Roles of N-glycans in the polymerization-dependent aggregation of mutant Ig-μ chains in the early secretory pathway

The polymeric structure of secretory IgM allows efficient antigen binding and complement fixation. The available structural models place the N-glycans bound to asparagines 402 and 563 of Ig-μ chains within a densely packed core of native IgM. These glycans are found in the high mannose state also in secreted IgM, suggesting that polymerization hinders them to Golgi processing enzymes. Their absence alters polymerization. Here we investigate their role following the fate of aggregation-prone mutant μ chains lacking the Cμ1 domain (μ∆). Our data reveal that μ∆ lacking 563 glycans (μ∆5) form larger intracellular aggregates than μ∆ and are not secreted. Like μ∆, they sequester ERGIC-53, a lectin previously shown to promote polymerization. In contrast, μ∆ lacking 402 glycans (μ∆4) remain detergent soluble and accumulate in the ER, as does a double mutant devoid of both (μ∆4–5). These results suggest that the two C-terminal Ig-μ glycans shape the polymerization-dependent aggregation by engaging lectins and acting as spacers in the alignment of individual IgM subunits in native polymers.

Over one third of the proteome starts folding in the endoplasmic reticulum (ER) 1,2 . The ER teams up with the Golgi and Intermediate Compartment to form a functional unit -the early secretory pathway (ESP)-acting coordinately to couple fidelity and efficiency of protein secretion. Key players are resident ESP chaperones and enzymes that favour and time glycoprotein quality control and transport 3 . Despite the existence of sophisticated proteostatic systems, however, mutations, lack of folding assistants or the unbalanced production of different subunits can generate conditions in which proteins that enter ESP (synthesis and translocation) exceed those exiting from it (secretion and/or degradation), causing traffic jams as in ER Storage Disorders (ERSD) 4 .
Secretory IgM are complex molecules, whose assembly occurs stepwise in the secretory pathway. The first step requires the formation of μ 2 L 2 "monomers" (Fig. 1), covalently linked by inter-chain disulfide bonds. These rapidly assemble in the ER. μ 2 L 2 that pass the BiP-dependent checkpoints must then form covalent polymers to negotiate secretion 1,4 . In the absence of Ig-J chains, hexamers are formed 5 , in which six monomers are bound via homotypic covalent bonds between cysteines 414 and 575 (Fig. 1). The addition and processing of N-glycans is important for IgM biogenesis and quality control. Ig-μ chains contain 5 N-glycans (171, 332, 395, 402 and 563). While the first three are found in a processed state, N402 and N563 are modified by high-mannose sugars in secreted IgM [6][7][8] , suggesting that they remain hidden to the glycan processing enzymes as polymers travel through the secretory pathway 9 . Exposure of high-mannose moieties upon antigen binding could be important for the clearance of serum immune complexes 7 .
For polymerization to take place, intra-subunit bonds ought to be prevented. At the same time, μ 2 L 2 subunits should be aligned to form circular polymers of limited size. Previous studies in reconstituted HeLa cells pointed at ERGIC-53, a hexameric lectin that assists ER-Golgi transport of selected glycoproteins 10 , as a platform for IgM polymerization 11 . Moreover, Ig-μ lacking N563 glycans were shown to form higher order polymers devoid of J chains 12 , suggesting that binding to hexameric ERGIC-53 may favour the closure of planar pentamers with a J chain or hexamers. However, since N563 oligosaccharides become inaccessible upon polymerization 9 , they Scientific RepoRts | 7:41815 | DOI: 10.1038/srep41815 may also act as spacers limiting the number of subunits that can be incorporated into a polymer. Conversely, the absence of N402 glycans inhibits polymerization 12,13 .
Owing to the high mutation rate of immunoglobulins and their abundant production by cells of the B lineage, transport-incompetent variants often accumulate in dilated ESP cisternae, called Russell Bodies (RB) 14 , particularly in Mott myelomas and other plasma cell dyscrasias [15][16][17] . Over the last years, we developed RB models based on the inducible expression of mutant Ig-μ chains lacking the first constant domain (μ ∆ ) 18,19 . In all Ig classes, CH1 domains mediate the association with Ig-L chains. In the absence of L, they bind the ER chaperone BiP 20 . Unassembled H chains are secreted in Heavy Chain Diseases, because they lack CH1 and escape BiP-dependent quality control. HCD can cause kidney damage 21 because CH1 deletion facilitates aggregation 22 .
