No abstract

Glycoproteins expressing the Lutheran blood group antigens were isolated from human erythrocyte membranes and from human fetal liver. Amino acid sequence analyses allowed the design of redundant oligonucleotides that were used to generate a 459-bp, sequence-specific probe by PCR. A cDNA clone of 2400 bp was isolated from a human placental lambda gt 11 library and sequenced, and the deduced amino acid sequence was studied. The predicted mature protein is a type I membrane protein of 597 amino acids with five potential N-glycosylation sites. There are five disulfide-bonded, extracellular, immunoglobulin superfamily domains (two variable-region set and three constant-region set), a single hydrophobic, membrane-spanning domain, and a cytoplasmic domain of 59 residues. The overall structure is similar to that of the human tumor marker MUC 18 and the chicken neural adhesion molecule SC1. The extracellular domains and cytoplasmic domain contain consensus motifs for the binding of integrin and Src homology 3 domains, respectively, suggesting possible receptor and signal-transduction function. Immunostaining of human tissues demonstrated a wide distribution and provided evidence that the glycoprotein is under developmental control in liver and may also be regulated during differentiation in other tissues.

The CD47 glycoprotein was isolated from human erythrocytes by immunoprecipitation using monoclonal antibody (mAb) BRIC-125. Enzymic deglycosylation of the protein showed it contained N-linked oligosaccharides, and trypsin proteolysis of the protein in situ in the erythrocyte membrane cleaved it into two portions, one of which was glycosylated. Both the intact protein and the glycosylated fragment had blocked N-termini. Amino acid sequence was obtained from several proteolytic fragments of CD47. Comparison with the sequence database showed the protein to be very similar to or identical with OA3, a multispanning membrane protein. The protein also appears to be the same as the integrin-associated protein, which has a role in cell adhesion in non-erythroid cells. CD47 has six potential N-glycosylation sites, five of which are in an Ig superfamily domain. We show that three of these sites carry N-glycans in erythrocytes. Immunocytochemical staining of human tissues showed that CD47 was broadly distributed on mesenchyme and epithelia at multiple sites. Reactivity was particularly prominent in surface and ductular epithelia, and in the brain. The possible roles of the CD47 glycoprotein are discussed.

We constructed cDNA clones encoding fragments of band 3 in which the membrane domain was truncated from either the N or the C terminus within each of the first four exofacial loops. The truncations containing the C terminus of the protein were fused with the cleavable N-terminal signal sequence of glycophorin A to facilitate the correct orientation of the most N-terminal band 3 membrane span. Cleavage of the glycophorin A signal sequence was observed, except when the truncation was in the first exofacial loop where the signal peptidase cleavage site was probably too close to the membrane. The anion transport activity of co-expressed complementary pairs of truncations which together contained the entire band 3 membrane domain was examined. The pairs of fragments divided in the third and fourth exofacial loops yielded transport activity, but the pair separated within the second exofacial loop was not active. We conclude that the integrity of the second exofacial loop, but not the third and fourth exofacial loops, is necessary for transport activity. The unusually stable association between the fragments divided in the second exofacial loop suggests that interactions may occur between polar surfaces on amphiphilic portions of the third and fifth transmembrane spans.

1. We have studied the inherited changes occurring in the sialoglycoproteins of membranes from erythrocytes of type Miltenberger Class III (Mi.III), Miltenberger Class IV (Mi.IV) and Miltenberger Class V (Mi.V) by using sodium dodecyl sulphate/polyacrylamide gel electrophoresis and lactoperoxidase radioiodination. 2. Mi.III erythrocytes lack the normal blood-group-Ss-active sialoglycoprotein but contain an unusual s-active sialoglycoprotein of higher apparent molecular weight. A similar abnormal S-active sialoglycoprotein appears to occur in Mi.IV erythrocytes. 3. The Mi.V condition is associated with the hemizygous absence of both the normal blood-group-MN-active sialoglycoprotein and the normal Ss-active sialoglycorprotein. However, a new sialoglycoprotein component is present in these cells that has properties characteristic of both the MN-active and Ss-active sialoglycoproteins. 4. Our results suggest that the new sialoglycorportein present in Mi.V erythrocytes is a hybrid of the normal MN sialoglycoprotein and an s-active sialoglycoprotein that has properties similar to the s-active sialoglycoprotein found in Mi.III erythrocytes. We suggest that the unusual Mi.V sialoglycoprotein is derived from chromosomal misalignment with unequal crossing-over between the genes for the MN- and Ss-active sialoglycoproteins in a manner similar to that which gives rise to haemoglobin Lepore. 5. Further studies of S-s-erythrocytes confirm that these cells lack normal Ss-active sialoglycoprotein, but contain an unusual component that shows some of the properties of the normal Ss-active sialoglycoprotein. 6. Analysis of erythrocytes of type Mk/Mi.III confirms that, in addition to the known hemizygous lack of the MN-active sialoglycoprotein, the Mk condition is also associated with a loss of the Ss-active sialoglycoprotein. 7. In order to facilitate discussion of the complex changes that occur in these variant erythrocytes, a new unified nomenclature is used for the erythrocyte sialoglycoproteins.