RESUMEN
Sialoglycosphingolipids (gangliosides) are membrane components of eukaryotic cells that modulate cell signal transduction events. Discrepancies exist in the published descriptions of the gangliosides present in the human peripheral monocyte/macrophage. Macrophages were isolated from healthy human volunteers by two different methods. Their ganglioside fractions were isolated and examined by 2D thin-layer mobility, enzymatic susceptibility, and mass spectral-collision induced dissociation-mass spectral analyses. Thin-layer ganglioside chromatographic patterns displayed four major doublets and were similar for monocytes/macrophages isolated by either apheresis/elutriation or density gradient centrifugation. All gangliosides were resistant to beta-galactosidase but sensitive to Clostridium perfringens sialidase, indicating the absence of terminal galactose residues and sialidase-resistant sialic acid moieties. Mass spectra indicated only three major sets of glycolipid components with mass heterogeneity in the ceramide portion of each set. In all the gangliosides, the ceramide moiety contained only C18 sphingosine with the heterogeneity produced by the presence of C16 or C24 fatty acid. One doublet was resistant to Newcastle disease virus sialidase, indicating the presence of an alpha(2-6)-linked sialic acid residue with the same mass as another doublet. All data was consistent with the following structures as the major gangliosides of human peripheral monocyte/macrophages: II(3)NeuAcLacCer (sialolactosyl ceramide, GM3), IV(3)- and IV(6)NeuAcnLcOse(4)Cer (sialoparagloboside, nLM1), and IV(3)NeuAcnLcOse(6)Cer (a sialohexosylceramide).
Asunto(s)
Gangliósidos/sangre , Macrófagos/metabolismo , Monocitos/metabolismo , Secuencia de Carbohidratos , Cromatografía en Capa Delgada , Gangliósidos/química , Humanos , Espectrometría de Masas , Datos de Secuencia Molecular , Neuraminidasa/metabolismo , Virus de la Enfermedad de Newcastle/enzimologíaRESUMEN
NOR is a rare inheritable polyagglutination phenomenon that has been described in two families. Our recent studies on these erythrocytes showed they contained at least two unique neutral glycosphingolipids, and based on their reactivity with Griffonia simplicifolia IB4 (GSL-IB4) isolectin (Kusnierz-Alejska, G., Duk, M., Storry, J. R., Reid, M. E., Wiecek, B., Seyfried, H., and Lisowska, E. (1999) Transfusion 39, 32-38), both oligosaccharide chains terminated with an alpha-galactose residue. The reactivity with GSL-IB4 suggested that these oligosaccharide chains terminated with a Galalpha1-->3Gal- sequence and that anti-NOR agglutinins were common human anti-Galalpha1-->3Gal xenoantibodies. In this report we describe the structure of one NOR component (NOR1) that migrated on thin-layer chromatographic plates in the region of pentaglycosylceramides. Treatment of this sample with alpha-galactosidase and beta-N-acetylhexosaminidase was followed by high-performance thin-layer chromatography with product detection by lectins and the anti-Gb4 monoclonal antibody. The results suggested that NOR1 was an alpha-galactosylated Gb4Cer with a beta-N-acetylhexosaminidase-resistant GalNAc residue. Gas phase disassembly by ion trap mass spectrometry analysis showed the sequence to be Hex1-->4HexN1-->3Hex1-->4Hex1-->4Hex linked to a ceramide composed of C18 sphingosine and a C24 monounsaturated fatty acid. Together these data indicate NOR1 to be a novel Galalpha1-->4GalNAcbeta1-->3Galalpha1-->4Galbeta1-->4 Glc-Cer structure. Additionally it has been shown that NOR glycolipids are recognized by human antibodies that were distinct from the known anti-Galalpha1-->3Gal xenoantibodies.
