Browsing by keyword "glycosylation"
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Dengue Virus Hijacks a Noncanonical Oxidoreductase Function of a Cellular Oligosaccharyltransferase ComplexDengue virus (DENV) is the most common arboviral infection globally, infecting an estimated 390 million people each year. We employed a genome-wide clustered regularly interspaced short palindromic repeat (CRISPR) screen to identify host dependency factors required for DENV propagation and identified the oligosaccharyltransferase (OST) complex as an essential host factor for DENV infection. Mammalian cells express two OSTs containing either STT3A or STT3B. We found that the canonical catalytic function of the OSTs as oligosaccharyltransferases is not necessary for DENV infection, as cells expressing catalytically inactive STT3A or STT3B are able to support DENV propagation. However, the OST subunit MAGT1, which associates with STT3B, is also required for DENV propagation. MAGT1 expression requires STT3B, and a catalytically inactive STT3B also rescues MAGT1 expression, supporting the hypothesis that STT3B serves to stabilize MAGT1 in the context of DENV infection. We found that the oxidoreductase CXXC active site motif of MAGT1 was necessary for DENV propagation, as cells expressing an AXXA MAGT1 mutant were unable to support DENV infection. Interestingly, cells expressing single-cysteine CXXA or AXXC mutants of MAGT1 were able to support DENV propagation. Utilizing the engineered peroxidase APEX2, we demonstrate the close proximity between MAGT1 and NS1 or NS4B during DENV infection. These results reveal that the oxidoreductase activity of the STT3B-containing OST is necessary for DENV infection, which may guide the development of antiviral agents targeting DENV. IMPORTANCE The host oligosaccharyltransferase (OST) complexes have been identified as essential host factors for dengue virus (DENV) replication; however, their functions during DENV infection are unclear. A previous study showed that the canonical OST activity was dispensable for DENV replication, suggesting that the OST complexes serve as scaffolds for DENV replication. However, our work demonstrates that one function of the OST complex during DENV infection is to provide oxidoreductase activity via the OST subunit MAGT1. We also show that MAGT1 associates with DENV NS1 and NS4B during viral infection, suggesting that these nonstructural proteins may be targets of MAGT1 oxidoreductase activity. These results provide insight into the cell biology of DENV infection, which may guide the development of antivirals against DENV.
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Leveraging the Cancer Stem Cell Glycome to Identify Aggressive Tumor Populations in Breast CancerIntratumor heterogeneity poses a significant challenge for the diagnosis and treatment of patients with breast cancer because distinct sub-populations of tumor cells contribute significantly more to therapy resistance and tumor recurrence than others. Consequently, understanding the mechanisms that contribute to this heterogeneity and identifying sub-populations responsible for aggressive behavior is a significant and timely problem. Considerable evidence indicates that a subpopulation of tumor cells with stem/progenitor-like characteristics, termed cancer stem cells (CSCs), is responsible for therapy resistance and recurrence, sparking interest in characterizing novel biomarkers and therapeutic targets for this aggressive population of cells. Unfortunately, CSCs share many protein markers with normal mammary stem/progenitor populations, minimizing potential targets for diagnostic and therapeutic purposes. Therefore, in my thesis research, I investigated novel ways to identify CSC populations based on their glycome. I observed that breast CSCs have a unique glycosylation pattern that can be used to distinguish them from other tumor populations. Specifically, I discovered a novel α2,3 sialoglycan on Core2 O-linked glycans expressed on CSCs that can identified using the lectin SLBR-N. I found that SLBR-N can be used to distinguish CSCs from bulk tumor cells in multiple in vitro and in vivo models. I also discovered that the CSC marker, CD44s, expresses O-linked α2,3 sialoglycan and that this glycan alters CD44s function by promoting the activation of the PDGFRβ/STAT3 pathway. In contrast, the fucosyltransferase FUT3 and its glycan sialyl Lewis X (sLeX) are expressed on non-CSCs and they function to impede stemness by inhibiting CD44s-mediated PDGFRβ/STAT3 signaling. In summary, this thesis provides insights into glycan heterogeneity in breast cancer and novel ways to identify CSCs using the glycome.
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Mutations in STT3A and STT3B cause two congenital disorders of glycosylationWe describe two unreported types of congenital disorders of glycosylation (CDG) which are caused by mutations in different isoforms of the catalytic subunit of the oligosaccharyltransferase (OST). Each isoform is encoded by a different gene (STT3A or STT3B), resides in a different OST complex and has distinct donor and acceptor substrate specificities with partially overlapping functions in N-glycosylation. The two cases from unrelated consanguineous families both show neurologic abnormalities, hypotonia, intellectual disability, failure to thrive and feeding problems. A homozygous mutation (c.1877T > C) in STT3A causes a p.Val626Ala change and a homozygous intronic mutation (c.1539 + 20G > T) in STT3B causes the other disorder. Both mutations impair glycosylation of a GFP biomarker and are rescued with the corresponding cDNA. Glycosylation of STT3A- and STT3B-specific acceptors is decreased in fibroblasts carrying the corresponding mutated gene and expression of the STT3A (p.Val626Ala) allele in STT3A-deficient HeLa cells does not rescue glycosylation. No additional cases were found in our collection or in reviewing various databases. The STT3A mutation significantly impairs glycosylation of the biomarker transferrin, but the STT3B mutation only slightly affects its glycosylation. Additional cases of STT3B-CDG may be missed by transferrin analysis and will require exome or genome sequencing.
