Publications by authors named "Stuart M Haslam"

158 Publications

A mutation in SLC37A4 causes a dominantly inherited congenital disorder of glycosylation characterized by liver dysfunction.

Am J Hum Genet 2021 Jun 7;108(6):1040-1052. Epub 2021 May 7.

Complex Carbohydrate Research Center, Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602, USA.

SLC37A4 encodes an endoplasmic reticulum (ER)-localized multitransmembrane protein required for transporting glucose-6-phosphate (Glc-6P) into the ER. Once transported into the ER, Glc-6P is subsequently hydrolyzed by tissue-specific phosphatases to glucose and inorganic phosphate during times of glucose depletion. Pathogenic variants in SLC37A4 cause an established recessive disorder known as glycogen storage disorder 1b characterized by liver and kidney dysfunction with neutropenia. We report seven individuals who presented with liver dysfunction multifactorial coagulation deficiency and cardiac issues and were heterozygous for the same variant, c.1267C>T (p.Arg423), in SLC37A4; the affected individuals were from four unrelated families. Serum samples from affected individuals showed profound accumulation of both high mannose and hybrid type N-glycans, while N-glycans in fibroblasts and undifferentiated iPSC were normal. Due to the liver-specific nature of this disorder, we generated a CRISPR base-edited hepatoma cell line harboring the c.1267C>T (p.Arg423) variant. These cells replicated the secreted abnormalities seen in serum N-glycosylation, and a portion of the mutant protein appears to relocate to a distinct, non-Golgi compartment, possibly ER exit sites. These cells also show a gene dosage-dependent alteration in the Golgi morphology and reduced intraluminal pH that may account for the altered glycosylation. In summary, we identify a recurrent mutation in SLC37A4 that causes a dominantly inherited congenital disorder of glycosylation characterized by coagulopathy and liver dysfunction with abnormal serum N-glycans.
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http://dx.doi.org/10.1016/j.ajhg.2021.04.013DOI Listing
June 2021

Loss of α2-6 sialylation promotes the transformation of synovial fibroblasts into a pro-inflammatory phenotype in arthritis.

Nat Commun 2021 04 20;12(1):2343. Epub 2021 Apr 20.

Institute of Infection, Immunity and Inflammation, University of Glasgow, Glasgow, UK.

In healthy joints, synovial fibroblasts (SFs) provide the microenvironment required to mediate homeostasis, but these cells adopt a pathological function in rheumatoid arthritis (RA). Carbohydrates (glycans) on cell surfaces are fundamental regulators of the interactions between stromal and immune cells, but little is known about the role of the SF glycome in joint inflammation. Here we study stromal guided pathophysiology by mapping SFs glycosylation pathways. Combining transcriptomic and glycomic analysis, we show that transformation of fibroblasts into pro-inflammatory cells is associated with glycan remodeling, a process that involves TNF-dependent inhibition of the glycosyltransferase ST6Gal1 and α2-6 sialylation. SF sialylation correlates with distinct functional subsets in murine experimental arthritis and remission stages in human RA. We propose that pro-inflammatory cytokines remodel the SF-glycome, converting the synovium into an under-sialylated and highly pro-inflammatory microenvironment. These results highlight the importance of glycosylation in stromal immunology and joint inflammation.
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http://dx.doi.org/10.1038/s41467-021-22365-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8058094PMC
April 2021

Red blood cell mannoses as phagocytic ligands mediating both sickle cell anaemia and malaria resistance.

Nat Commun 2021 03 19;12(1):1792. Epub 2021 Mar 19.

School of Medicine, Medical Sciences and Nutrition, University of Aberdeen, Aberdeen, UK.

