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1.
Proc Natl Acad Sci U S A ; 116(28): 13729-13737, 2019 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-31213534

RESUMO

Cell surface carbohydrates, termed "glycans," are ubiquitous posttranslational effectors that can tune cancer progression. Often aberrantly displayed or found at atypical levels on cancer cells, glycans can impact essentially all progressive steps, from malignant transformation to metastases formation. Glycans are structural entities that can directly bind promalignant glycan-binding proteins and help elicit optimal receptor-ligand activity of growth factor receptors, integrins, integrin ligands, lectins, and other type-1 transmembrane proteins. Because glycans play an integral role in a cancer cell's malignant activity and are frequently uniquely expressed, preclinical studies on the suitability of glycans as anticancer therapeutic targets and their promise as biomarkers of disease progression continue to intensify. While sialylation and fucosylation have predominated the focus of cancer-associated glycan modifications, the emergence of blood group I antigens (or I-branched glycans) as key cell surface moieties capable of modulating cancer virulence has reenergized investigations into the role of the glycome in malignant progression. I-branched glycans catalyzed principally by the I-branching enzyme GCNT2 are now indicated in several malignancies. In this Perspective, the putative role of GCNT2/I-branching in cancer progression is discussed, including exciting insights on how I-branches can potentially antagonize the cancer-promoting activity of ß-galactose-binding galectins.


Assuntos
Galectinas/genética , N-Acetilexosaminiltransferases/genética , Neoplasias/genética , Polissacarídeos/genética , Carboidratos/química , Carboidratos/genética , Proteínas de Transporte/genética , Progressão da Doença , Glicosilação , Humanos , Integrinas/genética , Lectinas/genética , Neoplasias/metabolismo , Neoplasias/patologia , Polissacarídeos/metabolismo , Receptores de Fatores de Crescimento/genética , Transdução de Sinais
2.
Cell Biochem Funct ; 37(1): 42-51, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30575058

RESUMO

Esophageal squamous cell carcinoma (ESCC) is one of the most common cancers in the world. The prognosis of patients with ESCC is dismal with a 5-year survival of about 15%. Thus, identification of novel diagnostic and prognostic biomarkers for ESCC patients is urgently needed. Here, we found that manipulation of I-branching N-acetylglucosaminyltransferase (GCNT2) expression had no effect on cell proliferation. Notably, overexpression of GCNT2 promoted the migration and invasion, and this effect was associated with increased expression of N-cadherin and vimentin and decreased expression of E-cadherin in KYSE30 and EC9706 cells. Knockdown of GCNT2 decreased the expression of N-cadherin and vimentin, increased the expression of E-cadherin, and inhibited the migration and invasion in KYSE150 and EC109 cells. The expression of GCNT2 was significantly higher in tumour tissues than in paratumour tissues through tissue microarray analysis. More importantly, overall survival was significantly lower in patients with high GCNT2 expression than those with low GCNT2 expression. Collectively, our findings establish GCNT2 as a novel regulator of epithelial-mesenchymal transition (EMT) and a candidate prognostic indicator of outcome in ESCC patients. SIGNIFICANCE OF THE STUDY: Our study suggested that GCNT2 was highly expressed in patients with ESCC and predicted adverse outcome. Overexpression of GCNT2 induces EMT and promotes migration and invasion in ESCC cells. Therefore, GCNT2 may act as a candidate prognostic indicator of outcome and a novel target in ESCC patients.


Assuntos
Movimento Celular , Transição Epitelial-Mesenquimal , Neoplasias Esofágicas/metabolismo , Carcinoma de Células Escamosas do Esôfago/metabolismo , N-Acetilexosaminiltransferases/metabolismo , Antineoplásicos/farmacologia , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , N-Acetilexosaminiltransferases/antagonistas & inibidores , N-Acetilexosaminiltransferases/genética , Invasividade Neoplásica , RNA Interferente Pequeno/farmacologia , Análise Serial de Tecidos , Células Tumorais Cultivadas , Cicatrização/efeitos dos fármacos
3.
Nat Commun ; 9(1): 3368, 2018 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-30135430

RESUMO

Cancer cells often display altered cell-surface glycans compared to their nontransformed counterparts. However, functional contributions of glycans to cancer initiation and progression remain poorly understood. Here, from expression-based analyses across cancer lineages, we found that melanomas exhibit significant transcriptional changes in glycosylation-related genes. This gene signature revealed that, compared to normal melanocytes, melanomas downregulate I-branching glycosyltransferase, GCNT2, leading to a loss of cell-surface I-branched glycans. We found that GCNT2 inversely correlated with clinical progression and that loss of GCNT2 increased melanoma xenograft growth, promoted colony formation, and enhanced cell survival. Conversely, overexpression of GCNT2 decreased melanoma xenograft growth, inhibited colony formation, and increased cell death. More focused analyses revealed reduced signaling responses of two representative glycoprotein families modified by GCNT2, insulin-like growth factor receptor and integrins. Overall, these studies reveal how subtle changes in glycan structure can regulate several malignancy-associated pathways and alter melanoma signaling, growth, and survival.


