Paracellular barrier properties of tissues are mainly determined by the composition of claudin hetero-polymers. To analyze the molecular organization of tight junctions (TJ), we investigated the ability of claudins (Cld) to form homo- and heteromers. Cld1, -2, -3, -5, and -12 expressed in cerebral barriers were investigated. TJ-strands were reconstituted by claudin-transfection of HEK293-cells. cis-Interactions and/or spatial proximity were analyzed by fluorescence resonance energy transfer inside and outside of strands and ranked: Cld5/Cld5 > Cld5/Cld1 > Cld3/Cld1 > Cld3/Cld3 > Cld3/Cld5, no Cld3/Cld2. Classic Cld1, -3, and -5 but not non-classic Cld12 showed homophilic trans-interaction. Freeze-fracture electron microscopy revealed that, in contrast to classic claudins, YFP-tagged Cld12 does not form homopolymers. Heterophilic trans-interactions were analyzed in cocultures of differently monotransfected cells. trans-Interaction of Cld3/Cld5 was less pronounced than that of Cld3/Cld1, Cld5/Cld1, Cld5/Cld5 or Cld3/Cld3. The barrier function of reconstituted TJ-strands was demonstrated by a novel imaging assay. A model of the molecular organization of TJ was generated.

Influenza viruses are respiratory pathogens and can cause severe disease. The best protection against influenza is provided by annual vaccination. These vaccines are produced in embryonated chicken eggs or using continuous animal cell lines. The latter processes are more flexible and scalable to meet the growing global demand. However, virus production in cell cultures is more expensive. Hence, further research is needed to make these processes more cost-effective and robust. We studied influenza virus replication dynamics to identify factors that limit the virus yield in adherent Madin-Darby canine kidney (MDCK) cells. The cell cycle stage of MDCK cells had no impact during early infection. Yet, our results showed that the influenza virus RNA synthesis levels out already 4 h post infection at a time when viral genome segments are exported from the nucleus. Nevertheless, virus release occurred at a constant rate in the following 16 h. Thereafter, the production of infectious viruses dramatically decreased, but cells continued to produce particles contributing to the hemagglutination (HA) titer. The majority of these particles from the late phase of infection were deformed or broken virus particles as well as large membranous structures decorated with viral surface proteins. These changes in particle characteristics and morphology need to be considered for the optimization of influenza virus production and vaccine purification steps. Moreover, our data suggest that in order to achieve higher cell-specific yields, a prolonged phase of viral RNA synthesis and/or a more efficient release of influenza virus particles is required.Electronic supplementary materialThe online version of this article (doi:10.1007/s00253-016-7542-4) contains supplementary material, which is available to authorized users.

Major histocompatibility complex (MHC) class I molecules present cell internally derived peptides at the plasma membrane for surveillance by cytotoxic T lymphocytes. The surface expression of most class I molecules at least partially depends on the endoplasmic reticulum protein, tapasin, which helps them to bind peptides of the right length and sequence. To determine what makes a class I molecule dependent on support by tapasin, we have conducted in silico molecular dynamics (MD) studies and laboratory experiments to assess the conformational state of tapasin-dependent and -independent class I molecules. We find that in the absence of peptide, the region around the F pocket of the peptide binding groove of the tapasin-dependent molecule HLA-B*44:02 is in a disordered conformational state and that it is converted to a conformationally stable state by tapasin. This novel chaperone function of tapasin has not been described previously. We demonstrate that the disordered state of class I is caused by the presence of two adjacent acidic residues in the bottom of the F pocket of class I, and we suggest that conformational disorder is a common feature of tapasin-dependent class I molecules, making them essentially unable to bind peptides on their own. MD simulations are a useful tool to predict such conformational disorder of class I molecules.

Calreticulin is a lectin chaperone of the endoplasmic reticulum (ER). In calreticulin-deficient cells, major histocompatibility complex (MHC) class I molecules travel to the cell surface in association with a sub-optimal peptide load. Here, we show that calreticulin exits the ER to accumulate in the ER-Golgi intermediate compartment (ERGIC) and the cis-Golgi, together with sub-optimally loaded class I molecules. Calreticulin that lacks its C-terminal KDEL retrieval sequence assembles with the peptide-loading complex but neither retrieves sub-optimally loaded class I molecules from the cis-Golgi to the ER, nor supports optimal peptide loading. Our study, to the best of our knowledge, demonstrates for the first time a functional role of intracellular transport in the optimal loading of MHC class I molecules with antigenic peptide.

