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1.
J Biol Chem ; 289(37): 25750-63, 2014 Sep 12.
Artículo en Inglés | MEDLINE | ID: mdl-25074926

RESUMEN

TYRO3, AXL, and MER receptors (TAMs) are three homologous type I receptor-tyrosine kinases that are activated by endogenous ligands, protein S (PROS1) and growth arrest-specific gene 6 (GAS6). These ligands can either activate TAMs as soluble factors, or, in turn, opsonize phosphatidylserine (PS) on apoptotic cells (ACs) and serve as bridging molecules between ACs and TAMs. Abnormal expression and activation of TAMs have been implicated in promoting proliferation and survival of cancer cells, as well as in suppressing anti-tumor immunity. Despite the fact that TAM receptors share significant similarity, little is known about the specificity of interaction between TAM receptors and their ligands, particularly in the context of ACs, and about the functional diversity of TAM receptors. To study ligand-mediated activation of TAMs, we generated a series of reporter cell lines expressing chimeric TAM receptors. Using this system, we found that each TAM receptor has a unique pattern of interaction with and activation by GAS6 and PROS1, which is also differentially affected by the presence of ACs, PS-containing lipid vesicles and enveloped virus. We also demonstrated that γ-carboxylation of ligands is essential for the full activation of TAMs and that soluble immunoglobulin-like TAM domains act as specific ligand antagonists. These studies demonstrate that, despite their similarity, TYRO3, AXL, and MER are likely to perform distinct functions in both immunoregulation and the recognition and removal of ACs.


Asunto(s)
Apoptosis/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Sanguíneas/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Células Jurkat , Proteína S , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Transducción de Señal , Estomatitis Vesicular/genética , Tirosina Quinasa c-Mer , Tirosina Quinasa del Receptor Axl
2.
J Biol Chem ; 289(37): 25737-49, 2014 Sep 12.
Artículo en Inglés | MEDLINE | ID: mdl-25074939

RESUMEN

MERTK, a member of the TAM (TYRO3, AXL, and MERTK) receptor tyrosine kinases, has complex and diverse roles in cell biology. On the one hand, knock-out of MERTK results in age-dependent autoimmunity characterized by failure of apoptotic cell clearance, while on the other, MERTK overexpression in cancer drives classical oncogene pathways leading to cell transformation. To better understand the interplay between cell transformation and efferocytosis, we stably expressed MERTK in human MCF10A cells, a non-tumorigenic breast epithelial cell line devoid of endogenous MERTK. While stable expression of MERTK in MCF10A resulted in enhanced motility and AKT-mediated chemoprotection, MERTK-10A cells did not form stable colonies in soft agar, or enhance proliferation compared with parental MCF10A cells. Concomitant to chemoresistance, MERTK also stimulated efferocytosis in a gain-of-function capacity. However, unlike AXL, MERTK activation was highly dependent on apoptotic cells, suggesting MERTK may preferentially interface with phosphatidylserine. Consistent with this idea, knockdown of MERTK in breast cancer cells MDA-MB 231 reduced efferocytosis, while transient or stable expression of MERTK stimulated apoptotic cell clearance in all cell lines tested. Moreover, human breast cancer cells with higher endogenous MERTK showed higher levels of efferocytosis that could be blocked by soluble TAM receptors. Finally, through MERTK, apoptotic cells induced PD-L1 expression, an immune checkpoint blockade, suggesting that cancer cells may adopt MERTK-driven efferocytosis as an immune suppression mechanism for their advantage. These data collectively identify MERTK as a significant link between cancer progression and efferocytosis, and a potentially unrealized tumor-promoting event when MERTK is overexpressed in epithelial cells.