Assuntos
Melanoma/metabolismo , Melanoma/patologia , N-Acetilexosaminiltransferases/metabolismo , Polissacarídeos/metabolismo , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Sobrevivência Celular/fisiologia , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Humanos , Melanoma/genética , Camundongos , Camundongos Knockout , N-Acetilglucosaminiltransferases/genética , N-Acetilglucosaminiltransferases/metabolismo , N-Acetilexosaminiltransferases/genética , Receptores de Interleucina-2/genética , Receptores de Interleucina-2/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/fisiologia
4.
Nat Commun ; 9(1): 3287, 2018 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-30120234

RESUMO

Leukocytes are coated with a layer of heterogeneous carbohydrates (glycans) that modulate immune function, in part by governing specific interactions with glycan-binding proteins (lectins). Although nearly all membrane proteins bear glycans, the identity and function of most of these sugars on leukocytes remain unexplored. Here, we characterize the N-glycan repertoire (N-glycome) of human tonsillar B cells. We observe that naive and memory B cells express an N-glycan repertoire conferring strong binding to the immunoregulatory lectin galectin-9 (Gal-9). Germinal center B cells, by contrast, show sharply diminished binding to Gal-9 due to upregulation of I-branched N-glycans, catalyzed by the ß1,6-N-acetylglucosaminyltransferase GCNT2. Functionally, we find that Gal-9 is autologously produced by naive B cells, binds CD45, suppresses calcium signaling via a Lyn-CD22-SHP-1 dependent mechanism, and blunts B cell activation. Thus, our findings suggest Gal-9 intrinsically regulates B cell activation and may differentially modulate BCR signaling at steady state and within germinal centers.


Assuntos
Linfócitos B/metabolismo , Galectinas/metabolismo , Polissacarídeos/química , Polissacarídeos/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Transdução de Sinais , Amino Açúcares/química , Sinalização do Cálcio , Linhagem Celular , Núcleo Celular/metabolismo , Proliferação de Células , Endocitose , Galectinas/sangue , Centro Germinativo/metabolismo , Humanos , Fatores Imunológicos/metabolismo , Memória Imunológica , Antígenos Comuns de Leucócito/metabolismo , Ativação Linfocitária/imunologia , Tecido Linfoide/metabolismo , Modelos Biológicos , N-Acetilexosaminiltransferases/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Fosforilação , Ligação Proteica , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico/metabolismo , Quinases da Família src/metabolismo
5.
G3 (Bethesda) ; 7(10): 3257-3268, 2017 10 05.
Artigo em Inglês | MEDLINE | ID: mdl-28839118

RESUMO

Pediatric cataract is a leading cause of childhood blindness. This study aimed to determine the genetic cause of pediatric cataract in Australian families by screening known disease-associated genes using massively parallel sequencing technology. We sequenced 51 previously reported pediatric cataract genes in 33 affected individuals with a family history (cases with previously known or published mutations were excluded) using the Ion Torrent Personal Genome Machine. Variants were prioritized for validation if they were predicted to alter the protein sequence and were absent or rare with minor allele frequency <1% in public databases. Confirmed mutations were assessed for segregation with the phenotype in all available family members. All identified novel or previously reported cataract-causing mutations were screened in 326 unrelated Australian controls. We detected 11 novel mutations in GJA3, GJA8, CRYAA, CRYBB2, CRYGS, CRYGA, GCNT2, CRYGA, and MIP; and three previously reported cataract-causing mutations in GJA8, CRYAA, and CRYBB2 The most commonly mutated genes were those coding for gap junctions and crystallin proteins. Including previous reports of pediatric cataract-associated mutations in our Australian cohort, known genes account for >60% of familial pediatric cataract in Australia, indicating that still more causative genes remain to be identified.


