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Planta Medica Jun 2024Hyaluronic acid is composed of repeating sugar units, glucuronic acid and N-acetylglucosamine, which are often associated with increased tumor progression. agglutinin...
Hyaluronic acid is composed of repeating sugar units, glucuronic acid and N-acetylglucosamine, which are often associated with increased tumor progression. agglutinin is a potential component that exhibits a high affinity for binding to N-acetylglucosamine. This study aimed to investigate Agglutinin's potential to inhibit the proliferation and migration of prostate cancer cells with high expression of hyaluronic acid through molecular docking and studies. The expression of hyaluronan synthase genes in prostate tissue and cell lines was checked by an study, and the interaction between hyaluronic acid with both CD44 transmembrane glycoprotein and agglutinin was analyzed through molecular docking. Agglutinin's effect on cell viability (neutral red uptake assay), migration (scratch wound healing assays), and both and expression (quantitative real-time polymerase chain reaction) were assessed . The results showed that in prostate cancer cell lines, the PC3 cell line has the highest expression of hyaluronan synthase genes. agglutinin exhibits an interaction of six specific residues on CD44 compared to hyaluronic acid's singular residue. While agglutinin alone effectively reduced cell viability and wound closer (≥ 150 µg/mL), combining it with hyaluronic acid significantly shifted the effective concentration to a higher dose (≥ 350 µg/mL). These results, together with low and high gene expression, suggest that agglutinin may impair the CD44-HA pathway in PC3 cells. This possibility is supported by Agglutinin's ability to compete with hyaluronic acid for binding to CD44. Based on this, agglutinin as a plant lectin shows promise in inhibiting cancer proliferation and migration by targeting its dependence on hyaluronic acid.
PubMed: 38942031
DOI: 10.1055/a-2324-2250 -
Stem Cell Reports Jun 2024Understanding the regulation of human embryonic stem cells (hESCs) pluripotency is critical to advance the field of developmental biology and regenerative medicine....
Understanding the regulation of human embryonic stem cells (hESCs) pluripotency is critical to advance the field of developmental biology and regenerative medicine. Despite the recent progress, molecular events regulating hESC pluripotency, especially the transition between naive and primed states, still remain unclear. Here we show that naive hESCs display lower levels of O-linked N-acetylglucosamine (O-GlcNAcylation) than primed hESCs. O-GlcNAcase (OGA), the key enzyme catalyzing the removal of O-GlcNAc from proteins, is highly expressed in naive hESCs and is important for naive pluripotency. Depletion of OGA accelerates naive-to-primed pluripotency transition. OGA is transcriptionally regulated by EP300 and acts as a transcription regulator of genes important for maintaining naive pluripotency. Moreover, we profile protein O-GlcNAcylation of the two pluripotency states by quantitative proteomics. Together, this study identifies OGA as an important factor of naive pluripotency in hESCs and suggests that O-GlcNAcylation has a broad effect on hESCs homeostasis.
PubMed: 38942028
DOI: 10.1016/j.stemcr.2024.05.009 -
Journal of Chromatography. B,... Jun 2024Hyaluronic acid (HA), a glycosaminoglycan, is comprised of alternating units of D-glucuronic acid and N-acetylglucosamine. This compound harbors numerous biomedical...
Hyaluronic acid (HA), a glycosaminoglycan, is comprised of alternating units of D-glucuronic acid and N-acetylglucosamine. This compound harbors numerous biomedical applications, including its use in pharmaceuticals, wound healing, osteoarthritis treatment, and drug delivery. Its unique composition and exceptional features, such as its high water-absorbing and retaining capacity, have also led to its use in the cosmetics industry. The employment of this biopolymer has given rise to an escalation in the request for its manufacture. The present investigation has explored the correlation between hyaluronic acid and chitosan and silica for the purpose of separation. Consequently, Iron oxide magnetic nano particles and micro particles were produced via co-precipitation method and were layered with chitosan and silica to purify the hyaluronic acid from the fermentation broth that was generated by Streptococcus Zooepidemicus. The size distribution and zeta potentials of the two kinds of particles were gauged with the aid of a dynamic laser light scattering apparatus and zeta potential meter (Malvern, Zeta master) respectively. The confirmation of the chemical structure of the FeO nanoparticles and FeO particles conjugated with chitosan and silica was accomplished through the utilization of Fourier Transform Infrared Spectroscopy (FT-IR). Protein contamination was thoroughly characterized by means of sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Nanodrop 2000/2000c spectrophotometers protein estimation method. The maximum HA adsorption capacity, under optimal pH conditions of 4, was determined to be 87 mg/g, 112 mg/g, 51 mg/g, and 44 mg/g for FeO -chitosan nanoparticle, FeO -chitosan micro particle, FeO -silica microparticle, and FeO -silica nanoparticle, respectively.
