Anna M. Fra

Developmental regulation of IgM secretion: The role of the carboxy-terminal cysteine

B lymphocytes do not secrete IgM, and plasma cells only secrete IgM polymers. Here we show that both events are attributable to the tailpiece found at the carboxyl terminus of mus chains, and we specifically implicate Cys-575. Thus, if Cys-575 was mutated, IgM was secreted by B cells. Similarly, a mutant IgG containing a mus tailpiece became largely retained within the cell; secretion was restored upon mutation of the tailpiece cysteine. Removal of Cys-575 also allowed hypersecretion of monomeric IgM by plasmacytoma cells. Following further removal of Cmu1, heavy chains were secreted in the absence of light chains. Thus, in B and plasma cells, Cys-575 is involved both in the polymerization of IgM and in intracellular retention of unpolymerized intermediates.

A photo-reactive derivative of ganglioside GM1 specifically cross-links VIP21-caveolin on the cell surface

Previous studies have shown that sphingolipids may be enriched in caveolae, plasmalemmal invaginations implicated in endocytosis and signal transduction. We synthesised a radiolabeled derivative of ganglioside GM1 bearing a photo‐reactive cross‐linker at the end of its acyl chain. After insertion in the plasma membrane of cultured A431 or MDCK cells and photoactivation, the main protein cross‐linked by the GM1 derivative was VIP21‐caveolin, an essential structural component of caveolae. This result shows close proximity between GM1 molecules and VIP21‐caveolin in the caveolar membrane and strongly implicates sphingolipid segregation in the biogenesis of caveolae.

Cutting Edge: Scavenging of Inflammatory CC Chemokines by the Promiscuous Putatively Silent Chemokine Receptor D6

In an effort to define the actual function of the promiscuous putatively silent chemokine receptor D6, transfectants were generated in different cell types. Engagement of D6 by inflammatory CC chemokines elicited no calcium response nor chemotaxis, but resulted in efficient agonist internalization and degradation. Also in lymphatic endothelium, where this receptor is expressed in vivo, D6 did not elicit cellular responses other than ligand internalization and degradation. In particular, no evidence was obtained for D6-mediated transcytosis of chemokines in the apical-to-basal or basal-to-apical directions. These results indicate that D6 acts as an inflammatory chemokine scavenging nonactivatory decoy receptors and suggest that in lymphatic vessels D6 may function as a gatekeeper for inflammatory CC chemokines, by clearing them and preventing excessive diffusion via afferent lymphatics to lymph nodes.

Quality control of ER synthesized proteins: an exposed thiol group as a three-way switch mediating assembly, retention and degradation.

Plasma cells secrete IgM only in the polymeric form: the C‐terminal cysteine of the mu heavy chain (Cys575) is responsible for both intracellular retention and assembly of IgM subunits. Polymerization is not quantitative, and part of IgM is degraded intracellularly. Neither chloroquine nor brefeldin A (BFA) inhibits degradation, suggesting that this process occurs in a pre‐Golgi compartment. Degradation of IgM assembly intermediates requires Cys575: the monomeric IgMala575 mutant is stable also when endoplasmic reticulum (ER) to Golgi transport is blocked by BFA. Addition of the 20 C‐terminal residues of mu to the lysosomal protease cathepsin D is sufficient to induce pre‐Golgi retention and degradation of the chimeric protein: the small amounts of molecules which exit from the ER are mostly covalent dimers. By contrast, when retained by the KDEL sequence, cathepsin D is stable in the ER, indicating that retention is not sufficient to cause degradation. Replacing the C‐terminal cysteine with serine restores transport through the Golgi. As all chimeric cathepsin D constructs display comparable protease activity in vitro, their different fates are not determined by gross alterations in folding. Thus, also out of its normal context, the mu chain Cys575 plays a crucial role in quality control, mediating assembly, retention and degradation.

