Regulation of the receptor specificity and function of the chemokine RANTES (regulated on activation, normal T cell expressed and secreted) by dipeptidyl peptidase IV (CD26)-mediated cleavage.

CD26 is a leukocyte activation marker that possesses dipeptidyl peptidase IV activity but whose natural substrates and immunological functions have not been clearly defined. Several chemo-kines, including RANTES (regulated on activation, normal T cell expressed and secreted), have now been shown to be substrates for recombinant soluble human CD26. The truncated RANTES(3-68) lacked the ability of native RANTES(1-68) to increase the cytosolic calcium concentration in human monocytes, but still induced this response in macrophages activated with macrophage colony-stimulating factor. Analysis of chemokine receptor messenger RNAs and patterns of desensitization of chemokine responses showed that the differential activity of the truncated molecule results from an altered receptor specificity. RANTES(3-68) showed a reduced activity, relative to that of RANTES(1-68), with cells expressing the recombinant CCR1 chemokine receptor, but retained the ability to stimulate CCR5 receptors and to inhibit the cytopathic effects of HIV-1. Our results indicate that CD26-mediated processing together with cell activation-induced changes in receptor expression provides an integrated mechanism for differential cell recruitment and for the regulation of target cell specificity of RANTES, and possibly other chemokines.

Synthesis and construction of a novel multiple peptide conjugate system: strategy for a subunit vaccine design.

We describe the design and synthesis of a novel well characterized multi-peptide conjugate (MPC) system containing antigens from human malaria parasite and the Tat protein of HIV type-1 (HIV-1-Tat). Construction of the MPC utilizes Fmoc solid-phase peptide synthesis coupled with solution chemistry. In the first phase, a core template that serves as primary anchor for the synthesis and attachment of multiple antigens is synthesized. Serine(trityl) and multiple lysine branches with epsilon groups blocked during chain assembly are incorporated forming a tetrameric core. Cysteine whose side chain thiol serves to couple haloacetyl or S-protected haloacetyl peptides is added to complete assembly of the core template. Modification to the coupling solvent, addition of key amino acid derivatives (N-[1-hydroxy-4-methoxybenzyl]) in the peptide sequence allows the synthesis of base peptides on the core template with molecular mass greater than 7500 kDa. Base peptides are then reacted with high performance liquid chromatography purified haloacetyl peptides to generate multiple peptide conjugates with molecular masses of 10 to 13 kDa. MPC constructs thus formed are further characterized by matrix assisted laser desorption-time of flight mass spectroscopy (MALDI-MS), amino acid analysis, size exclusion chromatography, and SDS-polyacrylamide gel electrophoresis (PAGE). To our knowledge, this is the first report describing a chemically well defined multiple conjugate system with potential for development of synthetic subunit vaccines.

Immunization with a novel HIV-1-Tat multiple-peptide conjugate induces effective immune response in mice.

We report here a novel, highly immunogenic synthetic, multiple-peptide conjugate comprising functional domains Tat(21-40) and Tat(53-68) from HIV-1 group M plus Tat(9-20) from HIV-1 group O of the HIV-Tat protein (HIV-1-Tat-MPC). Vaccination of mice with HIV-1-Tat-MPC induced an effective immune response to all three functional domains. The anti-HIV-1-Tat-MPC antibodies efficiently inhibited Tat-induced viral activation in monocytes infected with HIV(Ba-L) as well as with various clinical HIV-1 isolates, and reduced Tat-mediated cytopathicity in infected cells by 60-75%. Our results indicate that anti-HIV-1-Tat-MPC antibodies inhibit viral pathogenesis, possibly by blocking functional determinants of Tat and disrupting autocrine and paracrine actions of secreted Tat protein. This epitope-specific, synthetic Tat construct may, therefore, provide a subunit AIDS vaccine candidate for inducing an effective immunoprophylaxis response to reduce progression of HIV infection.

Micro-analysis of amino acids.

