ABSTRACT
Elevated circulating levels of the tachykinin, neurokinin B (NKB), have been observed in women with pre-eclampsia during the third trimester of pregnancy. Currently, the molecular mechanisms responsible for these increased levels remain unknown. To understand the molecular regulation, we have compared the differences in gene expression of the tachykinins and their receptors in control and pre-eclamptic placentae and the responses of the TAC3 gene encoding NKB to proposed physiological triggers of pre-eclampsia including hypoxia and oxidative stress using real-time quantitative PCR. We have determined the placenta to be the main site of TAC3 expression with levels 2.6-fold higher than the brain. TAC3 expression was found to be significantly higher in pre-eclamptic placenta (1.7-fold, P < 0.05) than in normal controls. No evidence was found that hypoxia and oxidative stress were responsible for increases in TAC3 expression. In rat placenta, a longitudinal study in normal late pregnancy was associated with a significant down-regulation of the NKB/NK3 ligand-receptor pair (P < 0.05). The present data suggest that the increased placental expression of TAC3 is part of the mechanism leading to the increased circulating levels of NKB in pre-eclampsia.
Subject(s)
Gene Expression Regulation/genetics , Neurokinin B/genetics , Pre-Eclampsia/genetics , Receptors, Neurokinin-3/genetics , Animals , Cells, Cultured , Female , Humans , Hypoxia/physiopathology , Male , Placenta/metabolism , Pre-Eclampsia/metabolism , Pre-Eclampsia/pathology , Pregnancy , Pregnancy Trimesters , Rats , Receptors, Tachykinin/genetics , Tachykinins/genetics , Trophoblasts/cytology , Trophoblasts/metabolismABSTRACT
Differential techniques have revealed several novel genes and peptides involved in trophoblast development including PL74/gdf15/MIC-1, a TGFbeta family cytokine that controls apoptosis and differentiation, PL48, a new serine-threonine protein kinase, serum and glucocorticoid-induced kinase, PBK-1, a tunicamycin-responsive gene, a cathepsin D-like gene (DAP-1) and hypoxia- regulated genes HRF-1,2,6,8 and HIF-1alpha, HIF-1beta, and hEPAS-1. Syncytin, a cell fusion- inducing gene, has been cloned from placenta where it regulates cell fusion. ERV-3 has also been demonstrated to promote cell fusion. These two genes represent the first demonstrated functions of endogenous retroviral sequences in human tissues. Endoglin, PlGF, TGFbeta3, IGF-II, IGFBP-1, and a placental IGFBP protease have found new roles in regulating cytotrophoblast proliferation and invasiveness. A specific placental p105 rasGAP protein has been identified. The homeobox genes DLX4, HB24, MSX2 and MOX2 also likely play a role in development at the epithelial-mesenchymal boundary. Transcription factors such as TEF-5, Hand1, HEB, HASH-2 and two genes represented by ESTs may have regulatory roles in placental development. Evidence suggests that the placenta has an unusual two-cell system for apoptosis regulation in which the cytotrophoblast may direct later apoptotic events in the syncytium, and with syncytialization possibly triggered by the "phosphatidylserine flip". Thus, the placenta is both a rich source of new growth-regulatory substances, and a model system for originating new paradigms of developmental biology.
