Biology subjects

Wang, L.-X.

Publications and source records attributed to Wang, L.-X..

7 recordsLinked to original sources

bioRxiv2024

MPCD Index for Hepatocellular Carcinoma Patients Based on Mitochondrial Function and Cell Death Patterns

Hepatocellular carcinoma (HCC) is a highly heterogeneous cancer with a poor prognosis. During the development of cancer cells, mitochondria influence various cell death patterns by regulating metabolic pathways such as oxidative phosphorylation. However, the relationship between mitochondrial function and cell death patterns in HCC remains unclear. In this study, we used a comprehensive machine learning framework to construct a mitochondrial functional activity-associated programmed cell death index (MPCDI) based on scRNA-seq and RNA-seq data from TCGA, GEO, and ICGC datasets. The index signature was used to classify HCC patients, and studied the multi-omics features, immune microenvironment, and drug sensitivity of the subtypes. Finally, we constructed the MPCDI signature consisting of four genes (S100A9, FYN, LGALS3, and HMOX1), which was one of the independent risk factors for the prognosis of HCC patients. The HCC patients were divided into high- and low-MPCDI groups, and the immune status was different between the two groups. Patients with high MPCDI had higher TIDE scores and poorer responses to immunotherapy, suggesting that high-MPCDI patients might not be suitable for immunotherapy. By analyzing the drug sensitivity data of CTRP, GDSC, and PRISM databases, it was found that staurosporine has potential therapeutic significance for patients with high MPCDI. In summary, based on the characteristics of mitochondria function and PCD patterns, we used single-cell and transcriptome data to identify four genes and construct the MPCDI signature, which provided new perspectives and directions for the clinical diagnosis and personalized treatment of HCC patients.

bioinformatics↗

bioRxiv2024

Discovery of a single-subunit oligosaccharyltransferase that enables glycosylation of full-length IgG antibodies in Escherichia coli

Human immunoglobulin G (IgG) antibodies are one of the most important classes of biotherapeutic agents and undergo glycosylation at the conserved N297 site in the CH2 domain, which is critical for IgG Fc effector functions and anti-inflammatory activity. Hence, technologies for producing authentically glycosylated IgGs are in high demand. While attempts to engineer Escherichia coli for this purpose have been described, they have met limited success due in part to the lack of available oligosaccharyltransferase (OST) enzymes that can install N-linked glycans within the QYNST sequon of the IgG CH2 domain. Here, we identified a previously uncharacterized single-subunit OST (ssOST) from the bacterium Desulfovibrio marinus that exhibited greatly relaxed substrate specificity and, as a result, was able to catalyze glycosylation of native CH2 domains in the context of both a hinge-Fc fragment and a full-length IgG. Although the attached glycans were bacterial in origin, conversion to a homogeneous, asialo complex-type G2 N-glycan at the QYNST sequon of the E. coli-derived hinge-Fc was achieved via chemoenzymatic glycan remodeling. Importantly, the resulting G2-hinge-Fc exhibited strong binding to human Fc{gamma}RIIIa (CD16a), one of the most potent receptors for eliciting antibody-dependent cellular cytotoxicity (ADCC). Taken together, the discovery of a unique ssOST from D. marinus provides previously unavailable biocatalytic capabilities to the bacterial glycoprotein engineering toolbox and opens the door to using E. coli for the production and glycoengineering of human IgGs and fragments derived thereof.

synthetic biology↗

bioRxiv2023

Ghost fragment ions in structure and site-specific glycoproteomics analysis

Mass spectrometry (MS) can unlock crucial insights into the intricate world of glycosylation analysis. Despite its immense potential, the qualitative and quantitative analysis of isobaric glycopeptide structures remains one of the most daunting hurdles in the field of glycoproteomics. The ability to distinguish between these complex glycan structures poses a significant challenge, hindering our ability to accurately measure and understand the role of glycoproteins in biological systems. A few recent publications described the use of collision energy (CE) modulation to improve structural elucidation, especially for qualitative purposes. Different linkages of glycan units usually demonstrate different stabilities under CID/HCD fragmentation conditions. Fragmentation of the glycan moiety produces low molecular weight ions (oxonium ions) that can serve as a structure-specific signature for specific glycan moieties, however, specificity of these fragments has never been examined closely. Here, we investigated fragmentation specificity using synthetic stable isotope-labelled glycopeptide standards. These standards were isotopically labelled at the reducing terminal GlcNAc, which allowed us to resolve fragments produced by oligomannose core moiety and fragments generated from outer antennary structures. Our research identified the potential for false positive structure assignments due to the occurrence of "Ghost" fragments resulting from single glyco unit rearrangement or mannose core fragmentation within the collision cell. To mitigate this issue, we have established a minimal intensity threshold for these fragments to prevent the misidentification of structure-specific fragments in glycoproteomics analysis. Our findings provide a crucial step forward in the quest for more accurate and reliable glycoproteomics measurements. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/541150v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@1a5bb21org.highwire.dtl.DTLVardef@1a09b5aorg.highwire.dtl.DTLVardef@137504borg.highwire.dtl.DTLVardef@78831e_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2023

Structure of a fungal 1,3-β-glucan synthase

1,3-{beta}-Glucan is the major component of the fungal cell wall and is synthesized by 1,3-{beta}-glucan synthase located in the plasma membrane, which is a molecular target of anti-fungal drugs echinocandins and the triterpenoid ibrexafungerp. In this study, we report the 3.0-[A] resolution cryo-EM structure of Saccharomyces cerevisiae 1,3-{beta}-glucan synthase, Fks1. The structure reveals a central catalytic region adopting a cellulose synthase fold with a cytosolic conserved GT-A type glycosyltransferase domain and a closed transmembrane glucan-transporting channel. We found that two extracellular disulfide bonds are crucial for Fks1 enzymatic activity. Structural comparison between Fks1 and cellulose synthases and structure-guided mutagenesis studies provided novel insights into the molecular mechanisms of the fungal 1,3-{beta}-glucan synthase.