In both sickle cell disease and malaria, red blood cells (RBCs) are phagocytosed in the spleen, but receptor-ligand pairs mediating uptake have not been identified. Here, we report that patches of high mannose N-glycans (ManGlcNAc), expressed on diseased or oxidized RBC surfaces, bind the mannose receptor (CD206) on phagocytes to mediate clearance. We find that extravascular hemolysis in sickle cell disease correlates with high mannose glycan levels on RBCs. Furthermore, Plasmodium falciparum-infected RBCs expose surface mannose N-glycans, which occur at significantly higher levels on infected RBCs from sickle cell trait subjects compared to those lacking hemoglobin S. The glycans are associated with high molecular weight complexes and protease-resistant, lower molecular weight fragments containing spectrin. Recognition of surface N-linked high mannose glycans as a response to cellular stress is a molecular mechanism common to both the pathogenesis of sickle cell disease and resistance to severe malaria in sickle cell trait.
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http://dx.doi.org/10.1038/s41467-021-21814-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7979802PMC
March 2021

Glycoengineering Chinese hamster ovary cells: a short history.

Biochem Soc Trans 2021 Apr;49(2):915-931

Department of Chemical Engineering, Imperial College London, London SW7 2AZ, U.K.

Biotherapeutic glycoproteins have revolutionised the field of pharmaceuticals, with new discoveries and continuous improvements underpinning the rapid growth of this industry. N-glycosylation is a critical quality attribute of biotherapeutic glycoproteins that influences the efficacy, half-life and immunogenicity of these drugs. This review will focus on the advances and future directions of remodelling N-glycosylation in Chinese hamster ovary (CHO) cells, which are the workhorse of recombinant biotherapeutic production, with particular emphasis on antibody products, using strategies such as cell line and protein backbone engineering.
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http://dx.doi.org/10.1042/BST20200840DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8106501PMC
April 2021

Efficient inhibition of O-glycan biosynthesis using the hexosamine analog AcGalNTGc.

Cell Chem Biol 2021 May 19;28(5):699-710.e5. Epub 2021 Feb 19.

Department of Chemical and Biological Engineering, State University of New York, 906 Furnas Hall, Buffalo, NY, USA; Department of Medicine, State University of New York, Buffalo, NY, USA. Electronic address:

There is a critical need to develop small-molecule inhibitors of mucin-type O-linked glycosylation. The best-known reagent currently is benzyl-GalNAc, but it is effective only at millimolar concentrations. This article demonstrates that AcGalNTGc, a peracetylated C-2 sulfhydryl-substituted GalNAc, fulfills this unmet need. When added to cultured leukocytes, breast cells, and prostate cells, AcGalNTGc increased cell-surface VVA binding by ∼10-fold, indicating truncation of O-glycan biosynthesis. Cytometry, mass spectrometry, and western blot analysis of HL-60 promyelocytes demonstrated that 50-80 μM AcGalNTGc prevented elaboration of 30%-60% of the O-glycans beyond the Tn-antigen (GalNAcα1-Ser/Thr) stage. The effect of the compound on N-glycans and glycosphingolipids was small. Glycan inhibition induced by AcGalNTGc resulted in 50%-80% reduction in leukocyte sialyl-Lewis X expression and L-/P-selectin-mediated rolling under flow conditions. AcGalNTGc was pharmacologically active in mouse. It reduced neutrophil infiltration to sites of inflammation by ∼60%. Overall, AcGalNTGc may find diverse applications as a potent inhibitor of O-glycosylation.
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http://dx.doi.org/10.1016/j.chembiol.2021.01.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8140985PMC
May 2021

Vulpeculin: a novel and abundant lipocalin in the urine of the common brushtail possum, .

Open Biol 2020 10 7;10(10):200218. Epub 2020 Oct 7.

Centre for Proteome Research, Institute of Systems, Molecular and Integrative Biology, University of Liverpool, Liverpool L69 7ZB, UK.

Lipocalins are a family of secreted proteins. They are capable of binding small lipophilic compounds and have been extensively studied for their role in chemosignalling in rodent urine. Urine of the common brushtail possum () contains a prominent glycoprotein of 20 kDa, expressed in both sexes. We have isolated this protein and determined its primary sequence by mass spectrometry, including the use of metabolic labelling to resolve the leucine/isoleucine isobaric ambiguity. The protein sequence was identified as a lipocalin, and phylogenetic analysis grouped the protein with other marsupial lipocalin sequences in a phylogenetic clade distinct from established cross-species lipocalin sub-families. The pattern of expression in possum urine and the similarity in sequence and structure to other lipocalins suggests this protein may have a role in brushtail possum chemosignalling.
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http://dx.doi.org/10.1098/rsob.200218DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7653361PMC
October 2020

Glycosylation of Trypanosoma cruzi TcI antigen reveals recognition by chagasic sera.

