Mapping cyclosporine-induced changes in protein secretion by renal cells using stable isotope labeling with amino acids in cell culture (SILAC).

Nephrotoxicity is an adverse event that strongly limits the use of the immunosuppressant cyclosporine in solid organ transplantation and the precise molecular mechanisms underlying this toxicity remain unclear. MS-based proteomic analysis of the secretome of HEK-293 renal cells exposed to cyclosporine was performed to identify changes in protein secretion, as a first step to discover potential biomarkers of such nephrotoxicity. To detect and quantify the perturbed proteins in the culture medium we used SILAC and nano-scale liquid chromatography followed by MALDI-TOF/TOF mass spectrometry. Among 106 proteins identified, 80 were quantified in both forward/reverse SILAC experiments and quantitative proteomic analysis revealed altered levels of expression for 24 secreted proteins. These included the down-regulation of a number of extracellular matrix/cell adhesion components, and the up-regulation of secreted cyclophilins A and B, macrophage inhibition factor and phosphatidylethanolamine-binding protein 1. These changes in protein secretion were not prevented by co-incubation with the antioxidant N-acetylcysteine, suggesting that they were not triggered by cyclosporine-induced oxidative stress. The results from the present study provide important new knowledge to gain insights into the molecular mechanisms of cyclosporine-related toxicity. Some of the proteins identified here should be tested as potential biomarkers of cyclosporine nephrotoxicity in subsequent clinical studies.

Quantitative proteomic analysis of cyclosporine-induced toxicity in a human kidney cell line and comparison with tacrolimus.

The calcineurin-inhibitors (CNIs) cyclosporine (CsA) and tacrolimus (TAC) remain the pillars of modern immunosuppression regimens used in solid organ transplantation. Nephrotoxicity is an adverse effect that limits their successful use. The precise molecular mechanisms underlying this nephrotoxicity remain unclear. Using SILAC together with LC-MALDI-TOF/TOF, we investigated the CNIs-induced proteomic perturbations in renal cells. Among the 495 proteins quantifiable in both forward and reverse SILAC, 69 displayed CsA-induced perturbations: proteins involved in ER-stress/protein folding, apoptosis, metabolism/transport or cytoskeleton pathways were up-regulated, while cyclophilin B as well as nuclear and RNA-processing proteins were down-regulated. Co-administration of CsA with the antioxidant N-acetylcysteine significantly decreased lipid peroxidation and also partially corrected the CsA-induced unfolded protein response. TAC toxicity profile was apparently different from that of CsA, especially without perturbation of cyclophilins A and B, up-regulation of ER-chaperones nor down-regulation of a number of nuclear proteins. These results provide a new insight and are consistent with recent data regarding the molecular mechanisms of CNIs-induced nephrotoxicity. Our findings offer new directions for future research aiming to identify specific biomarkers of CsA nephrotoxicity.

Bovine alpha1,3-galactosyltransferase catalytic domain structure and its relationship with ABO histo-blood group and glycosphingolipid glycosyltransferases.

alpha1,3-galactosyltransferase (alpha3GalT, EC 2.4.1.151) is a Golgi-resident, type II transmembrane protein that transfers galactose from UDP-alpha-galactose to the terminal N:-acetyllactosamine unit of glycoconjugate glycans, producing the Galalpha1,3Galbeta1,4GlcNAc oligosaccharide structure present in most mammalian glycoproteins. Unlike most other mammals, humans and Old World primates do not possess alpha3GalT activity, which is relevant for the hyperacute rejection observed in pig-to-human xenotransplantation. The crystal structure of the catalytic domain of substrate-free bovine alpha3GalT, solved and refined to 2.3 A resolution, has a globular shape with an alpha/beta fold containing a narrow cleft on one face, and shares a UDP-binding domain (UBD) with the recently solved inverting glycosyltransferases. The substrate-bound complex, solved and refined to 2.5 A, allows the description of residues interacting directly with UDP-galactose. These structural data suggest that the strictly conserved residue E317 is likely to be the catalytic nucleophile involved in galactose transfer with retention of anomeric configuration as accomplished by this enzyme. Moreover, the alpha3GalT structure helps to identify amino acid residues that determine the specificities of the highly homologous ABO histo-blood group and glycosphingolipid glycosyltransferases.