Since μ ∆ variants that cannot polymerize (e.g. μ ∆ C575A) do not form RB and are secreted 14 , aggregation depends on polymerization. Accordingly, factors that impact polymerization, e.g. Ero1 or ERp44, modulate RB biogenesis 19 . Also elements working in cis play a role, including the N563 and N402 glycans, which are located 12 residues upstream the two cysteines involved in polymerization (C414 and C575 respectively). Since formation of disulphide bonds and polymerization are needed for μ ∆ aggregation, we set up to investigate the role of the N402 and N563 glycans in the formation of detergent-insoluble μ ∆ deposits. Our results show that their absence inhibits or favours aggregation, respectively. Mutants lacking both remain soluble, suggesting that the 402 glycans favour the accessibility of C575 and C414 and ultimately polymerization, possibly binding ERGIC-53. The 563 glycan could limit the number of μ ∆ 2 or μ 2 L 2 subunits that can be incorporated into planar polymers.

Results and Discussion
Deletion of the N563 glycan favours aggregation and prevents secretion of mutant Ig-μ lacking the Cμ1 domain (μ∆). The absence of the Cμ 1 domain, the main interactor with the chaperone BiP 20 , increases the tendency of Ig-μ chains to form detergent-insoluble intracellular deposits 14,18 , a phenomenon hereafter referred to as aggregation. In the absence of L chains, μ ∆ accumulate mainly in ribosome-free cisternae stained by ERGIC-53, called smooth Russell Bodies (sRB). Since disulfide bonding of different μ ∆ 2 dimers via C575 is crucial for aggregation, these findings support the notion that ERGIC-53 can promote IgM polymerization 11,23 . Rough RB (rRB) form instead upon assembly with L chains 18 . In the absence of a Cμ 1 to dock to, the tendency of the Ig-L constant domain (C L ) to form homodimers might promote interactions amongst μ ∆ 2 complexes in the ER, favouring inter-C575 bonding 18 .
When serine 565 is replaced by alanine to destroy the C-terminal NVS glycon, higher order IgM polymers are rapidly formed 9,12,13 . We thus hypothesized that a double mutant lacking both the Cμ 1 domain and the 563 glycan (μ ∆ 5) would show stronger tendency to aggregate. To test this prediction, we expressed μ ∆ or μ ∆ 5 in HeLa cells and analyzed their distribution. As expected, much larger amounts of the μ ∆ 5 double mutant accumulated than μ ∆ in the detergent-insoluble fraction: furthermore, aggregates contained higher molecular weight covalent complexes ( Fig. 2A).
Part of μ ∆ chains are secreted by the HeLa cells used in these experiments, either as soluble μ ∆ 2 homodimers or as detergent-insoluble high molecular weight complexes that stick to the culture dishes and can be readily stained by immunofluorescence 19 (see also Fig. 2D below). These complexes contain Endo-H sensitive 402 and 563 glycans, suggesting that they traversed the Golgi in the polymeric state ref. 19 and our unpublished data. Secretion via a Golgi-independent route is less likely, because the 171, 332, 395 glycans are Endo-H resistant.  with increasing amounts of plasmids encoding for μ Δ or μ Δ 5, as indicated, were loaded on SDS-page and decorated with anti-μ antibodies. The percentage of secreted μ relative to the total intracellular amount was determined by densitometric quantification and is shown in the right panel. (C) HeLa cells co-expressing μ Δ 5 and Ig-λ chains were fixed with PFA and stained with anti-μ (red) and anti-idiotypic antibodies (AC38, green). Co-localization of the AC38 (which recognizes only properly paired μ λ complexes) and anti-μ staining confirms that μ Δ 5 assembles with λ chains (bar: 15 μ m). (D) HeLa transfectants expressing μ ∆ or μ ∆ 5 were fixed with PFA, stained with anti-μ Alexa 488 antibodies and visualized with deconvolution microscopy (left panels, bar: 15 μ m). Immunogold analyses (middle and right panels) confirmed that the electron dense material contains condensed μ chains (see arrows) (bar: 500 nm). The enlargements shown in the insets confirm that μ ∆ 5-containing SupeRB are bigger than μ ∆ -containing sRB. Their diameters (+ /− standard deviation) were calculated as the average of 60 such structures analyzed. Only minute amounts of μ Δ 5 are released extracellularly. The fraction of secreted μ ∆ was higher than μ ∆ 5 at all the expression levels tested (Fig. 2B), hence excluding saturation of the retention mechanisms as the sole cause of μ ∆ secretion. These results suggest that μ Δ 5 condense more rapidly and form deposits that cannot be transported further along ESP. When coexpressed with murine Ig-λ , μ Δ 5 chains react with NP hapten or Ac38 anti-idiotype antibodies (Fig. 2C), confirming proper VH folding and pairing with Vλ . Moreover, in the context of wild-type Ig-μ the A565S mutation does not prevent secretion of hapten binding, Ac38 + higher molecular weight polymers, suggesting that the absence of this glycan does not induce gross protein unfolding. Rapid condensation could thus lead to the formation of transport-incompetent large complexes.
