An immunochemical approach for the analysis of membrane protein alterations in Ca²⁺‐loaded human erythrocytes

Abstract

An increase in the intracellular concentration of Ca2+ in human erythrocytes results in the formation of gamma-glutamyl-epsilon-lysine cross-linked membrane protein polymers. Following solubilization of the membranes with SDS, these polymers can be isolated on a Lubrol-containing sucrose gradient. Immunoelectrophoresis of the polymeric material with a polyspecific rabbit antibody against human ghosts gave rise to a single, but heterogeneous, precipitate. The polymer was amphiphilic and, on addition to Triton-solubilized erythrocyte membrane proteins, it coprecipitated with spectrin. When the antihost antibody was absorbed with the polymer prior to cross immunoelectrophoresis of normal erythrocyte membrane proteins, the precipitates of glycophorin, acetylcholinesterase, and hemoglobin were normal, whereas the antibody titers against band 3 protein, spectrin, and ankyrin became reduced. Furthermore, a rabbit antibody raised against the isolated human polymer reacted selectively with the same three membrane proteins. No reactions occurred with lysate proteins.

Transglutaminases and Disease: Lessons From Genetically Engineered Mouse Models and Inherited Disorders

“…Since neither chemical nor biochemical procedures are available at present to separate the N -(␥-glutamyl)lysinebonded structures into their polypeptide building blocks, indirect methods have been employed for assessing polymer composition, with good agreement between different modes of analyses. For example, immunological approaches have utilized cross-reactive antibodies for recognizing accessible epitopes both in monomers and polymers (46), whereas the identification of tryptic fragments from polymers has been accomplished by proteomics (L. Lorand, personal communication). Membrane skeletal as well as cytoplasmic proteins are incorporated into the polymers (e.g., spectrins, ankyrin, band 4.1, hemoglobin, among several others), and the anion transporter (band 3) and possibly other transmembrane proteins serve to anchor the polymers firmly into the membrane.…”

Intraerythrocytic calcium chelators inhibit the invasion of Plasmodium falciparum

“…One of such mechanisms, the most important, is an activation caused by binding of the Ca 2+ -calmodulin complex to the ATPase (Larsen and Vincenzi 1979;Falchetto et al 1991). An increase in the intraerythrocytic concentration of free calcium causes a series of metabolic and morphological changes; calcium-dependent processes of phosphorylation (Cohen and Foley 1986), proteolysis (Au et al 1988;Inomata et al 1989), and transglutamination (Bjerrum et al 1981), among others, are activated. The normal discocyte shape changes to equinocyte and, if the situation does not revert within a short period, the cell vesiculates and lyses (Anderson et al 1977).…”

The Intermediate Filament Protein, Vimentin, in the Lens Is a Target for Cross-linking by Transglutaminase

“…Based on experiments with human red blood cells (9,10) and with keratinocytes (11,12), it seemed reasonable to assume that in addition to the specific subsets of crystallins, some membrane and cytoskeletal elements in the lens might also become cross-linked by TGase. Therefore, we undertook to examine changes occurring in the skeletal proteins of the lens as the latent TGase becomes activated by Ca 2ϩ in the tissue.…”