Subject(s)
Gene Expression Regulation, Developmental/physiology , Placenta/physiology , Trophoblasts/physiology , Animals , Apoptosis/physiology , Cell Lineage , Embryo Implantation/physiology , Giant Cells/physiology , Humans , Transcription Factors/physiology , Trophoblasts/cytologyABSTRACT
The inflammatory cytokines tumour necrosis factor alpha (TNF-alpha) and immune interferon gamma (IFN-gamma) stimulate villous cytotrophoblast apoptosis while epidermal growth factor (EGF) protects. We hypothesize that TNF-alpha, IFN-gamma and EGF regulate apoptosis in part by modulating cellular expression levels of the anti-death gene bcl-2. While Bcl-2 is reported to be strongly expressed in villous syncytiotrophoblasts, it is not known whether the protein is expressed in cultured villous cytotrophoblasts (CT) and, if so, whether it is functional. We show by Northern blot analysis that bcl-2 mRNA is expressed in cultured CT and by immunoblot analysis that the protein is strongly expressed in highly purified first trimester and term villous cytotrophoblasts. The expression levels of Bcl-2 protein were the same in first trimester and term cytotrophoblasts. Culture with TNF-alpha/IFN-gamma and EGF did not alter expression of either Bcl-2 protein or of the pro-apoptotic Bcl-2 family member Bak. Double label flow cytometric analysis that measured apoptosis and Bcl-2 content simultaneously showed that cells expressing low levels of Bcl-2 underwent TNF-alpha/IFN-gamma-induced apoptosis at a higher frequency than cells expressing lower levels. We conclude that Bcl-2 is expressed in cytotrophoblasts, that its expression is constitutive and that modulation of its expression levels does not mediate cytokine and growth factor regulation of apoptosis in these cells.
Subject(s)
Chorionic Villi/drug effects , Cytokines/antagonists & inhibitors , Epidermal Growth Factor/pharmacology , Gene Expression Regulation, Developmental/physiology , Genes, bcl-2 , Trophoblasts/pathology , Apoptosis/drug effects , Cells, Cultured , Chorionic Villi/pathology , Female , Humans , Interferon-gamma/antagonists & inhibitors , Membrane Proteins/metabolism , Pregnancy , Pregnancy Proteins/metabolism , Pregnancy Trimester, First , Pregnancy Trimester, Third , Tumor Necrosis Factor-alpha/antagonists & inhibitors , bcl-2 Homologous Antagonist-Killer ProteinABSTRACT
Human placental cytotrophoblast cells differentiate by a process of fusion into a syncytium. This process is stimulated by EGF but also occurs spontaneously at a slower rate in cultured cytotrophoblast cells. To determine nuclear proto-oncogene changes mediating these events, c-myc, c-fos, c-jun and junB were measured in spontaneously differentiating cells and in cells exposed to EGF. c-myc showed a transient rise in expression at 4-8 h with augmented expression by EGF, occurring even in the absence of serum or attachment. c-myc and c-jun declined during culture, but c-fos and particularly junB showed increased expression by day 3 with marked responses to EGF stimulation. Syncytia induced to form by EGF exposure for 48 h demonstrated marked junB expression after rechallenge with 40 min EGF exposure, but negligible responses of c-fos and c-jun. c-myc showed increased expression after 6 h EGF exposure throughout the culture period and in syncytia. The results indicate EGF promotes a syncytial phenotype characterized by c-fos and junB expression during syncytial formation. EGF continues to elicit junB and c-myc responsiveness in more mature syncytium, indicative of continued EGF actions which may include acting as a survival factor, as an hCG secretagogue, and as an inducer of continued development of the syncytium.
