RESUMO
Protonated ions of fucose-containing oligosaccharides are prone to undergo internal glycan rearrangement which results in chimeric fragments that obfuscate mass-spectrometric analysis. Lack of accessible tools that would facilitate systematic analysis of glycans in the gas phase limits our understanding of this phenomenon. In this work, we use density functional theory modeling to interpret cryogenic IR spectra of Lewis a and blood group type H1 trisaccharides and to establish whether these trisaccharides undergo the rearrangement during gas-phase analysis. Structurally unconstrained search reveals that none of the parent ions constitute a thermodynamic global minimum. In contrast, predicted collision cross sections and anharmonic IR spectra provide a good match to available experimental data which allowed us to conclude that fucose migration does not occur in these antigens. By comparing the predicted structures with those obtained for Lewis x and blood group type H2 epitopes, we demonstrate that the availability of the mobile proton and a large difference in the relative stability of the parent ions and rearrangement products constitute the prerequisites for the rearrangement reaction.
Assuntos
Antígenos do Grupo Sanguíneo de Lewis , Antígenos do Grupo Sanguíneo de Lewis/química , Epitopos/química , Termodinâmica , Polissacarídeos/química , Teoria da Densidade Funcional , Antígenos de Grupos Sanguíneos/química , Espectrofotometria Infravermelho , Oligossacarídeos/química , Trissacarídeos/químicaRESUMO
Immunotherapy has been successful in treating many tumour types. The development of additional tumour-antigen binding monoclonal antibodies (mAbs) will help expand the range of immunotherapeutic targets. Lewis histo-blood group and related glycans are overexpressed on many carcinomas, including those of the colon, lung, breast, prostate and ovary, and can therefore be selectively targeted by mAbs. Here we examine the molecular and structural basis for recognition of extended Lea and Lex containing glycans by a chimeric mAb. Both the murine (FG88.2) IgG3 and a chimeric (ch88.2) IgG1 mAb variants showed reactivity to colorectal cancer cells leading to significantly reduced cell viability. We determined the X-ray structure of the unliganded ch88.2 fragment antigen-binding (Fab) containing two Fabs in the unit cell. A combination of molecular docking, glycan grafting and molecular dynamics simulations predicts two distinct subsites for recognition of Lea and Lex trisaccharides. While light chain residues were exclusively used for Lea binding, recognition of Lex involved both light and heavy chain residues. An extended groove is predicted to accommodate the Lea-Lex hexasaccharide with adjoining subsites for each trisaccharide. The molecular and structural details of the ch88.2 mAb presented here provide insight into its cross-reactivity for various Lea and Lex containing glycans. Furthermore, the predicted interactions with extended epitopes likely explains the selectivity of this antibody for targeting Lewis-positive tumours.
Assuntos
Anticorpos Monoclonais Murinos , Antineoplásicos Imunológicos , Fragmentos Fab das Imunoglobulinas , Antígenos do Grupo Sanguíneo de Lewis , Antígenos CD15 , Simulação de Acoplamento Molecular , Neoplasias , Oligossacarídeos , Animais , Anticorpos Monoclonais Murinos/química , Anticorpos Monoclonais Murinos/imunologia , Antineoplásicos Imunológicos/química , Antineoplásicos Imunológicos/imunologia , Linhagem Celular Tumoral , Humanos , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/imunologia , Antígenos do Grupo Sanguíneo de Lewis/química , Antígenos do Grupo Sanguíneo de Lewis/imunologia , Antígenos CD15/química , Antígenos CD15/imunologia , Camundongos , Neoplasias/química , Neoplasias/imunologia , Oligossacarídeos/química , Oligossacarídeos/imunologiaRESUMO
Initial cell attachment of rotavirus (RV) to specific cell surface glycan receptors, which is the essential first step in RV infection, is mediated by the VP8* domain of the spike protein VP4. Recently, human histo-blood group antigens (HBGAs) have been identified as receptors or attachment factors for human RV strains. RV strains in the P[4] and P[8] genotypes of the P[II] genogroup share common recognition of the Lewis b (Leb) and H type 1 antigens, however, the molecular basis of receptor recognition by the major human P[8] RVs remains unknown due to lack of experimental structural information. Here, we used nuclear magnetic resonance (NMR) spectroscopy-based titration experiments and NMR-derived high ambiguity driven docking (HADDOCK) methods to elucidate the molecular basis for P[8] VP8* recognition of the Leb (LNDFH I) and type 1 HBGAs. We also used X-ray crystallography to determine the molecular details underlying P[6] recognition of H type 1 HBGAs. Unlike P[6]/P[19] VP8*s that recognize H type 1 HBGAs in a binding surface composed of an α-helix and a ß-sheet, referred as the "ßα binding site", the P[8] and P[4] VP8*s bind Leb HBGAs in a previously undescribed pocket formed by the edges of two ß-sheets, referred to as the "ßß binding site". Importantly, the P[8] and P[4] VP8*s retain binding capability to non-Leb type 1 HBGAs using the ßα binding site. The presence of two distinct binding sites for Leb and non-Leb HBGA glycans in the P[8] and P[4] VP8* domains suggests host-pathogen co-evolution under structural and functional adaptation of RV pathogens to host glycan polymorphisms. Assessment and understanding of the precise impact of this co-evolutionary process in determining RV host ranges and cross-species RV transmission should facilitate improved RV vaccine development and prediction of future RV strain emergence and epidemics.
