RESUMO
Background: Beta-1,4-galactosyltransferase-3 (B4GALT3) belongs to the family of beta-1,4-galactosyltransferases (B4GALTs) and is responsible for the transfer of UDP-galactose to terminal N-acetylglucosamine. B4GALT3 is differentially expressed in tumors and adjacent normal tissues, and is correlated with clinical prognosis in several cancers, including neuroblastoma, cervical cancer, and bladder cancer. However, the exact role of B4GALT3 in the tumor immune microenvironment (TIME) remains unclear. Here, we aimed to elucidate the function of B4GALT3 in the TIME. Methods: To study the functions of B4GALT3 in cancer immunity, either weakly or strongly immunogenic tumor cells were subcutaneously transplanted into wild-type (WT) and B4galt3 knockout (KO) mice. Bone marrow transplantation and CD8+ T cell depletion experiments were conducted to elucidate the role of immune cells in suppressing tumor growth in B4galt3 KO mice. The cell types and gene expression in the tumor region and infiltrating CD8+ T cells were analyzed using flow cytometry and RNA sequencing. N-glycosylated proteins from WT and B4galt3 KO mice were compared using the liquid chromatography tandem mass spectrometry (LC-MS/MS)-based glycoproteomic approach. Results: B4galt3 KO mice exhibited suppressed growth of strongly immunogenic tumors with a notable increase in CD8+ T cell infiltration within tumors. Notably, B4galt3 deficiency led to changes in N-glycan modification of several proteins, including integrin alpha L (ITGAL), involved in T cell activity and proliferation. In vitro experiments suggested that B4galt3 KO CD8+ T cells were more susceptible to activation and displayed increased downstream phosphorylation of FAK linked to ITGAL. Conclusion: Our study demonstrates that B4galt3 deficiency can potentially boost anti-tumor immune responses, largely through enhancing the influx of CD8+ T cells. B4GALT3 might be suppressing cancer immunity by synthesizing the glycan structure of molecules on the CD8+ T cell surface, as evidenced by the changes in the glycan structure of ITGAL in immune cells. Importantly, B4galt3 KO mice showed no adverse effects on growth, development, or reproduction, underscoring the potential of B4GALT3 as a promising and safe therapeutic target for cancer treatment.
Assuntos
Linfócitos T CD8-Positivos , N-Acetil-Lactosamina Sintase , Neoplasias , Animais , Camundongos , Cromatografia Líquida , Camundongos Knockout , N-Acetil-Lactosamina Sintase/genética , Polissacarídeos , Espectrometria de Massas em Tandem , Neoplasias/imunologia , Neoplasias/patologiaRESUMO
IgG N-glycans levels change with advancing age, making it a potential biomarker of aging. ß-1,4-galactosyltransferase (B4GALT) gene expression levels also increase with aging. Ultra performance liquid chromatography (UPLC) was used to examine changes inserum IgG N-glycans at six time points during the aging process. Most serum IgG N-glycans changed with aging in WT but not in CD19-cre B4GALT1 floxed mice. The relative abundance of fucosylated biantennary glycans with or without Neu5Gc structures changed with aging in heterozygous B4GALT1 floxed mice but not in homozygous B4GALT1 floxed mice. Additionally, the aging phenotype was more apparent in WT mice than in B4GALT1 floxed mice. These results demonstrate that fucosylated biantennary glycans and fucosylated biantennary glycans containing N-glycolylneuraminic acid (Neu5Gc)-linked N-acetyllactosamine (LacNAc) were highly associated with aging and were affected by the B4GALT1 floxed mouse genotype. The changing levels of fucosylated monoantennary glycans observed with aging in WT mice was reversed in B4GALT1 floxed mice and was not sex specific. In summary, B-cell-specific ablation of B4GALT1 from a glycoproteomic perspective prevented age-related changes in IgG N-glycans in mice. SIGNIFICANCE: In this study, serum IgG glycoproteomic data in wild-type (WT) and B-cell-specific ablation of ß-1,4-galactosyltransferase 1 mice (B4GALT) were analyzed. Results showed that fucosylated biantennary glycans with or without N-glycolylneuraminic acid (Neu5Gc)-linked N-acetyllactosamine (LacNAc) were highly associated with aging and were also affected by the B4GALT1 floxed mouse genotype. In terms of gender-specific information, the trend towards elevated fucosylated monoantennary glycans in WT mice was not seen in CD19-cre B4GALT1 floxed mice in either sex. B-cell-specific ablation of B4GALT1 plays an important role in age-related glycan changes; its specific functions and mechanisms are worthy of in-depth study. Our data suggest that investigating the relationship between galactosylation and aging may help advance the field of glycoproteomics and aging research.