Assuntos
Catarata/genética , Adolescente , Adulto , Aquaporinas/genética , Austrália , Criança , Pré-Escolar , Conexinas/genética , Cristalinas/genética , Proteínas do Olho/genética , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Mutação , N-Acetilexosaminiltransferases/genética , Linhagem , Análise de Sequência de DNA , Adulto Jovem
6.
FEBS Lett ; 591(13): 1902-1917, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28542779

RESUMO

ß-1,6-N-acetylglucosaminyltransferase 2 (GCNT2), which encodes a key glycosyltransferase for blood group I antigen synthesis, is induced upon epithelial-mesenchymal transition (EMT). Our results indicate that GCNT2 is upregulated upon EMT induced with epidermal growth factor and basic FGF in cultured human colon cancer cells. GCNT2 knockdown or overexpression decreases or increases, respectively, malignancy-related characteristics of colon cancer cells and I antigen levels. MiR-199a/b-5p is markedly downregulated upon EMT in colon cancer cells. Here, we find that miR-199a/b-5p consistently regulates GCNT2 expression in reporter assays and that it binds directly to the GCNT2 3' untranslated region intracellularly in RNA-induced silencing complex-trap assays. Overexpression of miR-199a/b-5p decreases GCNT2 expression and suppresses I antigen production. Based on these findings, we propose that miR-199a/b-5p regulates GCNT2 and I antigen expression in colon cancer cells undergoing EMT.


Assuntos
Neoplasias do Colo/patologia , Regulação para Baixo/genética , Transição Epitelial-Mesenquimal/genética , MicroRNAs/genética , N-Acetilexosaminiltransferases/genética , Ativação Transcricional/genética , Animais , Sequência de Bases , Linhagem Celular Tumoral , Neoplasias do Colo/genética , Regulação para Baixo/efeitos dos fármacos , Fator de Crescimento Epidérmico/farmacologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Feminino , Fator 2 de Crescimento de Fibroblastos/farmacologia , Técnicas de Silenciamento de Genes , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Camundongos , Camundongos Endogâmicos BALB C , N-Acetilexosaminiltransferases/deficiência , Ativação Transcricional/efeitos dos fármacos
7.
PLoS One ; 11(12): e0167562, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27936067

RESUMO

PURPOSE: The aim of this study is to identify the molecular basis of autosomal recessive congenital cataracts (arCC) in a large consanguineous pedigree. METHODS: All participating individuals underwent a detailed ophthalmic examination. Each patient's medical history, particularly of cataracts and other ocular abnormalities, was compiled from available medical records and interviews with family elders. Blood samples were donated by all participating family members and used to extract genomic DNA. Genetic analysis was performed to rule out linkage to known arCC loci and genes. Whole-exome sequencing libraries were prepared and paired-end sequenced. A large deletion was found that segregated with arCC in the family, and chromosome walking was conducted to estimate the proximal and distal boundaries of the deletion mutation. RESULTS: Exclusion and linkage analysis suggested linkage to a region of chromosome 6p24 harboring GCNT2 (glucosaminyl (N-acetyl) transferase 2) with a two-point logarithm of odds score of 5.78. PCR amplifications of the coding exons of GCNT2 failed in individuals with arCC, and whole-exome data analysis revealed a large deletion on chromosome 6p in the region harboring GCNT2. Chromosomal walking using multiple primer pairs delineated the extent of the deletion to approximately 190 kb. Interestingly, a failure to amplify a junctional fragment of the deletion break strongly suggests an insertion in addition to the large deletion. CONCLUSION: Here, we report a novel insertion/deletion mutation at the GCNT2 locus that is responsible for congenital cataracts in a large consanguineous family.


Assuntos
Catarata/genética , N-Acetilexosaminiltransferases/genética , Deleção de Sequência , Animais , Catarata/congênito , Criança , Pré-Escolar , Consanguinidade , Feminino , Ligação Genética , Loci Gênicos , Humanos , Lactente , Masculino , Camundongos , Repetições de Microssatélites , N-Acetilglucosaminiltransferases/genética , Linhagem
8.
BMC Med Genet ; 17(1): 64, 2016 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-27609212

RESUMO

BACKGROUND: Congenital cataracts affect 3-6 per 10,000 live births and represent one of the leading causes of blindness in children. Congenital cataracts have a strong genetic component with high heterogeneity and variability. CASE PRESENTATION: Analysis of whole exome sequencing data in a patient affected with congenital cataracts identified a pathogenic deletion which was further defined by other techniques. A ~98-kb homozygous deletion of 6p24.3 involving the first three exons (two non-coding and one coding) of GCNT2 isoform A, the first exon (coding) of GCNT2 isoform B, and part of the intergenic region between GCNT2 and TFAP2A was identified in the patient and her brother while both parents were found to be heterozygous carriers of the deletion. The exact breakpoints were identified and revealed the presence of Alu elements at both sides of the deletion, thus indicating Alu-mediated non-homologous end-joining as the most plausible mechanism for this rearrangement. Recessive mutations in GCNT2 are known to cause an adult i blood group phenotype with congenital cataracts in some cases. The GCNT2 gene has three differentially expressed transcripts, with GCNT2B being the only isoform associated with lens function and GCNT2C being the only isoform expressed in red blood cells based on earlier studies; previously reported mutations/deletions have either affected all three isoforms (causing blood group and cataract phenotype) or the C isoform only (causing blood group phenotype only). Dominant mutations in TFAP2A are associated with syndromic anophthalmia/microphthalmia and other ocular phenotypes as part of Branchio-Ocular-Facial-Syndrome (BOFS). While the patients do not fit a diagnosis of BOFS, one sibling demonstrates mild overlap with the phenotypic spectrum, and therefore an effect of this deletion on the function of TFAP2A cannot be ruled out. CONCLUSIONS: To the best of our knowledge, this is the first case reported in which disruption of the GCNT2 gene does not involve the C isoform. The congenital cataracts phenotype in the affected patients is consistent with the previously defined isoform-specific roles of this gene. The GCNT2-TFAP2A region may be prone to rearrangements through Alu-mediated non-homologous end-joining.


