Birbeck granule-like "organized smooth endoplasmic reticulum" resulting from the expression of a cytoplasmic YFP-tagged langerin.

Langerin is required for the biogenesis of Birbeck granules (BGs), the characteristic organelles of Langerhans cells. We previously used a Langerin-YFP fusion protein having a C-terminal luminal YFP tag to dynamically decipher the molecular and cellular processes which accompany the traffic of Langerin. In order to elucidate the interactions of Langerin with its trafficking effectors and their structural impact on the biogenesis of BGs, we generated a YFP-Langerin chimera with an N-terminal, cytosolic YFP tag. This latter fusion protein induced the formation of YFP-positive large puncta. Live cell imaging coupled to a fluorescence recovery after photobleaching approach showed that this coalescence of proteins in newly formed compartments was static. In contrast, the YFP-positive structures present in the pericentriolar region of cells expressing Langerin-YFP chimera, displayed fluorescent recovery characteristics compatible with active membrane exchanges. Using correlative light-electron microscopy we showed that the coalescent structures represented highly organized stacks of membranes with a pentalaminar architecture typical of BGs. Continuities between these organelles and the rough endoplasmic reticulum allowed us to identify the stacks of membranes as a form of "Organized Smooth Endoplasmic Reticulum" (OSER), with distinct molecular and physiological properties. The involvement of homotypic interactions between cytoplasmic YFP molecules was demonstrated using an A206K variant of YFP, which restored most of the Langerin traffic and BG characteristics observed in Langerhans cells. Mutation of the carbohydrate recognition domain also blocked the formation of OSER. Hence, a "double-lock" mechanism governs the behavior of YFP-Langerin, where asymmetric homodimerization of the YFP tag and homotypic interactions between the lectin domains of Langerin molecules participate in its retention and the subsequent formation of BG-like OSER. These observations confirm that BG-like structures appear wherever Langerin accumulates and confirm that membrane trafficking effectors dictate their physiology and, illustrate the importance of molecular interactions in the architecture of intracellular membranes.

HLA-DQA2 and HLA-DQB2 genes are specifically expressed in human Langerhans cells and encode a new HLA class II molecule.

The precise role of human epidermal Langerhans cells (LCs) in immune response is highly controversial. While studying the gene expression profile of these cells, we were intrigued to identify the HLA-DQB2 gene as potentially expressed in LCs. Despite a strong evolutionary conservation of their sequences, the concomitant expression of the poorly polymorphic HLA-DQA2/HLA-DQB2 genes, paralogous to the HLA-DQA1/HLA-DQB1 genes, has never been detected in any cell type. We confirmed by RT-PCR that the HLA-DQA2 and -DQB2 genes are both expressed in LCs, but not in monocyte-derived dendritic cells, or in blood CD1c(+) or plasmacytoid dendritic cells. The presence of the HLA-DQß2 chain in LCs could be demonstrated by Western blotting, whereas immunofluorescence revealed its localization in early endosomes. As in the case of other HLA class II molecules, the HLA-DQα2 and -DQß2 chains formed heterodimers that had to associate with the invariant chain to reach endosomal compartments. HLA-DQα2/ß2 heterodimers were expressed at the cell surface, where they could mediate staphylococcal superantigen stimulation of T cells. Interestingly, HLA-DQα2 and HLA-DQß1 chains formed mixed heterodimers which efficiently left the endoplasmic reticulum. These observations strongly suggest that the poorly polymorphic HLA-DQA2 and -DQB2 genes should be considered to be of immunological importance. The HLA-DQα2/ß2 molecules could influence the complexity of the repertoire of Ags presented by LCs.

Increased flexibility and liposome-binding capacity of CD1e at endosomal pH.

The plasma membrane proteins CD1a, CD1b and CD1c are expressed by human dendritic cells, the professional antigen-presenting cells of the immune system, and present lipid antigens to T lymphocytes. CD1e belongs to the same family of molecules, but accumulates as a membrane-associated form in the Golgi compartments of immature dendritic cells and as a soluble cleaved form in the lysosomes of mature dendritic cells. In lysosomes, the N-terminal propeptide of CD1e is also cleaved, but the functional consequences of this step are unknown. Here, we investigated how the pH changes encountered during transport to lysosomes affect the structure of CD1e and its ligand-binding properties. Circular dichroism studies demonstrated that the secondary and tertiary structures of recombinant CD1e were barely altered by pH changes. Nevertheless, at acidic pH, guanidium chloride-induced unfolding of CD1e molecules required lower concentrations of denaturing agent. The nonfunctional L194P allelic variant was found to be structurally less stable at acidic pH than the functional forms, providing an explanation for the lack of its detection in lysosomes. The number of water-exposed hydrophobic patches that bind 8-anilinonaphthalene-1-sulfonate was higher in acidic conditions, especially for the L194P variant. CD1e molecules interacted with lipid surfaces enriched in anionic lipids, such as bis(monoacylglycero)phosphate, a late endosomal/lysosomal lipid, especially at acidic pH, or when the propeptide was present. Altogether, these data indicate that, in the late endosomes/lysosomes of DCs, the acid pH promotes the binding of lipid antigens to CD1e through increased hydrophobic and ionic interactions.

Cutting edge: a naturally occurring mutation in CD1e impairs lipid antigen presentation.