Subject(s)
Cell Differentiation , Epidermal Growth Factor/pharmacology , Gene Expression , Genes, jun/genetics , Genes, myc/genetics , Trophoblasts/physiology , Cell Adhesion , Cell Division , Cells, Cultured , Female , Fluorescent Antibody Technique , Genes, fos/genetics , Glycoprotein Hormones, alpha Subunit/genetics , Humans , Kinetics , Phenotype , Pregnancy , Proto-Oncogene Mas , RNA, Messenger/metabolism , Trophoblasts/cytologyABSTRACT
The human trophoblast differentiates from proximal cell column cytotrophoblasts into two lineages: a villous phenotype that results in cell fusion and formation of syncytium and an extravillous phenotype that adopts an invasive behavior and displays cell surface markers of an endothelial cell. Both phenotypes develop spontaneously in in vitro cultured cytotrophoblasts, but there is a clear gestational regulation by unknown genetic and/or maternal environmental factors that results in first trimester villous cytotrophoblasts entering the invasive pathway and term villous cytotrophoblasts entering the syncytial pathway. No genetic factors are known that induce the invasive pathway. First trimester cytotrophoblasts are induced to enter the invasive pathway by activin A, LIF and IL-1beta but inhibited from differentiating in this direction by TGFbeta1, TGFbeta3, glucocorticoids and hypoxia. Term villous cytotrophoblasts are stimulated by EGF, EGF-II, IGFBP-1, alpha1beta1 integrin (laminin receptor) and hypoxia. Term villous cytotrophoblasts are stimulated to form a syncytium by EGF, GM-CSF, CSF-1, dexamethasone, hCG, fibronectin, collagen I and PL48 and inhibited by TGFbeta1. As well, there is evidence that TNFalpha and interferon gamma induce and EGF inhibits apoptosis. This provides a mechanism for trophoblast turnover and renewal. Further research will be likely to uncover additional genetic, cytokine, extracellular matrix and physicochemical factors that regulate this complex process.
Subject(s)
Trophoblasts/physiology , Cell Differentiation , Cytokines/physiology , Epidermal Growth Factor/physiology , Female , Gene Expression Regulation, Developmental , Humans , Pregnancy , Pregnancy Trimester, First , Transforming Growth Factor beta/physiologyABSTRACT
Normal human term cytotrophoblast cells prepared by trypsin-DNAse I digestion with and without secondary immunological purification with CD9 antibodies were investigated for the expression of morphological and genetic markers of proliferation and differentiation. After 24 h of culture, the cell preparations demonstrated spontaneous formation of microvilli and formation of small syncytial units as assessed by desmoplakin staining and FITC-dextran microinjection. EGF was required for mature syncytial formation. Compared to log-phase proliferating HeLa cells, uptake of [3H]thymidine incorporation was low and quickly decreased to negligible levels. Expression of the proto-oncogenes c-myc, c-fos, and c-jun and histone 2A decreased rapidly in the first 24 h of culture in both cell preparations, followed by an increase in expression of c-fos and junB over the next 3 days of culture. Proto-oncogene changes were similar in attached and suspension cells. Spontaneous increases in alpha hCG, pregnancy-specific beta(1)-glycoprotein and 3 beta-hydroxysteroid dehydrogenase (3 beta OHSD) occurred within 1 day in both cell preparations. EGF receptor blocking antibodies did not inhibit minor degrees of spontaneous syncytial formation nor inhibit spontaneous expression of alpha hCG or 3 beta OHSD mRNA, but did prevent extensive synctialization induced by EGF. The results demonstrate that term cytotrophoblast cells even in serum-free conditions or suspension culture rapidly commit to a non-proliferative differentiation program in culture which includes limited syncytialization and marked hormone mRNA expression. However, EGF is required for extensive syncytial development.
Subject(s)
Cell Differentiation , Epidermal Growth Factor/pharmacology , Giant Cells/cytology , Models, Biological , Trophoblasts/cytology , 3-Hydroxysteroid Dehydrogenases/genetics , Cell Division , Cells, Cultured , Epithelial Cells , Female , Gene Expression , Humans , Microscopy, Electron, Scanning , Pregnancy , Proto-Oncogene Mas , Proto-Oncogenes/geneticsABSTRACT
The cDNA for a novel gene, PL48, isolated by subtractive hybridization between undifferentiated human term cytotrophoblast and differentiating cytotrophoblast, has been cloned and sequenced. PL48 contains an open reading frame coding for a 537-amino acid protein, has multiple potential PKC, casein kinase II, and cAMP/cGMP-dependent kinase phosphorylation sites, and N-linked glycosylation sites. It is not present in a wide variety of proliferating cancer cells, but PL48 mRNA shows marked expression during cytotrophoblast and granulocyte lineage-specific HL-60 promyelocytic cell differentiation induced by DMSO.
