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Merkel cell carcinoma is a rare, lethal cancer histopathologically composed of cells showing similarity with mechanoreceptor Merkel cells. Merkel cell tumors manifest in two distinct forms. While a virus called Merkel cell polyomavirus is involved in the pathogenesis of one form of Merkel tumors, the other is driven by ultraviolet (UV)-linked mutations. In this study we investigated 18 cases, from the Indian population, of Merkel cell carcinoma for immunohistochemical (IHC) expression of Merkel cell polyomavirus (MCV) T antigen, including 12 cases tested by PCR, to identify viral etiopathology. We tested the tumors with two sensitive antibodies (CM2B4 and Ab3), targeting the viral large T antigen protein and with PCR primers targeting the N terminus of T antigen. Overall, we observed 38.8% (7/18) tumors displaying positive IHC expression of Merkel cell polyomavirus T antigen and 25% (3/12) tumors showing positive results, by both, immunohistochemistry and PCR. This constitutes the first report from India showing implication of MCV in Merkel cell carcinomas. Moreover, this is one of the larger series of Merkel cell carcinomas, tested for MCV, by both immunohistochemistry and PCR, in this part of the world. These results further indicate that a slightly more number of such cases in India are likely to be caused by UV-linked damage, as opposed to Merkel cell polyomavirus mediated tumorigenesis, which is definitely implicated in a subset of cases. • We investigated 18 Merkel cell carcinoma cases, from the Indian population to identify viral etiopathology. • Overall, 38.8% (7/18) tumors displayed positive IHC expression for MCV T antigen using antibodies CM2B4 and Ab3. • Three of the twelve tumors, (25%) showed positive MCV presence, by both, immunohistochemistry and PCR. • Our study is the first report from India showing implication of Merkel cell polyomavirus (MCV) in Merkel cell carcinomas. [ABSTRACT FROM AUTHOR]

As the phylogenetic organization of mammalian polyomaviruses is complex and currently incompletely resolved, we aimed at a deeper insight into their evolution by identifying polyomaviruses in host orders and families that have either rarely or not been studied. Sixteen unknown and two known polyomaviruses were identified in animals that belong to 5 orders, 16 genera, and 16 species. From 11 novel polyomaviruses, full genomes could be determined. Splice sites were predicted for large and small T antigen (LTAg, STAg) coding sequences (CDS) and examined experimentally in transfected cell culture. In addition, splice sites of seven published polyomaviruses were analyzed. Based on these data, LTAg and STAg annotations were corrected for 10/86 and 74/86 published polyomaviruses, respectively. For 25 polyomaviruses, a spliced middle T CDS was observed or predicted. Splice sites that likely indicate expression of additional, alternative T antigens, were experimentally detected for six polyomaviruses. In contrast to all other mammalian polyomaviruses, three closely related cetartiodactyl polyomaviruses display two introns within their LTAg CDS. In addition, the VP2 of Glis glis (edible dormouse) polyomavirus 1 was observed to be encoded by a spliced transcript, a unique experimental finding within the Polyomaviridae family. Co-phylogenetic analyses based on LTAg CDS revealed a measurable signal of codivergence when considering all mammalian polyomaviruses, most likely driven by relatively recent codivergence events. Lineage duplication was the only other process whose influence on polyomavirus evolution was unambiguous. Finally, our analyses suggest that an update of the taxonomy of the family is required, including the creation of novel genera of mammalian and non-mammalian polyomaviruses. [ABSTRACT FROM AUTHOR]

Summary We have developed an Escherichia coli strain for the in vivo production of O-glycosylated proteins. This was achieved using a dual plasmid approach: one encoding a therapeutic protein target, and a second encoding the enzymatic machinery required for O-glycosylation. The latter plasmid encodes human polypeptide N-acetylgalactosaminyl transferase as well as a β1,3-galactosyl transferase and UDP-Glc(NAc)-4-epimerase, both from Campylobacter jejuni , and a disulfide bond isomerase of bacterial or human origin. The effectiveness of this two-plasmid synthetic operon system has been tested on three proteins with therapeutic potential: the native and an engineered version of the naturally O-glycosylated human interferon α-2b, as well as human growth hormone with one engineered site of glycosylation. Having established proof of principle for the addition of the core-1 glycan onto proteins, we are now developing this system as a platform for producing and modifying human protein therapeutics with more complex O-glycan structures in E. coli. Graphical Abstract Highlights • Designed and expressed a synthetic core-1 O-glycan synthesis operon in E. coli • Improved the O-glycosylation sequon through sequence manipulation • Produced glycosylated, biologically active interferon-α2b • Demonstrated a platform expression system for core-1 O-glycan addition Du et al. describe a synthetic biology approach to producing mammalian-like O-glycans in bacteria. They demonstrate that this glycan can be produced on two examples of human therapeutic proteins. This demonstration of a platform technology for glycosylation has the potential to improve serum half-life of therapeutic proteins made in bacteria. [ABSTRACT FROM AUTHOR]

