Micklisch Sven, Lin Yuchen, Jacob Saskia, Karlstetter Marcus, Dannhausen Katharina, Dasari Prasad, von der Heide Monika, Dahse Hans-Martin, Schmölz Lisa, Grassmann Felix, Alene Medhanie, Fauser Sascha, Neumann Harald, Lorkowski Stefan, Pauly Diana, Weber Bernhard H, Joussen Antonia M, Langmann Thomas, Zipfel Peter F, Skerka Christine
Department of Infection Biology, Leibniz Institute for Natural Product Research and Infection Biology, Beutenbergstrasse 11, 07745, Jena, Germany.
Department of Ophthalmology, Charité Universitätsmedizin Berlin, Augustenburger Platz 1, 13353, Berlin, Germany.
J Neuroinflammation. 2017 Jan 5;14(1):4. doi: 10.1186/s12974-016-0776-3.
Age-related macular degeneration (AMD) is the leading cause of blindness in developed countries. The polymorphism rs10490924 in the ARMS2 gene is highly associated with AMD and linked to an indel mutation (del443ins54), the latter inducing mRNA instability. At present, the function of the ARMS2 protein, the exact cellular sources in the retina and the biological consequences of the rs10490924 polymorphism are unclear.
Recombinant ARMS2 was expressed in Pichia pastoris, and protein functions were studied regarding cell surface binding and complement activation in human serum using fluoresence-activated cell sorting (FACS) as well as laser scanning microscopy (LSM). Biolayer interferometry defined protein interactions. Furthermore, endogenous ARMS2 gene expression was studied in human blood derived monocytes and in human induced pluripotent stem cell-derived microglia (iPSdM) by PCR and LSM. The ARMS2 protein was localized in human genotyped retinal sections and in purified monocytes derived from AMD patients without the ARMS2 risk variant by LSM. ARMS2 expression in monocytes under oxidative stress was determined by Western blot analysis.
Here, we demonstrate for the first time that ARMS2 functions as surface complement regulator. Recombinant ARMS2 binds to human apoptotic and necrotic cells and initiates complement activation by recruiting the complement activator properdin. ARMS2-properdin complexes augment C3b surface opsonization for phagocytosis. We also demonstrate for the first time expression of ARMS2 in human monocytes especially under oxidative stress and in microglia cells of the human retina. The ARMS2 protein is absent in monocytes and also in microglia cells, derived from patients homozygous for the ARMS2 AMD risk variant (rs10490924).
ARMS2 is likely involved in complement-mediated clearance of cellular debris. As AMD patients present with accumulated proteins and lipids on Bruch's membrane, ARMS2 protein deficiency due to the genetic risk variant might be involved in drusen formation.
年龄相关性黄斑变性（AMD）是发达国家失明的主要原因。ARMS2基因中的多态性rs10490924与AMD高度相关，并与一个插入缺失突变（del443ins54）相关联，后者会导致mRNA不稳定。目前，ARMS2蛋白的功能、视网膜中确切的细胞来源以及rs10490924多态性的生物学后果尚不清楚。
重组ARMS2在毕赤酵母中表达，并使用荧光激活细胞分选（FACS）以及激光扫描显微镜（LSM）研究其在人血清中关于细胞表面结合和补体激活的蛋白功能。生物层干涉术确定蛋白相互作用。此外，通过PCR和LSM研究人血来源的单核细胞和人诱导多能干细胞来源的小胶质细胞（iPSdM）中内源性ARMS2基因的表达。通过LSM将ARMS2蛋白定位在人类基因分型视网膜切片以及来自无ARMS2风险变异的AMD患者的纯化单核细胞中。通过蛋白质印迹分析确定氧化应激下单核细胞中ARMS2的表达。
在此，我们首次证明ARMS2作为表面补体调节因子发挥作用。重组ARMS2与人凋亡和坏死细胞结合，并通过募集补体激活剂备解素启动补体激活。ARMS2 - 备解素复合物增强C3b表面调理作用以促进吞噬作用。我们还首次证明ARMS2在人单核细胞中表达，尤其是在氧化应激下以及在人类视网膜的小胶质细胞中表达。ARMS2蛋白在来自ARMS2 AMD风险变异（rs10490924）纯合子患者的单核细胞和小胶质细胞中均不存在。
ARMS2可能参与补体介导的细胞碎片清除。由于AMD患者在布鲁赫膜上存在蛋白质和脂质积累，由遗传风险变异导致的ARMS2蛋白缺乏可能与玻璃膜疣形成有关。