Accordingly, immunofluorescence analyses revealed that μ ∆ 5 accumulate in roundish vesicles bigger than sRB (Fig. 2D). This behaviour was not cell specific, as similar detergent solubility and subcellular distribution were obtained in HepG2 or Hek293T transfectants (data not shown). Electron-microscopy analyses revealed that μ ∆ 5 accumulated in deposits with an average diameter larger than what observed for sRB (394 ± 104 vs 268 ± 52 nm), hence the name SupeRB (Fig. 2D). Notably, few if any ribosomes decorated the membrane of SupeRB. Immuno-electron microscopy with gold-coupled anti-μ confirmed the presence of mutant μ chains in both sRB and SupeRB (see arrows).
Thus, preventing the attachment of the most C-terminal N-glycan, which accelerates IgM polymerization, increases the accumulation of μ ∆ chains into detergent-insoluble, high molecular weight covalent complexes that deposit in ESP vesicles and are retained intracellularly.
Since the binding between ERGIC-53 and its glycoprotein ligands is calcium-dependent 27 , treatment with a reversible SERCA inhibitor (cyclopiazonic acid, CPA) weakens lectin-dependent interactions 13,18 . Accordingly, ERGIC-53 regained its normal localization upon CPA treatment, even if μ ∆ or μ ∆ 5 aggregates remained in place (Fig. 4A). ERGIC-53 associated again with SupeRB upon CPA removal, as previously described for μ ∆ 18 . The co-localization of ERGIC-53 with μ ∆ and μ ∆ 5 could reflect its binding to detergent-insoluble deposits. Whatever their origin, sRB and SupeRB seem to remain in communication with the mainstream secretory pathway: ERGIC-53 can diffuse into them when the affinity of its lectin domains for μ ∆ or μ ∆ 5 glycans is higher than the affinity for the cytosolic molecules that normally drive its subcellular localization. Considering the sensitivity of intracellular μ ∆ and μ ∆ 5 to Endo-H 19 , sRB and SupeRB seem to originate from the aggregation of soluble cargo molecules between the ER and the Golgi.
Mannosidase l-dependent trimming of the N563 glycan promotes μ∆ aggregation. We previously showed that kifunensine, an inhibitor of mannosidase I, an enzyme that removes the terminal mannose from the B branch in N-glycans 28 , prevented the aggregation of μ ∆ chains in both HeLa and plasma cells 18 . This result suggested a role for a kifunensine-sensitive factor in promoting aggregation, especially considering that the drug would increase the concentration of μ ∆ and μ ∆ 5 in ESP 29,30 by preventing their degradation. One such factor could be ERGIC-53 11,31 . Unexpectedly, kifunensine had only minor effects on μ Δ 5 aggregation (Fig. 4B) compared to μ ∆ , a much larger fraction of μ Δ 5 becoming detergent-insoluble also under kifunensine treatment. Moreover, in the presence of kifunensine ERGIC-53 lost its co-localization with μ Δ , but not with μ Δ 5 (Fig. 4C), implying the existence of other direct or indirect interactions that are insensitive to kifunensine.