Asunto(s)
Neoplasias de la Mama/genética , Transformación Celular Neoplásica/genética , Células Epiteliales/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Tirosina Quinasas Receptoras/genética , Apoptosis/genética , Neoplasias de la Mama/patología , Movimiento Celular/genética , Células Epiteliales/patología , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Fagocitosis/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Tirosina Quinasa c-Mer , Tirosina Quinasa del Receptor Axl
3.
FASEB J ; 22(5): 1380-92, 2008 May.
Artículo en Inglés | MEDLINE | ID: mdl-18198210

RESUMEN

MAb 2G12 neutralizes HIV-1 by binding with high affinity to a cluster of high-mannose oligosaccharides on the envelope glycoprotein, gp120. Screening of phage-displayed peptide libraries with 2G12 identified peptides that bind specifically, with K(d)s ranging from 0.4 to 200 microM. The crystal structure of a 21-mer peptide ligand in complex with 2G12 Fab was determined at 2.8 A resolution. Comparison of this structure with previous structures of 2G12-carbohydrate complexes revealed striking differences in the mechanism of 2G12 binding to peptide vs. carbohydrate. The peptide occupies a site different from, but adjacent to, the primary carbohydrate-binding site on 2G12, and makes only slightly fewer contacts to the Fab than Man(9)GlcNAc(2) (51 vs. 56, respectively). However, only two antibody contacts with the peptide are hydrogen bonds in contrast to six with Man(9)GlcNAc(2), and only three of the antibody residues that interact with Man(9)GlcNAc(2) also contact the peptide. Thus, this mechanism of peptide binding to 2G12 does not support structural mimicry of the native carbohydrate epitope on gp120, since it neither replicates the oligosaccharide footprint on the antibody nor most of the contact residues. Moreover, 2G12.1 peptide is not an immunogenic mimic of the 2G12 epitope, since antisera produced against it did not bind gp120.


Asunto(s)
Anticuerpos Monoclonales/metabolismo , Epítopos/química , Anticuerpos Anti-VIH/efectos de los fármacos , Proteína gp120 de Envoltorio del VIH/metabolismo , Imitación Molecular , Péptidos/antagonistas & inhibidores , Secuencia de Aminoácidos , Animales , Sitios de Unión de Anticuerpos , Anticuerpos ampliamente neutralizantes , Cristalización , Cristalografía por Rayos X , Proteína gp120 de Envoltorio del VIH/antagonistas & inhibidores , Humanos , Enlace de Hidrógeno , Modelos Moleculares , Oligosacáridos/química , Biblioteca de Péptidos , Péptidos/química , Péptidos/inmunología , Conejos
5.
Proc Natl Acad Sci U S A ; 102(38): 13372-7, 2005 Sep 20.
Artículo en Inglés | MEDLINE | ID: mdl-16174734

RESUMEN

Human antibody 2G12 neutralizes a broad range of HIV-1 isolates. Hence, molecular characterization of its epitope, which corresponds to a conserved cluster of oligomannoses on the viral envelope glycoprotein gp120, is a high priority in HIV vaccine design. A prior crystal structure of 2G12 in complex with Man(9)GlcNAc(2) highlighted the central importance of the D1 arm in antibody binding. To characterize the specificity of 2G12 more precisely, we performed solution-phase ELISA, carbohydrate microarray analysis, and cocrystallized Fab 2G12 with four different oligomannose derivatives (Man(4), Man(5), Man(7), and Man(8)) that compete with gp120 for binding to 2G12. Our combined studies reveal that 2G12 is capable of binding both the D1 and D3 arms of the Man(9)GlcNAc(2) moiety, which would provide more flexibility to make the required multivalent interactions between the antibody and the gp120 oligomannose cluster than thought previously. These results have important consequences for the design of immunogens to elicit 2G12-like neutralizing antibodies as a component of an HIV vaccine.


