Article

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: 9.69). 09/2008; 182(3):531-42. DOI: 10.1083/jcb.200711151
Source: PubMed

ABSTRACT 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.

0 Followers
 · 
109 Views
  • Source
    • "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. "
    [Show abstract] [Hide abstract]
    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.
    The EMBO Journal 04/2013; 32(10). DOI:10.1038/emboj.2013.79 · 10.75 Impact Factor
  • Source
    • "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]. "
    [Show abstract] [Hide abstract]
    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.
    FEBS letters 05/2011; 585(9):1276-80. DOI:10.1016/j.febslet.2011.04.010 · 3.34 Impact Factor
  • Source
    • "The protein concentration in cell extracts was determined by the BCA method (Pierce, Rockford, IL) following the manufacturer's instructions with bovine serum albumin as a standard. PCR was carried out with Phusion™ High Fidelity PCR Kit (New England Biolabs) as described previously (Ju, Lanneau et al. 2008). The PCR product was purified using the QIAquick Gel Extraction Kit (Qiagen) following the manufacturer's protocol and sequenced. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Loss of T-synthase (uridine diphosphate galactose:N-acetylgalactosaminyl-α1-Ser/Thr β3galactosyltransferase), a key enzyme required for the formation of mucin-type core 1 O-glycans, is observed in several human diseases, including cancer, Tn syndrome and IgA nephropathy, but current methods to assay the enzyme use radioactive substrates and complicated isolation of the product. Here we report the development of a novel fluorescent assay to measure its activity in a variety of tumor cell lines. Deficiencies in T-synthase activity correlate with mutations in the gene encoding the molecular chaperone Cosmc that is required for folding the T-synthase. This new high-throughput assay allows for facile screening of tumor specimens and other biological material for T-synthase activity and could be used diagnostically.
    Glycobiology 10/2010; 21(3):352-62. DOI:10.1093/glycob/cwq168 · 3.75 Impact Factor
Show more

Preview (2 Sources)

Download
1 Download
Available from

Similar Publications