Isolation of cDNA clones for human erythrocyte membrane sialoglycoproteins alpha and delta.
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ABSTRACT:
SUBMITTER: Tate CG
PROVIDER: S-EPMC1135146 | biostudies-other | 1988 Sep
REPOSITORIES: biostudies-other
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Project description:By using radioiodinated monoclonal antibodies we have estimated that there are about 600 000 copies of sialoglycoprotein alpha (synonym glycophorin A) and 80 000 copies of sialoglycoprotein delta (synonym glycophorin B) per normal human erythrocyte. Erythrocytes expressing the product of only one alpha gene contain about 300 000 copies of alpha/cell. Two erythrocyte types containing alpha-delta hybrid molecules were studied. Those with heterozygous expression of the (alpha-delta)Mi.V gene contain about 100 000 alpha-delta copies per cell, whereas those with heterozygous expression of the En(UK) gene contain about 80 000 alpha-delta copies/cell. Erythrocyte types containing delta-alpha hybrid molecules were also studied. About 200 000 copies of (delta-alpha)Dantu were measured in cells with heterozygous expression of the (delta-alpha)Dantu gene (donor M.P.), whereas about 315 000 copies of the putative (delta-alpha)Dantu hybrid were found on the erythrocytes of donor J.O. [which also have heterozygous expression of the putative (delta-alpha)Dantu gene]. The erythrocytes of donor M.P. have normal levels of alpha, whereas those of donor J.O. have only about half-normal levels. It is proposed that the hybrid sialoglycoprotein of donor J.O. is of alpha-delta-alpha composition [(alpha-delta-alpha)Dantu] rather than delta-alpha and results from a double cross-over analogous to that which gives rise to haemoglobin Parchman.
| S-EPMC1152989 | biostudies-other
Project description:The Rh blood-group antigens are associated with human erythrocyte membrane proteins of approx. 30 kDa (the Rh30 polypeptides). Heterogeneously glycosylated membrane proteins of 50 and 45 kDa (the Rh50 glycoproteins) are coprecipitated with the Rh30 polypeptides on immunoprecipitation with anti-Rh-specific mono- and poly-clonal antibodies. We have isolated cDNA clones representing a member of the Rh50 glycoprotein family (the Rh50A glycoprotein). We used PCR with degenerate primers based on the N-terminal amino acid sequence of the Rh50 glycoproteins and human genomic DNA as a template and cloned and sequenced three types of PCR product of the expected size. Two of these products, Rh50A and Rh50B, gave the same translated amino acid sequence which corresponded to the expected Rh50 glycoprotein sequence but had only 75% DNA sequence similarity. The third product (Rh50C) contained a single base deletion, and the translated amino acid sequence contained an in-frame stop codon. We have isolated cDNA clones containing the full coding sequence of the Rh50A glycoprotein. This sequence predicts that it is a 409-amino acid N-glycosylated membrane protein with up to 12 transmembrane domains. The Rh50A glycoprotein shows clear similarity to the Rh30A protein in both amino acid sequence and predicted topology. Our results are consistent with the Rh30 and Rh50 groups of proteins being different subunits of an oligomeric complex which is likely to have a transport or channel function in the erythrocyte membrane. We mapped the Rh50A gene to human chromosome 6p21-qter, showing that genetic differences in the Rh30 rather than the Rh50 genes specify the major polymorphic forms of the Rh antigens.
| S-EPMC1133147 | biostudies-other
Project description:Healthy blood neutrophils are functionally quiescent in the bloodstream, have a short lifespan, and exit the circulation to carry out innate immune functions, or undergo rapid apoptosis and macrophage-mediated clearance to mitigate host tissue damage. Limitation of unnecessary intravascular neutrophil activation is also important to prevent serious inflammatory pathologies. Because neutrophils become easily activated after purification, we carried out ex vivo comparisons with neutrophils maintained in whole blood. We found a difference in activation state, with purified neutrophils showing signs of increased reactivity: shedding of l-selectin, CD11b upregulation, increased oxidative burst, and faster progression to apoptosis. We discovered that erythrocytes suppressed neutrophil activation ex vivo and in vitro, including reduced l-selectin shedding, oxidative burst, chemotaxis, neutrophil extracellular trap formation, bacterial killing, and induction of apoptosis. Selective and specific modification of sialic acid side chains on erythrocyte surfaces with mild sodium metaperiodate oxidation followed by aldehyde quenching with 4-methyl-3-thiosemicarbazide reduced neutrophil binding to erythrocytes and restored neutrophil activation. By enzyme-linked immunosorbent assay and immunofluorescence, we found that glycophorin A, the most abundant sialoglycoprotein on erythrocytes, engaged neutrophil Siglec-9, a sialic acid-recognizing receptor known to dampen innate immune cell activation. These studies demonstrate a previously unsuspected role for erythrocytes in suppressing neutrophils ex vivo and in vitro and help explain why neutrophils become easily activated after separation from whole blood. We propose that a sialic acid-based "self-associated molecular pattern" on erythrocytes also helps maintain neutrophil quiescence in the bloodstream. Our findings may be relevant to some prior experimental and clinical studies of neutrophils.
