The endoplasmic reticulum chaperone Cosmc directly promotes in vitro folding of T-synthase

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.

FIGURE 1.

Cosmc-dependent reconstitution of active T-synthase from denatured T-synthase. A, SDS-PAGE analysis of purified recombinant human 6×His-sCosmc and HPC4-sT-syn. The human N-terminal 6×His-tagged soluble Cosmc (6×His-sCosmc) and the N-terminal HPC4 epitope-tagged soluble T-synthase (HPC4-sT-syn) were expressed in Hi-5 cells and purified directly from the medium. Protein (2 μg each) was subjected to polyacrylamide gel electrophoresis and stained by Coomassie Blue, showing one major band in each lane (arrows). The lanes represent protein standards (lane 1), 6×His-sCosmc (lane 2), and HPC4-sT-syn (lane 3). B, HPC4-sT-syn was heat-denatured in reconstitution buffer over time and percent specific activities of native, and each preparation of denatured, HPC4-sT-syn were determined. C, purified soluble human HPC4-sT-syn was treated with GnHCl, and the percent specific activities of both treated (DT-syn) and untreated (NT-syn) T-synthase were determined. D, reconstitution of the heat-denatured HPC4-sT-syn, which was heated for 1min at 54 °C, was initiated by the addition of 6×His-sCosmc and the percent of restored T-synthase activity was determined, whereas galectin-3 (Gal-3), a control protein for specificity, did not support reconstitution. E, reconstitution of the heat-denatured HPC4-sT-syn was initiated by the addition of ER general chaperone Bip, which did not support the reconstitution of heat-denatured HPC4-sT-syn whereas (F) reconstitution of Bip appears to restore the activity of heat-denatured luciferase. N-Luc, native luciferase, D-Luc, denatured luciferase. G, reconstitution of GnHCl-denatured HPC4-sT-syn was initiated by the addition of recombinant 6×His-sCosmc, Gal-3, or BSA, as indicated, and percent T-synthase activity was determined. H, restoration of activity of denatured HPC4-sT-syn heated over time by addition of 6×His-sCosmc was measured. 6×His-sCosmc was added to denatured HPC4-sT-syn at each time point. A parallel reconstitution experiment of denatured HPC4-sT-syn was initiated by the addition of Gal-3, and percent T-synthase activity was determined. C, 6×His-sCosmc alone. A vertical line separates experiments performed at two different times (B and H). In B, D, and H, each assay was performed in duplicate, three replicate experiments were performed, and data represent the average of all experiments. In C and H, each assay was performed in duplicate and four replicate experiments were performed. In E and F, at least three replicate experiments were performed, and data represent the average of all experiments. Error bars, ± 1 S.D. from the average. * and ** represent p values p < 0.01 and p = 0.62, respectively.

FIGURE 2.

Cosmc restoration of activity of heat-denatured T-synthase is concentration dependent. A, purified HPC4-sT-syn (NT-syn) was heat-denatured in reconstitution buffer. Renaturation of the activity of the heat-denatured HPC4-sT-syn (DT-syn) was initiated by the addition of increasing concentrations of 6×His-sCosmc as indicated to DT-syn preparations, and percent T-synthase activity of each reaction was determined. B, time dependence of restoration of T-synthase activity from DT-syn by 6×His-sCosmc, in which reconstitution and assay of T-synthase was conducted at 37 °C, and the percent restored activity was determined at different time points as indicated. In A, each assay was performed in duplicate, two replicate experiments were performed, and data represent the average of all experiments. In B, each assay was performed in duplicate, three replicate experiments were performed, and data represent the average of all experiments. Error bars, ± 1 S.D. from the average.

FIGURE 3.

Mutated Cosmc has little effect on restoration of denatured T-synthase activity. A, depiction of the 6×His-sCosmc and 6×His-msCosmc constructs, where the latter has a point mutation E152K. B, SDS-PAGE analysis of purified recombinant soluble human 6×His-sCosmc and 6×His-msCosmc, as indicated. Lane 1, molecular weight standards; lane 2, 6×His-msCosmc (∼8 μg); lane 3, human N-terminal 6×His-sCosmc (∼4 μg). A densitometry scan of the protein bands in lanes 2 and 3 showed that the amount of protein corresponding to the apparent molecular mass of ∼35 kDa for both 6×His-smCosmc and 6×His-sCosmc were similar. C, renaturation of heat-denatured HPC4-sT-syn (DT-syn) was initiated by addition of 6×His-msCosmc and with recombinant 6×His-sCosmc, and percent T-synthase activity was determined. In C, each assay was performed in duplicate, three replicate experiments were performed, and data represent the average of all experiments. Error bars, ± 1 S.D. from the average. The vertical line separates experiments done at two different time points.

FIGURE 4.

Cosmc can restore the activity of heat-denatured T-synthase independently of ATP in vitro. A, purified HPC4-sT-syn (NT-syn) was heat-denatured in reconstitution buffer. Reconstitution of denatured HPC4-sT-syn (DT-syn) activity was initiated by the addition of 6×His-sCosmc in reconstitution buffer with ATP, non-hydrolyzable ATP (ATPγS), or without nucleotide, where final concentration of nucleotides is 5 mm, and percent T-synthase activity was determined. B, reconstitution of heat-denatured HPC4-sT-syn by the addition of increasing concentration (μm) of 6×His-sCosmc in reconstitution buffer with ATP, ATPγS, or without nucleotide, where final concentration of nucleotides is 5 mm, and percent T-synthase activity was determined. Each assay was performed in duplicate, two replicate experiments were performed, and the data represent the average of all experiments. Error bars, ± 1 S.D. from the average.

FIGURE 5.

Cosmc is unable to restore activity to denatured β4-GalT. A, β4-GalT was heat-denatured and reconstitution of denatured β4-GalT (Dβ4-GalT) was initiated by the addition of recombinant 6×His-sCosmc or Gal-3, and percent β4-GalT activity was determined. In parallel, reconstitution of the heat-denatured HPC4-sT-syn (DT-syn) was initiated by the addition of recombinant 6×His-sCosmc, and percent T-synthase activity was determined. B, β4-GalT was heat-denatured over time and reconstitution of denatured β4-GalT was initiated by the addition of recombinant 6×His-sCosmc (C) or Gal-3, and percent β4-GalT activity was determined. Each assay was performed in duplicate, two replicate experiments were performed, and data represent the average of all experiments. Error bars, ± 1 S.D. from the average.