RESUMEN
Virtually all living cells are covered with glycans. Their structures are primarily controlled by the specificities of glycosyltransferases (GTs). GTs typically adopt one of the three folds, namely, GT-A, GT-B, and GT-C. However, what defines their specificities remain poorly understood. Here, we developed a genetic glycoengineering platform by reprogramming the capsular polysaccharide pathways in Streptococcus pneumoniae to interrogate GT specificity and manipulate glycan structures. Our findings suggest that the central cleft of GT-B enzymes is important for determining acceptor specificity. The constraint of the glycoengineering platform was partially alleviated when the specificity of the precursor transporter was reduced, indicating that the transporter contributes to the overall fidelity of glycan synthesis. We also modified the pneumococcal capsule to produce several medically important mammalian glycans, as well as demonstrated the importance of regiochemistry in a glycosidic linkage on binding lung epithelial cells. Our work provided mechanistic insights into GT specificity and an approach for investigating glycan functions.
Asunto(s)
Glicosiltransferasas , Streptococcus pneumoniae , Animales , Glicosiltransferasas/genética , Streptococcus pneumoniae/genética , Células Epiteliales , Glicósidos , Proteínas de Transporte de Membrana , MamíferosRESUMEN
Tarantula venoms provide a model system for studying toxin selectivity, structure-activity relationships and molecular evolution of peptide toxins. Previous studies have identified a large number of peptide toxins in the venom of the Chinese bird spider Haplopelma hainanum, generally regarded as a highly venomous spider. However, the lack of available RNA-seq transcriptomic and genomic data is an obstacle to understanding its venom at the molecular level. In this study, we investigated the venom gland transcriptome of H. hainanum by RNA-seq, in the absence of an available genomic sequence. We identified 201 potential toxins among 57 181 de novo assembled transcripts, including knottins, Kunitz-type toxins, enzymes and other proteins. We systematically identified most of the knottins and Kunitz-type toxins, some of which showed strongly biased expression in the venom gland, including members of the huwentoxin-1, huwentoxin-2 and magi-1 families. We also discovered several novel potential toxins. These data demonstrate the high molecular and structural diversity in the venom toxins of H. hainanum. This study offers a useful strategy for exploring the complex components of spider venoms.
Asunto(s)
Péptidos/genética , Venenos de Araña/genética , Arañas/genética , Animales , Evolución Molecular , Glándulas Exocrinas/metabolismo , Perfilación de la Expresión Génica , Péptidos/química , Péptidos/metabolismo , Filogenia , Venenos de Araña/química , Venenos de Araña/metabolismo , Arañas/metabolismoRESUMEN
The low selectivity of Kv1 peptide inhibitors for specific isoforms makes them poor candidates for the development of theraputics. Using combined approaches, we showed that the Kv1 turret is the critical determinant for ADWX-1 peptide inhibitor selectivity of Kv1.3 over Kv1.1. Mutation of Kv1.1 turret residues to match the sequence of Kv1.3 lead to increased inhibition of Kv1.1 activity. These studies may lead to improvements in peptide inhibitor drug development.