Increased immunogenicity of an anchor-modified tumor-associated antigen is due to the enhanced stability of the peptide/MHC complex: implications for vaccine design

The use of "anchor-fixed" altered peptide ligands is of considerable interest in the development of therapeutic vaccines for cancer and infectious diseases, but the mechanism by which successful altered peptide ligands elicit enhanced immunity is unclear. In this study, we have determined the crystallographic structure of a major tumor rejection Ag, gp100(209-217), in complex with the HLA-A*0201 (HLA-A2) molecule, as well as the structure of a modified version of the peptide which substitutes methionine for threonine at position 2 (T2M; gp100(209-2M)). The T2M-modified peptide, which is more immunogenic in vitro and in vivo, binds HLA-A2 with a approximately 9-fold greater affinity and has a approximately 7-fold slower dissociation rate at physiological temperature. Within the limit of the crystallographic data, the T2M substitution does not alter the structure of the peptide/HLA-A2 complex. Consistent with this finding, in peripheral blood from 95 human subjects, we were unable to identify higher frequencies of T cells specific for either the native or modified peptide. These data strongly support the conclusion that the greater immunogenicity of the gp100(209-2M) peptide is due to the enhanced stability of the peptide/MHC complex, validating the anchor-fixing approach for generating therapeutic vaccine candidates. Thermodynamic data suggest that the enhanced stability of the T2M-modified peptide/HLA-A2 complex is attributable to the increased hydrophobicity of the modified peptide, but the gain due to hydrophobicity is offset considerably by the loss of a hydrogen bond made by the native peptide to the HLA-A2 molecule. Our findings have broad implications for the optimization of current vaccine-design strategies.     

Cellular mechanism for potentiation of Ca2+-mediated Cl- secretion by the flavonoid baicalein in intestinal epithelia

Flavonoids belong to a large group of plant polyphenols that are consumed daily in large amounts. Our previous findings have shown that baicalein, a major flavonoid derived from the medicinal herb Scutellariae radix, induces Cl(-) secretion across rat colonic mucosa. The current study examines the effect of baicalein on Cl(-) secretion in human colonic epithelial (T84) cells and its interaction with Ca(2+)- and cAMP-dependent secretagogues. We have employed a technique that allows concurrent monitoring of short-circuit current (I(SC)) and [Ca(2+)](i) in polarized epithelium. Basolateral application of baicalein induced a concentration-dependent increase in I(SC). The increase in I(SC) was because of Cl(-) secretion and was not accompanied by any discernible increase in [Ca(2+)](i). Baicalein acted synergistically with Ca(2+)- but not cAMP-dependent secretagogues. In the presence of baicalein, the carbachol and histamine induced increases in I(SC) that were markedly potentiated while increases in [Ca(2+)](i) were not significantly enhanced. Baicalein treatment uncoupled Cl(-) secretion from inhibitory effects normally generated by muscarinic activation. Baicalein treatment also resulted in increased cAMP content and activated PKA activity. Nystatin permeabilization studies revealed that baicalein stimulated an apical Cl(-) current but did not activate any basolateral K(+) current. These data suggest that baicalein potentiates Ca(2+)-mediated Cl(-) secretion through a signaling pathway involving cAMP and protein kinase A, most likely through the cystic fibrosis transmembrane conductance regulator in the apical membrane.     

Electrostatic control of the tryptophan radical in cytochrome c peroxidase

Previously a K(+)-binding site, analogous to that found in ascorbate peroxidase (APX), was engineered into cytochrome c peroxidase (CcP) to test the hypothesis that the bound K(+) influences the stability of the Trp191 cation radical formed during the CcP catalytic cycle (Bonagura et al., (1996) Biochemistry 35, 6107 and Bonagura et al., (1999) Biochemistry 38, 5528). Characterization of this mutant, designated CcPK2, showed that the stability of the Trp191 cation radical is dependent on the occupancy of the engineered K(+) site and that the Trp191 radical was much less stable in this mutant than in wild-type CcP. The mutations Met230Leu, Met231Gln, and Met172Ser have now been constructed on the CcPK2 mutant template to test if the Met residues also contribute to the stabilization of the Trp191 cation radical. Crystal structures show that the mutations affect only the local structure near the sites of mutation. Removal of these electronegative residues located less than 8 A from the Trp radical results in a further destabilization of the Trp radical. The characteristic EPR signal associated with the Trp radical is significantly narrowed and is characteristic of a tyrosine radical signal. Double-mixing stopped-flow experiments, where the delay time between the formation of CcP compound I and its mixing with horse heart ferrocytochrome c is varied, show that the stability of the Trp radical decreases as the Met residues are removed from the proximal cavity. When taken together, these results demonstrate a strong correlation between the experimentally determined stability of the Trp191 radical, the enzyme activity, and the calculated electrostatic stabilization of the Trp191 radical.     