PubMed: 38936266
DOI: 10.1016/j.jchromb.2024.124212 -
Acta Crystallographica. Section F,... Jul 2024Mycobacterium tuberculosis can reside and persist in deep tissues; latent tuberculosis can evade immune detection and has a unique mechanism to convert it into active...
Mycobacterium tuberculosis can reside and persist in deep tissues; latent tuberculosis can evade immune detection and has a unique mechanism to convert it into active disease through reactivation. M. tuberculosis Rv1421 (MtRv1421) is a hypothetical protein that has been proposed to be involved in nucleotide binding-related metabolism in cell-growth and cell-division processes. However, due to a lack of structural information, the detailed function of MtRv1421 remains unclear. In this study, a truncated N-terminal domain (NTD) of MtRv1421, which contains a Walker A/B-like motif, was purified and crystallized using PEG 400 as a precipitant. The crystal of MtRv1421-NTD diffracted to a resolution of 1.7 Å and was considered to belong to either the C-centered monoclinic space group C2 or the I-centered orthorhombic space group I222, with unit-cell parameters a = 124.01, b = 58.55, c = 84.87 Å, β = 133.12° or a = 58.53, b = 84.86, c = 90.52 Å, respectively. The asymmetric units of the C2 or I222 crystals contained two or one monomers, respectively. In terms of the binding ability of MtRv1421-NTD to various ligands, uridine diphosphate (UDP) and UDP-N-acetylglucosamine significantly increased the melting temperature of MtRv1421-NTD, which indicates structural stabilization through the binding of these ligands. Altogether, the results reveal that a UDP moiety may be required for the interaction of MtRv1421-NTD as a nucleotide-binding protein with its ligand.
PubMed: 38935514
DOI: 10.1107/S2053230X24005831 -
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 -
Pharmaceuticals (Basel, Switzerland) May 2024The circulatory system is a closed conduit system throughout the body and consists of two parts as follows: the cardiovascular system and the lymphatic system.... (Review)
Review
The circulatory system is a closed conduit system throughout the body and consists of two parts as follows: the cardiovascular system and the lymphatic system. Hematological malignancies usually grow and multiply in the circulatory system, directly or indirectly affecting its function. These malignancies include multiple myeloma, leukemia, and lymphoma. -linked β-N-acetylglucosamine (-GlcNAc) transferase (OGT) regulates the function and stability of substrate proteins through -GlcNAc modification. Abnormally expressed OGT is strongly associated with tumorigenesis, including hematological malignancies, colorectal cancer, liver cancer, breast cancer, and prostate cancer. In cells, OGT can assemble with a variety of proteins to form complexes to exercise related biological functions, such as OGT/HCF-1, OGT/TET, NSL, and then regulate glucose metabolism, gene transcription, cell proliferation, and other biological processes, thus affecting the development of hematological malignancies. This review summarizes the complexes involved in the assembly of OGT in cells and the role of related OGT complexes in hematological malignancies. Unraveling the complex network regulated by the OGT complex will facilitate a better understanding of hematologic malignancy development and progression.
PubMed: 38931332
DOI: 10.3390/ph17060664 -
Archives of Biochemistry and Biophysics Jun 2024Bovine intestinal alkaline phosphatase (biALP), a membrane-bound plasma metalloenzyme, maintains intestinal homeostasis, regulates duodenal surface pH, and protects...