Assuntos
Proteínas do Capsídeo/química , Antígenos do Grupo Sanguíneo de Lewis/química , Simulação de Acoplamento Molecular , Rotavirus/química , Proteínas do Capsídeo/metabolismo , Células HT29 , Humanos , Antígenos do Grupo Sanguíneo de Lewis/metabolismo , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Rotavirus/metabolismoRESUMO
Carbohydrate sequences are important for various biological processes. It has recently been estimated to have 100,000-500,000 carbohydrate structures in mammalian glycome. However, the peripheral carbohydrate determinants on N- and O-glycoproteins, glycolipids, polysaccharides and secreted free sugars are limited in numbers. Among these blood-group-related antigens the ABO(H)- and Lewis-types are particularly important. Negative-ion MS/MS has been successfully used in assignment of these epitopes on free reducing sugars but cannot be applied to reduced sugars, e.g. O-glycans typically released from mucins as alditols, or in positive-ion detection of either reducing or reduced oligosaccharides. In the present study, we investigate the fragmentation features of permethylated reducing and reduced sugars under positive-ion conditions of multi-stage MALDI-MS, and propose the concept of epitope ion and epitope spectrum for determination of peripheral blood-group related epitopes on secreted human milk oligosaccharides and N-glycans as reducing sugars and O-glycans as reduced alditols in conjunction with MALDI-MS glycan profiling.
Assuntos
Epitopos/análise , Glicoproteínas/química , Oligossacarídeos/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Sistema ABO de Grupos Sanguíneos/química , Sistema ABO de Grupos Sanguíneos/metabolismo , Animais , Cromatografia Líquida de Alta Pressão , Mucosa Gástrica/metabolismo , Humanos , Íons/química , Antígenos do Grupo Sanguíneo de Lewis/química , Antígenos do Grupo Sanguíneo de Lewis/metabolismo , Leite Humano/química , Leite Humano/metabolismo , Mucinas/química , Mucinas/metabolismo , Álcoois Açúcares/química , SuínosRESUMO
Human ovarian cyst glycoproteins (HOC, cyst gps) isolated from pseudomucinous type of human ovarian cyst fluids is one of the richest and pioneer sources for studying biosynthesis, structures and functional roles of blood group ABH, Lea,b,x,y, sLea and sLex active glycoproteins. After 70+ years of exploration, four top highlights are shared. (i) an updated concept of glycotopes and their internal structures in cyst gps was composited; (ii) the unknown codes of new genes in secreted cyst gps were unlocked as Lex and Ley; (iii) recognition profiles of cyst glycans and a sialic acid-rich (18%) glycan with lectins and antibodies were shown. (iv) Co-expression of Blood Group A/ A-Leb/y and B/B-Leb/y active Glycotopes in the same glycan chains were isolated and illustrated. These are the most advanced achievements since 1980.
