Sommer Jeffrey R, Chavali Venkata R M, Simpson Sean G, Ayyagari Radha, Petters Robert M
Department of Animal Science, North Carolina State University, Raleigh, NC, USA.
Mol Vis. 2012;18:92-102. Epub 2012 Jan 17.
Autosomal dominant early-onset long anterior zonules (LAZs) and late-onset retinal degeneration (L-ORD) in humans are associated with the S163R mutation of the complement 1q-tumor necrosis factor related protein-5 (CTRP5) gene. For using the pig as an L-ORD model for the study of pathology, we cloned, characterized, and studied the expression profile of pig CTRP5 (pCTRP5).
The pCTRP5 was cloned and sequenced from porcine genomic DNA. Bioinformatic analysis was done to evaluate the functional domains present in the pCTRP5 using PROSITE tools. The V5 epitope-tagged constructs of pCTRP5 and the mammalian promoters, elongation factor 1-α (EF) promoter and 579 bp of the putative promoter located upstream to pCTRP5 DNA, were used for in vitro expression analysis. The pCTRP5 expression, protein size, and cellular localization were studied in transiently transfected Cos-7 or ARPE-19 cells by western blot analysis using anti-CTRP5 and anti-V5 epitope antibodies. Expression of pCTRP5 in the pig eye tissues was analyzed by western blot analysis, real-time PCR, and immunohistochemistry.
As predicted, pCTRP5 showed a 92% DNA homology and 98% amino acid homology with human CTRP5 (hCTRP5). Bioinformatic analysis revealed the presence of an alternate in-frame translational start site upstream to the presumed initiator codon. The presence of a putative promoter region upstream to the pCTRP5 was identified. The putative pCTRP5 promoter was found to be functional by western blot analysis. The size of the pCTRP5 protein (pCTRP5) was consistent with its predicted molecular weight, indicating that the potential alternative start site was not used. Western blot and RT-PCR analyses showed that pCTRP5 was predominantly expressed in RPE, a pattern of expression consistent with that found in mouse and human eyes.
The sequence and genomic organization of pCTRP5 was found to be similar to the human homolog. The DNA and protein sequence of pCTRP5 are highly homologous to hCTRP5, indicating that they are highly conserved. A putative promoter region (579 bp) present upstream to pCTRP5 was found to be functional and was able to drive the expression of the pCTRP5 gene cloned downstream. The tissue distribution in the eye and the expression profile of pCTRP5 in transiently transfected cells is consistent with hCTRP5 expression. Immunohistochemistry analysis of the pig retinal sections revealed localization of pCTRP5 to the apical and basolateral regions on the RPE and in the ciliary body. The potential in-frame alternate start site was found to be nonfunctional by western blot analysis of transiently transfected cells. Similarities between human and pig CTRP5 and the presence of an area centralis region in the pig similar to the human macula, together with its large eyeball size, makes the domestic pig a good model for the study of LAZs and L-ORD.
人类常染色体显性早发性长前 zonules（LAZs）和迟发性视网膜变性（L - ORD）与补体 1q - 肿瘤坏死因子相关蛋白 - 5（CTRP5）基因的 S163R 突变相关。为了将猪用作研究病理学的 L - ORD 模型，我们克隆、表征并研究了猪 CTRP5（pCTRP5）的表达谱。
从猪基因组 DNA 中克隆并测序 pCTRP5。使用 PROSITE 工具进行生物信息学分析，以评估 pCTRP5 中存在的功能域。pCTRP5 的 V5 表位标签构建体以及哺乳动物启动子、伸长因子 1 - α（EF）启动子和位于 pCTRP5 DNA 上游的 579 bp 推定启动子用于体外表达分析。通过使用抗 CTRP5 和抗 V5 表位抗体的蛋白质印迹分析，在瞬时转染的 Cos - 7 或 ARPE - 19 细胞中研究 pCTRP5 的表达、蛋白质大小和细胞定位。通过蛋白质印迹分析、实时 PCR 和免疫组织化学分析猪眼组织中 pCTRP5 的表达。
如预期的那样，pCTRP5 与人类 CTRP5（hCTRP5）显示出 92%的 DNA 同源性和 98%的氨基酸同源性。生物信息学分析揭示在假定的起始密码子上游存在一个框内翻译起始替代位点。鉴定出在 pCTRP5 上游存在一个推定的启动子区域。通过蛋白质印迹分析发现推定的 pCTRP5 启动子具有功能。pCTRP5 蛋白（pCTRP5）的大小与其预测的分子量一致，表明未使用潜在的替代起始位点。蛋白质印迹和 RT - PCR 分析表明 pCTRP5 主要在视网膜色素上皮（RPE）中表达，这种表达模式与在小鼠和人眼中发现的一致。
发现 pCTRP5 的序列和基因组组织与人类同源物相似。pCTRP5 的 DNA 和蛋白质序列与 hCTRP5 高度同源，表明它们高度保守。发现在 pCTRP5 上游存在的一个推定启动子区域（579 bp）具有功能，并且能够驱动下游克隆的 pCTRP5 基因的表达。pCTRP5 在眼中的组织分布以及在瞬时转染细胞中的表达谱与 hCTRP5 的表达一致。对猪视网膜切片的免疫组织化学分析揭示 pCTRP5 定位于 RPE 的顶端和基底外侧区域以及睫状体。通过对瞬时转染细胞的蛋白质印迹分析发现潜在的框内替代起始位点无功能。人类和猪 CTRP5 之间的相似性以及猪中存在与人类黄斑相似的中央区域，再加上其较大的眼球大小，使得家猪成为研究 LAZs 和 L - ORD 的良好模型。
