Disulfide-bonded Homodimer Research Articles

Periostin Is a Disulfide-Bonded Homodimer and Forms a Complex with Fibronectin in the Human Skin.

The protein periostin is a matricellular protein that is expressed in connective tissue. It is composed of five globular domains arranged in an elongated structure with an extensive disordered C-terminal tail. Periostin contains 11 cysteine residues, of which one is unpaired and the rest form five intramolecular disulfide bonds. Periostin plays an important role during wound healing and is also involved in driving the inflammatory state in atopic diseases. This study provides a comprehensive biochemical characterization of periostin in human skin and in dermal and pulmonary fibroblasts in vitro. Through the application of Western blotting, co-immunoprecipitation, and LC-MS/MS, we show for the first time that periostin is a disulfide-bonded homodimer and engages in a novel disulfide-bonded complex with fibronectin both in vivo and in vitro. This inherent characteristic of periostin holds the potential to redefine our approach to exploring and understanding its functional role in future research endeavors.

Structural and Aggregation Features of a Human κ-Casein Fragment with Antitumor and Cell-Penetrating Properties.

Intrinsically disordered proteins play a central role in dynamic regulatory and assembly processes in the cell. Recently, a human κ-casein proteolytic fragment called lactaptin (8.6 kDa) was found to induce apoptosis of human breast adenocarcinoma MCF-7 and MDA-MB-231 cells with no cytotoxic activity toward normal cells. Earlier, we had designed some recombinant analogs of lactaptin and compared their biological activity. Among these analogs, RL2 has the highest antitumor activity, but the amino acid residues and secondary structures that are responsible for RL2′s activity remain unclear. To elucidate the structure–activity relations of RL2, we studied the structural and aggregation features of this fairly large intrinsically disordered fragment of human milk κ-casein by a combination of physicochemical methods: NMR, paramagnetic relaxation enhancement (PRE), Electron Paramagnetic Resonance (EPR), circular dichroism, dynamic light scattering, atomic force microscopy, and a cytotoxic activity assay. It was found that in solution, RL2 exists as stand-alone monomeric particles and large aggregates. Whereas the disulfide-bonded homodimer turned out to be more prone to assembly into large aggregates, the monomer predominantly forms single particles. NMR relaxation analysis of spin-labeled RL2 showed that the RL2 N-terminal region, which is essential not only for multimerization of the peptide but also for its proapoptotic action on cancer cells, is more ordered than its C-terminal counterpart and contains a site with a propensity for α-helical secondary structure.

Cloning, expression and characterization of Bombyx mori α1,6-fucosyltransferase

Although core α1,6-fucosylation is commonly observed in N-glycans of both vertebrates and invertebrates, the responsible enzyme, α1,6-fucosyltransferase, has been much less characterized in invertebrates compared to vertebrates. To investigate the functions of α1,6-fucosyltransferase in insects, we cloned the cDNA for the α1,6-fucosyltransferase from Bombyx mori (Bmα1,6FucT) and characterized the recombinant enzyme prepared using insect cell lines. The coding region of Bmα1,6FucT consists of 1737bp that code for 578 amino acids of the deduced amino acid sequence, showing significant similarity to other α1,6-fucosyltransferases. Enzyme activity assays demonstrated that Bmα1,6FucT is enzymatically active in spite of being less active compared to the human enzyme. The findings also indicate that Bmα1,6FucT, unlike human enzyme, is N-glycosylated and forms a disulfide-bonded homodimer. These findings contribute to a better understanding of roles of α1,6-fucosylation in invertebrates and also to the development of the more efficient engineering of N-glycosylation of recombinant glycoproteins in insect cells.