Asunto(s)
Eritrocitos/inmunología , Glicoesfingolípidos/metabolismo , Animales , Conformación de Carbohidratos , Secuencia de Carbohidratos , Cromatografía en Capa Delgada , Glicoesfingolípidos/química , Pruebas de Hemaglutinación , Humanos , Espectrometría de Masas , Datos de Secuencia Molecular , Estructura Molecular , Fenotipo , ConejosRESUMEN
Antigen presenting cells (APCs) expressing CD1b mediate the specific T cell recognition of mycobacterial lipid antigens. These lipid antigens require internalization by APCs prior to presentation, but the detailed mechanisms of uptake and intracellular processing are not known. Here we have examined several steps in the presentation of two related classes of CD1b-presented antigens, free and glycosylated mycolates. T cell recognition of glucose monomycolate (GMM) was blocked by agents that fix APC membranes or neutralize the pH of endosomes, indicating a requirement for GMM uptake into an acidic compartment prior to recognition. Different T cell lines responded to free mycolate or GMM without crossreactivity, yet both antigens were taken up by APCs at the same rate. This demonstrated that differential recognition of these antigens resulted from T cell specificity for their hydrophilic caps and that APCs were unable to interconvert these antigens by enzymatic or chemical deglycosylation or glycosylation. APCs were also unable to cleave mycobacterial trehalose dimycolate (TDM) at its most chemically labile linkages to yield antigenic free mycolates or GMM. Our results indicate that these mycolate-containing antigens are resistant to chemical or enzymatic cleavage by APCs, suggesting that molecular trimming is not a universal feature of lipid antigen processing.
Asunto(s)
Presentación de Antígeno , Antígenos CD1/inmunología , Ácidos Micólicos/inmunología , Ácidos Micólicos/metabolismo , Linfocitos T/inmunología , Células Presentadoras de Antígenos/metabolismo , Antígenos Bacterianos/inmunología , Antígenos CD1/biosíntesis , Línea Celular , Endosomas/fisiología , Glucolípidos/inmunología , Glicosilación , Humanos , Mycobacterium/inmunologíaRESUMEN
Glycoprotein hormone alpha subunit, in its free form (free alpha), is a major placental product. Its glycosylation was found to change dramatically during the advancement of pregnancy. In this study, we have analyzed these glycosylation changes in five normal pregnancies. Binding to Lens culinaris lectin increased dramatically in all subjects between weeks 14 and 17 from the last menstrual period, indicating more core fucosylation as well as possible changes in branching of glycans. Studies using Datura stramonium agglutinin confirmed that the type of triantennary branching changed in this period of pregnancy. The precise structural nature of these changes was determined by high-pH anion-exchange chromatography and electrospray ionization mass spectrometry. Amounts of core fucosylation and of triantennary glycans increased substantially from early to late second trimester, and a shift was observed from 1-->4/1-->3- toward predominantly 1-->6/1-->6-branched triantennary structures. The glycosylation changes occurred in all five individuals at the same time period in gestation, suggesting developmental regulation of N-acetylglucosaminyltransferases IV and V and alpha6-fucosyltransferase during normal pregnancy. These enzymatic activities also appear to be affected in malignant transformation of the trophoblast. Our findings have important implications for the proposed use of specific forms of glycosylation as markers for cancer, as the relative amounts of these glycans in normal pregnancy will be determined by gestational age.
Asunto(s)
Hormonas Glicoproteicas de Subunidad alfa/orina , Secuencia de Carbohidratos , Cromatografía de Afinidad , Cromatografía Líquida de Alta Presión , Cromatografía por Intercambio Iónico , Femenino , Hormonas Glicoproteicas de Subunidad alfa/química , Hormonas Glicoproteicas de Subunidad alfa/aislamiento & purificación , Glicosilación , Humanos , Lectinas/química , Espectrometría de Masas , Datos de Secuencia Molecular , Polisacáridos/química , EmbarazoRESUMEN
Mass spectra of fragments of permethylated oligosaccharides are analyzed by Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry. Sustained off-resonance irradiation (SORI) collision-induced dissociation (CID), quadrupolar axialization, multiple stages of isolation and dissociation (MSn), and ion remeasurement are exploited for carbohydrate structural analyses. That SORI CID internal energies are adequate for linkage analysis of a permethylated glucose oligomer is demonstrated by identifying ring-opened fragment ions from MALDI-generated mass-isolated and collisionally activated ions. Ion remeasurement and axialization techniques enhance the sensitivity of ion fragmentation analysis. Multiple stages of isolation and dissociation of ion fragments (MSn) provide for structural analysis of an electrospray-ionized permethylated lacto-N-fucopentaose isomer (LNFP II). Compared to MS2 spectra taken with a triple quadrupole, FT-ICR MSn (n > 2) provides more extensive characterization of the parent molecular structure than is available from a single stage of ion isolation and dissociation (MS2).
