RESUMEN
When conducting proteomics experiments to detect missing proteins and protein isoforms in the human proteome, it is desirable to use a protease that can yield more unique peptides with properties amenable for mass spectrometry analysis. Though trypsin is currently the most widely used protease, some proteins can yield only a limited number of unique peptides by trypsin digestion. Other proteases and multiple proteases have been applied in reported studies to increase the number of identified proteins and protein sequence coverage. To facilitate the selection of proteases, we developed a web-based resource, called in silico Human Proteome Digestion Map (iHPDM), which contains a comprehensive proteolytic peptide database constructed from human proteins, including isoforms, in neXtProt digested by 15 protease combinations of one or two proteases. iHPDM provides convenient functions and graphical visualizations for users to examine and compare the digestion results of different proteases. Notably, it also supports users to input filtering criteria on digested peptides, e.g., peptide length and uniqueness, to select suitable proteases. iHPDM can facilitate protease selection for shotgun proteomics experiments to identify missing proteins, protein isoforms, and single amino acid variant peptides.
Asunto(s)
Péptido Hidrolasas/metabolismo , Mapeo Peptídico/métodos , Proteoma/metabolismo , Gráficos por Computador , Simulación por Computador , Visualización de Datos , Bases de Datos Factuales , Receptores ErbB/metabolismo , Humanos , Internet , MAP Quinasa Quinasa 1/metabolismo , N-Acetilhexosaminiltransferasas/metabolismo , Isoformas de Proteínas/metabolismo , Proteómica/métodos , Receptores Odorantes/metabolismo , Interfaz Usuario-Computador , gamma-Glutamiltransferasa/metabolismoRESUMEN
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.
Asunto(s)
Movimiento Celular , Transición Epitelial-Mesenquimal , Neoplasias Esofágicas/metabolismo , Carcinoma de Células Escamosas de Esófago/metabolismo , N-Acetilhexosaminiltransferasas/metabolismo , Antineoplásicos/farmacología , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Transición Epitelial-Mesenquimal/efectos de los fármacos , Neoplasias Esofágicas/patología , Carcinoma de Células Escamosas de Esófago/patología , Femenino , Humanos , Masculino , Persona de Mediana Edad , N-Acetilhexosaminiltransferasas/antagonistas & inhibidores , N-Acetilhexosaminiltransferasas/genética , Invasividad Neoplásica , ARN Interferente Pequeño/farmacología , Análisis de Matrices Tisulares , Células Tumorales Cultivadas , Cicatrización de Heridas/efectos de los fármacosRESUMEN
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.
Asunto(s)
Linfocitos B/metabolismo , Galectinas/metabolismo , Polisacáridos/química , Polisacáridos/metabolismo , Receptores de Antígenos de Linfocitos B/metabolismo , Transducción de Señal , Amino Azúcares/química , Señalización del Calcio , Línea Celular , Núcleo Celular/metabolismo , Proliferación Celular , Endocitosis , Galectinas/sangre , Centro Germinal/metabolismo , Humanos , Factores Inmunológicos/metabolismo , Memoria Inmunológica , Antígenos Comunes de Leucocito/metabolismo , Activación de Linfocitos/inmunología , Tejido Linfoide/metabolismo , Modelos Biológicos , N-Acetilhexosaminiltransferasas/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Fosforilación , Unión Proteica , Proteína Tirosina Fosfatasa no Receptora Tipo 6/metabolismo , Lectina 2 Similar a Ig de Unión al Ácido Siálico/metabolismo , Familia-src Quinasas/metabolismoRESUMEN
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.
Asunto(s)
Melanoma/metabolismo , Melanoma/patología , N-Acetilhexosaminiltransferasas/metabolismo , Polisacáridos/metabolismo , Animales , Línea Celular Tumoral , Supervivencia Celular/genética , Supervivencia Celular/fisiología , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Humanos , Melanoma/genética , Ratones , Ratones Noqueados , N-Acetilglucosaminiltransferasas/genética , N-Acetilglucosaminiltransferasas/metabolismo , N-Acetilhexosaminiltransferasas/genética , Receptores de Interleucina-2/genética , Receptores de Interleucina-2/metabolismo , Transducción de Señal/genética , Transducción de Señal/fisiologíaRESUMEN
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.
Asunto(s)
Regulación Enzimológica de la Expresión Génica/fisiología , N-Acetilglucosaminiltransferasas/metabolismo , N-Acetilhexosaminiltransferasas/metabolismo , Polisacáridos/biosíntesis , Animales , Embrión de Mamíferos/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica/fisiología , Células HEK293 , Humanos , Ratones , Ratones Noqueados , N-Acetilglucosaminiltransferasas/genética , N-Acetilhexosaminiltransferasas/genética , EmbarazoRESUMEN
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.
Asunto(s)
Aeromonas hydrophila/enzimología , N-Acetilhexosaminiltransferasas/metabolismo , Fosfatos de Poliisoprenilo/metabolismo , Uridina Difosfato N-Acetilgalactosamina/metabolismo , Secuencia de Carbohidratos , Modelos Moleculares , Datos de Secuencia Molecular , N-Acetilhexosaminiltransferasas/químicaRESUMEN
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.
