Department of Surgery, Beth Israel Deaconess Medical Center, National Center for Functional Glycomics, Harvard Medical School Center for Glycoscience, Harvard Medical School, Boston, MA, USA.
Department of Pathology and Laboratory Medicine, Emory Vaccine Center, Emory University, Atlanta, GA, USA.
Methods Mol Biol. 2022;2421:73-89. doi: 10.1007/978-1-0716-1944-5_5.
The jawless vertebrates (lamprey and hagfish) evolved a novel adaptive immune system with many similarities to that found in the jawed vertebrates, including the production of antigen-specific circulating antibodies in response to immunization. However, the jawless vertebrates use leucine-rich repeat (LRR)-based antigen receptors termed variable lymphocyte receptors (VLRs) for immune recognition, instead of immunoglobulin (Ig)-based receptors. VLR genes are assembled in developing lymphocytes through a gene conversion-like process, in which hundreds of LRR gene segments are randomly selected as template donors to generate a large repertoire of distinct antigen receptors, similar to that found within the mammalian adaptive immune system. Here we describe the development of a robust platform using immunized lampreys (Petromyzon marinus) for generating libraries of anti-carbohydrate (anti-glycan) variable lymphocyte receptor B, or VLRBs. The anti-carbohydrate VLRBs are isolated using a yeast surface display (YSD) expression platform and enriched by binding to glycan microarrays through the anti-glycan VLRB. This enables both the initial identification and enrichment of individual yeast clones against hundreds of glycans simultaneously. Through this enrichment strategy a broad array of glycan-specific VLRs can be isolated from the YSD library. Subsequently, the bound yeast cells are directly removed from the microarray, the VLR antibody clone is sequenced, and the end product is expressed as a VLR-IgG-Fc fusion protein that can be used for ELISA, Western blotting, flow cytometry, and immunomicroscopy. Thus, by combining yeast surface display with glycan microarray technology, we have developed a rapid, efficient, and novel method for generating chimeric VLR-IgG-Fc proteins that recognize a broad array of unique glycan structures with exquisite specificity.
无颌脊椎动物（七鳃鳗和盲鳗）进化出了一种新的适应性免疫系统，与有颌脊椎动物有许多相似之处，包括针对免疫接种产生抗原特异性循环抗体。然而，无颌脊椎动物使用富含亮氨酸重复序列（LRR）的抗原受体，称为可变淋巴细胞受体（VLR），用于免疫识别，而不是免疫球蛋白（Ig）受体。VLR 基因在发育中的淋巴细胞中通过类似于基因转换的过程组装，其中数百个 LRR 基因片段被随机选择作为模板供体，以产生大量独特的抗原受体，类似于哺乳动物适应性免疫系统中发现的情况。在这里，我们描述了一个使用免疫七鳃鳗（Petromyzon marinus）生成抗碳水化合物（抗聚糖）可变淋巴细胞受体 B 或 VLRB 文库的强大平台的开发。使用酵母表面展示（YSD）表达平台分离抗碳水化合物 VLRB，并通过与聚糖微阵列结合来富集抗聚糖 VLRB。这使得能够同时针对数百种聚糖对单个酵母克隆进行初始鉴定和富集。通过这种富集策略，可以从 YSD 文库中分离出广泛的糖特异性 VLR。随后，将结合的酵母细胞直接从微阵列上洗脱，对 VLR 抗体克隆进行测序，最终产物表达为 VLR-IgG-Fc 融合蛋白，可用于 ELISA、Western blot、流式细胞术和免疫显微镜。因此，通过将酵母表面展示与聚糖微阵列技术相结合，我们开发了一种快速、高效、新颖的方法，用于生成能够识别广泛独特聚糖结构的嵌合 VLR-IgG-Fc 蛋白，具有极高的特异性。