Assuntos
Sistema ABO de Grupos Sanguíneos/química , Gangliosídeos/química , Antígenos do Grupo Sanguíneo de Lewis/química , Polissacarídeos/química , Antígeno Sialil Lewis X/química , Sistema ABO de Grupos Sanguíneos/genética , Sequência de Carboidratos/genética , Gangliosídeos/genética , Glicoproteínas/química , Glicoproteínas/genética , Humanos , Antígenos do Grupo Sanguíneo de Lewis/genética , Polissacarídeos/genética , Ligação Proteica , Antígeno Sialil Lewis X/genéticaRESUMO
Analogues of cancer-associated Lewis Y (Ley) antigen with varying structures at the reducing end were synthesized by a highly efficient strategy involving one-pot preactivation-based iterative glycosylation to obtain the key tetra-/pentasaccharide intermediates, which was followed by stereoselective fucosylation. After global deprotection, these oligosaccharides were coupled with carrier protein keyhole limpet hemocyanin. The resultant glycan-protein conjugates were subjected to immunological studies in mice. It was disclosed that the conjugate of the pentasaccharide analogue of Lewis Y antigen was more immunogenic than that of the hexasaccharide analogue, but the antisera of both conjugates could indiscriminately recognize each carbohydrate hapten. These results suggested that the short Lewis Y analogue may be utilized to develop functional conjugate cancer vaccines. More importantly, the results also proved that the reducing-end glucose residue in the hexasaccharide analogue of Lewis Y was probably not involved in its interaction with the immune system, whose discovery can have a broad impact on the design of new cancer vaccines.
Assuntos
Imunoconjugados/química , Imunoconjugados/imunologia , Antígenos do Grupo Sanguíneo de Lewis/química , Antígenos do Grupo Sanguíneo de Lewis/imunologia , Animais , Técnicas de Química Sintética , Reações Cruzadas , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Relação Estrutura-AtividadeRESUMO
Mumps virus (MuV) is an important aerosol-transmitted human pathogen causing epidemic parotitis, meningitis, encephalitis, and deafness. MuV preferentially uses a trisaccharide containing α2,3-linked sialic acid as a receptor. However, given the MuV tropism toward glandular tissues and the central nervous system, an additional glycan motif(s) may also serve as a receptor. Here, we performed a large-scale glycan array screen with MuV hemagglutinin-neuraminidase (MuV-HN) attachment proteins by using 600 types of glycans from The Consortium for Functional Glycomics Protein-Glycan Interaction Core in an effort to find new glycan receptor motif(s). According to the results of the glycan array, we successfully determined the crystal structures of MuV-HN proteins bound to newly identified glycan motifs, sialyl LewisX (SLeX) and the oligosaccharide portion of the GM2 ganglioside (GM2-glycan). Interestingly, the complex structures showed that SLeX and GM2-glycan share the same configuration with the reported trisaccharide motif, 3'-sialyllactose (3'-SL), at the binding site of MuV-HN, while SLeX and GM2-glycan have several unique interactions compared with those of 3'-SL. Thus, MuV-HN protein can allow an additional spatial modification in GM2-glycan and SLeX at the second and third carbohydrates from the nonreducing terminus of the core trisaccharide structure, respectively. Importantly, MuV entry was efficiently inhibited in the presence of 3'-SL, SLeX, or GM2-glycan derivatives, which indicates that these motifs can serve as MuV receptors. The α2,3-sialylated oligosaccharides, such as SLeX and 3'-sialyllactosamine, are broadly expressed in various tissues, and GM2 exists mainly in neural tissues and the adrenal gland. The distribution of these glycan motifs in human tissues/organs may have bearing on MuV tropism.IMPORTANCE Mumps virus (MuV) infection is characterized by parotid gland swelling and can cause pancreatitis, orchitis, meningitis, and encephalitis. MuV-related hearing loss is also a serious complication because it is usually irreversible. MuV outbreaks have been reported in many countries, even in high-vaccine-coverage areas. MuV has tropism toward glandular tissues and the central nervous system. To understand the unique MuV tropism, revealing the mechanism of receptor recognition by MuV is very important. Here, using a large-scale glycan array and X-ray crystallography, we show that MuV recognizes sialyl LewisX and GM2 ganglioside as receptors, in addition to a previously reported MuV receptor, a trisaccharide containing an α2,3-linked sialic acid. The flexible recognition of these glycan receptors by MuV may explain the unique tropism and pathogenesis of MuV. Structures will also provide a template for the development of effective entry inhibitors targeting the receptor-binding site of MuV.
