RESUMO
Contrary to the mainstream blood group systems, P1PK continues to puzzle and generate controversies over its molecular background. The P1PK system comprises three glycosphingolipid antigens: Pk, P1 and NOR, all synthesised by a glycosyltransferase called Gb3/CD77 synthase. The Pk antigen is present in most individuals, whereas P1 frequency is lesser and varies regionally, thus underlying two common phenotypes: P1, if the P1 antigen is present, and P2, when P1 is absent. Null and NOR phenotypes are extremely rare. To date, several single nucleotide polymorphisms (SNPs) have been proposed to predict the P1/P2 status, but it has not been clear how important they are in general and in relation to each other, nor has it been clear how synthesis of NOR affects the P1 phenotype. Here, we quantitatively analysed the phenotypes and A4GALT transcription in relation to the previously proposed SNPs in a sample of 109 individuals, and addressed potential P1 antigen level confounders, most notably the red cell membrane cholesterol content. While all the SNPs were associated with the P1/P2 blood type and rs5751348 was the most reliable, we found large differences in P1 level within groups defined by their genotype and substantial intercohort overlaps, which shows that the P1PK blood group system still eludes full understanding.
Assuntos
Antígenos de Grupos Sanguíneos/genética , Galactosiltransferases/genética , Globosídeos/genética , Polimorfismo de Nucleotídeo Único , Anticorpos/química , Colesterol/química , Citometria de Fluxo , Genótipo , Glicoesfingolipídeos/química , Homozigoto , Humanos , Lipídeos/química , Fenótipo , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Human Gb3/CD77 synthase (α1,4-galactosyltransferase) is the only known glycosyltransferase that changes acceptor specificity because of a point mutation. The enzyme, encoded by A4GALT locus, is responsible for biosynthesis of Gal(α1-4)Gal moiety in Gb3 (CD77, Pk antigen) and P1 glycosphingolipids. We showed before that a single nucleotide substitution c.631C > G in the open reading frame of A4GALT, resulting in replacement of glutamine with glutamic acid at position 211 (substitution p. Q211E), broadens the enzyme acceptor specificity, so it can not only attach galactose to another galactose but also to N-acetylgalactosamine. The latter reaction leads to synthesis of NOR antigens, which are glycosphingolipids with terminal Gal(α1-4)GalNAc sequence, never before described in mammals. Because of the apparent importance of position 211 for enzyme activity, we stably transfected the 2102Ep cells with vectors encoding Gb3/CD77 synthase with glutamine substituted by aspartic acid or asparagine, and evaluated the cells by quantitative flow cytometry, high-performance thin-layer chromatography and real-time PCR. We found that cells transfected with vectors encoding Gb3/CD77 synthase with substitutions p. Q211D or p. Q211N did not express Pk, P1 and NOR antigens, suggesting complete loss of enzymatic activity. Thus, amino acid residue at position 211 of Gb3/CD77 synthase is critical for specificity and activity of the enzyme involved in formation of Pk, P1 and NOR antigens. Altogether, this approach affords a new insight into the mechanism of action of the human Gb3/CD77 synthase.