Group A Streptococcus (GAS) ( Streptococcus pyogenes ) is an important human pathogen associated with significant global morbidity and mortality for which there is no safe and efficacious vaccine. The T antigen, a protein that polymerizes to form the backbone of the GAS pilus structure, is a potential vaccine candidate. Previous surveys of the tee gene, which encodes the T antigen, have identified 21 different tee types and subtypes such that any T antigen-based vaccine must be multivalent and carefully designed to provide broad strain coverage. In this study, the crystal structures of three two-domain T antigens (T3.2, T13, and T18.1) were determined and found to have remarkable structural similarity to the previously reported T1 antigen, despite moderate overall sequence similarity. This has enabled reliable modeling of all major two-domain T antigens to reveal that T antigen sequence variation is distributed along the full length of the protein and shields a highly conserved core. Immunoassays performed with sera from immunized animals and commercial T-typing sera identified a significant cross-reactive antibody response between T18.1, T18.2, T3.2, and T13. The existence of shared epitopes between T antigens, combined with the remarkably conserved structure and high level of surface sequence divergence, has important implications for the design of multivalent T antigen-based vaccines., (Copyright © 2019 American Society for Microbiology.)

Aberrant display of the truncated core1 O-glycan T-antigen is a common feature of human cancer cells that correlates with metastasis. Here we show that T-antigen in Drosophila melanogaster macrophages is involved in their developmentally programmed tissue invasion. Higher macrophage T-antigen levels require an atypical major facilitator superfamily (MFS) member that we named Minerva which enables macrophage dissemination and invasion. We characterize for the first time the T and Tn glycoform O-glycoproteome of the Drosophila melanogaster embryo, and determine that Minerva increases the presence of T-antigen on proteins in pathways previously linked to cancer, most strongly on the sulfhydryl oxidase Qsox1 which we show is required for macrophage tissue entry. Minerva's vertebrate ortholog, MFSD1, rescues the minerva mutant's migration and T-antigen glycosylation defects. We thus identify a key conserved regulator that orchestrates O-glycosylation on a protein subset to activate a program governing migration steps important for both development and cancer metastasis., Competing Interests: KV, JB, MR, SE, AG, MM, AR, PR, KS, IL, SV, HC, DS No competing interests declared, (© 2019, Valoskova et al.)

Royal jelly has received attention because of its necessity for the development of queen honeybees as well as claims of benefits on human health; this product of the hypopharyngeal glands of worker bees contains a large number of proteins, some of which have been claimed to have various biological effects only in their glycosylated state. However, although there have been glycomic and glycoproteomic analyses in the past, none of the glycan structures previously defined would appear to have potential to trigger specific biological functions. In the current study, whole royal jelly as well as single protein bands were subject to off-line LC-MALDI-TOF MS glycomic analyses, complemented by permethylation, Western blotting and arraying data. Similarly to recent in-depth studies on other insect species, previously overlooked glucuronic acid termini, sulfation of mannose residues and core β-mannosylation of the N-glycans were found; additionally, a relatively rare zwitterionic modification with phosphoethanolamine is present, in contrast to the phosphorylcholine occurring in lepidopteran species. Indicative of tissue-specific remodelling of glycans in the Golgi apparatus of hypopharyngeal gland cells, only a low amount of fucosylated or paucimannosidic glycans were detected as compared with other insect samples or even bee venom. The unusual modifications of hybrid and multiantennary structures defined here may not only have a physiological role in honeybee development, but represent epitopes recognized by pentraxins with roles in animal innate immunity., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

