Glycan Structures Contain Information for the Spatial Arrangement of Glycoproteins in the Plasma Membrane

Glycoconjugates at the cell surface are crucial for cells to communicate with each other and the extracellular microenvironment. While it is generally accepted that glycans are vectorial biopolymers, their information content is unclear. This report provides evidence that distinct N-glycan structures influence the spatial arrangement of two integral membrane glycoproteins, Kv3.1 and E-cadherin, at the adherent membrane which in turn alter cellular properties. Distinct N-glycan structures were generated by heterologous expression of these glycoproteins in parental and glycosylation mutant Chinese hamster ovary cell lines. Unlike the N-linked glycans, the O-linked glycans of the mutant cell lines are similar to those of the parental cell line. Western and lectin blots of total membranes and GFP immunopurified samples, combined with glycosidase digestion reactions, were employed to verify the glycoproteins had predominantly complex, oligomannose, and bisecting type N-glycans from Pro(-)5, Lec1, and Lec10B cell lines, respectively. Based on total internal reflection fluorescence and differential interference contrast microscopy techniques, and cellular assays of live parental and glycosylation mutant CHO cells, we propose that glycoproteins with complex, oligomannose or bisecting type N-glycans relay information for localization of glycoproteins to various regions of the plasma membrane in both a glycan-specific and protein-specific manner, and furthermore cell-cell interactions are required for deciphering much of this information. These distinct spatial arrangements also impact cell adhesion and migration. Our findings provide direct evidence that N-glycan structures of glycoproteins contribute significantly to the information content of cells

Suppression of cancer progression by MGAT1 shRNA knockdown.

Oncogenic signaling promotes tumor invasion and metastasis, in part, by increasing the expression of tri- and tetra- branched N-glycans. The branched N-glycans bind to galectins forming a multivalent lattice that enhances cell surface residency of growth factor receptors, and focal adhesion turnover. N-acetylglucosaminyltransferase I (MGAT1), the first branching enzyme in the pathway, is required for the addition of all subsequent branches. Here we have introduced MGAT1 shRNA into human HeLa cervical and PC-3-Yellow prostate tumor cells lines, generating cell lines with reduced transcript, enzyme activity and branched N-glycans at the cell surface. MGAT1 knockdown inhibited HeLa cell migration and invasion, but did not alter cell proliferation rates. Swainsonine, an inhibitor of α-mannosidase II immediately downstream of MGAT1, also inhibited cell invasion and was not additive with MGAT1 shRNA, consistent with a common mechanism of action. Focal adhesion and microfilament organization in MGAT1 knockdown cells also indicate a less motile phenotype. In vivo, MGAT1 knockdown in the PC-3-Yellow orthotopic prostate cancer xenograft model significantly decreased primary tumor growth and the incidence of lung metastases. Our results demonstrate that blocking MGAT1 is a potential target for anti-cancer therapy

Identification of a small molecule inhibitor of 3-phosphoglycerate dehydrogenase to target serine biosynthesis in cancers

Cancer cells reprogram their metabolism to promote growth and proliferation. The genetic evidence pointing to the importance of the amino acid serine in tumorigenesis is striking. The gene encoding the enzyme 3-phosphoglycerate dehydrogenase (PHGDH), which catalyzes the first committed step of serine biosynthesis, is overexpressed in tumors and cancer cell lines via focal amplification and nuclear factor erythroid-2-related factor 2 (NRF2)-mediated up-regulation. PHGDH-overexpressing cells are exquisitely sensitive to genetic ablation of the pathway. Here, we report the discovery of a selective small molecule inhibitor of PHGDH, CBR-5884, identified by screening a library of 800,000 drug-like compounds. CBR-5884 inhibited de novo serine synthesis in cancer cells and was selectively toxic to cancer cell lines with high serine biosynthetic activity. Biochemical characterization of the inhibitor revealed that it was a noncompetitive inhibitor that showed a time-dependent onset of inhibition and disrupted the oligomerization state of PHGDH. The identification of a small molecule inhibitor of PHGDH not only enables thorough preclinical evaluation of PHGDH as a target in cancers, but also provides a tool with which to study serine metabolism.status: publishe

shRNA MGAT1 suppresses N-glycan branching.

