Stephan Uebel

Molecular mechanism and species specificity of tap inhibition by herpes simplex virus protein icp47

The immediate early protein ICP47 of herpes simplex virus (HSV) inhibits the transporter for antigen processing (TAP)‐mediated translocation of antigen‐derived peptides across the endoplasmic reticulum (ER) membrane. This interference prevents assembly of peptides with class I MHC molecules in the ER and ultimately recognition of HSV‐infected cells by cytotoxic T‐lymphocytes, potentially leading to immune evasion of the virus. Here, we demonstrate that recombinant, purified ICP47 containing a hexahistidine tag inhibits peptide import into microsomes of insect cells expressing human TAP, whereas inhibition of peptide transport by murine TAP was much less effective. This finding indicates an intrinsic species‐specificity of ICP47 and suggests that no additional proteins interacting specifically with either ICP47 or TAP are required for inhibition of peptide transport. Since neither purified nor induced ICP47 inhibited photocrosslinking of 8‐azido‐ATP to TAP1 and TAP2 it seems that ICP47 does not prevent ATP from binding to TAP. By contrast, peptide binding was completely blocked by ICP47 as shown both by photoaffinity crosslinking of peptides to TAP and peptide binding to microsomes from TAP‐transfected insect cells. Competition experiments indicated that ICP47 binds to human TAP with a higher affinity (50 nM) than peptides whereas the affinity to murine TAP was 100‐fold lower. Our data suggest that ICP47 prevents peptides from being translocated by blocking their binding to the substrate‐binding site of TAP.

Functional expression and purification of the ABC transporter complex associated with antigen processing (TAP) in insect cells

Using the baculovirus expression system the gene products of human tap1 and tap2 were over‐expressed as wild‐type as well as oligohistidine fusion proteins in Spodoptera frugiperda (Sf9) insect cells. Both gene products were co‐expressed within the same cells and were found enriched in microsomal membranes. Immunoprecipitation and immobilized metal affinity chromatography revealed complex formation between TAP1 and TAP2. The expressed TAP complex was shown to be functional by peptide translocation into microsomes of Sf9 cells. Peptide transport strictly requires TAP1 and TAP2 as well as ATP. For the first time the functional expression of the human TAP complex in insect cells has been demonstrated, indicating that additional cofactors of a highly developed immune system are not essential for peptide transport across microsomal membranes.

Specificity of the proteasome and the TAP transporter

The generation of antigenic peptides and their transport across the membrane of the endoplasmic reticulum for assembly with MHC class I molecules are essential steps in antigen presentation to cytotoxic T lymphocytes. Recent studies have characterized the substrate specificities of the proteasome and the transporter associated with antigen processing. It is interesting to compare the specificity of this transporter to the wide spectrum of peptides generated by the proteasome, to the binding motifs of MHC class I molecules and in particular to the principles of T cell recognition.

The Transporters Associated with Antigen Processing (TAP)

Class I molecules of the major histocompatibility complex (MHC) present peptides derived from endogenous proteins at the cell surface. During viral infection or malignant transformation a different set of peptides is displayed by MHC class I molecules. These antigen-loaded class I complexes are recognized by cytotoxic T cells via the T cell receptors as nonself, thus leading to the destruction of the abnormal cell.

Peptide binding and photo-crosslinking to detergent solubilized and to reconstituted transporter associated with antigen processing (TAP)

The transporter associated with antigen processing (TAP) is essential for peptide loading onto major histocompatibility (MHC) class I molecules by translocating peptides into the endoplasmic reticulum. We have explored the conditions for detergent solubilization of functionally active, heterologously expressed human TAP from microsomal membranes. The efficiency to solubilize TAP was tested for a variety of detergents as well as for different solubilization conditions. The activity of the solubilized TAP complex was analyzed over time, using a non‐radioactive crosslinking assay with a photo‐activateable peptide, in the presence or absence of external lipid. The detergent CHAPS was found optimally to retain activity and thus allowed us to reconstitute detergent‐solubilized, active TAP into proteoliposomes.

Fibronectins containing extradomain A or B enhance osteoblast differentiation via distinct integrins

Fibronectin is a multidomain protein secreted by various cell types. It forms a network of fibers within the extracellular matrix and impacts intracellular processes by binding to various molecules, primarily integrin receptors on the cells. Both the presence of several isoforms and the ability of the various domains and isoforms to bind to a variety of integrins result in a wide range of effects. In vivo findings suggest that fibronectin isoforms produced by the osteoblasts enhance their differentiation. Here we report that the isoform characterized by the presence of extradomain A activates α4β1 integrin and augments osteoblast differentiation. In addition, the isoform containing extradomain B enhances the binding of fibronectin through the RGD sequence to β3-containing integrin, resulting in increased mineralization by and differentiation of osteoblasts. Our study thus reveals novel functions for two fibronectin isoforms and the mediating receptors in osteoblast differentiation.