Asunto(s)
Oligosacáridos/análisis , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Secuencia de Carbohidratos , Análisis de Fourier , Glucanos/análisis , Metilación , Datos de Secuencia MolecularRESUMEN
The human CD1b protein presents lipid antigens to T cells, but the molecular mechanism is unknown. Identification of mycobacterial glucose monomycolate (GMM) as a CD1b-presented glycolipid allowed determination of the structural requirements for its recognition by T cells. Presentation of GMM to CD1b-restricted T cells was not affected by substantial variations in its lipid tails, but was extremely sensitive to chemical alterations in its carbohydrate or other polar substituents. These findings support the view that the recently demonstrated hydrophobic CD1 groove binds the acyl chains of lipid antigens relatively nonspecifically, thereby positioning the hydrophilic components for highly specific interactions with T cell antigen receptors.
Asunto(s)
Presentación de Antígeno , Antígenos CD1/inmunología , Glucolípidos/inmunología , Subgrupos de Linfocitos T/inmunología , Antígenos Bacterianos/inmunología , Antígenos CD1/química , Antígenos CD1/metabolismo , Epítopos/inmunología , Glucolípidos/química , Glucolípidos/metabolismo , Glicosilación , Humanos , Ligandos , Espectrometría de Masas , Mycobacterium/inmunología , Ácidos Micólicos/química , Ácidos Micólicos/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Receptores de Antígenos de Linfocitos T/metabolismoRESUMEN
We have established a differential peptide display method, based on a mass spectrometric technique, to detect peptides that show semiquantitative changes in the neurointermediate lobe (NIL) of individual rats subjected to salt-loading. We employed matrix-assisted laser desorption/ionization mass spectrometry, using a single-reference peptide in combination with careful scanning of the whole crystal rim of the matrix-analyte preparation, to detect in a semiquantitative manner the molecular ions present in the unfractionated NIL homogenate. Comparison of the mass spectra generated from NIL homogenates of salt-loaded and control rats revealed a selective and significant decrease in the intensities of several molecular ion species of the NIL homogenates from salt-loaded rats. These ion species, which have masses that correspond to the masses of oxytocin, vasopressin, neurophysins, and an unidentified putative peptide, were subsequently chemically characterized. We confirmed that the decreased molecular ion species are peptides derived exclusively from propressophysin and prooxyphysin (i.e., oxytocin, vasopressin, and various neurophysins). The putative peptide is carboxyl-terminal glycopeptide. The carbohydrate moiety of the latter peptide was determined by electrospray tandem MS as bisected biantennary Hex3HexNAc5Fuc. This posttranslational modification accounts for the mass difference between the predicted mass of the peptide based on cDNA studies and the measured mass of the mature peptide.
Asunto(s)
Proteínas del Tejido Nervioso/metabolismo , Hipófisis/metabolismo , Cloruro de Sodio Dietético/administración & dosificación , Secuencia de Aminoácidos , Animales , Espectrometría de Masas , Datos de Secuencia Molecular , RatasRESUMEN
In a previous study (Kollár, R., Petráková, E., Ashwell, G., Robbins, P. W., and Cabib, E. (1995) J. Biol. Chem. 270, 1170-1178), the linkage region between chitin and beta(1-->3)-glucan was solubilized and isolated in the form of oligosaccharides, after digestion of yeast cell walls with beta(1-->3)-glucanase, reduction with borotritide, and subsequent incubation with chitinase. In addition to the oligosaccharides, the solubilized fraction contained tritium-labeled high molecular weight material. We have now investigated the nature of this material and found that it represents areas in which all four structural components of the cell wall, beta(1-->3)-glucan, beta(1-->6)-glucan, chitin, and mannoprotein are linked together. Mannoprotein, with a protein moiety about 100 kDa in apparent size, is attached to beta(1-->6)-glucan through a remnant of a glycosylphosphatidylinositol anchor containing five alpha-linked mannosyl residues. The beta(1-->6)-glucan has some beta(1-->3)-linked branches, and it is to these branches that the reducing terminus of chitin chains appears to be attached in a beta(1-->4) or beta(1-->2) linkage. Finally, the reducing end of beta(1-->6)-glucan is connected to the nonreducing terminal glucose of beta(1-->3)-glucan through a linkage that remains to be established. A fraction of the isolated material has three of the main components but lacks mannoprotein. From these results and previous findings on the linkage between mannoproteins and beta(1-->6)-glucan, it is concluded that the latter polysaccharide has a central role in the organization of the yeast cell wall. The possible mechanism of synthesis and physiological significance of the cross-links is discussed.
