Access to defined glycans and glycoconjugates is pivotal for discovery, dissection, and harnessing of a range of biological functions orchestrated by cellular glycosylation processes and the glycome. We previously employed genetic glycoengineering by nuclease-based gene editing to develop sustainable production of designer glycoprotein therapeutics and cell-based glycan arrays that display glycans in their natural context at the cell surface. However, access to human glycans in formats and quantities that allow structural studies of molecular interactions and use of glycans in biomedical applications currently rely on chemical and chemoenzymatic syntheses associated with considerable labor, waste, and costs.
View Article and Find Full Text PDFAngew Chem Int Ed Engl
December 2024
Core fucosylation is catalyzed by α-1,6-fucosyltransferase (FUT8), which fucosylates the innermost GlcNAc of N-glycans. Given the association of FUT8 with various diseases, including cancer, selective FUT8 inhibitors applicable to in vivo or cell-based systems are highly sought-after. Herein, we report the discovery of a compound that selectively inhibits FUT8 in cell-based assays.
View Article and Find Full Text PDFN-terminal nonsynonymous single-nucleotide polymorphisms (SNPs) of G protein-coupled receptors (GPCRs) are common and often affect receptor post-translational modifications. Their functional implications are, however, largely unknown. We have previously shown that the human β-adrenergic receptor (βAR) is O-glycosylated in the N-terminal extracellular domain by polypeptide GalNAc transferase-2 that co-regulates receptor proteolytic cleavage.
View Article and Find Full Text PDFProtein O-linked mannose (O-Man) glycosylation is an evolutionary conserved posttranslational modification that fulfills important biological roles during embryonic development. Three nonredundant enzyme families, POMT1/POMT2, TMTC1-4, and TMEM260, selectively coordinate the initiation of protein O-Man glycosylation on distinct classes of transmembrane proteins, including α-dystroglycan, cadherins, and plexin receptors. However, a systematic investigation of their substrate specificities is lacking, in part due to the ubiquitous expression of O-Man glycosyltransferases in cells, which precludes analysis of pathway-specific O-Man glycosylation on a proteome-wide scale.
View Article and Find Full Text PDFEbola virus glycoprotein (EBOV GP) is one of the most heavily O-glycosylated viral glycoproteins, yet we still lack a fundamental understanding of the structure of its large O-glycosylated mucin-like domain and to what degree the host O-glycosylation capacity influences EBOV replication. Using tandem mass spectrometry, we identified 47 O-glycosites on EBOV GP and found similar glycosylation signatures on virus-like particle- and cell lysate-derived GP. Furthermore, we performed quantitative differential O-glycoproteomics on proteins produced in wild-type HEK293 cells and cell lines ablated for the three key initiators of O-linked glycosylation, GalNAc-T1, -T2, and -T3.
View Article and Find Full Text PDFMicroalgae are a renewable and promising biomass for large-scale biofuel, food and nutrient production. However, their efficient exploitation depends on our knowledge of the cell wall composition and organization as it can limit access to high-value molecules. Here we provide an atomic-level model of the non-crystalline and water-insoluble glycoprotein-rich cell wall of Chlamydomonas reinhardtii.
View Article and Find Full Text PDFJ Phys Condens Matter
December 2023
Aging of the relaxors and PbMg1/3Nb2/3Oin particular was extensively studied in last two decades. Most of the results were related to the low temperature glass-like region. No systematic data around the freezing temperatures were reported.
View Article and Find Full Text PDFCell surface glycans are essential for establishing cell communication, adhesion, and migration. However, it remains challenging to obtain cell surface-specific information about glycoconjugate structures. Acquiring this information is essential for unraveling the functional role of glycans and for exploiting them as clinical targets.
View Article and Find Full Text PDFGalectins are a group of carbohydrate-binding proteins with a presumed immunomodulatory role and an elusive function on antigen-presenting cells. Here we analyzed the expression of galectin-1 and found upregulation of galectin-1 in the extracellular matrix across multiple tumors. Performing an in-depth and dynamic proteomic and phosphoproteomic analysis of human macrophages stimulated with galectin-1, we show that galectin-1 induces a tumor-associated macrophage phenotype with increased expression of key immune checkpoint protein programmed cell death 1 ligand 1 (PD-L1/CD274) and immunomodulator indoleamine 2,3-dioxygenase-1 (IDO1).
