Malectin participates in a backup glycoprotein quality control pathway in the mammalian ER - PubMed (original) (raw)
Malectin participates in a backup glycoprotein quality control pathway in the mammalian ER
Carmela Galli et al. PLoS One. 2011.
Abstract
Malectin is a conserved, endoplasmic reticulum (ER)-resident lectin that recognizes high mannose oligosaccharides displaying terminal glucose residues. Here we show that Malectin is an ER stress-induced protein that selectively associates with glycopolypeptides without affecting their entry and their retention in the Calnexin chaperone system. Analysis of the obligate Calnexin client influenza virus hemagglutinin (HA) revealed that Calnexin and Malectin associated with different timing to different HA conformers and that Malectin associated with misfolded HA. Analysis of the facultative Calnexin clients NHK and α1-antitrypsin (α1AT) revealed that induction of Malectin expression to simulate conditions of ER stress resulted in persistent association between the ER lectin and the model cargo glycoproteins, interfered with processing of cargo-linked oligosaccharides and reduced cargo secretion. We propose that Malectin intervention is activated upon ER stress to inhibit secretion of defective gene products that might be generated under conditions of aberrant functioning of the ER quality control machinery.
Conflict of interest statement
Competing Interests: The authors have declared that no competing interests exist.
Figures
Figure 1. Subcellular localization, topology and ER stress induction of Malectin.
A The asparagine-linked core oligosaccharide is composed of two N-acetylglucosamine (squares), nine mannose (circles) and three glucose residues (triangles). B Ectopically expressed Malectin-HA was visualized with an anti-HA antibody (left panel), Calnexin with a polyclonal antibody recognizing the endogenous protein (right panel). C Luminal vs membrane localization of ER chaperones was assessed after carbonate extraction of microsomes. D Intact microsomes were incubated with or without (Mock) trypsin to remove cytosolic portions of the labeled polypeptide chains. Calnexin ΔC lacks the 90 cytosolic residues. Molecular weight markers (200, 116, 97, 66, 45, 30 kDa) are shown on the right. E Human Malectin comprises 292 residues. The signal sequence and the transmembrane anchor are in bold, the putative N-glycosylation site is underlined. F Thapsigargin (Tg) triggers ER stress (uXBP1 and sXBP1 are unspliced and spliced XBP1 transcripts, respectively). Fold-induction of BiP, Grp94, EDEM1 and Malectin transcripts as determined by real time PCR (in triplicate). Actin served as negative control.
Figure 2. Consequences of Malectin overexpression on HA maturation.
A Schematics showing the cysteine residues covalently linked in the native HA. B Influenza virus HA was immunoisolated from detergent lysates of influenza virus-infected cells with normal (lanes 1–3) or elevated levels of Malectin (stably transfected HEK293Mal cells, lanes 4–6). The folding intermediates IT1, IT2 and NT are shown. The viral, non-glycosylated nuclear protein (NP) is also recognized by the antibody used. Molecular weight markers (200, 116, 97, 66 kDa) are shown on the right. C Assessment of EndoH-sensitivity of oligosaccharides displayed on HA in HEK293 (lanes 1–6) and in HEK293Mal cells (lanes 7–12). Res, EndoH-resistant HA; Sens, EndoH-sensitive HA. The % of EndoH-resistant HA (Res in the upper panel) has been quantified. This gel is representative of a series of experiments in which HA maturation was monitored at different chase times. D Release of HA from Calnexin in HEK293 (lanes 1–3 (non reducing gel); lanes 7–9 (reducing gel)) and in HEK293Mal cells (lanes 4–6 (non reducing); lanes 10–12 (reducing)). Calnexin and associated substrates have been immunoisolated from the lysate of 200'000 cells. Lane αHA shows the ratio of IT1, IT2, NT in infected cells after 2 min chase and the mobility of NP, which does not co-immunoprecipitate with Cnx (specificity control). Viral proteins in the lane αHA have been immunoisolated from the lysate of 20'000 cells. Molecular weight markers (116, 97, 66 kDa) are shown on the right. E Same as D for endogenous Calnexin substrates. Select polypeptides are shown with A–I. F Same as D for ectopically expressed NHK. G Same as D for ectopically expressed α1AT. H Same as D for Malectin-associated HA. The specificity of the anti-Malectin immunoprecipitation is confirmed by the low cross reactivity in infected cells that did not contain ectopically expressed Malectin (lanes 1–3) and by the lack of co-precipitation of the non-glycosylated NP (lane 1) with Malectin (lanes 4–6). Quantitations of HA release from Calnexin (upper panel, reducing gel, Fig. 2D)) and from Malectin (lower panel, gel in Fig. 2H) are shown. Ectopically expressed Malectin-HA and the associated influenza virus HA have been immunoisolated from detergent extracts with an antibody to the HA-tag sequence (YPYDVPDYA). This sequence is not present in the X-31 influenza virus HA (as confirmed by the lack of influenza virus protein in the immunoisolates of cells not expressing Malectin-HA, lanes 1–3). I Same as C for the labelled HA immunoisolated from cells after 20 min chase (lanes 1–2) and for Malectin-associated HA (lanes 3–4) after 20 min chase. K HA immunoisolated after 2 min chase from cells in the absence (Mock, lane 1) or in the presence of 1 mM Castanospermine (Cst, lane 4). The same for Calnexin-associated HA (Mock, lane 2; Cst, lane 5). The same for Malectin-associated HA (Mock, lane 3; Cst, lane 6). The analysis was also performed for cells solubilised after 20 min chase (lanes 7–12). Since Cst inhibits removal of glucose residues from HA-bound oligosaccharides, HA has slower electrophoretic mobility in lanes 4–6 and 10–12. The experiments shown in panels I–K have been performed in HEK293Mal cells.
Figure 3. Malectin overexpression interferes with oligosaccharide processing and cargo protein secretion.
A Intracellular, labelled NHK in HEK293 cells (lanes 1–3) and in HEK293 cells co-transfected with a plasmid for expression of Malectin-HA (lanes 4–6). Malectin co-precipitates with NHK throughout the chase (lanes 4–6). Molecular weight markers (116, 97, 66, 45, 30 kDa) are shown on the right. Quantitations in panels A, B, E, G are for at least two independent experiments (error bars represent SD). Data have also been reproduced in other cell lines (CHO cells in panel D, HeLa cells transiently transfected for Malectin overexpression and HEK293Mal, not shown). B Same as A for secreted NHK. C Assessment of EndoH-sensitivity of oligosaccharides displayed on NHK secreted from HEK293 cells (lanes 1–2) or from HEK293 cells co-transfected for expression of Malectin-HA (lanes 3–4). The arrow shows the fraction of labelled NHK displaying EndoH-sensitive oligosaccharides in lane 4. D Same as C for NHK secreted from CHO cells. E–G Same as A–C for α1AT. Molecular weight markers (116, 97, 66, 45 kDa) are shown on the right.
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