Abstract
The T-synthase is the key beta 3-galactosyltransferase essential for biosynthesis of core 1 O-glycans (Gal beta 1-3GalNAc alpha 1-Ser/Thr) in animal cell glycoproteins. Here we describe the novel ability of an endoplasmic reticulum-localized molecular chaperone termed Cosmc to specifically interact with partly denatured T-synthase in vitro to cause partial restoration of activity. By contrast, a mutated form of Cosmc observed in patients with Tn syndrome has reduced chaperone function. The chaperone activity of Cosmc is specific, does not require ATP in vitro, and is effective toward T-synthase but not another beta-galactosyltransferase. Cosmc represents the first ER chaperone identified to be required for folding of a glycosyltransferase.
Highlights
Previous studies indicate that a unique and specific molecular chaperone in the ER is Cosmc, which is required for the formation of active core 1 3-galactosyltransferase [16, 17], an essential enzyme required
The denaturation status of HPC4-sT-syn was monitored by assaying T-synthase activity after being heated over time or being treated with 6 M guanidinium hydrochloride (GnHCl) for 90 min, as shown in Fig. 1, B and C, respectively
For HPC4-sT-syn denatured by GnHCl treatment, incubation with 6ϫHis-sCosmc caused restoration of activity, whereas addition of control proteins, BSA, or galectin-3, had little effect (Fig. 1G)
Summary
Previous studies indicate that a unique and specific molecular chaperone in the ER is Cosmc, which is required for the formation of active core 1 3-galactosyltransferase (core 1 3GalT, T-synthase) [16, 17], an essential enzyme required. Cosmc Promotes Renaturation of Denatured T-synthase in Vitro—To investigate whether the chaperone function of Cosmc can be measured in vitro, we expressed recombinant soluble 6ϫHis-tagged Cosmc (6ϫHis-sCosmc) and HPC4tagged soluble T-synthase (HPC4-sT-syn), and purified each to apparent homogeneity (Fig. 1A), of apparent molecular mass of 35 and 39 kDa, respectively.
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