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Molecular Plant Jun 2024Steroidal glycoalkaloids (SGAs) are specialized metabolites produced by hundreds of Solanum species, including important vegetable crops such as tomato, potato and...
Steroidal glycoalkaloids (SGAs) are specialized metabolites produced by hundreds of Solanum species, including important vegetable crops such as tomato, potato and eggplant. Though SGAs are better known for their role in defence in plants and 'anti-nutritional' effects (e.g., toxicity and bitterness) to humans, many of these molecules have documented anti-cancer, anti-microbial, anti-inflammatory, anti-viral and anti-pyretic activities. Among these, α-solasonine and α-solamargine isolated from black nightshade (Solanum nigrum), are reported to have potent anti-tumor, anti-proliferative and anti-inflammatory activities. Notably, α-solasonine and α-solamargine, along with the core steroidal aglycone solasodine are the most widespread SGAs produced among the Solanum plants. However, it is still unknown how plants synthesize these bioactive steroidal molecules. Through comparative metabolomic-transcriptome guided approach, biosynthetic logic, combinatorial expression in Nicotiana benthamiana and functional recombinant enzyme assays, here we report the discovery of 12 enzymes from S. nigrum that converts the staring cholesterol precursor to solasodine aglycone, and the downstream α-solasonine, α-solamargine and malonyl-solamargine SGA products. We further identified 6 enzymes from cultivated eggplant that catalyse the production of α-solasonine, α-solamargine and malonyl-solamargine SGAs from solasodine aglycone, via glycosylation and atypical malonylation decorations. Our work provides the gene tool box and platform for engineering the production of high value, steroidal bioactive molecules in heterologous hosts using synthetic biology.
PubMed: 38937971
DOI: 10.1016/j.molp.2024.06.013 -
Surgery Today Jun 2024Hepatocellular carcinoma (HCC) frequently recurs after radical resection, resulting in a poor prognosis. This study assessed the prognostic value of Mac-2 binding...
PURPOSE
Hepatocellular carcinoma (HCC) frequently recurs after radical resection, resulting in a poor prognosis. This study assessed the prognostic value of Mac-2 binding protein glycosylation isomer (M2BPGi) for early recurrence (ER) in patients with HCC.
METHODS
Patients who underwent radical resection for HCC between 2015 and 2021. HCC recurrence within one year after curative resection was defined as ER.
RESULTS
The 150 patients were divided into two groups: non-ER (116, 77.3%) and ER (34, 22.7%). The ER group had a lower overall survival rate (p < 0.0001) and significantly higher levels of M2BPGi (1.06 vs. 2.74 COI, p < 0.0001) than the non-ER group. High M2BPGi levels (odds ratio [OR] 1.78, 95% confidence interval [CI] 1.31-2.41, p < 0.0001) and a large tumor size (OR 1.31, 95% CI, 1.05-1.63; p = 0.0184) were identified as independent predictors of ER. M2BPGi was the best predictor of ER according to a receiver operating characteristic (ROC) analysis (area under the ROC curve 0.82, p < 0.0001).
CONCLUSIONS
M2BPGi can predict ER after surgery and is useful for risk stratification in patients with HCC.
PubMed: 38937354
DOI: 10.1007/s00595-024-02885-z -
Toxicology Mechanisms and Methods Jun 2024Cyclosporine A (CsA) has shown efficacy against immunity-related diseases despite its toxicity in various organs, including the liver, emphasizing the need to elucidate...
Cyclosporine A (CsA) has shown efficacy against immunity-related diseases despite its toxicity in various organs, including the liver, emphasizing the need to elucidate its underlying hepatotoxicity mechanism. This study aimed to capture the alterations in genome-wide expression over time and the subsequent perturbations of corresponding pathways across species. Six data from humans, mice, and rats, including animal liver tissue, human liver microtissues, and two liver cell lines exposed to CsA toxic dose, were used. The microtissue exposed to CsA for 10 d was analyzed to obtain dynamically differentially expressed genes (DEGs). Single-time points data at 1, 3, 5, 7, and 28 d of different species were used to provide additional evidence. Using liver microtissue-based longitudinal design, DEGs that were consistently up- or down-regulated over time were captured, and the well-known mechanism involved in CsA toxicity was elucidated. Thirty DEGs that consistently changed in longitudinal data were also altered in 28-d rat in-house data with concordant expression. Some genes (e.g. , , ) showed good concordance with identified DEGs in 1-d and 7-d mouse data. Pathway analysis revealed up-regulations of protein processing, asparagine N-linked glycosylation, and cargo concentration in the endoplasmic reticulum. Furthermore, the down-regulations of pathways related to biological oxidations and metabolite and lipid metabolism were elucidated. These pathways were also enriched in single-time-point data and conserved across species, implying their biological significance and generalizability. Overall, the human organoids-based longitudinal design coupled with cross-species validation provides temporal molecular change tracking, aiding mechanistic elucidation and biologically relevant biomarker discovery.
