Calnexin Discriminates between Protein Conformational States and Functions as a Molecular Chaperone In Vitro  Yoshito Ihara, Myrna F Cohen-Doyle, Yoshiro Saito, David B Williams  Molecular Cell  Volume 4, Issue 3, Pages 331-341 (September 1999) DOI: 10.1016/S1097-2765(00)80335-4

Figure 1 S-CNX Inhibits the Thermal Aggregation of Glycosylated and Nonglycosylated Proteins (A) SBA possessing glucosylated (G1M9) or native (M9) oligosaccharides, or only a single GlcNAc residue (Deglyco), was denatured in 6 M GdnHCl and diluted 120-fold to a final concentration of 0.6 μM in the presence of various concentrations of S-CNX (0.04–1.8 μM) or IgG (0.15–1.8 μM). Protein aggregation at 45°C was monitored by measuring light scattering at 360 nm. (B) CS dimer or MDH dimer (1 μM) were incubated at 45°C in the presence of various concentrations of S-CNX (0.25–2 μM) or IgG (2 μM), and aggregation was monitored at 360 nm. (C) MDH dimer (0.6 μM) was incubated at 45°C in the absence or presence of S-CNX or ERp57 (0.6 μM) as indicated. In addition, S-CNX and ERp57 were preincubated at 25°C for 30 min and then incubated at 45°C with MDH dimer (S-CNX/MDH/ERp57 sample). Aggregation was monitored at 360 nm. Molecular Cell 1999 4, 331-341DOI: (10.1016/S1097-2765(00)80335-4)

Figure 2 Detection of S-CNX-Substrate Complexes S-CNX (2 μM) was incubated with 1 μM MDH (A) or 1 μM CS (B) at 25°C or 45°C for the indicated times, and then aliquots were analyzed by SEC using a TosoHaas G4000SWxL column in TS buffer, 2 mM CaCl2. In (C), S-CNX (2 μM), MDH (1 μM), or CS (1 μM) was incubated alone at 45°C for 90 min and then similarly analyzed. The masses of molecular size markers are indicated in kilodaltons. Vo, void volume. Molecular Cell 1999 4, 331-341DOI: (10.1016/S1097-2765(00)80335-4)

Figure 3 Characterization of S-CNX-Substrate Complexes (A) Complex composition. S-CNX (2 μM) was incubated with 1 μM MDH or 1 μM CS at 45°C for 90 min, and then S-CNX-substrate complexes, S-CNX, and CS were isolated by SEC (retention times: 6.5 min, 11.2 min, and 12.5 min, respectively). MDH (13.2 min) was also isolated from the sample incubated with S-CNX at 25°C for 90 min. Fractions were analyzed by 10% SDS-PAGE, and proteins were visualized with Coomassie blue. Standards of S-CNX and MDH or CS (molar ratios, 2 S-CNX [monomer] : 1 substrate [dimer]) were included to obtain the following relative staining ratios by densitometry: S-CNX:MDH = 1.14:1 and S-CNX:CS = 0.83:1. Using these values, the directly measured staining ratios of S-CNX to MDH (0.32:1) or CS (0.25:1) in the complexes were corrected for differential staining intensities and the molar ratios obtained. Values are the average of two independent experiments. (B) Protease sensitivity of complexes. S-CNX (2 μM) was incubated with MDH (1 μM) at 25°C or 45°C for 90 min. After centrifugation at 5000 rpm for 1 min, the supernatant fraction was supplemented with the indicated amounts of trypsin and incubated on ice for 10 min. Digestion was terminated with 60 μg/ml AEBSF, 10 μg/ml antipain, and 1 μg/ml leupeptin, and samples were analyzed by 10% SDS-PAGE followed by Coomassie blue staining. Molecular Cell 1999 4, 331-341DOI: (10.1016/S1097-2765(00)80335-4)