Differential Recognition and Scavenging of Native and Truncated Macrophage-Derived Chemokine (Macrophage-Derived Chemokine/CC Chemokine Ligand 22) by the D6 Decoy Receptor

The promiscuous D6 receptor binds several inflammatory CC chemokines and has been recently proposed to act as a chemokine-scavenging decoy receptor. The present study was designed to better characterize the spectrum of CC chemokines scavenged by D6, focusing in particular on CCR4 ligands and analyzing the influence of NH2-terminal processing on recognition by this promiscuous receptor. Using D6 transfectants, it was found that D6 efficiently bound and scavenged most inflammatory CC chemokines (CCR1 through CCR5 agonists). Homeostatic CC chemokines (CCR6 and CCR7 agonists) were not recognized by D6. The CCR4 agonists CC chemokine ligand 17 (CCL17) and CCL22 bound to D6 with high affinity. CCL17 and CCL22 have no agonistic activity for D6 (chemotaxis and calcium fluxes), but were rapidly scavenged, resulting in reduced chemotactic activity on CCR4 transfectants. CD26 mediates NH2 terminus processing of CCL22, leading to the production of CCL22 (3–69) and CCL22 (5–69) that do not interact with CCR4. These NH2-terminal truncated forms of CCL22 were not recognized by D6. The results presented in this study show that D6 recognizes and scavenges a wide spectrum of inflammatory CC chemokines, including the CCR4 agonists CCL22 and CCL17. However, this promiscuous receptor is not engaged by CD26-processed, inactive, CCL22 variants. By recognizing intact CCL22, but not its truncated variants, D6 expressed on lymphatic endothelial cells may regulate the traffic of CCR4-expressing cells, such as dendritic cells.

Progressively impaired proteasomal capacity during terminal plasma cell differentiation

After few days of intense immunoglobulin (Ig) secretion, most plasma cells undergo apoptosis, thus ending the humoral immune response. We asked whether intrinsic factors link plasma cell lifespan to Ig secretion. Here we show that in the late phases of plasmacytic differentiation, when antibody production becomes maximal, proteasomal activity decreases. The excessive load for the reduced proteolytic capacity correlates with accumulation of polyubiquitinated proteins, stabilization of endogenous proteasomal substrates (including Xbp1s, IκBα, and Bax), onset of apoptosis, and sensitization to proteasome inhibitors (PI). These events can be reproduced by expressing Ig‐μ chain in nonlymphoid cells. Our results suggest that a developmental program links plasma cell death to protein production, and help explaining the peculiar sensitivity of normal and malignant plasma cells to PI.

Degradation of unassembled soluble Ig subunits by cytosolic proteasomes: evidence that retrotranslocation and degradation are coupled events

Many aberrant or unassembled proteins synthesized in the endoplasmic reticulum (ER) are degraded by cytosolic proteasomes. To investigate how soluble glycoproteins destined for degradation are retrotranslocated across the ER membrane, we analyzed the fate of two IgM subunits, μ and J, retained in the ER by myeloma cells that do not synthesize light chains. Degradation of μ and J is prevented by proteasome inhibitors, suggesting that both chains are retrotranslocated to be disposed of by proteasomes. Indeed, when proteasomes are inhibited, some deglycosylated J chains that no longer contain intrachain disulfide bonds accumulate in the cytosol. However, abundant glycosylated J chains are still present in the ER at time points in which degradation would have been almost complete in the absence of proteasome inhibitors, suggesting that retrotranslocation and degradation are coupled events. This was confirmed by protease protection and cell fractionation assays, which revealed that virtually all μ chains are retained in the ER lumen in a glycosylated state when proteasomes are inhibited. Association with calnexin correlated with the failure of μ chains to dislocate to the cytosol. Taken together, these results suggest that active proteasomes are required for the extraction of Ig subunits from the ER, though the requirements for retrotranslocation may differ among individual substrates.—Mancini, R., Fagioli, C., Fra, A. M,. Maggioni, C., Sitia, R. Degradation of unassembled soluble Ig subunits by cytosolic proteasomes: evidence that retrotranslocation and degradation are coupled events. FASEB J. 14, 769–778 (2000)