Assuntos
Catarata/congênito , Catarata/genética , N-Acetilglucosaminiltransferases/genética , N-Acetilexosaminiltransferases/genética , Deleção de Sequência , Fator de Transcrição AP-2/genética , Pontos de Quebra do Cromossomo , Consanguinidade , Éxons , Feminino , Homozigoto , Humanos , Lactente , Isoenzimas/genética , Masculino , Linhagem
9.
Transfusion ; 56(11): 2691-2702, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27600951

RESUMO

BACKGROUND: Phosphorylation status of the transcription factor CCAAT/enhancer binding protein α (C/EBPα) has been demonstrated in a human hematopoietic cell model to regulate the formation of branched I antigen by affecting its binding affinity to the promoter region of the IGnTC gene during erythroid and granulocytic differentiation. STUDY DESIGN AND METHODS: The K-562 cell line was induced to differentiate into red blood cells (RBCs) or granulocytes by sodium butyrate or retinoic acid, respectively, to study the involvement of three MAP kinase pathways in I antigen synthesis. The regulatory effects of the extracellular signal-regulated kinase (ERK)2-Src homology region 2 domain-containing phosphatase 2 (SHP2) pathway on phosphorylation status and binding affinities of C/EBPα as well as the subsequent activation of IGnTC and synthesis of surface I formation were studied in wild-type K-562 cells and in mutant cells that overexpress ERK2 and SHP2. RESULTS: We found that SHP2-ERK2 signaling regulates the phosphorylation status of C/EBPα to alter its binding affinity onto the IGnTC promoter region, thereby activating the synthesis of cell surface I antigen formation during erythropoiesis. CONCLUSION: SHP2-ERK2 signaling acts upstream of C/EBPα as a regulator of cell surface I antigen synthesis. Such regulation is specific for RBC but not for granulocyte differentiation.


Assuntos
Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Eritropoese , Sistema do Grupo Sanguíneo I/biossíntese , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , N-Acetilglucosaminiltransferases/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Transdução de Sinais , Humanos , Células K562 , N-Acetilglucosaminiltransferases/genética , N-Acetilexosaminiltransferases , Fosforilação , Regiões Promotoras Genéticas , Ligação Proteica
10.
J Cell Physiol ; 229(4): 471-8, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24105809

RESUMO

Poly-N-acetyllactosamine (PLN) is a unique glycan composed of repeating units of the common disaccharide (Galß1,4-GlcNAcß1,3)n . The expression of PLN on glycoprotein core structures minimally requires enzyme activities for ß1,4-galactosyltransferase (ß4GalT) and ß1,3-N-acetylglucosminyltransferase (ß3GnT). Because ß4GalTs are ubiquitous in most cells, PLN expression is generally ascribed to the tissue-specific transcription of eight known ß3GnT genes in mice. In the olfactory epithelium (OE), ß3GnT2 regulates expression of extended PLN chains that are essential for axon guidance and neuronal survival. N-glycan branching and core composition, however, can also modulate the extent of PLN modification. Here, we show for the first time that the ß1,6-branching glycosyltransferase GCNT2 (formerly known as IGnT) is expressed at high levels specifically in the OE and other sensory ganglia. Postnatally, GCNT2 is maintained in mature olfactory neurons that co-express ß3GnT2 and PLN. This highly specific co-expression suggests that GCNT2 and ß3GnT2 function cooperatively in PLN synthesis. In support of this, ß3GnT2 and GCNT2 co-transfection in HEK293T cells results in high levels of PLN expression on the cell surface and on adenylyl cyclase 3, a major carrier of PLN glycans in the OE. These data clearly suggest that GCNT2 functions in vivo together with ß3GnT2 to determine PLN levels in olfactory neurons by regulating ß1,6-branches that promote PLN extension.


