Plasma Membrane Glycoproteins Research Articles

Development and validation of the chromatography-mass spectrometry technique for polyprenols quantitative assessment

Introduction. Polyprenols are known as a class of natural long-chain isoprenoid alcohols, which are natural bioregulators that directly participate in the synthesis of cell membrane glycoproteins. Their hepatoprotective activity is proven, as well other types of their pharmacological effects are known, which is the reason of significant interest in these substances as a promising medicinal product. It is non-trivial task to determine the sum of polyprenols in extracts as include design and implementation of accurate reproducible analytical methods, which will subsequently be used in standardization.Aim. Development and validation of the chromatographic-mass spectrometric technique for polyprenols identification and their quantitative assessment.Materials and methods. Chromatographic separation of polyprenols was performed by using an HPLC Agilent 1260 Infinity II (Agilent Technologies, США); with the mixture of methanol, n-hexane, propanol-2, and aqueous ammonium acetate solution as eluent in gradient mode. An AB Sciex QTrap® 3200MD (AB Sciex Pte. Ltd., Singapore) triple quadrupole mass spectrometer was used as a detector, with the registration of polyprenols adducts.Results and discussion. The conditions for chromatographic separation and detection of polyprenols were identified. The developed technique was validated for the following characteristics: specificity, limit of detection, limit of quantification, linearity, accuracy, precision, range of application, and stability.Conclusion. It was determined the content of polyprenols in the substance recieved from Ginkgo biloba L. and Picea abies L. The developed technique can be used in the future to assess the content of polyprenols in drug products or pharmaceutical substances.

Abstract 1884: DXC006, Anti-CD56-CPT113 ADC displays favorable anti-tumor efficacy, pharmacokinetic and safety profiles in preclinical studies

Abstract CD56, also known as neural cell adhesion molecule 1 (NCAM1), is a type I plasma membrane glycoprotein involved in cell-cell and cell-matrix adhesion. It is overexpressed in almost all neuroblastomas, 95% of small cell lung cancers and 78% of multiple myeloma patients[1], whereas CD56 is less frequently or less abundantly expressed in normal tissues, making it a potential target for tumor therapy. So far, no CD56-targeting ADCs have entered late-stage clinical trials.DXC006 is an antibody-drug conjugate of a humanized anti-CD56 antibody (DXA006) linked to the topoisomerase I inhibitor CPT113 through a cleavable peptidyl linker and CROSSCONJU™ technology. The pharmacologic activity and mechanism of action of DXC006 were investigated in several human cancer cell lines and xenograft mouse models. Pharmacokinetic and safety profiles were also assessed in rats and cynomolgus monkeys.DXC006 binds specifically to CD56 and internalizes into tumor cells, after intracellular trafficking to lysosome and CPT113 releasing, induces DNA damage. In vitro cell based assays showed that DXC006 remarkably inhibited the cell growth of CD56-positive cell lines of IMR-32 (neuroblastoma), NCI-H526 (small cell lung cancer), and NCI-H929 (multiple myeloma), with IC50 of 0.0046~0.061 nmol/L. Although DXC006 also binds to CD56 expressed NK cells, we found that the inhibition of DXC006 to human NK cells was much less effective, with IC50 higher than 675 nmol/L, which is ten thousand-folds less potent than that of targeted abnormal cells. In vivo, DXC006 demonstrated good durable antitumor activities in three CD56-positive xenograft models of IMR-32, NCI-H526 and NCI-H929 cell lines, with a single dose of 2 mg/Kg, 3 mg/Kg, and 5 mg/Kg, respectively. No animal death or moribund outcome was observed in two months during preclinical safety studies in 150 mg/Kg Q2W dosed rats. For cynomolgus monkeys, the highest non-severely toxic dose (HNSTD) of DXC006 was 20 mg/Kg, in Q2W, four times of administration. PK profiles of both DXC006 the naked antibody DXA006 were comparable in both rats and monkeys. In summary, DXC006 demonstrated strong antitumor activity in various types of cancer cells both in vitro and in vivo, and good pharmacokinetic and safety profiles in rat and monkeys, a potential to be explored in clinical applications. [1] Yang Feng, Yanping Wang, Zhongyu Zhu, et al. Differential killing of CD56-expressing cells by drug-conjugated human antibodies targeting membrane-distal and membrane-proximal nonoverlapping epitopes. mAbs 2016; 799-810. Citation Format: Xiangfei Kong, Huihui Guo, Yong Du, Zhicang Ye, Yongxiang Chen, Zhixiang Guo, Lingli Zhang, Lu Bai, Junxiang Jia, Yunxia Zheng, Wei Zheng, Jun Zheng, Wenjun Li, Yuanyuan Huang, Zhongliang Fan, Mengmeng Liu, Binbin Chen, Meng Dai, Juan Wang, Fang Du, Miaomiao Chen, Qingliang Yang, Robert Y. Zhao. DXC006, Anti-CD56-CPT113 ADC displays favorable anti-tumor efficacy, pharmacokinetic and safety profiles in preclinical studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1884.

