Conformational requirements for glycoprotein reglucosylation in the endoplasmic reticulum

Abstract

Newly synthesized glycoproteins interact during folding and quality control in the ER with calnexin and calreticulin, two lectins specific for monoglucosylated oligosaccharides. Binding and release are regulated by two enzymes, glucosidase II and UDP-Glc:glycoprotein:glycosyltransferase (GT), which cyclically remove and reattach the essential glucose residues on the N-linked oligosaccharides. GT acts as a folding sensor in the cycle, selectively reglucosylating incompletely folded glycoproteins and promoting binding of its substrates to the lectins. To investigate how nonnative protein conformations are recognized and directed to this unique chaperone system, we analyzed the interaction of GT with a series of model substrates with well defined conformations derived from RNaseB. We found that conformations with slight perturbations were not reglucosylated by GT. In contrast, a partially structured nonnative form was efficiently recognized by the enzyme. When this form was converted back to a nativelike state, concomitant loss of recognition by GT occurred, reproducing the reglucosylation conditions observed in vivo with isolated components. Moreover, fully unfolded conformers were poorly recognized. The results indicated that GT is able to distinguish between different nonnative conformations with a distinct preference for partially structured conformers. The findings suggest that discrete populations of nonnative conformations are selectively reglucosylated to participate in the calnexin/calreticulin chaperone pathway.

Figure 1

RNaseB conformers. (A) Schematic representation of some of the RNaseB conformers generated including RNaseB, RNaseBS, RNaseBS-Prot, RNase-B5′ and RNase-B5′′. Shaded parts depict peptides added to regenerate RNase-B5′, and RNase-B5′′. (B) RNase conformers analyzed by SDS-PAGE were as follows: (A) native intact RNaseB, (B) reduced and alkylated RNaseB, (C) RNaseBS, (D) RNaseBS-Prot, (E) RNaseBS′, (F) RNaseBS′′, (G) α-mannosidase–digested RNaseBS-Prot, (H) EndoH-digested RNaseBS-Prot, (I) reduced and alkylated RNaseA, (J) RNaseS, and (K) RNaseS-Prot. (C) Ribonuclease activity of the indicated RNaseB conformers. “No Add.” indicates reactions where no enzyme was added.

Figure 1

RNaseB conformers. (A) Schematic representation of some of the RNaseB conformers generated including RNaseB, RNaseBS, RNaseBS-Prot, RNase-B5′ and RNase-B5′′. Shaded parts depict peptides added to regenerate RNase-B5′, and RNase-B5′′. (B) RNase conformers analyzed by SDS-PAGE were as follows: (A) native intact RNaseB, (B) reduced and alkylated RNaseB, (C) RNaseBS, (D) RNaseBS-Prot, (E) RNaseBS′, (F) RNaseBS′′, (G) α-mannosidase–digested RNaseBS-Prot, (H) EndoH-digested RNaseBS-Prot, (I) reduced and alkylated RNaseA, (J) RNaseS, and (K) RNaseS-Prot. (C) Ribonuclease activity of the indicated RNaseB conformers. “No Add.” indicates reactions where no enzyme was added.

Figure 1

RNaseB conformers. (A) Schematic representation of some of the RNaseB conformers generated including RNaseB, RNaseBS, RNaseBS-Prot, RNase-B5′ and RNase-B5′′. Shaded parts depict peptides added to regenerate RNase-B5′, and RNase-B5′′. (B) RNase conformers analyzed by SDS-PAGE were as follows: (A) native intact RNaseB, (B) reduced and alkylated RNaseB, (C) RNaseBS, (D) RNaseBS-Prot, (E) RNaseBS′, (F) RNaseBS′′, (G) α-mannosidase–digested RNaseBS-Prot, (H) EndoH-digested RNaseBS-Prot, (I) reduced and alkylated RNaseA, (J) RNaseS, and (K) RNaseS-Prot. (C) Ribonuclease activity of the indicated RNaseB conformers. “No Add.” indicates reactions where no enzyme was added.

Figure 2

Characterization of RNaseB conformers. Elution profiles from ion exchange chromatography on MonoS (A–E) and Gel filtration chromatography on Superdex S-75 (F–J). (A and F) Native RNaseB, (B and G) RNaseBS, (C and H) RNaseBS-Prot, (D and I) RNaseBS′, (E) RNaseBS′′, and (J) RNaseB reduced and alkylated with iodoacetamide. Arrows indicate the elution peaks for 15- and 25-kD markers.

Figure 3

Characterization of RNaseB conformers. Sensitivity of indicated RNaseB conformers to EndoH (A), N-glycanase (B), and to trypsin (C). RNase samples were as follows: (B) intact RNaseB, (BS) RNaseBS, and (B-IAcNH₂) RNaseB reduced and alkylated with iodoacetamide. Samples were digested as indicated in Materials and Methods and run in SDS-PAGE stained with Coomasie blue.

Figure 3

Characterization of RNaseB conformers. Sensitivity of indicated RNaseB conformers to EndoH (A), N-glycanase (B), and to trypsin (C). RNase samples were as follows: (B) intact RNaseB, (BS) RNaseBS, and (B-IAcNH₂) RNaseB reduced and alkylated with iodoacetamide. Samples were digested as indicated in Materials and Methods and run in SDS-PAGE stained with Coomasie blue.

Figure 3

Characterization of RNaseB conformers. Sensitivity of indicated RNaseB conformers to EndoH (A), N-glycanase (B), and to trypsin (C). RNase samples were as follows: (B) intact RNaseB, (BS) RNaseBS, and (B-IAcNH₂) RNaseB reduced and alkylated with iodoacetamide. Samples were digested as indicated in Materials and Methods and run in SDS-PAGE stained with Coomasie blue.

Figure 4

Recognition of RNaseB conformers by GT. (A) Different RNaseB conformers were tested as substrates of GT. In all samples, 350 μg of the indicated RNaseB conformers were used as substrates in glycoprotein reglucosylation reactions. (B) RNaseB conformers as inhibitors of GT. For inhibition experiments, the same incubations in A were conducted in the presence of 100 μg of denatured SBA.

Figure 5

Reversible recognition of RNaseBSProt by GT. The indicated amounts of S-peptides 1–20 (solid diamonds) or 1–15 (solid circles) were added to samples containing 500 μg of RNaseBS-Prot in a total volume of 45 μl of 10 mM CaCl₂, 50 mM NaCl, 20 mM Hepes, pH 7.5. After 5 min at 20°C, glucosylation reactions were started with 5 μl containing 0.3 μg of GT and 400,000 cpm of UDP[³H]Glc. Reactions were incubated for 5 min at 30°C, stopped with 1 ml of 10% TCA, and radioactivity incorporated in TCA insoluble material quantified. Parallel incubations lacking UDP[³H]Glc were used to measure ribonuclease activity (empty diamonds and circles) as described in Materials and Methods.