Sharma Shiwani, Chataway Tim, Klebe Sonja, Griggs Kim, Martin Sarah, Chegeni Nusha, Dave Alpana, Zhou Tiger, Ronci Maurizio, Voelcker Nicolas H, Mills Richard A, Craig Jamie E
Department of Ophthalmology, Flinders University, Bedford Park, SA, Australia.
Department of Human Physiology, Flinders University, Bedford Park, SA, Australia.
Mol Vis. 2018 Dec 28;24:801-817. eCollection 2018.
Pseudoexfoliation (PEX) syndrome is an age-related progressive disease of the extracellular matrix with ocular manifestations. PEX is clinically diagnosed by the presence of extracellular exfoliative deposits on the anterior surface of the ocular lens. PEX syndrome is a major risk factor for developing glaucoma, the leading cause of irreversible blindness in the world, and is often associated with the development of cataract. PEX reportedly coexists with Alzheimer disease and increases the risk of heart disease and stroke. PEX material deposited on the anterior surface of the ocular lens is highly proteinaceous, complex, and insoluble, making deciphering the protein composition of the material challenging. Thus, to date, only a small proportion of the protein composition of PEX material is known. The aim of this study was to decipher the protein composition of pathological PEX material deposited on the ocular lens in patients and advance the understanding of pathophysiology of PEX syndrome.
Liquid-chromatography and tandem mass spectrometry (LC-MS/MS) was employed to discover novel proteins in extracts of neat PEX material surgically isolated from patients (n = 4) with PEX syndrome undergoing cataract surgery. A sub-set of the identified proteins was validated with immunohistochemistry using lens capsule specimens from independent patients (n=3); lens capsules from patients with cataract but without PEX syndrome were used as controls (n=4). Expression of transcripts of the validated proteins in the human lens epithelium was analyzed with reverse transcription PCR (RT-PCR). Functional relationships among the proteins identified in this study and genes and proteins previously implicated in the disease were bioinformatically determined using InnateDB.
Peptides corresponding to 66 proteins, including ten proteins previously known to be present in PEX material, were identified. Thirteen newly identified proteins were chosen for validation. Of those proteins, 12 were found to be genuine components of the material. The novel protein constituents include apolipoproteins (APOA1 and APOA4), stress response proteins (CRYAA and PRDX2), and blood-related proteins (fibrinogen and hemoglobin subunits), including iron-free hemoglobin. The gene expression data suggest that the identified stress-response proteins and hemoglobin are contributed by the lens epithelium and apolipoproteins and fibrinogen by the aqueous humor to the PEX material. Pathway analysis of the identified novel protein constituents and genes or proteins previously implicated in the disease reiterated the involvement of extracellular matrix organization and degradation, elastic fiber formation, and complement cascade in PEX syndrome. Network analysis suggested a central role of fibronectin in the pathophysiology of the disease. The identified novel protein constituents of PEX material also shed light on the molecular basis of the association of PEX syndrome with heart disease, stroke, and Alzheimer disease.
This study expands the understanding of the protein composition of pathological PEX material deposited on the ocular lens in patients with PEX syndrome and provides useful insights into the pathophysiology of this disease. This study together with the previous study by our group (Sharma et al. Experimental Eye Research 2009;89(4):479-85) demonstrate that using neat PEX material, devoid of the underlying lens capsule, for proteomics analysis is an effective approach for deciphering the protein composition of complex and highly insoluble extracellular pathological ocular deposits present in patients with PEX syndrome.
假性剥脱（PEX）综合征是一种与年龄相关的细胞外基质进行性疾病，伴有眼部表现。PEX通过晶状体前表面存在细胞外剥脱沉积物进行临床诊断。PEX综合征是青光眼发生的主要危险因素，青光眼是全球不可逆性失明的主要原因，且常与白内障的发生相关。据报道，PEX与阿尔茨海默病共存，并增加心脏病和中风的风险。沉积在晶状体前表面的PEX物质富含蛋白质、成分复杂且不溶，这使得解读该物质的蛋白质组成具有挑战性。因此，迄今为止，已知的PEX物质蛋白质组成仅占一小部分。本研究的目的是解读患者晶状体上病理性PEX物质的蛋白质组成，并增进对PEX综合征病理生理学的理解。
采用液相色谱和串联质谱（LC-MS/MS）技术，从接受白内障手术的PEX综合征患者（n = 4）手术分离的纯PEX物质提取物中发现新蛋白质。使用来自独立患者（n = 3）的晶状体囊膜标本，通过免疫组织化学对部分鉴定出的蛋白质进行验证；将无PEX综合征的白内障患者的晶状体囊膜（n = 4）用作对照。用逆转录PCR（RT-PCR）分析验证蛋白质在人晶状体上皮中的转录本表达。使用InnateDB通过生物信息学方法确定本研究中鉴定的蛋白质与先前涉及该疾病的基因和蛋白质之间的功能关系。
鉴定出与66种蛋白质相对应的肽段，其中包括10种先前已知存在于PEX物质中的蛋白质。选择13种新鉴定的蛋白质进行验证。在这些蛋白质中，发现12种是该物质真正的成分。新的蛋白质成分包括载脂蛋白（APOA1和APOA4）、应激反应蛋白（CRYAA和PRDX2）以及血液相关蛋白（纤维蛋白原和血红蛋白亚基，包括无铁血红蛋白）。基因表达数据表明，鉴定出的应激反应蛋白和血红蛋白由晶状体上皮贡献，而载脂蛋白和纤维蛋白原由房水贡献至PEX物质。对鉴定出的新蛋白质成分以及先前涉及该疾病的基因或蛋白质进行通路分析，再次证实细胞外基质组织和降解、弹性纤维形成以及补体级联反应与PEX综合征有关。网络分析表明纤连蛋白在该疾病的病理生理学中起核心作用。鉴定出的PEX物质新蛋白质成分也揭示了PEX综合征与心脏病、中风和阿尔茨海默病关联的分子基础。
本研究扩展了对PEX综合征患者晶状体上病理性PEX物质蛋白质组成的理解，并为该疾病的病理生理学提供了有用的见解。本研究与我们团队之前的研究（Sharma等人，《实验眼研究》2009年；89(4):479 - 85）共同表明，使用不含下层晶状体囊膜的纯PEX物质进行蛋白质组学分析，是解读PEX综合征患者中存在的复杂且高度不溶的细胞外病理性眼部沉积物蛋白质组成的有效方法。