Subject(s)
HL-60 Cells/cytology , Proteins/genetics , Trophoblasts/cytology , Amino Acid Sequence , Animals , Base Sequence , Binding Sites , Cell Adhesion Molecules , Cell Differentiation/genetics , Cell Lineage/genetics , Cloning, Molecular , Dimethyl Sulfoxide/pharmacology , Female , Gene Expression Regulation, Developmental , Gene Expression Regulation, Neoplastic , Genes, Tumor Suppressor , HL-60 Cells/drug effects , HL-60 Cells/metabolism , Humans , Molecular Sequence Data , Phosphorylation , Placenta/metabolism , Pregnancy , Protein Conformation , Protein Kinase C/metabolism , Proteins/chemistry , Proteins/metabolism , RNA, Messenger/biosynthesis , RNA, Messenger/drug effects , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Transcription, Genetic , Trophoblasts/metabolism , Tumor Cells, CulturedABSTRACT
We have previously demonstrated that epidermal growth factor (EGF), colony stimulating factor-1 (CSF-I), and granulocyte-monocyte colony stimulating factor (GMCSF) stimulate, while transforming growth factor beta 1 (TGF beta 1) inhibits, cytotrophoblast differentiation. To identify genes mediating EGF induced differentiation, we constructed a subtracted cDNA library between undifferentiated cytotrophoblast and differentiating cytotrophoblast. We identified six novel genes and four known syncytial products alpha-human chorionic gonadotrophin (alpha hCG) pregnancy-specific beta 1-glycoprotein, 3 beta-hydroxysteroid dehydrogenase, and plasminogen activator inhibitor type 1 whose mRNAs increased during differentiation. Ten other genes were identified whose mRNAs increased during differentiation. Five of these (keratin 19, calcreticulin, heat shock protein 27, serum and glucocorticoid-regulated kinase and adrenomedullin) were not previously reported to be expressed in placenta. Five other genes known to be expressed in placenta were identified. keratin 8, fibronectin, mitochondrial ATP synthase, 1119, and cytosolic copper-zinc superoxide dismutase (SOD-1). Several of these genes may have regulatory functions in trophoblast differentiation.
Subject(s)
Cell Differentiation/genetics , Nucleic Acid Hybridization , Trophoblasts/cytology , 3-Hydroxysteroid Dehydrogenases/genetics , Epidermal Growth Factor/pharmacology , Female , Fibronectins/genetics , Glycoprotein Hormones, alpha Subunit/genetics , Humans , Molecular Sequence Data , Plasminogen Activator Inhibitor 1/genetics , Pregnancy , Pregnancy-Specific beta 1-Glycoproteins/genetics , RNA, Messenger/metabolism , Superoxide Dismutase/geneticsABSTRACT
From a subtracted library of differentiating human cytotrophoblast cells, we have cloned a gene termed PL33, a novel probable member of the G-protein-linked seven transmembrane domain receptor family. The 496 amino acid open reading frame of PL33 codes for a protein of predicted molecular weight of 54.7 kDa. Northern blot analysis shows that the two mRNA transcripts of PL33 are expressed in testes, keratinocytes, differentiating cytotrophoblast and in monocyte/macrophage lineage-specific HL-60 cell differentiation.
