Bessa Pereira Catarina, Bocková Markéta, Santos Rita F, Santos Ana Mafalda, Martins de Araújo Mafalda, Oliveira Liliana, Homola Jiří, Carmo Alexandre M
i3S-Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal; IBMC - Instituto de Biologia Molecular e Celular, Porto, Portugal; ICBAS - Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, Porto, Portugal.
Institute of Photonics and Electronics of the Czech Academy of Sciences , Prague , Czech Republic.
Front Immunol. 2016 Oct 13;7:416. doi: 10.3389/fimmu.2016.00416. eCollection 2016.
The scavenger receptor cysteine-rich (SRCR) family comprises a group of membrane-attached or secreted proteins that contain one or more modules/domains structurally similar to the membrane distal domain of type I macrophage scavenger receptor. Although no all-inclusive biological function has been ascribed to the SRCR family, some of these receptors have been shown to recognize pathogen-associated molecular patterns (PAMP) of bacteria, fungi, or other microbes. SSc5D is a recently described soluble SRCR receptor produced by monocytes/macrophages and T lymphocytes, consisting of an N-terminal portion, which contains five SRCR modules, and a large C-terminal mucin-like domain. Toward establishing a global common role for SRCR domains, we interrogated whether the set of five SRCR domains of SSc5D displayed pattern recognition receptor (PRR) properties. For that purpose, we have expressed in a mammalian expression system the N-terminal SRCR-containing moiety of SSc5D (N-SSc5D), thus excluding the mucin-like domain likely by nature to bind microorganisms, and tested the capacity of the SRCR functional groups to physically interact with bacteria. Using conventional protein-bacteria binding assays, we showed that N-SSc5D had a superior capacity to bind to strains RS218 and IHE3034 compared with that of the extracellular domains of the SRCR proteins CD5 and CD6 (sCD5 and sCD6, respectively), and similar -binding properties as Spα, a proven PRR of the SRCR family. We have further designed a more sensitive, real-time, and label-free surface plasmon resonance (SPR)-based assay and examined the capacity of N-SSc5D, Spα, sCD5, and sCD6 to bind to different bacteria. We demonstrated that N-SSc5D compares with Spα in the capacity to bind to and , and further that it can distinguish between pathogenic RS218 and IHE3034 strains and the non-pathogenic laboratory strain BL21(DE3). Our work thus advocates the utility of SPR-based assays as sensitive tools for the rapid screening of interactions between immune-related receptors and PAMP-bearing microbes. The analysis of our results suggests that SRCR domains of different members of the family have a differential capacity to interact with bacteria, and further that the same receptor can discriminate between different bacteria strains and species.
富含半胱氨酸的清道夫受体（SRCR）家族由一组膜附着或分泌的蛋白质组成，这些蛋白质包含一个或多个在结构上与I型巨噬细胞清道夫受体的膜远端结构域相似的模块/结构域。尽管尚未赋予SRCR家族全面的生物学功能，但已证明其中一些受体可识别细菌、真菌或其他微生物的病原体相关分子模式（PAMP）。SSc5D是一种最近描述的由单核细胞/巨噬细胞和T淋巴细胞产生的可溶性SRCR受体，由一个N端部分和一个大的C端粘蛋白样结构域组成，N端部分包含五个SRCR模块。为了确定SRCR结构域的普遍共同作用，我们研究了SSc5D的五个SRCR结构域是否具有模式识别受体（PRR）特性。为此，我们在哺乳动物表达系统中表达了SSc5D的含N端SRCR的部分（N-SSc5D），从而排除了可能天然就与微生物结合的粘蛋白样结构域，并测试了SRCR功能基团与细菌物理相互作用的能力。使用传统的蛋白质-细菌结合试验，我们发现与SRCR蛋白CD5和CD6的细胞外结构域（分别为sCD5和sCD6）相比，N-SSc5D与RS218和IHE3034菌株结合的能力更强，并且具有与SRCR家族中已证实的PRR Spα相似的结合特性。我们进一步设计了一种更灵敏、实时且无标记的基于表面等离子体共振（SPR）的试验，并检测了N-SSc5D、Spα、sCD5和sCD6与不同细菌结合的能力。我们证明，N-SSc5D在与[具体细菌名称1]和[具体细菌名称2]结合的能力上与Spα相当，并且它还能区分致病性RS218和IHE3034菌株与非致病性实验室菌株BL21(DE3)。因此，我们的工作提倡将基于SPR的试验作为快速筛选免疫相关受体与携带PAMP的微生物之间相互作用的灵敏工具。对我们结果的分析表明，该家族不同成员的SRCR结构域与细菌相互作用的能力存在差异，并且同一受体可以区分不同的细菌菌株和种类。