An automated amino acid analyzer has been developed for the analysis of amino acids with the sensitivity at the 10-100 pmol level except for proline which requires greater than 50 pmol. omicron-Phthalaldehyde, in the presence of 2-mercaptoethanol, is used for the fluorometric detection of amino groups (Roth, M. (1971) Anal. Chem. 43, 880-882). A post-column reaction of the amino acid with sodium hypochlorite (Bohlen, P. and Mellet, M. (1979) Anal. Biochem. 94, 313-321) gives oxidation products amenable to detection with omicron-phthalaldehyde. The instrument uses high-performance liquid chromatographic pumps capable of micro-flow rates with a minimum pulsation. The method is suitable for routine analyses of amino acids at picomole levels with reproducibility and accuracy comparable to the ninhydrin-based amino acid analysis.

Automatic analysis of neutral sugar components in glycoproteins and complex carbohydrates.

A simple and sensitive automated method has been developed to separate monosaccharides by reverse-phase chromatography using the amino acid analyzer column in the Li+ form with 90% alcohol as eluent. The column can be regenerated with LiBr and 90% ethanol to achieve highly reproducible resolution of sugar components. Glycoproteins and polysaccharides are hydrolyzed by a combination of methanesulfonic acid/Dowex 50 X 8 resin in water. Glucose and mannose are distinguished by treatment of the hydrolysate with glucose oxidase prior to analysis.

Structural and immunological studies of the Escherichia coli K7 (K56) capsular polysaccharide.

The structure of the Escherichia coli K7 capsular polysaccharide has been investigated by a combination of chemical and spectroscopic methods. The Structure of the repeating unit of the polymer was found to be goes to 3)-beta-D-ManNAcA-(1 leads to 4)-beta-D-Glc-(1 goes to ; the O-6 atom of the D-glucosyl residue in the repeating unit is acetylated. The K7 polysaccharide is cross-reactive with the Streptococcus pneumoniae type 3 polysaccharide, the structure of which had previously been determined; our n.m.r. studies of the S. pneumoniae type 3 polysaccharide are in accord with this structure. The E. coli K7 and K56 capsular antigens have been shown by serology and 13C-n.m.r. spectroscopy to be identical.

Structural and immunological studies of the Haemophilus influenzae type d capsular polysaccharide.

Employing a combination of chemical and spectroscopic techniques, the structure of the Haemophilus influenzae type d capsular polysaccharide was found to be leads to 4)-beta-D-GlcNAc-(1 leads to 3)-beta-D-ManNAcA-(1 leads to. L-Alanine, L-serine, and L-threonine, in the molar ratios of approximately 1.0:1.0:0.3, were linked to C-6 of the D-mannosyluronic residue as amides; the (serine + alanine + threonine) to ManNAcA ratio was approximately 0.95:1.0. Removal of the amino acids by mild hydrolysis with sodium hydroxide resulted in a material that was cross-reactive with the native, type d polymer. The base-treated, type d polysaccharide was not observed to cross-react with either the H. influenzae type e or Escherichia coli K7 capsular polysaccharide, both of which are structurally similar to type d.

Modification of glyceraldehyde-3-phosphate dehydrogenase, EC 1.2.1.12, isolated from rainbow trout during acclimation at 5 or 15 degrees C--II. Biochemical characterization of G3PDH muscle isolates.

G3PDH was isolated from the lateral muscle of rainbow trout (Salmo gairdneri) acclimated at 5 degrees C (cold) and 15 degrees C (warm). No differences were found in muscle concentration, molecular weights, isoelectric focusing patterns, amino acid compositions or peptide maps between cold and warm isolates. Cold and warm G3PDH contained mannose in variable concentration but no other prosthetic groups.

Hydrolysis of proteins and peptides in a hermetically sealed microcapillary tube: high recovery of labile amino acids.

