Separate functional domains of human MD-2 mediate Toll-like receptor 4-binding and lipopolysaccharide responsiveness

Cellular responses to LPS are mediated by a cell surface receptor complex consisting of Toll-like receptor 4 (TLR4), MD-2, and CD14. MD-2 is a secreted protein that interacts with the extracellular portion of TLR4. Site-directed mutagenesis was used to identify the regions of human MD-2 involved in its ability to bind TLR4 and confer LPS responsiveness. A separate region of MD-2 was found to mediate each function. MD-2 binding to TLR4 was dependent on Cys(95) and Cys(105), which might form an intramolecular disulfide bond. Hydrophilic and charged residues surrounding this area, such as R90, K91, D100, and Y102, also contributed to the formation of the TLR4-MD-2 complex. A different region of MD-2 was found to be responsible for conferring LPS responsiveness. This region is not involved in TLR4 binding and is rich in basic and aromatic residues, several of which cooperate for LPS responsiveness and might represent a LPS binding site. Disruption of the endogenous MD-2-TLR4 complex by expression of mutant MD-2 inhibited LPS responses in primary human endothelial cells. Thus, our data indicate that MD-2 interaction with TLR4 is necessary but not sufficient for cellular response to LPS. Either of the two functional domains of MD-2 can be disrupted to impair LPS responses and therefore represent attractive targets for therapeutic interventions.     

Expression, localization, and functional activity of TL1A, a novel Th1-polarizing cytokine in inflammatory bowel disease

TL1A is a novel TNF-like factor that acts as a costimulator of IFN-gamma secretion through binding to the death domain-containing receptor, DR3. The aim of this study was to test the hypothesis that TL1A may play an important role in inflammatory bowel disease (IBD) by functioning as a Th1-polarizing cytokine. The expression, cellular localization, and functional activity of TL1A and DR3 were studied in intestinal tissue specimens as well as isolated lamina propria mononuclear cells from IBD patients and controls. TL1A mRNA and protein expression was up-regulated in IBD, particularly in involved areas of Crohn's disease (CD; p < 0.03 vs control). TL1A production was localized to the intestinal lamina propria in macrophages and CD4(+) and CD8(+) lymphocytes from CD patients as well as in plasma cells from ulcerative colitis patients. The amount of TL1A protein and the number of TL1A-positive cells correlated with the severity of inflammation, most significantly in CD. Increased numbers of immunoreactive DR3-positive T lymphocytes were detected in the intestinal lamina propria from IBD patients. Addition of recombinant human TL1A to cultures of PHA-stimulated lamina propria mononuclear from CD patients significantly augmented IFN-gamma production by 4-fold, whereas a minimal effect was observed in control patients. Our study provides evidence for the first time that the novel cytokine TL1A may play an important role in a Th1-mediated disease such as CD.     

CC chemokine receptor 2 expression in donor cells serves an essential role in graft-versus-host-disease

The complete repertoire of cellular and molecular determinants that influence graft-vs-host disease (GVHD) is not known. Using a well-established murine model of GVHD (B6-->bm12 mice), we sought to elucidate the role of the donor non-T cell compartment and molecular determinants therein in the pathogenesis of GVHD. In this model the acute GVHD-inducing effects of purified B6 wild-type (wt) CD4(+) T cells was inhibited by wt non-T cells in a dose-dependent manner. Paradoxically, unlike the chronic GVHD phenotype observed in bm12 mice transplanted with B6wt unfractionated splenocytes, bm12 recipients of B6ccr2-null unfractionated splenocytes developed acute GVHD and died of IFN-gamma-mediated bone marrow aplasia. This switch from chronic to acute GVHD was associated with increased target organ infiltration of activated CD4(+) T cells as well as enhanced expression of Th1/Th2 cytokines, chemokines, and the antiapoptotic factor bfl1. In vitro, ccr2(-/-) CD4(+) T cells in unfractionated splenocytes underwent significantly less activation-induced cell death than B6wt CD4(+) T cells, providing another potential mechanistic basis along with enhanced expression of bfl1 for the increased numbers of activated T cells in target organs of B6ccr2(-/-) splenocyte-->bm12 mice. Collectively, these findings have important clinical implications, as they implicate the donor non-T cell compartment as a critical regulator of GVHD and suggest that ccr2 expression in this cellular compartment may be an important molecular determinant of activation-induced cell death and GVHD pathogenesis.     

Novel natural peptide substrates for endopeptidase 24.15, neurolysin,and angiotensin-converting enzyme

Endopeptidase 24.15 (EC; ep24.15), neurolysin (EC; ep24.16), and angiotensin-converting enzyme (EC; ACE) are metallopeptidases involved in neuropeptide metabolism in vertebrates. Using catalytically inactive forms of ep24.15 and ep24.16, we have identified new peptide substrates for these enzymes. The enzymatic activity of ep24.15 and ep24.16 was inactivated by site-directed mutagenesis of amino acid residues within their conserved HEXXH motifs, without disturbing their secondary structure or peptide binding ability, as shown by circular dichroism and binding assays. Fifteen of the peptides isolated were sequenced by electrospray ionization tandem mass spectrometry and shared homology with fragments of intracellular proteins such as hemoglobin. Three of these peptides (PVNFKFLSH, VVYPWTQRY, and LVVYPWTQRY) were synthesized and shown to interact with ep24.15, ep24.16, and ACE, with K(i) values ranging from 1.86 to 27.76 microm. The hemoglobin alpha-chain fragment PVNFKFLSH, which we have named hemopressin, produced dose-dependent hypotension in anesthetized rats, starting at 0.001 microg/kg. The hypotensive effect of the peptide was potentiated by enalapril only at the lowest peptide dose. These results suggest a role for hemopressin as a vasoactive substance in vivo. The identification of these putative intracellular substrates for ep24.15 and ep24.16 is an important step toward the elucidation of the role of these enzymes within cells.     