Sci Rep 2020 10 2;10(1):16395. Epub 2020 Oct 2.

Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, UK.

Chagas disease is considered the most important parasitic disease in Latin America. The protozoan agent, Trypanosoma cruzi, comprises six genetic lineages, TcI-TcVI. Genotyping to link lineage(s) to severity of cardiomyopathy and gastrointestinal pathology is impeded by the sequestration and replication of T. cruzi in host tissues. We describe serology specific for TcI, the predominant lineage north of the Amazon, based on expression of recombinant trypomastigote small surface antigen (gTSSA-I) in the eukaryote Leishmania tarentolae, to allow realistic glycosylation and structure of the antigen. Sera from TcI-endemic regions recognised gTSSA-I (74/146; 50.7%), with no cross reaction with common components of gTSSA-II/V/VI recombinant antigen. Antigenicity was abolished by chemical (periodate) oxidation of gTSSA-I glycosylation but retained after heat-denaturation of conformation. Conversely, non-specific recognition of gTSSA-I by non-endemic malaria sera was abolished by heat-denaturation. TcI-specific serology facilitates investigation between lineage and diverse clinical presentations. Glycosylation cannot be ignored in the search for immunogenic antigens.
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http://dx.doi.org/10.1038/s41598-020-73390-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7532467PMC
October 2020

Metabolic precision labeling enables selective probing of O-linked -acetylgalactosamine glycosylation.

Proc Natl Acad Sci U S A 2020 10 28;117(41):25293-25301. Epub 2020 Sep 28.

The Chemical Glycobiology Laboratory, The Francis Crick Institute, NW1 1AT London, United Kingdom;

Protein glycosylation events that happen early in the secretory pathway are often dysregulated during tumorigenesis. These events can be probed, in principle, by monosaccharides with bioorthogonal tags that would ideally be specific for distinct glycan subtypes. However, metabolic interconversion into other monosaccharides drastically reduces such specificity in the living cell. Here, we use a structure-based design process to develop the monosaccharide probe -()-azidopropionylgalactosamine (GalNAzMe) that is specific for cancer-relevant Ser/Thr(O)-linked -acetylgalactosamine (GalNAc) glycosylation. By virtue of a branched -acylamide side chain, GalNAzMe is not interconverted by epimerization to the corresponding -acetylglucosamine analog by the epimerase -acetylgalactosamine-4-epimerase (GALE) like conventional GalNAc-based probes. GalNAzMe enters O-GalNAc glycosylation but does not enter other major cell surface glycan types including Asn(N)-linked glycans. We transfect cells with the engineered pyrophosphorylase mut-AGX1 to biosynthesize the nucleotide-sugar donor uridine diphosphate (UDP)-GalNAzMe from a sugar-1-phosphate precursor. Tagged with a bioorthogonal azide group, GalNAzMe serves as an O-glycan-specific reporter in superresolution microscopy, chemical glycoproteomics, a genome-wide CRISPR-knockout (CRISPR-KO) screen, and imaging of intestinal organoids. Additional ectopic expression of an engineered glycosyltransferase, "bump-and-hole" (BH)-GalNAc-T2, boosts labeling in a programmable fashion by increasing incorporation of GalNAzMe into the cell surface glycoproteome. Alleviating the need for GALE-KO cells in metabolic labeling experiments, GalNAzMe is a precision tool that allows a detailed view into the biology of a major type of cancer-relevant protein glycosylation.
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http://dx.doi.org/10.1073/pnas.2007297117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7568240PMC
October 2020

Site-specific characterization of SARS-CoV-2 spike glycoprotein receptor-binding domain.

Glycobiology 2021 04;31(3):181-187

Department of Life Sciences, Imperial College London, London, SW7 2AZ, UK.

