Unique Glycoforms Research Articles

The biosynthesis of asiaticoside and madecassoside reveals tandem duplication–directed evolution of glycoside glycosyltransferases in the Apiales

Certain plant species within the Apiales order accumulate triterpenoid saponins that feature a distinctive glucose-glucose-rhamnose (G-G-R) sugar chain attached at the C-28 position of the pentacyclic triterpene skeleton. Until recently, the genomic basis underlying the biosynthesis and evolution of this sugar chain has remained elusive. In this study, we identified two novel glycoside glycosyltransferases (GGTs) that can sequentially install the sugar chain’s second D-glucose and third L-rhamnose during the biosynthesis of asiaticoside and madecassoside, two representative G-G-R sugar chain–containing triterpenoid saponins produced by Centella asiatica. Enzymatic assays revealed the remarkable substrate promiscuity of the two GGTs and the key residues crucial for sugar-donor selectivity of the glucosyltransferase and rhamnosyltransferase. We further identified syntenic tandem gene duplicates of the two GGTs in the Apiaceae and Araliaceae families, suggesting a well-conserved genomic basis underlying sugar chain assembly that likely has evolved in the early ancestors of the Apiales order. Moreover, expression patterns of the two GGTs in pierced leaves of C. asiatica were found to be correlated with the production of asiaticoside and madecassoside, implying their involvement in host defense against herbivores and pathogens. Our work sheds light on the biosynthesis and evolution of complex saponin sugars, paving the way for future engineering of diverse bioactive triterpenoids with unique glycoforms.

Identification of Core-Fucosylated Glycoproteins by Single-Step Truncation of N-Glycans.

Alpha-1,6 core fucosylation (CF) is a unique glycoform of N-glycans, and studies showed that CF modifications are involved in the occurrence and progression of various diseases and may provide potential disease biomarkers. Current strategies for the CF glycoproteome are often based on multistep enrichment of glycoproteins or glycopeptides and sequential cleavage with different glycosidases to truncate the N-glycans. Although the detection ability of low-abundance glycoproteins is improved, sample loss, high cost, and the time-consuming multistep operation also affect the reproducibility of results and the practicality of the method. Here we developed a single-step truncation (SST) strategy and evaluated its potential for the CF glycoproteome of human serum. The SST strategy has the advantages of fewer operational steps, lower cost, higher number of identifications, and better quantitative stability compared with previous approaches and provides an efficient solution for large-scale quantitative analysis of the CF glycoproteome. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Single-step truncation strategy for core fucosylation glycoproteome analysis in human serum Basic Protocol 2: Liquid chromatography-tandem mass spectrometry quantification of site-specific core fucosylation glycopeptides Alternate Protocol: Pretreatment of cellular samples of core fucosylation glycoproteome with single-step truncation strategy.

Plasma glycoproteomics delivers high-specificity disease biomarkers by detecting site-specific glycosylation abnormalities

IntroductionThe human plasma glycoproteome holds enormous potential to identify personalized biomarkers for diagnostics. Glycoproteomics has matured into a technology for plasma N-glycoproteome analysis but further evolution towards clinical applications depends on the clinical validity and understanding of protein- and site-specific glycosylation changes in disease. ObjectivesHere, we exploited the uniqueness of a patient cohort of genetic defects in well-defined glycosylation pathways to assess the clinical applicability of plasma N-glycoproteomics. MethodsComparative glycoproteomics was performed of blood plasma from 40 controls and 74 patients with 13 different genetic diseases that impact the protein N-glycosylation pathway. Baseline glycosylation in healthy individuals was compared to reference glycome and intact transferrin protein mass spectrometry data. Use of glycoproteomics data for biomarker discovery and sample stratification was evaluated by multivariate chemometrics and supervised machine learning. Clinical relevance of site-specific glycosylation changes were evaluated in the context of genetic defects that lead to distinct accumulation or loss of specific glycans. Integrated analysis of site-specific glycoproteome changes in disease was performed using chord diagrams and correlated with intact transferrin protein mass spectrometry data. ResultsGlycoproteomics identified 191 unique glycoforms from 58 unique peptide sequences of 34 plasma glycoproteins that span over 3 magnitudes of abundance in plasma. Chemometrics identified high-specificity biomarker signatures for each of the individual genetic defects with better stratification performance than the current diagnostic standard method. Bioinformatic analyses revealed site-specific glycosylation differences that could be explained by underlying glycobiology and protein-intrinsic factors. ConclusionOur work illustrates the strong potential of plasma glycoproteomics to significantly increase specificity of glycoprotein biomarkers with direct insights in site-specific glycosylation changes to better understand the glycobiological mechanisms underlying human disease.