Assuntos
Regulação Enzimológica da Expressão Gênica/fisiologia , N-Acetilglucosaminiltransferases/metabolismo , N-Acetilexosaminiltransferases/metabolismo , Polissacarídeos/biossíntese , Animais , Embrião de Mamíferos/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Células HEK293 , Humanos , Camundongos , Camundongos Knockout , N-Acetilglucosaminiltransferases/genética , N-Acetilexosaminiltransferases/genética , Gravidez
11.
J Bacteriol ; 193(8): 1943-52, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21335454

RESUMO

The Aeromonas hydrophila AH-3 WecP represents a new class of UDP-HexNAc:polyprenol-P HexNAc-1-P transferases. These enzymes use a membrane-associated polyprenol phosphate acceptor (undecaprenyl phosphate [Und-P]) and a cytoplasmic UDP-d-N-acetylhexosamine sugar nucleotide as the donor substrate. Until now, all the WecA enzymes tested were able to transfer UDP-GlcNAc to the Und-P. In this study, we present in vitro and in vivo proofs that A. hydrophila AH-3 WecP transfers GalNAc to Und-P and is unable to transfer GlcNAc to the same enzyme substrate. The molecular topology of WecP is more similar to that of WbaP (UDP-Gal polyprenol-P transferase) than to that of WecA (UDP-GlcNAc polyprenol-P transferase). WecP is the first UDP-HexNAc:polyprenol-P GalNAc-1-P transferase described.


Assuntos
Aeromonas hydrophila/enzimologia , N-Acetilexosaminiltransferases/metabolismo , Fosfatos de Poli-Isoprenil/metabolismo , Uridina Difosfato N-Acetilgalactosamina/metabolismo , Sequência de Carboidratos , Modelos Moleculares , Dados de Sequência Molecular , N-Acetilexosaminiltransferases/química
12.
J Bacteriol ; 191(12): 4030-4, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19376878

RESUMO

There have been considerable strides made in the characterization of the dispensability of teichoic acid biosynthesis genes in recent years. A notable omission thus far has been an early gene in teichoic acid synthesis encoding the N-acetylmannosamine transferase (tagA in Bacillus subtilis; tarA in Staphylococcus aureus), which adds N-acetylmannosamine to complete the synthesis of undecaprenol pyrophosphate-linked disaccharide. Here, we show that the N-acetylmannosamine transferases are dispensable for growth in vitro, making this biosynthetic enzyme the last dispensable gene in the pathway, suggesting that tagA (or tarA) encodes the first committed step in wall teichoic acid synthesis.


Assuntos
Bacillus subtilis/enzimologia , Proteínas de Bactérias/metabolismo , N-Acetilexosaminiltransferases/metabolismo , Staphylococcus aureus/enzimologia , Ácidos Teicoicos/biossíntese , Bacillus subtilis/genética , Bacillus subtilis/crescimento & desenvolvimento , Bacillus subtilis/metabolismo , Proteínas de Bactérias/genética , Catálise , N-Acetilexosaminiltransferases/genética , Staphylococcus aureus/genética , Staphylococcus aureus/crescimento & desenvolvimento , Staphylococcus aureus/metabolismo
13.
Biochemistry ; 44(20): 7526-34, 2005 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-15895995

RESUMO

Previous studies have reported that insect cell lines lack the capacity to generate endogenously the nucleotide sugar, CMP-Neu5Ac, required for sialylation of glycoconjugates. In this study, the biosynthesis of this activated form of sialic acid completely from endogenous metabolites is demonstrated for the first time in insect cells by expressing the mammalian genes required for the multistep conversion of endogenous UDP-GlcNAc to CMP-Neu5Ac. The genes for UDP-GlcNAc-2-epimerase/ManNAc kinase (EK), sialic acid 9-phosphate synthase (SAS), and CMP-sialic acid synthetase (CSAS) were coexpressed in insect cells using baculovirus expression vectors, but the CMP-Neu5Ac and precursor Neu5Ac levels synthesized were found to be lower than those achieved with ManNAc supplementation due to feedback inhibition of the EK enzyme by CMP-Neu5Ac. When sialuria-like mutant EK genes, in which the site for feedback regulation has been mutated, were used, CMP-Neu5Ac was synthesized at levels more than 4 times higher than that achieved with the wild-type EK and 2.5 times higher than that achieved with ManNAc feeding. Addition of N-acetylglucosamine (GlcNAc), a precursor for UDP-GlcNAc, to the media increased the levels of CMP-Neu5Ac even more to a level 7.5 times higher than that achieved with ManNAc supplementation, creating a bottleneck in the conversion of Neu5Ac to CMP-Neu5Ac at higher levels of UDP-GlcNAc. The present study provides a useful biochemical strategy to synthesize and enhance the levels of the sialylation donor molecule, CMP-Neu5Ac, a critical limiting substrate for the generation of complex glycoproteins in insect cells and other cell culture systems.