Assuntos
Galactosiltransferases , Glicoesfingolipídeos/biossíntese , Mutação de Sentido Incorreto , Acetilgalactosamina/genética , Acetilgalactosamina/metabolismo , Substituição de Aminoácidos , Antígenos Nucleares/genética , Antígenos Nucleares/metabolismo , Linhagem Celular Tumoral , Galactosiltransferases/genética , Galactosiltransferases/metabolismo , Glicoesfingolipídeos/genética , Humanos , Especificidade por SubstratoRESUMO
INTRODUCTION: Saccharomyces cerevisiae is an excellent model organism for studies of transcriptional regulation of metabolic processes in other eukaryotic cells including human cells. Cellular acid-base balance can be disturbed in pathologic situations such as renal acidosis or cancer. The extracellular pH of malignant solid tumors is acidic in the range of 6.5-6.9. EG07 and EG37 aci mutants of Saccharomyces cerevisiae excessively excrete carboxylic acids to glucose-containing media or distilled water. The excreted acids are Krebs and/or glyoxylate cycle intermediates. The genes restoring the wild-type phenotype have function that does not easily explain theAci+ phenotype. MATERIAL/METHODS: In this study, using real-time PCR we measured relative mRNA expression, in the mutants compared to the wild-type strain, of selected genes associated with both carboxylic acid cycles and two cell transporters, Pma1 and Pdr12, of organic acids. RESULTS: Unexpectedly, we found that the relative expression of the selected Krebs cycle and glyoxylate cycle genes did not change significantly. However, the expression of the two transporter genes was strongly elevated in EG37 and moderately increased in EG07. CONCLUSION: These results indicate that the induction of the two cell transporterg enes plays an important role in acid excretion by the aci mutants.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Ácidos Carboxílicos/metabolismo , Ciclo do Ácido Cítrico/genética , Mutação , ATPases Translocadoras de Prótons/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Ativação Transcricional/fisiologia , Transporte Biológico , Expressão Gênica , Glucose/metabolismo , Glioxilatos/metabolismo , Humanos , Proteínas de Membrana Transportadoras/metabolismo , RNA Mensageiro/análise , Saccharomyces cerevisiae/classificação , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Especificidade da EspécieRESUMO
The CEA related cell adhesion molecules (CEACAM) contain variable and constant immunoglobulin-like domains and are classified as a member of the immunoglobulin supergene family, IgSF. The seven CEACAM (CD66) antigens (CEACAM1, CEACAM3, CEACAM4, CEA, CEACAM6, CEACAM7 and CEACAM8) differ in the number of Ig-like domains, sugar content, presence of isoforms, tissue distribution and form of membrane attachment (transmembrane region or GPI anchor). CEACAMs with a transmembrane region possess a cytoplasmic domain with or without the immunoreceptor motifs. The structural diversity of CEACAMs results in their multifunctionality, especially displayed in calcium independent homo- and heterotypic adhesion interactions. The scientific data, collected mainly for CEA, strongly confirm involvement of this molecule in colorectal cancer. Recent research also indicates that CEACAMs play an important role in signal transduction, recognition and binding of pathogenic bacteria belonging to Neisseria and Escherichia genera.
Assuntos
Antígenos CD/química , Antígenos CD/metabolismo , Antígeno Carcinoembrionário/química , Antígeno Carcinoembrionário/imunologia , Moléculas de Adesão Celular/química , Moléculas de Adesão Celular/metabolismo , Neoplasias Colorretais/metabolismo , Escherichia/metabolismo , Humanos , Neisseria/metabolismo , Isoformas de Proteínas/metabolismo , Estrutura Terciária de Proteína , Transdução de Sinais/fisiologiaRESUMO
Carcinoembryonic antigen (CEA) was identified as a prominent tumor-associated antigen in human colorectal cancer and it is still intensively investigated. However, its physiological role remains unclear. The CEA molecule is composed of seven highly hydrophobic, immunoglobulin-like domains, six of which contain a single disulphide bridge. The production of recombinant protein containing Ig-like domains in bacterial expression systems often results in partial degradation or insolubility due to aggregation hampering the analysis of their native structure and function. Here, we present a new method of expression and purification of CEA N-terminal domains (N-A1) fused to MBP in Escherichia coli. In order to optimize the expression and purification of CEA N-A1 domains we evaluated bacteria cultivation conditions, the length of N-A1 domains, fusion systems (GST- and MBP-tag), IPTG concentrations and protein purification conditions. We have found that MBP-N-A1 fusion protein digested with TEV protease forms soluble aggregates composed of N-A1 domains and incompletely digested MBP-N-A1 fusion protein. Using 1.25 M guanidinium chloride (GdmCl) as a component of the elution buffer we were able to achieve an almost complete dissociation of the aggregates. The dissociation was monitored by circular dichroism and fluorescence measurements. The CD spectra and Ellman's assay suggest that the conformation of N-A1 domains and their disulphide bonds are correct.
