Metabolic C-Labeling of IgA Fc Fragment by Transient Mammalian Expression for NMR Analysis.

The atomic description of glycosylated proteins is essential for gaining an understanding of the physiological roles of protein glycosylation. X-ray crystallographic and cryo-EM analyses currently play a central role in the 3D structural determination of biological macromolecules. 3D structural information of glycosylated proteins remains limited due to the weak or absent electron densities of the glycan parts.

VarMeter: a prediction method for the impact of glycogene variants.

The clinical relevance of glycans, which play a wide array of physiological roles, is underscored by the emergence of congenital disorders of glycosylation, a group of rare inherited diseases caused by defects in glycan-related genes (glycogenes). Biochemical studies of recombinant proteins and phenotypic analyses in knockout mice are revealing critical insights into the roles of various glycosyltransferases, glycosidases, and glycan-binding proteins. However, the biological functions of numerous glycogenes and their role in disease remain incompletely understood, partly due to human-specific functions that are not recapitulated in model organisms, and partly due to the structural diversity and complexity of glycan modifications, which are difficult to fully assess by conventional methods.

Antagonistic effects of PU.1 on Gfi-1B-induced erythroid colony formation in human cord blood cells.

Gfi-1B is a hematopoietic transcription factor essential for growth and differentiation of the erythroid/megakaryocytic lineages, and PU.1 is a master regulator for myeloid development. Herein, we demonstrate that PU.

Complete assignment of H and C NMR signals of monoglucosylated high-mannose type glycan attached to asparagine.

GlcManGlcNAc (G1M9) glycan and other high mannose-type glycans play key roles in the quality control mechanisms of glycoprotein synthesis. The lectin-like proteins calnexin (CNX) and calreticulin (CRT) specifically recognize G1M9 glycan and assist newly synthesized glycoproteins to achieving correct folding. Nuclear magnetic resonance (NMR) spectroscopy is a unique method for analyzing the conformation, dynamics and interactions of glycans like G1M9 glycan and CNX/CRT.

VarMeter2: An enhanced structure-based method for predicting pathogenic missense variants through Mahalanobis distance.

Various computational methods have been developed to predict the pathogenicity of missense variants, which is crucial for diagnosing rare diseases. Recently, we introduced VarMeter, a diagnostic tool for predicting variant pathogenicity based on normalized solvent-accessible surface area (nSASA) and mutation energy calculated from AlphaFold 3D models, and validated it on arylsulfatase L. To evaluate the broader applicability of VarMeter and enhance its predictive accuracy, here we analyzed 296 pathogenic and 240 benign variants extracted from the ClinVar database.

Bacterial expression, purification and characterization of the human Dectin-1 lectin domain for structural and functional studies.

Dectin-1 (CLEC7A), a C-type lectin-like receptor that recognizes β-1,3 glucans, has a key role in the innate immune system. While the lectin domain of mouse Dectin-1 has been solubilized and refolded from inclusion bodies in Escherichia coli, similar refolding of the human Dectin-1 lectin domain is hindered by the formation of misfolded multimers with aberrant intermolecular disulfide bonds. The aim of this study was to develop a method for the large-scale production of the human Dectin-1 lectin domain.

Chemical extension and glycodendrimer formation of the matriglycan decasaccharide, -(3Xylα1-3GlcAβ1)- and its affinity for laminin.

Muscle tissue is stabilized by the strong interaction between laminin and matriglycan. Matriglycan is a polysaccharide composed of the repeating disaccharide, -3Xylα1-3GlcAβ1-, and is a pivotal part of the core M3 O-mannosyl glycan. Patients with muscular dystrophy cannot synthesize matriglycan or the core M3 O-mannosyl glycan due to a defect in or the lack of glycosyltransferases owing to glycan synthesis.

3D structural insights into the effect of N-glycosylation in human chitotriosidase variant G102S.