Assuntos
Envelhecimento , Imunoglobulina G , N-Acetil-Lactosamina Sintase , Polissacarídeos , Envelhecimento/genética , Envelhecimento/metabolismo , Animais , Linfócitos B/metabolismo , Imunoglobulina G/química , Imunoglobulina G/metabolismo , Camundongos , N-Acetil-Lactosamina Sintase/genética , N-Acetil-Lactosamina Sintase/metabolismo , Ácidos Neuramínicos , Fenótipo , Polissacarídeos/químicaRESUMO
ß-1,4-Galactosyltransferase 7 (ß4GalT7) is a key enzyme in the synthesis of two classes of glycosaminoglycans (GAG), i.e., heparan sulfate (HS) and chondroitin/dermatan sulfate (CS/DS). GAG chains are linear polysaccharides of alternating hexuronic acid and N-acetylhexosamine residues, commonly linked to core proteins to form proteoglycans with important roles in the regulation of a range of biological processes. The biosynthesis of GAGs is initiated by xylosylation of a serine residue of the core protein followed by galactosylation, catalyzed by ß4GalT7. The biosynthesis can also be initiated by xylosides carrying hydrophobic aglycons, such as 2-naphthyl ß-D-xylopyranoside. We have cloned and expressed ß4GalT7, and designed a cell-free assay to measure the activity of this enzyme. The assay employs a 96-well plate format for high throughput. In this chapter, we describe the cloning, expression, and purification of ß4GalT7, as well as assays proposed for development of substrates for GAG priming and for investigating inhibitors of ß4GalT7.
Assuntos
N-Acetil-Lactosamina Sintase/metabolismo , Sulfatos de Condroitina , Glicosaminoglicanos , N-Acetil-Lactosamina Sintase/genética , ProteoglicanasRESUMO
The communication between tumor-derived elements and stroma in the metastatic niche has a critical role in facilitating cancer metastasis. Yet, the mechanisms tumor cells use to control metastatic niche formation are not fully understood. Here we report that in the lung metastatic niche, high-metastatic hepatocellular carcinoma (HCC) cells exhibit a greater capacity to convert normal fibroblasts to cancer-associated fibroblasts (CAFs) than low-metastatic HCC cells. We show high-metastatic HCC cells secrete exosomal miR-1247-3p that directly targets B4GALT3, leading to activation of ß1-integrin-NF-κB signaling in fibroblasts. Activated CAFs further promote cancer progression by secreting pro-inflammatory cytokines, including IL-6 and IL-8. Clinical data show high serum exosomal miR-1247-3p levels correlate with lung metastasis in HCC patients. These results demonstrate intercellular crosstalk between tumor cells and fibroblasts is mediated by tumor-derived exosomes that control lung metastasis of HCC, providing potential targets for prevention and treatment of cancer metastasis.
Assuntos
Fibroblastos Associados a Câncer/metabolismo , Carcinoma Hepatocelular/metabolismo , Exossomos/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias Hepáticas/metabolismo , Neoplasias Pulmonares/metabolismo , MicroRNAs/genética , N-Acetil-Lactosamina Sintase/genética , Animais , Fibroblastos Associados a Câncer/patologia , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/secundário , Comunicação Celular , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Exossomos/química , Humanos , Integrina beta1/genética , Integrina beta1/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Interleucina-8/genética , Interleucina-8/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/secundário , Masculino , Camundongos , Camundongos Nus , MicroRNAs/metabolismo , N-Acetil-Lactosamina Sintase/metabolismo , Invasividade Neoplásica , Transplante de Neoplasias , Células Neoplásicas Circulantes/metabolismo , Células Neoplásicas Circulantes/patologia , Transdução de SinaisRESUMO
Long noncoding RNAs (lncRNAs) have been reported to serve a crucial role in renal diseases; however, their role in immunoglobulin A nephropathy (IgAN) remains unclear. In the present study, peripheral blood mononuclear cells (PBMCs) were collected from both patients with IgAN and healthy controls. A microarray analysis was then performed to identify differentially expressed lncRNAs and mRNAs in PBMCs, which were confirmed by quantitative polymerase chain reaction. In addition, Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and lncRNAmRNA coexpression network analyses were conducted. The present study identified 167 differentially expressed lncRNAs and 94 differentially expressed mRNAs. Numerous GO terms, including innate immune response, inflammatory response, IPAF inflammasome complex and UDPgalactose:ßNacetylglucosamine ß1, and 3galactosyltransferase activity, were significantly enriched in the differentially expressed mRNAs. The top five KEGG signaling pathways included nucleotidebinding oligomerization domainlike receptor signaling pathway, hematopoietic cell lineage, inflammatory bowel disease, tumor necrosis factor signaling pathway and other types of Oglycan biosynthesis. In addition, a total of 149 lncRNAs were shown to interact with 7 mRNAs that were associated with the 'innate immune response' GO term. The results of the present study demonstrated that differentially expressed lncRNAs and mRNAs may have a role in the development of IgAN. These results may aid in the elucidation of a basic pathogenic mechanism, the identification of possible biomarkers and the generation of potential novel treatment strategies for IgAN.