Unique Glycoform-Dependent Monoclonal Antibodies for Mouse Mucin 21.

Mucin 21(Muc21)/epiglycanin is expressed on apical surfaces of squamous epithelia and has potentially protective roles, which are thought to be associated with its unique glycoforms, whereas its aberrant glycosylation is implicated in the malignant behaviors of some carcinomas. Despite the importance of glycoforms, we lack tools to detect specific glycoforms of mouse Muc21. In this study, we generated two monoclonal antibodies (mAbs) that recognize different glycoforms of Muc21. We used membrane lysates of Muc21-expressing TA3-Ha cells or Chinese hamster ovary (CHO)-K1 cells transfected with Muc21 as antigens. Specificity testing, utilizing Muc21 glycosylation variant cells, showed that mAb 1A4-1 recognized Muc21 carrying glycans terminated with galactose residues, whereas mAb 18A11 recognized Muc21 carrying sialylated glycans. mAb 1A4-1 stained a majority of mouse mammary carcinoma TA3-Ha cells in vitro and in engrafted tumors in mice, whereas mAb 18A11 recognized only a subpopulation of these. mAb 1A4-1 was useful in immunohistochemically detecting Muc21 in normal squamous epithelia. In conclusion, these mAbs recognize distinct Muc21 epitopes formed by combinations of peptide portions and O-glycans.

Simultaneous enrichment and separation of neutral and sialyl glycopeptides of SARS-CoV-2 spike protein enabled by dual-functionalized Ti-IMAC material.

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) presents a serious threat to human health all over the world. The development of effective vaccines has been focusing on the spike (S) glycoprotein, which mediates viral invasion to human cells through its interaction with the angiotensin-converting enzyme 2 (ACE2) receptor. In this work, we perform analytical characterization of N- and O-linked glycosylation of the SARS-CoV-2 S glycoprotein. We explore the novel use of dual-functionalized titanium (IV)-immobilized metal affinity chromatography (Ti-IMAC) material for simultaneous enrichment and separation of neutral and sialyl glycopeptides of a recombinant SARS-CoV-2 S glycoprotein from HEK293 cells. This strategy helps eliminate signal suppression from neutral glycopeptides for the detection of sialyl glycopeptides and improves the glycoform coverage of the S protein. We profiled 19 of its 22 potential N-glycosylated sites with 398 unique glycoforms using the dual-functional Ti-IMAC approach, which exhibited improvement of coverage by 1.6-fold compared to the conventional hydrophilic interaction chromatography (HILIC) glycopeptide enrichment method. We also identified O-linked glycosylation site that was not found using the conventional HILIC approach. In addition, we reported on the identification of mannose-6-phosphate (M6P) glycosylation, which substantially expands the current knowledge of the spike protein’s glycosylation landscape and enables future investigation into the influence of M6P glycosylation of the spike protein on its cell entry.Graphical abstract Supplementary InformationThe online version contains supplementary material available at 10.1007/s00216-021-03433-1.

A streamlined pipeline for multiplexed quantitative site-specific N-glycoproteomics

Regulation of protein N-glycosylation is essential in human cells. However, large-scale, accurate, and site-specific quantification of glycosylation is still technically challenging. We here introduce SugarQuant, an integrated mass spectrometry-based pipeline comprising protein aggregation capture (PAC)-based sample preparation, multi-notch MS3 acquisition (Glyco-SPS-MS3) and a data-processing tool (GlycoBinder) that enables confident identification and quantification of intact glycopeptides in complex biological samples. PAC significantly reduces sample-handling time without compromising sensitivity. Glyco-SPS-MS3 combines high-resolution MS2 and MS3 scans, resulting in enhanced reporter signals of isobaric mass tags, improved detection of N-glycopeptide fragments, and lowered interference in multiplexed quantification. GlycoBinder enables streamlined processing of Glyco-SPS-MS3 data, followed by a two-step database search, which increases the identification rates of glycopeptides by 22% compared with conventional strategies. We apply SugarQuant to identify and quantify more than 5,000 unique glycoforms in Burkitt’s lymphoma cells, and determine site-specific glycosylation changes that occurred upon inhibition of fucosylation at high confidence.