The novel coronavirus SARS-CoV-2, the infective agent causing COVID-19, is having a global impact both in terms of human disease as well as socially and economically. Its heavily glycosylated spike glycoprotein is fundamental for the infection process, via its receptor-binding domains interaction with the glycoprotein angiotensin-converting enzyme 2 on human cell surfaces. We therefore utilized an integrated glycomic and glycoproteomic analytical strategy to characterize both N- and O- glycan site-specific glycosylation within the receptor-binding domain. We demonstrate the presence of complex-type N-glycans with unusual fucosylated LacdiNAc at both sites N331 and N343 and a single site of O-glycosylation on T323.
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http://dx.doi.org/10.1093/glycob/cwaa085DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7499654PMC
April 2021

The glycomic sialylation profile of GNE Myopathy muscle cells does not point to consistent hyposialylation of individual glycoconjugates.

Neuromuscul Disord 2020 08 4;30(8):621-630. Epub 2020 Jun 4.

Goldyne Savad Institute of Gene Therapy, Hadassah Hebrew University Medical Center, Jerusalem, Israel. Electronic address:

GNE Myopathy is a recessive neuromuscular disorder characterized by adult-onset, slowly progressive distal and proximal muscle weakness, and a typical muscle pathology. Although GNE, which is the mutated gene in the disease, is well known as the key enzyme in the biosynthesis pathway of sialic acid, the pathophysiological pathway leading from GNE mutations to the muscle phenotype in GNE Myopathy is still unclear. The obvious hypothesis of impaired sialylation in patients' skeletal muscle as the cause of the disease is still controversial. In the present study we have investigated whether a distinctive altered pattern of sialylation in GNE Myopathy cultured muscle cells could be attributed to a specific glycoconjugate. Mass spectrometry based glycomic methodologies have been utilized to assess the sialylation level of protein N- and O-linked glycans and glycolipid derived glycans from patient and matched control samples. No consistent change in sialylation was detected in glycoconjugates. These results suggest potential additional roles for GNE that could account for the disease pathology.
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http://dx.doi.org/10.1016/j.nmd.2020.05.008DOI Listing
August 2020

Role of galectin-glycan circuits in reproduction: from healthy pregnancy to preterm birth (PTB).

Semin Immunopathol 2020 08 29;42(4):469-486. Epub 2020 Jun 29.

Laboratory of Experimental Medicine, Hospital Alemán, School of Medicine, University of Buenos Aires, CONICET, Buenos Aires, Argentina.

Growing evidence suggests that galectins, an evolutionarily conserved family of glycan-binding proteins, fulfill key roles in pregnancy including blastocyst implantation, maternal-fetal immune tolerance, placental development, and maternal vascular expansion, thereby establishing a healthy environment for the growing fetus. In this review, we comprehensively present the function of galectins in shaping cellular circuits that characterize a healthy pregnancy. We describe the current understanding of galectins in term and preterm labor and discuss how the galectin-glycan circuits contribute to key immunological pathways sustaining maternal tolerance and preventing microbial infections. A deeper understanding of the glycoimmune pathways regulating early events in preterm birth could offer the broader translational potential for the treatment of this devastating syndrome.
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http://dx.doi.org/10.1007/s00281-020-00801-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7508936PMC
August 2020

Analysis of N- and O-Linked Glycosylation: Differential Glycosylation after Rat Spinal Cord Injury.

J Neurotrauma 2020 09 26;37(18):1954-1962. Epub 2020 Jun 26.

School of Pharmacy, University of Wyoming, Laramie, Wyoming, USA.

Glycosylation is a fundamental cellular process that has a dramatic impact on the functionality of glycoconjugates such as proteins or lipids and mediates many different biological interactions including cell migration, cellular signaling, and synaptic interactions in the nervous system. In spinal cord injury (SCI), all of these cellular processes are altered, but the potential contributions of glycosylation changes to these alterations has not been thoroughly investigated. We studied the glycosylation of injured spinal cord tissue from rats that received a contusion SCI. The N- and O-linked glycosylation was assessed at 3 and 14 days post-injury (DPI), and compared with uninjured control and time-matched sham spinal tissue. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and tandem MS (MS/MS) were performed to analyze carbohydrate structures. Results revealed diverse and abundant glycosylation in all groups, with some carbohydrate structures differentially produced in SCI animals compared with uninjured controls and shams. One such change occurred in the abundance of the Sda structure, Neu5Ac-α-(2,3)-[GalNAc-β-(1,4)-]Gal-β-(1,4)-GlcNAc, which was increased in SCI samples compared with shams and non-injured controls. Immunohistochemistry (IHC) and western blot were performed on SCI and sham samples using the CT1 antibody, which recognizes the terminal trisaccharide of Sda with high specificity. Both of these metrics confirmed elevated Sda structure in SCI tissue, where IHC further showed that Sda is expressed mainly by microglia. The results of these studies suggest that SCI causes a significant alteration in N- and O-linked glycosylation.
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http://dx.doi.org/10.1089/neu.2019.6974DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7470220PMC
September 2020