The human CD1a-d proteins are plasma membrane molecules involved in the presentation of lipid Ags to T cells. In contrast, CD1e is an intracellular protein present in a soluble form in late endosomes or lysosomes and is essential for the processing of complex glycolipid Ags such as hexamannosylated phosphatidyl-myo-inositol, PIM(6). CD1e is formed by the association of beta(2)-microglobulin with an alpha-chain encoded by a polymorphic gene. We report here that one variant of CD1e with a proline at position 194, encoded by allele 4, does not assist PIM(6) presentation to CD1b-restricted specific T cells. The immunological incompetence of this CD1e variant is mainly due to inefficient assembly and poor transport of this molecule to late endosomal compartments. Although the allele 4 of CD1E is not frequent in the population, our findings suggest that homozygous individuals might display an altered immune response to complex glycolipid Ags.

Control of the intracellular pathway of CD1e.

CD1e is a membrane-associated protein predominantly detected in the Golgi compartments of immature human dendritic cells. Without transiting through the plasma membrane, it is targeted to lysosomes (Ls) where it remains as a cleaved and soluble form and participates in the processing of glycolipidic antigens. The role of the cytoplasmic tail of CD1e in the control of its intracellular pathway was studied. Experiments with chimeric molecules demonstrated that the cytoplasmic domain determines a cellular pathway that conditions the endosomal cleavage of these molecules. Other experiments showed that the C-terminal half of the cytoplasmic tail mediates the accumulation of CD1e in Golgi compartments. The cytoplasmic domain of CD1e undergoes monoubiquitinations, and its ubiquitination profile is maintained when its N- or C-terminal half is deleted. Replacement of the eight cytoplasmic lysines by arginines results in a marked accumulation of CD1e in trans Golgi network 46+ compartments, its expression on the plasma membrane and a moderate slowing of its transport to Ls. Fusion of this mutated form with ubiquitin abolishes the accumulation of CD1e molecules in the Golgi compartments and restores the kinetics of their transport to Ls. Thus, ubiquitination of CD1e appears to trigger its exit from Golgi compartments and its transport to endosomes. This ubiquitin-dependent pathway may explain several features of the very particular intracellular traffic of CD1e in dendritic cells compared with other CD1 molecules.

Mutations on the FG surface loop of human papillomavirus type 16 major capsid protein affect recognition by both type-specific neutralizing antibodies and cross-reactive antibodies.

The aim of this study was to further characterize the conformational neutralizing epitopes present on the surface-exposed FG loop of human papillomavirus (HPV) type 16 L1 major capsid protein. We have generated previously two chimeric L1 proteins by insertion of a foreign peptide encoding an epitope of the hepatitis B core (HBc) antigen within the FG loop. In addition, three other chimeric L1 proteins were obtained by replacing three different FG loop sequences by the HBc motif and three others by point mutations. All these chimeric L1 proteins retained the ability to self-assemble into virus-like particles (VLPs), with the exception of the mutant with substitution of the L1 sequence 274-279 by the HBc motif. The eight chimeric VLPs were then analyzed for differential reactivity with a set of six HPV-16 and HPV-31 monoclonal antibodies that bound to conformational and linear epitopes. The binding patterns of these monoclonal antibodies confirmed that the FG loop contained or contributed to neutralizing conformational epitopes. The results obtained suggested that the H31.F7 antibody, an anti-HPV-31 cross-reacting and neutralizing antibody, recognized a conformational epitope situated before the 266-271 sequence. In addition, H16.E70 neutralizing antibody reactivity was reduced with L1 VLPs with an Asn to Ala point mutation at position 270, suggesting that Asn is a part of the epitope recognized by this antibody. This study contributes to the understanding of the antigenic structure of HPV-16 and -31 L1 proteins by confirming that the FG loop contributes to neutralizing epitopes and suggesting the existence of both type-specific and cross-reactive conformational epitopes within the FG loop.

Insertion of a foreign sequence on capsid surface loops of human papillomavirus type 16 virus-like particles reduces their capacity to induce neutralizing antibodies and delineates a conformational neutralizing epitope.

The aims of this study were to generate chimeric human papillomavirus (HPV)-16 L1 virus-like particles (VLPs) in order to identify immunogenic domains and conformational neutralizing epitopes, and to characterize the regions where a foreign epitope could be introduced. We hypothesized that these regions could be on L1 protein loops since they are exposed on the surface of VLPs. The aims of this study were achieved by mutating HPV-16 L1 proteins. Six amino acids encoding for the epitope 78-83 (DPASRE) of the hepatitis B core (HBc) antigen were introduced within the different loops of the L1 protein at positions 56/57, 140/141, 179/180, 266/267, 283/284 or 352/353. All these chimeric L1 proteins were capable of self-assembly into VLPs. The antigenicity and immunogenicity of some of these VLPs were reduced compared to the levels observed with wild-type VLPs. All were nevertheless able to induce neutralizing antibodies. VLPs with insertion at position 266/267 induced lower levels of neutralizing antibodies, suggesting the involvement of residues situated on FG loop in L1 neutralizing epitopes. All the chimeric L1 proteins except the one with insertion at position 56/57 were also able to induce anti-HBc antibodies, thus suggesting exposure of the HBc epitope on the VLP surface. Taken together, our findings indicate the possibility of designing HPV-derived vectors that are less immunogenic and suggest positions for insertion of defined immune epitopes or cell ligands into L1 protein to be exposed on the surface of VLPs.