Identification of abundant proteins and potential allergens in Culicoides nubeculosus salivary glands.

IgE-mediated type 1 hypersensitivity reactions to the bites of insects are a common cause of skin disease in horses. Insect bite hypersensitivity (IBH) is most frequently associated with bites of Culicoides spp. and occurs in all parts of the world where horses and Culicoides coexist. The main allergens that cause IBH are probably some of the abundant proteins in the saliva of Culicoides associated with blood feeding. Western blots of Culicoides proteins separated by 1D gel-electrophoresis detected strong IgE responses in all horses with IBH to antigens in protein extracts from wild caught Culicoides, but only weak responses to salivary antigens from captive bred C. nubeculosus which may reflect important differences among allergens from different species of Culicoides or differences between thorax and salivary gland antigens. 2D electrophoresis and mass spectrometry were used to identify several of the abundant proteins in the saliva of C. nubeculosus. These included maltase, members of the D7 family, and several small, basic proteins associated with blood feeding. The most frequently detected IgE-binding proteins were in a group of proteins with pI>8.5 and mass 40-50kDa. Mass spectrometry identified two of these allergenic proteins as similar to hyaluronidase and a heavily glycosylated protein of unknown function that have previously been identified in salivary glands of C. sonorensis.

The low-frequency MNS blood group antigens Ny(a) (MNS18) and Os(a) (MNS38) are associated with GPA amino acid substitutions.

BACKGROUND: Antigens of the MNS blood group system are located on two sialoglycoproteins, GPA and GPB, encoded by GYPA and GYPB. The molecular backgrounds of the low-frequency antigens Ny(a) and Os(a) are not known. STUDY DESIGN AND METHODS: Immunoblotting and a monoclonal antibody-specific immobilization of erythrocyte antigens (MAIEA) assay were used to analyze Os(a). PCR-amplified products of the coding exons of GYPA were studied by single-strand conformation polymorphism analysis, and exon 3 was sequenced. Synthetic peptides were used in hemagglutination-inhibition tests. RESULTS: Sequencing of GYPA exon 3 of two unrelated Ny(a+) persons revealed heterozygosity for a T194A base change encoding an Asp27Glu substitution. Immunoblotting with anti-Os(a) and an MAIEA assay with MoAbs to GPA showed that Os(a) is on GPA. Sequencing exon 3 of an Os(a+) person from the only family with Os(a) revealed heterozygosity for a C273T base change encoding a Pro54Ser substitution. A synthetic peptide representing part of GPA with the Os(a) mutation (VRTVYPSEEETGE) completely inhibited anti-Os(a), whereas the control peptide (VRTVYPPEEETGE) did not inhibit anti-Os(a). CONCLUSION: Ny(a) and Os(a) are low-frequency antigens of the MNS blood group system that represent Asp27Glu and Pro54Ser substitutions in GPA, respectively.

Complementation studies with Co-expressed fragments of the human red cell anion transporter (Band 3; AE1). The role of some exofacial loops in anion transport.

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.

The Oka blood group antigen is a marker for the M6 leukocyte activation antigen, the human homolog of OX-47 antigen, basigin and neurothelin, an immunoglobulin superfamily molecule that is widely expressed in human cells and tissues.

The high-frequency blood group antigen Ok(a) is carried on a red cell membrane glycoprotein (gp) of 35-69 kDa that is widely distributed on malignant cells of different origins. Immunostaining of hemopoietic cells and a range of normal human tissues demonstrated a wide distribution of the Ok(a) gp that appears to be nonlineage-restricted, although certain tissues show differentiation-related expression. Ok(a) gp was purified from red cell membranes by immunoaffinity chromatography using mAb A103 and amino acid sequence analysis was performed. The N-terminal 30 amino acids are identical to the predicted sequence of M6 leukocyte activation antigen (M6), a member of the Ig superfamily (IgSF) with two IgSF domains. There are homologs in rat (MRC OX-47 or CE9), in mouse (basigin or gp42), and in chicken (HT7 or neurothelin). The molecular basis of the Ok(a) mutation was established by sequencing M6 cDNA derived from normal and Ok(a-) EBV-transformed B cell lines. A point mutation in the translated portion of M6 cDNA, G331AG-->AAG gives rise to a predicted E92-->K amino acid change in the first Ig-like domain of the Ok(a-) form of the protein. Transfection of mouse NS-0 cells with normal or Ok(a-) cDNA confirmed the identity of the protein and only the Ok(a-) transfectants failed to react with monoclonal anti-Ok(a) Ab.