Assuntos
Proteína HN/metabolismo , Antígenos do Grupo Sanguíneo de Lewis/metabolismo , Vírus da Caxumba/fisiologia , Receptores Virais/metabolismo , Ácidos Siálicos/metabolismo , Tropismo Viral , Ligação Viral , Cristalografia por Raios X , Proteína HN/química , Antígenos do Grupo Sanguíneo de Lewis/química , Análise em Microsséries , Ligação Proteica , Conformação Proteica , Ácidos Siálicos/químicaRESUMO
Mass spectrometry (MS) has become the primary method for high-sensitivity structural determination of oligosaccharides. Fragmentation in the negative-ion MS can provide a wealth of structural information and these can be used for sequence determination. However, although negative-ion MS of neutral oligosaccharide using the deprotonated molecule [M-H]- as the precursor has been very successful for electrospray ionization (ESI), it has only limited success for matrix-assisted laser desorption/ionization (MALDI). In the present study, the features of negative-ion MALDI primary spectra were investigated in detail and the product-ion spectra using [M-H]- and [M+Cl]- as the precursors were carefully compared. The formation of [M-H]- was the main difficulty for MALDI while [M+Cl]- was proved to be useful as alternative precursor anion for MALDI-MS/MS to produce similar fragmentation for sequencing of neutral oligosaccharides. N-(1-naphthyl)ethylenediamine dihydrochloride was then used as both the matrix and the Cl- dopant to evaluate the extent of structural information that can be obtained by negative-ion fragmentation from [M+Cl]- using laser-induced dissociation (LID)-MS/MS for linkage assignment of gluco-oligosaccharides and for typing of blood-group ABO(H) and Lewis antigens on either type 1 or type 2 backbone-chains.
Assuntos
Sistema ABO de Grupos Sanguíneos/análise , Glucanos/análise , Antígenos do Grupo Sanguíneo de Lewis/análise , Oligossacarídeos/análise , Sistema ABO de Grupos Sanguíneos/química , Tipagem e Reações Cruzadas Sanguíneas/métodos , Sequência de Carboidratos , Glucanos/química , Antígenos do Grupo Sanguíneo de Lewis/química , Oligossacarídeos/química , Análise de Sequência , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Espectrometria de Massas em Tandem/métodosRESUMO
The caudal-related homeobox protein 1 (CDX1) is a transcription factor, which is important in the development, differentiation, and homeostasis of the gut. Although the involvement of CDX genes in the regulation of the expression levels of a few glycosyltransferases has been shown, associations between glycosylation phenotypes and CDX1 mRNA expression have hitherto not been well studied. Triggered by our previous study, we here characterized the N-glycomic phenotype of 16 colon cancer cell lines, selected for their differential CDX1 mRNA expression levels. We found that high CDX1 mRNA expression associated with a higher degree of multi-fucosylation on N-glycans, which is in line with our previous results and was supported by up-regulated gene expression of fucosyltransferases involved in antenna fucosylation. Interestingly, hepatocyte nuclear factors (HNF)4A and HNF1A were, among others, positively associated with high CDX1 mRNA expression and have been previously proven to regulate antenna fucosylation. Besides fucosylation, we found that high CDX1 mRNA expression in cancer cell lines also associated with low levels of sialylation and galactosylation and high levels of bisection on N-glycans. Altogether, our data highlight a possible role of CDX1 in altering the N-glycosylation of colorectal cancer cells, which is a hallmark of tumor development.
