Regulation of protein O-glycosylation by the endoplasmic reticulum–localized molecular chaperone Cosmc

Department of Biochemistry, Emory University School of Medicine, Atlanta, GA 30322, USA.
The Journal of Cell Biology (Impact Factor: 9.83). 09/2008; 182(3):531-42. DOI: 10.1083/jcb.200711151
Source: PubMed


Regulatory pathways for protein glycosylation are poorly understood, but expression of branchpoint enzymes is critical. A key branchpoint enzyme is the T-synthase, which directs synthesis of the common core 1 O-glycan structure (T-antigen), the precursor structure for most mucin-type O-glycans in a wide variety of glycoproteins. Formation of active T-synthase, which resides in the Golgi apparatus, requires a unique molecular chaperone, Cosmc, encoded on Xq24. Cosmc is the only molecular chaperone known to be lost through somatic acquired mutations in cells. We show that Cosmc is an endoplasmic reticulum (ER)-localized adenosine triphosphate binding chaperone that binds directly to human T-synthase. Cosmc prevents the aggregation and ubiquitin-mediated degradation of the T-synthase. These results demonstrate that Cosmc is a molecular chaperone in the ER required for this branchpoint glycosyltransferase function and show that expression of the disease-related Tn antigen can result from deregulation or loss of Cosmc function.

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    • "The human GalNAc-type O-glycoproteome C Steentoft et al studies demonstrating that cells without a functional COSMC chaperone lack the C1GalT1 synthase activity and O-glycan elongation (Ju et al, 2008). We generated 12 human SimpleCell lines from diverse organs to be able to probe cell and organ variation in the O-glycoproteome Figure 1A. "
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    ABSTRACT: Glycosylation is the most abundant and diverse posttranslational modification of proteins. While several types of glycosylation can be predicted by the protein sequence context, and substantial knowledge of these glycoproteomes is available, our knowledge of the GalNAc-type O-glycosylation is highly limited. This type of glycosylation is unique in being regulated by 20 polypeptide GalNAc-transferases attaching the initiating GalNAc monosaccharides to Ser and Thr (and likely some Tyr) residues. We have developed a genetic engineering approach using human cell lines to simplify O-glycosylation (SimpleCells) that enables proteome-wide discovery of O-glycan sites using 'bottom-up' ETD-based mass spectrometric analysis. We implemented this on 12 human cell lines from different organs, and present a first map of the human O-glycoproteome with almost 3000 glycosites in over 600 O-glycoproteins as well as an improved NetOGlyc4.0 model for prediction of O-glycosylation. The finding of unique subsets of O-glycoproteins in each cell line provides evidence that the O-glycoproteome is differentially regulated and dynamic. The greatly expanded view of the O-glycoproteome should facilitate the exploration of how site-specific O-glycosylation regulates protein function.
    Full-text · Article · Apr 2013 · The EMBO Journal
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    • "Human endothelial cell line EA.hy926 was a gift from Drs. Shu-Huei Wang and Hsiu-Ni Kung (Graduate Institute of Anatomy and Cell Biology, National Taiwan University College of Medicine, Taiwan) and was maintained in Dulbecco's modified Eagle's medium (DMEM; Thermo scientific, Barrington IL) containing 10% FBS, 100 IU/mL penicillin, and 100 µg/mL streptomycin (Invitrogen, Carlsbad, CA) in a humidified tissue culture incubator at 37°C and 5% CO2 atmosphere. COSMC/pcDNA3.1 [15] (a gift from Dr. Richard D. Cummings at the Emory University School of Medicine, Atlanta, USA) and control pcDNA3.1 (Mock) (Invitrogen, Carlsbad, CA) were transfected into HUVECs (3-5 passages) using an Amaxa Nucleofector™ (Lonza, Walkersville, MD) and the HUVEC Nucleofector kit (Lonza, Walkersville, MD) according to the manufacturer's instructions. For COSMC knockdown, duplex siRNA against COSMC and non-targeting control siRNA were purchased from Invitrogen. "
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    ABSTRACT: Infantile hemangiomas are localized lesions comprised primarily of aberrant endothelial cells. COSMC plays a crucial role in blood vessel formation and is characterized as a molecular chaperone of T-synthase which catalyzes the synthesis of T antigen (Galβ1,3GalNAc). T antigen expression is associated with tumor malignancy in many cancers. However, roles of COSMC in infantile hemangioma are still unclear. In this study, immunohistochemistry showed that COSMC was upregulated in proliferating hemangiomas compared with involuted hemangiomas. Higher levels of T antigen expression were also observed in the proliferating hemangioma. Overexpression of COSMC significantly enhanced cell growth and phosphorylation of AKT and ERK in human umbilical vein endothelial cells (HUVECs). Conversely, knockdown of COSMC with siRNA inhibited endothelial cell growth. Mechanistic investigation showed that O-glycans were present on VEGFR2 and these structures were modulated by COSMC. Furthermore, VEGFR2 degradation was delayed by COSMC overexpression and facilitated by COSMC knockdown. We also showed that COSMC was able to regulate VEGF-triggered phosphorylation of VEGFR2. Our results suggest that COSMC is a novel regulator for VEGFR2 signaling in endothelial cells and dysregulation of COSMC expression may contribute to the pathogenesis of hemangioma.
    Full-text · Article · Feb 2013 · PLoS ONE
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    • "Cosmc supports the folding and stabilization of C1GalT in the endoplasmic reticulum (ER) [6] [9]. In cells lacking Cosmc, such as the human T leukemic cell line, Jurkat, and the human colon cancer cell line, LSC, C1GalT aggregates and is subsequently degraded in the proteasome [6] [8] [9]. Furthermore, Cosmc knock-out mice [10] are embryonic lethal, owing to brain hemorrhage similar to what is observed in C1GalT knock-out mice [11]. "
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    ABSTRACT: The core 1 structure of the mucin type O-glycan is synthesized by core 1 β1,3-galactosyltransferase (C1GalT). Core 1 synthase specific molecular chaperone (Cosmc), a molecular chaperone specific for C1GalT, is essential for the expression of functional C1GalT in mammalian cells. In this study, we have established a procedure for detecting the chaperone activity of Cosmc by using a wheat germ cell-free translation system. Active C1GalT was expressed following simultaneous translation with Cosmc or translation in the presence of recombinant Cosmc protein. Moreover, we show that recombinant Cosmc must be present during the translation of C1GalT, as it is ineffective when added after translation. These results indicate that Cosmc mediates the co-translational activation of C1GalT and that it may prevent the unfavorable aggregation of C1GalT.
    Full-text · Article · May 2011 · FEBS letters
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