The inability of vaccinia virus A33R protein to form intermolecular disulfide-bonded homodimers does not affect the production of infectious extracellular virus

The orthopoxvirus protein A33 forms a disulfide-bonded high molecular weight species that could be either a homodimer or a heteromultimer. The protein is a major target for neutralizing antibodies and the majority of antibodies raised against A33 only recognize the disulfide-bonded form. Here, we report that A33 is present as a disulfide-bonded homodimer during infection. Additionally, we examined the function of intermolecular disulfide bonding in A33 homodimerization during infection. We show that the cysteine at amino acid 62 is required for intermolecular disulfide bonding, but not dimerization as this mutant was still able to form homodimers. To investigate the role of disulfide-bonded homodimers during viral morphogenesis, recombinant viruses that express an A33R with cysteine 62 mutated to serine were generated. The recombinant viruses had growth characteristics similar to their parental viruses, indicating that intermolecular disulfide-bonded homodimerization of A33 is not required for its function.

The High Reactivity of Peroxiredoxin 2 with H2O2 Is Not Reflected in Its Reaction with Other Oxidants and Thiol Reagents

Peroxiredoxin 2 is a member of the mammalian peroxiredoxin family of thiol proteins that is important in antioxidant defense and redox signaling. We have examined its reactivity with various biological oxidants, in order to assess its ability to act as a direct physiological target for these species. Human erythrocyte peroxiredoxin 2 was oxidized stoichiometrically to its disulfide-bonded homodimer by hydrogen peroxide, as monitored electrophoretically under nonreducing conditions. The protein was highly susceptible to oxidation by adventitious peroxide, which could be prevented by treating buffers with low concentrations of catalase. However, this did not protect peroxiredoxin 2 against oxidation by added H(2)O(2). Experiments measuring inhibition of dimerization indicated that at pH 7.4 catalase and peroxiredoxin 2 react with hydrogen peroxide at comparable rates. A rate constant of 1.3 x 10(7) M(-1) s(-1) for the peroxiredoxin reaction was obtained from competition kinetic studies with horseradish peroxidase. This is 100-fold faster than is generally assumed. It is sufficiently high for peroxiredoxin to be a favored cellular target for hydrogen peroxide, even in competition with catalase or glutathione peroxidase. Reactions of t-butyl and cumene hydroperoxides with peroxiredoxin were also fast, but amino acid chloramines reacted much more slowly. This contrasts with other thiol compounds that react many times faster with chloramines than with hydrogen peroxide. The alkylating agent iodoacetamide also reacted extremely slowly with peroxiredoxin 2. These results demonstrate that peroxiredoxin 2 has a tertiary structure that facilitates reaction of the active site thiol with hydrogen peroxide while restricting its reactivity with other thiol reagents.

Evidence Supporting Thiol-Disulfide Exchange as a Novel Mechanism of Platelet TGF-ß1 Activation.