Assuntos
Neoplasias Colorretais/genética , Regulação Neoplásica da Expressão Gênica , Glicômica , Proteínas de Homeodomínio/genética , Transcriptoma/genética , Linhagem Celular Tumoral , Fucose/metabolismo , Glicosilação , Hexosaminas/metabolismo , Proteínas de Homeodomínio/metabolismo , Humanos , Antígenos do Grupo Sanguíneo de Lewis/química , Antígenos do Grupo Sanguíneo de Lewis/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Fenótipo , Polissacarídeos/química , Polissacarídeos/metabolismo , Análise de Componente Principal , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
An efficient streamlined chemoenzymatic approach has been developed for gram-scale synthesis of Lewis a angtigen (LeaßProN3) and a library of sialyl Lewis a antigens (sLeaßProN3) containing different sialic acid forms. Intially, commercially available inexpensive N-acetylglucosamine (GlcNAc) was converted to its N'-glycosyl p-toluenesulfonohydrazide in one step. Followed by chemical glycosylation, GlcNAcßProN3 was synthesized using this protecting group-free method in high yield (82%). Sequential one-pot multienzyme (OPME) ß1-3-galactosylation of GlcNAcßProN3 followed by OPME α1-4-fucosylation reactions produced target LeaßProN3 in gram-scale. Structurally diverse sialic acid forms was successfully introduced using a OPME sialylation reation containing a CMP-sialic acid synthetase and Pasteurella multocida α2-3-sialyltransferase 1 (PmST1) mutant PmST1 M144D with or without a sialic acid aldolase to form sLeaßProN3 containing naturally occurring or non-natural sialic acid forms in preparative scales.
Assuntos
Antígenos do Grupo Sanguíneo de Lewis/química , N-Acilneuraminato Citidililtransferase/metabolismo , Ácidos Siálicos/química , Sialiltransferases/metabolismo , Acetilglucosamina/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Mutação , N-Acilneuraminato Citidililtransferase/genética , Pasteurella multocida/enzimologia , Sialiltransferases/genética , Compostos de Tosil/químicaRESUMO
Prolactin-inducible protein (PIP) is a glycoprotein found in body secretions from exocrine glands like saliva and seminal plasma. Important biological functions of PIP concentrations have been demonstrated, e.g. in tumor diagnosis and progression. PIP quantity has been also found useful to determine the success of chemotherapy of mammary carcinoma. Here, we present the analysis of the N-glycosylation of PIP isolated from different sources by LC-MS(/MS) and 1H-NMR. We found a very uncommon N-type glycosylation of PIP in healthy individuals from both, seminal fluid and saliva. PIP carries unusual highly fucosylated N-linked glycans with multiple Lewisy (Ley) epitopes on bi-, tri- and tetraantennary structures resulting in up to nine fucosyl residues on a tetraantennary glycan. In most organs, Ley epitopes are not present on N-glycans except in case of a tumor when it is highly up-regulated and important for prognosis. Here, for the first time on a specific glycoprotein Ley antigens are unambiguously characterized on an N-type glycan by NMR spectroscopy. So far, for specific glycoproteins Ley epitopes had only been reported on O-glycans. Furthermore, a correlation between a nonsynonymous single nucleotide polymorphism (SNP) and glycosylation pattern was detected: individuals heterozygous for the SNP causing the amino acid exchange 51Gln to 51His have glycan structures with a higher degree of sialylation compared to individuals lacking the SNP.
Assuntos
Proteínas de Transporte/química , Epitopos/química , Glicoproteínas/química , Antígenos do Grupo Sanguíneo de Lewis/química , Configuração de Carboidratos , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Epitopos/genética , Epitopos/metabolismo , Glicoproteínas/genética , Glicoproteínas/metabolismo , Glicosilação , Humanos , Antígenos do Grupo Sanguíneo de Lewis/genética , Antígenos do Grupo Sanguíneo de Lewis/metabolismo , Proteínas de Membrana Transportadoras , Polimorfismo de Nucleotídeo ÚnicoRESUMO
There is considerable potential for the use of ion mobility mass spectrometry in structural glycobiology due in large part to the gas-phase separation attributes not typically observed by orthogonal methods. Here, we evaluate the capability of traveling wave ion mobility combined with negative ion collision-induced dissociation to provide structural information on N-linked glycans containing multiple fucose residues forming the Lewisx and Lewisy epitopes. These epitopes are involved in processes such as cell-cell recognition and are important as cancer biomarkers. Specific information that could be obtained from the intact N-glycans by negative ion CID included the general topology of the glycan such as the presence or absence of a bisecting GlcNAc residue and the branching pattern of the triantennary glycans. Information on the location of the fucose residues was also readily obtainable from ions specific to each antenna. Some isobaric fragment ions produced prior to ion mobility could subsequently be separated and, in some cases, provided additional valuable structural information that was missing from the CID spectra alone. Graphical abstract á .