Assuntos
Ácido N-Acetilneuramínico Citidina Monofosfato/química , Ácido N-Acetilneuramínico Citidina Monofosfato/metabolismo , Líquido Intracelular/química , Líquido Intracelular/metabolismo , Mutagênese Sítio-Dirigida , N-Acilneuraminato Citidililtransferase/biossíntese , Spodoptera/enzimologia , Spodoptera/genética , Animais , Arginina/genética , Baculoviridae/enzimologia , Baculoviridae/genética , Carboidratos Epimerases/antagonistas & inibidores , Carboidratos Epimerases/biossíntese , Carboidratos Epimerases/genética , Células Cultivadas , Hexosaminas/química , Hexosaminas/metabolismo , Humanos , Leucina/genética , Manosefosfatos , Mariposas/virologia , N-Acetilexosaminiltransferases/biossíntese , N-Acetilexosaminiltransferases/genética , N-Acilneuraminato Citidililtransferase/genética , Ratos , Doença do Armazenamento de Ácido Siálico/genética , Especificidade por Substrato/genética
14.
Biochemistry ; 43(42): 13248-55, 2004 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-15491132

RESUMO

Tunicamycins are potent inhibitors of UDP-N-acetyl-D-hexosamine:polyprenol-phosphate N-acetylhexosamine-1-phosphate translocases (D-HexNAc-1-P translocases), a family of enzymes involved in bacterial cell wall synthesis and eukaryotic protein N-glycosylation. Structurally, tunicamycins consist of an 11-carbon dialdose core sugar called tunicamine that is N-linked at C-1' to uracil and O-linked at C-11' to N-acetylglucosamine (GlcNAc). The C-11' O-glycosidic linkage is highly unusual because it forms an alpha/beta anomeric-to-anomeric linkage to the 1-position of the GlcNAc residue. We have assigned the (1)H and (13)C NMR spectra of tunicamycin and have undertaken a conformational analysis from rotating angle nuclear Overhauser effect (ROESY) data. In addition, chirally deuterated tunicamycins produced by fermentation of Streptomyces chartreusis on chemically synthesized, monodeuterated (S-6)-[(2)H(1)]glucose have been used to assign the geminal H-6'a, H-6'b methylene bridge of the 11-carbon dialdose sugar, tunicamine. The tunicamine residue is shown to assume pseudo-D-ribofuranose and (4)C(1) pseudo-D-galactopyranosaminyl ring conformers. Conformation about the C-6' methylene bridge determines the relative orientation of these rings. The model predicts that tunicamycin forms a right-handed cupped structure, with the potential for divalent metal ion coordination at 5'-OH, 8'-OH, and the pseudogalactopyranosyl 7'-O ring oxygen. The formation of tunicamycin complexes with various divalent metal ions was confirmed experimentally by MALDI-TOF mass spectrometry. Our data support the hypothesis that tunicamycin is a structural analogue of the UDP-D-HexNAc substrate and is reversibly coordinated to the divalent metal cofactor in the D-HexNAc-1-P translocase active site.


Assuntos
Medição da Troca de Deutério , Galactosamina/análogos & derivados , Metano/análogos & derivados , Sondas Moleculares/metabolismo , N-Acetilexosaminiltransferases/química , N-Acetilexosaminiltransferases/metabolismo , Tunicamicina/química , Tunicamicina/metabolismo , Sítios de Ligação , Configuração de Carboidratos , Cátions Bivalentes/metabolismo , Medição da Troca de Deutério/métodos , Dissacarídeos/química , Galactosamina/química , Glicosídeos/química , Hidrocarbonetos , Isomerismo , Magnésio/metabolismo , Metano/química , Ressonância Magnética Nuclear Biomolecular/métodos , Ligação Proteica , Conformação Proteica , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Streptomyces/enzimologia , Especificidade por Substrato , Uracila/química
15.
J Biol Chem ; 279(53): 55722-7, 2004 Dec 31.
Artigo em Inglês | MEDLINE | ID: mdl-15498763