Platelets are a rich source of latent TGF-β1, a cytokine with potent profibrotic and immunomodulatory effects, yet little is known about how platelet TGF-β1 becomes activated. Current theory is that TGF-β1 (a 25 kD disulfide-bonded homodimer produced by furin cleavage of a conjoint LAP-TGF-β1 precursor) must dissociate from the LAP (a 80 kD disulfide-bonded homodimer) to be active.Methods: Platelet-rich plasma (PRP), whole blood, or 5x108 washed platelets in HEPES-buffered modified Tyrode's buffer with 1 mM each Ca2+ and Mg2+, was placed in an aggregometer cuvette at 37°C with thrombin (0.125 U/ml) or TRAP (10 μM) for 2 h. Samples were then centrifuged and both the pellet and the supernatants were analyzed. TGF-β1 levels were quantified by: (1) Quantikine TGF-β1 assays in which recombinant TGF-β receptor-II is used to capture active TGF-β1. Active TGF-β1 was measured in untreated samples while total TGF-β1 was measured by converting latent TGF-β1 to active by acidification, (2) Mink lung epithelial cell (MLEC) assay in which active TGF-β1 binding results in expression of a luciferase reporter gene fused to a truncated PAI-1 promoter. Samples were incubated with MLECs overnight and cell lysates were assessed for luciferase activity. Non-reduced and reduced samples were also analyzed by SDS-PAGE and immunoblotting with antibodies to TGF-β1, LAP (present in large latent complexes [LLCs] and small latent complexes [SLCs]), and LTBP-1 (present in LLCs).Results: Latent TGF-β1 was maximally released within 5 minutes after platelet stimulation with thrombin (130 ± 15 ng/ml, n=9), whereas TGF-β1 activity increased slowly and progressively, reaching approximately 1% of total TGF-β1 at 2 hours (1.44 ± 0.69 ng/ml, n=14). A polyclonal antibody reported to be specific for active TGF-β1 reacted with a 25 kD band in nonreduced samples at all time points (5-120 mins) as expected, since SDS dissociates TGF-β1 from LAP in both SLCs and LLCs. There was also, however, an unexpected time-dependent increase in the binding of this antibody to several closely spaced bands of ~110 kD and ~260 kD representing SLCs and LLCs, respectively. Antibodies to LAP also reacted with the ~110 and ~260 kD bands. Reduced samples immunoblotted with the LAP antibody demonstrated a prominent band at 40 kD at all time points representing the LAP monomer. Preincubation of platelets with reagents that inhibit thiol-disulfide exchange reduced the peak generation of active TGF-β1 by 62–81%: bacitracin (165 ± 71 pg/ml vs. control, 848 ± 448 pg/ml, n=8, p=0.005), CMPS (781 ± 362 pg/ml vs. 2060 ± 420 pg/ml, n=5, p=0.01), DTNB (570 ± 140 pg/ml vs. 1810 ± 222 pg/ml; n=3, p=0.005). A maleimide/biotin probe (BMCC) demonstrated time-dependent incorporation into SLCs and LLCs. Chromatography of 2 h platelet supernatants using Superdex-200 demonstrated that high molecular weight fractions containing LLCs had TGF-β1 activity on Quantikine assays (n=4).Conclusions: 1) Inhibitors of thiol-disulfide exchange inhibit the activation of platelet TGF-β1, (2) Molecular species containing TGF-β1 covalently attached to LAP or other proteins via disulfide bonds appear in the platelet supernatant over time, (3) TGF-β1 activity can be detected in high molecular weight species. Our data suggest that thiol-disulfide exchange may contribute to TGF-β1 activation; this mechanism may have implications for TGF-β activation in other cells.

Peptide profiling of cells with multiple gene products: combining immunochemistry and MALDI mass spectrometry with on-plate microextraction.

Due to the intracellular chemical complexity and a wide range of transmitter concentrations, the detection of the complete set of peptide transmitters in a single cell is problematic. In the current study, a multidisciplinary approach combining single-cell MALDI-MS peptide profiling, northern analysis, in situ hybridization, and immunocytochemistry allows characterization of a more complete set of neurotransmitters than individual approaches in the Aplysia californica B1 and B2 motor neurons. Because different results were obtained using both in situ and immunohistochemical techniques compared to previous reports, MALDI-MS assays have been used to examine CP1-related gene products in these cells. However, MALDI with standard sample preparation does not detect the presence of the CP1 gene products. A novel on-plate microextraction approach using concentrated MALDI matrix 2,5-dihydroxybenzoic acid with a mixture of acetone and water as the solvent has been developed to allow the detection of trace-level gene expression products. Both neuropeptide precursors in the B1 and B2 neurons-the SCP and CP1 prohormones-end with large peptides that have multiple cysteine residues. For SCP, MALDI-MS verifies the presence of a novel 9325 Da SCP-related peptide. In the case of CP1, a disulfide-bonded homodimer is detected and the disulfide bonding pattern elucidated using MALDI-MS coupled with on-plate enzymatic digestion.

Prenatal cocaine exposure reduces glial cell line-derived neurotrophic factor (GDNF) in the striatum and the carotid body of the rat: implications for DA neurodevelopment