The human MHC class I protein HLA-B*27:05 is statistically associated with ankylosing spondylitis, unlike HLA-B*27:09, which differs in a single amino acid in the F pocket of the peptide-binding groove. To understand how this unique amino acid difference leads to a different behavior of the proteins in the cell, we have investigated the conformational stability of both proteins using a combination of in silico and experimental approaches. Here, we show that the binding site of B*27:05 is conformationally disordered in the absence of peptide due to a charge repulsion at the bottom of the F pocket. In agreement with this, B*27:05 requires the chaperone protein tapasin to a greater extent than the conformationally stable B*27:09 in order to remain structured and to bind peptide. Taken together, our data demonstrate a method to predict tapasin dependence and physiological behavior from the sequence and crystal structure of a particular class I allotype.Keywords: Ankylosing spondylitis r HLA-B27 r Major histocompatibility complex r Molecular dynamics r Natively unstructured proteins r Protein folding r Simulations Additional supporting information may be found in the online version of this article at the publisher's web-site Introduction MHC class I molecules are heterotrimeric proteins that transport antigenic peptides to the cell surface and present them to cytotoxic T cells. They consist of the transmembrane heavy chain (HC), the noncovalently associated light chain beta-2 microglobulin (β 2 m), and an antigenic peptide of eight to ten amino acids. The Correspondence: Prof. Sebastian Springer e-mail: s.springer@jacobs-university.de extracellular part of the heavy chain comprises the α 1 , α 2 , and α 3 domains. The α 1 and α 2 domains form the peptide-binding groove, a superdomain that consists of an antiparallel beta sheet surmounted by two alpha helices, between which the peptide binds [1,2].In the cell, peptide binding to class I is a multistep process within the ER. It involves the peptide-loading complex, which consists of the peptide transporter associated with antigen processing (TAP) that transports peptides from the cytosol into the ER lumen [3] and several chaperone proteins such as tapasin, which binds both to the class I molecule and TAP [4,5].C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu Eur. J. Immunol. 2015. 45: 1248-1257 Molecular immunology 1249The sequence of peptides that can bind to class I is determined by interactions with the amino acid side chains of the peptide-binding groove. The high sequence polymorphism of the peptide-binding groove in human class I molecules (human leukocyte antigens, HLA-A, -B, and -C) means that different allotypes bind different peptides; thus, individual HLA allotypes-such as HLA-B27-support-specific immune responses and are statistically associated with disease [6,7]. Among the subtypes of HLA-B27 [8,9], HLA-B*27:05 (B*27:05) shows a very strong statistical association with spondyloarthropathies such as ankylosing spondylitis (AS), in contr...

Major histocompatibility complex class I proteins, which present antigenic peptides to cytotoxic T lymphocytes at the surface of all nucleated cells, are endocytosed and destroyed rapidly once their peptide ligand has dissociated. The molecular mechanism of this cellular quality control process, which prevents rebinding of exogenous peptides and thus erroneous immune responses, is unknown. To identify the nature of the decisive step in endocytic sorting of class I molecules and its location, we have followed the removal of optimally and suboptimally peptide-loaded murine H-2K(b) class I proteins from the cell surface. We find that the binding of their light chain, β2-microglobulin (β2m), protects them from endocytic destruction. Thus, the extended survival of suboptimally loaded K(b) molecules at 25°C is attributed to decreased dissociation of β2m. Because all forms of K(b) are constantly internalized but little β2m-receptive heavy chain is present at the cell surface, it is likely that β2m dissociation and recognition of the heavy chain for lysosomal degradation take place in an endocytic compartment.

In the presence of the murine cytomegalovirus (mCMV) gp40 (m152) protein, murine major histocompatibility complex (MHC) class I molecules do not reach the cell surface but are retained in an early compartment of the secretory pathway. We find that gp40 does not impair the folding or high-affinity peptide binding of the class I molecules but binds to them, leading to their retention in the endoplasmic reticulum (ER), the ER-Golgi intermediate compartment (ERGIC) and the cis-Golgi, most likely by retrieval from the cis-Golgi to the ER. We identify a sequence in gp40 that is required for both its own retention in the early secretory pathway and for that of class I molecules.

The translocation of cytosolic peptides into the lumen of the endoplasmic reticulum (ER) is a crucial step in the presentation of intracellular antigen to T cells by major histocompatibility complex (MHC) class I molecules. It is mediated by the transporter associated with antigen processing (TAP) protein, which binds to peptide-receptive MHC class I molecules to form the MHC class I peptide-loading complex (PLC). We investigated whether TAP is present and active in compartments downstream of the ER. By fluorescence microscopy, we found that TAP is localized to the ERGIC (ER-Golgi intermediate compartment) and the Golgi of both fibroblasts and lymphocytes. Using an in vitro vesicle formation assay, we show that COPII vesicles, which carry secretory cargo out of the ER, contain functional TAP that is associated with MHC class I molecules. Together with our previous work on post-ER localization of peptide-receptive class I molecules, our results suggest that loading of peptides onto class I molecules in the context of the peptide-loading complex can occur outside the ER.