Molecular identification and functional characterization of a novel protein that mediates the attachment of erythroblasts to macrophages.

We have previously identified a novel protein that mediates the attachment of erythroblasts to macrophages in vitro. This attachment promotes terminal maturation and enucleation of erythroblasts (Hanspal and Hanspal, Blood 84:3494, 1994). This protein is referred to here as Emp for erythroblast macrophage protein. Two immunologically related isoforms of Emp with apparent molecular weights of 33 kD and 36 kD were detected in macrophage membranes. The complete amino acid sequence of the larger isoform of Emp was deduced from the nucleotide sequence of a full-length 2.0-kb cDNA that was isolated from a human macrophage cDNA library using affinity-purified anti-Emp antibodies. Of the 2,005 bp, 1,185 bp encode for 395 amino acids representing 43 kD (the sodium dodecyl sulfate-polyacrylamide gel electrophoresis [SDS-PAGE] molecular mass is 36 kD). Northern blot analysis of human macrophage poly(A) RNA detected a message for Emp of 2.1 kb. The deduced amino acid sequence contains a putative transmembrane domain near the N-terminus. To investigate the structure/function relationships of Emp, recombinant fusion proteins of full-length and truncated Emp were produced in bacteria, COS-7, and HeLa cells. Cell binding assays showed that the N-terminus is exposed on the cell surface. The recombinant Emp functions as a cell attachment molecule when expressed in heterologous cells. Furthermore, we showed that the demise of erythroblasts in the absence of Emp-mediated erythroblast-macrophage association is accompanied by apoptosis. We postulate that Emp-mediated contact between erythroblasts and macrophages promotes terminal maturation of erythroid cells by suppressing apoptosis.

Temporal synthesis of band 3 oligomers during terminal maturation of mouse erythroblasts. Dimers and tetramers exist in the membrane as preformed stable species.

Band 3, the anion transport protein of the erythrocyte membrane, exists in the membrane as a mixture of dimers (B3D) and tetramers (B3T). The dimers are not linked to the skeleton and constitute the free mobile band 3 fraction. The tetramers are linked to the skeleton by their interaction with ankyrin. In this report we have examined the temporal synthesis and assembly of band 3 oligomers into the plasma membrane during red cell maturation. The oligomeric state of newly synthesized band 3 in early and late erythroblasts was analyzed by size-exclusion high-pressure liquid chromatography of band 3 extracts derived by mild extraction of plasma membranes with the nonionic detergent C12E8 (octaethylene glycol n-dodecyl monoether). This analysis revealed that at the early erythroblast stage, the newly synthesized band 3 is present predominantly as tetramers, whereas at the late stages of erythroid maturation, it is present exclusively as dimers. To examine whether the dimers and tetramers exist in the membrane as preformed stable species or whether they are interconvertible, the fate of band 3 species synthesized during erythroblast maturation was examined by pulse-chase analysis. We showed that the newly synthesized band 3 dimers and tetramers are stable and that there is no interconversion between these species in erythroblast membranes. Pulse-chase analysis followed by cellular fractionation showed that, in early erythroblasts, the newly synthesized band 3 tetramers are initially present in the microsomal fraction and later incorporated stably into the plasma membrane fraction. In contrast, in late erythroblasts the newly synthesized band 3 dimers move rapidly to the plasma membrane fraction but then recycle between the plasma membrane and microsomal fractions. Fluorescence photobleaching recovery studies showed that significant fractions of B3T and B3D are laterally mobile in early and late erythroblast plasma membranes, respectively, suggesting that many B3T-ankyrin complexes are unattached to the membrane skeleton in early erythroblasts and that the membrane skeleton has yet to become tightly organized in late erythroblasts. We postulate that in early erythroblasts, band 3 tetramers are transported through microsomes and stably incorporated into the plasma membrane. However, when ankyrin synthesis is downregulated in late erythroblasts, it appears that B3D are rapidly transported to the plasma membrane but then recycled between the plasma membrane and microsomal compartments. These observations may suggest novel roles for membrane skeletal proteins in stabilizing integral membrane protein oligomers at the plasma membrane and in regulating the endocytosis of such proteins.