Merkel cell carcinoma (MCC) is a rare, aggressive cutaneous neuroendocrine malignancy. Merkel cell polyomavirus, a tumorigenic DNA virus, is present in most MCC tumors, with implications for tumor biology, diagnosis, and management. Merkel cell polyomavirus-negative tumors have a high burden of UV-signature mutations, similar to melanoma. The histopathologic diagnosis of MCC requires immunohistochemistry to exclude morphologically similar entities. Therapies for advanced disease are currently lacking. Here, the features of MCC are reviewed, including recent molecular discoveries with implications for improved therapy for advanced disease., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Immortalization of chondrocytes enables long term in vitro culture; however, the chondrogenic capacity of transformed cells varies, thus highlighting the need to develop a proliferative and tuneable chondrocyte cell line where hypertrophic differentiation can be controlled. In this study the SV40 large T antigen and human telomerase reverse transcriptase were employed to immortalize pooled equine chondrocytes through lentiviral vector mediated transduction either singly or on combination. Transformed chondrocytes proliferated stably over multiple passages, but resulted in significantly lower expression of chondrocyte specific collagen II mRNA (P < 0.0001) and up regulation of the hypertrophic marker collagen X (P < 0.0001) in three dimensional cultures. A Col2a1 promoter driven GFP reporter was constructed for real time monitoring of chondrogenic differentiation and a significant increase in promoter activation was observed in cultures treated with the growth factor TGFβ-3 (P < 0.05). To recapitulate the native articular chondrocyte phenotype we further transduced large T antigen immortalized chondrocytes with lentiviral vectors allowing either constitutive or doxycycline inducible expression of Sox9. In 3D cultures, the Sox9 over-expressing chondrocytes secreted significantly higher levels of extracellular matrix polysaccharide glycosaminoglycan (P < 0.05), while up-regulating collagen II and Aggrecan mRNA (P < 0.05) in both expression systems with a similar patterns observed with imunohistochemical staining. High levels of collagen X mRNA and protein were maintained with constitutive sox9 reflecting hypetrophic differentiation but significantly lower expression could be achieved with inducible Sox9. In conclusion, immortalization of equine chondrocytes results in stable proliferation but a reduction of chondrogenic potential whilst modulation of sox9 expression enabled control of hypertrophic characteristics. J. Cell. Biochem. 118: 1201-1215, 2017. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Quantitative PCR is a diagnostic mainstay of clinical virology, and accurate quantitation of viral load among labs requires the use of international standards. However, the use of multiple passages of viral isolates to obtain sufficient material for international standards may result in genomic changes that complicate their use as quantitative standards. We performed next-generation sequencing to obtain single-nucleotide resolution and relative copy number of JC virus (JCV) clinical standards. Strikingly, the WHO international standard and the Exact v1/v2 prototype standards for JCV showed 8-fold and 4-fold variation in genomic coverage between different loci in the viral genome, respectively, due to large deletions in the large T antigen region. Intriguingly, several of the JCV standards sequenced in this study with large T antigen deletions were cultured in cell lines immortalized using simian virus 40 (SV40) T antigen, suggesting the possibility of transcomplementation in cell culture. Using a cutoff 5% allele fraction for junctional reads, 7 different rearrangements were present in the JC virus sequences present in the WHO standard across multiple library preparations and sequencing runs. Neither the copy number differences nor the rearrangements were observed in a clinical sample with a high copy number of JCV or a plasmid control. These results were also confirmed by the quantitative real-time PCR (qPCR), droplet digital PCR (ddPCR), and Sanger sequencing of multiple rearrangements. In summary, targeting different regions of the same international standard can result in up to an 8-fold difference in quantitation. We recommend the use of next-generation sequencing to validate standards in clinical virology., (Copyright © 2017 Greninger et al.)

The usefulness of T antigen in the diagnosis of lung cancer (LCA), including early, was assessed by determining the in vitro delayed type hypersensitivity response to T(DTHR-T), and by measuring with a solid phase immunofluorescent assay the serum anti-T IgM response. Sensitivity of DTHR-T was 89% for 73 patients with LCA including 8/9 with Stage TNM disease, overall specificity was 95% for 212 healthy persons and those with non-CA disease. The humoral immune assay detected 31/35 (89%) LCA patients including 4/5 Stage TNM patients. Overall specificity was >90% among 116 persons without CA. [ABSTRACT FROM AUTHOR]