Assuntos
Redes Reguladoras de Genes/imunologia , Glomerulonefrite por IGA/genética , Imunidade Inata , RNA Longo não Codificante/genética , RNA Mensageiro/genética , Adulto , Estudos de Casos e Controles , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Ontologia Genética , Glomerulonefrite por IGA/imunologia , Glomerulonefrite por IGA/patologia , Humanos , Masculino , Análise em Microsséries , Pessoa de Meia-Idade , Anotação de Sequência Molecular , N-Acetil-Lactosamina Sintase/genética , N-Acetil-Lactosamina Sintase/imunologia , Proteína Adaptadora de Sinalização NOD1/genética , Proteína Adaptadora de Sinalização NOD1/imunologia , RNA Longo não Codificante/imunologia , RNA Mensageiro/imunologia , Transdução de Sinais , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Nicotiana tabacum BY-2 suspension cells have several advantages that make them suitable for the production of full-size monoclonal antibodies which can be purified directly from the culture medium. Carbohydrate characterization of an antibody (Lo-BM2) expressed in N. tabacum BY-2 cells showed that the purified Lo-BM2 displays N-glycan homogeneity with a high proportion (>70%) of the complex GnGnXF glycoform. The stable co-expression of a human ß-1,4-galactosyltransferase targeted to different Golgi sub-compartments altered Lo-BM2N-glycosylation and resulted in the production of an antibody that exhibited either hybrid structures containing a low abundance of the plant epitopes (α-1,3-fucose and ß-1,2-xylose), or a large amount of galactose-extended N-glycan structures. These results demonstrate the suitability of stable N-glycoengineered N. tabacum BY-2 cell lines for the production of human-like antibodies.
Assuntos
Imunoglobulina G/metabolismo , N-Acetil-Lactosamina Sintase/genética , Nicotiana/genética , Plantas Geneticamente Modificadas/genética , Cromatografia de Afinidade , Regulação da Expressão Gênica , Glicosilação , Complexo de Golgi/metabolismo , Humanos , Imunoglobulina G/genética , Imunoglobulina G/isolamento & purificação , N-Acetil-Lactosamina Sintase/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Nicotiana/metabolismoRESUMO
Proteoglycans and glycosaminoglycans modulate numerous cellular processes relevant to tumour progression, including cell proliferation, cell-matrix interactions, cell motility and invasive growth. Among the glycosaminoglycans with a well-documented role in tumour progression are heparan sulphate, chondroitin/dermatan sulphate and hyaluronic acid/hyaluronan. While the mode of biosynthesis differs for sulphated glycosaminoglycans, which are synthesised in the ER and Golgi compartments, and hyaluronan, which is synthesized at the plasma membrane, these polysaccharides partially compete for common substrates. In this study, we employed a siRNA knockdown approach for heparan sulphate (EXT1) and heparan/chondroitin/dermatan sulphate-biosynthetic enzymes (ß4GalT7) in the aggressive human breast cancer cell line MDA-MB-231 to study the impact on cell behaviour and hyaluronan biosynthesis. Knockdown of ß4GalT7 expression resulted in a decrease in cell viability, motility and adhesion to fibronectin, while these parameters were unchanged in EXT1-silenced cells. Importantly, these changes were associated with a decreased expression of syndecan-1, decreased signalling response to HGF and an increase in the synthesis of hyaluronan, due to an upregulation of the hyaluronan synthases HAS2 and HAS3. Interestingly, EXT1-depleted cells showed a downregulation of the UDP-sugar transporter SLC35D1, whereas SLC35D2 was downregulated in ß4GalT7-depleted cells, indicating an intricate regulatory network that connects all glycosaminoglycans synthesis. The results of our in vitro study suggest that a modulation of breast cancer cell behaviour via interference with heparan sulphate biosynthesis may result in a compensatory upregulation of hyaluronan biosynthesis. These findings have important implications for the development of glycosaminoglycan-targeted therapeutic approaches for malignant diseases.
