RESUMEN
Members of the RegIII family of intestinal C-type lectins are directly antibacterial proteins that play a vital role in maintaining host-bacterial homeostasis in the mammalian gut, yet little is known about the mechanisms that regulate their biological activity. Here we show that the antibacterial activities of mouse RegIIIgamma and its human ortholog, HIP/PAP, are tightly controlled by an inhibitory N-terminal prosegment that is removed by trypsin in vivo. NMR spectroscopy revealed a high degree of conformational flexibility in the HIP/PAP inhibitory prosegment, and mutation of either acidic prosegment residues or basic core protein residues disrupted prosegment inhibitory activity. NMR analyses of pro-HIP/PAP variants revealed distinctive colinear backbone amide chemical shift changes that correlated with antibacterial activity, suggesting that prosegment-HIP/PAP interactions are linked to a two-state conformational switch between biologically active and inactive protein states. These findings reveal a novel regulatory mechanism governing C-type lectin biological function and yield new insight into the control of intestinal innate immunity.
Asunto(s)
Antibacterianos/química , Antígenos de Neoplasias/química , Biomarcadores de Tumor/química , Homeostasis/fisiología , Lectinas Tipo C/química , Proteínas/química , Animales , Antibacterianos/inmunología , Antibacterianos/metabolismo , Antígenos de Neoplasias/inmunología , Antígenos de Neoplasias/metabolismo , Biomarcadores de Tumor/inmunología , Biomarcadores de Tumor/metabolismo , Humanos , Inmunidad Innata/fisiología , Mucosa Intestinal/metabolismo , Intestinos/química , Intestinos/inmunología , Lectinas Tipo C/inmunología , Lectinas Tipo C/metabolismo , Ratones , Ratones Noqueados , Resonancia Magnética Nuclear Biomolecular , Proteínas Asociadas a Pancreatitis , Estructura Secundaria de Proteína/fisiología , Estructura Terciaria de Proteína/fisiología , Proteínas/inmunología , Proteínas/metabolismoRESUMEN
The regenerating (Reg) family comprises an extensive, diversified group of proteins with homology to C-type lectins. Several members of this family are highly expressed in the gastrointestinal tract under normal conditions, and often show increased expression in inflammatory bowel disease. However, little is known about Reg protein function, and the carbohydrate ligands for these proteins are poorly characterized. We report here the first expression and purification of Reg proteins using a bacterial system. Mouse RegIIIgamma and its human counterpart, HIP/PAP, were expressed in Escherichia coli, resulting in the accumulation of aggregated recombinant protein. Both proteins were renatured by arginine-assisted procedures and were further purified using cation-exchange chromatography. The identities of the purified proteins were confirmed by SDS-PAGE, N-terminal sequencing, and MALDI-TOF mass spectrometry. Size exclusion chromatography revealed that both proteins exist as monomers, and circular dichroism showed that their secondary structures exhibit a predominance of beta-strands which is typical of C-type lectins. Finally, both RegIIIgamma and human HIP/PAP bind to mannan but not to monomeric mannose, giving initial insights into their carbohydrate ligands. These studies thus provide an essential foundation for further analyses of human and mouse RegIII protein function.
Asunto(s)
Antígenos de Neoplasias/genética , Biomarcadores de Tumor/genética , Escherichia coli/genética , Lectinas Tipo C/genética , Proteínas/genética , Animales , Antígenos de Neoplasias/química , Antígenos de Neoplasias/aislamiento & purificación , Sitios de Unión , Biomarcadores de Tumor/química , Biomarcadores de Tumor/aislamiento & purificación , Biopolímeros/metabolismo , Cromatografía por Intercambio Iónico , Dicroismo Circular , Escherichia coli/metabolismo , Humanos , Cuerpos de Inclusión/genética , Cuerpos de Inclusión/metabolismo , Lectinas Tipo C/química , Lectinas Tipo C/aislamiento & purificación , Manosa/metabolismo , Espectrometría de Masas , Ratones , Proteínas Asociadas a Pancreatitis , Pliegue de Proteína , Proteínas/química , Proteínas/aislamiento & purificación , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismoRESUMEN
The mammalian intestine harbors complex societies of beneficial bacteria that are maintained in the lumen with minimal penetration of mucosal surfaces. Microbial colonization of germ-free mice triggers epithelial expression of RegIIIgamma, a secreted C-type lectin. RegIIIgamma binds intestinal bacteria but lacks the complement recruitment domains present in other microbe-binding mammalian C-type lectins. We show that RegIIIgamma and its human counterpart, HIP/PAP, are directly antimicrobial proteins that bind their bacterial targets via interactions with peptidoglycan carbohydrate. We propose that these proteins represent an evolutionarily primitive form of lectin-mediated innate immunity, and that they reveal intestinal strategies for maintaining symbiotic host-microbial relationships.