Abstract 5436: A therapeutically targetable dysadherin/YAP axis promotes cancer stem features and tumor aggressiveness in hepatocellular carcinoma

Abstract Hepatocellular carcinoma (HCC) remains one of the most challenging tumors to treat. Cancer stem-like cells (CSCs) are believed to be important in HCC development, immune evasion, and treatment resistance. Dysadherin is a cell membrane glycoprotein linked to cancer progression by endowing HCC with CSC features. However, the underlying mechanism of increasing CSC characteristics in refractory advanced HCC is not entirely understood. A comprehensive bioinformatics study revealed that dysadherin-expressing HCC cells were associated with cancer stemness and the YAP signaling activation. Dysadherin knockdown or overexpression in liver cancer cell lines attenuated or increased cancer stemness and aggressiveness via YAP signaling, respectively. Correspondingly, dysadherin deficiency lowered cancer stemness and aggressiveness in the HCC mouse model by blocking the YAP pathway. Mechanistically, dysadherin promoted YAP nuclear localization, which activated stem cell transcription factors such as OCT4, KLF4, and MYC. Of note, our comprehensive gain and loss of function studies in CSCs demonstrated that targeting the dysadherin/YAP axis could boost sorafenib’s therapeutic effect and suppress PD-L1 expression, which helps tumor cells escape from immune surveillance. Thus, our results highlight that the dysadherin/YAP axis drives the aggressive phenotype of HCC, providing a potential therapeutic strategy for HCC. Citation Format: So-El Jeon, Tae-Young Jang, Choong-Jae Lee, Hyeon-Ji Yun, Yeong-Hoon Cho, Da-Ye Lim, Jeong-Seok Nam. A therapeutically targetable dysadherin/YAP axis promotes cancer stem features and tumor aggressiveness in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5436.

Physiological Function of Oligosaccharides in Teleost Blood

Oligosaccharides in human blood are involved in various blood group antigens. Sialo-oligosaccharide-rich glycoproteins (GPs) in red blood cell (RBC) membranes also carry M, N, Ss and other blood group antigens. However, teleost blood is not expressed as a blood group antigen. Our study showed that carp, yellow tail and red sea bream GPs exhibit broad-spectrum antibiotic activity. This bacteriostatic activity is caused by the sialo-oligosaccharides from GPs. Based on NMR and GC–MS results, the structure of the bacteriostatic sialo-oligosaccharide from carp GP was determined to be NeuGcα2→6(Fucα1→4) (Glcα1→3) Galβ1→4GalNAc-ol. Compared to carp pentose, the structure of human GP O-linked tetra- or tri-oligosaccharides is simpler. In addition, NeuAc is simpler in human GPs than in carp NeuGc. In contrast to human blood, teleost blood does not contain IgG, and other antibodies occur at low levels. Teleost GPs may act as substitutes for antibodies, such as IgG, in the immune system. In this review, the transformation of the physiological function of oligosaccharides with respect to their structure is described.

Generally applicable circulating tumor cell enrichment and identification through a membrane glycoprotein-targeting strategy combining magnetic isolation and biological orthogonality labeling.

Although the importance of circulating tumor cells (CTCs) has been widely recognized, it is still a challenge to realize high-efficiency and accurate enrichment and identification of highly heterogeneous CTCs derived from various types of tumors in complex cancer processes. Currently, the most widely used methods follow the general idea of sequential immunoaffinitive capture and immunostaining to achieve the abovementioned goal. However, different organ/tissue origins as well as the inherent heterogeneity of CTCs would lead to the missed detection of certain CTC subtypes using such methods. Further, immunocytochemistry (ICC) immunostaining disrupts the physiological structure of cells, severely limiting the detection and application scenarios that require the participation of live cells. To address these limitations, we have developed a generally applicable strategy for the isolation and labeling of CTCs. This strategy focuses on targeting the universal characteristics of all tumor cells, specifically the abnormally expressed cell membrane glycoproteins, such as the transferrin receptor and sialic acid. Strategically, transferrin-functionalized magnetic beads (TMBs) were applied to enrich CTCs, and azide-based bioorthogonal chemistry was employed to label target CTCs. Accordingly, the membrane glycoprotein-targeting strategy achieved unbiased enrichment and labeling of broad-spectrum CTCs that were both epithelial and non-epithelial phenotypic populations with varied organ/tissue origins (MCF-7, HepG2, A549, Jurkat, and B16), with a capture efficiency of >95% and a detection limit as low as 5 cells per mL in artificial blood. In particular, our developed strategy displayed excellent specificity, and the CTCs under capture and fluorescence labelling remained with good viability and could be further cultivated and analyzed. Finally, the membrane glycoprotein-targeting strategy successfully detected and identified 33-223 CTCs from 1 mL patient blood samples.