Interaction of glycoprotein H of human herpesvirus 6 with the cellular receptor CD46

Human herpesvirus 6 (HHV-6) employs the complement regulator CD46 (membrane cofactor protein) as a receptor for fusion and entry into target cells. Like other known herpesviruses, HHV-6 encodes multiple glycoproteins, several of which have been implicated in the entry process. In this report, we present evidence that glycoprotein H (gH) is the viral component responsible for binding to CD46. Antibodies to CD46 co-immunoprecipitated an approximately 110-kDa protein band specifically associated with HHV-6-infected cells. This protein was identified as gH by selective depletion with an anti-gH monoclonal antibody, as well as by immunoblot analysis with a rabbit hyperimmune serum directed against a gH synthetic peptide. In reciprocal experiments, a monoclonal antibody against HHV-6 gH was found to co-immunoprecipitate CD46. Studies using monoclonal antibodies directed against specific CD46 domains, as well as engineered constructs lacking defined CD46 regions, demonstrated a close correspondence between the CD46 domains involved in the interaction with gH and those previously shown to be critical for HHV-6 fusion (i.e. short consensus repeats 2 and 3).     

Methyl-beta-cyclodextrin-enhanced solubilization and aerobic biodegradation of polychlorinated biphenyls in two aged-contaminated soils

The bioremediation of aged polychlorinated biphenyl (PCB)-contaminated soils is adversely affected by the low bioavailability of the pollutants. Randomly methylated-beta-cyclodextrins (RAMEB) were tested as a potential PCB-bioavailability-enhancing agent in the aerobic treatment of two aged-contaminated soils. The soils, contaminated by about 890 and 8500 mg/kg of Aroclor 1260 PCBs, were amended with biphenyl (4 g/kg), inorganic nutrients (to adjust their C:N ratio to 20:1), and variable amounts of RAMEB (0%, 0.5%, or 1.0% [w/w]) and treated in both aerobic 3-L solid-phase reactors and 1.5-L packed-bed loop reactors for 6 months. Notably, significant enhancement of the PCB biodegradation and dechlorination, along with a detectable depletion of the initial soil ecotoxicity, were generally observed in the RAMEB-treated reactors of both soils. RAMEB effects were different in the two soils, depending upon the treatment conditions employed, and generally increased proportionally with the concentration at which RAMEB was applied. RAMEB, which was slowly metabolized by the soil's aerobic microorganisms, was found to markedly enhance the occurrence of the indigenous aerobic, cultivable biphenyl-growing bacteria harboring genes homologous to those of two highly specialized PCB degraders (i.e., bphABC genes of Pseudomonas pseudoalcaligenes KF707 and bphA1A2A3A4BC1 genes of Rhodococcus globerulus P6) and chlorobenzoic acid-degrading bacteria as well as the occurrence of PCBs in the water phase of the soil reactors. These findings indicate that RAMEB enhanced the aerobic bioremediation of the two soils by increasing the bioavailability of PCBs and the occurrence of specialized bacteria in the soil reactors.     

Heterologous expression and characterization of mouse spermine oxidase

Polyamine oxidases are key enzymes responsible of the polyamine interconversion metabolism in animal cells. Recently, a novel enzyme belonging to this class of enzymes has been characterized for its capability to oxidize preferentially spermine and designated as spermine oxidase. This is a flavin adenine dinucleotide-containing enzyme, and it has been expressed both in vitro and in vivo systems. The primary structure of mouse spermine oxidase (mSMO) was deduced from a cDNA clone (Image Clone 264769) recovered by a data base search utilizing the human counterpart of polyamine oxidases, PAOh1. The open reading frame predicts a 555-amino acid protein with a calculated M(r) of 61,852.30, which shows a 95.1% identity with PAOh1. To understand the biochemical properties of mSMO and its structure/function relationship, the mSMO cDNA has been subcloned and expressed in secreted and secreted-tagged forms into Escherichia coli BL21 DE3 cells. The recombinant enzyme shows an optimal pH value of 8.0 and is able to oxidize rapidly spermine to spermidine and 3-aminopropanal and fails to act upon spermidine and N(1)-acetylpolyamines. The purified recombinant-tagged form enzyme (M(r) approximately 68,000) has K(m) and k(cat) values of 90 microm and 4.5 s(-1), respectively, using spermine as substrate at pH 8.0. Molecular modeling of mSMO protein based on maize polyamine oxidase three-dimensional structure suggests that the general features of maize polyamine oxidase active site are conserved in mSMO.