Glycan characterization of pregnancy-specific glycoprotein 1 and its identification as a novel Galectin-1 ligand.

Glycobiology 2020 10;30(11):895-909

Department of Pathology, Uniformed Services University, 4301 Jones Bridge Rd, Bethesda, MD 20814, USA.

Pregnancy-specific beta 1 glycoprotein (PSG1) is secreted from trophoblast cells of the human placenta in increasing concentrations as pregnancy progresses, becoming one of the most abundant proteins in maternal serum in the third trimester. PSG1 has seven potential N-linked glycosylation sites across its four domains. We carried out glycomic and glycoproteomic studies to characterize the glycan composition of PSG1 purified from serum of pregnant women and identified the presence of complex N-glycans containing poly LacNAc epitopes with α2,3 sialyation at four sites. Using different techniques, we explored whether PSG1 can bind to galectin-1 (Gal-1) as these two proteins were previously shown to participate in processes required for a successful pregnancy. We confirmed that PSG1 binds to Gal-1 in a carbohydrate-dependent manner with an affinity of the interaction of 0.13 μM. In addition, we determined that out of the three N-glycosylation-carrying domains, only the N and A2 domains of recombinant PSG1 interact with Gal-1. Lastly, we observed that the interaction between PSG1 and Gal-1 protects this lectin from oxidative inactivation and that PSG1 competes the ability of Gal-1 to bind to some but not all of its glycoprotein ligands.
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http://dx.doi.org/10.1093/glycob/cwaa034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7581653PMC
October 2020

Altered glycosylation of glycodelin in endometrial carcinoma.

Lab Invest 2020 07 23;100(7):1014-1025. Epub 2020 Mar 23.

Department of Clinical Chemistry, University of Helsinki and Helsinki University Hospital, Helsinki, Finland.

Glycodelin is a major glycoprotein expressed in reproductive tissues, like secretory and decidualized endometrium. It has several reproduction related functions that are dependent on specific glycosylation, but it has also been found to drive differentiation of endometrial carcinoma cells toward a less malignant phenotype. Here we aimed to elucidate whether the glycosylation and function of glycodelin is altered in endometrial carcinoma as compared with a normal endometrium. We carried out glycan structure analysis of glycodelin expressed in HEC-1B human endometrial carcinoma cells (HEC-1B Gd) by mass spectrometry glycomics strategies. Glycans of HEC-1B Gd were found to comprise a typical mixture of high-mannose, hybrid, and complex-type N-glycans, often containing undecorated LacNAc (Galβ1-4GlcNAc) antennae. However, several differences, as compared with previously reported glycan structures of normal human decidualized endometrium-derived glycodelin isoform, glycodelin-A (GdA), were also found. These included a lower level of sialylation and more abundant poly-LacNAc antennae, some of which are fucosylated. This allowed us to select lectins that showed different binding to these classes of glycodelin. Despite the differences in glycosylation between HEC-1B Gd and GdA, both showed similar inhibitory activity on trophoblast cell invasion and peripheral blood mononuclear cell proliferation. For the detection of cancer associated glycodelin, we established a novel in situ proximity-ligation based histochemical staining method using a specific glycodelin antibody and UEAI lectin. We found that the UEAI reactive glycodelin was abundant in endometrial carcinoma, but virtually absent in normal endometrial tissue even when glycodelin was strongly expressed. In conclusion, we established a histochemical staining method for the detection of endometrial carcinoma-associated glycodelin and showed that this specific glycodelin is exclusively expressed in cancer, not in normal endometrium. Similar methods can be used for studies of other glycoproteins.
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http://dx.doi.org/10.1038/s41374-020-0411-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7312397PMC
July 2020

Discovery of O-Linked Carbohydrate on HIV-1 Envelope and Its Role in Shielding against One Category of Broadly Neutralizing Antibodies.