Subject(s)
Neoplasm Proteins , Receptors, Cell Surface/genetics , Trophoblasts/metabolism , Amino Acid Sequence , Antigens, Differentiation/genetics , Base Sequence , Cell Differentiation , Cell Lineage , Cloning, Molecular , HL-60 Cells , Humans , Membrane Glycoproteins , Molecular Sequence DataABSTRACT
Melanogenesis is a cascade of events significantly controlled by regulatory genes which are associated with the melanosomal membrane. This report introduces our current research efforts dealing with (a) the gene and protein expressions of tyrosinase and Lamp (lysosome-associated membrane protein) families by human melanoma cells after repeated exposures to UV light, (b) the coordinated alterations in the expression of the Lamp family gene and its encoding product after transfection of two genes of the tyrosinase family in human melanoma cells and (c) cloning and sequencing of a Ca(2+)-binding phosphoprotein, calnexin, which could be a candidate as a chaperone for sorting and maturation of tyrosinase and Lamp family glycoproteins in melanogenesis cascade. Our UV exposure study, as well as gene transfection and antisense hybridization experiments, has clearly indicated a marked and coordinated interaction of the Lamp-1 gene with the tyrosinase and TRP-1 genes in this process. We propose that melanogenesis is controlled at least by two major gene family products, i.e., (a) the tyrosinase family of tyrosinase, TRP-1 and TRP-2, and the Lamp family of Lamp-1, Lamp-2 and Lamp-3. These two gene families probably derived from primordial melanogenesis-associated genes which are common or closely related to each other.
Subject(s)
Antigens, CD , Gene Expression Regulation, Neoplastic/radiation effects , Melanoma/genetics , Membrane Glycoproteins/physiology , Oxidoreductases , Proteins/physiology , Skin Neoplasms/genetics , Calcium-Binding Proteins/metabolism , Calnexin , Humans , Lysosomal Membrane Proteins , Melanins/metabolism , Melanocytes/metabolism , Melanocytes/ultrastructure , Melanoma/metabolism , Membrane Glycoproteins/genetics , Membrane Glycoproteins/radiation effects , Polymerase Chain Reaction , Proteins/genetics , Proteins/radiation effects , RNA-Directed DNA Polymerase , Skin Neoplasms/metabolism , Transfection , Ultraviolet Rays/adverse effectsABSTRACT
In order to have a proper biosynthesis and secretion of the melanin-pigment granules (melanosomes) the melanocyte may require a melanosome-associated molecule that provides a signal for assembly and organization of melanogenic enzymes and proteins within the compartment of melanosomes. This study reports the presence of a Ca(2+)-binding phosphoprotein, p90, which can be engaged in such melanogenic function, located on the melanosomal membrane of human melanocytes. A human melanoma cDNA expression library in lambda Zap II was screened with a rabbit polyclonal antibody raised against human melanosomes isolated from cultured human melanoma cells, SK MEL 23. A cDNA encoding a melanosomal protein, M(r) 90 kDa, was identified through this immunoscreening. A partial sequencing of nucleotides (822 bp from the N-terminal domain) of this clone (3.8 kb) and predicted amino acids showed more than 90% homology with dog calnexin, a previously reported endoplasmic reticulum (ER) transmembrane protein. A fusion protein of this p90 with beta-galactosidase expressed in Escherichia coli revealed both the immuno-cross-reactivity with anti-dog calnexin and anti-human melanosome antibodies and the Ca(2+)-binding property. Upon immunohistochemistry, the anti-dog calnexin antibody revealed the positive immunoreactivities with both normal and malignant human melanocytes, showing a much higher expression of antigenic epitope than nonmelanocytic human cells. The laser scanning confocal immunofluorescence, using an antibody against a human melanosome-specific antigen (HMSA-5), and immunoelectron microscopy, using immunogold, confirmed the major localization of anti-dog calnexin antibody epitope on the melanosomes and ER.