Can CD133 Be Regarded as a Prognostic Biomarker in Oncology: Pros and Cons.

The CD133 cell membrane glycoprotein, also termed prominin-1, is expressed on some of the tumor cells of both solid and blood malignancies. The CD133-positive tumor cells were shown to exhibit higher proliferative activity, greater chemo- and radioresistance, and enhanced tumorigenicity compared to their CD133-negative counterparts. For this reason, CD133 is regarded as a potential prognostic biomarker in oncology. The CD133-positive cells are related to the cancer stem cell subpopulation in many types of cancer. Recent studies demonstrated the involvement of CD133 in the regulation of proliferation, autophagy, and apoptosis in cancer cells. There is also evidence of its participation in the epithelial-mesenchymal transition associated with tumor progression. For a number of malignant tumor types, high CD133 expression is associated with poor prognosis, and the prognostic significance of CD133 has been confirmed in a number of meta-analyses. However, some published papers suggest that CD133 has no prognostic significance or even demonstrate a certain correlation between high CD133 levels and a positive prognosis. This review summarizes and discusses the existing evidence for and against the prognostic significance of CD133 in cancer. We also consider possible reasons for conflicting findings from the studies of the clinical significance of CD133.

The Antipsychotic Drug Penfluridol Inhibits N-Linked Glycoprotein Processing and Enhances T-cell-Mediated Tumor Immunity.

Aberrant N-linked glycosylation is a prominent feature of cancers. Perturbance of oligosaccharide structure on cell surfaces directly affects key processes in tumor development and progression. In spite of the critical role played by N-linked glycans in tumor biology, the discovery of small molecules that specifically disturbs the N-linked glycans is still under investigation. To identify more saccharide-structure-perturbing compounds, a repurposed drug screen by using a library consisting of 1530 FDA-approved drugs was performed. Interestingly, an antipsychotic drug, penfluridol, was identified as being able to decrease cell surface wheat germ agglutinin staining. In the presence of penfluridol, cell membrane glycoproteins programmed death-ligand 1 (PD-L1) shifted to a lower molecular weight. Further studies demonstrated that penfluridol treatment caused an accumulation of high-mannose oligosaccharides, especially Man5-7GlcNAc2 glycan structures. Mechanistically, this effect is due to direct targeting of MAN1A1 mannosidase, a Golgi enzyme involved in N-glycan maturation. Moreover, we found that altered glycosylation of PD-L1 caused by penfluridol disrupted interactions between programmed cell death protein 1 and PD-L1, resulting in activation of T-cell tumor immunity. In a mouse xenograft and glioma model, penfluridol enhanced the antitumor effect of the anti-PD-L1 antibody in vivo. Overall, these findings revealed an important biological activity of the antipsychotic drug penfluridol as an inhibitor of glycan processing and proposed a repurposed use of penfluridol in antitumor therapy through activation of T-cell immunity.

Antibody binding reports spatial heterogeneities in cell membrane organization

The spatial organization of cell membrane glycoproteins and glycolipids is critical for mediating the binding of ligands, receptors, and macromolecules on the plasma membrane. However, we currently do not have the methods to quantify the spatial heterogeneities of macromolecular crowding on live cell surfaces. In this work, we combine experiment and simulation to report crowding heterogeneities on reconstituted membranes and live cell membranes with nanometer spatial resolution. By quantifying the effective binding affinity of IgG monoclonal antibodies to engineered antigen sensors, we discover sharp gradients in crowding within a few nanometers of the crowded membrane surface. Our measurements on human cancer cells support the hypothesis that raft-like membrane domains exclude bulky membrane proteins and glycoproteins. Our facile and high-throughput method to quantify spatial crowding heterogeneities on live cell membranes may facilitate monoclonal antibody design and provide a mechanistic understanding of plasma membrane biophysical organization.