Assuntos
Sulfatos de Condroitina/biossíntese , Dermatan Sulfato/análogos & derivados , Células Epiteliais/metabolismo , Regulação Neoplásica da Expressão Gênica , Heparitina Sulfato/biossíntese , Ácido Hialurônico/biossíntese , Adesão Celular , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Sobrevivência Celular , Sulfatos de Condroitina/antagonistas & inibidores , Sulfatos de Condroitina/genética , Dermatan Sulfato/antagonistas & inibidores , Dermatan Sulfato/biossíntese , Dermatan Sulfato/genética , Células Epiteliais/patologia , Matriz Extracelular/química , Matriz Extracelular/metabolismo , Heparitina Sulfato/antagonistas & inibidores , Heparitina Sulfato/genética , Humanos , Hialuronan Sintases/antagonistas & inibidores , Hialuronan Sintases/genética , Hialuronan Sintases/metabolismo , Ácido Hialurônico/antagonistas & inibidores , Ácido Hialurônico/genética , Isoenzimas/antagonistas & inibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Glândulas Mamárias Humanas/metabolismo , Glândulas Mamárias Humanas/patologia , Proteínas de Transporte de Monossacarídeos/antagonistas & inibidores , Proteínas de Transporte de Monossacarídeos/genética , Proteínas de Transporte de Monossacarídeos/metabolismo , N-Acetilglucosaminiltransferases/antagonistas & inibidores , N-Acetilglucosaminiltransferases/genética , N-Acetilglucosaminiltransferases/metabolismo , N-Acetil-Lactosamina Sintase/antagonistas & inibidores , N-Acetil-Lactosamina Sintase/genética , N-Acetil-Lactosamina Sintase/metabolismo , Proteínas de Transporte de Nucleotídeos/antagonistas & inibidores , Proteínas de Transporte de Nucleotídeos/genética , Proteínas de Transporte de Nucleotídeos/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de SinaisRESUMO
BACKGROUND: Glaucoma is a major cause of irreversible blindness worldwide. There is evidence showing that a subset of the disease is genetically determined. In this study, we screened for mutations in chromosome 1q-linked open-angle glaucoma (GLC1A) in a Chinese family with primary open-angle glaucoma (POAG). METHODS: A total of 23 members from five generations of a family were enrolled and underwent thorough ophthalmologic examinations. In addition, 200 unrelated healthy Chinese controls were also recruited as normal control. GLC1A gene was amplified by polymerase chain reaction, and DNA sequencing was performed to screen for mutations. RESULTS: Six members were diagnosed as POAG, with severe clinical manifestations, and history of high intraocular pressures. The mean age of disease onset was 26.3 years. However, the others were asymptomatic. In six affected and three asymptomatic members, gene sequencing revealed a mutation c.C1456T in exon 3 of myocilin gene (MYOC). Furthermore, we also identified a novel mutation c.G322A in beta-1,4-galactosyltransferase 3 (B4GALT3) gene in all six affected and three asymptomatic members, which was not reported previously in POAG patients. The two newly identified variants were absent in other family members as well as controls. CONCLUSION: The mutations c.1456C < T (p.L486F) in MYOC and c.322G < A (p.V108I) in B4GALT3 are likely responsible for the pathogenesis of POAG in this family.
Assuntos
Proteínas do Citoesqueleto/genética , Proteínas do Olho/genética , Glaucoma de Ângulo Aberto/genética , Glicoproteínas/genética , N-Acetil-Lactosamina Sintase/genética , Adulto , Biologia Computacional , Feminino , Predisposição Genética para Doença , Testes Genéticos , Humanos , Masculino , Mutação/genética , Linhagem , Análise de Sequência de DNA , Adulto JovemRESUMO
Most insect cells have a simple N-glycosylation process and consequently paucimannosidic or simple core glycans predominate. It has been proposed that ß-N-acetylglucosaminidase (GlcNAcase), a hexosaminidase in the Golgi membrane which removes a terminal N-acetylglucosamine (GlcNAc), might contribute to simple N-glycosylation profile in several insect cells including Drosophila S2. Here, we describe GlcNAcase suppression strategy using RNA interference (RNAi) to avoid the formation of paucimannosidic glycans in insect S2 cells. In addition, we describe coexpression of ß(1,4)-galactosyltransferase (GalT) as a strategy to improve N-glycosylation pattern and enable recombinant therapeutic proteins to be produced in S2 cells with more complex N-glycans.