Asunto(s)
Bacillus subtilis/enzimología , Proteínas Bacterianas/metabolismo , N-Acetilhexosaminiltransferasas/metabolismo , Staphylococcus aureus/enzimología , Ácidos Teicoicos/biosíntesis , Bacillus subtilis/genética , Bacillus subtilis/crecimiento & desarrollo , Bacillus subtilis/metabolismo , Proteínas Bacterianas/genética , Catálisis , N-Acetilhexosaminiltransferasas/genética , Staphylococcus aureus/genética , Staphylococcus aureus/crecimiento & desarrollo , Staphylococcus aureus/metabolismoRESUMEN
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.
Asunto(s)
Medición de Intercambio de Deuterio , Galactosamina/análogos & derivados , Metano/análogos & derivados , Sondas Moleculares/metabolismo , N-Acetilhexosaminiltransferasas/química , N-Acetilhexosaminiltransferasas/metabolismo , Tunicamicina/química , Tunicamicina/metabolismo , Sitios de Unión , Conformación de Carbohidratos , Cationes Bivalentes/metabolismo , Medición de Intercambio de Deuterio/métodos , Disacáridos/química , Galactosamina/química , Glicósidos/química , Hidrocarburos , Isomerismo , Magnesio/metabolismo , Metano/química , Resonancia Magnética Nuclear Biomolecular/métodos , Unión Proteica , Conformación Proteica , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Streptomyces/enzimología , Especificidad por Sustrato , Uracilo/químicaRESUMEN
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.
Asunto(s)
Glicosiltransferasas/metabolismo , Polisacáridos Bacterianos/biosíntesis , Streptococcus agalactiae/genética , Secuencia de Aminoácidos , Cápsulas Bacterianas/química , Cápsulas Bacterianas/metabolismo , Secuencia de Carbohidratos , Adhesión Celular/efectos de los fármacos , Línea Celular Tumoral , Bases de Datos Genéticas , Escherichia coli/genética , Orden Génico/genética , Glicosiltransferasas/genética , Hexosas/metabolismo , Hexosiltransferasas/genética , Hexosiltransferasas/metabolismo , Humanos , Datos de Secuencia Molecular , N-Acetilhexosaminiltransferasas/genética , N-Acetilhexosaminiltransferasas/metabolismo , Sistemas de Lectura Abierta/genética , Polisacáridos Bacterianos/farmacología , Homología de Secuencia , Sialiltransferasas/genética , Sialiltransferasas/metabolismo , Streptococcus agalactiae/química , Streptococcus agalactiae/enzimología , Streptococcus pneumoniae/genética , Streptococcus pyogenes/genéticaRESUMEN
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.
Asunto(s)
Glicósidos/biosíntesis , N-Acetilhexosaminiltransferasas/metabolismo , Serratia marcescens/enzimología , Secuencia de Aminoácidos , Glicosilación , Concentración de Iones de Hidrógeno , Cinética , Metanol/metabolismo , N-Acetilhexosaminiltransferasas/química , N-Acetilhexosaminiltransferasas/aislamiento & purificación , Especificidad por Sustrato , TemperaturaRESUMEN
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.
Asunto(s)
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans/enzimología , Proteínas del Helminto/fisiología , Heparitina Sulfato/biosíntesis , Proteínas de la Membrana , N-Acetilglucosaminiltransferasas/fisiología , Animales , Células COS , Genes Supresores de Tumor , Proteínas del Helminto/genética , N-Acetilglucosaminiltransferasas/genética , N-Acetilglucosaminiltransferasas/metabolismo , N-Acetilhexosaminiltransferasas/metabolismo , Proteínas/metabolismo , Especificidad por Sustrato , TransfecciónRESUMEN
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.
Asunto(s)
Proteínas de la Membrana , N-Acetilglucosaminiltransferasas , N-Acetilhexosaminiltransferasas/genética , Secuencia de Aminoácidos , Animales , Northern Blotting , ADN/química , ADN/genética , ADN Complementario/genética , ADN Complementario/aislamiento & purificación , Exones , Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Genes/genética , Intrones , Masculino , Ratones , Datos de Secuencia Molecular , N-Acetilhexosaminiltransferasas/metabolismo , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Distribución TisularRESUMEN
Hereditary multiple exostoses (HME) is a genetically heterogeneous disease characterized by the development of bony protuberances at the ends of all long bones. Genetic analyses have revealed HME to be a multigenic disorder linked to three loci on chromosomes 8q24 (EXT1), 11p11-13 (EXT2), and 19p (EXT3). The EXT1 and EXT2 genes have been cloned and defined as glycosyltransferases involved in the synthesis of heparan sulfate. EST database analysis has demonstrated additional gene family members, EXT-like genes (EXTL1, EXTL2, and EXTL3), not associated with a HME locus. The mouse homologs of EXT1 and EXT2 have also been cloned and shown to be 99% and 95% identical to their human counterparts, respectively. Here, we report the identification of the mouse EXTL1 gene and show it is 74% identical to the human EXTL1 gene. Expression studies of all three mouse EXT genes throughout various stages of embryonic development were carried out and whole-mount in situ hybridization in the developing limb buds showed high levels of expression of all three EXT genes. However, in situ hybridization of sectioned embryos revealed remarkable differences in expression profiles of EXT1, EXT2, and EXTL1. The identical expression patterns found for the EXT1 and EXT2 genes support the recent observation that both proteins form a glycosyltransferase complex. We suggest a model for exostoses formation based on the involvement of EXT1 and EXT2 in the Indian hedgehog/parathyroid hormone-related peptide (PTHrP) signaling pathway, an important regulator of the chondrocyte maturation process.