Assuntos
Fucose/análise , Espectrometria de Mobilidade Iônica/métodos , Polissacarídeos/química , Ânions/química , Sequência de Carboidratos , Epitopos/química , Humanos , Antígenos do Grupo Sanguíneo de Lewis/química , Antígenos CD15/química , Glândula Parótida/químicaRESUMO
Helicobacter pylori α1-3/4-fucosyltransferase (Hp3/4FT) was expressed in Escherichia coli at a level of 30 mg L-1 culture and used as a diverse catalyst in a one-pot multienzyme (OPME) system for high-yield production of l-fucose-containing carbohydrates including Lewis antigens such as Lewis a, b, and x, O-sulfated Lewis x, and sialyl Lewis x and human milk fucosides such as 3-fucosyllactose (3-FL), lacto-N-fucopentaose (LNFP) III, and lacto-N-difuco-hexaose (LNDFH) II and III. Noticeably, while difucosylation of tetrasaccharides was readily achieved using an excess amount of donor, the synthesis of LNFP III was achieved by Hp3/4FT-catalyzed selective fucosylation of the N-acetyllactosamine (LacNAc) component in lacto-N-neotetraose (LNnT).
Assuntos
Fucose/biossíntese , Fucosiltransferases/metabolismo , Helicobacter pylori/enzimologia , Antígenos do Grupo Sanguíneo de Lewis/biossíntese , Leite Humano/metabolismo , Biocatálise , Configuração de Carboidratos , Fucose/química , Humanos , Antígenos do Grupo Sanguíneo de Lewis/química , Leite Humano/químicaRESUMO
Helicobacter pylori infect millions of people around the world. It occupies a niche in the human gastrointestinal tract characterized by high expression of a repertoire of carbohydrates. ABO and Lewis histo-blood group systems are controlled by genes coding for functional glycosyltransferases which synthesize great diversity of related fucosylated carbohydrate in different tissues, including gastrointestinal mucosa, and exocrine secretions. The structural diversity of histo-blood group carbohydrates is highly complex and depends on epistatic interactions among gene-encoding glycosyltransferases. The histo-blood group glycosyltransferases act in the glycosylation of proteins and lipids in the human gastrointestinal tract allowing the expression of a variety of potential receptors in which H. pylori can adhere. These oligosaccharide molecules are part of the gastrointestinal repertoire of carbohydrates which act as potential receptors for microorganisms, including H. pylori. This Gram-negative bacillus is one of the main causes of the gastrointestinal diseases such as chronic active gastritis, peptic ulcer, and cancer of stomach. Previous reports showed that some H. pylori strains use carbohydrates as receptors to adhere to the gastric and duodenal mucosa. Since some histo-blood group carbohydrates are highly expressed in one but not in others histo-blood group phenotypes it has pointed out that quantitative differences among them influence the susceptibility to diseases caused by H. pylori. Additionally, some experiments using animal model are helping us to understand how this bacillus explore histo-blood group carbohydrates as potential receptors, offering possibility to explore new strategies of management of infection, disease treatment, and prevention. This text highlights the importance of structural diversity of ABO and Lewis histo-blood group carbohydrates as facilitators for H. pylori infection.