Background: N-glycosylation is a key post-translational modification critical for protein function and stability. Chitotriosidase-1 (CHIT1), belonging to glycoside hydrolase family 18, is clinically utilized as a biomarker of Gaucher disease. A G102S variant is common in some populations, but the implications of this missense mutation on CHIT1 function and in disease pathology are unknown.

Lysate of bovine adipose-derived stem cells accelerates in-vitro development and increases cryotolerance through reduced content of lipid in the in vitro fertilized embryos.

Mesenchymal stem cells such as adipose-derived stem cells (ADSCs) are known to secrete factors that stimulate cell division and promote regeneration in neighboring cells. While conditioned medium from stem cells has been used in blastocyst production, no studies have examined the use of cell lysates. In this study we investigated the effects of adding ADSC lysate to in vitro culture (IVC) medium.

Comparative Conformational Analysis of Acyclic Sugar Alcohols Ribitol, Xylitol and d-Arabitol by Solution NMR and Molecular Dynamics Simulations.

Ribitol (CHO) is an acyclic sugar alcohol that was recently identified in -mannose glycan on mammalian α-dystroglycan. The conformation and dynamics of acyclic sugar alcohols such as ribitol are dependent on the stereochemistry of the hydroxyl groups; however, the dynamics are not fully understood. To gain insights into the conformation and dynamics of sugar alcohols, we carried out comparative analyses of ribitol, d-arabitol and xylitol by a crystal structure database search, solution NMR analysis and molecular dynamics (MD) simulations.

Molecular Dynamics Simulation and Docking of MUC1 O-Glycopeptide.

Advances in computer performance and computational simulations allow increasing sophistication in applications in biological systems. Molecular dynamics (MD) simulations are especially suitable for studying conformation, dynamics, and interaction of flexible biomolecules such as free glycans and glycopeptides. Computer simulations are best performed when the scope and limitations in performance have been thoroughly assessed.

Solution NMR Analysis of O-Glycopeptide-Antibody Interaction.

O-Linked glycans potentially play a functional role in cellular recognition events. Recent structural analyses suggest that O-glycosylation can be a specific signal for a lectin receptor which recognizes both the O-glycan and the adjacent polypeptide region. Further, certain antibodies specifically bind to the O-glycosylated peptide.

A convenient assay for soluble Dectin-1 lectin domain binding to insoluble β-glucans.

β-Glucan is a homopolymer with a backbone of β-1,3-linked glucose residues. The solubility and biological activity of β-glucan can be influenced by the length of the backbone and the length/interval of the β-1,6 branches. Dectin-1 is crucial in innate immunity through its binding to exogenous β-glucans.

NMR characterization of uniformly C- and/or N-labeled, unsulfated chondroitins with high molecular weights.

Solution nuclear magnetic resonance (NMR) analysis of polysaccharides can provide valuable information not only on their primary structures but also on their conformation, dynamics, and interactions under physiological conditions. One of the main problems is that non-anomeric H signals typically overlap, and this often hinders detailed NMR analysis. Isotope enrichment, such as with C and N, will add a new dimension to the NMR spectra of polysaccharides, and spectral analysis can be performed with enhanced sensitivity using isolated peaks.

Prediction of protein structure and AI.

AlphaFold, an artificial intelligence (AI)-based tool for predicting the 3D structure of proteins, is now widely recognized for its high accuracy and versatility in the folding of human proteins. AlphaFold is useful for understanding structure-function relationships from protein 3D structure models and can serve as a template or a reference for experimental structural analysis including X-ray crystallography, NMR and cryo-EM analysis. Its use is expanding among researchers, not only in structural biology but also in other research fields.

Each N-glycan on human IgA and J-chain uniquely affects oligomericity and stability.

Background: Immunoglobulin A (IgA) plays a pivotal role in various immune responses, especially that of mucosal immunity. IgA is usually assembled into dimers with the contribution of J-chains. There are two N-glycosylation sites in human IgA1-Fc and one in the J-chain.