Oligomerization and regulated proteolytic processing of angiopoietin-like protein 4

Angiopoietin-like protein 4 (Angptl4) is a recently identified circulating protein expressed primarily in adipose tissue and liver. Also known as peroxisome proliferator-activated receptor (PPAR)-gamma angiopoietin-related, fasting induced adipose factor, and hepatic fibrinogen/angiopoietin-related protein, recombinant Angptl4 causes increase of plasma very low density lipoprotein levels by inhibition of lipoprotein lipase activity. Similar to angiopoietins and other angiopoietin-like proteins, Angptl4 contains an amino-terminal coiled-coil domain and a carboxyl-terminal fibrinogen-like domain. We report here that Angptl4 is evolutionarily conserved among several mammalian species and that full-length Angptl4 protein is an oligomer containing intermolecular disulfide bonds. Oligomerized Angptl4 undergoes proteolytic processing to release its carboxyl fibrinogen-like domain, which circulates as a monomer. Angptl4's N-terminal coiled-coil domain mediates its oligomerization, which by itself is sufficient to form higher order oligomeric structure. Adenovirus-mediated overexpression of Angptl4 in 293 cells shows that conversion of full-length, oligomerized Angptl4 is mediated by a cell-associated protease activity induced by serum. These findings demonstrate a novel property of angiopoietin-like proteins and suggest that oligomerization and proteolytic processing of Angptl4 may regulate its biological activities in vivo.     

Buffalograss germplasm resistance to Blissus occiduus (Hemiptera: Lygaeidae)

Plant germplasm collections may offer genetic variability useful in identifying insect resistance. The goal of this project was to evaluate buffalograss genotypes [Buchlo? dactyloides (Nutt.) Engelm.] for resistance to the chinch bug, Blissus occiduus Barber (Hemiptera: Lygaeidae), and to relate resistance to ploidy level, chinch bug number, and pubescence. Forty-eight buffalograss genotypes from diverse geographic locations were evaluated in replicated studies under greenhouse conditions. Of the genotypes studied, four were highly resistant, 22 were moderately resistant, 19 were moderately susceptible, and three were highly susceptible to chinch bug damage. The mean number of chinch bugs was significantly different among the 48 genotypes. There was no significant correlation between chinch bug resistance and ploidy level or chinch bug resistance and pubescence. These results indicate the genetic source of resistance to chinch bugs exists in buffalograss germplasm. Highly resistant genotypes can be used in breeding programs to further improve buffalograss cultivars.     

Turfgrass, crop, and weed hosts of Blissus occiduus (Hemiptera: Lygaeidae)

Blissus occiduus Barber is an important pest of buffalograss, Buchlo? dactyloides (Nuttall) Engelmann, turf. No-choice studies documented the susceptibility of selected turfgrasses, crops, and weeds to B. occiduus feeding. Highly to moderately susceptible grasses included buffalograss; yellow Setaria glauca (L.) and green foxtail Setaria viridis (L.); Kentucky bluegrass, Poa pratensis L.; perennial ryegrass, Lolium perenne L.; brome, Bromus spp. Leyss.; zoysiagrass, Zoysia japonica Steudel; Bermuda grass, Cynodon dactylon (L.) Pers.; sorghum, Sorghum bicolor (L.) Moench; tall fescue, Festuca arundinacea Schreb.; and barley Hordeum vulgare (L.). Slightly to nonsusceptible grasses included fine fescue, Festuca ovina hirtula L.; rye, Secale cereale L.; crabgrass Digitaria sanguinalis (L.); bentgrass, Agrostis palustris Huds.; wheat, Tritium aestivun L.; corn, Zea mays L.; fall panicum Panicum dichotomiflorum Michx.; and St. Augustinegrass, Stenotaphrum secundatum (Walt.) Kuntze. The reproductive potential of B. occiduus was also investigated on these same grasses. B. occiduus produced offspring on 15 of the 18 turfgrass, crop, and weed species evaluated. No reproduction occurred on either Bermuda grass or St. Augustinegrass, and buffalograss plants were killed by B. occiduus feeding before offspring could be produced.     