Bovine intestinal alkaline phosphatase (biALP), a membrane-bound plasma metalloenzyme, maintains intestinal homeostasis, regulates duodenal surface pH, and protects against infections caused by pathogenic bacteria. The N-glycans of biALP regulate its enzymatic activity, protein folding, and thermostability, but their structures are not fully reported. In this study, the structures and quantities of the N-glycans of biALP were analyzed by liquid chromatography-electrospray ionization-high energy collision dissociation-tandem mass spectrometry. In total, 48 N-glycans were identified and quantified, comprising high-mannose [6 N-glycans, 33.1% (sum of relative quantities of each N-glycan)], hybrid (6, 11.9%), and complex (36, 55.0%) structures [bi- (13, 26.1%), tri- (16, 21.5%), and tetra-antennary (7, 7.4%)]. These included bisecting N-acetylglucosamine (33, 56.6%), mono- to tri-fucosylation (32, 53.3%), mono- to tri-α-galactosylation (16, 20.7%), and mono- to tetra-β-galactosylation (36, 58.5%). No sialylation was identified. N-glycans with non-bisecting GlcNAc (9, 10.3%), non-fucosylation (10, 13.6%), non-α-galactosylation (26, 46.2%), and non-β-galactosylation (6, 8.4%) were also identified. The activity (100%) of biALP was reduced to 37.3 ± 0.2% (by de-fucosylation), 32.7 ± 2.9% (by de-α-galactosylation), and 0.2 ± 0.2% (by de-β-galactosylation), comparable to inhibition by 10 to 10 mM EDTA, a biALP inhibitor. These results indicate that fucosylated and galactosylated N-glycans, especially β-galactosylation, affected the activity of biALP. This study is the first to identify 48 diverse N-glycan structures and quantities of bovine as well as human intestinal ALP and to demonstrate the importance of the role of fucosylation and galactosylation for maintaining the activity of biALP.
PubMed: 38914216
DOI: 10.1016/j.abb.2024.110069 -
JCI Insight May 2024The regulated glycosylation of the proteome has widespread effects on biological processes that cancer cells can exploit. Expression of N-acetylglucosaminyltransferase V...
The regulated glycosylation of the proteome has widespread effects on biological processes that cancer cells can exploit. Expression of N-acetylglucosaminyltransferase V (encoded by Mgat5 or GnT-V), which catalyzes the addition of β1,6-linked N-acetylglucosamine to form complex N-glycans, has been linked to tumor growth and metastasis across tumor types. Using a panel of murine pancreatic ductal adenocarcinoma (PDAC) clonal cell lines that recapitulate the immune heterogeneity of PDAC, we found that Mgat5 is required for tumor growth in vivo but not in vitro. Loss of Mgat5 results in tumor clearance that is dependent on T cells and dendritic cells, with NK cells playing an early role. Analysis of extrinsic cell death pathways revealed Mgat5-deficient cells have increased sensitivity to cell death mediated by the TNF superfamily, a property that was shared with other non-PDAC Mgat5-deficient cell lines. Finally, Mgat5 knockout in an immunotherapy-resistant PDAC line significantly decreased tumor growth and increased survival upon immune checkpoint blockade. These findings demonstrate a role for N-glycosylation in regulating the sensitivity of cancer cells to T cell killing through classical cell death pathways.
Topics: Animals; Glycosylation; Mice; N-Acetylglucosaminyltransferases; Carcinoma, Pancreatic Ductal; Pancreatic Neoplasms; Cell Line, Tumor; Humans; T-Lymphocytes; Dendritic Cells; Killer Cells, Natural; Mice, Knockout
PubMed: 38912584
DOI: 10.1172/jci.insight.178804 -
Disease Models & Mechanisms Jun 2024Pathogenic variants in GFPT1, encoding a key enzyme to synthesize UDP-N-acetylglucosamine (UDP-GlcNAc), cause congenital myasthenic syndrome (CMS). We made a knock-in...
Pathogenic variants in GFPT1, encoding a key enzyme to synthesize UDP-N-acetylglucosamine (UDP-GlcNAc), cause congenital myasthenic syndrome (CMS). We made a knock-in (KI) mouse model carrying a frameshift variant in Gfpt1 exon 9 simulating a CMS patient. As Gfpt1 exon 9 is exclusively included in striated muscles, Gfpt1-KI mice were deficient for Gfpt1 only in skeletal muscles. In Gfpt1-KI mice, (i) UDP-HexNAc, CMP-NeuAc, and protein O-GlcNAcylations were reduced in skeletal muscles; (ii) aged Gfpt1-KI mice showed poor exercise performance and abnormal neuromuscular junction structures; and (iii) markers for unfolded protein response (UPR) were elevated in skeletal muscles. Denervation-mediated enhancement of ER stress in Gfpt1-KI mice facilitated protein folding, ubiquitin-proteasome degradation, and apoptosis, whereas autophagy was not induced and protein aggregates were markedly increased. Lack of autophagy was accounted for by enhanced degradation of FoxO1 by increased Xbp1-s/u proteins. Similarly, in Gfpt1-silenced C2C12 myotubes, ER stress exacerbated protein aggregates and activated apoptosis, but autophagy was attenuated. In both skeletal muscles in Gfpt1-KI mice and Gfpt1-silenced C2C12 myotubes, maladaptive UPR failed to eliminate protein aggregates and provoked apoptosis.
PubMed: 38903011
DOI: 10.1242/dmm.050768