An O‐Glycosylation of Fibronectin Mediates Hepatic Osteodystrophy Through α4β1 Integrin

Patients with cholestatic liver disease experience increased fracture risk. Higher circulating levels of a fibronectin isoform called oncofetal fibronectin (oFN) were detected in a subset of such patients. Administering this isoform to mice suppresses osteoblast differentiation and diminishes bone mineral density in vivo, suggesting it is responsible for bone loss in cholestatic liver disease. The aim of this study was to define the mechanism by which oFN affects osteoblast function and evaluate possible modifiers in experimental hepatic osteodystrophy. The fibronectin isoform oFN is characterized by the presence of various glycosylations. In line with this, adding oFN that underwent enzymatic O‐deglycosylation to osteoblasts normalized nodule formation in vitro. Of three possible O‐glycosylation sites in oFN, only a mutation at AA 33 of the variable region or binding of this glycosylated site with an antibody normalized osteoblast differentiation. Because the responsible site is located in the variable region of fibronectin, which binds to α4β1 or α4β7 integrins, these integrins were evaluated. We show that integrin α4β1 mediates the inhibitory effect of oFN both in vitro as well as in vivo. In a hepatic osteodystrophy mouse model, we demonstrate that liver fibrosis is associated with increased circulating oFN and diminished BMD. In addition, trabecular bone loss induced by oFN injection or fibrosis induction could be prevented by either administering an antibody that binds to α4 integrin (PS/2) or the CS1 peptide, which contains a binding site for α4β1 integrin. In summary, oFN inhibits osteoblast activity. This is because of an O‐glycosylation in the variable region that results in decreased integrin‐mediated signaling. This deleterious effect can be thwarted by binding α4β1 integrin. Thus, we have characterized the defect and the receptor mediating bone loss in patients with hepatic osteodystrophy and evaluated possible therapeutic interventions in a murine model.

Peptide libraries in cellular immune recognition.

Protective immunity against microbial pathogens is connected with the ability to discriminate between self and non-self. Vertebrates have evolved an adaptive immune system in which specialized white blood cells, the T cells, fulfill this role. These cells are trained to recognize proteolytic fragments of viral, bacterial or parasitic antigens in the context of specialized antigen presenting proteins, the major histocompatibility complex (MHC) molecules. Recognition of a non-self peptide in complex with the self component, the MHC molecule, ultimately leads to destruction of the infected cell or to the production of neutralizing antibodies.

An O-Glycosylation of Fibronectin Mediates Hepatic Osteodystrophy Through α4β1 Integrin: OFN FIBRONECTIN INHIBITS OSTEOBLAST DIFFERENTIATION

Patients with cholestatic liver disease experience increased fracture risk. Higher circulating levels of a fibronectin isoform called oncofetal fibronectin (oFN) were detected in a subset of such patients. Administering this isoform to mice suppresses osteoblast differentiation and diminishes bone mineral density in vivo, suggesting it is responsible for bone loss in cholestatic liver disease. The aim of this study was to define the mechanism by which oFN affects osteoblast function and evaluate possible modifiers in experimental hepatic osteodystrophy. The fibronectin isoform oFN is characterized by the presence of various glycosylations. In line with this, adding oFN that underwent enzymatic O‐deglycosylation to osteoblasts normalized nodule formation in vitro. Of three possible O‐glycosylation sites in oFN, only a mutation at AA 33 of the variable region or binding of this glycosylated site with an antibody normalized osteoblast differentiation. Because the responsible site is located in the variable region of fibronectin, which binds to α4β1 or α4β7 integrins, these integrins were evaluated. We show that integrin α4β1 mediates the inhibitory effect of oFN both in vitro as well as in vivo. In a hepatic osteodystrophy mouse model, we demonstrate that liver fibrosis is associated with increased circulating oFN and diminished BMD. In addition, trabecular bone loss induced by oFN injection or fibrosis induction could be prevented by either administering an antibody that binds to α4 integrin (PS/2) or the CS1 peptide, which contains a binding site for α4β1 integrin. In summary, oFN inhibits osteoblast activity. This is because of an O‐glycosylation in the variable region that results in decreased integrin‐mediated signaling. This deleterious effect can be thwarted by binding α4β1 integrin. Thus, we have characterized the defect and the receptor mediating bone loss in patients with hepatic osteodystrophy and evaluated possible therapeutic interventions in a murine model.

An O-Glycosylation of Fibronectin Mediates Hepatic Osteodystrophy Through alpha 4 beta 1 Integrin

Patients with cholestatic liver disease experience increased fracture risk. Higher circulating levels of a fibronectin isoform called oncofetal fibronectin (oFN) were detected in a subset of such patients. Administering this isoform to mice suppresses osteoblast differentiation and diminishes bone mineral density in vivo, suggesting it is responsible for bone loss in cholestatic liver disease. The aim of this study was to define the mechanism by which oFN affects osteoblast function and evaluate possible modifiers in experimental hepatic osteodystrophy. The fibronectin isoform oFN is characterized by the presence of various glycosylations. In line with this, adding oFN that underwent enzymatic O‐deglycosylation to osteoblasts normalized nodule formation in vitro. Of three possible O‐glycosylation sites in oFN, only a mutation at AA 33 of the variable region or binding of this glycosylated site with an antibody normalized osteoblast differentiation. Because the responsible site is located in the variable region of fibronectin, which binds to α4β1 or α4β7 integrins, these integrins were evaluated. We show that integrin α4β1 mediates the inhibitory effect of oFN both in vitro as well as in vivo. In a hepatic osteodystrophy mouse model, we demonstrate that liver fibrosis is associated with increased circulating oFN and diminished BMD. In addition, trabecular bone loss induced by oFN injection or fibrosis induction could be prevented by either administering an antibody that binds to α4 integrin (PS/2) or the CS1 peptide, which contains a binding site for α4β1 integrin. In summary, oFN inhibits osteoblast activity. This is because of an O‐glycosylation in the variable region that results in decreased integrin‐mediated signaling. This deleterious effect can be thwarted by binding α4β1 integrin. Thus, we have characterized the defect and the receptor mediating bone loss in patients with hepatic osteodystrophy and evaluated possible therapeutic interventions in a murine model.