Cell Rep 2020 02;30(6):1862-1869.e4

Department of Pathology, Miller School of Medicine, University of Miami, Miami, FL, USA. Electronic address:

Approximately 50% of the mass of the Envelope (Env) glycoprotein surface subunit (gp120) of human immunodeficiency virus type 1 (HIV-1) is composed of N-linked carbohydrate. Until now, the dogma has been that HIV-1 lacks O-linked carbohydrate on Env. Here we show that a subset of patient-derived HIV-1 isolates contain O-linked carbohydrate on the variable 1 (V1) domain of Env gp120. We demonstrate the presence of this O-glycosylation both on virions and on gp120 expressed as a secreted protein. Further, we establish that these O-linked glycans can confer a more than 1,000-fold decrease in neutralization sensitivity (IC) to V3-glycan broadly neutralizing antibodies. These findings uncover a structural modification to the HIV-1 Env and suggest a functional role in promoting viral escape from one category of broadly neutralizing antibodies.
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http://dx.doi.org/10.1016/j.celrep.2020.01.056DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7904042PMC
February 2020

Insights into the hyperglycosylation of human chorionic gonadotropin revealed by glycomics analysis.

PLoS One 2020 11;15(2):e0228507. Epub 2020 Feb 11.

Department of Life Sciences, Imperial College London, London, United Kingdom.

Human chorionic gonadotropin (hCG) is a glycoprotein hormone that is essential for the maintenance of pregnancy. Glycosylation of hCG is known to be essential for its biological activity. "Hyperglycosylated" variants secreted during early pregnancy have been proposed to be involved in initial implantation of the embryo and as a potential diagnostic marker for gestational diseases. However, what constitutes "hyperglycosylation" is not yet fully understood. In this study, we perform comparative N-glycomic analysis of hCG expressed in the same individuals during early and late pregnancy to help provide new insights into hCG function, reveal new targets for diagnostics and clarify the identity of hyperglycosylated hCG. hCG was isolated in urine collected from women at 7 weeks and 20 weeks' gestation. hCG was also isolated in urine from women diagnosed with gestational trophoblastic disease (GTD). We used glycomics methodologies including matrix assisted laser desorption/ionisation-time of flight (MALDI-TOF) mass spectrometry (MS) and MS/MS methods to characterise the N-glycans associated with hCG purified from the individual samples. The structures identified on the early pregnancy (EP-hCG) and late pregnancy (LP-hCG) samples corresponded to mono-, bi-, tri-, and tetra-antennary N-glycans. A novel finding was the presence of substantial amounts of bisected type N-glycans in pregnancy hCG samples, which were present at much lower levels in GTD samples. A second novel observation was the presence of abundant LewisX antigens on the bisected N-glycans. GTD-hCG had fewer glycoforms which constituted a subset of those found in normal pregnancy. When compared to EP-hCG, GTD-hCG samples had decreased signals for tri- and tetra-antennary N-glycans. In terms of terminal epitopes, GTD-hCG had increased signals for sialylated structures, while LewisX antigens were of very minor abundance. hCG carries the same N-glycans throughout pregnancy but in different proportions. The N-glycan repertoire is more diverse than previously reported. Bisected and LewisX structures are potential targets for diagnostics. hCG isolated from pregnancy urine inhibits NK cell cytotoxicity in vitro at nanomolar levels and bisected type glycans have previously been implicated in the suppression of NK cell cytotoxicity, suggesting that hCG-related bisected type N-glycans may directly suppress NK cell cytotoxicity.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0228507PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7012436PMC
May 2020

Glycan biomarkers for Alzheimer disease correlate with T-tau and P-tau in cerebrospinal fluid in subjective cognitive impairment.

FEBS J 2020 08 14;287(15):3221-3234. Epub 2020 Jan 14.