Assuntos
Acetilglucosaminidase/genética , Insetos/genética , N-Acetil-Lactosamina Sintase/genética , beta-N-Acetil-Hexosaminidases/genética , Animais , Glicosilação , Polissacarídeos/genética , Interferência de RNA/fisiologia , Proteínas Recombinantes/genéticaRESUMO
The presence of α2,6-sialic acids on the Fc N-glycan provides anti-inflammatory properties to the IgGs through a mechanism that remains unclear. Fc-sialylated IgGs are rare in humans as well as in industrial host cell lines such as Chinese hamster ovary (CHO) cells. Facilitated access to well-characterized α2,6-sialylated IgGs would help elucidate the mechanism of this intriguing IgG's effector function. This study presents a method for the efficient Fc glycan α2,6-sialylation of a wild-type and a F243A IgG1 mutant by transient co-expression with the human α2,6-sialyltransferase 1 (ST6) and ß1,4-galactosyltransferase 1 (GT) in CHO cells. Overexpression of ST6 alone only had a moderate effect on the glycoprofiles, whereas GT alone greatly enhanced Fc-galactosylation, but not sialylation. Overexpression of both GT and ST6 was necessary to obtain a glycoprofile dominated by α2,6-sialylated glycans in both antibodies. The wild-type was composed of the G2FS(6)1 glycan (38%) with remaining unsialylated glycans, while the mutant glycoprofile was essentially composed of G2FS(6)1 (25%), G2FS(3,6)2 (16%) and G2FS(6,6)2 (37%). The α2,6-linked sialic acids represented over 85% of all sialic acids in both antibodies. We discuss how the limited sialylation level in the wild-type IgG1 expressed alone or with GT results from the glycan interaction with Fc's amino acid residues or from intrinsic galactosyl- and sialyl-transferases substrate specificities.
Assuntos
Anticorpos Monoclonais , Imunoglobulina G , Mutação de Sentido Incorreto , Ácidos Siálicos/metabolismo , Substituição de Aminoácidos , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/genética , Antígenos CD/biossíntese , Antígenos CD/genética , Células CHO , Cricetinae , Cricetulus , Humanos , Imunoglobulina G/biossíntese , Imunoglobulina G/genética , N-Acetil-Lactosamina Sintase/biossíntese , N-Acetil-Lactosamina Sintase/genética , Sialiltransferases/biossíntese , Sialiltransferases/genéticaRESUMO
Modification of the plant N-glycosylation pathway towards human type structures is an important strategy to implement plants as expression systems for therapeutic proteins. Nevertheless, relatively little is known about the overall impact of non-plant glycosylation enzymes in stable transformed plants. Here, we analyzed transgenic lines (Nicotiana benthamiana and Arabidopsis thaliana) that stably express a modified version of human ß1,4-galactosyltransferase ((ST)GalT). While some transgenic plants grew normally, other lines exhibited a severe phenotype associated with stunted growth and developmental retardation. The severity of the phenotype correlated with both increased (ST)GalT mRNA and protein levels but no differences were observed between N-glycosylation profiles of plants with and without the phenotype. In contrast to non-transgenic plants, all (ST)GalT expressing plants synthesized significant amounts of incompletely processed (largely depleted of core fucose) N-glycans with up to 40% terminally galactosylated structures. While transgenic plants showed no differences in nucleotide sugar composition and cell wall monosaccharide content, alterations in the reactivity of cell wall carbohydrate epitopes associated with arabinogalactan-proteins and pectic homogalacturonan were detected in (ST)GalT expressing plants. Notably, plants with phenotypic alterations showed increased levels of hydrogen peroxide, most probably a consequence of hypersensitive reactions. Our data demonstrate that unfavorable phenotypical modifications may occur upon stable in planta expression of non-native glycosyltransferases. Such important issues need to be taken into consideration in respect to stable glycan engineering in plants.
Assuntos
Arabidopsis/genética , N-Acetil-Lactosamina Sintase/genética , Nicotiana/genética , Fenótipo , Plantas Geneticamente Modificadas , Polissacarídeos/biossíntese , Arabidopsis/metabolismo , Parede Celular/metabolismo , Epitopos , Galactosiltransferases/metabolismo , Engenharia Genética , Glicosilação , Humanos , Peróxido de Hidrogênio/metabolismo , Mucoproteínas/metabolismo , N-Acetil-Lactosamina Sintase/metabolismo , Pectinas/metabolismo , Folhas de Planta/metabolismo , Proteínas de Plantas/metabolismo , RNA Mensageiro/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Nicotiana/crescimento & desenvolvimento , Nicotiana/metabolismoRESUMO
ß1,4-Galactosyltransferase I (ß1,4-GalT-I), a key enzyme in glycobiology, mediates several biological mechanisms. However, the correlation between ß-1,4-GalT-I expression in the uterine endometrium and embryo implantation remains unclear. This study aims to elucidate the relationship between ß1,4-GalT-I and Lewis(Y) (Le(Y)) glycan during embryo implantation. So far, using green fluorescent protein as an indicator, ß1,4-GalT-I interference plasmid (pcDNA3.0-siGalT I), overexpression plasmid (pcDNA3.0-HA-GalT I), interference control plasmid (control pcDNA3.0-siGalT I), and empty vector (pcDNA3.0) were transfected into human uterine epithelial RL95-2 cells that imitate the receptive endometrium. Invasive embryos at pre-implantation and treated RL95-2 cells were co-cultured to determine embryo attachment in each of the transfection groups. The results showed that plasmid transfection was successful in all the groups. ß1,4-GalT-I and Fucosyltransferase 1 (FUT1) gene expression declined in the interference group, and the synthesis of Le(Y) decreased accordingly, but the expression of this antigen increased in the overexpression group. After co-culturing of the embryos and 36h transfection of RL95-2, the results of these in vitro implantation models showed that the attachment rate was lower in the interference group (30.0 ± 0.2%) than in the untreated group (50.0 ± 0.6%), empty vector group (50.0 ± 0.2%), and interference control group (46.7 ± 0.6%), however, it was highest in the overexpression group (70.0 ± 0.2%). These results indicated that ß1,4-galactosyltransferase I possibly regulate mutual uterus-embryo adhesion and embryo implantation by regulating cell surface Le(Y) glycan expression.