Asunto(s)
Anticuerpos Monoclonales/química , Epítopos/química , Anticuerpos Anti-VIH/química , Proteína gp120 de Envoltorio del VIH , VIH-1 , Manosa/química , Vacunas contra el SIDA/inmunología , Anticuerpos Monoclonales/inmunología , Sitios de Unión de Anticuerpos/inmunología , Secuencia de Carbohidratos , Cristalografía por Rayos X , Epítopos/inmunología , Anticuerpos Anti-VIH/inmunología , Proteína gp120 de Envoltorio del VIH/inmunología , VIH-1/química , VIH-1/inmunología , Manosa/inmunología , Datos de Secuencia Molecular , Oligosacáridos de Cadena Ramificada/química , Oligosacáridos de Cadena Ramificada/inmunología
6.
J Am Chem Soc ; 126(28): 8640-1, 2004 Jul 21.
Artículo en Inglés | MEDLINE | ID: mdl-15250702

RESUMEN

A covalent array for the display of complex oligosaccharides in microtiter plates has been developed. This strategy is conducive to the display of carbohydrates to proteins of interest such as lectins and antibodies, including the broadly neutralizing antibody 2G12 against HIV envelope oligomannose and can be cleaved from the surface for further characterization by mass spectrometry. The system was used to probe the multivalent interaction of 2G12 with an optimal epitope (Kd 0.1 muM).


Asunto(s)
Técnicas Químicas Combinatorias , Oligosacáridos/química , Secuencia de Carbohidratos , Ditiotreitol/química , Datos de Secuencia Molecular , Estructura Molecular , Oligosacáridos/síntesis química , Espectrometría de Masa por Ionización de Electrospray , Triazoles/química
7.
Science ; 300(5628): 2065-71, 2003 Jun 27.
Artículo en Inglés | MEDLINE | ID: mdl-12829775

RESUMEN

Human antibody 2G12 neutralizes a broad range of human immunodeficiency virus type 1 (HIV-1) isolates by binding an unusually dense cluster of carbohydrate moieties on the "silent" face of the gp120 envelope glycoprotein. Crystal structures of Fab 2G12 and its complexes with the disaccharide Manalpha1-2Man and with the oligosaccharide Man9GlcNAc2 revealed that two Fabs assemble into an interlocked VH domain-swapped dimer. Further biochemical, biophysical, and mutagenesis data strongly support a Fab-dimerized antibody as the prevalent form that recognizes gp120. The extraordinary configuration of this antibody provides an extended surface, with newly described binding sites, for multivalent interaction with a conserved cluster of oligomannose type sugars on the surface of gp120. The unique interdigitation of Fab domains within an antibody uncovers a previously unappreciated mechanism for high-affinity recognition of carbohydrate or other repeating epitopes on cell or microbial surfaces.


Asunto(s)
Anticuerpos Anti-VIH/química , Anticuerpos Anti-VIH/inmunología , Proteína gp120 de Envoltorio del VIH/inmunología , VIH-1/inmunología , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/inmunología , Oligosacáridos/inmunología , Secuencia de Aminoácidos , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , Afinidad de Anticuerpos , Especificidad de Anticuerpos , Sitios de Unión de Anticuerpos , Moléculas de Adhesión Celular/metabolismo , Centrifugación por Gradiente de Densidad , Cristalización , Cristalografía por Rayos X , Dimerización , Disacáridos/química , Disacáridos/metabolismo , Epítopos , Anticuerpos Anti-VIH/genética , Anticuerpos Anti-VIH/metabolismo , Humanos , Enlace de Hidrógeno , Fragmentos Fab de Inmunoglobulinas/genética , Fragmentos Fab de Inmunoglobulinas/metabolismo , Cadenas Pesadas de Inmunoglobulina/química , Cadenas Pesadas de Inmunoglobulina/inmunología , Cadenas Ligeras de Inmunoglobulina/química , Cadenas Ligeras de Inmunoglobulina/inmunología , Región Variable de Inmunoglobulina/química , Región Variable de Inmunoglobulina/inmunología , Lectinas/química , Lectinas/inmunología , Lectinas/metabolismo , Lectinas Tipo C/metabolismo , Ligandos , Mananos/química , Mananos/metabolismo , Manósidos/química , Manósidos/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis , Oligosacáridos/química , Oligosacáridos/metabolismo , Conformación Proteica , Estructura Terciaria de Proteína , Receptores de Superficie Celular/metabolismo
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