Immunochemical analysis of the human erythrocyte Rh polypeptides.

We have used rabbit polyclonal antisera raised against synthetic peptides complementary to different domains of the Rh polypeptides and Rh glycoprotein to examine the topography and organization of these proteins in the human erythrocyte membrane. Previously unrecognized exofacial protease sites have been identified on Rh CcEe, D proteins, and Rh glycoprotein. The Rh D protein has two specific bromelain cleavage sites located within the first and sixth predicted external domains, with the site of cleavage localized in the sixth domain to lie between residues 353 and 354. All Rh polypeptide species were found to be susceptible to cleavage with trypsin and subtilisin within the first external domain of these proteins. The Rh glycoprotein has two bromelain cleavage sites within the first external domain. These flank the single N-glycosylation site (Asn37), with the cleavage site toward the C-terminal side of this residue being between residues 39 and 40. Bromelain treatment was found to deglycosylate the Rh glycoprotein. Immunoprecipitation experiments have revealed that anti-C, -c,E, -e, and -D immune complexes are reactive with antisera raised against the fourth predicted external loop of the Rh proteins and the C-terminal domain. These data indicate that the hypothesis that suggests Rh C/c antigens are expressed on truncated Rh polypeptides by a mechanism of alternate splicing is incorrect and support the hypothesis that Rh Cc and Ee antigens are expressed on a single polypeptide chain.

The Lutheran blood group glycoprotein, another member of the immunoglobulin superfamily, is widely expressed in human tissues and is developmentally regulated in human liver.

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.

Isolation and characterization of CD47 glycoprotein: a multispanning membrane protein which is the same as integrin-associated protein (IAP) and the ovarian tumour marker OA3.

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.

Localization of the protein 4.1-binding site on human erythrocyte glycophorins C and D.

The flexibility of the human erythrocyte membrane is mediated by an underlying network of skeletal proteins which interact with the membrane through ankyrin and protein 4.1. The nature of the membrane attachment site(s) for protein 4.1 has yet to be fully elucidated. In this paper we show that purified protein 4.1 binds much less strongly to alkali-stripped membranes from erythrocytes of individuals with total glycophorin C and D deficiency (Leach phenotype) than to alkali-stripped normal membranes. We further show that a synthetic peptide corresponding to amino acid residues 82-98 of the cytoplasmic domain of glycophorin C specifically binds to purified protein 4.1 and inhibits protein 4.1 binding to alkali-stripped normal membranes. The same synthetic peptide binds directly to membranes from individuals with glycophorin C and D deficiency but not to normal membranes. These results indicate that glycophorins C and D provide major membrane attachment sites for protein 4.1 in normal erythrocytes and that this interaction is mediated by protein 4.1 binding to amino acid residues 82-98 of glycophorin C and 61-77 of glycophorin D.

Topology and organization of human Rh (rhesus) blood group-related polypeptides.

The Rh blood group antigens are associated with nonglycosylated human erythrocyte membrane proteins of molecular mass 30 kDa (the Rh30 polypeptides) and a glycoprotein of 40-100 kDa (the Rh glycoprotein). We have studied the topology of this family of proteins in the erythrocyte membrane. We confirmed the predicted cytosolic localization of the C and N termini of the Rh protein family. We located Lys-196 and Arg-323 of the Rh glycoprotein to the cytosol, and Glu-34 to the extracellular side of the plasma membrane in erythrocytes, by N-terminal sequencing of Rh glycoprotein peptides produced by proteolysis at the cytoplasmic or extracellular side of the membrane. We also show that a glycan chain is present on only one (Asn-37) of the three potential N-glycan addition sites in the Rh glycoprotein. Studies of the Rh glycoprotein fragments that co-immunoprecipitated with the Rh30 polypeptides suggest there is an interaction between the Rh30 polypeptides and amino acids 35-196 of the Rh glycoprotein. A model for the organization of the components of the Rh complex in the red cell membrane is proposed.

Studies on the glycoprotein associated with Rh (rhesus) blood group antigen expression in the human red blood cell membrane.

