In vivo antagonism of a T cell response by an endogenously expressed ligand

3.L2 T cell receptor transgenic T cells are activated by the 64–76 peptide of the mouse hemoglobin d β chain [Hb(64–76)], and their response is antagonized by the position 72 alanine substitution of this peptide (A72). To test the effect of this altered peptide ligand (APL) on 3.L2 T cell function in vivo, a transgene expressing A72 in major histocompatibility complex II positive cells (A72tg) has been introduced into mice. We demonstrate that 3.L2 T cells, when transferred to A72tg+ mice show a dramatically reduced proliferative response to Hb(64–76). Identical decreased responses were observed using T cells that developed in either A72tg+ or A72tg− hosts. This affect was not attributable to diminished precursor frequency, anergy, or competition for binding to I-Ek molecules. These results unequivocally demonstrate in vivo antagonism by an endogenous APL and characterize a class of self-peptides that, although inefficient in causing deletion in the thymus, effectively modulate T cell responses in the periphery.

Structural changes in hemoglobin during adsorption to solid surfaces: Effects of pH, ionic strength, and ligand binding

We have studied the adsorption of two structurally similar forms of hemoglobin (met-Hb and HbCO) to a hydrophobic self-assembled methyl-terminated thiol monolayer on a gold surface, by using a Quartz Crystal Microbalance (QCM) technique. This technique allows time-resolved simultaneous measurements of changes in frequency (f) (c.f. mass) and energy dissipation (D) (c.f. rigidity/viscoelastic properties) of the QCM during the adsorption process, which makes it possible to investigate the viscoelastic properties of the different protein layers during the adsorption process. Below the isoelectric points of both met-Hb and HbCO, the ΔD vs. Δf graphs displayed two phases with significantly different slopes, which indicates two states of the adsorbed proteins with different visco-elastic properties. The slope of the first phase was smaller than that of the second phase, which indicates that the first phase was associated with binding of a more rigidly attached, presumably denatured protein layer, whereas the second phase was associated with formation of a second layer of more loosely bound proteins. This second layer desorbed, e.g., upon reduction of Fe3+ of adsorbed met-Hb and subsequent binding of carbon monoxide (CO) forming HbCO. Thus, the results suggest that the adsorbed proteins in the second layer were in a native-like state. This information could only be obtained from simultaneous, time-resolved measurements of changes in both D and f, demonstrating that the QCM technique provides unique information about the mechanisms of protein adsorption to solid surfaces.

Structure and inhibition of plasmepsin II, a hemoglobin-degrading enzyme from Plasmodium falciparum.

Plasmodium falciparum is the major causative agent of malaria, a disease of worldwide importance. Resistance to current drugs such as chloroquine and mefloquine is spreading at an alarming rate, and our antimalarial armamentarium is almost depleted. The malarial parasite encodes two homologous aspartic proteases, plasmepsins I and II, which are essential components of its hemoglobin-degradation pathway and are novel targets for antimalarial drug development. We have determined the crystal structure of recombinant plasmepsin II complexed with pepstatin A. This represents the first reported crystal structure of a protein from P. falciparum. The crystals contain molecules in two different conformations, revealing a remarkable degree of interdomain flexibility of the enzyme. The structure was used to design a series of selective low molecular weight compounds that inhibit both plasmepsin II and the growth of P. falciparum in culture.

A new hemoglobin gene from soybean: a role for hemoglobin in all plants.

We have isolated a new hemoglobin gene from soybean. It is expressed in cotyledons, stems of seedlings, roots, young leaves, and in some cells in the nodules that are associated with the nitrogen-fixing Bradyrhizobium symbiont. This contrasts with the expression of the leghemoglobins, which are active only in the infected cells of the nodules. The deduced protein sequence of the new gene shows only 58% similarity to one of the soybean leghemoglobins, but 85-87% similarity to hemoglobins from the nonlegumes Parasponia, Casuarina, and barley. The pattern of expression and the gene sequence indicate that this new gene is a nonsymbiotic legume hemoglobin. The finding of this gene in legumes and similar genes in other species strengthens our previous suggestion that genomes of all plants contain hemoglobin genes. The specialized leghemoglobin gene family may have arisen from a preexisting nonsymbiotic hemoglobin by gene duplication.

Transfection of the human heme oxygenase gene into rabbit coronary microvessel endothelial cells: protective effect against heme and hemoglobin toxicity.

Heme oxygenase (HO) is a stress protein and has been suggested to participate in defense mechanisms against agents that may induce oxidative injury such as metals, endotoxin, heme/hemoglobin, and various cytokines. Overexpression of HO in cells might therefore protect against oxidative stress produced by certain of these agents, specifically heme and hemoglobin, by catalyzing their degradation to bilirubin, which itself has antioxidant properties. We report here the successful in vitro transfection of rabbit coronary microvessel endothelial cells with a functioning gene encoding the human HO enzyme. A plasmid containing the cytomegalovirus promoter and the human HO cDNA complexed to cationic liposomes (Lipofectin) was used to transfect rabbit endothelial cells. Cells transfected with human HO exhibited an approximately 3.0-fold increase in enzyme activity and expressed a severalfold induction of human HO mRNA as compared with endogenous rabbit HO mRNA. Transfected and nontransfected cells expressed factor VIII antigen and exhibited similar acetylated low-density lipoprotein uptake (two important features that characterize endothelial cells) with > 85% of cells staining positive for each marker. Moreover, cells transfected with the human HO gene acquired substantial resistance to toxicity produced by exposure to recombinant hemoglobin and heme as compared with nontransfected cells. The protective effect of HO overexpression against heme/hemoglobin toxicity in endothelial cells shown in these studies provides direct evidence that the inductive response of human HO to such injurious stimuli represents an important tissue adaptive mechanism for moderating the severity of cell damage produced by these blood components.

The structure of Ascaris hemoglobin domain I at 2.2 A resolution: molecular features of oxygen avidity.

The perienteric hemoglobin of the parasitic nematode Ascaris has an exceptionally high affinity for oxygen. It is an octameric protein containing two similar heme-binding domains per subunit, but recombinant constructs expressing a single, monomeric heme-binding domain (domain 1; D1) retain full oxygen avidity. We have solved the crystal structure of D1 at 2.2 A resolution. Analysis of the structure reveals a characteristic globin fold and illuminates molecular features involved in oxygen avidity of Ascaris perienteric hemoglobin. A strong hydrogen bond between tyrosine at position 10 in the B helix (tyrosine-B10) and the distal oxygen of the ligand, combined with a weak hydrogen bond between glutamine-E7 and the proximal oxygen, grips the ligand in the binding pocket. A third hydrogen bond between these two amino acids appears to stabilize the structure. The B helix of D1 is displaced laterally by 2.5 A when compared with sperm whale myoglobin. This shifts the tyrosine-B10 hydroxyl far enough from liganded oxygen to form a strong hydrogen bond without steric hindrance. Changes in the F helix compared with myoglobin contribute to a tilted heme that may also be important for oxygen affinity.