Measurements of Na+-occluded intermediates during the catalytic cycle of the Na+/K+-ATPase provide novel insights into the mechanism of Na+ transport

The Na+/K+-ATPase is an integral plasma membrane glycoprotein of all animal cells that couples the exchange of intracellular Na+ for extracellular K+ to the hydrolysis of ATP. The asymmetric distribution of Na+ and K+ is essential for cellular life and constitutes the physical basis of a series of fundamental biological phenomena. The pumping mechanism is explained by the Albers-Post model. It involves the presence of gates alternatively exposing Na+/K+-ATPase transport sites to the intracellular and extracellular sides and includes occluded states in which both gates are simultaneously closed. Unlike for K+, information is lacking about Na+-occluded intermediates, as occluded Na+ was only detected in states incapable of performing a catalytic cycle, including two Na+-containing crystallographic structures. The current knowledge is that intracellular Na+ must bind to the transport sites and become occluded upon phosphorylation by ATP to be transported to the extracellular medium. Here, taking advantage of epigallocatechin-3-gallate to instantaneously stabilize native Na+-occluded intermediates, we isolated species with tightly bound Na+ in an enzyme able to perform a catalytic cycle, consistent with a genuine occluded state. We found that Na+ becomes spontaneously occluded in the E1 dephosphorylated form of the Na+/K+-ATPase, exhibiting positive interactions between binding sites. In fact, the addition of ATP does not produce an increase in Na+ occlusion as it would have been expected; on the contrary, occluded Na+ transiently decreases, whereas ATP lasts. These results reveal new properties of E1 intermediates of the Albers-Post model for explaining the Na+ transport pathway.

Antiviral Activity of Syzygium polyanthum Extract against Herpes Simplex Virus-Type 1 (HSV-1)

Herpes simplex virus type 1 (HSV-1) is part of the alpha subfamily of human herpesviruses. Several antiviral medications can be prescribed for treating HSV-1 which is usually first line antiviral agents such as acyclovir (ACV). But there are several cases that have been reported on ACV resistance to HSV, thus, researchers tend to develop the antiviral agent from plants extract. Syzygium polyanthum is one of the plants that has been traditionally used as a drug which is rich in bioactive compounds, including essential oils, terpenoids, tannins, and flavonoids [1]. These phytochemical compounds, which have strong antioxidant action, can aid in the inhibition of viral genome replication which disables the viral lipid envelope [2].
  
 The objective of this study is to determine the cytotoxic concentration (CC50) and antiviral activity of S. polyanthum methanolic extract. Cytotoxicity was performed using the method described by [3]. The absorbance readings were analysed using microplate reader (Infinite M200 Pro). The 50% cytotoxicity concentration (CC50) was defined as the sample concentration that able to reduce 50% of cell viability compared to the untreated cells. Antiviral activity was performed by using plaque reduction assay with three different treatments [3] which are post- treatment, pre-treatment and virucidal assay.
  
 Based on Figure 1, cytotoxicity screening against Vero cells using MTT assay showed that the CC50 values for extract was 0.135 mg/mL which considered not in the range of cytotoxic compounds. Any CC50 or IC50 of a substance less than 4 µg/mL was considered an active cytotoxic effect [4].
 Based on Figure 2, post treatment assay showed the most anti-HSV-1 activity of S. polyanthum extract which the EC50 and SI value were 0.015mg/mL and 9.03, respectively. The extract showed to be slightly effective in reducing HSV-1 replication after 2-hour incubation which might be due to the inhibition in the early phase of the attachment. For pre-treatment, EC50 and SI values were 0.019 mg/mL and 7.12, which the extract showed to be moderately effective in inhibiting the attachment of HSV-1 before the replication can occur. This might occur as Vero cells bind to the extract that could interfere with the cell membrane's glycoprotein receptors which prevented HSV-1 from attaching to the cell surface [5]. The EC50 and SI value for virucidal assay was 0.018 mg/mL and 7.28 as the result showed moderately effective inhibited the viral replication. S. polyanthum has been shown to directly inactivate HSV-1 virions within 30 minutes of exposure in this study. An SI value greater than 10 (SI>10) indicates that any antimicrobial compound has the potency to become an antiviral treatment agent [6].
 In conclusion, the findings of this study demonstrate that S. polyanthum extract seems to have moderate antiviral  effects and non-toxic to Vero cells.