Structure-activity relationships of 111In- and 99mTc-labeled quinolin-4-one peptidomimetics as ligands for the vitronectin receptor: potential tumor imaging agents

The integrin receptor alpha(v)beta(3) is overexpressed on the endothelial cells of growing tumors and on some tumor cells themselves. Radiolabeled alpha(v)beta(3) antagonists have demonstrated potential application as tumor imaging agents and as radiotherapeutic agents. This report describes the total synthesis of eight new HYNIC and DOTA conjugates of receptor alpha(v)beta(3) antagonists belonging to the quinolin-4-one class of peptidomimetics, and their radiolabeling with (99m)Tc (for HYNIC) and (111)In (for DOTA). Tethering of the radionuclide-chelator complexes was achieved at two different sites on the quinolin-4-one molecule. All such derivatives maintained high affinity for receptor alpha(v)beta(3) and high selectivity versus receptors alpha(IIb)beta(3), alpha(v)beta(5), alpha(5)beta(1). Biodistribution of the radiolabeled compounds was evaluated in the c-neu Oncomouse mammary adenocarcinoma model. DOTA conjugate (111)In-TA138 presented the best biodistribution profile. Tumor uptake at 2 h postinjection was 9.39% of injected dose/g of tissue (%ID/g). Activity levels in selected organs was as follows: blood, 0.54% ID/g; liver, 1.94% ID/g; kidney, 2.33% ID/g; lung, 2.74% ID/g; bone, 1.56% ID/g. A complete biodistribution analysis of (111)In-TA138 and the other radiolabeled compounds of this study are presented and discussed. A scintigraphic imaging study with (111)In-TA138 showed a clear delineation of the tumors and rapid clearance of activity from nontarget tissues.     

Characterization of HIV-1 envelope gp41 genetic diversity and functional domains following perinatal transmission

Background

HIV-1 envelope gp41 is a transmembrane protein that promotes fusion of the virus with the plasma membrane of the host cells required for virus entry. In addition, gp41 is an important target for the immune response and development of antiviral and vaccine strategies, especially when targeting the highly variable envelope gp120 has not met with resounding success. Mutations in gp41 may affect HIV-1 entry, replication, pathogenesis, and transmission. We, therefore, characterized the molecular properties of gp41, including genetic diversity, functional motifs, and evolutionary dynamics from five mother-infant pairs following perinatal transmission.

Results

The gp41 open reading frame (ORF) was maintained with a frequency of 84.17% in five mother-infant pairs' sequences following perinatal transmission. There was a low degree of viral heterogeneity and estimates of genetic diversity in gp41 sequences. Both mother and infant gp41 sequences were under positive selection pressure, as determined by ratios of non-synonymous to synonymous substitutions. Phylogenetic analysis of 157 mother-infant gp41 sequences revealed distinct clusters for each mother-infant pair, suggesting that the epidemiologically linked mother-infant pairs were evolutionarily closer to each other as compared with epidemiologically unlinked sequences. The functional domains of gp41, including fusion peptide, heptad repeats, glycosylation sites and lentiviral lytic peptides were mostly conserved in gp41 sequences analyzed in this study. The CTL recognition epitopes and motifs recognized by fusion inhibitors were also conserved in the five mother-infant pairs.

Conclusion

The maintenance of an intact envelope gp41 ORF with conserved functional domains and a low degree of genetic variability as well as positive selection pressure for adaptive evolution following perinatal transmission is consistent with an indispensable role of envelope gp41 in HIV-1 replication and pathogenesis.