Generation and characterization of a mouse lymphatic endothelial cell line

Lymphatic vessels, by channeling fluid and leukocytes from the periphery into lymph nodes, play a central role in the development of the immune response. Despite their importance in homeostasis and disease, the difficulties in enriching and culturing lymphatic endothelial cells limit studies of their biology. Here, we report the isolation, stabilization, and characterization of a mouse lymphatic endothelial cell line (MELC) and the generated clones thereof. Cells were isolated from benign lymphangiomas induced by intraperitoneal injections of incomplete Freund’s adjuvant. The MELC line expressed molecules typical of lymphatic endothelium, including VEGFR3/Flt-4, podoplanin, Prox-1, and D6, but not LYVE-1. It also expressed CD34, ICAM-1, VCAM, and JAM-A, but not CD31, VE-cadherin, E-selectin, or CX3CL1/fractalkine (both TNFα-induced), at variance with vascular endothelial cells tested in parallel. The inflammatory cytokines TNFα and IL-4 regulated production of selected adhesion molecules (VCAM), cytokines (IL-6), and chemokines (CCL2/JE). Whole genome transcriptional profiling identified a set of 150 known genes differentially expressed in MELC versus vascular endothelial cells. Thus, the MELC line may represent an invaluable source of lymphatic endothelium.

CHOP-independent apoptosis and pathway-selective induction of the UPR in developing plasma cells

Upon antigen stimulation, B lymphocytes differentiate into antibody secreting cells (ASC), most of which undergo apoptosis after a few days of intense Ig production. Differentiation entails expansion of the endoplasmic reticulum (ER) and requires XBP1 but not other elements of the unfolded protein response, like PERK. Moreover, normal and malignant ASC are exquisitely sensitive to proteasome inhibitors, but the underlying mechanisms are poorly understood. Here we analyze the role of C/EBP homologous protein (CHOP), a transcription factor mediating apoptosis in many cell types that experience high levels of ER stress. CHOP is transiently induced early upon B cell stimulation: covalent IgM aggregates form more readily and IgM secretion is slower in chop(-/-) cells. Despite these subtle changes, ASC differentiation and lifespan are normal in chop(-/-) mice. Unlike fibroblasts and other cell types, chop(-/-) ASC are equally or slightly more sensitive to proteasome inhibitors and ER stressors, implying tissue-specific roles for CHOP in differentiation and stress.

Three New Alpha1-Antitrypsin Deficiency Variants Help to Define a C-Terminal Region Regulating Conformational Change and Polymerization

Alpha1-antitrypsin (AAT) deficiency is a hereditary disorder associated with reduced AAT plasma levels, predisposing adults to pulmonary emphysema. The most common genetic AAT variants found in patients are the mildly deficient S and the severely deficient Z alleles, but several other pathogenic rare alleles have been reported. While the plasma AAT deficiency is a common trait of the disease, only a few AAT variants, including the prototypic Z AAT and some rare variants, form cytotoxic polymers in the endoplasmic reticulum of hepatocytes and predispose to liver disease. Here we report the identification of three new rare AAT variants associated to reduced plasma levels and characterize their molecular behaviour in cellular models. The variants, called Mpisa (Lys259Ile), Etaurisano (Lys368Glu) and Yorzinuovi (Pro391His), showed reduced secretion compared to control M AAT, and accumulated to different extents in the cells as ordered polymeric structures resembling those formed by the Z variant. Structural analysis of the mutations showed that they may facilitate polymerization both by loosening ‘latch’ interactions constraining the AAT reactive loop and through effects on core packing. In conclusion, the new AAT deficiency variants, besides increasing the risk of lung disease, may predispose to liver disease, particularly if associated with the common Z variant. The new mutations cluster structurally, thus defining a region of the AAT molecule critical for regulating its conformational state.