A method for the hydrolysis of peptides and proteins in a hermetically sealed microcapillary tube has been developed. The method is based on the concept that oxidative degradation of labile amino acids during acid hydrolysis of proteins and peptides at high temperature can be reduced to a minimum by limiting the ratio of air to liquid (v/v, less than 1:10) in a microcapillary tube. Furthermore, the physical constraints imposed by the capillary tube will restrict the exposure of the protein solution to air at a very limited area at the meniscus of the liquid. This method eliminates the necessity of time-consuming sealing under vacuum and/or flushing with nitrogen to remove oxygen in the hydrolysis tube. High recovery of labile amino acids can be obtained in a reproducible manner. Because of the simplicity and high reproducibility of the method described, it could be the method of choice for the hydrolysis of protein and peptide intended for quantitative amino acid analysis. Performic acid oxidation is performed at 50 degrees C for 10 min instead of 4 to 20 h at 0 degrees C to achieve an equally good yield of cysteic acid and methionine sulfone from peptides and proteins.

Heterogeneity and variation among Neisseria meningitidis lipopolysaccharides.

Eight immunotype lipopolysaccharides (LPSs) of Neisseria meningitidis were prepared by the phenol-water procedure and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and sugar analyses. By SDS-PAGE and a highly sensitive silver strain. N. meningitidis LPSs from cells grown in tryptic soy broth were shown to contain one or two predominant components and a few minor, somewhat higher-molecular-weight components. The molecular sizes of the two predominant components were approximately the same as those of two E. coli rough-type LPSs, one with a complete core and the other with an incomplete core. The molecular weight of the major LPS component varied somewhat among different immunotypes but was estimated to be in the range of 4,200 to 5,000. By sugar analyses, the eight immunotype LPSs were different in their monosaccharide compositions. All contained glucose, galactose, heptose, glucosamine, and 2-keto-3-deoxyoctonate, but in different molar ratios. The growth of N. meningitidis in tryptic soy broth under different levels of aeration resulted in a change in the two major LPS components seen on the SDS-PAGE gel. High aeration increased the amount of the smaller component, whereas low aeration increased the amount of the larger component. Sugar analyses of LPSs from high and low aeration indicated that the larger LPS component contained more galactose residues per molecule. Use of different media for cell growth may also result in small, but noticeable, variations in the LPS components and in the galactose content of the LPS. The observed heterogeneity of N. meningitidis LPS may explain why many strains of N. meningitidis appear to possess more than one immunotype.

Chemical synthesis and characterization of chemokine RANTES and its analogues.

RANTES, a polypeptide of 68 amino acid residues, is a member of the C-C chemokine subfamily including other monocyte chemoattractants such as MIP-1alpha, MIP-1beta, MCP-1, MCP-2 and MCP-3. To provide a chemically defined RANTES in quantity suitable for structure-function studies, RANTES and its analogues were synthesized, using a modified solid-phase chemistry approach. The fully protected RANTES and RANTES(3-68) were assembled by automated solid-phase methodology using Fmoc chemistry. Deprotection and cleavage of the resin bound peptides yielded crude peptides, which were then folded and further purified by reverse-phase HPLC. The chemically synthesized RANTES with its identity and purity established, was found to be immunochemically and functionally indistinguishable from the recombinant human RANTES. RANTES and its analog, RANTES(3-68), have recently been used as the substrate in the study of dipeptidyl peptidase IV (CD26)-mediated processing of RANTES and its effect on receptor specificity.

Effect of culture conditions on the structure of Streptococcus pneumoniae type 19A(57) capsular polysaccharide.