Glial cell line-derived neurotrophic factor (GDNF) is a glycosylated, disulfide-bonded homodimer, and a member of the transforming growth factor-beta superfamily. GDNF has been shown to promote the survival and morphological differentiation of dopamine (DA) neurons and increase their high-affinity dopamine uptake. In order to determine whether the mechanism for our previously observed cocaine-induced DA reductions in brain and carotid body were GDNF-mediated, we exposed Sprague–Dawley rat fetuses to cocaine via maternal subcutaneous injections (30 mg/kg b.i.d., E7–E19). Brains and carotid bodies of fetuses were excised and processed for assessment of GDNF levels using an Enzyme-Linked ImmunoadSorbent Assay (ELISA). ANOVA indicated that cocaine reduced carotid body GDNF by 36% ( F (1,5)=28.11, p<0.05) and striatal GDNF by 41% ( F (1,5)=41.77, p<0.01). Although there was no interaction between drug exposure and fetal uterine position, post-hoc pairwise comparisons indicated that reductions in GDNF in the cocaine groups were due to differences at more distal positions (positions 4–8). The magnitude of the reductions in striatal GDNF (but not carotid body GDNF) in both cocaine-exposed and control fetuses followed a cervical (smallest GDNF reductions) to ovarian (greatest GDNF reductions) uterine position gradient. This pattern was similar to that which we observed in prior studies examining DA reductions in brain following prenatal cocaine exposure. The finding that cocaine reduces GDNF levels in striatum and carotid body support the hypothesis that cocaine's ability to reduce striatal and carotid body DA may be indirect through its ability to reduce GDNF. These data along with previous findings support the hypothesis that cocaine's effects on DA neurons are at least partially due to its indirect effects on trophic activity. The possible mechanisms whereby cocaine affects trophic activity are discussed.

The isocitrate dehydrogenase kinase/phosphatase from Escherichia coli is highly sensitive to in-vitro oxidative conditions role of cysteine67 and cysteine108 in the formation of a disulfide-bonded homodimer.

Isocitrate dehydrogenase kinase/phosphatase (IDHK/P) is a homodimeric enzyme which controls the oxidative metabolism of Escherichia coli, and exibits a high intrinsic ATPase activity. When subjected to electrophoresis under nonreducing conditions, the purified enzyme migrates partially as a dimer. The proportion of the dimer over the monomer is greatly increased by treatment with cupric 1,10 phenanthrolinate or 5,5'-dithio-bis(2-nitrobenzoic acid), and fully reversed by dithiothreitol, indicating that covalent dimerization is produced by a disulfide bond. To identify the residue(s) involved in this intermolecular disulfide-bond, each of the eight cysteines of the enzyme was individually mutated into a serine. It was found that, under nonreducing conditions, the electrophoretic patterns of all corresponding mutants are identical to that of the wild-type, except for the Cys67-->Ser which migrates exclusively as a monomer and for the Cys108-->Ser which migrates preferentially as a dimer. Furthermore, in contrast to the wild-type enzyme and all the other mutants, the Cys67-->Ser mutant still migrates as a monomer after treatment with cupric 1,10 phenanthrolinate. This result indicates that the intermolecular disulfide bond involves only Cys67 in each IDHK/P wild-type monomer. This was further supported by mass spectrum analysis of the tryptic peptides derived from either the cupric 1,10 phenanthrolinate-treated wild-type enzyme or the native Cys108-->Ser mutant, which show that they both contain a Cys67-Cys67 disulfide bond. Moreover, both the cupric 1,10 phenanthrolinate-treated wild-type enzyme and the native Cys108-->Ser mutant contain another disulfide bond between Cys356 and Cys480. Previous results have shown that this additional Cys356-Cys480 disulfide bond is intramolecular [Oudot, C., Jault, J.-M., Jaquinod, M., Negre, D., Prost, J.-F., Cozzone, A.J. & Cortay, J.-C. (1998) Eur. J. Biochem. 258, 579-585].

Cutting Edge: HLA-B27 Can Form a Novel β2-Microglobulin-Free Heavy Chain Homodimer Structure

AbstractHLA-B27 has a striking association with inflammatory arthritis. We show that free HLA-B27 heavy chains can form a disulfide-bonded homodimer, dependent on residue Cys67 in their extracellular α1 domain. Despite the absence of β2-microglobulin, HLA-B27 heavy chain homodimers (termed HC-B27) were stabilized by a known peptide epitope. HC-B27 complexes were recognized by the conformation-specific Ab W6/32, but not the ME1 Ab. Surface labeling and immunoprecipitation demonstrated the presence of similar W6/32-reactive free heavy chains at the surface of HLA-B27-transfected T2 cells. HC-B27 homodimer formation might explain the ability of HLA-B27 to induce spondyloarthropathy in β2-microglobulin-deficient mice.

