Stefi V Benjamin, Sabine A F Jégouzo, Chloe Lieng, Connor Daniels, Marine Coispeau, Rikin J Lau, Suyeon Kim, Yasmine Metaxa, James Philpott, Tiannuo Li, Chao Dai, Xin Wang, Maddy L Newby, Gerald B Pier, Max Crispin, Abigail Clements, Maureen E Taylor, Kurt Drickamer
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引用次数: 0
摘要
为了探究先天性免疫受体与病原微生物和共生微生物之间的相互作用,我们开发了一种人类凝集素阵列。继成功推出包含所有奶牛 C 型碳水化合物识别结构域(CRDs)的凝集素阵列之后,这里介绍的人类凝集素阵列包含 C 型 CRDs 以及其他糖结合受体类别的 CRDs,包括 galectins、siglecs、R 型 CRDs、ficolins、intelectins 和类几丁质酶凝集素。该阵列是用单位生物素标签修饰的 CRDs 构建的,确保 CRDs 中的糖结合位点以确定的方向显示在链霉亲和素涂层表面上,并可接触到微生物的表面。在表达和展示所有不同结构类别的糖结合受体的 CRDs 时,采用了一种通用的方法,这样就可以对不同凝集素家族进行比较。除了先前记录的荧光标记细菌结合方案外,还开发了通过 DNA 结合染料反染色来检测与阵列结合的未标记细菌的方法。此外,还对病毒糖蛋白以及细菌和真菌多糖进行了筛选。该阵列可无偏见地筛选出与受体相互作用的糖配体,其中许多配体的结合是早期研究未曾预料到的。例如,一些半凝集素与缺乏乳糖或 N-乙酰半乳糖胺的细菌糖结合的亲和力很高。研究结果表明,人类凝集素阵列可以提供先天免疫系统中多类糖结合蛋白与不同类型微生物相互作用的独特概况。
A human lectin array for characterizing host-pathogen interactions.
A human lectin array has been developed to probe the interactions of innate immune receptors with pathogenic and commensal microorganisms. Following the successful introduction of a lectin array containing all of the cow C-type carbohydrate-recognition domains (CRDs), a human array described here contains the C-type CRDs as well as CRDs from other classes of sugar-binding receptors, including galectins, siglecs, R-type CRDs, ficolins, intelectins, and chitinase-like lectins. The array is constructed with CRDs modified with single-site biotin tags, ensuring that the sugar-binding sites in CRDs are displayed on a streptavidin-coated surface in a defined orientation and are accessible to the surfaces of microbes. A common approach used for expression and display of CRDs from all of the different structural categories of glycan-binding receptors allows comparisons across lectin families. In addition to previously documented protocols for binding of fluorescently labeled bacteria, methods have been developed for detecting unlabeled bacteria bound to the array by counter-staining with DNA-binding dye. Screening has also been undertaken with viral glycoproteins and bacterial and fungal polysaccharides. The array provides an unbiased screen for sugar ligands that interact with receptors and many show binding not anticipated from earlier studies. For example, some of the galectins bind with high affinity to bacterial glycans that lack lactose or N-acetyllactosamine. The results demonstrate the utility of the human lectin array for providing a unique overview of the interactions of multiple classes of glycan-binding proteins in the innate immune system with different types of microorganisms.
期刊介绍:
The Journal of Biological Chemistry welcomes high-quality science that seeks to elucidate the molecular and cellular basis of biological processes. Papers published in JBC can therefore fall under the umbrellas of not only biological chemistry, chemical biology, or biochemistry, but also allied disciplines such as biophysics, systems biology, RNA biology, immunology, microbiology, neurobiology, epigenetics, computational biology, ’omics, and many more. The outcome of our focus on papers that contribute novel and important mechanistic insights, rather than on a particular topic area, is that JBC is truly a melting pot for scientists across disciplines. In addition, JBC welcomes papers that describe methods that will help scientists push their biochemical inquiries forward and resources that will be of use to the research community.