CT-167 Using the Sodium Iodide Symporter to Visualize CAR-T Cell Trafficking

Chimeric antigen receptor T (CAR-T) cell therapy revolutionized the treatment of refractory B cell malignancies, but relapses occur through a variety of incompletely understood mechanisms. Currently, tracking CAR-T expansion and persistence in patients is limited to peripheral blood sampling, but this approach provides no insights into how and where CAR-T cells traffic beyond the peripheral blood or how cellular localization may impact persistence and efficacy. Furthermore, costimulatory endodomains that are known to influence CAR-T cell phenotype have unclear downstream effects on how CAR-T cells traffic. The sodium iodide symporter (NIS) is a plasma membrane glycoprotein that actively uptakes iodide and its analogs, and it can be transduced into various cell types in preclinical models allowing sensitive and specific PET/SPECT imaging. Coexpression of NIS with CAR on T cells would allow real-time, in vivo imaging of CAR-T cells and aid in understanding CAR-T cell biology and trafficking. Using Golden Gate modular vector construction, multiple anti-CD20 CAR constructs with specificity for both human and non-human primate (NHP) CD20 antigens were generated. Each construct included one of several costimulatory domains (4-1BB, CD28, or CD27), a standard CD3-zeta signaling domain, and either the rhesus-NIS reporter or truncated EGFR following a short ribosomal skip sequence peptide, allowing transduction under control of the same EF1a promoter. CAR was effectively cotransduced with NIS in both human and NHP primary T cells with no effect of NIS on CAR-T cell-mediated cytotoxicity against CD20-expressing Raji cells in vitro compared to control. Cytotoxicity was antigen-specific as no difference was seen against CD20- control cells. Functional iodide and [18F]-TFB, an iodide analog, uptake via NIS was specific to NIS-transduced cells and was appropriately inhibited by coincubation with KClO4. An NSG mouse xenograft model will be used to track the localization, expansion, efficacy, and persistence of CAR-T constructs against Raji lymphoma cells. Different costimulatory domains appear to elicit only slight differences in cytokine production in vitro but will be further studied for differences in efficacy and trafficking in vivo in mice and later in a syngeneic NHP model.

The Significance of Cell Surface N-Glycosylation for Internalization and Potency of Cytotoxic Conjugates Targeting Receptor Tyrosine Kinases.

Precise anticancer therapies employing cytotoxic conjugates constitute a side-effect-limited, highly attractive alternative to commonly used cancer treatment modalities, such as conventional chemotherapy, radiotherapy or surgical interventions. Receptor tyrosine kinases are a large family of N-glycoproteins intensively studied as molecular targets for cytotoxic conjugates in various cancers. At the cell surface, these receptors are embedded in a dense carbohydrate layer formed by numerous plasma membrane glycoproteins. The complexity of the cell surface architecture is further increased by galectins, secreted lectins capable of recognizing and clustering glycoconjugates, affecting their motility and activity. Cell surface N-glycosylation is intensively remodeled by cancer cells; however, the contribution of this phenomenon to the efficiency of treatment with cytotoxic conjugates is largely unknown. Here, we evaluated the significance of N-glycosylation for the internalization and toxicity of conjugates targeting two model receptor tyrosine kinases strongly implicated in cancer: HER2 and FGFR1. We employed three conjugates of distinct molecular architecture and specificity: AffibodyHER2-vcMMAE (targeting HER2), vcMMAE-KCK-FGF1.E and T-Fc-vcMMAE (recognizing different epitopes within FGFR1). We demonstrated that inhibition of N-glycosylation reduced the cellular uptake of all conjugates tested and provided evidence for a role of the galectin network in conjugate internalization. In vitro binding studies revealed that the reduced uptake of conjugates is not due to impaired HER2 and FGFR1 binding. Importantly, we demonstrated that alteration of N-glycosylation can affect the cytotoxic potential of conjugates. Our data implicate a key role for cell surface N-glycosylation in the delivery of cytotoxic conjugates into cancer cells.

MO071: Hemolysis is Associated with Altered Heparan Sulfate of Endothelial Glycocalyx And with Local Complement Activation in Thrombotic Microangiopathies