Asunto(s)
Pared Celular/metabolismo , Quitina/metabolismo , Glucanos/metabolismo , Glicoproteínas de Membrana/metabolismo , beta-Glucanos , Amidohidrolasas/metabolismo , Secuencia de Aminoácidos , Conformación de Carbohidratos , Secuencia de Carbohidratos , Cromatografía de Afinidad , Cromatografía Líquida de Alta Presión , Concanavalina A/metabolismo , Espectroscopía de Resonancia Magnética , Mananos/metabolismo , Espectrometría de Masas , Datos de Secuencia Molecular , Peso Molecular , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa , Saccharomyces cerevisiae , beta-Glucosidasa/metabolismoRESUMEN
A novel saccharide was synthesized by incubating globo-N-tetraose, GalNAc beta1-3Gal alpha1-4Gal beta1-4Glc, and UDP[3H]GlcNAc with hog gastric mucosal microsomes, known to contain beta1,6-N-acetylglucosaminyltransferase activity of a broad acceptor specificity. Chromatography and MALDI-TOF mass spectrometry of the product, as well as the amount of incorporated radioactivity indicated that one [3H]GlcNAc residue was transferred to the acceptor saccharide. One- and two-dimensional 1H NMR-spectroscopic analysis of the product and ESI-CID mass spectrometry of the pentasaccharide in permethylated form established its structure as GalNAc beta1-3([3H]GlcNAc beta1-6)Gal alpha1-4Gal beta1-4Glc. The new enzyme activity possesses substrate specificity features common to a purified beta1,6-GlcNAc-transferase from bovine tracheal epithelium, which forms branches at the subterminal beta1,3-substituted galactose and accepts both GlcNAc- and Gal-configuration at the terminal residue of the acceptor (Ropp et al. (1991) J. Biol. Chem., 266, 23863-23871). The new beta1,6-GlcNAc-branch was readily galactosylated by bovine milk beta1,4-galactosyltransferase, revealing a pathway to novel hybrid type glycans with N-acetyllactosamine chains on globotype saccharides. This pathway may lead to the rare IP blood-group antigen and to globoside-like molecules mediating cell adhesion.
Asunto(s)
Mucosa Gástrica/enzimología , Globósidos/metabolismo , Microsomas/enzimología , N-Acetilglucosaminiltransferasas/metabolismo , Oligosacáridos/síntesis química , Animales , Conformación de Carbohidratos , Secuencia de Carbohidratos , Bovinos , Epitelio/enzimología , Globósidos/química , Espectroscopía de Resonancia Magnética , Leche/enzimología , Datos de Secuencia Molecular , Oligosacáridos/química , Oligosacáridos/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Especificidad por Sustrato , Porcinos , Tráquea/enzimología , Tritio , Uridina Difosfato N-Acetilglucosamina/metabolismoRESUMEN
Sialoglycosphingolipids (gangliosides) have been increasingly implicated as regulators of membrane signaling events. Macrophage ganglioside patterns dramatically increase in complexity when murine peritoneal macrophages are stimulated in vivo with the appearance of the sialidase-sensitive monosialoganglioside GM1b (cisGM1) as a major component. Gangliosides from stimulated murine peritoneal macrophages were separated into monosialo and polysialo fractions and the polysialo fraction structurally characterized by enzymatic, chemical, and mass spectra methods. All detectable components of the polysialo fraction were determined to be disialogangliosides. Treatment of the polysialo fraction with Clostridium perfringens sialidase produced mostly the sialidase-resistant monosialoganglioside, GM1a, and a minor amount of asialoGM1. Periodate oxidation and mass spectrometry analyses demonstrated the lack of tandem disialo moieties which indicated the absence of GD1b or GD1c (GD1) entities. The combined data showed the major disialogangliosides consisted of GD1a entities comprising IV3-NeuAc,II3NeuAc-GgOse4Cer, IV3-NeuGc,II3NeuAc-GgOse4Cer, IV3NeuAc,II3NeuGc-GgOse4Cer, and IV3-NeuGc,II3NeuGc-GgOse4Cer. Minor components consisted of GD1alpha entities, IV3NeuAc, III6NeuAcGgOse4Cer, IV3NeuGc, III6NeuGc-GgOse4Cer, and also positional isomer(s) of GD1alpha(NeuAc, NeuGc). These isomeric components were identified by collision analysis and tandem mass spectrometry. Consistent with previous analyses, the ceramide portion of all polysialo (disialo) gangliosides contained solely C18 sphingosine with C16 and C24 fatty acid moieties. These results, combined with the previous characterization of macrophage monosialogangliosides, indicate normal murine macrophage ganglioside biosynthesis proceeds along the "a" ganglioside pathway, e.g., GM3-->GM2-->GM1a-->GD1a, and the proposed asialoganglioside or "alpha" pathway, asialoGM1-->GM1b-->GD1alpha. The presence of totally sialidase-sensitive gangliosides appears to be characteristic of functional murine peritoneal macrophages while they are reduced in genetically impaired cells.