Division of Neurogeriatrics, Center for Alzheimer Research, Department of Neurobiology, Care Sciences, and Society, Karolinska Institutet, Solna, Sweden.

Alzheimer disease (AD) is a devastating disease and a global health problem, and current treatments are only symptomatic. A wealth of clinical studies support that the disease starts to develop decades before the first symptoms appear, emphasizing the importance of studying early changes for improving early diagnosis and guiding toward novel treatment strategies. Protein glycosylation is altered in AD but it remains to be clarified why these alterations occur and how they affect the disease development. Here, we used a glycomics approach to search for alterations in protein glycosylation in cerebrospinal fluid (CSF) in AD compared with nondemented controls. Using both matrix-assisted laser desorption ionization-time of flight and liquid chromatography-electrospray mass spectrometry, we observed an increase in N-glycans carrying bisecting N-acetylglucosamine in AD. Based on those findings, we designed an enzyme-linked multiwell plate assay to quantify N-glycans binding to the lectin Phaseolus vulgaris Erythroagglutinin (PHA-E), which is specific for N-glycans containing bisecting N-acetylglucosamine. Using this assay, we found a similar increase in CSF in AD compared with controls. Further analysis of CSF from 242 patients with subjective cognitive impairment (SCI), mild cognitive impairment (MCI), or AD dementia revealed significantly increased binding to PHA-E in MCI and AD compared to SCI. Interestingly, PHA-E binding correlated with CSF levels of phosphorylated tau and total tau and this correlation was most prominent in the SCI group (R = 0.53-0.54). This study supports a link between N-glycosylation, neurodegeneration, and tau pathology in AD and suggests that glycan biomarkers have potential to identify SCI cases at risk of developing AD.
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http://dx.doi.org/10.1111/febs.15197DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7496940PMC
August 2020

East-Asian Helicobacter pylori strains synthesize heptan-deficient lipopolysaccharide.

PLoS Genet 2019 11 20;15(11):e1008497. Epub 2019 Nov 20.

West China Marshall Research Center for Infectious Diseases, Center of Infectious Diseases, Division of Infectious Diseases, State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu, China.

The lipopolysaccharide O-antigen structure expressed by the European Helicobacter pylori model strain G27 encompasses a trisaccharide, an intervening glucan-heptan and distal Lewis antigens that promote immune escape. However, several gaps still remain in the corresponding biosynthetic pathway. Here, systematic mutagenesis of glycosyltransferase genes in G27 combined with lipopolysaccharide structural analysis, uncovered HP0102 as the trisaccharide fucosyltransferase, HP1283 as the heptan transferase, and HP1578 as the GlcNAc transferase that initiates the synthesis of Lewis antigens onto the heptan motif. Comparative genomic analysis of G27 lipopolysaccharide biosynthetic genes in strains of different ethnic origin revealed that East-Asian strains lack the HP1283/HP1578 genes but contain an additional copy of HP1105 and JHP0562. Further correlation of different lipopolysaccharide structures with corresponding gene contents led us to propose that the second copy of HP1105 and the JHP0562 may function as the GlcNAc and Gal transferase, respectively, to initiate synthesis of the Lewis antigen onto the Glc-Trio-Core in East-Asian strains lacking the HP1283/HP1578 genes. In view of the high gastric cancer rate in East Asia, the absence of the HP1283/HP1578 genes in East-Asian H. pylori strains warrants future studies addressing the role of the lipopolysaccharide heptan in pathogenesis.
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http://dx.doi.org/10.1371/journal.pgen.1008497DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6892558PMC
November 2019

Site-specific glycoproteomic characterization of ES-62: The major secreted product of the parasitic worm Acanthocheilonema viteae.

Glycobiology 2019 07;29(8):562-571

Department of Life Sciences, Imperial College London, London SW7 2AZ, United Kingdom.