The structural modifications and immunochemical activities of several Streptococcus pneumoniae type 19A polysaccharide (PS) preparations have been studied by sugar compositional analysis and immunodiffusion. The 19A PS preparations Lab-A-1 and Lab-A-3 and one PS isolated from 19A strain OB contained fucose (Fuc) and galactose (Gal) in addition to rhamnose (Rha) and glucose (Glc). In contrast, 19A PSs Lab-A-2 and Lab-B contained only Rha and Glc. Despite their different sugar compositions, these 19A preparations appeared to be identical in serologic activity as measured by immunodiffusion with rabbit 19A and 19F antisera. The 19A PS Lab-A-1 was separated into three fractions by DEAE-Sepharose CL-6B column chromatography with a NaCl gradient. Fraction II was the major peak with a yield of 72.9%. Fraction Ia contained Fuc and Gal, while fraction II contained Fuc, Gal, Rha, and Glc. Fractions Ia and Ib did not react with rabbit 19A antiserum. In contrast, 19A PS Lab-A-2 displayed only one peak, which was eluted by a NaCl gradient (0 to 0.6 M NaCl), and contained only Rha and Glc. The 19A PSs prepared from Lab-A and Centers for Disease Control (CDC) strains and grown in pneumococcal inoculum medium (PIM) and modified Holt medium were chromatographed on a DEAE-Sepharose CL-6B column, and the separated fractions were examined for their sugar composition. The fractions obtained from the 19A PSs Lab-A-PIM and CDC-PIM exhibited four sugar components, as observed for the PS Lab-A-1, while the separated fractions from the 19A PSs Lab-A-Holt and CDC-Holt displayed two sugar components, a pattern similar to that of PS Lab-A-2. Thus, the sugar compositions of 19A PS appeared to vary according to the type of culture medium used to grow the 19A organisms.

The amino acid sequence of Limulus C-reactive protein. Evidence of polymorphism.

The amino acid sequence, the positions of the disulfide bonds, and the site of glycosylation for the three subunits of Limulus C-reactive proteins (CRPs) 1.1, 1.4, and 3.3 have been established. The three subunits were shown to exist approximately in equimolar amount and are tightly associated. The hexagonal structure of Limulus CRP, as revealed by electron microscopic studies of Fernandez-Moran et al. (Fernandez-Moran, H., Marchalonis, J., and Edelman, G. M. (1968) J. Mol. Biol. 32, 467-469) might consist of two each of the subunits. The three subunits share an identical amino-terminal sequence of 44 residues and a carboxyl-terminal sequence from residues 206 to 218. Microheterogeneity exists to the extent of 10 to 11% for the entire protein. The positions of 6 half-cystines that form the three disulfide bonds and the site of glycosylation are constant in all subunits. Sequence analyses of peptides derived from enzymatic and chemical cleavages of affinity purified Limulus CRP indicate that subunits other than the three mentioned above exist in the hemolymph. Limulus CRP is therefore polymorphic. Topological analyses of Limulus CRPs, human CRP, rabbit CRP, human amyloid P-component, and Syrian hamster female protein indicate that the seven proteins may originate from the same ancestral gene. Using the topological data generated from the amino acid sequences of the proteins, we calculate that human and Limulus CRPs diverged about 500 million years ago. This figure is in general agreement with the evolutionary distance postulated by anthropological estimation of 400-500 million years.

Inhibition of interferon-gamma signaling by Leishmania donovani.

Leishmania infection causes marked down-regulation of interferon (IFN)-gamma-induced gene activity in macrophages, but the mechanism of the blockade has not been fully defined. The IFN-gamma signal transduction pathway was analyzed in Leishmania donovani-infected phorbol-differentiated U937 human promonocytic cells. IFN-gamma stimulation induced marked phosphorylation of its own receptor (IFN-gammaR)-alpha chain. Phosphorylation of the receptor subunit was significantly inhibited after 24 h of infection with the parasite, apparently because of decreased amounts of the receptor subunit. Formation of the IFN-gammaR complex, as assessed by tyrosine phosphorylation and association of Jak2, was strongly inhibited in cells infected for 24 h. Inhibition of the IFN-gammaR complex formation correlated with inhibition of STAT1alpha binding to the IFN-gamma response region. Pretreatment with purified parasite lipophosphoglycan before IFN-gamma stimulation had no effect on tyrosine phosphorylation. Thus, inhibition of tyrosine phosphorylation of the IFN-gammaR-alpha chain and subsequent signal transduction are most likely due to the decreased amount of IFN-gammaR-alpha protein after infection.