Assuntos
Antígeno Carcinoembrionário/isolamento & purificação , Proteínas Recombinantes de Fusão/isolamento & purificação , Antígeno Carcinoembrionário/biossíntese , Antígeno Carcinoembrionário/química , Cromatografia em Gel , Dicroísmo Circular , Dissulfetos , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Escherichia coli/metabolismo , Guanidina , Humanos , Proteínas Ligantes de Maltose/química , Proteínas Ligantes de Maltose/metabolismo , Estabilidade Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , SolubilidadeRESUMO
The pComb3H vector system is used for constructing and panning recombinant antibody libraries. It allows for expression of monovalent Fab fragments, either on the surface of M13 phage, or in the form of soluble proteins secreted into the periplasmic space of bacteria. We constructed a modified pComb3H vector containing cDNA encoding for a 23-amino acid fragment of the Escherichia coli biotin carboxy carrier protein (BCCP), which is an acceptor sequence for biotinylation. The vector was used to express the Fab fragment recognizing human glycophorin A. The purified Fab fragment containing this biotin acceptor sequence was effectively biotinylated in vitro using biotin ligase (BirA). The specificity and avidity of the biotinylated Fab fragments were similar to the previously produced, unmodified Fab fragments. An avidin-alkaline phosphatase conjugate was used to detect the recombinant Fab fragments, instead of secondary antibody. In addition, when biotinylated Fab fragments were mixed with avidin, red blood cells were directly agglutinated.
Assuntos
Testes de Aglutinação/métodos , Hemaglutinação/fisiologia , Fragmentos Fab das Imunoglobulinas/metabolismo , Proteínas Recombinantes/metabolismo , Avidina/metabolismo , Biotinilação , Western Blotting , Cromatografia em Gel , Eletroforese em Gel de Poliacrilamida , Eritrócitos/citologia , Eritrócitos/metabolismo , Glicoforinas/metabolismo , Humanos , Ligação ProteicaRESUMO
The ABO human blood group system consists of A antigens, B antigens, and antibodies against these antigens. The antigenic determinants are synthesized in the Golgi apparatus by specific glycosyltransferases which transfer proper sugars to an oligosaccharide acceptor, called H antigen. N-acetylgalactosaminotransferase (transferase A) uses a UDP-GalNac donor to convert the H antigen to A antigen, whereas galactosyltransferase (transferase B) uses a UDP-galactose donor to convert the H antigen to B antigen. The amino-acid sequences of transferases A and B differ by four residues, of which only two cause a change in enzyme specificity. These residues are Leu/Met266 and Gly/Ala268 in transferases A and B, respectively. Structural studies revealed that the presence of amino acids with bulky side chains (methionine and alanine) in transferase B cause its inability to bind N-acetylgalactosamine. The recessive trait O, in which antigens A and B are not present, is caused by the expression of an incomplete enzyme as a result of a base deletion and a subsequent reading frame change. In addition to the basic ABO gene variants, several alleles are rarely found that may lead to the expression of enzymes with different specificities. In this article the mechanism of the synthesis of A and B antigens, the molecular background of ABO gene variablity, their allelic variants, and possible mechanisms by which they emerge are described.
Assuntos
Sistema ABO de Grupos Sanguíneos/biossíntese , Galactosiltransferases/genética , Regulação Enzimológica da Expressão Gênica , N-Acetilgalactosaminiltransferases/genética , Alelos , Sequência de Aminoácidos , Humanos , Dados de Sequência MolecularRESUMO
The Duffy antigen/receptor for chemokines (DARC) is a seven-transmembrane glycoprotein carrying the Duffy (Fy) blood group antigen. The polypeptide chain of DARC contains two NSS motifs at positions 16 and 27 and one NDS motif at position 33 that represent canonical sequences for efficient N-glycosylation. To verify whether all of these three sites are occupied by a sugar chain, we generated mutants in which potential N-glycosylation sites (AsnXSer) were removed by replacement of serine by alanine. Seven DARC glycosylation variants, missing one (S18A, S29A, S35A), two (S18A.S29A, S18A.S35A, S29A.S35A), or three (S18A.S29A.S35A) glycosylation sites, were obtained. cDNA encoding DARC mutants was cloned into the eukaryotic expression vector pcDNA3.1/myc-HisA and expressed in human K562 cells. Stable transfectants expressing wild-type or mutated forms of Duffy were then lysed, purified by metal-affinity chromatography, and subjected to Western blots with an anti-Duffy monoclonal antibody. The gel electrophoresis data indicate that all three canonical sites are used for sugar attachment.