Interleukin-10 immunoadhesin production by a replication-defective adenovirus

The present study examined the use of replication-defective adenovirus for in vitro production of an immunoadhesin. A recombinant adenovirus, rendered replication defective by deletion of the E1 gene, was constructed to contain the murine interleukin-10 gene fused in frame with the hinge, CH2, and CH3 domains of the murine immunoglobulin gamma 1 heavy chain constant region gene under the control of the human cytomegalovirus promoter. The resultant recombinant virus, Ad5.hCMV.mIL-10:HFc, was used to transduce several cell types. The expressed protein, mIL-10:HFc, is secreted as a disulfide-bonded homodimer. In vitro, a murine pro-B-cell line expressing transfected recombinant murine interleukin-10 receptor proliferated in response to purified mIL-10:HFc. The results obtained demonstrate the relative ease of production of an immunoadhesin using replication-defective adenovirus.

Functional role of CD101 on skin dendritic cells.

CD101 is a cell surface protein with a molecular weight of about 140 kDa and seven Ig-like loops in its extra-cellular domain, that was first described in our laboratory, using two different monoclonal antibodies (mAb), BB27 and BA27 (1,2). The two anti-CD101 mAbs BB27 and BA27 were generated by immunization of BALB/c mice with the CD4+CD8+ thymic clone B12 (3). These mAbs recognize CD101, a disulfide bonded-homodimer, on subsets of circulating T lymphocytes.

A Disulfide-bonded Dimer of the Golgi β-Galactoside α2,6-Sialyltransferase Is Catalytically Inactive yet Still Retains the Ability to Bind Galactose

The alpha2,6-sialyltransferase is a terminal glycosyltransferase localized in the trans Golgi and trans Golgi network. Here we show that 30% of the total rat liver Golgi alpha2,6-sialyltransferase forms a disulfide-bonded 100-kDa species that can be converted to the 50-kDa monomer form of the enzyme upon reduction. Limited proteolysis of both enzyme forms demonstrates that the 100-kDa species is a disulfide-bonded homodimer of the alpha2,6-sialyltransferase. The alpha2,6-sialyltransferase disulfide-bonded dimer is found in bovine liver Golgi membranes and in Golgi membranes prepared and solubilized in the presence of 100 mM iodoacetamide, suggesting that it is not unique to rat liver or formed aberrantly upon membrane lysis. The dimer form of the enzyme possesses no significant catalytic activity and has a much lower affinity for CDP-hexanolamine-agarose compared with the monomer form. In contrast, both the alpha2,6-sialyltransferase monomer and the disulfide-bonded dimer bind strongly to galactose and galactose-terminated substrates. These results suggest that the alpha2,6-sialyltransferase disulfide-bonded dimer lacks catalytic activity due to a weak affinity for its sugar nucleotide donor, CMP-NeuAc, and that this catalytically inactive form of the enzyme may act as a galactose-specific lectin in the Golgi.

Molecular and biological properties of an interleukin-1 receptor immunoadhesin

Overproduction of the cytokine interleukin 1 (IL-1) is an important factor in the pathogenesis of several autoimmune and inflammatory diseases. To develop a recombinant inhibitor of IL-1 with an extended pharmacologic half-life, we constructed an IL-1 receptor immunoadhesin (IL-1R-IgG), by fusing the extracellular domain of the type IL-1 receptor with the hinge and Fc regions of human IgG1 heavy chain. Transfected human 293 cells express IL-1R-IgG as a secreted, disulfide-bonded homodimer. The secreted protein contains an intact antibody Fc region, as indicated by immunoblotting, and a functional IL-1 receptor region, as indicated by ligand-blotting. Saturation binding analysis indicates an equilibrium dissociation constant ( K D ) of 350 pM for the binding of IL-1R-IgG to its ligand, IL-1 β. Kinetic analysis of the binding reveals an off rate of 0.1 min −1 and an on rate of 1.5 × 10 8min −1 M −1, yielding a calculated K D of 770 pM. These binding properties are similar to those of cell-surface type I IL-1 receptor. IL-1R-IgG is capable of inhibiting the biological activity of IL-1 β in vitro, as evidenced in a thymocyte proliferation assay. Pharmacokinetic analysis in mice indicates that IL-1R-IgG has a terminal half-life of 91 hr in the blood circulation. This half-life is markedly longer than the values reported for other recombinant inhibitors of IL-1 such as the IL-1 receptor antagonist or soluble IL-1 receptor. Thus, IL-1R-IgG may be useful for investigating the interaction of IL-1 with its receptor and the role of IL-1 in disease, as well as for potential intervention in pathological situations involving overproduction of IL-1.