Removal of the N402 glycan prevents polymerization and aggregation. The higher avidity and larger size of the aggregates formed by μ ∆ 5 could compensate for the lower affinity of non-processed 402 glycans in recruiting ERGIC-53. To establish whether this was the case, we replaced N402 for glutamine in either μ ∆ or μ Δ 5, to generate the μ Δ 4 and μ Δ 4-5 mutants, respectively. Immunofluorescence assays demonstrated that removal of the 402 glycan almost completely prevents aggregation. Both μ Δ 4 and μ Δ 4-5 yielded a reticular staining pattern (Fig. 5A) overlapping with ER markers (not shown). Interestingly, ERGIC-53 is still recruited by μ Δ 4 but not by μ Δ 4-5 (Fig. 5B), indicating that either sugar can be recognized singularly by the lectin. Biochemical analyses confirmed that, while μ Δ and μ Δ 5 accumulated abundantly in the non-soluble fraction, mutants lacking 402 glycans formed few HMW species and remained soluble (Fig. 5A). Taken together, these findings confirm that in vivo the presence of μ 4 sugars is important for efficient IgM polymerization 13 and s(upe)RB formation.
The molecular weight shifts clearly detectable in western blot analyses (Supplementary Figure 2) confirmed that kifunensine inhibited mannose trimming. The mobility differences were attenuated in μ Δ 5, μ Δ 4 and above all μ Δ 4-5 (Supplementary Figure 2). As previously noted in our imaging analyses, kifunensine had little if any effect on the aggregation of μ Δ 5. Slightly more μ Δ 4 accumulated in the insoluble fraction upon mannosidase I inhibition: a possible explanation is that under these conditions degradation of this mutant is partly inhibited. However, kifunensine had no effect on the distribution of μ ∆ 4-5 between the soluble and non-soluble fractions.
Concluding remarks. So far, no crystallographic data are available for polymeric IgM. Current models predict a mushroom shape with tightly packed Cμ 3 and Cμ 4 domains. Upon antigen binding, these undergo conformational changes, allowing efficient complement fixation 32,33 . Since μ ∆ aggregation and polymerization are faces of the same coin, our experiments confirm that the two C-terminal N-glycans (402 and 563) are crucial for IgM biogenesis. They can act in at least two non-alternative ways. Firstly, by engaging ERGIC-53, or additional lectins present in differentiating B cells, so as to time and shape compaction of the mushroom stem. Secondly, they could act as spacers, N402 facilitating the exposure of C414 and C575 and hence polymer formation, N563 limiting instead the number of subunits that can be inserted into planar polymers (Fig. 6). Accordingly, the absence of the 402 glycan is dominant on CH1 deletion, preventing μ ∆ aggregation. Replacing the Cμ 1 with different tags (GFP, RFP, Halo) yielded different aggregation and localization patterns (our unpublished results) suggesting that additional factors are in play to assist polymerization in cis as well as in trans. Considering the biotechnological relevance of a portable polymerization module, further experiments are needed to dissect the intrinsic and extrinsic factors that control IgM biogenesis.

Materials and Methods
Cells, plasmids and reagents. Unless otherwise indicated, chemicals were from Sigma Chemical Co (ST. Louis, MO). HeLa, HepG2 and Hek293 cells were obtained from ATCC, Hela-off from Clonthech, and cultured in DMEM (GIBCO Life Technologies) containing 2 mM glutamine and 5% FCS (GIBCO Life Technologies).