The blood group Rh antigens are associated with non-glycosylated 30-kDa erythrocyte membrane proteins (the Rh30 polypeptides) and the Rh glycoprotein. We used antipeptide antibodies to study the Rh glycoprotein in human erythrocyte membranes. The Rh glycoprotein was present in Rhnull U+ve cells. However, the N-glycan chain of the Rh glycoprotein in Rhnull U+ve cells was smaller than in normal cells. In contrast, the N-glycan chain of the Rh glycoprotein was larger than normal in glycophorin B-deficient red cells. We suggest that this observation reflects a lower rate of movement of newly synthesized Rh glycoprotein through intracellular membranes to the cell surface in the absence of glycophorin B, and that in normal red cells glycophorin B facilitates the movement of the Rh protein complex to the cell surface. Our results provide evidence for the intracellular interaction of at least three proteins, the Rh glycoprotein, Rh30 polypeptides, and glycophorin B during the biosynthesis and cell surface expression of the Rh complex. These observations are likely to be important for the successful design of expression systems for the blood group Rh antigens.

Localization of the C termini of the Rh (rhesus) polypeptides to the cytoplasmic face of the human erythrocyte membrane.

We have raised a rabbit antiserum to a synthetic peptide corresponding to the C terminus (residues 400-416) of the Rh30A polypeptide. The rabbit antiserum reacted with the Rh30B (D30) polypeptide in addition to the Rh30A (C/c and/or E/e) polypeptide(s), indicating that these proteins share homology at their C termini. The antiserum did not react with erythrocyte membranes from an individual with Rh(null) syndrome. The rabbit antiserum immunoprecipitated Rh polypeptides from erythrocyte membranes and alkali-stripped membranes, but not from intact erythrocytes. Treatment of intact red cells with carboxypeptidase Y did not affect the reactivity of the antiserum, whereas treatment of alkali-stripped and unsealed erythrocyte ghost membranes resulted in the loss of antibody binding. Carboxypeptidase A treatment of intact erythrocytes and alkali-stripped membranes had no effect on antibody binding, indicating that the C-terminal domains of the Rh polypeptides contain lysine, arginine, proline, or histidine residues. These results show that the C termini of the Rh polypeptides are located toward the cytoplasmic face of the erythrocyte membrane. Treatment of intact radioiodinated erythrocytes with bromelain followed by immunoprecipitation with monoclonal anti-D gave a band of M(r) 24,000-25,000, indicating that the Rh30B (D30) polypeptide is cleaved at an extracellular domain close to the N or C terminus, with loss of the major radioiodinated domain. Immunoblotting of bromelain treated D-positive erythrocyte membranes with the rabbit antiserum to the C-terminal peptide revealed a new band of M(r) 6000-6500, indicating that the extracellular bromelain cleavage site is located near the C terminus of the molecule. The band of M(r) 6000-6500 was not obtained in erythrocyte membranes derived from bromelain treated D-negative erythrocytes. Erythrocytes of the rare -D- phenotype appear to either totally lack, or have gross alterations in, the Cc/Ee polypeptide(s), since the bromelain treatment of these cells resulted in the total loss of staining in the M(r) 35,000-37,000 region and the concomitant appearance of the new band of M(r) 6000-6500.

The abundance and organization of polypeptides associated with antigens of the Rh blood group system.

Twelve murine monoclonal antibodies, which react with human red cells of common Rh phenotype but give weak or negative reactions with Rh null erythrocytes, were used in quantitative binding assays and competitive binding assays to investigate the abundance and organization of polypeptides involved in the expression of antigens of the Rh blood group system. Antibodies of the R6A-type (R6A, BRIC-69, BRIC-207) and the 2D10-type (MB-2D10, LA18.18, LA23.40) recognize related structures and 100,000-200,000 molecules of each antibody bind maximally to erythrocytes of common Rh phenotype. Antibodies of the BRIC-125 type (BRICs 32, 122, 125, 126, 168, 211) recognize structures that are unrelated to those recognized by R6A-type and 2D10-type antibodies and between 10,000 and 50,000 antibody molecules bind maximally to erythrocytes of the common Rh phenotype. The binding of antibodies of the R6A-type and the 2D10-type, but not of antibodies of the BRIC-125-type could be partially inhibited by human anti-D antibodies (polyclonal and monoclonal) and a murine anti-e-like antibody. These results are consistent with evidence (Moore & Green 1987; Avent et al., 1988b) that the Rh blood group antigens are associated with a complex that comprises two groups of related polypeptides of M(r) 30,000 and M(r) 35,000-100,000, respectively, and suggest that there are 1-2 x 10(5) copies of this complex per erythrocyte. The polypeptide recognized by antibodies of the BRIC-125 type is likely to be associated with this complex.