| S-EPMC5465837 | biostudies-literature
Project description:The Mammalian Gene Collection (MGC) consortium (http://mgc.nci.nih.gov) seeks to establish publicly available collections of full-ORF cDNAs for several organisms of significance to biomedical research, including human. To date over 15,200 human cDNA clones containing full-length open reading frames (ORFs) have been identified via systematic expressed sequence tag (EST) analysis of a diverse set of cDNA libraries; however, further systematic EST analysis is no longer an efficient method for identifying new cDNAs. As part of our involvement in the MGC program, we have developed a scalable method for targeted recovery of cDNA clones to facilitate recovery of genes absent from the MGC collection. First, cDNA is synthesized from various RNAs, followed by polymerase chain reaction (PCR) amplification of transcripts in 96-well plates using gene-specific primer pairs flanking the ORFs. Amplicons are cloned into a sequencing vector, and full-length sequences are obtained. Sequences are processed and assembled using Phred and Phrap, and analyzed using Consed and a number of bioinformatics methods we have developed. Sequences are compared with the Reference Sequence (RefSeq) database, and validation of sequence discrepancies is attempted using other sequence databases including dbEST and dbSNP. Clones with identical sequence to RefSeq or containing only validated changes will become part of the MGC human gene collection. Clones containing novel splice variants or polymorphisms have also been identified. Our approach to clone recovery, applied at large scale, has the potential to recover many and possibly most of the genes absent from the MGC collection.
| S-EPMC528924 | biostudies-literature
Project description:cDNA clones of ski and the ski-related gene, sno, were obtained by screening human cDNA libraries. The predicted open reading frame of h-ski could encode a protein of 728 amino acid residues. The h-ski protein is highly homologous with the v-ski protein. The overall homology between h-ski and v-ski is 91% at the amino acid level. DNA sequencing analysis revealed two types of cDNA clones from the sno (ski-related novel gene) gene, possibly due to alternative splicing. The first type, named snoN (non Alu-containing), encoded a protein of 684 amino acid residues. The second type, named snoA (Alu-containing), encoded a protein of 415 amino acid residues. The first 366 amino acid residues of snoN and snoA are the same, but subsequent amino acids show divergence. Several transcripts of h-ski (6.0, 4.7, 3.8, 3.0, 2.1 and 1.8 kb) were detected. The mRNAs of h-sno were 6.2, 4.4 and 3.2kb.
| S-EPMC318172 | biostudies-other
Project description:alpha-L-Iduronidase (IDUA; EC 3.2.1.76) is a lysosomal hydrolase in the metabolic pathway responsible for the degradation of the glycosaminoglycans heparan sulfate and dermatan sulfate. A deficiency of IDUA in humans leads to the accumulation of these glycosaminoglycans and results in the lysosomal storage disorder mucopolysaccharidosis type I. We have isolated and sequenced cDNA clones containing part of the human IDUA coding region and used PCR from reverse-transcribed RNA to obtain the full IDUA sequence. Analysis of the predicted 653-amino acid precursor protein shows that IDUA has a 26-amino acid signal peptide that is cleaved immediately prior to the amino terminus of the 74-kDa polypeptide present in human liver IDUA. The protein sequence contains six potential N-glycosylation sites. Northern blot analysis with IDUA cDNA detected only a single 2.3-kilobase mRNA species in human placental RNA; however, PCR analysis of fibroblast, liver, kidney, and placental RNA showed the existence of alternatively spliced mRNA from the IDUA gene. Southern blot analysis failed to detect major deletions or gene rearrangements in any of the 40 mucopolysaccharidosis type I patients studied. Expression of a full-length IDUA cDNA construct in Chinese hamster ovary cells produced human IDUA protein at a level 13-fold higher than, and with a specific activity comparable to, IDUA present in normal human fibroblasts.
| S-EPMC52785 | biostudies-other
Project description:The human erythrocyte membrane sialoglycoproteins beta and gamma are important for the maintenance of the discoid shape of the normal erythrocyte. In this paper we show that the human erythrocyte sialoglycoproteins beta and gamma (hereafter called beta and gamma) are structurally related. Rabbit antisera produced against purified beta and beta 1 and rendered specific to the cytoplasmic portion of these proteins also react with the cytoplasmic portion of gamma. Some human anti-Gerbich (Ge) sera react with the extracellular portion of both beta and gamma. This reactivity is shown to be directed towards a common epitope on beta and gamma. However, most anti-Ge sera do not react with beta, but react with an extracellular epitope only present on gamma. All individuals who lack the Ge antigens lack beta and gamma. In some cases abnormal sialoglycoproteins are present in the erythrocytes, and these are shown to be structurally related to beta and gamma. Rabbit antisera raised against the purified abnormal sialoglycoprotein from a Ge-negative erythrocyte type reacted with the cytoplasmic portion of both beta and gamma. Unlike normal beta and gamma, the abnormal sialoglycoproteins found in Ge-negative erythrocytes migrate as a diffuse band on SDS/polyacrylamide-gel electrophoresis. Studies using endoglycosidases suggest that the diffuse nature of these bands results from carbohydrate heterogeneity and that the abnormal sialoglycoproteins contain N-glycosidically linked oligosaccharides with repeating lactosamine units. Such polylactosamine chains are not present on normal beta or gamma.