Asunto(s)
Gangliósidos/análisis , Gangliósidos/química , Macrófagos Peritoneales/química , Animales , Secuencia de Carbohidratos , Cromatografía Líquida de Alta Presión , Femenino , Gangliósido G(M1)/análisis , Espectrometría de Masas , Metilación , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Datos de Secuencia Molecular , Estructura Molecular , Peso Molecular , Neuraminidasa/farmacologíaRESUMEN
We have reported that transglycosylation activity of endo-beta-N-acetylglucosaminidase from Arthrobacter protophormiae (endo-A) can be enhanced to near completion using GlcNAc as an acceptor in a medium containing 30% acetone (Fan J-Q, Takegawa K, Iwahara S, Kondo A, Kato I, Abeygunawardana C, Lee YC (1995) J Biol Chem 270: 17723-29). In this paper, we found that the endo-A can also transfer an oligosaccharide, Man9GlcNAc, to L-Fuc using Man9GlcNAc2Asn as donor substrate in a medium containing 35% acetone. The transglycosylation yield was greater than 25% when 0.2 M L-Fuc was used as acceptor. The transglycosylation produce was purified by high performance liquid chromatography on a graphitized carbon column and the presence of L-Fuc was confirmed by sugar composition analysis and electrospray mass spectrometry. Sequential exo-glycosidase digestion of pyridyl-2-aminated transglycosylation product, Man9GlcNAc-L-Fuc-PA, revealed that a beta-anomeric configuration linkage was formed between GlcNAc and L-Fuc. The GlcNAc was found to be 1,2-linked to L-Fuc by two methods: i) collision-induced decomposition on electrospray mass spectrometry after periodate oxidation, reduction and permethylation of Man9GlcNAc-L-Fuc; and ii) preparation of Man9GlcNAc-L-Fuc-PA, its periodate oxidation and reduction, followed by hydrolysis and HPLC analysis. Thus, the structure of the oligosaccharide synthesized by endo-A transglycosylation was determined to be Man9GlcNAc beta (1,2)-L-Fuc. Methyl-beta-L-fucopyranoside, L-Gal are also acceptors for the enzymic transglycosylation. However, transglycosylation failed when methyl alpha-L-fucopyranoside, D-Fuc and D-Gal were used. These results indicate that the endo-A requires not only 3-OH and 4-OH to be equatorial but also a 4C1-conformation or equivalent conformation of the acceptor to perform transglycosylation.
Asunto(s)
Acetilglucosaminidasa/química , Arthrobacter/enzimología , Fucosa/química , Mananos/química , Acetilglucosaminidasa/metabolismo , Glicosilación , Espectrometría de Masas , Especificidad por SustratoRESUMEN
Previous studies suggested that sialosyl-Le(x) (SLex) is a ligand expressed in human neutrophils and myelogenous leukemia HL60 cells which binds to E-selectin and possibly P-selectin. However, clear data on structures of carbohydrate epitopes in these cells were lacking. A systematic study was therefore initiated, employing a large quantity of HL60 cells (> or = 1200 mL packed) and human leukocytes (approximately 100 mL packed). Gangliosides were extracted, followed by extensive fractionation and examination of the E- and P-selectin binding ability of each fraction. The following results were of particular interest: (i) Only monosialogangliosides having a polylactosamine core with > 10 monosaccharide units (or > 4 N-acetyllactosamine units) showed E-selectin binding under static conditions with thin-layer chromatography overlay technique employing 32P-labeled E-selectin-expressing CHO cells. (ii) Sulfate groups were not detectable in the binding fractions, and di- and trisialoganglioside fractions did not show E-selectin binding under these conditions. (iii) None of the fractions showed P-selectin binding under a similar assay system using 32P-labeled P-selectin-expressing CHO cells. (iv) Major gangliosides of HL60 cells were structures I-XI (shown in Table 1 of text), none of which showed E-selectin binding under the above conditions. (v) SLex gangliosides having tetraosyl to octaosyl ceramide core, which are the major gangliosides of epithelial tumors (shown in Table 2), were completely absent from HL60 cells and neutrophils. Isolation and chemical characterization of ganglioside structures I-XI are described in this paper.