Assuntos
Sistema ABO de Grupos Sanguíneos/metabolismo , Carboidratos/química , Epistasia Genética , Infecções por Helicobacter/genética , Helicobacter pylori/genética , Antígenos do Grupo Sanguíneo de Lewis/metabolismo , Sistema ABO de Grupos Sanguíneos/química , Sistema ABO de Grupos Sanguíneos/genética , Animais , Sequência de Carboidratos , Gastrite/enzimologia , Gastrite/genética , Gastrite/microbiologia , Gastrite/patologia , Glicosilação , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Infecções por Helicobacter/enzimologia , Infecções por Helicobacter/microbiologia , Infecções por Helicobacter/patologia , Helicobacter pylori/crescimento & desenvolvimento , Helicobacter pylori/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Antígenos do Grupo Sanguíneo de Lewis/química , Antígenos do Grupo Sanguíneo de Lewis/genética , Úlcera Péptica/enzimologia , Úlcera Péptica/genética , Úlcera Péptica/microbiologia , Úlcera Péptica/patologia , Neoplasias Gástricas/enzimologia , Neoplasias Gástricas/genética , Neoplasias Gástricas/microbiologia , Neoplasias Gástricas/patologiaRESUMO
Molecular assemblies varying morphologies in a wide range from spherical micelle, nanosheet, curved sheet, nanotube and vesicle were prepared and loaded with Lewis y (Ley ) tumor-associated carbohydrate antigen on the assembly surface. The molecular assemblies were composed of poly(sarcosine)m -block-poly(L-lactic acid)30 (m = 15 or 50, Lactosome), poly(sarcosine)m -block-(D/L-Leu-Aib)n (m = 22 or 30, n = 6 or 8) and their combinations. The molecular assemblies carrying Ley on the surface were administered in BALB/c nu/nu mice. The major epitopes of the molecular assemblies are commonly Ley and poly(sarcosine). IgM productions upon administrations of the molecular assemblies were assayed by ELISA, showing that anti-poly(sarcosine) IgM was highly produced by Lactosome of spherical micelle but with a negligible amount of anti-Ley IgM. On the other hand, the nanosheet of the interdigitated monolayer triggered the production of anti-Ley IgM but with less anti-poly(sarcosine) IgM production. Taken together, IgM specificity differs according to the molecular environment of the epitopes in the molecular assemblies. The antigenicity of poly(sarcosine) was augmented in polymeric micelle providing loose environment for B cells to penetrate in, whereas a high density of Ley on the molecular assembly was required for anti-Ley IgM production. The antigenicity of Ley is therefore dependent on the molecular assemblies on which Ley is displayed on the surface. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.
Assuntos
Linfócitos B/efeitos dos fármacos , Imunoglobulina M/biossíntese , Antígenos do Grupo Sanguíneo de Lewis/farmacologia , Nanotubos/química , Peptídeos/química , Poliésteres/química , Sarcosina/análogos & derivados , Animais , Linfócitos B/imunologia , Composição de Medicamentos , Ensaio de Imunoadsorção Enzimática , Humanos , Imunidade Inata , Imunização , Antígenos do Grupo Sanguíneo de Lewis/química , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Micelas , Sarcosina/química , Propriedades de SuperfícieRESUMO
The Japanese cedar pollen allergen (Cry j1) and the mountain cedar pollen allergen (Jun a1) are glycosylated with plant complex type N-glycans bearing Lewis a epitope(s) (Galß1-3[Fucα1-4]GlcNAc-). The biological significance of Lewis a type plant N-glycans and their effects on the human immune system remain to be elucidated. Since a substantial amount of such plant specific N-glycans are required to evaluate immunological activity, we have searched for good plant-glycan sources to characterize Lewis a epitope-containing plant N-glycans. In this study, we have found that three water plants, Elodea nuttallii, Egeria densa, and Ceratophyllum demersum, produce glycoproteins bearing Lewis a units. Structural analysis of the N-glycans revealed that almost all glycoproteins expressed in these three water plants predominantly carry plant complex type N-glycans including the Lewis a type, suggesting that these water plants are good sources for preparation of Lewis a type plant N-glycans in substantial amounts.
Assuntos
Cryptomeria/química , Epitopos/química , Antígenos do Grupo Sanguíneo de Lewis/química , Polissacarídeos/química , Alérgenos/química , Sequência de Carboidratos , Humanos , Pólen/química , Pólen/imunologiaRESUMO
Seven lipophilic constructs containing Lewis (Lea, Leb, Ley) or chimeric Lewis/ABH (ALeb, BLeb, ALey, BLey) glycans were obtained starting from corresponding oligosaccharides in form of 3-aminopropyl glycosides. ALeb and BLeb pentasaccharides were synthesized via [3 + 1] blockwise approach. The constructs (neoglycolipids, or FSLs) were inserted in erythrocyte membrane, and obtained "kodecytes" were used to map the immunochemical specificity of historical and contemporary monoclonal and polyclonal blood group system Lewis reagents.