Human Notch-1 inhibits NF-kappa B activity in the nucleus through a direct interaction involving a novel domain.

Notch participates in diverse cell fate decisions throughout embryonic development and postnatal life. Members of the NF-kappaB/Rel family of transcription factors are involved in the regulation of a variety of genes important for immune function. The biological activity of the NF-kappaB transcription factors is controlled by IkappaB proteins. Our previous work demonstrated that an intracellular, constitutively active form of human Notch-1/translocation-associated Notch homologue-1 (Notch(IC)) functions as an IkappaB molecule with specificity for the NF-kappaB p50 subunit and physically interacts with NF-kappaB in T cells. In the current study, we investigated the roles of different domains of Notch(IC) in the regulation of NF-kappaB-directed gene expression and NF-kappaB DNA binding activity. We found that Notch(IC) localizes to the nucleus and that a region in the N-terminal portion of Notch(IC), not the six ankyrin repeats, is responsible for the inhibitory effects of Notch on NF-kappaB-directed gene expression and NF-kappaB DNA binding activity. The N-terminal portion of Notch(IC) inhibited p50 DNA binding and interacted specifically with p50 subunit, not p65 of NF-kappaB. The interaction between Notch and NF-kappaB indicates that in addition to its role in the development of the immune system, Notch-1 may also have critical functions in the immune response, inflammation, viral infection, and apoptosis through control of NF-kappaB-mediated gene expression.

Purification and characterization of a peptidoglycan-associated lipoprotein from Haemophilus influenzae.

We have purified to homogeneity a peptidoglycan-associated protein from Haemophilus influenzae. Our purification process used differential extraction of cell envelopes with nondenaturing detergents. Solubilization of this protein was accomplished by heating a peptidoglycan-enriched subcellular fraction in the presence of one of several nondenaturing detergents at 55-60 degrees C. The purified protein migrated as a single band, with a Mr approximately 15,000, following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This protein contains covalently linked fatty acids, is rich in tyrosine, but lacks methionine and tryptophan. Amino acid analysis also revealed the presence of glycerylcysteine, which has been shown to be the site of fatty acylation in other bacterial lipoproteins. Over 87% of the primary structure has been determined by sequencing high pressure liquid chromatography purified fragments derived from several endoproteinase digests. This protein belongs to a family of proteins, known as peptidoglycan associated lipoproteins, which appear to be components of the outer membranes of most Gram-negative bacteria.

Chemical evidence for covalent linkages of a semi-synthetic glycoconjugate vaccine for Haemophilus influenzae type B disease.

We have defined the nature of the covalent linkages in a Haemophilus influenzae type b oligosaccharide-CRM197 conjugate vaccine, designated HbOC. The conjugate was acid hydrolyzed to release a novel amino-acid derivative, N epsilon-(2-hydroxyethyl)lysine (OHEt-Lys), identifiable with an amino-acid analyzer. This amino-acid derivative was formed by reduction of Schiff bases formed between H. influenzae type b oligosaccharides (HbO) and the lysyl epsilon-amino groups of CRM197 (a non-toxic, cross-reactive variant of diphtheria toxin), followed by acid hydrolysis of HbOC. Quantification of OHEt-Lys per CRM197 molecule allowed the determination of a covalency ratio, a useful parameter for evaluating the stoichiometry and consistency of HbOC preparations. Covalent association between HbO and CRM197 was also demonstrated by the coincidence of immunoreactivity of gel-electrophoresed HbOC on a Western blot probed with anti-CRM197 and anti-saccharide antisera.

Biology of malarial liver stages: implications for vaccine design.