Asunto(s)
Desarrollo Óseo/fisiología , Cartílago/embriología , Condrocitos/metabolismo , Exostosis Múltiple Hereditaria/genética , N-Acetilglucosaminiltransferasas , N-Acetilhexosaminiltransferasas/genética , Proteínas/genética , Proteínas Supresoras de Tumor , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Células COS , Cartílago/metabolismo , Retículo Endoplásmico/metabolismo , Exostosis Múltiple Hereditaria/patología , Expresión Génica , Perfilación de la Expresión Génica , Humanos , Hibridación in Situ , Ratones , Modelos Biológicos , Datos de Secuencia Molecular , N-Acetilhexosaminiltransferasas/metabolismo , Reacción en Cadena de la Polimerasa , Proteínas/metabolismo , Análisis de Secuencia de ADNRESUMEN
This review covers discoveries made over the past 30-35 years that were important to our understanding of the synthetic pathway required for initiation of the antennae or branches on complex N-glycans and O-glycans. The review deals primarily with the author's contributions but the relevant work of other laboratories is also discussed. The focus of the review is almost entirely on the glycosyltransferases involved in the process. The following topics are discussed. (1) The localization of the synthesis of complex N-glycan antennae to the Golgi apparatus. (2) The "evolutionary boundary" at the stage in N-glycan processing where there is a change from oligomannose to complex N-glycans; this switch correlates with the appearance of multicellular organisms. (3) The discovery of the three enzymes which play a key role in this switch, N-acetylglucosaminyltransferases I and II and mannosidase II. (4) The "yellow brick road" which leads from oligomannose to highly branched complex N-glycans with emphasis on the enzymes involved in the process and the factors which control the routes of synthesis. (5) A short discussion of the characteristics of the enzymes involved and of the genes that encode them. (6) The role of complex N-glycans in mammalian and Caenorhabditis elegans development. (7) The crystal structure of N-acetylglucosaminyltransferase I. (8) The discovery of the enzymes which synthesize O-glycan cores 1, 2, 3 and 4 and their elongation.
Asunto(s)
Polisacáridos/biosíntesis , Animales , Caenorhabditis elegans/crecimiento & desarrollo , Caenorhabditis elegans/metabolismo , Canadá , Secuencia de Carbohidratos , Historia del Siglo XX , Humanos , Mamíferos , Datos de Secuencia Molecular , N-Acetilglucosaminiltransferasas/genética , N-Acetilglucosaminiltransferasas/metabolismo , N-Acetilhexosaminiltransferasas/metabolismo , Polisacáridos/química , Polisacáridos/historiaRESUMEN
We previously demonstrated a unique alpha-N-acetylgalactosaminyltransferase that transferred N-acetylgalactosamine (GalNAc) to the tetrasaccharide-serine, GlcAbeta1-3Galbeta1-3Galbeta1-4Xylbeta1-O-Ser (GlcA represents glucuronic acid), derived from the common glycosaminoglycan-protein linkage region, through an alpha1,4-linkage. In this study, we purified the enzyme from the serum-free culture medium of a human sarcoma cell line. Peptide sequence analysis of the purified enzyme revealed 100% identity to the multiple exostoses-like gene EXTL2/EXTR2, a member of the hereditary multiple exostoses (EXT) gene family of tumor suppressors. The expression of a soluble recombinant form of the protein produced an active enzyme, which transferred alpha-GalNAc from UDP-[3H]GalNAc to various acceptor substrates including GlcAbeta1-3Galbeta1-3Galbeta1-4Xylbeta1-O-Ser. Interestingly, the enzyme also catalyzed the transfer of N-acetylglucosamine (GlcNAc) from UDP-[3H]GlcNAc to GlcAbeta1-3Galbeta1-O-naphthalenemethanol, which was the acceptor substrate for the previously described GlcNAc transferase I involved in the biosynthetic initiation of heparan sulfate. The GlcNAc transferase reaction product was sensitive to the action of heparitinase I, establishing the identity of the enzyme to be alpha1, 4-GlcNAc transferase. These results altogether indicate that EXTL2/EXTR2 encodes the alpha1,4-N-acetylhexosaminyltransferase that transfers GalNAc/GlcNAc to the tetrasaccharide representing the common glycosaminoglycan-protein linkage region and that is most likely the critical enzyme that determines and initiates the heparin/heparan sulfate synthesis, separating it from the chondroitin sulfate/dermatan sulfate synthesis.