RESUMO

N-Acetylmannosamine (ManNAc) is the physiological precursors to all sialic acids that occur in nature. As variations in the sialic acid decoration of cell surfaces can profoundly affect cell-cell, pathogen-cell, or drug-cell interactions, the enzymes that convert ManNAc into sialic acid are attractive targets for the development of drugs that specifically interrupt sialic acid biosynthesis or lead to modified sialic acids on the surface of cells. The first step in the enzymatic conversion of ManNAc into sialic acid is phosphorylation, yielding N-acetylmannosamine-6-phosphate. The enzyme that catalyzes this conversion is the N-acetylmannosamine kinase (ManNAc kinase) as part of the bifunctional enzyme UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase. Here, we employed saturation transfer difference (STD) NMR experiments to study the binding of ManNAc and related ligands to the ManNAc kinase. It is shown that the configuration of C1 and C4 of ManNAc is crucial for binding to the enzyme, whereas the C2 position not only accepts variations in the attached N-acyl side chain but also tolerates inversion of configuration. Our experiments also show that ManNAc kinase maintains its functionality, even in the absence of Mg(2+). From the analysis of the STD NMR-derived binding epitopes, it is concluded that the binding mode of the N-acylmannosamines critically depends on the N-acyl side chain. In conjunction with the relative binding affinities of the ligands obtained from STD NMR titrations, it is possible to derive a structure-binding affinity relationship. This provides a cornerstone for the rational design of drugs for novel therapeutic applications by altering the sialic acid decorations of cell walls.


Assuntos
Espectroscopia de Ressonância Magnética/métodos , N-Acetilexosaminiltransferases/química , Ácido N-Acetilneuramínico/biossíntese , Trifosfato de Adenosina/química , Animais , Carboidratos Epimerases/química , Linhagem Celular , Membrana Celular/metabolismo , Epitopos/química , Hexosaminas/química , Insetos , Cinética , Ligantes , Magnésio/química , Modelos Químicos , Modelos Moleculares , Fosfatos/química , Ligação Proteica , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , Fatores de Tempo
16.
Adv Biochem Eng Biotechnol ; 90: 89-111, 2004.
Artigo em Inglês | MEDLINE | ID: mdl-15453186

RESUMO

Capsular polysaccharides (CPs) of several pathogenic bacteria are thought to be good materials for the development of new therapeutic reagents. These polysaccharides can be used as vaccines against infection of pathogenic bacteria and are also useful as inhibitors for disease caused by aberrant and abnormal cell-cell interaction, such as cancer metastasis and inflammation. Since bacterial CPs are diverse in structure and these bacteria have a variety of sugar transferases responsible for the synthesis of CPs, bacterial CP synthesis (cps) genes have attracted much interest as a source of glycosyltransferases useful for glycoengineering. In this review, we describe physiological effects of the bacterial CPs on mammalian cells, and the structure and function of the cps genes, by focusing on group B streptococci, Streptococcus agalactiae type Ia and Ib, that produce high-molecular weight polysaccharides consisting of the following pentasaccharide repeating units: -->4)-[alpha-D-NeupNAc-(2-->3)-beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->3)]-beta-D-Galp-(1-->4)-beta-D-Glcp-(1--> and -->4)-[alpha-D-NeupNAc-(2-->3)-beta-D-Galp-(1-->3)-beta-D-GlcpNAc-(1-->3)]-beta-D-Galp-(1-->4)beta-D-Glcp-(1-->, respectively.


Assuntos
Glicosiltransferases/metabolismo , Polissacarídeos Bacterianos/biossíntese , Streptococcus agalactiae/genética , Sequência de Aminoácidos , Cápsulas Bacterianas/química , Cápsulas Bacterianas/metabolismo , Sequência de Carboidratos , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Bases de Dados Genéticas , Escherichia coli/genética , Ordem dos Genes/genética , Glicosiltransferases/genética , Hexoses/metabolismo , Hexosiltransferases/genética , Hexosiltransferases/metabolismo , Humanos , Dados de Sequência Molecular , N-Acetilexosaminiltransferases/genética , N-Acetilexosaminiltransferases/metabolismo , Fases de Leitura Aberta/genética , Polissacarídeos Bacterianos/farmacologia , Homologia de Sequência , Sialiltransferases/genética , Sialiltransferases/metabolismo , Streptococcus agalactiae/química , Streptococcus agalactiae/enzimologia , Streptococcus pneumoniae/genética , Streptococcus pyogenes/genética
17.
J Agric Food Chem ; 51(6): 1701-5, 2003 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-12617608