Abstract BACKGROUND AND AIMS The complement system plays a key role in the pathophysiology of renal thrombotic microangiopathies (TMA), as illustrated by atypical hemolytic uremic syndrome (HUS) characterized by a high rate of mutations in complement proteins. However, complement abnormalities are not fully sufficient to drive to TMA lesions. Among other suspected pathophysiological actors, we hypothesize that the alteration of heparan sulfate (HS) of endothelial glycocalyx—a cell membrane glycoaminoglycans and glycoproteins matrix able of regulating complement by binding factor H—may play a significant role. METHOD We recorded clinical and histological features in native kidney biopsies from a monocentric retrospective cohort of 72 TMA-patients. Glomerular HS integrity and local complement activation markers (C3 and C5b9 staining) were analyzed by immunofluorescence (IF) and immunohistochemistry. Using an in vitro endothelial cells model (human umbilical vein endothelial cells, HUVECs), we studied the impact of heme exposure (30’, 37°C)—a major product of hemolysis—on endothelial HS integrity by IF and flow cytometry (FCM). Heme-induced HS disruption was studied by slot blot of HS core-protein Syndecan (SDC) 1 and 4 in cells supernatant. Artificial enzymatic degradation of HS was achieved after exposure of HUVECs to a combination of heparinases I, II and III (30', 37°C), and we studied its effect on the ability of the HS-free cell membrane to locally activate complement by analyzing C3b deposition by IF and FCM, and the release of complement anaphylatoxins C3a and soluble attack complex membrane s-C5b9 in the cell supernatants by ELISA. The HS remodeling process induced by heme exposure (16 h, 37°C) were indirectly analyzed via a transcriptional profiling of the expression of the key enzymes involved in HS synthesis by RT–qPCR. RESULTS Glomerular HS staining decreased in 57 TMA-patients (79%), moderately (2+) or strongly (0–1+) in 20 (28%) and 37 (51%) cases, respectively. Reduced HS density was significantly associated with hemolysis at the time of biopsy (P = 0.008) and local complement activation (C3d and/or C5b-9 deposits, P < 0.001). Hemolysis at the time of biopsy was associated with more severe glomerular TMA lesions (P = 0.025), the presence of vascular thrombi (P = 0.007) and local complement activation (C3d and/or C5b-9 deposits, P = 0.006). On endothelial cells (HUVECs), we confirmed that HS expression was decreased after heme exposure in IF and FCM (P < 0.01). Coherently, HS core-proteins SDC-1 and 4 were significantly increased in HUVECs supernatant (P < 0.05), suggesting a negative heme-driven impact on HS integrity. Artificial HS degradation increased C3b membrane deposition on HUVECs on average ∼ 3 fold by FCM and ∼ 10 by IF, and we observed an increased release of complement C3a and s-C5b9 in cells supernatant (P < 0.05). Finally, we showed that prolonged exposure to heme induced a decreased of genic expression of C5-Epimerase and an increase in 6-OST1 and 3-OST1 expression (P < 0.05), the very three crucial genes of HS remodeling specifically and sequentially involved in the generation of anti-thrombin pattern, the well-known key regulator of the HS/heparin-driven inhibition of coagulation. CONCLUSION This study highlights the impact of hemolysis on the integrity of endothelial glycocalyx HS, both in kidney biopsy of TMA-patients and in an in vitro endothelial cell model, and suggests that acute alteration of HS is a mechanism of heme-induced complement activation. The endothelial glycocalyx appears then to be an important key in the understanding of TMA and could lead to the development of therapeutics targeting its preservation and/or reconstitution, in order to better control the disease.

The Role of CD200-CD200 Receptor in Human Blood and Lymphatic Endothelial Cells in the Regulation of Skin Tissue Inflammation.

CD200 is a cell membrane glycoprotein that interacts with its structurally related receptor (CD200R) expressed on immune cells. We characterized CD200–CD200R interactions in human adult/juvenile (j/a) and fetal (f) skin and in in vivo prevascularized skin substitutes (vascDESS) prepared by co-culturing human dermal microvascular endothelial cells (HDMEC), containing both blood (BEC) and lymphatic (LEC) EC. We detected the highest expression of CD200 on lymphatic capillaries in j/a and f skin as well as in vascDESS in vivo, whereas it was only weakly expressed on blood capillaries. Notably, the highest CD200 levels were detected on LEC with enhanced Podoplanin expression, while reduced expression was observed on Podoplanin-low LEC. Further, qRT-PCR analysis revealed upregulated expression of some chemokines, including CC-chemokine ligand 21 (CCL21) in j/aCD200+ LEC, as compared to j/aCD200− LEC. The expression of CD200R was mainly detected on myeloid cells such as granulocytes, monocytes/macrophages, T cells in human peripheral blood, and human and rat skin. Functional immunoassays demonstrated specific binding of skin-derived CD200+ HDMEC to myeloid CD200R+ cells in vitro. Importantly, we confirmed enhanced CD200–CD200R interaction in vascDESS in vivo. We concluded that the CD200–CD200R axis plays a crucial role in regulating tissue inflammation during skin wound healing.