Predicting the pathogenicity of missense variants based on protein instability to support diagnosis of patients with novel variants of ARSL.

Rare diseases are estimated to affect 3.5%-5.9% of the population worldwide and are difficult to diagnose.

Synthesis of the matriglycan hexasaccharide, -3Xylα1-3GlcAβ1-trimer and its interaction with laminin.

Matriglycan, a polysaccharide that is a pivotal part of the core M3 -mannosyl glycan composed of the repeating disaccharide -3Xylα1-3GlcAβ1-, interacts with laminin to stabilize muscle tissue. We herein report the synthesis of matriglycan-repeating hexasaccharides equipped with an alkyne linker to form glycoconjugates. The key step in the formation of an α-linked xylosyl glycoside was resolved by solvent-specific separation from an anomeric mixture.

A Data Set of Ion Mobility Collision Cross Sections and Liquid Chromatography Retention Times from 71 Pyridylaminated N-Linked Oligosaccharides.

Determination of the glycan structure is an essential step in understanding structure-function relationships of glycans and glycoconjugates including biopharmaceuticals. Mass spectrometry, because of its high sensitivity and mass resolution, is an excellent means of analyzing glycan structures. We previously proposed a method for rapid and precise identification of -glycan structures by ultraperformance liquid chromatography-connected ion mobility mass spectrometry (UPLC/IM-MS).

-Glycan-Dependent Interaction between MUC1 Glycopeptide and MY.1E12 Antibody by NMR, Molecular Dynamics and Docking Simulations.

Anti-mucin1 (MUC1) antibodies have been widely used for breast cancer diagnosis and treatment. This is based on the fact that MUC1 undergoes aberrant glycosylation upon cancer progression, and anti-MUC1 antibodies differentiate changes in glycan structure. MY.

Discovery of a lectin domain that regulates enzyme activity in mouse N-acetylglucosaminyltransferase-IVa (MGAT4A).

N-Glycosylation is a common post-translational modification, and the number of GlcNAc branches in N-glycans impacts glycoprotein functions. N-Acetylglucosaminyltransferase-IVa (GnT-IVa, also designated as MGAT4A) forms a β1-4 GlcNAc branch on the α1-3 mannose arm in N-glycans. Downregulation or loss of GnT-IVa causes diabetic phenotypes by dysregulating glucose transporter-2 in pancreatic β-cells.

Functional validation of novel variants in B4GALNT1 associated with early-onset complex hereditary spastic paraplegia with impaired ganglioside synthesis.

Childhood-onset forms of hereditary spastic paraplegia are ultra-rare diseases and often present with complex features. Next-generation-sequencing allows for an accurate diagnosis in many cases but the interpretation of novel variants remains challenging, particularly for missense mutations. Where sufficient knowledge of the protein function and/or downstream pathways exists, functional studies in patient-derived cells can aid the interpretation of molecular findings.

Chemical and Chemo-Enzymatic Syntheses of Glycans Containing Ribitol Phosphate Scaffolding of Matriglycan.

Ribitol phosphate modifications to the core M3 -mannosyl glycan are important for the functional maturation of α-dystroglycan. Three sequentially extended partial structures of the core M3 -mannosyl glycan including a tandem ribitol phosphate were regio- and stereo-selectively synthesized: Rbo5P-3GalNAcβ, Rbo5P-1Rbo5P-3GalNAcβ, and Xylβ1-4Rbo5P-1Rbo5P-3GalNAcβ (Rbo5P, d-ribitol-5-phosphate; GalNAc, -acetyl-d-galactosamine; Xyl, d-xylose). Rbo5P-3GalNAcβ with -nitrophenyl at the aglycon part served as a substrate for ribitol phosphate transferase (FKRP, fukutin-related protein), and its product was glycosylated by the actions of a series of glycosyltransferases, namely, ribitol xylosyltransferase 1 (RXYLT1), β1,4-glucuronyltransferase 1 (B4GAT1), and like-acetyl-glucosaminyltransferase (LARGE).