Development of Systems for the Production of Plant-Derived Biopharmaceuticals.

Over the last several decades, plants have been developed as a platform for the production of useful recombinant proteins due to a number of advantages, including rapid production and scalability, the ability to produce unique glycoforms, and the intrinsic safety of food crops. The expression methods used to produce target proteins are divided into stable and transient systems depending on applications that use whole plants or minimally processed forms. In the early stages of research, stable expression systems were mostly used; however, in recent years, transient expression systems have been preferred. The production of the plant itself, which produces recombinant proteins, is currently divided into two major approaches, open-field cultivation and closed-indoor systems. The latter encompasses such regimes as greenhouses, vertical farming units, cell bioreactors, and hydroponic systems. Various aspects of each system will be discussed in this review, which focuses mainly on practical examples and commercially feasible approaches.

Frequent Detection of Pituitary-Derived PrPres in Human Prion Diseases.

Human prion diseases including sporadic Creutzfeldt-Jakob disease (sCJD), inherited prion diseases, and acquired human prion diseases are lethal neurodegenerative diseases. One of the major sources of iatrogenic Creutzfeldt-Jakob disease was human growth hormone (hGH-iCJD) derived from contaminated cadaveric pituitaries. The incidence of hGH-iCJD has decreased since changing from growth hormone extracted from human cadaveric pituitaries to recombinant pituitary hormones. However, extensive analysis on the localization and detecting of abnormal prion protein in the pituitary gland are limited. In this study, we examined 9 autopsied brains and pituitary glands from 6 patients with prion disease (3 Gerstmann-Sträussler-Scheinker disease, 2 sCJD, and 1 dura mater graft-associated CJD) and 3 individuals with nonprion diseases. Western blot analysis of pituitary samples demonstrated unique glycoforms of normal cellular prion protein with molecular weights of 30-40 kDa, which was higher than the typical 25-35 kDa prion protein in brains. Proteomic analysis also revealed prion protein approximately the molecular weight of 40 kDa in pituitary samples. Moreover, proteinase K-resistant Prion protein was frequently detected in pituitary samples of the prion diseases. Immunohistochemistry for Prion protein revealed mosaic cellular distribution preferentially in growth hormone- or prolactin-producing cells.

Altered N-Linked Glycosylation in Follicular Lymphoma and Chronic Lymphocytic Leukemia: Involvement in Pathogenesis and Potential Therapeutic Targeting.

B-cell antigen receptor (BCR) expression is indispensable for survival of most B-cell malignancies. In follicular lymphoma (FL), N-linked glycosylation sites are introduced in the immunoglobulin (Ig) variable region genes. Oligosaccharides added to the acquired sites are unusually of the high-mannose type. These glycans interact with mannose-specific lectins, especially with dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN). Lectin binding to FL triggers persistent activating signals, suggesting that lectins within the tumor microenvironment promote cell survival and proliferation. Insertion of N-glycosylation sites in Ig variable region genes has been detected in other germinal center-associated lymphomas, specifically in subsets of diffuse large B-cell lymphomas and Burkitt’s lymphomas, suggesting involvement of altered glycans in pathogenesis of these malignancies as well. Furthermore, the BCR in chronic lymphocytic leukemia (CLL) carries high-mannose oligosaccharides, albeit in the heavy chain constant rather than variable region. The high expression level of the unique glycoform, particularly in the more aggressive unmutated CLL subset, suggests a functional significance for this glycan in CLL. As lectin interaction with the BCR is critical for FL and probably for some other lymphomas, targeting this interaction is considered to be an interesting therapeutic strategy. Reagents for blockade of lectin–BCR interaction may include antibodies against high-mannose glycans and mannose-based oligosaccharide mimics or non-carbohydrate glycomimetics. Moreover, as this interaction triggers signaling pathways similar to those demonstrated for BCR engagement by antigen, BCR signal transduction inhibitors may emerge as effective therapeutics for lectin-driven malignancies.