Subject(s)
Calcium-Binding Proteins/metabolism , Melanocytes/metabolism , Phosphoproteins/genetics , Amino Acid Sequence , Base Sequence , Calcium-Binding Proteins/genetics , Calnexin , Cell Compartmentation , Cloning, Molecular , DNA, Complementary/genetics , Gene Library , Humans , Immunohistochemistry , In Vitro Techniques , Microscopy, Electron , Molecular Sequence Data , Precipitin Tests , Sequence Alignment , Sequence Homology, Amino Acid , Tumor Cells, CulturedABSTRACT
Monoclonal antibodies against melanosomal components (human melanosome specific antigens; HMSAs) have been developed in our laboratory. HMSA-1-4 recognizes structural matrix proteins of melanosomes. HMSA-5 is identical to TRP-1, equivalent to the b (brown) locus of murine melanocytes and expressed in early stages of melanosomal maturation. HMSA-6 is a protein associated with melanosomes but its role is still unclarified, and HMSA-7 is identical to the lysosomal protein CD63. We have also recently identified p90 calnexin-like, Ca(2+)-binding protein p97 melanotransferrin, and p64 beta-D-galactosidase-like protein associated with melanosomes through immunological screening of our melanocytes (melanoma cells) cDNA library. Approximately 150 genes and 60 loci are known to influence eye, skin and hair colour in mammals. Tyrosinase is a rate-limiting enzyme responsible for melanin synthesis. In addition, tyrosine-related proteins (TRPs) and their genes have been identified and cloned. Tyrosinase and TRPS (e.g., TRP-1; b-locus protein identical to HMSA-5 and TRP-2; dopachrome tautomerase) are synthesized according to underlying genetic programmes, and are up- and/or down-regulated to create various forms of abnormal melanin pigmentation. We herein propose the importance of investigating the role of non-tyrosinase related proteins such as those which we have recently identified.
Subject(s)
DNA, Neoplasm/genetics , Melanoma/genetics , Melanoma/immunology , Neoplasm Proteins/genetics , Neoplasm Proteins/immunology , Animals , Antibodies, Monoclonal , Antigens, Neoplasm/genetics , Antigens, Neoplasm/immunology , Humans , Immunohistochemistry , Melanoma-Specific Antigens , Precipitin TestsABSTRACT
We have recently identified a gene encoding a calnexin-like protein (p90) by the immunoscreening of a human melanoma cDNA library, using a rabbit anti-human melanosomal antibody. This p90 protein was highly expressed by human melanocytes and associated with melanosomal membrane and endoplasmic reticulum. In this study we report the computer analysis of the predicted amino acid sequence of this calnexin-like melanosomal protein. We found that p90 is a membrane-bound protein whose large N-terminal domain is located within the melanosomal compartment; its shorter C-terminal is exposed to the cytosol and separated by a short transmembrane region. This p90 protein was found to have consensus sequences of a Ca(2+)-binding loop and a protein kinase C phosphorylation site at the N-terminal domain. The C-terminal domain, on the other hand, contained sequences of a casein kinase II phosphorylation site and two protein kinase A phosphorylation sites. Such functional motifs could provide signal transduction across the melanosomal membrane, the reception of melanogenic protein via carriers at the melanosomal membrane and the translocation of melanosomes in the melanocyte.
Subject(s)
Calcium-Binding Proteins/genetics , DNA, Complementary/metabolism , Melanoma/genetics , Melanoma/metabolism , Membrane Proteins/genetics , Amino Acid Sequence , Animals , Calnexin , Cell Membrane/metabolism , Cell Membrane/ultrastructure , Dogs , Humans , Membrane Proteins/biosynthesis , Membrane Proteins/chemistry , Models, Molecular , Molecular Sequence Data , Protein Structure, Secondary , Sequence Homology, Amino AcidABSTRACT
This report introduces some aspects of our current basic research focus on the unique metabolic pathways within the melanocyte. Using this approach, we hope to gain a better understanding of the pathophysiology of malignant melanoma and develop early laboratory diagnostic tests for this disease. Specifically, we will discuss that: 1) the synthesis of pheomelanin is markedly increased in malignant melanoma and dysplastic melanocytic nevi; 2) high levels of metabolites of pheomelanin and eumelanin can be detected in the urine and blood of patients with metastatic melanoma; 3) this release of melanin metabolites appears to correlate with tumor thickness and tumor load, including the extent of metastasis; 4) the synthesis of melanosomal proteins also becomes aberrant in malignant melanoma; and 5) this abnormal melanosome synthesis can be utilized in the identification of antigenic epitopes that are uniquely expressed in malignant melanoma. We believe that this synthesis and secretion of abnormal melanin pigment and melanosomal proteins (human melanosome-specific antigen) would be useful for the development of early laboratory diagnostic and monitoring tools for malignant melanoma. In addition, we also report the detection of pheomelanin component in "normal" unexposed skin; however, the relative amount of pheomelanin in the skin does not reflect hair color (e.g., red hair). The nature of this pheomelanin component in the skin needs to be further clarified.