PubMed: 38937256
DOI: 10.1080/15376516.2024.2371894 -
Biochimica Et Biophysica Acta. General... Jun 2024N-Acetylglucosaminyltransferase-III (GnT-III, also designated MGAT3) catalyzes the formation of a specific N-glycan branch, bisecting GlcNAc, in the Golgi apparatus....
BACKGROUND
N-Acetylglucosaminyltransferase-III (GnT-III, also designated MGAT3) catalyzes the formation of a specific N-glycan branch, bisecting GlcNAc, in the Golgi apparatus. Bisecting GlcNAc is a key residue that suppresses N-glycan maturation and is associated with the pathogenesis of cancer and Alzheimer's disease. However, it remains unclear how GnT-III recognizes its substrates and how GnT-III activity is regulated in cells.
METHODS
Using AlphaFold2 and structural comparisons, we predicted the key amino acid residues in GnT-III that interact with substrates in the catalytic pocket. We also performed in vitro activity assay, lectin blotting analysis and N-glycomic analysis using point mutants to assess their activity.
RESULTS
Our data suggested that E320 of human GnT-III is the catalytic center. More interestingly, we found a unique mutant, K346T, that exhibited lower in vitro activity and higher intracellular activity than wild-type GnT-III. The enzyme assays using various substrates showed that the substrate specificity of K346T was unchanged, whereas cycloheximide chase experiments revealed that the K346T mutant has a slightly shorter half-life, suggesting that the mutant is unstable possibly due to a partial misfolding. Furthermore, TurboID-based proximity labeling showed that the localization of the K346T mutant is shifted slightly to the cis side of the Golgi, probably allowing for prior action to competing galactosyltransferases.
CONCLUSIONS
The slight difference in K346T localization may be responsible for the higher biosynthetic activity despite the reduced activity.
GENERAL SIGNIFICANCE
Our findings underscore the importance of fine intra-Golgi localization and reaction orders of glycosyltransferases for the biosynthesis of complex glycan structures in cells.
PubMed: 38936637
DOI: 10.1016/j.bbagen.2024.130663 -
Life Sciences Jun 2024Membrane trafficking within the Golgi apparatus plays a pivotal role in the intracellular transportation of lipids and proteins. Dysregulation of this process can give... (Review)
Review
Membrane trafficking within the Golgi apparatus plays a pivotal role in the intracellular transportation of lipids and proteins. Dysregulation of this process can give rise to various pathological manifestations, including cancer. Exploiting Golgi defects, cancer cells capitalise on aberrant membrane trafficking to facilitate signal transduction, proliferation, invasion, immune modulation, angiogenesis, and metastasis. Despite the identification of several molecular signalling pathways associated with Golgi abnormalities, there remains a lack of approved drugs specifically targeting cancer cells through the manipulation of the Golgi apparatus. In the initial section of this comprehensive review, the focus is directed towards delineating the abnormal Golgi genes and proteins implicated in carcinogenesis. Subsequently, a thorough examination is conducted on the impact of these variations on Golgi function, encompassing aspects such as vesicular trafficking, glycosylation, autophagy, oxidative mechanisms, and pH alterations. Lastly, the review provides a current update on promising Golgi apparatus-targeted inhibitors undergoing preclinical and/or clinical trials, offering insights into their potential as therapeutic interventions. Significantly more effort is required to advance these potential inhibitors to benefit patients in clinical settings.
PubMed: 38936604
DOI: 10.1016/j.lfs.2024.122868 -
Pathology, Research and Practice Jun 2024Nasopharyngeal carcinoma (NPC) is closely related to Epstein-Barr virus (EBV) infection, and glycosylation of proteins is associated with precancerous lesions and... (Review)
Review
Nasopharyngeal carcinoma (NPC) is closely related to Epstein-Barr virus (EBV) infection, and glycosylation of proteins is associated with precancerous lesions and carcinogenesis of NPC, and viral glycoproteins mediates the fusion of viruses with B cells or epithelial cells in the infection stage, promoting the conversion of normal epithelial cells into cancer cells. In the process of occurrence and development of NPC, various glycoproteins in the body promote or inhibit the proliferation, invasion, metastasis, and drug resistance of tumor cells, such as the tumor inhibitory effect of NGX6 and inhibin B (INHBB); the cancer-promoting effect of tenascin-C (TNC), fibronectin 1 (FN1), insulin-like growth factor binding protein-3 (IGFBP3), serglycin, and its core protein; and some effects of glycosylation of immune proteins on immunotherapy in NPC. This article provides an overview of the research progress on the interaction of glycoproteins associated with EBV infection with the occurrence and development of NPC.
PubMed: 38936091
DOI: 10.1016/j.prp.2024.155427 -
Analytical Chemistry Jun 2024Tandem mass spectrometry coupled with liquid chromatography (LC-MS/MS) has proven a versatile tool for the identification and quantification of proteins and their...