A model of photoprobe docking with beta1,4-galactosyltransferase identifies a possible carboxylate involved in glycosylation steps.

A molecular docking study has been performed on the interaction of beta1,4-galactosyltransferase with an acceptor site photoprobe. This is based on an acceptor site peptide fragment which was recently identified by the use of a photoprobe. The present model strongly suggests that the carboxylate group of Asp318 could be involved in the activation of the acceptor sugar 4-OH for the efficient galactosyltransfer. The result also exemplified that the combination of photoaffinity labeling with crystallography is a powerful method for the detailed structural analysis of ligand protein complex.

Crystal structures of the bovine beta4galactosyltransferase catalytic domain and its complex with uridine diphosphogalactose.

beta1,4-galactosyltransferase T1 (beta4Gal-T1, EC 2.4.1.90/38), a Golgi resident membrane-bound enzyme, transfers galactose from uridine diphosphogalactose to the terminal beta-N-acetylglucosamine residues forming the poly-N-acetyllactosamine core structures present in glycoproteins and glycosphingolipids. In mammals, beta4Gal-T1 binds to alpha-lactalbumin, a protein that is structurally homologous to lyzozyme, to produce lactose. beta4Gal-T1 is a member of a large family of homologous beta4galactosyltransferases that use different types of glycoproteins and glycolipids as substrates. Here we solved and refined the crystal structures of recombinant bovine beta4Gal-T1 to 2.4 A resolution in the presence and absence of the substrate uridine diphosphogalactose. The crystal structure of the bovine substrate-free beta4Gal-T1 catalytic domain showed a new fold consisting of a single conical domain with a large open pocket at its base. In the substrate-bound complex, the pocket encompassed residues interacting with uridine diphosphogalactose. The structure of the complex contained clear regions of electron density for the uridine diphosphate portion of the substrate, where its beta-phosphate group was stabilized by hydrogen-bonding contacts with conserved residues including the Asp252ValAsp254 motif. These results help the interpretation of engineered beta4Gal-T1 point mutations. They suggest a mechanism possibly involved in galactose transfer and enable identification of the critical amino acids involved in alpha-lactalbumin interactions.

Crystal structure of hemolin: a horseshoe shape with implications for homophilic adhesion.

Hemolin, an insect immunoglobulin superfamily member, is a lipopolysaccharide-binding immune protein induced during bacterial infection. The 3.1 angstrom crystal structure reveals a bound phosphate and patches of positive charge, which may represent the lipopolysaccharide binding site, and a new and unexpected arrangement of four immunoglobulin-like domains forming a horseshoe. Sequence analysis and analytical ultracentrifugation suggest that the domain arrangement is a feature of the L1 family of neural cell adhesion molecules related to hemolin. These results are relevant to interpretation of human L1 mutations in neurological diseases and suggest a domain swapping model for how L1 family proteins mediate homophilic adhesion.

In vivo calcineurin crystals formed using the baculovirus expression system.

Calcineurin is a heterodimeric phosphatase involved in the signal transduction of antigen-activated T cells. Coexpression of its two subunits, the regulatory subunit from human and the catalytic subunit from Neurospora crassa in cultured insect cells using the baculovirus expression system results in the formation of very large crystals in the cytoplasm. The crystals are formed initially in vesicles, but their subsequent growth appears to be uninhibited and continues without the need of an enclosing membrane until the host cell lyses. Although these in vivo crystals are low in population, ranging only 0-3 per cell, they are extremely large, over 10 mu m in some cases. Biochemical assays confirm their calcineurin origin, with the regulatory subunit incorporated being myristoylated, although both the myristoylated and unmyristoylated forms are expressed. The lattice structure of the in vivo crystals, with a spacing of 5.5 nm, is preserved with the regular electron microscopic (EM) specimen preparation procedure.

Crystal structure at 2.2 A resolution of the MHC-related neonatal Fc receptor.

The three-dimensional structure of the rat neonatal Fc receptor (FcRn) is similar to the structure of molecules of the major histocompatibility complex (MHC). The counterpart of the MHC peptide-binding site is closed in FcRn, making the FcRn groove incapable of binding peptides. A dimer of FcRn heterodimers seen in the crystals may represent a receptor dimer that forms when the Fc portion of a single immunoglobulin binds. An alternative use of the MHC fold for immune recognition is indicated by the FcRn and FcRn/Fc co-crystal structures.