Assuntos
Sistema do Grupo Sanguíneo Duffy/genética , Receptores de Superfície Celular/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Anticorpos Monoclonais , Western Blotting , Cromatografia de Afinidade , Análise Mutacional de DNA , Sistema do Grupo Sanguíneo Duffy/química , Sistema do Grupo Sanguíneo Duffy/imunologia , Eletroforese em Gel de Poliacrilamida , Glicosilação , Humanos , Células K562 , Dados de Sequência Molecular , Receptores de Superfície Celular/química , Receptores de Superfície Celular/imunologiaRESUMO
Well-characterized mouse models of allo-immune antibody-mediated hemolysis would provide a valuable approach for gaining greater insight into the pathophysiology of hemolytic transfusion reactions. To this end, mouse red blood cells (mRBCs) from human glycophorin A transgenic (hGPA-Tg) donor mice were transfused into non-Tg recipients that had been passively immunized with IgG or IgM hGPA-specific monoclonal antibodies (mAbs). In this novel murine "blood group system," mRBCs from hGPA-Tg mice are "antigen positive" and mRBCs from non-Tg mice are "antigen negative." Passive immunization of non-Tg mice with the IgG1 10F7 and IgG3 NaM10-2H12 anti-hGPA mAbs each induced rapid clearance of incompatible transfused hGPA-Tg-mRBCs in a dose-response manner. Using various knockout mice as transfusion recipients, both the complement system and activating Fcgamma receptors were found to be important in the clearance of incompatible mRBCs by each of these IgG mAbs. In addition, the IgM E4 anti-hGPA mAb induced complement-dependent intravascular hemolysis of transfused incompatible hGPA-Tg-mRBCs accompanied by gross hemoglobinuria. These initial studies validate the relevance of these new mouse models for addressing important questions in the field of transfusion medicine.
Assuntos
Hemólise/imunologia , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Reação Transfusional , Animais , Anticorpos Monoclonais , Proteínas do Sistema Complemento/metabolismo , Modelos Animais de Doenças , Glicoforinas/genética , Humanos , Imunização Passiva , Técnicas In Vitro , Isoanticorpos/sangue , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Receptores de IgG/sangueRESUMO
The immunoglobulins of the Camelidae family belonging to subclasses IgG2 and IgG3 consist of heavy chains only. The lack of light chains is caused by a point mutation in the heavy-chain gene, resulting in the loss of the splice consensus signal and the removal of the entire CH1 domain together with introns. The heavy-chains antibodies also contain longer hinge regions and conservative amino-acid substitutions in the framework regions. Despite the lack of light chains, the heavy-chain antibodies reveal normal antigen binding ability and effector functions. The heavy-chain antibodies are relatively easy to clone and possess good stability, high specificity, low molecular weight, and the ability to recognize unique epitopes. Possible areas of application of heavy-chain antibodies include their use as in vivo imaging reagents and sources of peptide-based drugs.
Assuntos
Camelídeos Americanos/imunologia , Imunoglobulina G/imunologia , Sequência de Aminoácidos , Animais , Humanos , Imunoglobulina G/química , Imunoglobulina G/genética , Dados de Sequência Molecular , Biblioteca de Peptídeos , Especificidade da EspécieRESUMO
It was shown that CEACAM5 can mediate cell-cell adhesion through homotypic and heterotypic interactions; however, its role in the expression of the malignant phenotype remains obscure. To study whether the formation of both primary tumors and metastases is directly related to the presence or absence of CEACAM5, we applied the antisense RNA strategy. By transfecting human CX-1.1 colon carcinoma cells with CEACAM5 antisense-expressing vector or with the vector itself, cell variants with a highly decreased expression of CEACAM5 were obtained. Profound differences in proliferative abilities among parental and obtained subclones of CX-1.1 cells were revealed when cells were implanted subcutaneously into nude mice. In contrast to their highly tumorigenic parental CX-1.1 cells (with high expression of membrane-bound and secreted CEACAM5), two subclones (3E and AS6Q) with substantially decreased expression of membrane-bound and secreted CEA showed a considerably diminished growth rate. Even more striking results were obtained with AS8Q cells, producing a residual amount of this glycoprotein. However, 3B cells (producing a large amount of secreted CEACAM5) did not differ significantly in their tumorigenic properties from CX-1.1 cells. Our experiments performed in nu/nu mice suggest that CEACAM5 supports the growth of primary tumors, but is not involved in the formation of metastases by colon cancer cells.