Tandem mass spectrometry coupled with liquid chromatography (LC-MS/MS) has proven a versatile tool for the identification and quantification of proteins and their post-translational modifications (PTMs). Protein glycosylation is a critical PTM for the stability and biological function of many proteins, but full characterization of site-specific glycosylation of proteins remains analytically challenging. Collision-induced dissociation (CID) is the most common fragmentation method used in LC-MS/MS workflows, but the loss of labile modifications renders CID inappropriate for detailed characterization of site-specific glycosylation. Electron-based dissociation methods provide alternatives that retain intact glycopeptide fragments for unambiguous site localization, but these methods often underperform CID due to increased reaction times and reduced efficiency. Electron-activated dissociation (EAD) is another strategy for glycopeptide fragmentation. Here, we use a ZenoTOF 7600 SCIEX instrument to compare the performance of various fragmentation techniques for the analysis of a complex mixture of mammalian - and -glycopeptides. We found CID fragmentation identified the most glycopeptides and generally produced higher quality spectra, but EAD provided improved confidence in glycosylation site localization. Supplementing EAD with CID fragmentation (EAciD) further increased the number and quality of glycopeptide identifications, while retaining localization confidence. These methods will be useful for glycoproteomics workflows for either optimal glycopeptide identification or characterization.
PubMed: 38935274
DOI: 10.1021/acs.analchem.4c01450 -
ACS Chemical Biology Jun 2024-linked glycosylation plays a key role in the efficacy of many therapeutic proteins. One limitation to the bacterial glycoengineering of human -linked glycans is the...
-linked glycosylation plays a key role in the efficacy of many therapeutic proteins. One limitation to the bacterial glycoengineering of human -linked glycans is the difficulty of installing a single -acetylglucosamine (GlcNAc), the reducing end sugar of many human-type glycans, onto asparagine in a single step (-GlcNAcylation). Here, we develop an method for -GlcNAcylating proteins using the oligosaccharyltransferase PglB from . We use cell-free protein synthesis (CFPS) to test promiscuous PglB variants previously reported in the literature for the ability to produce -GlcNAc and successfully determine that PglB with an N311V mutation (PglB) exhibits increased GlcNAc transferase activity relative to the wild-type enzyme. We then improve the transfer efficiency by producing CFPS extracts enriched with PglB and further optimize the reaction conditions, achieving a 98.6 ± 0.5% glycosylation efficiency. We anticipate this method will expand the glycoengineering toolbox for therapeutic research and biomanufacturing.
PubMed: 38934647
DOI: 10.1021/acschembio.4c00228 -
Organic & Biomolecular Chemistry Jun 2024Sialyl Lewis (sLe), also known as cancer antigen 19-9, is a tumor-associated carbohydrate antigen. In this article, chemical and chemoenzymatic syntheses of a...
Sialyl Lewis (sLe), also known as cancer antigen 19-9, is a tumor-associated carbohydrate antigen. In this article, chemical and chemoenzymatic syntheses of a tetrasaccharide glycan 1 structurally derived from sLe are reported. Challenges involved in the chemical synthesis include the highly stereoselective construction of 1,2--α-L-fucoside and α-D-sialoside, as well as the assembly of the 3,4-disubstituted -acetylglucosamine subunit. Perbenzylated thiofucoside and -acetyl-5-,4--oxazolidinone protected sialic acid thioglycoside were employed as glycosyl donors, respectively, for the efficient preparation of the desired α-fucoside and α-sialoside. The 3,4-branched glucosamine backbone was established through a 3- and then 4- glycosylation sequence in which the 3-hydroxyl group of the glucosamine moiety was glycosylated first and then the 4-hydroxyl. A facile chemoenzymatic approach was also exploited to synthesize the target molecule. The chemically obtained free disaccharide 30 was sequentially sialylated and fucosylated in an enzyme-catalyzed regio- and stereospecific manner to form 1 in high yields. The linker appended 1 can be covalently attached to a carrier protein for further immunological studies.
PubMed: 38934561
DOI: 10.1039/d4ob00809j -
Mass Spectrometry Reviews Jun 2024With implications in several medical conditions, N-linked glycosylation is one of the most important posttranslation modifications present in all living organisms. Due... (Review)
Review
With implications in several medical conditions, N-linked glycosylation is one of the most important posttranslation modifications present in all living organisms. Due to their nontemplate synthesis, glycan structures are extraordinarily complex and require multiple analytical techniques for complete structural elucidation. Mass spectrometry is the most common way to investigate N-linked glycans; however, with techniques such as liquid-chromatography mass spectrometry, there is complete loss of spatial information. Mass spectrometry imaging is a transformative analytical technique that can visualize the spatial distribution of ions within a biological sample and has been shown to be a powerful tool to investigate N-linked glycosylation. This review covers the fundamentals of mass spectrometry imaging and N-linked glycosylation and highlights important findings of recent key studies aimed at expanding and improving the glycomics imaging field.
PubMed: 38934211
DOI: 10.1002/mas.21895