Development of Neutrophile Regulatory Peptides with Receptor Association Structures

中性粒细胞的功能包括趋化性、脱颗粒和超氧阴离子的产生，由多种细胞外激动剂（如n -甲酰基-甲硫基-酰基-苯丙氨酸（fMLP））调节。甲酰基肽受体（FPR）和甲酰基肽受体样1 （FPRL1）是一个由7个跨膜（TM）蛋白组成的超家族，作为fMLP结合受体在人中性粒细胞上表达。我们发现用人甲酰基肽受体跨膜（hFPRTM）肽预处理的人中性粒细胞在fMLP刺激下产生的超氧阴离子增加。然而，尚未发现与hFPRTM肽相互作用的膜蛋白。为探究TM多肽对中性粒细胞示踪活性的序列依赖性，采用固相法和Fmoc化学合成了具有甲酰基肽受体（FPR）、FPR样1受体（FPRL1）和GABA受体TM序列的多肽。通过HPLC和MALDI-TOF ms证实了合成肽的均匀性和结构。合成肽的生物活性作为人类中性粒细胞的超氧化物产物进行了研究。由FPR和FPRL1提取的TM肽处理的中性粒细胞在中性粒细胞激动剂fMLP处理后产生2-3倍的超氧阴离子。GABA受体的TM肽无明显的启动活性。说明TM肽对中性粒细胞的引物作用是序列依赖的，需要FPR相关序列。

Neutrophil functions including chemotaxis, degranulation, and generation of superoxide anion are modulated by diverse extracellular agonists such as N-formyl-methionyl-leucyl-phenylalanine (fMLP). Formyl peptide receptor (FPR) and formyl peptide receptor-like 1 (FPRL1), a superfamily of seven transmembrane (TM) proteins, are expressed on human neutrophils as the fMLP binding receptors. We found that human neutrophils pretreated with human formyl peptide receptor transmembrane (hFPRTM) peptides were enhanced superoxide anion production when stimulated with fMLP. However, a membrane protein interacting with hFPRTM peptides is not identified. To explore the sequence dependences of the TM peptides for neutrophil proming activities, peptides possess the TM sequences of formyl peptide receptor (FPR), FPR like 1 receptor (FPRL1), and GABA receptor were synthesized by solid-phase method with Fmoc chemistry. Homogeneities and structures of synthetic peptides were confirmed by HPLC and MALDI-TOF MS. The biological activities of synthetic peptides were carried out as superoxide production for human neutrophils. Neutrophils treated with TM peptides from FPR and FPRL1 produced 2-3 folds of superoxide anion by the treatment of fMLP, neutrophil agonist. TM peptide from GABA receptor exhibited no significant priming activity. These results suggested the priming effect of TM peptide for neutrophil was sequence dependence and it required the FPR related sequences.