View Article and Find Full Text PDFMutations in the gene cause structural heart defects and renal anomalies syndrome, but the function of the encoded protein remains unknown. We previously reported wide occurrence of O-mannose glycans on extracellular immunoglobulin, plexin, transcription factor (IPT) domains found in the hepatocyte growth factor receptor (cMET), macrophage-stimulating protein receptor (RON), and plexin receptors, and further demonstrated that two known protein O-mannosylation systems orchestrated by the POMT1/2 and transmembrane and tetratricopeptide repeat-containing proteins 1-4 gene families were not required for glycosylation of these IPT domains. Here, we report that the gene encodes an ER-located protein O-mannosyltransferase that selectively glycosylates IPT domains.
View Article and Find Full Text PDFFront Bioeng Biotechnol
February 2023
Currently available enzyme replacement therapies for lysosomal storage diseases are limited in their effectiveness due in part to short circulation times and suboptimal biodistribution of the therapeutic enzymes. We previously engineered Chinese hamster ovary (CHO) cells to produce α-galactosidase A (GLA) with various N-glycan structures and demonstrated that elimination of mannose-6-phosphate (M6P) and conversion to homogeneous sialylated N-glycans prolonged circulation time and improved biodistribution of the enzyme following a single-dose infusion into Fabry mice. Here, we confirmed these findings using repeated infusions of the glycoengineered GLA into Fabry mice and further tested whether this glycoengineering approach, Long-Acting-GlycoDesign (LAGD), could be implemented on other lysosomal enzymes.
View Article and Find Full Text PDFSmall molecule inhibitors of glycosylation enzymes are valuable tools for dissecting glycan functions and potential drug candidates. Screening for inhibitors of glycosyltransferases are mainly performed by in vitro enzyme assays with difficulties moving candidates to cells and animals. Here, we circumvent this by employing a cell-based screening assay using glycoengineered cells expressing tailored reporter glycoproteins.
View Article and Find Full Text PDFThe article presents modern approaches to mastoid obliteration in cases of chronic purulent otitis media with cholesteatoma, published in peer-reviewed russian and foreign journals from 2017 to 2021. The review tells us about effect of mastoid cavity obliteration on reducing the recurrence of cholesteatoma, improving the quality of life of patients according to the results of international questionnaires and the effect of surgery on hearing in the long-term postoperative period. An analysis of the use of autologous and biocompatible materials in middle ear surgery is presented.
View Article and Find Full Text PDFTransforming growth factor-β (TGF-β) signaling regulates various aspects of cell growth and differentiation and is often dysregulated in human cancers. We combined genetic engineering of a human organotypic three-dimensional (3D) skin model with global quantitative proteomics and phosphoproteomics to dissect the importance of essential components of the TGF-β signaling pathway, including the ligands TGF-β1, TGF-β2, and TGF-β3, the receptor TGF-βRII, and the intracellular effector SMAD4. Consistent with the antiproliferative effects of TGF-β signaling, the loss of TGF-β1 or SMAD4 promoted cell cycling and delayed epidermal differentiation.
View Article and Find Full Text PDFMucin-type-O-glycosylation on proteins is integrally involved in human health and disease and is coordinated by an enzyme family of 20 N-acetylgalactosaminyltransferases (GalNAc-Ts). Detailed knowledge on the biological effects of site-specific O-glycosylation is limited due to lack of information on specific glycosylation enzyme activities and O-glycosylation site-occupancies. Here we present a systematic analysis of the isoform-specific targets of all GalNAc-Ts expressed within a tissue-forming human skin cell line, and demonstrate biologically significant effects of O-glycan initiation on epithelial formation.