Rabbit polyclonal anti-calreticulin and anti-ERGIC-53 were purchased from SIGMA Aldrich; goat anti-mouse (IgM) μ -chain antibodies (Alexa 546, 647, 680 and 700) were purchased from Invitrogen; mouse anti-GM130 from BD Transduction Laboratories; the monoclonal AC38 antibody was previously described 14,34 . Rabbit ant-p115, rabbit anti-Sec31 and mouse monoclonal anti-ERGIC53 antibodies were kind gifts from Drs. De Matteis (TIGEM, Naples, IT), Hong (Institute of Molecular and Cell Biology, Singapore) Appenzeller-Herzog and Hauri (Biozentrum, University of Basel, Switzerland). Cells were transfected using calcium phosphate or polyethylene imine (PEI, Polysciences, Inc.) or silenced with specific siRNAs targeted by Lipofectamine RNAimax (Invitrogen, Eugene, Oregon, USA), following the manufacturers' instructions. ERp44 duplexes sequences were previously described 19 . Immunofluorescence. Cells were cultured and transfected on glass coverslips. 48 hours after transfection, cells were fixed with 4% paraformaldehyde for 10 min at RT, permeabilized with PBS containing 0.1% Tx100 for 5 min at RT, washed in PBS, saturated with 2% FCS and then stained with the indicated primary and secondary antibody as described 19 . Slides were mounted in 90% glycerol and images acquired with an Olympus inverted fluorescence microscope (model IX70) with DeltaVision RT Deconvolution System (Alembic, HSR). Deconvoluted images were processed with Adobe Photoshop 7.0 (Adobe Systems Inc.). In other cases, images were taken with a Leica TCS SP2 laser Scanning Confocal Microscope.
Electron microscopy. For cryo-electron microscopy, HeLa cells expressing μ Δ or μ Δ 5 were fixed for 1 hour at room temperature (0.2% glutaraldehyde/2% paraformaldehyde in cacodylate Buffer 0.1 M) and processed as described 35 . Briefly, samples were embedded in 12% gelatin, infiltrated in 2.3 M sucrose and frozen in liquid nitrogen. Cryosections were obtained using a Leica EM FC7 ultramicrotome (Leica microsystem, Vienna, Austria) and collected on 150 mesh formvar carbon coated copper grids. Grids were then incubated with 0.1 μ g/μ l rabbit anti-μ (Zymed Laboratories, San Francisco, CA) followed by goat anti-rabbit IgG coupled to 15 nm gold beads.
Grids were contrasted in a solution of uranyl acetate and methylcellulose, air-dried and observed in a Leo 912AB transmission electron microscope (Carl Zeiss, Oberkochen, Germany).
Images were analysed with ImageJ in order to determine the size of the μ -containing vesicles. At least two perpendicular measurements were performed for each structure; 60 structures were analysed for each sample and the diameter averaged.
Cell Lysis and Western Blotting. 48 hours after transfection, cells were washed and lysed at the concentration of 1 × 10 4 cells/μ l in buffer A (0.2% Tx100, 50 mM Tris-HCl pH 7.5), 150 mM NaCl, 5 mM EDTA, 10 mM N-ethylmaleimide and a cocktail of protease inhibitors (Roche, San Francisco, CA, USA). The Tx100-insoluble fraction (insol) was separated by centrifugation at 3,400 g for 10 minutes and solubilized in lysis buffer B (1% SDS, 50 mM Tris-HCl pH 7.5, 10 mM NEM) for 10 minutes at RT, diluted in 50 mM Tris-HCl pH 7.5, 0.2% Tx100, to keep the volume of the soluble and insoluble fractions equal, and sonicated for 10 seconds. In order to collect the secreted material, 48 hours after transfection, cells were washed three times with PBS and incubated for 4 hours in pre-warmed OPTIMEM. After 4 hours, cell culture supernatant was collected (SN), cells were detached from the plate with PBS containing 10 mM EDTA. The secreted material attached to the plate (plate) was then scraped from the plate in 2% SDS, 0.1 M Tris pH 7.4, 10 mM N-ethylmaleimide and a cocktail of protease inhibitors (Roche, San Francisco, CA, USA). Samples were resolved under reducing or non-reducing conditions by pre-casted 10% Interactions with ERGIC-53 favour and control IgM polymerization, the N563-glycan (blue circles) and N402 (red circles) being the main binding sites in μ chains. (B) Without the N402 glycan, μ 2 L 2 subunits may adopt a closed, transport-incompetent conformation, C575 becoming inaccessible for polymerization. (C) In the absence of the N563 glycan, high molecular weight polymers are formed, suggesting that this sugar acts as a spacer between adjacent subunits or/and that the interaction with ERGIC-53 only via N402 can cause aberrant polymerization.