Truncated-semaphorin3A is a potential regulatory molecule to restore immune homeostasis in immune-mediated diseases.
Truncated-semaphorin3A is a potential regulatory molecule to restore immune homeostasis in immune-mediated diseases.
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DOI：
10.3389/fphar.2022.1085892
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发表时间：
2022
影响因子：
5.6
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中科院分区：
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调节分子最近被认为在免疫介导疾病的治疗中具有有益作用，而不是使用与许多不良副作用相关的细胞毒性免疫抑制药物。信号蛋白3a （sema3A）是一种独特的免疫系统调节大师，在系统性红斑狼疮（SLE）患者的血清中显示出与疾病严重程度相关的降低。后来，我们在NZB/W小鼠模型中显示了其治疗狼疮性肾炎的极有益效果。sema3A维持其调节作用的机制是通过与B细胞上的调节受体CD72结合，从而降低B细胞上BCR信号传导的阈值，减少促炎细胞因子的产生。本研究的目的是产生一种稳定的sema3A分子，易于生产，与CD72受体的结合能力比与Neuropilin-1 （nlp -1）受体的结合能力更高，在许多细胞类型中表达。利用亲本sema3A的晶体结构，我们合成了一个新的分泌（较短）sema3A衍生物，我们称之为截断sema3A （T-sema3A）。新分子缺乏NRP-1结合域（c -末端），在257位有一个人工二聚化位点（丝氨酸残基与半胱氨酸残基交换）。为了便于该分子的纯化，我们在框架上游的一个停止密码子上添加了组氨酸表位标签。利用病毒载体将该构建体转染到293HEK细胞中，生成稳定表达T-sema3A的细胞。T-sema3A与CD72的结合能力比与NRP-1的结合能力强。此外，T- sema3a被证明是一种调节剂，可以诱导人T淋巴细胞和b淋巴细胞IL-10和TGF-β的表达，减少IL-6、IFN-γ和IL-17A等促炎细胞因子的分泌。考虑到这一点，T-sema3A在维持免疫稳态方面非常有效，因此，它成为恢复免疫系统在免疫介导疾病中的调节状态的潜在药物。
Regulatory molecules have recently been recognized for their beneficial effects in the treatment of immune-mediated diseases, rather than using cytotoxic immune-suppressing drugs, which are associated with many unwanted side effects. Semaphorin3A (sema3A), a unique regulatory master of the immune system, was shown to be decreased in the serum of systemic lupus erythematosus (SLE) patients, in association with disease severity. Later, we were able to show its extremely beneficial effect in treating lupus nephritis in the NZB/W mice model. The mechanisms by which sema3A maintains its regulatory effect is by binding the regulatory receptor CD72 on B cells, thereby reducing the threshold of BCR signaling on B cells and reducing the production of pro-inflammatory cytokines. The aim of this study was to generate a stable sema3A molecule, easy to produce with a higher binding capacity to CD72 receptor rather than to Neuropilin-1 (NRP-1) receptor, which is expressed in many cell types. Using the crystallographic structure of parental sema3A, we synthesized a new secreted (shorter) sema3A derivative, which we called truncated sema3A (T-sema3A). The new molecule lacked the NRP-1 binding domain (the C-terminal site) and has an artificial dimerization site at position 257 (serine residue was exchanged with a cysteine residue). To facilitate the purification of this molecule we added Histidine epitope tag in frame upstream to a stop codon. This construct was transfected using a viral vector to 293HEK cells to generate cells stably expressing T-sema3A. T-sema3A is shown to be with a higher binding ability to CD72 than to NRP-1 as demonstrated by a homemade ELISA. In addition, T-sema3A was shown to be a regulatory agent which can induce the expression of IL-10 and TGF-β and reduce the secretion of pro-inflammatory cytokines such as IL-6, IFN-γ, and IL-17A from human T and B-lymphocytes. Keeping this in mind, T-sema3A is highly effective in maintaining immune homeostasis, therefore, becoming a potential agent in restoring the regulatory status of the immune system in immune-mediated diseases.