View Article and Find Full Text PDFImmunoglobulins G (IgG) and their Fc gamma receptors (FcγRs) play important roles in our immune system. The conserved glycan in the Fc region of IgG1 impacts interaction of IgG with FcγRs and the resulting effector functions, which has led to the design of antibody therapeutics with greatly improved antibody-dependent cell cytotoxicity (ADCC) activities. Studies have suggested that also glycosylation of the FcγRIII affects receptor interactions with IgG, but detailed studies of the interaction of IgG1 and FcγRIIIa with distinct -glycans have been hindered by the natural heterogeneity in glycosylation.
View Article and Find Full Text PDFPrader−Willi syndrome (PWS) is a complex neurodevelopmental disorder caused by the deletion or inactivation of paternally expressed imprinted genes at the chromosomal region 15q11−q13. The PWS-critical region (PWScr) harbors tandemly repeated non-protein coding IPW-A exons hosting the intronic SNORD116 snoRNA gene array that is predominantly expressed in brain. Paternal deletion of PWScr is associated with key PWS symptoms in humans and growth retardation in mice (PWScr model).
View Article and Find Full Text PDFFUT8 is an essential α-1,6-fucosyltransferase that fucosylates the innermost GlcNAc of N-glycans, a process called core fucosylation. , FUT8 exhibits substrate preference for the biantennary complex N-glycan oligosaccharide (G0), but the role of the underlying protein/peptide to which N-glycans are attached remains unclear. Here, we explored the FUT8 enzyme with a series of N-glycan oligosaccharides, N-glycopeptides, and an Asn-linked oligosaccharide.
View Article and Find Full Text PDFMass spectrometry (MS) easily detects C-mannosylated peptides from purified proteins but not from complex biological samples. Enrichment of specific glycopeptides by lectin affinity prior to MS analysis has been widely applied to support glycopeptide identification but was until now not available for C-mannosylated peptides. Here, we used the α-mannose-specific lectin A (BC2L-A) and show that, in addition to its previously demonstrated high-mannose N-glycan binding capability, this lectin is able to retain C- and O-mannosylated peptides.
View Article and Find Full Text PDFThe large family of polypeptide GalNAc-transferases (GalNAc-Ts) controls with precision how GalNAc -glycans are added in the tandem repeat regions of mucins (, MUC1). However, the structural features behind the creation of well-defined and clustered patterns of -glycans in mucins are poorly understood. In this context, herein, we disclose the full process of MUC1 -glycosylation by GalNAc-T2/T3/T4 isoforms by NMR spectroscopy assisted by molecular modeling protocols.
View Article and Find Full Text PDFMucins and glycoproteins with mucin-like regions contain densely O-glycosylated domains often found in tandem repeat (TR) sequences. These O-glycodomains have traditionally been difficult to characterize because of their resistance to proteolytic digestion, and knowledge of the precise positions of O-glycans is particularly limited for these regions. Here, we took advantage of a recently developed glycoengineered cell-based platform for the display and production of mucin TR reporters with custom-designed O-glycosylation to characterize O-glycodomains derived from mucins and mucin-like glycoproteins.
View Article and Find Full Text PDF-Glycosylation is an omnipresent modification of the human proteome affecting many cellular functions, including protein cleavage, protein folding, and cellular signaling, interactions, and trafficking. The functions are governed by differentially regulated -glycan types and terminal structures. It is therefore essential to develop analytical methods that facilitate the annotation of -glycans in biological material.
View Article and Find Full Text PDFDNA mismatch repair (MMR) is a highly conserved pathway that corrects both base-base mispairs and insertion-deletion loops (IDLs) generated during DNA replication. Defects in MMR have been linked to carcinogenesis and drug resistance. However, the regulation of MMR is poorly understood.
View Article and Find Full Text PDFThe question of the microscopic origin of the M-superstructure and additional satellite peaks in the Zr-rich lead zirconate-titanate is discussed for nearly 50 years. Clear contradiction between the selection rules of the critical scattering and the superstructure was found preventing unambiguous attributing of the observed superstructure either to the rotation of the oxygen octahedra or to the antiparallel displacements of the lead cations. Detailed analysis of the satellite pattern explained it as the result of the incommensurate phase transition rather than antiphase domains.
View Article and Find Full Text PDF