Purification and Initial Characterization of Rat B49 Glial Cell Line‐Derived Neurotrophic Factor

The rat glial cell line B49 releases into its culture medium a potent neurotrophic factor that exhibits relative specificity for the dopaminergic neurons in dissociated cultures of rat embryonic midbrain. This factor is a heparin-binding, basic protein that is heterogeneously glycosylated and migrates on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and on molecular sieve chromatography with an apparent mass of approximately 33-45 kDa. The factor behaves like a disulfide-bonded homodimer, whose biological activity is destroyed by reduction of disulfide bonds but not by SDS-PAGE or reversed-phase (RP)-HPLC. The apparent mass of the monomer is approximately 16 kDa after deglycosylation with N-Glycanase. This factor has been purified 34,000-fold to apparent homogeneity by a combination of heparin-affinity chromatography, molecular sieving chromatography, SDS-PAGE, and RP-HPLC. The purified rat protein promotes the survival, morphological differentiation, and high-affinity dopamine reuptake of dopaminergic neurons in midbrain cultures, without obvious effects on total neurons or glia and without increasing high-affinity GABA or serotonin reuptake. The purified protein exhibits an EC50 in midbrain cultures at approximately 40 pg/ml, or 1 pM, and has unique amino-terminal and internal amino acid sequences. The sequences provide a basis for cloning and expression of the gene for rat and human glial cell line-derived neurotrophic factor (GDNF), confirming that the protein purified as reported here is GDNF.

Analysis of the Structure and Synthesis of GRP94, an Abundant Stress Protein of the Endoplasmic Reticulum

The synthesis and cotranslational modification of GRP94, an abundant, resident protein of the endoplasmic reticulum belonging to the hsp90 family of stress proteins, has been studied in transient expression studies in COS cells. A fraction of the expressed murine GRP94 was more highly glycosylated than normal, having a greater number of endoglycosidase H (Endo H)-sensitive oligosaccharide moieties than authentic GRP94. To understand better the basis for the appearance of the hyperglycosylated form and determine the acceptor sites that were used for this extra glycosylation, we have used in vitro mutagenesis techniques to construct a set of point mutants and deletion mutants of GRP94. Analysis of the expression of wild-type GRP94 and the mutant proteins has revealed that Asn-196 is the acceptor site used in normal glycosylation of GRP94 and that hyperglycosylation is dependent upon the level of expression of the GRP94 and is occurring at acceptor sites in the carboxy-terminal region of the protein. We have shown, in addition, that Cys-117 is involved in the formation of a disulfide-bonded homodimer of GRP94. Finally, analysis of deletions made from the amino terminus of the mature protein has demonstrated that these alterations change the pattern of usage of the remaining N-glycosylation sites in the mutants.

Characterization of CTLA-4 structure and expression on human T cells.

