Fuller John A, Brun-Zinkernagel Anne-Marie, Clark Abbot F, Wordinger Robert J
Department of Cell Biology and Genetics, University of North Texas Health Science Center, Fort Worth, Texas 76107, USA.
Invest Ophthalmol Vis Sci. 2009 Dec;50(12):5759-68. doi: 10.1167/iovs.08-2616. Epub 2009 Apr 1.
Subtilisin-like proprotein convertases (SPCs) are a family of calcium-dependent cleavage enzymes that act on dibasic sites of various peptide/protein substrates. The purpose of this study was to investigate the expression, localization, and activity of SPCs in the human retina and optic nerve head.
mRNA expression of the SPC family in the human retina and optic nerve head tissues was evaluated by quantitative reverse transcription polymerase chain reaction (QRT-PCR). Double immunofluorescence staining was performed on paraffin-embedded human posterior sections to localize SPC family members. Western blot analysis was used to identify PACE4 isoform expression within the optic nerve head and retina. In addition, a fluorogenic SPC substrate-based assay was used to elucidate SPC enzyme activity within human retina and optic nerve head (ONH) tissues.
QPCR results indicated that PC1 and PC2 were expressed 4.1- and 5.7-fold higher in retina compared to optic nerve head, whereas PACE4 was expressed 4.1-fold higher in the ONH. PC1 and PC2 were localized primarily in neuronal cells, whereas PACE4 and PC5 were limited to the glia of the retina and optic nerve head. SPC activity in ONH lysate was significantly higher than that of retinal lysate; however, when an SPC inhibitor was added, activity in ONH decreased more than that in retina.
These results indicate that the SPCs are expressed in distinct patterns throughout the human retina and ONH. PC1 and PC2 were primarily expressed in neurons, whereas PACE4 appeared to be largely restricted to glia. Thus, elevated PACE4 may modulate the bioactivity of proteins secreted in the ONH and retina.
枯草杆菌蛋白酶样前体蛋白转化酶（SPCs）是一类钙依赖性裂解酶，作用于各种肽/蛋白质底物的双碱性位点。本研究旨在探讨SPCs在人视网膜和视神经乳头中的表达、定位及活性。
采用定量逆转录聚合酶链反应（QRT-PCR）评估人视网膜和视神经乳头组织中SPC家族的mRNA表达。对石蜡包埋的人眼后部切片进行双重免疫荧光染色，以定位SPC家族成员。采用蛋白质免疫印迹分析来鉴定视神经乳头和视网膜中PACE4同工型的表达。此外，使用基于荧光SPC底物的检测方法来阐明人视网膜和视神经乳头（ONH）组织中的SPC酶活性。
QPCR结果表明，与视神经乳头相比，PC1和PC2在视网膜中的表达分别高4.1倍和5.7倍，而PACE4在视神经乳头中的表达高4.1倍。PC1和PC2主要定位于神经元细胞，而PACE4和PC5仅限于视网膜和视神经乳头的神经胶质细胞。视神经乳头裂解物中的SPC活性显著高于视网膜裂解物；然而，添加SPC抑制剂后，视神经乳头中的活性下降幅度大于视网膜。
这些结果表明，SPCs在整个人视网膜和视神经乳头中以不同模式表达。PC1和PC2主要在神经元中表达，而PACE4似乎主要局限于神经胶质细胞。因此，升高的PACE4可能调节视神经乳头和视网膜中分泌蛋白的生物活性。
