More Than One Glycan Is Needed for ER Glucosidase II to Allow Entry of Glycoproteins into the Calnexin/Calreticulin Cycle  Paola Deprez, Matthias Gautschi, Ari Helenius  Molecular Cell  Volume 19, Issue 2, Pages 183-195 (July 2005) DOI: 10.1016/j.molcel.2005.05.029 Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 1 The N-Linked Core Glycan and the Model Protein (A) In the core N-glycan, mannoses are organized in a 3′ or A branch capped by glucoses and a 6′-pentamannosyl branch subdivided into B and C branches. The cleavage sites of glycosidases are indicated. (B) Our model protein for in vitro translation consists of the signal peptide of VSV G, the SFV capsid protease domain, and the SFV p62 protein with two glycosylation sites at Asn12 and Asn59. We used a construct with both glycosylation sites (NN) and one in which the second site was eliminated (N). Molecular Cell 2005 19, 183-195DOI: (10.1016/j.molcel.2005.05.029) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 2 Glycosylation Occurs When Asn12 and Asn59 Are 72 Residues from the P Site Truncated mRNAs derived from NN construct were translated in the presence of Cst, processed for SDS-PAGE ([A] and [E]), or further treated with PNGase F (B) or puromycin (C). In the latter case, after 15 min of translation, puromycin was added for 5 more min. Arrowheads indicate when the singly (A) and doubly (E) glycosylated-arrested chains were first detected. In this and subsequent figures, the capsid protease domain is indicated by an asterisk and the p62 peptides by a drawing showing the number and likely glycan configuration. (D) After quantification, glycosylation was expressed as the percentage of glycosylated peptides relative to the total p62 peptides. Half-maximal glycosylation values of 71.5 amino acids for Asn12 and 71.7 for Asn59 were obtained from the sigmoidal equation fit to the data: mean (X) ± standard deviation (SD); n = 3–41. Molecular Cell 2005 19, 183-195DOI: (10.1016/j.molcel.2005.05.029) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 3 Trimming by GI Occurs Immediately after Glycosylation (A) Band migration pattern and schematic representation of the glycans obtained after sequential treatment with purified GII and α-man. Lanes were numbered according to the glycan structure generated. (B and D) p76 and p72 were translated for different times in the presence or the absence of Cst. After solubilization of the microsomes, the ribosome-arrested chain complexes were collected by centrifugation, digested with purified GII and α-man, and analyzed by SDS-PAGE and autoradiography. (C) GI trimming was calculated as the percentage of the faster migrating band (GII sensitive) relative to the total glycosylated peptide (GII sensitive + GII resistant). The data were fit to a sigmoidal equation: X ± SD; n = 5 (p76) and n = 6 (p72). Molecular Cell 2005 19, 183-195DOI: (10.1016/j.molcel.2005.05.029) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 4 No GII Trimming of Singly Glycosylated-Arrested Chains (A) Translation was done in the presence or absence of Cst, and ribosome-arrested chain complexes were isolated. Cst-treated samples (Glc3, lane 1) were digested with GI to generate Glc2 peptide markers (lanes 2, 5, 8, 11, and 14). Treatment with purified GII partially inhibited with Cst gave a mixture of Glc3, Glc2, Glc1, and Glc0 peptides (lanes 4, 7, 10, 13, and 16). Test samples were run in lanes 3, 6, 9, 12, and 15. Digestion with α-man further increased the molecular weight differences. (B) After translation as in (A), microsomes were solubilized and subjected to IP with α-Cnx antibodies. 1/5 of the same translation reaction (Total) was run together with the IP samples. Cst-treated samples were used to control for nonspecific or glycan-independent Cnx Co-IP. (C) Translation of p76/29 with or without Cst. After 15 min, puromycin was added as indicated and incubated for 5 more min. The samples were subjected to α-Cnx IP. A glucose colored in gray in the drawings indicates that the precise configuration of this glycan is unknown. Both Glc1-Glc1 and Glc2-Glc1 doubly glycosylated peptides are possible. Molecular Cell 2005 19, 183-195DOI: (10.1016/j.molcel.2005.05.029) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 5 GII Trimming Occurs When Two Glycans Are Present (A) p144 and p144/97 were translated with and without Cst. After 18 min, puromycin was added as indicated and incubated for 5 more min. α-Cnx IPs in (A), (C), and (D) were performed as in Figure 4. (B) Cnx Co-IP expressed as a percentage relative to the total microsomal lysate. X ± SD, n = 3–14, and p > 0.1 ± Cst, except for doubly glycosylated peptides p < 0.001. (C) p80 was translated with or without Cst. After 10 min, puromycin was added as indicated, and incubation proceeded for 5–50 min. For samples treated with puromycin, the times indicated correspond to total incubation time. Arrowheads show the position of monoglucosylated peptides. (D) Translation of p116/69 to p140/93 with or without Cst and Cnx IP as in (A). (E) p144 and p144/97 were translated with or without Cst. A fraction from each sample was saved (Total), and the rest was split in two for IP with α-Cnx and α-GII. (F) The band intensity from mock-precipitated samples was subtracted prior to normalization. X ± SD, n = 6, and p < 0.0001 ± Cst for doubly glycosylated peptides coprecipitated with GII. Molecular Cell 2005 19, 183-195DOI: (10.1016/j.molcel.2005.05.029) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 6 Pulse-Chase Analysis of RNase RNase was transiently expressed in CHO cells, and pulse-chase experiments were done in the presence and the absence of Cst. After IP, samples were analyzed by SDS-PAGE. Band labels indicate the number of glycans present in RNase. (A) Cell lysates were subjected to IP with α-RNase, split in two, and PNGase F treated as indicated. (B) After 4 min pulse, cells were chased for the indicated times. Cell lysates were split in three and subjected to IP with α-RNase, α-Cnx, and α-Crt. (C) After pulse chase as in (B), a 1/10 aliquot of the cell lysate was used for IP with α-RNase, and the rest was subjected to a sequential IP with α-Cnx and α-RNase. (D) RNase Co-IP with Cnx in (C) expressed as a percentage relative to the total cell lysate. The values from Cst-treated samples were subtracted. X ± SD, n = 2–4, and p < 0.0002 between RNase with one and two glycans at 4 min chase. (E) Cells were pulsed for 8 min and chased for the indicated times. The media were recovered and subjected to IP with α-RNase. (F) Quantification of secreted RNase with 0, 1, 2 and 3 glycans from (E) in the presence or the absence of Cst. Secretion half-times shown in the plots with early time points (middle and right) were obtained from the sigmoidal equation fit to the data up to 120 min (left plot). X ± SD, n = 3–8. p > 0.1 ± Cst for RNase with no and one glycan, and p < 0.0001 for RNase with two and three glycans. Molecular Cell 2005 19, 183-195DOI: (10.1016/j.molcel.2005.05.029) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 7 A Model for GII Trimming We propose that GIIα has a mannose-independent basal activity. In addition, it can be activated by binding of the 6′-pentamannosyl branch to the MRH domain in GIIβ. For cleavage 1, due to the orientation of Glcα3Glc recognition epitope on the external side of the 3′ branch, GII can only be activated by mannoses from a second glycan (trans activation). For cleavage 2, which occurs from the inner side of the 3′ branch, GII can be activated by the mannoses of the same glycan (cis activation). Drawings of the N-glycan are based on the NMR structure by Petrescu et al. (1997). Molecular Cell 2005 19, 183-195DOI: (10.1016/j.molcel.2005.05.029) Copyright © 2005 Elsevier Inc. Terms and Conditions