Investigation of the interaction between the class I MHC-related Fc receptor and its immunoglobulin G ligand.

The neonatal Fc receptor (FcRn) is structurally similar to class I major histocompatibility molecules. FcRn transports maternal immunoglobulin G (IgG) from ingested milk into the blood. IgG is bound at the pH of milk (pH 6.0-6.5) in the gut and released at the pH of blood (pH 7.5). We find that alteration of a histidine pair within the alpha 3 domain of FcRn and of a nearby loop (the FcRn counterpart of the class I CD8-binding loop) affects the affinity for IgG. Inhibition studies suggest the involvement of the FcRn B2-microglobulin domain in IgG binding. Fragment B of protein A inhibits FcRn binding to IgG, localizing the binding site on Fc for FcRn to the CH2-CH3 domain interface. Three histidines present at the CH2-CH3 domain interface of Fc could be partially responsible for the pH-dependent interaction between FcRn and IgG.

The class I major histocompatibility complex related Fc receptor shows pH-dependent stability differences correlating with immunoglobulin binding and release.

Maternal immunoglobulin G (IgG) in milk is transported to the bloodstream of newborn rodents via an Fc receptor (FcRn) expressed in the gut. The receptor shows a striking structural similarity to class I major histocompatibility complex (MHC) molecules, being composed of a related heavy chain and the identical light chain (beta 2-microglobulin). FcRn binds IgG at the pH of milk in the proximal intestine (pH 6.0-6.5) and releases it at the pH of blood (pH approximately 7.5). We have compared the stability of a soluble form of FcRn in these two pH ranges and find that the heterodimer is markedly more stable at the permissive pH for IgG binding. Using the rate of beta 2m exchange as a correlate of heterodimer stability, we find that exchange is more than 10 times slower at pH 6.1 compared to pH 7.8. Thermal denaturation profiles of FcRn heterodimers at pH 8.0 indicate a two-step, sequential heavy-chain (Tm = 52 degrees C) and beta 2m (Tm = 67 degrees C) denaturation. By contrast, at pH 6.0, a single transition is observed, centered at 62 degrees C, corresponding to denaturation of both chains. The striking difference in stability does not appear to be correlated with the binding of peptide as in class I MHC molecules, because analysis of purified FcRn by acid dissociation and sequencing suggests that FcRn is not associated with cellular peptides. These results are indicative of pH-dependent conformational changes in the FcRn heterodimer, which may be related to its physiological function.

Crystallization and stoichiometry of binding of a complex between a rat intestinal Fc receptor and Fc.

Fc receptors expressed in the gut of newborn rodents bind to maternal immunoglobulin in milk at pH 6.5, and transport it to the bloodstream of the neonate, where it dissociates at pH 7.4. The rat intestinal Fc receptor (FcRn) consists of a heavy chain, with significant sequence similarity to the heavy chain of class I MHC molecules, complexed to the class I light chain, beta 2-microglobulin. Although FcRn is predicted to contain a groove analogous to that which serves as the MHC peptide-binding site, the immunoglobulin ligand of FcRn is a macromolecule instead of a peptide. We have expressed and crystallized a secreted form of FcRn, and here report the crystallization of a complex between FcRn and its Fc ligand. Isolated FcRn-Fc complexes crystallize in space group I222 or I2(1)2(1)2(1) with unit cell dimensions a = 125 A, b = 152 A and c = 216 A. The crystals diffract to 5.5 A resolution with anisotropic diffraction to 3.5 A. Data collection from cryopreserved crystals may allow the resolution limit to be extended, since the major reason for the poor resolution appears to be radiation decay. Even a low-resolution view of how FcRn binds Fc would be of interest to see if the binding site corresponds to the functional part of an MHC molecule. Since the structure of Fc is known, and a structure determination of FcRn is underway, it may be possible to locate the Fc binding site on FcRn at low resolution. As an initial characterization of the FcRn-Fc mode of interaction, and to facilitate the structure determination, we have determined the stoichiometry of binding of FcRn to Fc. We show that two FcRn molecules bind per Fc, as determined by analysis of gels of washed crystals, a column binding assay, and isothermal titration calorimetry.