Assuntos
Implantação do Embrião , Antígenos do Grupo Sanguíneo de Lewis/metabolismo , N-Acetil-Lactosamina Sintase/metabolismo , Animais , Técnicas de Cocultura , Técnicas de Cultura Embrionária , Feminino , Fucosiltransferases/genética , Fucosiltransferases/metabolismo , Glicosilação , Humanos , Masculino , Camundongos , N-Acetil-Lactosamina Sintase/genética , Galactosídeo 2-alfa-L-FucosiltransferaseRESUMO
INTRODUCTION: Glycosylation controls diverse protein functions and regulates various cellular phenotypes. Trophoblast invasion is essential for normal placental development. However, the role of glycosylation in human placenta throughout pregnancy is still unclear. The ß-1,4-galactosyltransferase III (B4GALT3) has been found to regulate cancer cell invasion. We therefore investigated the expression of B4GALT3 in placenta and its roles in trophoblast. METHODS: B4GALT3 protein expression was examined by quantitative Western blotting analysis in human placentas. For identification of B4GALT3-positive cells in normal human placenta, immunohistochemistry and immunofluorescence methods were used. To investigate effects of B4GALT3 on extravillous trophoblast (EVT)-like cell and primary EVT cells, we analyzed cell growth, adhesion, migration, and invasion in mock and B4GALT3-transfected cell. RESULTS: B4GALT3 expression significantly increased in third trimester human placenta. Immunostaining revealed that B4GALT3 expressed in placental villous cytotrophoblast, syncytiotrophoblast, and a subpopulation of EVT cells throughout pregnancy. Interestingly, we found increases in the expression level and percentage of B4GALT3-positive cells in third trimester EVT, but not in syncytiotrophoblasts and cytotrophoblasts of placental villi. Overexpression of B4GALT3 in HTR8/SVneo cells and primary trophoblast cells significantly suppressed cell migration. In addition, B4GALT3 suppressed cell invasion, and enhanced cell adhesion to laminin in HTR8/SVneo cells. Notably, we found that B4GALT3 modified glycans on ß1-integrin, suppressed focal adhesion kinase (FAK) signaling, and enhanced ß1-integrin degradation. DISCUSSION: We propose that B4GALT3-mediated glycosylation change not only enhances ß1-integrin binding to laminin, but also attenuates ß1-integrin stability. Our findings suggest that B4GALT3 is a critical regulator for suppressing EVT invasion in the late stages of pregnancy.
Assuntos
Regulação para Baixo , Regulação da Expressão Gênica no Desenvolvimento , Integrina beta1/metabolismo , N-Acetil-Lactosamina Sintase/metabolismo , Placentação , Processamento de Proteína Pós-Traducional , Trofoblastos/metabolismo , Adulto , Adesão Celular , Linhagem Celular , Movimento Celular , Células Cultivadas , Feminino , Glicosilação , Humanos , Imuno-Histoquímica , Integrina beta1/química , Isoenzimas/genética , Isoenzimas/metabolismo , N-Acetil-Lactosamina Sintase/genética , Gravidez , Estabilidade Proteica , Proteínas Recombinantes/metabolismo , Trofoblastos/citologia , Trofoblastos/enzimologiaRESUMO
The ß(1,4)-galactosyltransferase-I gene (ß4galt1) encodes the catalytic part of the enzyme lactose synthase, responsible of lactose synthesis in the mammary gland. The complete coding region of the gene was screened for the presence of allelic variation among a sample of 1,200 Iranian Holstein cows, using PCR-SSCP technique followed by sequencing. Nine polymorphic nucleotide sites were identified-one in exons I and VI, two in exons II and III, and three in exon V. Altogether 18 different genotypes were assigned. Statistical analysis showed that the genotypes of Β4GALT1 significantly affect milk, lactose, protein and total solid productions in both the first and second lactation (P < 0.001). Variance component analysis considering restricted maximum likelihood showed that the major factor making differences in milk, lactose, protein and total solid productions among the studied cow is the ß4galt1 genotype. We concluded that the ß4galt1 gene is potentially associated with milk production traits in dairy cows and should be considered for further studies on genetics of the milk production traits.