ОКИСЛИТЕЛЬНЫЙ СТРЕСС И АНТИОКСИДАНТНАЯ СИСТЕМА ПРИ НЕСПЕЦИФИЧЕСКОМ АОРТОАРТЕРИИТЕ I ТИПА

Objective: To study the features of lipid peroxidation (LPO) and the antioxidant system (AOS) in blood in Takayasu arteritis (NF) with a predominant lesion of brachiocephalic vessels. Methods: Several markers of LPO (malondialdehyde – MDA), AOS (superoxide dismutase – SOD, ascorbic acid – AA) and inflammation (sialic acid – SA) were studied in 16 patients with type I TA, where the inflammatory process was localized mainly in the aortic arch and its branches. Most commonly TA was observed among females (n=14; 87.5%), the average age of patients was 20.8±0.8 years. The study of the above markers was carried out in two stages: upon admission of patients in the stage of exacerbation of inflammation and after targeted anti-inflammatory, immunomodulatory and antioxidant therapy – in the stage of remission of the inflammatory process. Results: The study of LPO and AOS showed that in all patients, upon admission, there was a considerable oxidant-antioxidant imbalance due to an acute inflammatory process. MDA was increased by 2.6 times constituting 3.9±0.1 μmol/l. After pathogenetically targeted therapy, its significant decrease to 2.9±0.1 μmol/l (р˂0.001) was achieved. Activation of LPO was paralleled with a significant decrease of the body's AOS markers. The activity of SOD and concentration of AA during acute inflammatory process was reduced by 1.4 and 1.3 times, and amounted to 13.3±0.1 c.u./l and 57.8±0.3 μmol/l, respectively. After pulse therapy these indicators showed a significant increase compared with the initial data: up to 15.9±0.2 c.u./l and 63.9±0.4 μmol/l, respectively. The content of SA, which is one of the important components of cell membrane glycoproteins and glycolipids, was increased by 2.3 times (6.1±0.1 mmol/l), while after treatment it significantly decreased to 3.4±0.1 mmol/l (p˂0.001). Gender and age did not affect the dynamics of the specified markers, except for SA, which showed higher values in women (p<0.05). Conclusion: With TA in the stage of acute inflammation, increased content of MDA was found, indicating development of the oxidative stress. On this background the AOS indicators were decreased, which should be taken into account during preoperative preparation of patients for upcoming revascularization. The inclusion antioxidants in the protocol of standard TA therapy may improve the patients’ outcomes. Keywords: Takayasu arteritis, lipid peroxidation , antioxidant system, malondialdehyde, superoxide dismutase, ascorbic acid, sialic acid.

Expression and Impact of C1GalT1 in Cancer Development and Progression.

Simple SummaryC1GalT1 is one of the enzymes that catalyze the addition of sugar residues to proteins (protein glycosylation). It specifically controls the synthesis and formation of a special disaccharide structure Galβ1,3GalNAcα-, which occurs predominately in cancer but rarely in normal cells. Recent studies have shown that C1GalT1 is overexpressed in many common cancers including colon, breast, gastric, lung, head and neck, pancreatic, esophageal, prostate, and hepatocellular cancer. C1GalT1 overexpression is also often associated with poorer prognosis and poorer patient survival. This review summarizes our current understanding of the expression of C1GalT1 in various cancers and discusses the impact of C1GalT change on cancer cell activities in cancer development and progression.C1GalT1 (T-synthase) is one of the key glycosyltransferases in the biosynthesis of O-linked mucin-type glycans of glycoproteins. It controls the formation of Core-1 disaccharide Galβ1,3GalNAcα- (Thomsen–Friedenreich oncofetal antigen, T or TF antigen) and Core-1-associated carbohydrate structures. Recent studies have shown that C1GalT1 is overexpressed in many cancers of epithelial origin including colon, breast, gastric, head and neck, pancreatic, esophageal, prostate, and hepatocellular cancer. Overexpression of C1GalT1 is often seen to also be associated with poorer prognosis and poorer patient survival. Change of C1GalT1 expression causes glycosylation changes of many cell membrane glycoproteins including mucin proteins, growth factor receptors, adhesion molecules, and death receptors. This leads to alteration of the interactions of these cell surface molecules with their binding ligands, resulting in changes of cancer cell activity and behaviors. This review summarizes our current understanding of the expression of C1GalT1 in various cancers and discusses the impact of C1GalT change on cancer cell activities in cancer development and progression.

Plasma membrane N-glycoproteome analysis of wheat seedling leaves under drought stress

Protein glycosylation is one of the ubiquitous post-translational modifications in eukaryotic cells, which play important roles in plant growth and adverse response. In this study, we performed the first comprehensive wheat plasma membrane N-glycoproteome analysis under drought stress via glycopeptide HILIC enrichment and LC-MS/MS identification. In total, 414 glycosylated sites corresponding to 407 glycopeptides and 312 unique glycoproteins were identified, of which 173 plasma membrane glycoproteins with 215 N-glycosylation sites were significantly regulated by drought stress. Functional enrichment analysis reveals that the significantly regulated N-glycosylation proteins were particularly related to protein kinase activity involved in the reception and transduction of extracellular signal and plant cell wall remolding. The motifs and sequence structures analysis showed that the significantly regulated N-glycosylation sites were concentrated within [NxT] motif, and 79.5% of them were located on the random coil that is always on the protein surface and flexible regions, which could facilitate protein glycosylated modification and enhance protein structural stability via reducing protein flexibility. PNGase F enzyme digestion and glycosylation site mutation further indicated that N-glycosylated modification could increase protein stability. Therefore, N-glycosylated modification is involved in plant adaptation to drought stress by improving the stability of cell wall remodeling related plasma membrane proteins.

