Accumulation of altered aspartyl residues in erythrocyte proteins from patients with Down's syndrome

Spontaneous protein deamidation of labile Asn residues, generating L-isoaspartates and D-aspartates, is associated with cell aging and is enhanced by an oxidative microenvironment; to minimize the damage, the isoaspartate residues can be 'repaired' by a specific L-isoaspartate (D-aspartate) protein O-methyltransferase (PIMT). As both premature aging and chronic oxidative stress are typical features of Down's syndrome (DS), we tested the hypothesis that deamidated proteins may build up in trisomic patients. Blood samples were obtained from children with karyotypically confirmed full trisomy 21 and from age-matched healthy controls. Using recombinant PIMT as a probe, we demonstrated a dramatic rise of L-isoaspartates in erythrocyte membrane proteins from DS patients. The content of D-aspartate was also significantly increased. The integrity of the repair system was checked by evaluating methionine transport, PIMT specific activity, and intracellular concentrations of adenosylmethionine and adenosylhomocysteine. The overall methylation pathway was directly monitored by incubating fresh red blood cells with methyl-labeled methionine; a three-fold increase of protein methyl esters was detected in trisomic children. Deamidated species include ankyrin, band 4.1, band 4.2 and the integral membrane protein band 3; ankyrin and band 4.1 were significantly hypermethylated in DS. When DS red blood cells were subjected to oxidative treatment in vitro, the increase of protein deamidation paralleled lipid peroxidation and free radical generation. We observed a similar pattern in Epstein-Barr virus B-lymphocytes from trisomic patients. In conclusion, our findings support the hypothesis that protein instability at asparagine sites is a biochemical feature of DS, presumably depending upon the oxidative microenvironment. The possible pathophysiological implications are discussed.     

Aging of the erythrocyte. XX. Decreased red cell life span in Down's syndrome

Decreased content of glycosylated hemoglobin in erythrocytes of normoglycemic patients with Down's syndrome (DS) evidences a decreased life span of red blood cells in DS, supposed previously.     

Ultrastructural localization of erythrocyte cytoskeletal and integral membrane proteins in Plasmodium falciparum-infected erythrocytes

The distributions of ankyrin, spectrin, band 3, and glycophorin A were examined in Plasmodium falciparum-infected erythrocytes by immunoelectron microscopy to determine whether movement of parasite proteins and membrane vesicles between the parasitophorous vacuole membrane and erythrocyte surface membrane involves internalization of host membrane skeleton proteins. Monospecific rabbit antisera to spectrin, band 3 and ankyrin and a mouse monoclonal antibody to glycophorin A reacted with these erythrocyte proteins in infected and uninfected human erythrocytes by immunoblotting. Cross-reacting malarial proteins were not detected. The rabbit sera also failed to immunoprecipitate [3H]isoleucine labeled malarial proteins from Triton X-100 and sodium dodecyl sulfate (SDS) extracts of infected erythrocytes. These three antibodies as well as the monoclonal antibody to glycophorin A bound to the membrane skeleton of infected and uninfected erythrocytes. The parasitophorous vacuole membrane was devoid of bound antibody, a result indicating that this membrane contains little, if any, of these host membrane proteins. With ring-, trophozoite- and schizont-infected erythrocytes, spectrin, band 3 and glycophorin A were absent from intracellular membranes including Maurer's clefts and other vesicles in the erythrocyte cytoplasm. In contrast, Maurer's clefts were specifically labeled by anti-ankyrin antibody. There was a slight, corresponding decrease in labeling of the membrane skeleton of infected erythrocytes. A second, morphologically distinct population of circular, vesicle-like membranes in the erythrocyte cytoplasm was not labeled with anti-ankyrin antibody. We conclude that membrane movement between the host erythrocyte surface membrane and parasitophorous vacuole membrane involves preferential sorting of ankyrin into a subpopulation of cytoplasmic membranes.     

Alterations in erythrocyte membrane lipid and fatty acid composition in Chediak-Higashi syndrome

Chediak-Higashi syndrome (CHS) is an autosomal recessive disease characterized by the presence of abnormally large cytoplasmic organelles in all body granule producing cells. The molecular mechanism for this disease is still unknown. Functional disorders in membrane-related processes have been reported. Erythrocyte membranes from four CHS patients and 15 relatives including obligatory heterozygous were studied to examine potential alterations in the lipid and fatty acid profile of erythrocyte membranes associated with this syndrome. Plasma concentrations of cholesterol, triglycerides, phospholipids, and apolipoproteins AI and B100, and the lipid components of very low-, intermediate-, low- and high-density lipoproteins were also determined. CHS erythrocyte membranes were found to be enriched with lipids in relation to protein and to show: (1) an increase in cholesterol and choline-containing phospholipids (sphingomyelin and phosphatidylcholine) that predominate in the outer monolayer, which is higher than the increase in phosphatidylserine and phosphatidylethanolamine, that are chiefly limited to the inner monolayer in normal red blood cells; (2) a relative palmitic acid and saturated fatty acid increase and arachidonic acid and unsaturated fatty acid decrease, this resulting in a lower unsaturation index than controls. Changes in CHS erythrocyte membrane lipids seem to be unrelated to serum lipid disorders as plasma lipid and apolipoprotein concentrations were apparently in the normal range, with the exception of a modest hypertriglyceridemia in patients and relatives and a decreased concentration of HDL cholesterol in patients. These findings indicate that CHS erythrocyte membranes contain an abnormal lipid matrix with which membrane proteins are defectively associated. The anomalous CHS membrane composition can be explained on the postulated effects of the CHS1/Lyst gene.