Glycoprotein profiling by electrospray mass spectrometry

This work compares several different methods of site-specific analysis of glycoproteins using electrospray mass spectrometry. The glycoprotein, oLHα (ovine luteinizing hormone, α-subunit) was chosen as an appropriate example protein for these studies because of its biological relevance and extreme microheterogeneity. More than 20 unique glycoforms were detected for this glycoprotein at the Asn 56 site of oLHα. The carbohydrates present at this site affect receptor binding affinity, so understanding the great variety in the composition of these carbohydrates is important in studying ligand binding interactions. MS data was acquired on a quadrupole ion trap, a triple quadrupole, and a quadrupole time of flight mass spectrometer, and carbohydrate composition at the Asn 56 site of oLHα was determined using these instruments. Additionally, neutral loss and precursor ion scanning modes were also used to identify the glycoforms present, and these techniques were compared to the standard MS data. Of the three instruments compared in the study, the qTOF mass spectrometer achieved the lowest sample consumption, but all three instruments were useful in profiling the glycopeptide composition.

Proteomic and Postproteomic Characterization of Keratan Sulfate-Glycanated Isoforms of Thyroglobulin and Transferrin Uniquely Elaborated by Papillary Thyroid Carcinomas

Previous studies have suggested that surface components of papillary thyroid carcinoma (PTC) cells may be aberrantly glycanated, but the precise nature of these molecules has not been unveiled nor documented to be of clinical relevance. A monoclonal antibody was raised against a unique keratan sulfate (KS) determinant and used to differentially screen benign and malignant thyroid tissue for the expression of components carrying these moieties. In a total of 349 cases of benign and malignant thyroid lesions, 100% of the 115 PTC cases examined (including various histological subtypes) were found to contain KS-bearing molecules, whereas these were virtually absent from benign tissues and other thyroid tumors, with the exception of 21% of the follicular carcinoma cases analyzed. A composite immunoaffinity chromatography, immunochemistry, and mass spectrometric approach revealed that the PTC-specific KS-bearing macromolecules were unique glycoforms of thyroglobulin and transferrin. Combined, reciprocal immunoprecipitation and Western blotting further indicated that the former glycoform predominated and that most of the transferrin produced by PTC was glycanated with KS moieties. Fluorescent keratanase II-based fingerprinting of the KS moieties bound to these isoforms further demonstrated several PTC-specific peculiarities: 1) that a considerable portion of the moieties was covalently attached via a novel core protein linkage structure; 2) they had an unusual extended average length; 3) an unusual relative ratio of highly sulfated disaccharides terminating with alpha (2-3)-linked N-acetylneuraminic acid capping residues; and 4) a novel unidentified oligosaccharide moiety at the nonreducing terminus. Comparative analysis of the relative distribution of transferrin in benign versus PTC tissues highlighted a marked malignancy-associated abundance of the molecule, with a >75% frequency in expression in PTC. These findings demonstrate that PTC cells synthesize unique post-translationally modified thyroglobulin and transferrin variants in situ that may be directly exploitable for diagnosis, through histological and noninvasive cytological procedures; for devising novel strategies for antibody-guided imaging of this tumor in vivo; and for postsurgery follow-up of PTC patients.

CD34 expression on platelets

Flow cytometric analysis of human platelets from healthy volunteers by high-resolution immunophenotyping accomplished with enzymatic amplification staining revealed the expression of CD34. Assessment of the platelets with five different monoclonal antibodies to the three different classes of CD34 epitopes suggested that platelets express a unique glycoform of the molecule. Expression of CD34 was verified by immunoblotting. Platelet activation resulted in a marked decrease in surface CD34. CD34 expression has significant implications for the function of platelets as well as for the determination of hematopoietic progenitor cells.

Identification of the Ulex europaeus agglutinin-I-binding protein as a unique glycoform of the neural cell adhesion molecule in the olfactory sensory axons of adult rats

Histochemical localization of two lectins, Ulex europaeus agglutinin-I (UEA-I) and Tetragonolobus purpureus (TPA), was studied in the olfactory bulb of adult rats. In contrast to TPA, UEA-I detected a fucosylated glycoprotein that is only present in the surface membranes of olfactory sensory cells including the whole course of their neurites up to the final arborization in glomeruli. Immunoblotting revealed that UEA-I binds specifically to a protein of 205 kDa, while TPA stains several other glycoproteins. Affinity chromatography with the use of a UEA-I column identified the 205 kDa protein as a glycoform of neural cell adhesion molecule (N-CAM), specific for the rat olfactory sensory nerves.