Oncogene-mediated metabolic gene signature predicts breast cancer outcome

Breast cancer remains the second most lethal cancer among women in the United States and triple-negative breast cancer is the most aggressive subtype with limited treatment options. Trop2, a cell membrane glycoprotein, is overexpressed in almost all epithelial cancers. In this study, we demonstrate that Trop2 is overexpressed in triple-negative breast cancer (TNBC), and downregulation of Trop2 delays TNBC cell and tumor growth supporting the oncogenic role of Trop2 in breast cancer. Through proteomic profiling, we discovered a metabolic signature comprised of TALDO1, GPI, LDHA, SHMT2, and ADK proteins that were downregulated in Trop2-depleted breast cancer tumors. The identified oncogene-mediated metabolic gene signature is significantly upregulated in TNBC patients across multiple RNA-expression clinical datasets. Our study further reveals that the metabolic gene signature reliably predicts poor survival of breast cancer patients with early stages of the disease. Taken together, our study identified a new five-gene metabolic signature as an accurate predictor of breast cancer outcome.

Global Diversification and Distribution of Coronaviruses With Furin Cleavage Sites.

Knowledge about coronaviruses (CoVs) with furin cleavage sites is extremely limited, although these sites mediate the hydrolysis of glycoproteins in plasma membranes required for MERS-CoV or SARS-CoV-2 to enter cells and infect humans. Thus, we have examined the global epidemiology and evolutionary history of SARS-CoV-2 and 248 other CoVs with 86 diversified furin cleavage sites that have been detected in 24 animal hosts in 28 countries since 1954. Besides MERS-CoV and SARS-CoV-2, two of five other CoVs known to infect humans (HCoV-OC43 and HCoV-HKU1) also have furin cleavage sites. In addition, human enteric coronavirus (HECV-4408) has a furin cleavage site and has been detected in humans (first in Germany in 1988), probably via spillover events from bovine sources. In conclusion, the presence of furin cleavage sites might explain the polytropic nature of SARS-CoV-2- and SARS-CoV-2-like CoVs, which would be helpful for ending the COVID-19 pandemic and preventing outbreaks of novel CoVs.

Abstract 2324: A novel oncogene mediated metabolic gene signature predicts breast cancer outcome

Abstract Introduction and Objectives: Breast cancer remains the second most lethal cancer among women in the US, with triple negative breast cancer (TNBC) as the most aggressive subtype with limited treatment options. Trop2, a cell membrane glycoprotein, is overexpressed nearly all epithelial cancers including triple negative breast cancer. In this study, we demonstrate that loss of Trop2 derives downregulation of metabolic genes. Trop2 oncogene-driven metabolic gene signature is highly elevated in breast cancer patients and predicts poor outcome. Methods: Trop2 expression levels in two independent patient cohorts were analyzed with immunohistochemistry. Trop2 was knockdown in TNBC cell line HCC1806 with shRNA and knock-out in HCC1806 with CRISPR Cas9 system. Trop2 downregulation or gene deletion effect was tested in vitro with colony formation and 3D Matrigel invasion assays. The effect of Trop2 downregulation in tumorigenesis was evaluated in vivo by subcutaneous implantation of control and Trop2 knockdown xenografts. Proteomic changes were analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS). Trop2 led to downregulation of TALDO1 associated metabolic cluster genes which expression levels were further investigated in four independent breast cancer cohort. Finally, the correlation of Trop2 oncogene-driven metabolic gene signature with breast cancer outcome was analyzed in 13 independent breast cancer patient datasets. Results: We demonstrate Trop2 is overexpressed in TNBC, and downregulation of Trop2 disrupts TNBC cell and tumor growth. Through whole proteomic profiling, we discovered a novel metabolic cluster that was downregulated in Trop2-depleted breast cancer tumors. The identified oncogene-driven metabolic gene signature is significantly upregulated in TNBC patients across multiple clinical datasets. Our study further reveals that the metabolic gene signature reliably predicts poor survival of breast cancer patients with early stage of the disease. Conclusion: Our study identifies a new five gene metabolic oncogene-driven signature as a prognostic predictor of breast cancer. Citation Format: Merve Aslan, En-Chi Hsu, Fernando J. Garcia Marques, Abel Bermudez, Michelle Shen, Meghan A. Rice, Shiqin Liu, Robert West, Sharon J. Pitteri, Balázs Győrffy, Tanya Stoyanova. A novel oncogene mediated metabolic gene signature predicts breast cancer outcome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2324.