Background: Ovarian cancer is the most lethal gynecological malignancy due to its frequent recurrence and drug resistance even after successful initial treatment. Accumulating scientific evidence indicates that subpopulations of cancer cells with stem cell-like properties, such as so-called side population (SP) cells, are primarily responsible for these recurrences. A better understanding of SP cells may provide new clues for detecting and targeting these cancer-initiating cells and ultimately help to eradicate cancer. Changes in glycosylation patterns are remarkable features of SP cells. Here, we isolated SP cells from ovarian cancer cell lines and analyzed their glycosylation patterns using multiple glycomic strategies., Methods: Six high-grade serous ovarian cancer cell lines were used for SP cell isolation. Among them, HO8910 pm, which contained the highest proportion of SP cells, was used for glycomic analysis of SP cells. Cell lysate of SP cells and main population cells was applied to lectin microarray and mass spectrometry for glycan profiling. Differently expressed glycan structures were further verified by lectin blot, flow cytometry, and real-time PCR analysis of their relevant enzymes., Results: Expression of core fucosylated N-glycan and tumor-associated Tn, T and sT antigens were increased in SP cells. By contrast, SP cells exhibited decreased hybrid glycan, α2,3-linked sialic glycan and multivalent sialyl-glycan., Conclusions: Glycan structures, such as Tn, T, sT antigens, and core fucosylation may serve as biomarkers of ovarian cancer stem cells.

JC virus (JCV), a ubiquitous polyoma virus that commonly infects the human, is identified as the etiologic agent for progressive multifocal leukoencephalopathy and some malignancies. To clarify the oncogenic role of JCV T antigen, we established two transgenic mice of T antigen using either α-crystallin A (αAT) or cytokeratin 19(KT) promoter. Lens tumors were found in high-copy αAT mice with the immunopositivity of T antigen, p53, β-catenin and N-cadherin. Enlarged eyeballs were observed and tumor invaded into the brain by magnetic resonance imaging and hematoxylin-and-eosin staining. The overall survival time of homozygous mice was shorter than that of hemizygous mice (p<0.01), the latter than wild-type mice (p<0.01). The spontaneous salivary tumor and hepatocellular carcinoma were seen in αAT5 transgenic mice with no positivity of T antigen. KT7 mice suffered from lung tumor although JCV T antigen was strongly expressed in gastric epithelial cells. The alternative splicing of T antigen intron was detectable in the lens tumor of αAT mice, gastric mucosa of KT mice, and various cells transfected with pEGFP-N1-T antigen. It was suggested that JCV T antigen might induce carcinogenesis at a manner of cell specificity, which is not linked to alternative splicing of its intron. Both spontaneous lens and lung tumor models provide good tools to investigate the oncogenic role of JCV T antigen.

Aberrant glycosylation is frequently observed in cancers. Core 1 β1,3-galactosyltransferase (C1GALT1) is an exclusive enzyme in humans that catalyzes the biosynthesis of core 1 O-glycan structure, Gal-GalNAc-O-Ser/Thr, whose expression is commonly up-regulated during tumorigenesis. Little is known about the function of C1GALT1 in breast cancer. This study aims to determine the correlation between C1GALT1 expression and breast cancer clinicopathological features and roles of C1GALT1 in breast cancer malignant phenotypes. Public databases and our data showed that C1GALT1 mRNA and C1GALT1 protein are frequently up-regulated in breast cancer; and increased C1GALT1 expression correlates with higher histological grade and advanced tumor stage. Overexpression of C1GALT1 enhanced breast cancer cell growth, migration, and invasion in vitro as well as tumor growth in vivo. Conversely, C1GALT1 knockdown suppressed these malignant phenotypes. Furthermore, C1GALT1 modulates O-glycan structures on Mucin (MUC) 1 and promotes MUC1-C/β-catenin signaling in breast cancer cells. These findings suggest that C1GALT1 enhances breast cancer malignant progression through promoting MUC1-C/β-catenin signaling pathway. Unveiling the function of C1GALT1 in breast cancer opens new insights to the roles of C1GALT1 and O-glycosylation in tumorigenesis and renders the potential of C1GALT1 as a target of novel therapeutic agent development.

Heparan sulfate (HS) is a polysaccharide fundamentally important for biologically activities. T/Tn antigens are universal carbohydrate cancer markers. Here, we report the specific imaging of these carbohydrates using a mesenchymal stem cell line and human umbilical vein endothelial cells (HUVEC). The staining specificities were demonstrated by comparing imaging of different glycans and validated by either removal of target glycans, which results in loss of signal, or installation of target glycans, which results in gain of signal. As controls, representative key glycans including O-GlcNAc, lactosaminyl glycans and hyaluronan were also imaged. HS staining revealed novel architectural features of the extracellular matrix (ECM) of HUVEC cells. Results from T/Tn antigen staining suggest that O-GalNAcylation is a rate-limiting step for O-glycan synthesis. Overall, these highly specific approaches for HS and T/Tn antigen imaging should greatly facilitate the detection and functional characterization of these biologically important glycans.