Assuntos
Antígenos do Grupo Sanguíneo de Lewis/química , Polissacarídeos/síntese química , Polissacarídeos/imunologia , Anticorpos Monoclonais/metabolismo , Membrana Eritrocítica/imunologia , Humanos , Antígenos do Grupo Sanguíneo de Lewis/imunologia , Estrutura Molecular , Polissacarídeos/químicaRESUMO
The novel and efficient synthesis of type 2 Lewis antigens is reported in this study. The rationally designed lactosamine-3,2'-diol derivative with an orthogonal set of protecting groups is efficiently glycosylated with a benzyl protected 1-thio-l-fucoside donor in a unique regioselective manner to produce Lewis x (Le(x)) and Lewis y (Le(y)) derivatives in good yields. These derivatives can be prepared not only exclusively but also synchronously by choosing the appropriate reaction temperature and donor-acceptor molar ratio. The Le(x) derivatives are easily converted into sulfated or non-sulfated Le(x) bearing a terminal azido functionalized oligo-(ethyleneoxide) linker; the Le(y) derivative having the same linker can also be prepared, all of which can be further used for the chemical modification of other compounds and materials.
Assuntos
Amino Açúcares/química , Glicóis/química , Antígenos do Grupo Sanguíneo de Lewis/química , Azidas/química , Técnicas de Química Sintética , Glicosilação , Cinética , EstereoisomerismoRESUMO
Blood group oligosaccharides are one of the most clinically important antigen families and they may also act as secondary ligands for bacterial toxins from Escherichia coli and Vibrio cholerae. Herein we report the synthesis of spacered (sp = CH2CH2CH2NH2) glycosides of A antigen {α-D-GalNAc-(lâ3)-[α-L-Fuc-(lâ2)]-ß-D-Gal-}, B antigen{α-D-Gal-(lâ3)-[α-L-Fuc-(lâ2)]-ß-D-Gal-}, LewisX{α-D-Gal-(lâ4)-[α-L-Fuc-(lâ3)]-ß-D-GlcNAc-}, A type-II {α-D-GalNAc-(lâ3)-[α-L-Fuc-(lâ2)]-ß-D-Gal-(1â4)-ß-D-GlcNAc-}, B type-II {α-D-Gal-(lâ3)-[α-L-Fuc-(lâ2)]-ß-D-Gal-(1â4)-ß-D-GlcNAc-}, H type-II{α-L-Fuc-(lâ2)-ß-D-Gal-(1â4)-ß-D-GlcNAc-}, xenoantigen {α-D-Gal-(lâ3)-ß-D-Gal-(1â4)-[α-L-Fuc-(lâ2)]-ß-D-GlcNAc-} and Linear B Type II {α-D-Gal-(lâ3)-ß-D-Gal-(1â4)-ß-D-GlcNAc-} useful for a range of biochemical investigations. This linker was chosen so as to facilitate the future conjugation of the antigens to proteins or other molecules. We also measured the affinities of some synthesized oligosaccharides against El Tor CTB strain from V. cholera.
Assuntos
Sistema ABO de Grupos Sanguíneos/química , Antígenos Heterófilos/química , Oligossacarídeos/química , Sequência de Carboidratos , Humanos , Antígenos do Grupo Sanguíneo de Lewis/química , Dados de Sequência MolecularRESUMO
BACKGROUND: Non-secretor individuals lack ABO blood group antigens in their secretions like saliva; these carbohydrate structures play an important role in protection of the oral cavity from exogenous pathogens; therefore these individuals are more susceptible to mucous membrane damages. The aim was to assess the secretory state of patients with oral lichen planus (OLP) in comparison with healthy controls. METHODS: Fifty patients and 100 age-gender matched control subjects were recruited to the study. Patients were visited in the outpatient clinic of dermatology at Shohada-e-Tajrish Hospital, Shahid Beheshti University of Medical Sciences from 2012 - 2014. Two-milliliter (mL) blood was collected from each subject to detect Lewis phenotypes. According to Lewis phenotype of each subject, secretory state was determined except in subjects with Le (a-b-) phenotype, in whom saliva was collected to determine the secretor status. RESULTS: Non-secretor status in patients with OLP was more frequent compared with healthy controls (37 out of 50 patients (74%) vs. 24 out of 100 healthy controls (24%), (P < 0.001)). There was no association between secretory state, and type of OLP and disease duration (P > 0.05). CONCLUSION: This study supported the possible role of cell surface histo-blood group antigens in protection of mucosal surface from exogenous pathogens. Therefore, it appears that non-secretor individuals are more prone to oral lichen planus.