Subject(s)
Biomarkers/analysis , Melanins/analysis , Melanocytes/chemistry , Melanoma/chemistry , Proteins/analysis , Animals , Cell Differentiation , Humans , Melanocytes/cytologyABSTRACT
A high-performance liquid affinity chromatography column that contains immobilized anti-A monoclonal antibody specifically retards blood group A-active oligosaccharides and can be used to detect the product(s) of the reaction catalyzed by alpha-1,3-N-acetyl-D-galactosaminyltransferase: [formula: see text] After a brief incubation (15 min) of an assay mixture containing 1-100 microliters human serum, the sugar nucleotide donor UDP-GalNAc, and radiolabeled oligosaccharide acceptors 2'-fucosyllactose and/or lacto-N-fucpentaose I blood group A-active products are isolated and quantitated in a single affinity chromatographic step that takes less than 30 min. Kinetic studies to determine the pH optima for serum alpha-3-GalNAc transferase from individuals of blood groups A1 and A2 and the Km value for UDP-GalNAc for the A1 transferase agree with previous determinations. As monoclonal antibodies against many different complex carbohydrate antigens are now available, the method described could be adapted to give rapid, inexpensive assays for a variety of glycosyltransferases.
Subject(s)
Chromatography, Affinity/methods , Galactosyltransferases/analysis , N-Acetylgalactosaminyltransferases , Carbohydrate Sequence , Galactosyltransferases/blood , Humans , Kinetics , Molecular Sequence Data , Oligosaccharides/chemistry , Substrate SpecificityABSTRACT
A bifunctional hapten was synthesized consisting of a blood group A active tetrasaccharide (A-tetra) and a blood group Le(a) active pentasaccharide lacto-N-fucopentaose II (LNF II), linked to each other with a phenylaminothiourea spacer connecting the reducing ends (A-tetra-LNF II). The hapten was demonstrated to retain both blood group A and Le(a) activity and could be easily bound to both monoclonal anti-A and anti-Le(a) affinity columns. Due to the strong temperature dependence of the two antibodies their binding to oligosaccharides, the bifunctional hapten could be utilized to achieve easy desorption in the final step of affinity purification of either monoclonal anti-Le(a) or anti-A. The system is postulated to have general applicability in affinity purification of any ligate that binds with an avidity too high to achieve non-denaturing desorption.
Subject(s)
Antibodies, Monoclonal/isolation & purification , Cross-Linking Reagents , Haptens , Oligosaccharides , Carbohydrate Sequence , Chromatography, Affinity , Electrophoresis, Polyacrylamide Gel , Enzyme-Linked Immunosorbent Assay , Molecular Sequence DataSubject(s)
Oligosaccharides/analysis , Antigen-Antibody Complex , Carbohydrate Conformation , Carbohydrate Sequence , Chromatography, Affinity/methods , Chromatography, High Pressure Liquid/methods , Feces/analysis , Humans , Immunoglobulin M , Infant, Newborn , Kinetics , Lectins , Molecular Sequence Data , Oligosaccharides/isolation & purification , Oligosaccharides/urine , TritiumABSTRACT
Nine neutral and five acidic oligosaccharides were isolated from feces of a preterm (30th postmenstrual week) blood group A nonsecretor infant fed on pooled breast milk. Structural analyses were carried out using sugar and methylation analyses, fast atom bombardment mass spectrometry, and 1H NMR. The acidic oligosaccharides are well-known components of human milk. The neutral oligosaccharides are characteristic of nonsecretor milk. Surprisingly, no secretor gene-dependent oligosaccharides were present in the feces. Another preterm (27th postmenstrual week) blood group A, secretor infant fed on pooled breast milk showed the same fecal oligosaccharide pattern as above during the first week after birth, despite being a secretor individual. Also notable was the absence of blood group A-active oligosaccharides in this sample. Another sample of feces collected 8 weeks later from the latter infant contained the expected blood group A-active oligosaccharides. Furthermore, free sialic acid was present at the cost of the sialyl oligosaccharides seen earlier. Thus, infants born prematurely do not show the same degree of development of oligosaccharide metabolism as their more mature counterparts.
