I-branched carbohydrates as emerging effectors of malignant progression.

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.

GCNT2 induces epithelial-mesenchymal transition and promotes migration and invasion in esophageal squamous cell carcinoma cells.

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.

Loss of GCNT2/I-branched glycans enhances melanoma growth and survival.

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.

High-Throughput Genetic Screening of 51 Pediatric Cataract Genes Identifies Causative Mutations in Inherited Pediatric Cataract in South Eastern Australia.

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.

Downregulation of miR-199a/b-5p is associated with GCNT2 induction upon epithelial-mesenchymal transition in colon cancer.

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.

Deletion at the GCNT2 Locus Causes Autosomal Recessive Congenital Cataracts.

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.

Case report of homozygous deletion involving the first coding exons of GCNT2 isoforms A and B and part of the upstream region of TFAP2A in congenital cataract.

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.

N-linked polylactosamine glycan synthesis is regulated by co-expression of ß3GnT2 and GCNT2.

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.

The N-acetylmannosamine transferase catalyzes the first committed step of teichoic acid assembly in Bacillus subtilis and Staphylococcus aureus.

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.

Engineering intracellular CMP-sialic acid metabolism into insect cells and methods to enhance its generation.

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.

Bacterial capsular polysaccharide and sugar transferases.

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.

Characterization and genomic localization of the mouse Extl2 gene.

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.

EXT genes are differentially expressed in bone and cartilage during mouse embryogenesis.

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.

Refined physical mapping and genomic structure of the EXTL1 gene.

Recently, the EXTL1 gene, a member of the EXT tumor suppressor gene family, has been mapped to 1p36, a chromosome region which is frequently implicated in a wide variety of malignancies, including breast carcinoma, colorectal cancer and neuroblastoma. In this study, we show that the EXTL1 gene is located between the genetic markers D1S511 and D1S234 within 200 kb of the LAP18 gene on chromosome 1p36. 1, a region which has been proposed to harbor a tumor suppressor gene implicated in MYCN-amplified neuroblastomas. In addition, we determined the genomic structure of the EXTL1 gene, revealing that the EXTL1 coding sequence spans 11 exons within a 50-kb region.

Mutation analysis of hereditary multiple exostoses in the Chinese.

Hereditary multiple exostoses (EXT; MIM 133700) is an autosomal dominant bone disorder. It is genetically heterogeneous with at least three chromosomal loci: EXT1 on 8q24.1, EXT2 on 11p11, and EXT3 on 19p. EXT1 and EXT2, the two genes responsible for EXT1 and EXT2, respectively, have been cloned. Recently, three other members of the EXT gene family, named the EXT-like genes (EXTL: EXTL1, EXTL2, and EXTL3), have been isolated. EXT1, EXT2, and the three EXTLs are homologous with one another. We have identified the intron-exon boundaries of EXTL1 and EXTL3 and analyzed EXT1, EXT2, EXTL1, and EXTL3, in 36 Chinese families with EXT, to identify underlying disease-related mutations in the Chinese population. Of the 36 families, five and 12 family groups have mutations in EXT1 and EXT2, respectively. No disease-related mutation has been found in either EXTL1 or EXTL2, although one polymorphism has been detected in EXTL1. Of the 15 different mutations (three families share a common mutation in EXT2), 12 are novel. Most of the mutations are either frameshift or nonsense mutations (12/15). These mutations lead directly or indirectly to premature stop codons, and the mutations generate truncated proteins. This finding is consistent with the hypothesis that the development of EXT is mainly attributable to loss of gene function. Missense mutations are rare in our families, but these mutations may reflect some functionally crucial regions of these proteins. EXT1 is the most frequent single cause of EXT in the Caucasian population in Europe and North America. It accounts for about 40% of cases of EXT. Our study of 36 EXT Chinese families has found that EXT1 seems much less common in the Chinese population, although the frequency of the EXT2 mutation is similar in the Caucasian and Chinese populations. Our findings suggest a possibly different genetic spectrum of this disease in different populations.

The tumor suppressor EXT-like gene EXTL2 encodes an alpha1, 4-N-acetylhexosaminyltransferase that transfers N-acetylgalactosamine and N-acetylglucosamine to the common glycosaminoglycan-protein linkage region. The key enzyme for the chain initiation of heparan sulfate.

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.