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Smith McCort (SMC) dysplasia is a rare, autosomal recessive, osteochondrodysplasia that can be caused by pathogenic variants in either RAB33B or DYM genes. These genes codes for proteins that are located at the Golgi apparatus and have a role in intracellular vesicle trafficking. We generated mice that carry a Rab33b disease-causing variant, c.136A>C (p.Lys46Gln), which is identical to that of members from a consanguineous family diagnosed with SMC. In male mice at 4 months of age, the Rab33b variant caused a mild increase in trabecular bone thickness in the spine and femur and in femoral mid-shaft cortical thickness with a concomitant reduction of the femoral medullary area, suggesting a bone resorption defect. In spite of the increase in trabecular and cortical thickness, bone histomorphometry showed a 4-fold increase in osteoclast parameters in homozygous Rab33b mice suggesting a putative impairment in osteoclast function, while dynamic parameters of bone formation were similar in mutant versus control mice. Femur biomechanical tests showed an increased in yield load and a progressive elevation, from WT to heterozygote to homozygous mutants, of bone intrinsic properties. These findings suggest an overall impact on bone material properties which may be caused by disturbed protein glycosylation in cells contributing to skeletal formation, supported by the altered and variable pattern of lectin staining in murine and human tissue cultured cells and in liver and bone murine tissues. The mouse model only reproduced some of the features of the human disease and was sex-specific, manifesting in male but not female mice. Our data reveal a potential novel role of RAB33B in osteoclast function and protein glycosylation and their dysregulation in SMC and lay the foundation for future studies. [ABSTRACT FROM AUTHOR]

Conserved Oligomeric Golgi (COG) complex controls Golgi trafficking and glycosylation, but the precise COG mechanism is unknown. The auxin‐inducible acute degradation system was employed to investigate initial defects resulting from COG dysfunction. We found that acute COG inactivation caused a massive accumulation of COG‐dependent (CCD) vesicles that carry the bulk of Golgi enzymes and resident proteins. v‐SNAREs (GS15, GS28) and v‐tethers (giantin, golgin84, and TMF1) were relocalized into CCD vesicles, while t‐SNAREs (STX5, YKT6), t‐tethers (GM130, p115), and most of Rab proteins remained Golgi‐associated. Airyscan microscopy and velocity gradient analysis revealed that different Golgi residents are segregated into different populations of CCD vesicles. Acute COG depletion significantly affected three Golgi‐based vesicular coats—COPI, AP1, and GGA, suggesting that COG uniquely orchestrates tethering of multiple types of intra‐Golgi CCD vesicles produced by different coat machineries. This study provided the first detailed view of primary cellular defects associated with COG dysfunction in human cells. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Cell surface lectin staining, examination of Golgi glycosyltransferases stability and localization, and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analysis were employed to investigate conserved oligomeric Golgi (COG)-dependent glycosylation defects in HeLa cells. Both Griffonia simplicifolia lectin-II and Galanthus nivalus lectins were specifically bound to the plasma membrane glycoconjugates of COG-depleted cells, indicating defects in activity of medial- and trans-Golgi-localized enzymes. In response to siRNA-induced depletion of COG complex subunits, several key components of Golgi glycosylation machinery, including MAN2A1, MGAT1, B4GALT1 and ST6GAL1, were severely mislocalized. MALDI-TOF analysis of total N-linked glycoconjugates indicated a decrease in the relative amount of sialylated glycans in both COG3 KD and COG4 KD cells. In agreement to a proposed role of the COG complex in retrograde membrane trafficking, all types of COG-depleted HeLa cells were deficient in the Brefeldin A- and Sar1 DN-induced redistribution of Golgi resident glycosyltransferases to the endoplasmic reticulum. The retrograde trafficking of medial- and trans-Golgi-localized glycosylation enzymes was affected to a larger extent, strongly indicating that the COG complex regulates the intra-Golgi protein movement. COG complex-deficient cells were not defective in Golgi re-assembly after the Brefeldin A washout, confirming specificity in the retrograde trafficking block. The lobe B COG subcomplex subunits COG6 and COG8 were localized on trafficking intermediates that carry Golgi glycosyltransferases, indicating that the COG complex is directly involved in trafficking and maintenance of Golgi glycosylation machinery. [ABSTRACT FROM AUTHOR]