Asunto(s)
Selectina E/metabolismo , Gangliósidos/química , Células HL-60/química , Antígeno Lewis X/química , Neutrófilos/química , Animales , Células CHO , Secuencia de Carbohidratos , Cricetinae , Epítopos , Gangliósidos/inmunología , Gangliósidos/metabolismo , Humanos , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Selectina-P/metabolismo , Espectrometría de Masa Bombardeada por Átomos VelocesRESUMEN
E-selectin binding gangliosides were isolated from myelogenous leukemia HL60 cells, and the E-selectin binding pattern was compared with that of human neutrophils as described in the preceding paper in this issue. The binding fractions were identified as monosialogangliosides having a series of unbranched polylactosamine cores. Structures of fractions 12-3, 13-1, 13-2, and 14, which showed clear binding to E-selectin under the conditions described in the preceding paper, were characterized by functional group analysis by application of monoclonal antibodies, 1H-NMR, FAB-MS, and electrospray mass spectrometry with collision-induced dissociation of permethylated fractions. Fractions 12-3, 13-1, and 13-2 were characterized by the presence of a major ganglioside with the following structure: NeuAc alpha 2-->3Gal beta 1-->4 GlcNAc beta 1-->3Gal beta 1-->4(Fuc alpha 1-->3) GlcNAc beta 1-->3Gal beta 1-->4(Fuc alpha 1-->3)-GlcNAc beta 1-->3Gal beta 1-->4GlcNAc beta 1-->3 Gal beta 1-->4 Glc beta Cer. Fractions 12-3 and 13-2 contained, in addition, small quantities (10-15%) of extended SLex with internally fucosylated structures: NeuAc alpha 2-->3 Gal beta 1-->4-(Fuc alpha 1-->3) GlcNAc beta 1-->3 Gal beta 1-->4(Fuc alpha 1-->3) GlcNAc beta 1-->3 Gal beta 1-->4 (+/- Fuc alpha 1-->3)GlcNA c beta 1-->3 Gal beta beta 1-->4GlcNAc beta 1-->3 Gal beta 1-->Glc Beta Cer. Fraction 13-1, showing stronger E-selectin binding activity than 12-3 and 13-2, contained only a trace quantity (< 1%) of SLex. Fraction 14, which also showed clear binding to E-selectin, was characterized by the presence of the following structures, in addition to two internally monofucosylated structures (XX and XXI, Table 2, text): NeuAc alpha 2-->3Gal beta 1-->4GlcNAc beta 1-->3Gal beta 1-->4(Fuc alpha 1-->3)GlcNAc beta 1-->3 Gal beta 1-->4(Fuc alpha 1-->3)GlcNAc beta 1-->3Gal beta 1-->4 GlcNAc beta 1-->3 Gal beta 1-->4 GlcNAc beta 1-->3 Gal beta 1-->4 Glc beta Cer; andNeuAc alpha 2-->3Gal beta 1-->4GlcNAc beta 1-->3 Gal beta 1-->4(Fuc alpha 1-->3)GlcNAc beta 1-->3Gal beta 1-->4GlcNAc beta 1-->3Gal beta 1-->4 (Fuc alpha 1--3)-GlcNAc beta 1-->3Gal beta 1-->4GlcNAc beta 1-->3Gal beta 1--4Glc beta Cer. SLex determinant was completely absent. Thus, the E-selectin binding epitope in HL60 cells is carried by unbranched terminally alpha 2-->3 sialylated polylactosamine having at least 10 monosaccharide units (4 N-acetyllactosamine units) with internal multiple fucosylation at GlcNAc. These structures are hereby collectively called "myeloglycan". Monosialogangliosides from normal human neutrophils showed an essentially identical pattern of gangliosides with selectin binding property. Myeloglycan, rather than SLex, provides a major physiological epitope in E-selectin-dependent binding of leukocytes and HL60 cells.
