Robert A. Boykins

Human Notch-1 Inhibits NF-κB 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-κB/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-κB transcription factors is controlled by IκB proteins. Our previous work demonstrated that an intracellular, constitutively active form of human Notch-1/translocation-associated Notch homologue-1 (NotchIC) functions as an IκB molecule with specificity for the NF-κB p50 subunit and physically interacts with NF-κB in T cells. In the current study, we investigated the roles of different domains of NotchIC in the regulation of NF-κB-directed gene expression and NF-κB DNA binding activity. We found that NotchIC localizes to the nucleus and that a region in the N-terminal portion of NotchIC, not the six ankyrin repeats, is responsible for the inhibitory effects of Notch on NF-κB-directed gene expression and NF-κB DNA binding activity. The N-terminal portion of NotchIC inhibited p50 DNA binding and interacted specifically with p50 subunit, not p65 of NF-κB. The interaction between Notch and NF-κB 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-κB-mediated gene expression.

Structural Basis of Peroxide-mediated Changes in Human Hemoglobin A NOVEL OXIDATIVE PATHWAY

Hydrogen peroxide (H2O2) triggers a redox cycle between ferric and ferryl hemoglobin (Hb) leading to the formation of a transient protein radical and a covalent hemeprotein cross-link. Addition of H2O2 to highly purified human hemoglobin (HbA0) induced structural changes that primarily resided within β subunits followed by the internalization of the heme moiety within α subunits. These modifications were observed when an equal molar concentration of H2O2 was added to HbA0 yet became more abundant with greater concentrations of H2O2. Mass spectrometric and amino acid analysis revealed for the first time that βCys-93 and βCys-112 were oxidized extensively and irreversibly to cysteic acid when HbA0 was treated with H2O2. Oxidation of further amino acids in HbA0 exclusive to the β-globin chain included modification of βTrp-15 to oxyindolyl and kynureninyl products as well as βMet-55 to methionine sulfoxide. These findings may therefore explain the premature collapse of the β subunits as a result of the H2O2 attack. Analysis of a tryptic digest of the main reversed phase-high pressure liquid chromatography fraction revealed two α-peptide fragments (α128 - α139) and a heme moiety with the loss of iron, cross-linked between αSer-138 and the porphyrin ring. The novel oxidative pathway of HbA0 modification detailed here may explain the diverse oxidative, toxic, and potentially immunogenic effects associated with the release of hemoglobin from red blood cells during hemolytic diseases and/or when cell-free Hb is used as a blood substitute.

Multiple Antigen Peptide Vaccines against Plasmodium falciparum Malaria

ABSTRACT The multiple antigen peptide (MAP) approach is an effective method to chemically synthesize and deliver multiple T-cell and B-cell epitopes as the constituents of a single immunogen. Here we report on the design, chemical synthesis, and immunogenicity of three Plasmodium falciparum MAP vaccines that incorporated antigenic epitopes from the sporozoite, liver, and blood stages of the life cycle. Antibody and cellular responses were determined in three inbred (C57BL/6, BALB/c, and A/J) strains, one congenic (HLA-A2 on the C57BL/6 background) strain, and one outbred strain (CD1) of mice. All three MAPs were immunogenic and induced both antibody and cellular responses, albeit in a somewhat genetically restricted manner. Antibodies against MAP-1, MAP-2, and MAP-3 had an antiparasite effect that was also dependent on the mouse major histocompatibility complex background. Anti-MAP-1 (CSP-based) antibodies blocked the invasion of HepG2 liver cells by P. falciparum sporozoites (highest, 95.16% in HLA-A2 C57BL/6; lowest, 11.21% in BALB/c). Furthermore, antibodies generated following immunizations with the MAP-2 (PfCSP, PfLSA-1, PfMSP-142, and PfMSP-3b) and MAP-3 (PfRAP-1, PfRAP-2, PfSERA, and PfMSP-142) vaccines were able to reduce the growth of blood stage parasites in erythrocyte cultures to various degrees. Thus, MAP-based vaccines remain a viable option to induce effective antibody and cellular responses. These results warrant further development and preclinical and clinical testing of the next generation of candidate MAP vaccines that are based on the conserved protective epitopes from Plasmodium antigens that are widely recognized by populations of divergent HLA types from around the world.

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.

Immunogenicity of Well-Characterized Synthetic Plasmodium falciparum Multiple Antigen Peptide Conjugates

ABSTRACT 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 fromPlasmodium 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 × 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.