Assuntos
Lactação/genética , Leite/metabolismo , N-Acetil-Lactosamina Sintase/genética , Polimorfismo de Nucleotídeo Único , Substituição de Aminoácidos , Animais , Sequência de Bases , Bovinos , Feminino , Estudos de Associação Genética , Genótipo , Polimorfismo Conformacional de Fita Simples , Análise de Sequência de DNARESUMO
Previously, we have shown that simple paucimannosidic N-glycan structures in insect Drosophila S2 cells arise mainly because of ß-N-acetylglucosaminidase (GlcNAcase) action. Thus, in an earlier report, we suppressed GlcNAcase activity and clearly demonstrated that more complex N-glycans with two terminal N-acetylglucosamine (GlcNAc) residues were then synthesized. In the present work, we investigated the synergistic effects of ß-1,4-galactosyltransferase (GalT) expression and GlcNAcase suppression on N-glycan patterns. We found that the N-glycan pattern of human erythropoietin secreted by engineered S2 cells expressing GalT but not GlcNAcase was complete, even in small portion, except for sialylation; the N-glycan structures had two terminal galactose (Gal) residues. When GalT was expressed but GlcNAcase was not inhibited, N-glycan with GlcNAc and Gal at only one branch end was synthesized. Therefore, it will be possible to express a complete functional human glycoprotein in engineered Drosophila S2 cells by suppressing GlcNAcase and co-expressing additional glycosyltransferases of N-glycosylation pathway.
Assuntos
Acetilglucosaminidase/antagonistas & inibidores , Drosophila melanogaster/metabolismo , N-Acetil-Lactosamina Sintase/biossíntese , Polissacarídeos/biossíntese , Engenharia de Proteínas/métodos , Acetilglucosaminidase/biossíntese , Acetilglucosaminidase/genética , Acetilglucosaminidase/metabolismo , Animais , Western Blotting , Células CHO , Linhagem Celular , Cromatografia Líquida de Alta Pressão , Cricetinae , Cricetulus , Drosophila melanogaster/genética , Eritropoetina/química , Eritropoetina/genética , Eritropoetina/metabolismo , Humanos , Microscopia de Fluorescência , N-Acetil-Lactosamina Sintase/genética , N-Acetil-Lactosamina Sintase/metabolismo , Polissacarídeos/química , Polissacarídeos/metabolismo , Interferência de RNA , Proteínas RecombinantesRESUMO
Proteoglycans (PGs), including heparan sulfate forms, are important regulators of tumor progression. In the PGs biosynthetic process, the core protein is synthesized on a ribosomal template and the sugar chains are assembled post-translationally, one sugar at a time, starting with the linkage of xylose to a serine residue of the core protein and followed by galactosidation of the xylosylprotein. Hydrophobic xylopyranosides have been previously shown to prime heparan sulfate synthesis, a property that was required to cause growth inhibition of tumor cells. To know if the antiproliferative activity of synthetic xylopyranosides is related to their ability to act as "decoy acceptors" of xylosylprotein 4-ß-galactosyltransferase, we have heterologously expressed the catalytic domain of the human ß-1,4-GalT 7 and studied the ability of a variety of synthetic xylopyranoside derivatives to act as substrates or inhibitors of the recombinant enzyme.
