Minor histocompatibility antigens as determinants for graft-versus-host disease after allogeneic haematopoietic stem cell transplantation.

Minor histocompatibility antigens (minor H antigens) are genetically polymorphic peptides that have been shown to elicit immune response when mismatched between donor and recipient of haematopoietic stem cell transplantation (HSCT). Depending on the expression profiles, mismatches in these genes may either lead to harmful graft-versus-host (GvH) reaction or desired graft-versus-leukaemia (GvL) effect. We analysed retrospectively the effect of HLA-restricted matching 11 established autosomal minor H antigens on the risk of graft-versus-host disease and relapse in 311 HLA-matched sibling HSCT of a single centre. Increased incidence of chronic GvH disease was shown to be associated with mismatches in the HA-8 and ACC-1. The mRNA expression profiles in a large set of healthy and malignant tissue samples of minor H antigen genes demonstrated in silico that the expression profiles of HA-8 and ACC-1 were surprisingly different: HA-8 gene was expressed in practically all tissues, whereas ACC-1 gene had a restricted profile. The results demonstrated that mismatches in minor H antigens HA-8 and ACC-1 predisposed to chronic graft-versus-host disease (GvHD).

Acyl chain-dependent effect of lysophosphatidylcholine on human neutrophils.

Lysophosphatidylcholine (LPC) is the most abundant lysophospholipid in plasma and tissues, and its level increases in ischemia and inflammation. LPC induces various proinflammatory actions in leukocytes, endothelial cells, and smooth muscle cells, but its effects may vary, depending on the acyl chain. In the present study, we identified the molecular species of LPC in human plasma and studied their effects on human neutrophils. Unsaturated LPC species over a wide concentration range (5-200 microM) induced long-lasting superoxide production in neutrophils. The response was preceded by a >10-min lag time and lasted for 60-90 min. Superoxide production was prevented when albumin was added together with LPC at a molar ratio of 1:2 or higher, and significant inhibition was observed even when albumin was added 4-8 min after LPC. Saturation of albumin by fivefold molar excess of stearic acid reduced the inhibitory effect significantly. Saturated LPCs, particularly the most abundant 16:0 species, induced significantly less superoxide production than the unsaturated species and only at 5-10 microM concentrations. Saturated LPC species elicited a several-fold higher increase in cytoplasmic calcium and at >20 microM, increased plasma membrane permeability. A mixture of LPCs mimicking the plasma LPC composition induced nearly similar superoxide production as the most active LPC18:1 alone. These results indicate remarkable acyl chain-dependent differences in the cellular effects of LPC. Elevation of LPC level may increase inflammation through activation of neutrophil NADPH oxidase, particularly when the simultaneous increase of free fatty acids diminishes the ability of albumin to scavenge LPCs.

Identification of yeast cofilin residues specific for actin monomer and PIP2 binding.

Cofilin/ADF is a ubiquitous actin-binding protein that is important for rapid actin dynamics in vivo. The long alpha-helix (helix 3 in yeast cofilin) forms the most highly conserved region in cofilin/ADF proteins, and residues in the NH2-terminal half of this alpha-helix have been shown to be essential for actin binding in cofilin/ADF. Recent studies also suggested that the basic residues in the COOH-terminal half of this alpha-helix would play an important role in F-actin binding. In contrast to these studies, we show here that the charged residues in the COOH-terminal half of helix 3 are not important for actin filament binding in yeast cofilin. Mutations in these residues, however, result in a small defect in actin monomer interactions. We also show that yeast cofilin can differentiate between various phosphatidylinositides, and mapped the PI(4,5)P2 binding site by using a collection of cofilin mutants. The PI(4,5)P2 binding site of yeast cofilin is a large positively charged surface that consists of residues in helix 3 as well as residues in other parts of the cofilin molecule. This suggests that cofilin/ADF proteins probably interact simultaneously with more than one PI(4,5)P2 molecule. The PI(4,5)P2-binding site overlaps with areas that are important for F-actin binding, explaining why the actin-related activities of cofilin/ADF are inhibited by PI(4,5)P2. The biological roles of actin and PI(4,5)P2 interactions of cofilin are discussed in light of phenotypes of specific yeast strains carrying mutations in residues that are important for actin and PI(4,5)P2 binding.

Interactions with PIP2, ADP-actin monomers, and capping protein regulate the activity and localization of yeast twinfilin.

Twinfilin is a ubiquitous actin monomer-binding protein that regulates actin filament turnover in yeast and mammalian cells. To elucidate the mechanism by which twinfilin contributes to actin filament dynamics, we carried out an analysis of yeast twinfilin, and we show here that twinfilin is an abundant protein that localizes to cortical actin patches in wild-type yeast cells. Native gel assays demonstrate that twinfilin binds ADP-actin monomers with higher affinity than ATP-actin monomers. A mutant twinfilin that does not interact with actin monomers in vitro no longer localizes to cortical actin patches when expressed in yeast, suggesting that the ability to interact with actin monomers may be essential for the localization of twinfilin. The localization of twinfilin to the cortical actin cytoskeleton is also disrupted in yeast strains where either the CAP1 or CAP2 gene, encoding for the alpha and beta subunits of capping protein, is deleted. Purified twinfilin and capping protein form a complex on native gels. Twinfilin also interacts with phosphatidylinositol 4,5-bisphosphate (PI[4,5]P2), and its actin monomer-sequestering activity is inhibited by PI(4,5)P2. Based on these results, we propose a model for the biological role of twinfilin as a protein that localizes actin monomers to the sites of rapid filament assembly in cells.

Mouse A6/twinfilin is an actin monomer-binding protein that localizes to the regions of rapid actin dynamics.

In our database searches, we have identified mammalian homologues of yeast actin-binding protein, twinfilin. Previous studies suggested that these mammalian proteins were tyrosine kinases, and therefore they were named A6 protein tyrosine kinase. In contrast to these earlier studies, we did not find any tyrosine kinase activity in our recombinant protein. However, biochemical analysis showed that mouse A6/twinfilin forms a complex with actin monomer and prevents actin filament assembly in vitro. A6/twinfilin mRNA is expressed in most adult tissues but not in skeletal muscle and spleen. In mouse cells, A6/twinfilin protein is concentrated to the areas at the cell cortex which overlap with G-actin-rich actin structures. A6/twinfilin also colocalizes with the activated forms of small GTPases Rac1 and Cdc42 to membrane ruffles and to cell-cell contacts, respectively. Furthermore, expression of the activated Rac1(V12) in NIH 3T3 cells leads to an increased A6/twinfilin localization to nucleus and cell cortex, whereas a dominant negative form of Rac1(V12,N17) induces A6/twinfilin localization to cytoplasm. Taken together, these studies show that mouse A6/twinfilin is an actin monomer-binding protein whose localization to cortical G-actin-rich structures may be regulated by the small GTPase Rac1.