Interleukin 3 promotes histamine synthesis in hematopoietic progenitors by increasing histidine decarboxylase mRNA expression.

Interleukin 3 (IL-3) is a potent stimulator of histamine production by cells from murine hematopoietic organs. We demonstrate herein that this phenomenon results from increased histidine decarboxylase (HDC: EC 4.1.1.22) activity in progenitor-enriched bone marrow cells (around 5% of the total bone marrow) isolated from the low density layers of a discontinuous Ficoll gradient. HDC levels are markedly enhanced after a 24 h incubation with IL-3 while a 4 h exposure results only in a slight activation. It results from increased expression of the mRNA coding for HDC, as assessed by Northern blot analysis after a 24 h incubation with IL-3. At the same time point and after a 4 h stimulation, we have evaluated the percentage of cells in this population which express HDC mRNA in response to IL-3, using in situ hybridization with the antisense riboprobe. We have thus established that enhanced HDC mRNA expression occurs in a small immature subset representing from 5 to 8% of the progenitor-enriched bone marrow cells.

Expression and crystallization of a soluble and functional form of an Fc receptor related to class I histocompatibility molecules.

Maternal transport of immunoglobulin to the newborn mammal is important for immune defense during the first weeks of independent life. Receptors for the Fc portion of IgG mediate the transfer of immunoglobulin from milk to the bloodstream of newborn mice and rats, by passage through intestinal epithelial cells. Neonatal Fc receptors (FcRn) isolated from intestinal epithelial cells of suckling rats bear a striking resemblance to class I histocompatibility molecules. The heavy chain of FcRn has sequence similarity in three extracellular domains to the corresponding domains of class I molecules, and the light chain of both types of molecules is beta 2-microglobulin. To facilitate biochemical characterization and crystallization of FcRn, we have expressed a secreted form, as well as two different lipid-linked forms solubilizable by phospholipase treatment. The lipid-linked forms are heterodimers consisting of beta 2-microglobulin and the extracellular portion of the heavy chain and are anchored to the membrane by a phosphatidylinositol linkage attached to either the heavy chain or beta 2-microglobulin. Cells expressing either lipid-linked form bind rat Fc, reproducing the known physiological pH dependence of binding. Secreted FcRn has been purified in yields up to 40 mg/liter from cell supernatants. Circular dichroism spectra of soluble FcRn appear similar to spectra of class I MHC molecules, suggesting that the similarities in primary sequence extend also to a similarity in secondary structure. Soluble FcRn crystallizes in a form amenable to a structure determination by x-ray diffraction methods, which will ultimately allow a detailed comparison of the two types of molecules.

Mutations in the D strand of the human CD4 V1 domain affect CD4 interactions with the human immunodeficiency virus envelope glycoprotein gp120 and HLA class II antigens similarly.

CD4, a cell surface glycoprotein expressed primarily by T lymphocytes and monocytes, interacts with HLA class II antigens to regulate the immune response. In AIDS, CD4 is the receptor for the human immunodeficiency virus, which binds to CD4 through envelope glycoprotein gp120. Delineation of the ligand-binding sites of CD4 is necessary for the development of immunomodulators and antiviral agents. Although the gp120 binding site has been characterized in detail, much less is known about the class II binding site, and it is as yet uncertain whether they partially or fully overlap. To investigate CD4 binding sites, a cellular adhesion assay between COS cells transiently transfected with CD4 and B lymphocytes expressing HLA class II antigens has been developed that is strictly dependent on the CD4--class II interaction, quantitative, and highly reproducible. Mutants of CD4 expressing amino acids with distinct physicochemical properties at positions Arg-54, Ala-55, Asp-56, and Ser-57 in V1, the first extracellular immunoglobulin-like domain, have been generated and studied qualitatively and quantitatively for interaction with HLA class II antigens, for membrane expression, for the integrity of CD4 epitopes recognized by a panel of monoclonal antibodies, and for gp120 binding. The results obtained show that the mutations in this tetrapeptide, which forms the core of a synthetic peptide previously shown to have immunosuppressive properties, affect the two binding functions of CD4 similarly, lending support to the hypothesis that the human immunodeficiency virus mimicks HLA class II binding to CD4.

