Development and Initial Characterization of Cellular Models for COG Complex-Related CDG-II Diseases

Area of Science:

• Cell Biology
• Genetics
• Biochemistry

Background:

• Conserved Oligomeric Golgi (COG) complex is crucial for intra-Golgi trafficking of glycosylation enzymes.
• Congenital Disorders of Glycosylation (CDGs) linked to COG mutations present heterogeneous neurological, skeletal, and hepatic abnormalities.
• Patient fibroblasts have limitations in representing the full spectrum of COG mutation effects across various cell types and organ systems.

Purpose of the Study:

• To develop and characterize novel cellular models for human COG4 mutations using RPE1 and HEK293T cell lines.
• To investigate the molecular mechanisms underlying COG4-related defects in glycosylation and protein trafficking.
• To establish a more comprehensive model for studying COG-CDGs beyond patient fibroblasts.

Main Methods:

• Utilized CRISPR/Cas9 and lentiviral transduction to create isogenic cell lines expressing wild-type and mutant (G516R, R729W) COG4 proteins under the endogenous promoter.
• Employed biochemical assays, super-resolution and electron microscopy, and proteomics for comprehensive characterization.
• Analyzed plasma membrane glycoconjugate binding, heparin sulfate proteoglycan levels, and secreted proteins via quantitative mass spectrometry.

Main Results:

• Developed and validated COG4 mutant cell lines (G516R, R729W) with normal Golgi morphology and COG complex subunit stability.
• Identified specific glycosylation defects: COG4-G516R cells showed increased HPA-647 binding, while COG4-R729W cells exhibited high GNL-647 binding.
• Observed elevated heparin sulfate proteoglycans in both mutant cell lines and abnormal secretion of SIL1 and ERGIC-53 proteins in COG4-G516R cells.

Conclusions:

• The developed cellular models provide a robust platform for studying COG4 mutations and their impact on cellular processes.
• The findings highlight specific O- and N-glycosylation defects and aberrant protein secretion as key molecular consequences of COG4 mutations.
• This research offers insights into the pathogenesis of COG-CDGs and may guide the development of therapeutic strategies, particularly noting the overlap with Marinesco-Sjogren syndrome.