Assuntos
Adenocarcinoma/metabolismo , Antígenos de Neoplasias/genética , Moléculas de Adesão Celular/genética , Neoplasias do Colo/metabolismo , Animais , Antígenos de Neoplasias/biossíntese , Moléculas de Adesão Celular/biossíntese , Humanos , Camundongos , Camundongos Nus , Plasmídeos , RNA Antissenso , TransfecçãoRESUMO
Carcinoembryonic antigen (CEA) is an oncofoetal cell surface glycoprotein that serves as an important tumour marker for colorectal and some other carcinomas. Its immunoglobulin-like structure places CEA within the immunoglobulin superfamily. CEA functions in several biological roles including homotypic and heterotypic (with other CEA family members) cell adhesion. Cell-cell interaction can be modulated by different factors, e.g., post-translational modifications such as glycosylation. The purpose of this study was to examine whether changes in carbohydrate composition of CEA oligosaccharides can influence homotypic (CEA-CEA) interactions. In order to modulate glycosylation of CEA we used two different glycosylation mutants of Chinese hamster ovary (CHO) cells, Lec2 and Lec8. Lec2 cells should produce CEA with nonsialylated N-glycans, while Lec8 cells should yield more truncated sugar structures than Lec2. Parental CHO (Pro5) cells and the glycosylation deficient mutants were stably transfected with CEA cDNA. All three CEA glycoforms, tested in a solid-phase cell adhesion assay, showed an ability to mediate CEA-dependent cell adhesion, and no qualitative differences in the adhesion between the glycoforms were observed. Thus, it may be assumed that carbohydrates do not play a role in homotypic adhesion, and the interactions between CEA molecules depend solely on the polypeptide structure.
Assuntos
Antígeno Carcinoembrionário/metabolismo , Animais , Células CHO , Adesão Celular/fisiologia , Cricetinae , Glicosilação , Oligossacarídeos/genética , Oligossacarídeos/metabolismo , TransfecçãoRESUMO
BACKGROUND: When expressed in Escherichia coli, recombinant F(ab) contain a heavy-chain Fd fragment and a complete light-chain fragment. Because these F(ab) are monovalent, their avidity is significantly lower than that of a corresponding bivalent IgG antibody. In addition, when monovalent F(ab) are used in hemagglutination assays, antiglobulin reagents are required. Therefore, it would be useful to develop a system that expresses recombinant bivalent F(ab) in E. coli. STUDY DESIGN AND METHODS: Three modified vectors were constructed. Each contained cDNA sequences encoding a peptide linked to the C terminus of a heavy-chain CH1 region: an IgG1 hinge region (Hinge), a leucine zipper (Zip), or a peptide containing the Hinge and Zip sequences in tandem (HingeZip). The vectors were used to express two cloned F(ab) recognizing human antigens M and N: NNA7 (anti-N) and 425/2B (anti-M). The recombinant proteins were expressed in E. coli and were purified and evaluated by ELISA and hemagglutination. RESULTS: By gel filtration chromatography, 35, 90, and 70 percent of the purified F(ab) expressing the Hinge, Zip, and HingeZip tails, respectively, were dimers. By ELISA, the avidity of F(ab) containing the Zip or HingeZip tails was six to eight times higher than that of the corresponding monovalent F(ab). In addition, the dimeric F(ab) directly agglutinated RBCs in concentrations similar to those of corresponding bivalent IgG antibodies. CONCLUSIONS: An introduction of dimer-inducing peptides allowed the isolation of bacterially produced, bivalent F(ab). This approach could be useful for obtaining inexpensive, serologic reagents that may replace or complement conventional MoAbs produced by mammalian tissue culture methods.