| S-EPMC1147962 | biostudies-other
Project description:We have studied the erythrocytes of two individuals (P. L. and K. W.) who lack the Gerbich (Ge) blood-group antigen. The erythrocytes of P. L. and K. W. were not reactive with two monoclonal antibodies (NBTS/BRIC 4 and NBTS/BRIC 10) which reacted with normal erythrocytes. The membranes of P. L. and K. W. erythrocytes appeared to lack three minor sialoglycoproteins (beta, beta 1 and gamma). These three minor sialoglycoproteins were found to be associated with the cytoskeletons of normal erythrocytes. Approx. 10% of the erythrocytes of P. L. and K. W. were frankly elliptocytic. We suggest that one or more of the minor sialoglycoproteins may play a part in maintaining the discoid shape of the human erythrocyte.
| S-EPMC1153379 | biostudies-other
Project description:Membranes from erythrocytes with a new Gerbich (Ge)-negative phenotype (Leach phenotype), as well as those from two other Ge-negative phenotypes, were examined. Whereas cells of the Leach phenotype apparently lack three minor sialoglycoproteins (beta, beta 1 and gamma), the membranes of Ge- Yus- and Ge- Yus+ erythrocytes apparently lack beta- and gamma-sialoglycoproteins but contain additional diffusely migrating components of apparent Mr 30 500-34 500 and 32 500-36 500 respectively. Immunoprecipitation experiments showed that the abnormal components of both Ge- Yus- and Ge- Yus+ erythrocytes reacted with two monoclonal antibodies, BRIC 4 and BRIC 10. These antibodies have been shown to react with sialoglycoproteins beta and beta 1 in normal erythrocytes. Cytoskeletal preparations from Ge- Yus- and Ge- Yus+ erythrocyte membranes contained the abnormal components. In contrast with cells of the Leach phenotype, which are elliptocytic, Ge- Yus- and Ge- Yus+ were of normal shape, despite their apparent lack of beta- and gamma-sialoglycoproteins. It seems likely that the abnormal components in these cells contribute to their normal shape. Ovalocytic erythrocytes were shown to incorporate more radioactivity in the sialoglycoprotein-beta 1 region than normal erythrocytes after labelling by the periodate/NaB3H4 technique. It is suggested that abnormal components in Ge- Yus- and Ge- Yus+ erythrocytes result from chromosomal misalignment with unequal crossing-over at meiosis between the genes giving rise to beta-, beta 1- and gamma-sialoglycoproteins.
| S-EPMC1144007 | biostudies-other
Project description:The human genome sequence defines our inherent biological potential; the realization of the biology encoded therein requires knowledge of the function of each gene. Currently, our knowledge in this area is still limited. Several lines of investigation have been used to elucidate the structure and function of the genes in the human genome. Even so, gene prediction remains a difficult task, as the varieties of transcripts of a gene may vary to a great extent. We thus performed an exhaustive integrative characterization of 41,118 full-length cDNAs that capture the gene transcripts as complete functional cassettes, providing an unequivocal report of structural and functional diversity at the gene level. Our international collaboration has validated 21,037 human gene candidates by analysis of high-quality full-length cDNA clones through curation using unified criteria. This led to the identification of 5,155 new gene candidates. It also manifested the most reliable way to control the quality of the cDNA clones. We have developed a human gene database, called the H-Invitational Database (H-InvDB; http://www.h-invitational.jp/). It provides the following: integrative annotation of human genes, description of gene structures, details of novel alternative splicing isoforms, non-protein-coding RNAs, functional domains, subcellular localizations, metabolic pathways, predictions of protein three-dimensional structure, mapping of known single nucleotide polymorphisms (SNPs), identification of polymorphic microsatellite repeats within human genes, and comparative results with mouse full-length cDNAs. The H-InvDB analysis has shown that up to 4% of the human genome sequence (National Center for Biotechnology Information build 34 assembly) may contain misassembled or missing regions. We found that 6.5% of the human gene candidates (1,377 loci) did not have a good protein-coding open reading frame, of which 296 loci are strong candidates for non-protein-coding RNA genes. In addition, among 72,027 uniquely mapped SNPs and insertions/deletions localized within human genes, 13,215 nonsynonymous SNPs, 315 nonsense SNPs, and 452 indels occurred in coding regions. Together with 25 polymorphic microsatellite repeats present in coding regions, they may alter protein structure, causing phenotypic effects or resulting in disease. The H-InvDB platform represents a substantial contribution to resources needed for the exploration of human biology and pathology.
| S-EPMC393292 | biostudies-literature