The membrane domain of the human erythrocyte anion transport protein. Epitope mapping of a monoclonal antibody defines the location of a cytoplasmic loop near the C-terminus of the protein.

We have used synthetic peptides to study the location of the amino acid sequences in the human erythrocyte anion transport protein (band 3) which are recognized by four murine monoclonal antibodies, BRIC 130, 132, 154 and 155. These antibodies are known to react with epitopes in the protein which are on the cytoplasmic side of the membrane. The results suggest that the amino acid residues important for the reaction of BRIC 130 and BRIC 154/155 are located within amino acids 899-908 and 895-901 respectively in the cytoplasmic tail of the protein. The BRIC 132 epitope is located within amino acid residues 813-824. This is part of a surface loop in the protein which probably extends from residue 814 to residue 832 and is located on the cytoplasmic side of the membrane. These results provide direct evidence for the topographical location of a sequence in a poorly understood region of the protein.

Protein-sequence studies on Rh-related polypeptides suggest the presence of at least two groups of proteins which associate in the human red-cell membrane.

The Rh D blood-group antigen forms part of a complex, involving several other polypeptides, that is deficient in the red cells of individuals who lack all the antigens of the Rh blood-group system (Rhnull red cells). These include components recognized by anti-(Rh D) antibodies and the murine monoclonal antibodies R6A and BRIC 125. We have carried out protein-sequence studies on the components immunoprecipitated by these antibodies. Anti-(Rh D) antibodies immunoprecipitate an Mr-30,000-32,000 polypeptide (the D30 polypeptide) and an Mr-45,000-100,000 glycoprotein (D50 polypeptide). Antibody R6A immunoprecipitates two glycoproteins of Mr 31,000-34,000 (R6A32 polypeptide) and Mr 35,000-52,000 (R6A45 polypeptide). The D30 and R6A32 polypeptides were found to have the same N-terminal amino acid sequences, showing that they are closely related proteins. The D50 polypeptide and the R6A45 polypeptide also had indistinguishable N-terminal amino acid sequences that differed from that of the D30 and R6A32 polypeptides. The putative N-terminal membrane-spanning segments of the two groups of proteins showed homology in their amino acid sequence, which may account for the association of each of the pairs of proteins during co-precipitation by the antibodies. Supplementary data related to the protein sequence have been deposited as Supplementary Publication SUP 50417 (6 pages) at the British Library Document Supply Centre, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1988) 249, 5.

Incomplete glycosylation of erythrocyte membrane proteins in congenital dyserythropoietic anaemia type II (CDA II).

The alterations in the erythrocyte membrane proteins of individuals with congenital dyserythropoietic anaemia (CDA II) were studied. Alterations were observed in both the erythrocyte sialoglycoproteins and erythrocyte anion transport protein (Band 3). There was a decrease in the apparent molecular weight of the major sialoglycoprotein alpha (glycophorin A) as well as a general reduction in the intensity of staining of all the sialoglycoproteins by the PAS stain. Sialoglycoprotein alpha isolated from CDA II erythrocytes contained 30% less sialic acid than normal alpha. The anion transport protein of CDA II erythrocytes migrated as a band with a lower molecular weight than the normal protein on SDS-gel electrophoresis. The CDA II anion transport protein had a substantially reduced content of N-acetylglucosamine and galactose, which probably reflects a reduction in the number of N-acetyl-lactosamine units carried by the protein. Our results suggest that there is a general defect in glycosylation of the major membrane glycoproteins of CDA II erythrocytes. We suggest that this glycosylation defect is a consequence of bone marrow stress.

Characterization and partial sequence of di-iodosulphophenyl isothiocyanate-binding peptide from human erythrocyte anion-transport protein.

