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

Ju T, Aryal RP, Stowell CJ, Cummings RD - J. Cell Biol. (2008)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Emory University School of Medicine, Atlanta, GA 30322, USA. tju@emory.edu

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.

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Cosmc has ATP-binding activity. (A) Hi-5 insect cells were infected with Baculovirus encoding either Cosmc-HA or HPC4-sT-synthase plus wild-type Cosmc. Cells for Cosmc-HA and media from cell coexpressing HPC4–sT-synthase and wild-type Cosmc were harvested. The cell extracts and media were loaded on ATP-Sepharose column for chromatography and eluted with 50 mM ATP, respectively. The washes and eluates were analyzed by Western blot with anti-HA for Cosmc and anti-HPC4 for T-synthase, as indicated. (B) HPC4-sCosmc and HPC4–sT-synthase (coexpressed with wtCosmc) were expressed in Hi-5 cells and purified from the media. 3 μg of Cosmc and T-synthase were photolabeled with 8-Azido α-[32P]ATP, analyzed on SDS-PAGE, and transferred to nitrocellulose membrane for radioautogram and Western blot with anti-HPC4. Black line indicates that intervening lanes have been spliced out.
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fig3: Cosmc has ATP-binding activity. (A) Hi-5 insect cells were infected with Baculovirus encoding either Cosmc-HA or HPC4-sT-synthase plus wild-type Cosmc. Cells for Cosmc-HA and media from cell coexpressing HPC4–sT-synthase and wild-type Cosmc were harvested. The cell extracts and media were loaded on ATP-Sepharose column for chromatography and eluted with 50 mM ATP, respectively. The washes and eluates were analyzed by Western blot with anti-HA for Cosmc and anti-HPC4 for T-synthase, as indicated. (B) HPC4-sCosmc and HPC4–sT-synthase (coexpressed with wtCosmc) were expressed in Hi-5 cells and purified from the media. 3 μg of Cosmc and T-synthase were photolabeled with 8-Azido α-[32P]ATP, analyzed on SDS-PAGE, and transferred to nitrocellulose membrane for radioautogram and Western blot with anti-HPC4. Black line indicates that intervening lanes have been spliced out.

Mentions: Many chaperones have ATP binding or ATPase activity, some with low affinity. Thus, we tested whether Cosmc has ATP binding activity (Itoh et al., 1995). Cosmc binds to ATP-Sepharose and can be eluted with ATP, although the protein does not bind well (Fig. 3 A). In contrast, there was no significant binding of recombinant T-synthase to ATP-Sepharose. To further test whether Cosmc binds ATP directly, we performed photolabeling using 8-azido α-[32P]ATP. Under ultraviolet exposure, the activated azido group directly cross-links to the peptide bond in the ATP binding region of Cosmc. Purified Cosmc, but not T-synthase, could be photo–cross-linked with 8-azido α-[32P]ATP. This binding is specific because it was significantly inhibited by excesses of cold ATP or GTP (Fig. 3 B). Typically, either ATP or GTP can inhibit specific ATP binding to molecular chaperones (Csermely and Kahn, 1991; Saira Mian, 1993; Soti et al., 2002). These results demonstrate that Cosmc can bind ATP, which is similar to other chaperones, whereas T-synthase does not bind ATP.


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

Ju T, Aryal RP, Stowell CJ, Cummings RD - J. Cell Biol. (2008)

Cosmc has ATP-binding activity. (A) Hi-5 insect cells were infected with Baculovirus encoding either Cosmc-HA or HPC4-sT-synthase plus wild-type Cosmc. Cells for Cosmc-HA and media from cell coexpressing HPC4–sT-synthase and wild-type Cosmc were harvested. The cell extracts and media were loaded on ATP-Sepharose column for chromatography and eluted with 50 mM ATP, respectively. The washes and eluates were analyzed by Western blot with anti-HA for Cosmc and anti-HPC4 for T-synthase, as indicated. (B) HPC4-sCosmc and HPC4–sT-synthase (coexpressed with wtCosmc) were expressed in Hi-5 cells and purified from the media. 3 μg of Cosmc and T-synthase were photolabeled with 8-Azido α-[32P]ATP, analyzed on SDS-PAGE, and transferred to nitrocellulose membrane for radioautogram and Western blot with anti-HPC4. Black line indicates that intervening lanes have been spliced out.
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2500138&req=5

fig3: Cosmc has ATP-binding activity. (A) Hi-5 insect cells were infected with Baculovirus encoding either Cosmc-HA or HPC4-sT-synthase plus wild-type Cosmc. Cells for Cosmc-HA and media from cell coexpressing HPC4–sT-synthase and wild-type Cosmc were harvested. The cell extracts and media were loaded on ATP-Sepharose column for chromatography and eluted with 50 mM ATP, respectively. The washes and eluates were analyzed by Western blot with anti-HA for Cosmc and anti-HPC4 for T-synthase, as indicated. (B) HPC4-sCosmc and HPC4–sT-synthase (coexpressed with wtCosmc) were expressed in Hi-5 cells and purified from the media. 3 μg of Cosmc and T-synthase were photolabeled with 8-Azido α-[32P]ATP, analyzed on SDS-PAGE, and transferred to nitrocellulose membrane for radioautogram and Western blot with anti-HPC4. Black line indicates that intervening lanes have been spliced out.
Mentions: Many chaperones have ATP binding or ATPase activity, some with low affinity. Thus, we tested whether Cosmc has ATP binding activity (Itoh et al., 1995). Cosmc binds to ATP-Sepharose and can be eluted with ATP, although the protein does not bind well (Fig. 3 A). In contrast, there was no significant binding of recombinant T-synthase to ATP-Sepharose. To further test whether Cosmc binds ATP directly, we performed photolabeling using 8-azido α-[32P]ATP. Under ultraviolet exposure, the activated azido group directly cross-links to the peptide bond in the ATP binding region of Cosmc. Purified Cosmc, but not T-synthase, could be photo–cross-linked with 8-azido α-[32P]ATP. This binding is specific because it was significantly inhibited by excesses of cold ATP or GTP (Fig. 3 B). Typically, either ATP or GTP can inhibit specific ATP binding to molecular chaperones (Csermely and Kahn, 1991; Saira Mian, 1993; Soti et al., 2002). These results demonstrate that Cosmc can bind ATP, which is similar to other chaperones, whereas T-synthase does not bind ATP.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Emory University School of Medicine, Atlanta, GA 30322, USA. tju@emory.edu

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.

Show MeSH