Subject(s)
Feces/analysis , Infant, Premature/metabolism , Milk, Human/metabolism , Oligosaccharides/analysis , Carbohydrate Conformation , Chromatography, Affinity , Disaccharides/analysis , Female , Gas Chromatography-Mass Spectrometry , Gestational Age , Hexoses/analysis , Humans , Infant, Newborn , Magnetic Resonance Spectroscopy , Male , Monosaccharides/analysis , N-Acetylneuraminic Acid , Sialic Acids/analysisABSTRACT
Affinity columns prepared by immobilizing monoclonal antibodies that specifically recognize the Lea or the Leb blood group antigens can be used for analytical or preparative isolation of oligosaccharides with the corresponding reactivities. The number of immobilized functional antibody combining sites on a column and the dissociation constants for standard oligosaccharides are determined by frontal analysis. By employing a simple approximation [K.-I. Kasai et al. (1986) J. Chromatogr. 376, 33-47] these parameters can be used to rationally design columns with properties appropriate for zonal affinity chromatography. The affinity for binding of the Lea-active oligosaccharide lacto-N-fucopentaose II (LNF II) by the anti-Lea antibody CO-514 doubles for each 8 degrees C downward shift in temperature between 37 and 4 degrees C. By zonal chromatography, Lea- or Leb-active oligosaccharides are recovered from a complex mixture of milk oligosaccharides containing more than a 20-fold molar excess of structurally similar but antigenically distinct oligosaccharides. The capacity for preparative isolation of an oligosaccharide increases in a linear fashion with the amount of antibody loaded on the solid support. The monoclonal antibodies used in these studies are products of hybridomas derived from mice immunized with human colorectal carcinoma cell lines [M. Blaszczyk et al. (1984) Arch. Biochem. Biophys. 233, 161-168]. The experiments establish that affinity chromatography applied to mixtures of oligosaccharides released by enzymatic or chemical cleavage of glycoconjugates may simplify the task of isolating and characterizing biologically interesting target antigens of monoclonal antibodies.
Subject(s)
Antibodies, Monoclonal , Chromatography, Affinity , Lewis Blood Group Antigens , Oligosaccharides/isolation & purification , Animals , Antigen-Antibody Reactions , Binding Sites, Antibody , Chromatography, Affinity/methods , Feces/analysis , Humans , Infant , Kinetics , Lewis Blood Group Antigens/immunology , Mice , Mice, Inbred BALB C , Milk/analysis , Oligosaccharides/immunologyABSTRACT
A column for high-pressure liquid affinity chromatography is prepared by binding a murine monoclonal anti-blood group A antibody of IgM isotype to concanavalin A-coated silica particles. The column specifically retards blood group A-active oligosaccharides with the nonreducing immunodominant trisaccharide sequence, GalNAc alpha 1-3(Fuc alpha 1-2)Gal beta 1- ..., and separates three A-active oligosaccharides with different core structures. Retention of the oligosaccharides on the column diminishes with increasing temperatures, permitting thermal elution in the range 25-50 degrees C.