Figure 4 Electron Microscopic Analysis of S-CNX, MDH, and S-CNX-MDH Complexes S-CNX (2 μM) and MDH (1 μM) were incubated alone or together at 25°C or 45°C for 60 min. The samples were negatively stained using uranyl acetate. Note the lower magnification for MDH alone at 45°C (magnification 6,600 versus 66,000 in other panels). Molecular Cell 1999 4, 331-341DOI: (10.1016/S1097-2765(00)80335-4)

Figure 5 Effects of ATP and Oligosaccharide on S-CNX-Substrate Interactions (A) Effect of ATP. CS (1 μM) was incubated in TS buffer, 5 mM CaCl2 with 0.5 μM S-CNX at 45°C in the presence of 0–1 mM ATP and 10 mM MgCl2. After 60 min of incubation, protein aggregation was estimated by measuring light scattering at 360 nm. Additionally, GdnHCl-denatured α-mannosidase (0.35 μM) was incubated with S-CNX (1 μM) at 25°C under the same buffer conditions, and aggregation was measured after 5 min. (B) Effect of oligosaccharide. CS (1 μM) was incubated in TS buffer, 5 mM CaCl2 with 0.5 μM S-CNX at 45°C in the absence or presence of the indicated oligosaccharides (40 μM), and aggregation was measured after 60 min. G, glucose; M, mannose. (C) Effect of oligosaccharide and ATP on preformed complexes of CS and S-CNX. CS (1 μM) was incubated at 45°C in the absence or presence of 1 μM S-CNX, and after 15 min of incubation (arrow), either 100 μM G1M3, 1 mM Mg-ATP, or both were added to the samples containing S-CNX, and the incubations were continued for an additional 45 min. Aggregation was measured throughout the incubations. (D) G1M3 oligosaccharide causes dissociation of S-CNX-CS complexes. Aggregation assays were performed as in (C), and samples containing CS and S-CNX were analyzed by SEC after 0, 15, and 40 min of incubation at 45°C. A similar sample in which G1M3 oligosaccharide was added at the 15 min time point and then further incubated for a total duration of 40 min was also analyzed by SEC. Dashed line, A230 of 0.015. Molecular Cell 1999 4, 331-341DOI: (10.1016/S1097-2765(00)80335-4)

Figure 6 Conformational Changes in S-CNX Induced by ATP and Oligosaccharide (A) Tryptophan fluorescence. The fluorescence emission spectra of S-CNX (1 μM) in the absence or presence of the indicated concentrations of ATP, AMP, or the oligosaccharides G1M3 and G1M were monitored as described in Experimental Procedures (excitation, 290 nm). (B) ANS binding. The binding of ANS to S-CNX in the absence or presence of ATP, AMP, or the G1M3 and G1M oligosaccharides was monitored by its fluorescence emission (excitation, 370 nm). Molecular Cell 1999 4, 331-341DOI: (10.1016/S1097-2765(00)80335-4)

Figure 7 Effects of S-CNX on the Inactivation and Reactivation of CS (A) S-CNX prevents the thermal inactivation of CS. CS (1 μM) was incubated at 43°C in the absence or presence of various concentrations of S-CNX or 4 μM IgG. CS activity was assayed at the indicated times. (B) Refolding of heat-inactivated CS. CS (1 μM) was partially inactivated by incubation with S-CNX (1 μM) or IgG (1 μM) at 43°C for 60 min. Reactivation of CS was initiated by diluting the sample in buffer containing various additions and by shifting the temperature to 25°C. Additions included 1 mM ATP, 100 μM G1M3 oligosaccharide, or rabbit reticulocyte lysate (RRL) either alone or supplemented with ATP or ATP plus G1M3. CS activity was assayed at the indicated times. Molecular Cell 1999 4, 331-341DOI: (10.1016/S1097-2765(00)80335-4)

Figure 8 Revised Dual-Binding Model for the Interaction of CNX with Folding Glycoproteins Molecular Cell 1999 4, 331-341DOI: (10.1016/S1097-2765(00)80335-4)