<p><b>A</b>) HeLa cells were infected with lentiviral vectors targeting MGAT1(shRNA1 or shRNA2) or the control shRNA sequences. Stable cell populations were selected by the addition of puromycin (1 µg/mL). <b>A</b>)MGAT1 mRNA were measured by qRT-PCR, <b>B</b>) MGAT1 enzyme activity, <b>C</b>) L-PHA reactive surface N-glycans by Array scan microscope, <b>D</b>) Proliferation over 4 days Data represent the mean ± SD relative expression of mRNA relative to control sequence (n = 3 independent experiments performed in triplicate).</p

MGAT1 knockdown decreases tumor migration, and invasion in prostate cancer cells.

<p>PC-3-Yellow cells with MGAT1-shRNA2 or the control shRNA sequences were assessed for <b>A</b>) MGAT1 mRNA levels by qRT-PCR. <b>B</b>)MGAT1 enzyme activity, <b>C</b>) L-PHA reactive surface N-glycans by Arrayscan microscope, <b>D</b>) invasion through a Matrigel barrier and migration (<b>E</b>) using the xCELLigence Real-Time Cell Analyzer. Data represents mean ±SD cell index (3 independent experiments with quadruplicates). (<b>F</b>) Tumor size and (<b>G</b>) metastasis in mice injected with PC-3-Yellow cells with MGAT1-shRNA2 or the control shRNA sequences. The mice were injected orthotopically, with 0.5×10⁶ cell per mouse, into the prostate of the sub-lethally irradiated SCID mice. Four weeks after injection, mice were sacrificed and their organs were imaged using a fluorescent microscope. The numbers of metastatic nodules in all five lobes of the lungs were quantified using image analysis software. Each point represents one mouse.</p

MGAT1 shRNA inhibits HeLa cell migration and invasion <i>in vitro</i>.

<p><b>A</b>) HeLa cells were plated into chambers with 8-µm pores and 10% FBS was used as a chemoattractant. <b>B</b>) HeLa cells were plated into invasion chambers with Matrigel. After 48 h, cells that had migrated through the pores were fixed, stained and counted automatically. C) migration and D) invasion assays with or without pretreatment for 72 h with 2 µM swainsonine (SW) for 72 hours prior. The mean number of migrated cells ± SD of 3 independent experiments performed in triplicate were graphed. (<b>E</b>) Cell morphology by staining with phospho-paxillin and TRITC-conjugated phalloidin for F-actin, shown as a merged image.</p

A Large Retrospective Study of Epidemiological Characteristics of COVID-19 Patients in the North of Iran: Association between SARS-CoV-2 RT-PCR Ct Values with Demographic Data

Objectives: To avoid worsening from mild, moderate, and severe diseases and to reduce mortality, it is necessary to identify the subpopulation that is more vulnerable to the development of COVID-19 unfavorable consequences. This study aims to investigate the demographic information, prevalence rates of common comorbidities among negative and positive real-time reverse-transcriptase polymerase chain reaction (rRT-PCR) patients, and the association between SARS-CoV-2 cycle threshold (Ct) at hospital admission, demographic data, and outcomes of the patients in a large population in Northern Iran. Methods: This large retrospective cross-sectional study was performed from 7 March to 20 December 2020. Demographic data, including gender, age, underlying diseases, clinical outcomes, and Ct values, were obtained from 8,318 cases suspected of COVID-19, who were admitted to four teaching hospitals affiliated to Babol University of Medical Sciences (MUBABOL), in the north of Iran. Results: Since 7 March 2020, the data were collected from 8,318 cases suspected of COVID-19 (48.5 female and 51.5 male) with a mean age of 53�±�25.3 years. Among 8,318 suspected COVID-19 patients, 3,250 (39.1) had a positive rRT-PCR result; 1,632 (50.2) patients were male and 335 (10.3) patients died during their hospital stay. The distribution of positive rRT-PCR revealed that most patients (464 (75.7)) had a Ct between 21 and 30 (Group B). Conclusion: Elderly patients, lower Ct, patients having at least one comorbidity, and male cases were significantly associated with increased risk for COVID-19-related mortality. Moreover, mortality was significantly higher in patients with diabetes, kidney disease, and respiratory disease. Copyright © 2022 Farzin Sadeghi et al