Lipid Ligands of SPLUNC1 Proteins

描述（由申请人提供）：自十多年前发现PLUNC（腭、肺和鼻上皮克隆）蛋白家族以来，同源脂质配体的身份一直难以捉摸。该家族中最具特征的成员是短腭、肺和鼻上皮克隆1 （SPLUNC1）蛋白，该蛋白在正常气道上皮细胞中大量表达。由于其在过敏性气道中的分布以及与BPI（杀菌/通透性增加蛋白）序列的相似性，其在婴儿免疫系统中的功能已引起人们的极大兴趣。SPLUNC1在宿主防御病原体感染中起着至关重要的作用。SPLUNC1对肺炎支原体具有杀菌活性，抑制铜绿假单胞菌的生长；n端结构域抑制上皮钠通道（ENaC），也有报道称SPLUNC1作为表面活性剂降低气道表面张力并干扰病原体的生物膜形成。然而，没有明确的证据表明SPLUNC1的功能与BPI相似，包括对大多数革兰氏阴性菌或革兰氏阳性菌没有明显的杀菌活性，不中和LPS，没有调理素活性。此外，关于SPLUNC1与LPS的结合也有争议的报道。为了更好地了解SPLUNC1在宿主防御和先天免疫系统中的功能作用，我们最近确定了SPLUNC1的高分辨率结构。令我们惊讶的是，在BPI和SPLUNC1之间存在着巨大的结构差异。其整体结构与屋尘螨过敏原Der p7相似。特别是SPLUNC1的表面覆盖着带负电荷的斑块，而BPI的表面是带正电荷的，这对于结合带负电荷的LPS头基团是必不可少的。我们的体外结合实验显示SPLUNC1与LPS之间没有结合。为了确定可能与SPLUNC1结合的潜在脂质，我们对来自人293细胞的SPLUNC1进行了质谱分析，发现SPLUNC1主要饱和鞘磷脂（SM），少量饱和磷脂酰胆碱（PC）。体外结合实验证明SPLUNC1可以结合鞘磷脂，但不能结合最常见的PC（来自鸡蛋的POPC）。脂质结合筛选显示肺中最丰富的脂质表面活性剂DPPC与SPLUNC1结合。此外，DPPC是人类和小鼠BAL液脂质提取物中唯一与SPLUNC1结合的脂质，而SM则检测不到。有趣的是，SPLUNC1是第一个发现的DPPC蛋白受体，DPPC是肺表面活性剂中唯一能够将表面张力降至接近零的活性表面成分。我们假设DPPC可能是SPLUNC1的同源脂质配体，而不是LPS。因此，本提案的第一个目标是鉴定、验证和确认SPLUNC的同源脂质配体。另一方面

DESCRIPTION (provided by applicant): The identity of the cognate lipid ligands for the PLUNC (palate, lung, and nasal epithelium clone) protein family have remained elusive since the family was discovered more than one decade ago. The most characterized member of the family, the short palate, lung, and nasal epithelium clone 1 (SPLUNC1) protein is abundantly expressed by normal airway epithelial cells. Due to its distribution in allergic airways and sequence similarity to BPI (bactericidal/permeability increasing protein), its function in the innae immune system has become of great interest. SPLUNC1 plays crucial roles in host defense against pathogen infections. SPLUNC1 has bactericidal activity against Mycoplasma pneumoniae and arrests the growth of Pseudomonas aeruginosa; the n-terminal domain inhibits the epithelial sodium channel (ENaC), and it is also reported that SPLUNC1 acts as surfactant to reduce airway surface tension and interfere with biofilm formation by pathogens. However, no definitive evidence has shown that SPLUNC1 functions similarly to BPI, including no apparent bactericidal activity against most Gram-negative bacteria or Gram-positive bacteria, no neutralization of LPS, and no opsonin activity. Furthermore, there are controversial reports about the binding between SPLUNC1 and LPS. To better understand the function roles of SPLUNC1 in host defense and innate immune system, we recently determined the high-resolution structure of SPLUNC1. To our surprise, there are dramatic structural differences between BPI and SPLUNC1. The overall structure is similar to that of Der p 7, a house-dust mite allergen. In particular, the surface of SPLUNC1 is covered with negatively charged patches, in contrast to the positively charged surface of BPI, which is essential to bind the negatively charged head group of LPS. Our in vitro binding assays showed no binding between SPLUNC1 and LPS. To identify potential lipids that might bind to SPLUNC1, we subjected SPLUNC1 derived from human 293 cells to mass spectrometry analysis and found that SPLUNC1 was saturated mainly with Sphingomyelin(SM) and minorly with Phosphatidyl choline (PC). In vitro binding assays proved that SPLUNC1 could bind Sphingomyelin but not the most common PC (POPC from chicken egg). Lipid binding screen revealed that DPPC, the most rich lipid surfactant in lung, binds to SPLUNC1. Furthermore, DPPC is the only lipid of lipid extracts from human and mouse BAL fluid that binds SPLUNC1 while SM is undetectable. Interestingly, SPLUNC1 is the first identified protein receptor of DPPC, which is the only active surface component of lung surfactant capable of lowering surface tension to near zero. We hypothesize that DPPC could be the cognate lipid ligand for SPLUNC1 but not LPS. Thus, the first goal of this proposal is to identify, verify, and confirm the cognate lipid ligands of SPLUNC. On the other hand, information regarding the specific binding between proteins and lipids is also limited. We have carried out systemic screening of potential targets for SPLUNC1. Our preliminary data showed that the specificity between ligand and protein is not only determined by the head group of the lipid but also the by the fatty acid chains. Accordingly, the second goal of this proposal i to elucidate the structural basis of the specificity determinants between SPLUNC1 and lipid(s). Based on structural features of the lipid identified and the general specific interaction determinants of SPLUNC1 with lipids, we will proceed with investigating potential lipid ligands for other PLUNC family members. Antimicrobial peptides (AMP) are key weapons by which eukaryotes protect themselves against infection and represent a major component of the innate immune system. This proposal will investigate the SPLUNC1 protein, a member of the poorly characterized PLUNC family of AMPs. SPLUNC1 is dramatically down regulated in patients with Asthma, COPD, and Cystic Fibrosis diseases. DPPC is highly elevated in Asthma. Impaired sphingolipid synthesis causes airway hyperreactivity while SM is a major product of sphingolipid metabolism process. Determining the novel mechanisms of SPLUNC1 regulation and function in the airways will improve our understanding of impaired innate immunity in allergic airways. By translating our research discoveries into therapies through manipulation of SPLUNC1, DPPC, and SM, our proposed work will ultimately provide opportunities to treat bacterial or other pathogen infections in chronic lung diseases such as asthma and other pulmonary diseases. Our discovery of DPPC as the cognate receptor of SPLUNC1 while ruling out LPS will also bring great impact in the PLUNC field, which may completely change the direction of the research focus of the field.