biophysics↗

bioRxiv2022

LC-MS/MS-PRM Quantification of IgG glycoforms using stable isotope labeled IgG1 Fc glycopeptide standard

Targeted quantification of proteins is a standard methodology with broad utility, but targeted quantification of glycoproteins has not reached its full potential. The lack of optimized workflows and isotopically labeled standards limits the acceptance of glycoproteomics quantification. In this paper, we introduce an efficient and streamlined chemoenzymatic synthesis of a library of isotopically labeled glycopeptides of IgG1 which we use for quantification in an energy optimized LC-MS/MS-PRM workflow. Incorporation of the stable isotope labeled N-acetylglucosamine enables an efficient monitoring of all major fragment ions of the glycopeptides generated under the soft collision induced dissociation (CID) conditions which reduces the CVs of the quantification to 0.7-2.8%. Our results document, for the first time, that the workflow using a combination of stable isotope labeled standards with intra-scan normalization enables quantification of the glycopeptides by an electron transfer dissociation (ETD) workflow as well as the CID workflow with the highest sensitivity compared to traditional workflows., This was exemplified by a rapid quantification (13-minute) of IgG1 Fc glycoforms from COVID-19 patients. Graphic Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=115 SRC="FIGDIR/small/501850v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@b5b18corg.highwire.dtl.DTLVardef@9b87b9org.highwire.dtl.DTLVardef@eba7ecorg.highwire.dtl.DTLVardef@15da596_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2021

An intranasally administrated SARS-CoV-2 beta variant subunit booster vaccine prevents beta variant viral replication in rhesus macaques

Emerging of SARS-CoV-2 variants and waning of vaccine/infection-induced immunity poses threats to curbing the COVID-19 pandemic. An effective, safe, and convenient booster vaccine will be needed. We hypothesized that a variant-modified mucosal booster vaccine might induce local immunity to prevent SARS-CoV-2 infection at the port of entry. The beta-variant is hardest to cross-neutralize. Herein we assessed the protective efficacy of an intranasal booster composed of beta variant-spike protein S1 with IL-15 and TLR agonists in previously immunized macaques. The macaques were first vaccinated with Wuhan strain S1 with the same adjuvant. One year later, negligibly detectable SARS-CoV-2-specific antibody remained. Nevertheless, the booster induced vigorous humoral immunity including serum- and bronchoalveolar lavage (BAL)-IgG, secretory nasal- and BAL-IgA, and neutralizing antibody against the original strain and/or beta variant. Beta-variant S1-specifc CD4+ and CD8+ T cell responses were also elicited in PBMC and BAL. Following SARS-CoV-2 beta variant challenge, the vaccinated group demonstrated significant protection against viral replication in the upper and lower respiratory tracts, with almost full protection in the nasal cavity. The fact that one intranasal beta-variant booster administrated one year after the first vaccination provoked protective immunity against beta variant infections may inform future SARS-CoV-2 booster design and administration timing.

immunology↗

bioRxiv2020

FcRn, but not FcgRs, drives maternal-fetal transplacental transport of human IgGantibodies

The IgG Fc domain has the capacity to interact with diverse types of receptors, including FcRn and Fc{gamma}Rs, which confer pleiotropic biological activities. Whereas FcRn regulates IgG epithelial transport and recycling, Fc effector activities, such as ADCC and phagocytosis are mediated by Fc{gamma}Rs, which upon crosslinking transduce signals that modulate the function of effector leukocytes. Despite the well-defined and non-overlapping functional properties of FcRn and Fc{gamma}Rs, recent studies have suggested that Fc{gamma}Rs mediate transplacental IgG transport, as certain Fc glycoforms were reported to be enriched in fetal circulation. To determine the contribution of Fc{gamma}Rs and FcRn to the maternal-fetal transport of IgG, we characterized the IgG Fc glycosylation in paired maternal-fetal samples from patient cohorts from Uganda and Nicaragua. No differences in IgG1 Fc glycan profiles and minimal differences in IgG2 Fc glycans were noted, whereas the presence or absence of galactose on the Fc glycan of IgG1 did not alter Fc{gamma}RIIIA or FcRn binding, half-life, or their ability to deplete target cells in Fc{gamma}R/FcRn humanized mice. Modeling maternal/fetal transport in Fc{gamma}R/FcRn humanized mice confirmed that only FcRn contributed to transplacental transport of IgG; IgG selectively enhanced for FcRn binding resulted in enhanced accumulation of maternal antibody in the fetus. In contrast, enhancing Fc{gamma}RIIIA binding did not result in enhanced maternal/fetal transport. These results argue against a role for Fc{gamma}Rs in IgG transplacental transport, suggesting Fc engineering of maternally administered antibody to only enhance FcRn binding as a means to improve maternal/fetal transport of IgG. Significance StatementTransport of IgG antibodies from the maternal to the fetal circulation is a key process for neonatal immunity, as neonates cannot sufficiently generate IgG antibodies to reach protective levels during the first months after birth. In humans and other primates, maternal to fetal transport of IgG antibodies is largely mediated through the placental tissue. FcRn has been previously identified as the major driver of IgG transplacental transport. Here we examined whether other receptors, such as Fc{gamma}Rs, also contribute to the maternal-fetal IgG transfer. By characterizing the Fc domain structure of paired maternal-fetal IgG samples and modeling transplacental IgG transport in genetically engineered mouse strains, we determined that FcRn, but not Fc{gamma}Rs, is the major receptor that mediates transplacental IgG transport.

immunology↗