Inactivation of thrombomodulin by ionizing radiation in a cell-free system: possible implications for radiation responses in vascular endothelium.

Normal tissue radiation injury is associated with loss of vascular thromboresistance, notably because of deficient levels of endothelial thrombomodulin (TM). TM is located on the luminal surface of most endothelial cells and has critical anticoagulant and anti-inflammatory functions. Chemical oxidation of a specific methionine residue (Met388) at the thrombin-binding site in TM reduces its main functional activity, i.e., the ability to activate protein C. We examined whether exposure to ionizing radiation affects TM in a similar manner. Full-length recombinant human TM, a construct of epidermal growth factor-like domains 4-6, which are involved in protein C activation, and a synthetic peptide containing the methionine of interest were exposed to gamma radiation in a cell-free system, i.e., a system not confounded by TM turnover or ectodomain shedding. The influence of radiation on functional activity was assessed with the protein C activation assay; formation of a TM-thrombin complex was assessed with surface plasmon resonance (Biacore), and oxidation of Met388 was assessed by HPLC and confirmed by mass spectroscopy. Exposure to radiation caused a dose-dependent reduction in protein C activation, impaired TM-thrombin complex formation, and oxidation of Met388. These results demonstrate that ionizing radiation adversely affects the TM molecule. Our findings may have relevance to normal tissue toxicity in clinical radiation therapy as well as to the development of radiation syndromes in the non-therapeutic radiation exposure setting.

Multiple antigenic mimotopes of HIV carbohydrate antigens: relating structure and antigenicity.

Carbohydrate mimetic peptides are designable, and they can carry T-cell epitopes and circumvent tolerance. A mimic-based human immunodeficiency virus (HIV) vaccine can be a viable alternative to carbohydrate-based antigens if the diversity of epitopes found on gp120 can be recapitulated. To improve existing mimics, an attempt was made to study the structural correlates of the observed polyspecificity of carbohydrate mimetic peptides based on the Y(P/R)Y motif in more detail. A carbohydrate mimetic peptide, D002 (RGGLCYCRYRYCVCVGR), bound a number of lectins with different specificities. Although this peptide reacted strongly with both lotus and concanavalin A (ConA) lectins, it bound to lotus stronger than ConA. By varying the central motif RYRY, five versions were produced in multiple antigen peptide format, and their avidity for lotus and ConA lectins was tested by surface plasmon resonance. Although the kinetic parameters were similar, the version based on the sequence YPYRY had an optimal affinity for both lectins as well as improved avidity for wheat germ agglutinin and phytohemagglutinin. Thus, as far as lectin specificity is concerned, YPYRY had improved multiple antigenic properties. Both RYRY and YPYRY precipitated antibodies from human IgG for intravenous use that bound to gp120 in vitro and immunoprecipitated gp120 from transfected CHO-PI cells. Thus, Y(P/R)Y motifs mimic multiple carbohydrate epitopes, many of which are found on HIV, and preimmune human IgG antibodies that bind to HIV carbohydrates cross-react to a comparable extent with both RYRY and YPYRY carbohydrate mimetic peptides.

Targeting carbohydrate antigens in HIV vaccine development.

Peptide mimotopes provide a strategy to augment human immunodeficiency virus 1 (HIV-1) specific carbohydrate reactive immune responses. Their antigenic and immunological properties will depend on the optimization of motif clustering and multimerization. We observe that structural variants of the same mimetic motif, linear versus cyclic, can be used to tune the properties of the antibodies elicited. The expansion of the database of mimotope sequence motifs can be increased by analyzing structures that bind to HIV directed monoclonal antibody 2G12 and the lectin Concanavalin A (Con A), fostering new mimotope designs. Such analysis indicates that these reagents bind to subsets of mannosyl antigens on the envelope (env) protein.