CTLA-4 is an adhesion receptor expressed on activated T cells. The amino acid sequence of CTLA-4 is related to CD28, and although the function of CTLA-4 remains unknown, it shares several features with CD28, including a common counter-receptor, B7, that is present on Ag-presenting cells. In a recent study we found that CD28 and CTLA-4 were coexpressed at the mRNA level on activated T cells but that only CD28 was expressed on resting T cells. Here we show that within the T cell population, CTLA-4 expression is restricted to the subset of T cells that also express cell surface CD28. CTLA-4 mRNA expression can be induced on quiescent T cells via phorbol ester-mediated activation of protein kinase C but not with calcium ionophore treatment alone. Phorbol ester-induced expression of CTLA-4 mRNA could be enhanced with calcium ionophore treatment, and treatment of cells in this manner resulted in a reciprocal decrease in expression of CD28 mRNA. Ligation of CD28 with monoclonal antibody also resulted in the specific and rapid induction of CTLA-4 mRNA. To study the expression of CTLA-4 at the protein level, a rabbit antiserum against a recombinant protein derived from CTLA-4 cDNA was generated. When activated T cells were labeled with [35S]methionine, the rabbit antiserum precipitated a 41- to 43-kDa protein from whole cell lysates. Similar results were found when detergent-soluble lysates from 125I surface-labeled resting and activated T cells were analyzed by SDS-PAGE. Surprisingly, under the conditions tested, CTLA-4 migrated primarily as a monomer at the cell surface, and could not be shown to exist as a disulfide-bonded homodimer or as a heterodimer consisting of CTLA-4 and CD28. These results suggest that B7 can bind to T cells via distinct receptor complexes consisting of either CD28 or CTLA-4, and that these complexes may potentially mediate distinct biologic functions. Further, the present results suggest that noncovalent interactions might mediate association of CTLA-4 and/or CD28 at the cell surface.

Identification of a structural glycoprotein of an RNA virus as a ribonuclease.

One of the three structural glycoproteins of classical swine fever virus (CSFV) is E0, a disulfide-bonded homodimer that induces virus-neutralizing antibodies and occurs in a virion-bound as well as a secreted form. E0 was shown to be similar to a family of fungal and plant ribonucleases. Purified E0 from CSFV-infected cells was a potent ribonuclease specific for uridine and inhibitable by zinc ions.

Crystallization and Preliminary X-ray Diffraction Studies of Recombinant Human Interleukin-5

Recombinant human interleukin-5 (rhIL-5) has been crystallized by the hanging drop vapor diffusion method using 0·1 M-Tris·HCL buffer (pH 8·5) containing 0·2 to 0·25 M-sodium acetate and 26 to 30% PEG 4000 at 22°C. The parallelepiped crystals belong to the space group C 2 with unit cell dimensions of a = 122·1 Å, b = 36·11 Å, c = 56·42 Å, β = 98·59°. They diffract to at least 2·0 Å resolution on a rotating anode X-ray source. The molecular mass weight of the protein and the volume of the unit cell suggest that the asymmetric unit contains one intermolecular disulfide-bonded homodimer.

Protection against endotoxic shock by a tumor necrosis factor receptor immunoadhesin.

Tumor necrosis factors (TNF) alpha and beta are structurally related cytokines that mediate a wide range of immunological, inflammatory, and cytotoxic effects. During bacterial infection of the bloodstream (sepsis), TNF-alpha induction by bacterial endotoxin is thought to be a major factor contributing to the cardiovascular collapse and critical organ failure that can develop. Despite antibiotic therapy, these consequences of sepsis continue to have a high mortality rate in humans. Here we describe a potent TNF antagonist, a TNF receptor (TNFR) immunoadhesin, constructed by gene fusion of the extracellular portion of human type 1 TNFR with the constant domains of human IgG heavy chain (TNFR-IgG). When expressed in transfected human cells, TNFR-IgG is secreted as a disulfide-bonded homodimer. Purified TNFR-IgG binds to both TNF-alpha and TNF-beta and exhibits 6- to 8-fold higher affinity for TNF-alpha than cell surface or soluble TNF receptors. In vitro, TNFR-IgG blocks completely the cytolytic effect of TNF-alpha or TNF-beta on actinomycin D-treated cells and is markedly more efficient than soluble TNFR (24-fold) or monoclonal anti-TNF-alpha antibodies (4-fold) in inhibiting TNF-alpha. In vitro, TNFR-IgG prevents endotoxin-induced lethality in mice when given 0.5 hr prior to endotoxin and provides significant protection when given up to 1 hr after endotoxin challenge. These results confirm the importance of TNF-alpha in the pathogenesis of septic shock and suggest a clinical potential for TNFR-IgG as a preventive and therapeutic treatment in sepsis.