, TgMIC1, and its homologue Neospora caninum MIC1 (NcMIC1) share a preference for alpha2-3- over alpha2-6-linked sialyl-N-acetyllactosamine sequences. However, the three lectins also display differences in binding preferences. Intense binding of TgMIC13 to alpha2-9-linked disialyl sequence reported on embryonal cells and relatively strong binding to 4-O-acetylated-Sia found on gut epithelium and binding of NcMIC1 to 6'sulfo-sialyl Lewis(x) might have implications for tissue tropism.
Assuntos
Lectinas/química , Lectinas/metabolismo , Análise em Microsséries , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Toxoplasma/citologia , Toxoplasma/fisiologia , Animais , Células CHO , Metabolismo dos Carboidratos , Sequência Conservada , Cricetinae , Cricetulus , Cristalografia por Raios X , Técnicas de Inativação de Genes , Glicoconjugados/metabolismo , Humanos , Lectinas/genética , Modelos Moleculares , Ácido N-Acetilneuramínico/metabolismo , Estrutura Terciária de Proteína , Proteínas de Protozoários/genética , Homologia de Sequência de Aminoácidos , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico , Especificidade por Substrato , Toxoplasma/genética , Toxoplasma/metabolismo , Toxoplasmose/metabolismoRESUMO
The eukaryotic mismatch repair (MMR) protein MSH6 exhibits a core region structurally and functionally similar to bacterial MutS. However, it possesses an additional N-terminal region (NTR), comprising a PCNA binding motif, a large region of unknown function and a nonspecific DNA binding fragment. Yeast NTR was recently described as an extended tether between PCNA and the core of MSH6 . In contrast, we show that human NTR presents a globular PWWP domain in the region of unknown function. We demonstrate that this PWWP domain binds double-stranded DNA, without any preference for mismatches or nicks, whereas its apparent affinity for single-stranded DNA is about 20 times lower. The S144I mutation, which in human MSH6 causes inherited somatic defects in MMR resulting in increased development of hereditary non polyposis colorectal cancer , is located in the DNA binding surface of the PWWP domain. However, it only moderately affects domain stability, and it does not perturb DNA binding in vitro.