RESUMO

Serratia marcescens YS-1, a chitin-degrading microorganism, produced mainly N-acetylhexosaminidase. The purified enzyme had an optimal pH of approximately 8-9 and remained stable at 40 degrees C for 60 min at pH 6-8. The optimum temperature was around 50 degrees C, and enzyme activity was relatively stable below 50 degrees C. YS-1 N-acetylhexosaminidase hydrolyzed p-nitrophenyl beta-N-acetylgalactosamide by 28.1% relative to p-nitrophenyl beta-N-acetylglucosamide. The N-acetylchitooligosaccharides were hydrolyzed more rapidly, but the cellobiose and chitobiose of disaccharides that had the same beta-1,4 glycosidic bond as di-N-acetylchitobiose were not hydrolyzed. YS-1 N-acetylhexosaminidase efficiently transferred the N-acetylglucosamine residue from di-N-acetylchitobiose (substrate) to alcohols (acceptor). The ratio of transfer to methanol increased to 86% in a reaction with 32% methanol. N-Acetylglucosamine was transferred to the hydroxyl group at C1 of monoalcohols. A dialcohol was used as an acceptor when the carbon number was more than 4 and a hydroxyl group existed on each of the two outside carbons. Sugar alcohols with hydroxyl groups in all carbon positions were not proper acceptors.


Assuntos
Glicosídeos/biossíntese , N-Acetilexosaminiltransferases/metabolismo , Serratia marcescens/enzimologia , Sequência de Aminoácidos , Glicosilação , Concentração de Íons de Hidrogênio , Cinética , Metanol/metabolismo , N-Acetilexosaminiltransferases/química , N-Acetilexosaminiltransferases/isolamento & purificação , Especificidade por Substrato , Temperatura
19.
J Biol Chem ; 276(7): 4834-8, 2001 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-11121397

RESUMO

The proteins encoded by the EXT1, EXT2, and EXTL2 genes, members of the hereditary multiple exostoses gene family of tumor suppressors, are glycosyltransferases required for the heparan sulfate biosynthesis. Only two homologous genes, rib-1 and rib-2, of the mammalian EXT genes were identified in the Caenorhabditis elegans genome. Although heparan sulfate is found in C. elegans, the involvement of the rib-1 and rib-2 proteins in heparan sulfate biosynthesis remains unclear. In the present study, the substrate specificity of a soluble recombinant form of the rib-2 protein was determined and compared with those of the recombinant forms of the mammalian EXT1, EXT2, and EXTL2 proteins. The present findings revealed that the rib-2 protein was a unique alpha1,4-N-acetylglucosaminyltransferase involved in the biosynthetic initiation and elongation of heparan sulfate. In contrast, the findings confirmed the previous observations that both the EXT1 and EXT2 proteins were heparan sulfate copolymerases with both alpha1,4-N-acetylglucosaminyltransferase and beta1,4-glucuronyltransferase activities, which are involved only in the elongation step of the heparan sulfate chain, and that the EXTL2 protein was an alpha1,4-N-acetylglucosaminyltransferase involved only in the initiation of heparan sulfate synthesis. These findings suggest that the biosynthetic mechanism of heparan sulfate in C. elegans is distinct from that reported for the mammalian system.


Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans/enzimologia , Proteínas de Helminto/fisiologia , Heparitina Sulfato/biossíntese , Proteínas de Membrana , N-Acetilglucosaminiltransferases/fisiologia , Animais , Células COS , Genes Supressores de Tumor , Proteínas de Helminto/genética , N-Acetilglucosaminiltransferases/genética , N-Acetilglucosaminiltransferases/metabolismo , N-Acetilexosaminiltransferases/metabolismo , Proteínas/metabolismo , Especificidade por Substrato , Transfecção
20.
Cytogenet Cell Genet ; 89(3-4): 185-8, 2000.
Artigo em Inglês | MEDLINE | ID: mdl-10965119

RESUMO

Human EXTL2 is an alpha1,4-N-acetylhexosaminyltransferase involved in the biosynthesis of heparin/heparan sulfate. We have cloned and characterized the mouse homolog of this gene. Mouse Extl2 encodes a 330 amino acid protein that is 87% identical to its human counterpart. Expression analysis showed that Extl2 is ubiquitously expressed in adult mouse tissues and that the Extl2 transcript is already present in early stages of embryonic development. Determination of the genomic structure revealed that the Extl2 gene spans five exons within a 10-kb region and that the genomic organization between mouse and man is well preserved, with conservation of the number and position of all five exons. By radiation hybrid analysis, Extl2 was mapped to mouse chromosome 3, in a region homologous to the human EXTL2 region on chromosome 1.


Assuntos
Proteínas de Membrana , N-Acetilglucosaminiltransferases , N-Acetilexosaminiltransferases/genética , Sequência de Aminoácidos , Animais , Northern Blotting , DNA/química , DNA/genética , DNA Complementar/genética , DNA Complementar/isolamento & purificação , Éxons , Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Genes/genética , Íntrons , Masculino , Camundongos , Dados de Sequência Molecular , N-Acetilexosaminiltransferases/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Distribuição Tecidual
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