We investigated the presumed anion-binding domain of the anion-transport protein from human erythrocyte membranes, using 2,6-di-iodo-4-sulphophenyl isothiocyanate, an inhibitor of anion transport. The 125I-labelled reagent binds covalently to the protein with a half-maximal inhibitory concentration of 86 microM. Treatment of unsealed erythrocyte 'ghosts' with chymotrypsin yielded a membrane-bound fragment (mol.wt. 14 500 +/- 1000) that contained all the protein-bound radioactivity. The binding of the inhibitor to this peptide gave a pattern very similar to that obtained for the effect of the compound on phosphate transport into erythrocytes. The peptide is therefore presumed to be intimately involved in the mediation of anion exchange. Cleavage of the 14 500-mol.wt. transmembrane fragment with CNBr resulted in the production of two peptides with apparent molecular weights of 8800 and 4700. The 4700-mol.wt. peptide is the N-terminal portion of the 14 500-mol.wt. peptide. The attachment site for 2,6-di-iodo-4-sulphophenyl isothiocyanate is situated near the C-terminal of the 8800-mol.wt. peptide. This locates the inhibitor-binding site near the chymotrypsin cleavage point at the extracellular surface of the membrane. A partial sequence (residues 1--38) of the 8800-mol.wt. peptide was obtained.

Immunochemical evidence for hybrid sialoglycoproteins of human erythrocytes.

The two major sialoglycoproteins of the human erythrocyte membrane (alpha and delta, glycophorins A and B) have identical amino acid sequences for the first 26 residues from the amino terminus, except that alpha expresses M or N blood group antigen activity whereas deta carries only blood group N activity. In addition, the asparagine at position 26 on alpha carries an oligosaccharide chain which is absent from the same position on delta. The two sialoglycoproteins differ in their remaining amino acid sequence and delta expresses blood group Ss activity. There are also variant sialoglycoproteins which have properties of both the alpha and delta molecules and may be hybrids of these. Using antibodies directed against different structural regions of the major sialoglycoprotein alpha, we confirm here and two variant erythrocytes (Miltenberger class V (MiV) and Ph) contain hybrid sialoglycoprotein molecules (Fig. 1). These hybrid sialoglycoproteins arise from cross-over events between the genes coding for alpha and delta. It is suggested that the two genes are closely associated in the order alpha, delta (5' leads to 3') on the chromosome.

Studies on the blood of an MiV homozygote.

An individual, whose parents are third cousins, has been shown to be homozygous for the rare Mi.V. condition. The proposita's red blood cells type as M-, N+(weak), S-, s+(strong), U+, Mi(a-), Vw-, Hil+; Wr(a-b-). The cells react, albeit less strongly than most other samples, with anti-Ena. However, from studies on the red blood cells of the proposita and on those of another person of the En(a+), Wr(a-b-) phenotype, it is apparent that the term "anti-Ena" actually describes a number of antibodies of differing specificities. Inhibition studies with sialoglycoprotein (SGP) isolates, and tests on protease-modified red blood cells illustrate some of the differences in specificity. Biochemical analyses of the SGPs of the red blood cells of the MiV homozygote and those of her parents confirm that the Mi.V condition is associated with the absence of normal MN SGP (alpha) and normal Ss SGP (delta), the appearance of a hybrid SGP molecule comprised of a portion of the MN SGP at its NH2 terminal end, and a portion of the Ss SGP at its C terminal end.

A new human erythrocyte variant (Ph) containing an abnormal membrane sialoglycoprotein.

1. A new human erythrocyte variant (Ph) is described. The variant contains an unusual sialic acid-rich glycoprotein in addition to the blood-group-MN([unk])- and blood-group-Ss(delta)-active sialoglycoproteins found in normal erythrocytes. 2. The unusual component Ph has an apparent mol.wt. of 32000 on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The Ph component is not degraded during trypsin treatment of intact erythrocytes. 3. The Ph component was labelled by lacto-peroxidase-mediated radioiodination of intact erythrocytes and was found to be present in amounts approximately equimolar to alpha-sialoglycoprotein in the variant erythrocytes. 4. The Ph component had receptors for the lectins from Maclura aurantiaca (osage orange) and Triticum vulgaris (wheat-germ), but lacked a receptor for the Phaseolus vulgaris (red kidney bean) lectin, suggesting that it carries only O-linked oligosaccharides. 5. The presence of the Ph component in these erythrocytes does not correspond to any of the known blood-group-MNSs-related antigens examined. 6. We suggest that this component may be a hybrid polypeptide containing the N-terminal portion derived from normal delta-sialoglycoprotein, and the C-terminal portion from normal alpha-sialoglycoprotein, in a manner similar to the anti-Lepore haemoglobin.