Conserved Oligomeric Golgi (COG) is an octameric protein complex that orchestrates intra-Golgi trafficking of glycosylation enzymes. Over a hundred individuals with 31 different COG mutations have been identified until now. The cellular phenotypes and clinical presentations of COG-CDGs are heterogeneous, and patients primarily represent neurological, skeletal, and hepatic abnormalities. The establishment of a cellular COG disease model will benefit the molecular study of the disease, explaining the detailed sequence of the interplay between the COG complex and the trafficking machinery. Moreover, patient fibroblasts are not a good representative of all the organ systems and cell types that are affected by COG mutations. We developed and characterized cellular models for human COG4 mutations, specifically in RPE1 and HEK293T cell lines. Using a combination of CRISPR/Cas9 and lentiviral transduction technologies, both myc-tagged wild-type and mutant (G516R and R729W) COG4 proteins were expressed under the endogenous COG4 promoter. Constructed isogenic cell lines were comprehensively characterized using biochemical, microscopy (superresolution and electron), and proteomics approaches. The analysis revealed similar stability and localization of COG complex subunits, wild-type cell growth, and normal Golgi morphology in all three cell lines. Importantly, COG4-G516R cells demonstrated increased HPA-647 binding to the plasma membrane glycoconjugates, while COG4-R729W cells revealed high GNL-647 binding, indicating specific defects in O- and N-glycosylation. Both mutant cell lines express an elevated level of heparin sulfate proteoglycans. Moreover, a quantitative mass-spectrometry analysis of proteins secreted by COG-deficient cell lines revealed abnormal secretion of SIL1 and ERGIC-53 proteins by COG4-G516R cells. Interestingly, the clinical phenotype of patients with congenital mutations in the SIL1 gene (Marinesco-Sjogren syndrome) overlaps with the phenotype of COG4-G516R patients (Saul-Wilson syndrome). Our work is the first compressive study involving the creation of different COG mutations in different cell lines other than the patient’s fibroblast. It may help to address the underlying cause of the phenotypic defects leading to the discovery of a proper treatment guideline for COG-CDGs.

保守寡聚高尔基体 (COG) 是一种八聚体蛋白质复合物，可协调高尔基体内糖基化酶的运输。迄今为止，已鉴定出一百多个具有 31 种不同 COG 突变的个体。 COG-CDG 的细胞表型和临床表现是异质的，患者主要表现为神经、骨骼和肝脏异常。细胞COG疾病模型的建立将有利于该疾病的分子研究，解释COG复合物与运输机制之间相互作用的详细序列。此外，患者成纤维细胞并不能很好地代表受 COG 突变影响的所有器官系统和细胞类型。我们开发并表征了人类 COG4 突变的细胞模型，特别是在 RPE1 和 HEK293T 细胞系中。结合使用 CRISPR/Cas9 和慢病毒转导技术，myc 标记的野生型和突变型（G516R 和 R729W）COG4 蛋白在内源 COG4 启动子下表达。使用生化、显微镜（超分辨率和电子）和蛋白质组学方法对构建的同基因细胞系进行全面表征。分析显示，所有三种细胞系中 COG 复合体亚基、野生型细胞生长和正常高尔基体形态具有相似的稳定性和定位。重要的是，COG4-G516R 细胞表现出 HPA-647 与质膜糖缀合物的结合增加，而 COG4-R729W 细胞表现出高 GNL-647 结合，表明 O-和 N-糖基化的特定缺陷。两种突变细胞系均表达升高水平的硫酸肝素蛋白聚糖。 此外，对 COG 缺陷细胞系分泌的蛋白质进行定量质谱分析显示，COG4-G516R 细胞异常分泌 SIL1 和 ERGIC-53 蛋白质。有趣的是，SIL1 基因先天性突变患者（Marinesco-Sjogren 综合征）的临床表型与 COG4-G516R 患者（Saul-Wilson 综合征）的表型重叠。我们的工作是第一个压缩研究，涉及在患者成纤维细胞以外的不同细胞系中产生不同的 COG 突变。它可能有助于解决表型缺陷的根本原因，从而发现 COG-CDG 的正确治疗指南。