Interaction of CD4 with HLA class II antigens and HIV gp120.

We have developed a cellular adhesion assay in which B lymphocytes expressing HLA class II antigens form rosettes with COS cells expressing high levels of cell surface CD4 upon transient transfection with a CDM8-CD4 plasmid construct. The assay is specific, quantitative, and overcomes the difficulties encountered with a previously described system using an SV40 viral vector. Rosette formation was inhibited by a series of CD4- and HLA-DR-specific antibodies, as well as by human immunodeficiency virus (HIV) gp 120, and a synthetic peptide derived from part of its binding site for CD4 (amino acid residues 414-434), but not by a variety of other effectors, including several soluble CD4 derivatives. The comparison of this pattern of inhibition with those observed in other systems further emphasizes the great similarity, but incomplete identity, in the CD4 binding sites for HLA class II antigens and HIV gp120, and supports a model in which CD4 is considered as an allosteric servomodulator of T-cell adhesion and function which probably is induced to interact with HLA class II antigens when associated with the Tcr/CD3 complex.

Histamine-producing cell-stimulating activity. A biological activity shared by interleukin 3 and granulocyte-macrophage colony-stimulating factor.

The histamine-producing cell-stimulating factor (HCSF) was first described as a lymphokine which is produced during secondary mixed leukocyte culture and which induces increased histamine synthesis by murine hematopoietic cells. It has been shown that it is different from interleukin 3 (IL 3), despite the fact that pure IL 3 expresses HCSF activity. Our results provide evidence that this factor (constitutively produced by the P388 D1 cell line) is identical with granulocyte-macrophage colony-stimulating factor (GM-CSF) i.e.: (a) physiochemical properties of HCSF and GM-CSF, such as molecular weight, isoelectric charge, hydrophobicity and behavior during affinity chromatography, are indistinguishable and both activities coelute during all biochemical purification procedures; (b) increased bone marrow cell histamine synthesis induced by P388 D1-derived HCSF is inhibited by anti-GM-CSF antiserum; (c) the GM-CSF cDNA probe hybridizes with a poly(A)+RNA from P388 D1 cells while no hybridizing signal was obtained with poly(A)+RNA from WEHI-3 and from P815 cells. On the other hand, the IL 3 cDNA probe hybridizes with a 1.0-kb poly(A)+RNA from WEHI-3 but not with those from P388 D1 and P815. Moreover, well known sources of GM-CSF, such as lung conditioned medium and semi-purified GM-CSF from phytohemagglutinin-induced supernatant of the murine T lymphoma LBRM-33-5 A4 (preparation devoid of IL 3), as well as recombinant murine GM-CSF, induce increased histamine synthesis by hematopoietic cells. All these results demonstrate that, in our culture conditions, the P388 D1 cell line spontaneously produces GM-CSF which is responsible for the P388 D1-induced HCS activity. Consequently, the latter is a property shared by the two distinct hematopoietic growth factors acting on the less committed cells, i.e. IL 3 and GM-CSF, whereas M-CSF or G-CSF are unable to induce histamine production. Interestingly, IL-4 which is known to support established mast cell line proliferation cannot induce HCS activity. In addition, none of the other cytokines tested, such as IL 1, IL 2, interferons or tumor necrosis factor can express HCS activity. This expression seems to be a specific property of IL 3 and GM-CSF.

Synthesis and biological activity of eight thymulin analogues.

Eight analogues of thymulin, a thymic nonapeptide involved in several aspects of T-cell differentiation, were synthesized by the conventional method in solution. Four were modified in residue 7 (Ala, D-Ala, D-Leu or Sar instead of Gly) and two in residue 8 (D-Ser or Thr instead of Ser); in the others, the Gly6-Gly7 sequence was replaced either by a single glycyl residue or by a triglycyl sequence. The biological activity of the analogues was determined in the rosette assay: five exhibited a prolonged activity in vivo with respect to thymulin. All the analogues inhibited the binding of tritiated thymulin to thymulin receptors on three human lymphoblastoid T-cell lines (HSB2, 1301 and CEM) with the same order of magnitude as non-labelled thymulin.