Assuntos
Galactosiltransferases , Glicopeptídeos , Glicosídeos , N-Acetil-Lactosamina Sintase/metabolismo , Naftóis , Proteínas Recombinantes/metabolismo , Sequência de Aminoácidos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Galactosiltransferases/síntese química , Galactosiltransferases/metabolismo , Galactosiltransferases/farmacologia , Regulação Bacteriana da Expressão Gênica , Glicopeptídeos/síntese química , Glicopeptídeos/metabolismo , Glicopeptídeos/farmacologia , Glicosídeos/síntese química , Glicosídeos/metabolismo , Glicosídeos/farmacologia , Humanos , Dados de Sequência Molecular , N-Acetil-Lactosamina Sintase/genética , Naftóis/síntese química , Naftóis/metabolismo , Naftóis/farmacologia , Proteínas Recombinantes/genética , SolubilidadeAssuntos
Fucosiltransferases/química , N-Acetil-Lactosamina Sintase/química , Peptídeos/química , Sequência de Aminoácidos , Membrana Celular/química , Membrana Celular/metabolismo , Dicroísmo Circular , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Fucosiltransferases/metabolismo , Helicobacter pylori/enzimologia , Humanos , Dados de Sequência Molecular , N-Acetil-Lactosamina Sintase/genética , N-Acetil-Lactosamina Sintase/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Cell surface glycans play important cellular functions and are synthesized by glycosyltransferases. Structure and function studies show that the donor sugar specificity of the invertebrate ß1,4-N-acetyl-glactosaminyltransferase (ß4GalNAc-T) and the vertebrate ß1,4-galactosyltransferase I (ß4Gal-T1) are related by a single amino acid residue change. Comparison of the catalytic domain crystal structures of the ß4Gal-T1 and the α-polypeptidyl-GalNAc-T (αppGalNAc-T) shows that their protein structure and sequences are similar. Therefore, it seems that the invertebrate ß4GalNAc-T and the catalytic domain of αppGalNAc-T might have emerged from a common primordial gene. When vertebrates emerged from invertebrates, the amino acid that determines the donor sugar specificity of the invertebrate ß4GalNAc-T might have mutated, thus converting the enzyme to a ß4Gal-T1 in vertebrates.
Assuntos
Evolução Molecular , Glicosiltransferases/química , Glicosiltransferases/metabolismo , Invertebrados/enzimologia , Vertebrados , Sequência de Aminoácidos , Animais , Glicosiltransferases/genética , Humanos , Invertebrados/genética , Dados de Sequência Molecular , N-Acetilgalactosaminiltransferases/química , N-Acetilgalactosaminiltransferases/genética , N-Acetilgalactosaminiltransferases/metabolismo , N-Acetil-Lactosamina Sintase/química , N-Acetil-Lactosamina Sintase/genética , N-Acetil-Lactosamina Sintase/metabolismo , Vertebrados/genética , Polipeptídeo N-AcetilgalactosaminiltransferaseRESUMO
Two bacterial beta1-4-galactosyltransferases, NmLgtB and Hp1-4GalT, exhibit promiscuous and complementary acceptor substrate specificity. They have been used in an efficient one-pot multienzyme system to synthesize LacNAc, lactose, and their derivatives including those containing negatively charged 6-O-sulfated GlcNAc and C2-substituted GlcNAc or Glc, from monosaccharide derivatives and inexpensive Glc-1-P.
Assuntos
Galactosídeos/síntese química , Helicobacter pylori/enzimologia , N-Acetil-Lactosamina Sintase/química , Neisseria meningitidis/enzimologia , Pasteurella multocida/enzimologia , Proteínas Recombinantes de Fusão/química , Configuração de Carboidratos , Sequência de Carboidratos , Clonagem Molecular , Escherichia coli/genética , Galactosídeos/química , N-Acetil-Lactosamina Sintase/genética , Proteínas Recombinantes de Fusão/genética , Especificidade por SubstratoRESUMO
The beta1,4-galactosyltransferase-7 (beta4Gal-T7) enzyme, one of seven members of the beta4Gal-T family, transfers in the presence of manganese Gal from UDP-Gal to an acceptor sugar (xylose) that is attached to a side chain hydroxyl group of Ser/Thr residues of proteoglycan proteins. It exhibits the least protein sequence similarity with the other family members, including the well studied family member beta4Gal-T1, which, in the presence of manganese, transfers Gal from UDP-Gal to GlcNAc. We report here the crystal structure of the catalytic domain of beta4Gal-T7 from Drosophila in the presence of manganese and UDP at 1.81 A resolution. In the crystal structure, a new manganese ion-binding motif (HXH) has been observed. Superposition of the crystal structures of beta4Gal-T7 and beta4Gal-T1 shows that the catalytic pocket and the substrate-binding sites in these proteins are similar. Compared with GlcNAc, xylose has a hydroxyl group (instead of an N-acetyl group) at C2 and lacks the CH(2)OH group at C5; thus, these protein structures show significant differences in their acceptor-binding site. Modeling of xylose in the acceptor-binding site of the beta4Gal-T7 crystal structure shows that the aromatic side chain of Tyr(177) interacts strongly with the C5 atom of xylose, causing steric hindrance to any additional group at C5. Because Drosophila Cd7 has a 73% protein sequence similarity to human Cd7, the present crystal structure offers a structure-based explanation for the mutations in human Cd7 that have been linked to Ehlers-Danlos syndrome.