Expression of T antigen (Galbeta1, 3GalNAc) is associated with enhanced metastatic potential and poor prognosis in colorectal cancer. Cosmc is a molecular chaperone required for the formation of an active T-synthase, which catalyzes the synthesis of T antigen. However, the expression and role of Cosmc in colorectal cancer are still unclear. Here, real-time PCR showed that overexpression of Cosmc mRNA in colorectal tumors compared with paired non-tumorous tissues was associated with increased American Joint Committee on Cancer (AJCC) tumor stage. Forced expression of Cosmc in HCT116 cells significantly increased T antigen expression and enhanced cell growth, migration, and invasion, which was associated with increased phosphorylation of focal adhesion kinase (FAK), ERK, and Akt. These Cosmc-enhanced malignant phenotypes were significantly suppressed by specific inhibitor of MEK or PI3K. We also found that Cosmc overexpression increased tumor growth and decreased survival of tumor-bearing SCID mice. Conversely, knockdown of Cosmc with siRNA in SW480 cells decreased malignant behaviors and the signaling pathways, which were substantially reversed by constitutively active Akt or MEK. Taken together, these results suggest that Cosmc promotes malignant phenotypes of colon cancer cells mainly via activation of MEK/ERK and PI3K/Akt signaling pathways, and that Cosmc may serve as a potential target for colorectal cancer treatment., (© 2013 Wiley Periodicals, Inc.)

A series of STn-MUC1 and ST-MUC1 glycopeptides containing naturally occurring and non-natural sialic acids have been chemoenzymatically synthesized from Tn-MUC1 glycopeptide using one-pot multienzyme (OPME) approaches. In situ generation of the sialyltransferase donor cytidine 5'-monophosphate-sialic acid (CMP-Sia) using a CMP-sialic acid synthetase in the presence of an extra amount of cytidine 5'-triphosphate (CTP) and removal of CMP from the reaction mixture by flash C18 cartridge purification allow the complete consumption of Tn-MUC1 glycopeptide for quantitative synthesis of STn-MUC1. A Campylobacter jejuni β1-3GalT (CjCgtBΔ30-His6) mutant has been found to catalyze the transfer of one or more galactose residues to Tn-MUC1 for the synthesis of T-MUC1 and galactosylated T-MUC1. Sialylation of T-MUC1 using Pasteurella multocida α2-3-sialyltransferase 3 (PmST3) with Neisseria meningitidis CMP-sialic acid synthetase (NmCSS) and Escherichia coli sialic acid aldolase in one pot produced ST-MUC1 efficiently. These glycopeptides are potential cancer vaccine candidates., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The Merkel cell polyomavirus (MCPyV), identified in humans in 2008, is associated with a relatively rare but aggressive neuroendocrine skin cancer, the Merkel cell carcinoma (MCC). MCC incidence is increasing due to the advancing age of the population, the increase in damaging sun exposure and in the number of immunocompromised individuals. MCPyV must be considered as the etiological agent of MCC and thus is the first example of a human oncogenic polyomavirus. MCPyV infection is common, and seroprevalence studies indicate that widespread exposure begins early in life. The majority of adults have anti-MCPyV antibodies and there is a growing body of evidence that healthy human skin harbors resident or transient MCPyV suggesting that MCPyV infection persists throughout life. However, the mode of transmission, the host cells, and the latency characteristics of this virus remain to be elucidated. In addition, it is still not clear whether MCPyV is associated with diseases or lesions other than Merkel cell carcinoma. The etiologic role of MCPyV in MCC opens up opportunities to improve the understanding of this cancer and to potentially improve its treatment., (© 2013 APMIS Published by John Wiley & Sons Ltd.)

Merkel cell carcinoma (MCC) is the most aggressive skin cancer. Recently, it was demonstrated that human Merkel cell polyomavirus (MCV) is clonally integrated in 80% of MCC tumors. Genetic studies of MCV have shown that T antigen protein is responsible for replication of genome and play a foremost role in viral infection. Therefore, T antigen protein may be used as suitable target for disease diagnosis. Viral activity can be restrained through RNA interference (RNAi) technology, an influential method for post transcriptional gene silencing in a sequence specific manner. In current study four effective siRNA molecules for silencing of MCV were rationally designed and validated using computational methods, which may lead to knockdown the activity of virus. Thus, this approach may provide an insight for the chemical synthesis of antiviral RNA molecule for the treatment of MCC at genome level.