O‐raffinose crosslinked hemoglobin lacks site‐specific chemistry in the central cavity: Structural and functional consequences of β93Cys modification

Reacting human deoxyHbA0 with oxidized raffinose (O‐raffinose), a trisaccharide, results in a low oxygen affinity “blood substitute,” stabilized in a noncooperative T‐conformation and possesses readily oxidizable rhombic heme. In this study, we fractionated the O‐raffinose–modified HbA0 heterogeneous polymer (O‐R‐PolyHbA0) into six distinct fractions with a molecular weight distribution ranging from 64 to ∼600 kDa using size‐exclusion chromatography (SEC). Oxygen equilibrium and kinetics binding parameters of all fractions were nearly identical, reflecting a lack of heterogeneity in ligand binding properties among O‐R‐PolyHbA0 species (Hill coefficient n equal to 1.0). Several mass spectrometry techniques were used to evaluate undigested and digested HbA0, O‐R‐PolyHbA0, and O‐R‐PolyHbA0 fractions. Proposed sites of intramolecular crosslinking (i.e., β1Lys82, β2Lys82, and β1Val1) were not found to be the predominant site of crosslinking within the central cavity. Intermolecular crosslinking with O‐raffinose results in no discernible site of amino acids modifications with the exception of β93Cys and α104Cys. Based on accessible surface area (ASA) calculations in intact deoxyHbA0, slight conformational changes are required to allow for the S on α104Cys to be modified during the reaction with O‐raffinose or its partially oxidized product(s). The stabilization of HbA0 in the T‐conformation may not be a direct correlate of O‐raffinose induced changes, but an indirect consequence of changing hydration in the water‐filled central cavity and/or the distal heme pocket leading in the latter case to accelerated iron oxidation. Structural data presented here when taken together with the oxidative instability of O‐R‐PolyHbA0 may provide some basis for the reported toxicity of this oxygen carrier. Proteins 2005. Published 2005 Wiley‐Liss, Inc.

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.

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.

Comparison of protein structure and genomic structure of human, rabbit, and limulus C-reactive proteins: Possible implications for function and evolution

The primary structures of human, rabbit, and Limulus C-reactive proteins (CRPs) have been compared by a computer program. Based on these data, a PAMs matrix (accepted point mutation per 100 residues) was constructed to generate topologies for the three proteins. Five trees with the shortest absolute length were generated, but only one positive tree was found. Using the relatively well-established distance between human and rabbit of 150 million years, we calculate that human and Limulus CRPs diverged at least 500 million years ago. The data indicate that the amino acid sequence indentity between Limulus CRPs and their mammalian counterparts is about 25%, strongly suggesting that human CRP, rabbit CRP, and Limulus CRPs share common ancestral genes. There are two highly conserved regions in the primary structures among the CRPs. Residues 52–67 in Limulus CRP and residues 51–66 in human CRP show identity in 10 of 16 positions, with 3 additional conservative replacements. This region of the molecule is thought to be involved in the binding of phosphorylcholine ligand. Residues 139–153 in Limulus CRP and residues 133–147 in human CRP show identity in 9 of 15 positions, with 5 additional conservative replacements. The biological function of this stretch of amino acid sequence is thought to be associated with the CA2+ binding of the CRPs.

Antibodies against a multiple-peptide conjugate comprising chemically modified human immunodeficiency virus type-1 functional Tat peptides inhibit infection

We demonstrated recently that selective side-chain modification of functional cysteine-rich (Tat(21-40)) and arginine-rich (Tat(53-68)) domains of the HIV-1 Tat protein blocks pathogenic activities of these peptides while retaining their immunological characteristics. In the present study, we have synthesized a multiple-peptide conjugate system comprising modified Tat(21-40) and Tat(53-68) peptides (HIV-1-Tat-MPC). Immunization of mice with this highly homogeneous 10.7 kDa HIV-1-Tat-MPC synthetic construct induced an effective immune response in mice. The antibodies generated against HIV-1-Tat-MPC efficiently suppressed Tat-induced viral replication and significantly reduced HIV-associated cytopathic effects in human monocytes. These results indicate that epitope-specific antibodies directed against functional sites of Tat protein using non-pathogenic peptides inhibit HIV pathogenesis. The HIV-1-Tat-MPC, therefore, has potential for the development of a safe, effective, and economical therapeutic vaccine to reduce the progression of HIV infection.