Asunto(s)
Selectina E/metabolismo , Gangliósidos/química , Células HL-60/química , Neutrófilos/química , Sitios de Unión , Secuencia de Carbohidratos , Gangliósidos/inmunología , Gangliósidos/metabolismo , Humanos , Antígeno Lewis X/metabolismo , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Espectrometría de Masa Bombardeada por Átomos VelocesRESUMEN
This section summarizes several strategies for a more complete understanding of carbohydrate structure with a focus on glycolipids and glycoprotein glycans. The techniques include periodate oxidation to impart greater molecular specificity to isomeric glycans, methylation to improve sensitivity and the information content within CID spectra, electrospray for "soft" and efficient ionization, and CID to obtain structural detail. The lipophilicity of the products following derivatization contributes to product cleanup by solvent extraction and enhances sensitivity during ES. When combined with CID information, this yields sequence, linkage, and branching information. Oxidation and reduction preceding methylation augments CID analysis with an altered structure that can be profiled at the same sensitivity. Within the context of established motifs, these contrasting profiles corroborate glycan structure and specifically identify isobaric elements transparent in the initial profile. An earlier report indicating ring-opening fragments were essentially absent in low-energy collisions of methylated and natriated oligosaccharides contrasts our observations. However, as this report used a methylated oligomer containing an internal N-acetylhexose as an illustration, the conclusion is plausible (cf., Figure 9). The poor ionization efficiency of FAB and the high matrix background limit the dynamic range in the CID spectrum and, thereby, the ability to unambiguously identify weaker peaks. It would be expected that high-energy CID affords a broader range of fragment types, including ring-opening fragments. In terms of a structural methodology, this is ambivalent since the increase in fragmentation pathways also applies to small molecule eliminations which are usually less informative. In ES-CID-MS, the carbohydrate chemist has a powerful new tool in hand for structural elucidations that can be conducted at the low-picomole level. Parallel developments can be expected to continue for other ionization methods, in particular matrix-assisted desorption/ionization on linear and reflectron time of flight mass spectrometers, and improvement in the performance and sensitivity of high-resolution mass analyzers through the use of focal plane detectors and more sophisticated hardware and software for Fourier transform ion cyclotron resonance mass measurements. These have, as yet, only begun to be applied to carbohydrate structural analysis but should add still more versatility to experimental design in the future.
Asunto(s)
Carbohidratos/química , Conformación de Carbohidratos , Secuencia de Carbohidratos , Glicoconjugados/química , Espectrometría de Masas/métodos , Datos de Secuencia Molecular , Peso Molecular , Polisacáridos/químicaRESUMEN
In the preceding paper, preliminary analysis revealed a new type of O-linked oligosaccharide of 1244 Da at each of two proposed glycosylation sites on several proteins secreted by the Gram-negative bacterium Flavobacterium meningosepticum (Plummer, T. H., Jr., Tarentino, A. L., and Hauer, C. R. (1995) J. Biol. Chem. 270, 13192-13196). In this report we detail the linkage, sequence, and branching of this unusual heptasaccharide by electrospray (ES) ionization mass spectrometry (MS), and collision-induced dissociation (CID). The proposed structure was supported by a combination of isotopic labeling, composition and methylation analysis, and the preparation of several chemical analogs and derivatives with each product evaluated by MS and CID. The singly branched structure contained seven residues, including three different uronyl analogs: a methylated rhamnose and mannose, a glucose, and a reducing terminal mannose. Only pyranose ring forms were detected ((2-OMe)Man1-4GlcNAcU1-4GlcU1-4Glc1-4(2-OMe)G lcU-4 [(2-OMe)Rham1-2]Man).
Asunto(s)
Flavobacterium/química , Polisacáridos/química , Conformación de Carbohidratos , Secuencia de Carbohidratos , Ésteres , Glicopéptidos/química , Glicosilación , Espectrometría de Masas/métodos , Datos de Secuencia Molecular , Oxidación-ReducciónRESUMEN
Sialosyl-Lex (SLex) is assumed to be the binding epitope of E- and P-selectin in normal human neutrophils and myelocytic leukemia HL60 cells. Glycosphingolipid (GSL) fractions from large quantities of normal human neutrophils and HL60 cell extract did not contain SLex GSLs having 6-10 sugar residues, as commonly found in solid tumor cells and tissues. Instead, the binding target of E-selectin was revealed to be a series of long-chain, unbranched polylactosamine GSLs with terminally sialylated, internally alpha 1-->3 polyfucosylated structure as the major component, or having SLex at the terminus and internally polyfucosylated structure as a minor component. These GSLs are hereby collectively termed "myeloglycan." Regardless of the site of fucosylation, all myeloglycans cross-react strongly with "anti-SLex" monoclonal antibodies such as CSLEX, FH6, SNH3, and SNH4.
Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Glicoesfingolípidos/metabolismo , Neutrófilos/metabolismo , Acetilglucosamina/análisis , Animales , Anticuerpos Monoclonales , Sitios de Unión , Células CHO , Secuencia de Carbohidratos , Moléculas de Adhesión Celular/biosíntesis , Línea Celular , Cromatografía Líquida de Alta Presión , Cromatografía en Capa Delgada , Cricetinae , Selectina E , Epítopos/análisis , Glicoconjugados/química , Glicoconjugados/aislamiento & purificación , Glicoesfingolípidos/química , Glicoesfingolípidos/aislamiento & purificación , Humanos , Leucemia Promielocítica Aguda , Espectrometría de Masas , Datos de Secuencia Molecular , Selectina-P , Glicoproteínas de Membrana Plaquetaria/biosíntesis , Glicoproteínas de Membrana Plaquetaria/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/metabolismo , Transfección , Células Tumorales CultivadasRESUMEN
The improved sensitivity and soft ionization characteristics of electrospray (ES) mass spectrometry (MS) has been applied to the glycan structures of recombinant erythropoietin (rEPO). The four glycopeptides (O-126, N-24, N-38, N-83) were mapped, isolated, deglycosylated, and the glycans profiled (without desialylation) by ES-MS as their methyl derivatives. The O-linked glycopeptide was also analyzed directly. In this fraction seven glycans were identified (two previously unreported) in addition to the unglycosylated peptide. The three N-linked fractions were divided with one fraction preceded by periodate oxidation and reduction to resolve isomeric structures, linkage, and branching patterns, and confirm the overall structural motif. All were methylated and profiled. Two biantennary, 8 triantennary, and 10 tetraantennary structures were identified with approximately 13% of each N-linked glycan possessing a single N-glycolylneuraminyl analog. The minimal energies of ionization, the absence of a matrix background, and enhanced sensitivity brings an improved technology for studying carbohydrate structural detail.
Asunto(s)
Eritropoyetina/química , Glicopéptidos/química , Espectrometría de Masas/métodos , Oligosacáridos/química , Polisacáridos/química , Conformación de Carbohidratos , Secuencia de Carbohidratos , Cromatografía Líquida de Alta Presión , Glicopéptidos/aislamiento & purificación , Glicósido Hidrolasas , Indicadores y Reactivos , Datos de Secuencia Molecular , Oligosacáridos/aislamiento & purificación , Fragmentos de Péptidos/química , Fragmentos de Péptidos/aislamiento & purificación , Polisacáridos/aislamiento & purificación , Proteínas Recombinantes/química , Sensibilidad y Especificidad , TripsinaRESUMEN
The detailed structure of the symbiotically important exopolysaccharide succinoglycan from Rhizobium meliloti Rm1021 was determined by mass spectrometry with electrospray ionization and collision-induced dissociation of the octameric oligosaccharide repeating unit. Previously undetermined locations of the succinyl and acetyl modifications were determined, in respect to both residue locations within the octamer and the carbon positions within the pyranose ring. Glycosidic linkages determined previously by methylation analysis were also verified.
Asunto(s)
Oligosacáridos/química , Polisacáridos Bacterianos/química , Sinorhizobium meliloti/química , Secuencia de Carbohidratos , Cromatografía de Gases y Espectrometría de Masas , Datos de Secuencia MolecularRESUMEN
Identification of the substrate activation site of beta-trypsin by a 1:1 reaction with p-diazoniumbenzamidine chloride was confirmed by spectral analysis. Proteolysis of Cm-p-benzamidino-azo-beta-trypsin provided peptides containing modified tyrosine residues. The major product, Ser-146 to Lys-156, which corresponded to labeling at Tyr-151, was recovered in 35% yield, and its structure was demonstrated by amino acid analysis, Edman degradation, and mass spectrometry. Yields of labeled Tyr-151, Tyr-39, and Tyr-172, identified by peptide analysis, were in the proportion of 100:7:3. Tyr-151-(p-benzamidino)-azo-beta-trypsin is permanently activated, but can be further activated by substrates. Values of kcat, Ks', and kcat' vary from two to three times the equivalent values for trypsin. Berenil (4,4'-diazoamino-bis-benzamidine), a parabolic competitive inhibitor of beta-trypsin, was a hyperbolic competitive inhibitor of azo-beta-trypsin. Thus, Tyr-151, part of subsite S'2, affects the catalytic process and, when modified covalently, permanently activates trypsin. Equilibrium binding with berenil supported the kinetic data obtained with substrates. This permits the integration of protein modification, kinetics, equilibrium binding, and crystallographic data to demonstrate a fine interaction between subsites S1-S3 and S'2 in trypsin and azo-beta-trypsin, resulting in subtle structural changes when the native enzyme is covalently modified at Tyr-151.