The molecular events controlling sporozoite invasion and exo-erythrocytic (EE) development within hepatocytes are largely not understood, and EE parasites are probably better defined immunologically than biologically. The observation that the Plasmodium falciparum sporozoite antigen TRAP (thrombospondin-related anonymous protein) contains multiple adhesive domains that recognize endothelial and hepatocyte receptors indicates that, like leucocyte passage across capillaries, sporozoite invasion probably involves a co-ordinated interaction between sporozoite and hepatic molecules. The parallel with leucocyte extravasation is strengthened by the finding that TRAP contains a functional, integrin-like, I domain. EE parasites are an important target of immunity elicited by irradiated sporozoites, and much current effort is focused on developing malaria vaccines targeting EE parasites. Only one EE-specific antigen, liver-stage antigen 1 (LSA-1), is known to be expressed during EE development and may contribute to protective immunity elicited by irradiated P. falciparum sporozoites. In a study in Papua New Guinea, resistance to P. falciparum infection correlated with CD8+ T-cell interferon-gamma responses to an LSA-1 epitope that contains an HLA A11-restricted sequence. Since A11 is > 40% frequent in this population it is reasonable to suggest that, as with B53 responses to LSA-1 in The Gambia, P. falciparum has driven genetic selection of certain HLA haplotypes, as proposed by Haldane nearly 50 years ago. LSA-1 is thus an important vaccine candidate, and is being expressed in bacterial and phage vectors.

Immunogenicity of well-characterized synthetic Plasmodium falciparum multiple antigen peptide conjugates.

Given the emerging difficulties with malaria drug resistance and vector control, as well as the persistent lack of an effective vaccine, new malaria vaccine development strategies are needed. We used a novel methodology to synthesize and fully characterize multiple antigen peptide (MAP) conjugates containing protective epitopes from Plasmodium falciparum and evaluated their immunogenicity in four different strains of mice. A di-epitope MAP (T3-T1) containing two T-cell epitopes of liver stage antigen-1 (LSA-1), a di-epitope MAP containing T-cell epitopes from LSA-1 and from merozoite surface protein-1, and a tri-epitope MAP (T3-CS-T1) containing T3-T1 and a potent B-cell epitope from the circumsporozoite protein central repeat region were tested in this study. Mice of all four strains produced peptide-specific antibodies; however, the magnitude of the humoral response indicated strong genetic restriction between the different strains of mice. Anti-MAP antibodies recognized stage-specific proteins on the malaria parasites in an immunofluorescence assay. In addition, serum from hybrid BALB/cJ x A/J CAF1 mice that had been immunized with the tri-epitope MAP T3-CS-T1 successfully inhibited the malaria sporozoite invasion of hepatoma cells in vitro. Spleen cells from immunized mice also showed a genetically restricted cellular immune response when stimulated with the immunogen in vitro. This study indicates that well-characterized MAPs combining solid-phase synthesis and conjugation chemistries are potent immunogens and that this approach can be utilized for the development of subunit vaccines.

Cutting edge: a short polypeptide domain of HIV-1-Tat protein mediates pathogenesis.

HIV-1 encodes the transactivating protein Tat, which is essential for virus replication and progression of HIV disease. However, Tat has multiple domains, and consequently the molecular mechanisms by which it acts remain unclear. In this report, we provide evidence that cellular activation by Tat involves a short core domain, Tat21-40, containing only 20 aa including seven cysteine residues highly conserved in most HIV-1 subtypes. Effective induction by Tat21-40 of both NF-kappaB-mediated HIV replication and TAR-dependent transactivation of HIV-long terminal repeat indicates that this short sequence is sufficient to promote HIV infection. Moreover, Tat21-40 possesses potent angiogenic activity, further underscoring its role in HIV pathogenesis. These data provide the first demonstration that a 20-residue core domain sequence of Tat is sufficient to transactivate, induce HIV replication, and trigger angiogenesis. This short peptide sequence provides a potential novel therapeutic target for disrupting the functions of Tat and inhibiting progression of HIV disease.