ES-62 is the major secreted product of the parasitic filarial nematode Acanthocheilonema viteae and has potent anti-inflammatory activities as a consequence of posttranslational decoration by phosphorylcholine (PC). Previously, we showed that ES-62's PC was attached to N-linked glycans, and using fast atom bombardment mass spectrometry, we characterized the structure of the glycans. However, it was unknown at this time which of ES-62's four potential N-glycosylation sites carries the PC-modified glycans. In the present study, we now employ more advanced analytical tools-nano-flow liquid chromatography with high-definition electrospray mass spectrometry-to show that PC-modified glycans are found at all four potential N-glycosylation sites. Also, our earlier studies showed that up to two PC groups were detected per glycan, and we are now able to characterize N-glycans with up to five PC groups. The number per glycan varies in three of the four glycosylation sites, and in addition, for the first time, we have detected PC on the N-glycan chitobiose core in addition to terminal GlcNAc. Nevertheless, the majority of PC is detected on terminal GlcNAc, enabling it to interact with the cells and molecules of the immune system. Such expression may explain the potent immunomodulatory effects of a molecule that is considered to have significant therapeutic potential in the treatment of certain human allergic and autoimmune conditions.
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http://dx.doi.org/10.1093/glycob/cwz035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6639541PMC
July 2019

Serum IgA1 shows increased levels of 2,6-linked sialic acid in breast cancer.

Interface Focus 2019 Apr 15;9(2):20180079. Epub 2019 Feb 15.

School of Life Sciences, University of Westminster, 115 New Cavendish Street, London W1W 6UW, UK.

The lectin agglutinin (HPA) recognizes altered glycosylation in solid cancers and the identification of HPA binding partners in tumour tissue and serum is an important aim. Among the many HPA binding proteins, IgA1 has been reported to be the most abundant in liver metastases. In this study, the glycosylation of IgA1 was evaluated using serum samples from patients with breast cancer (BCa) and the utility of IgA1 glycosylation as a biomarker was assessed. Detailed mass spectrometric structural analysis showed an increase in disialo-biantennary linked glycans on IgA1 from BCa patients ( < 0.0001: non-core fucosylated; = 0.0345: core fucosylated) and increased asialo-Thomsen-Friedenreich antigen (TF) and disialo-TF antigens in the linked glycan preparations from IgA1 of cancer patients compared with healthy control individuals. An increase in binding was observed, suggestive of increased 2,6-linked sialic acid on IgA1 in BCa. Logistic regression analysis showed HPA binding to IgA1 and tumour size to be significant independent predictors of distant metastases ( 13.359; = 114; = 0.020) with positive and negative predictive values of 65.7% and 64.6%, respectively. Immunohistochemical analysis of tumour tissue samples showed IgA1 to be detectable in BCa tissue. This report provides a detailed analysis of serum IgA1 glycosylation in BCa and illustrates the potential utility of IgA1 glycosylation as a biomarker for BCa prognostication.
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http://dx.doi.org/10.1098/rsfs.2018.0079DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6388022PMC
April 2019

Human B Cell Differentiation Is Characterized by Progressive Remodeling of O-Linked Glycans.

Front Immunol 2018 14;9:2857. Epub 2018 Dec 14.

Department of Dermatology, Brigham and Women's Hospital, Boston MA, United States.

Germinal centers (GC) are microanatomical niches where B cells proliferate, undergo antibody affinity maturation, and differentiate to long-lived memory B cells and antibody-secreting plasma cells. For decades, GC B cells have been defined by their reactivity to the plant lectin peanut agglutinin (PNA), which binds serine/threonine (O-linked) glycans containing the asialylated disaccharide Gal-β1,3-GalNAc-Ser/Thr (also called T-antigen). In T cells, acquisition of PNA binding by activated T cells and thymocytes has been linked with altered tissue homing patterns, cell signaling, and survival. Yet, in GC B cells, the glycobiological basis and significance of PNA binding remains surprisingly unresolved. Here, we investigated the basis for PNA reactivity of GC B cells. We found that GC B cell binding to PNA is associated with downregulation of the α2,3 sialyltransferase, (ST3Gal1), and overexpression of ST3Gal1 was sufficient to reverse PNA binding in B cell lines. Moreover, we found that the primary scaffold for PNA-reactive O-glycans in B cells is the B cell receptor-associated receptor-type tyrosine phosphatase CD45, suggesting a role for altered O-glycosylation in antigen receptor signaling. Consistent with similar reports in T cells, ST3Gal1 overexpression in B cells induced drastic shortening in O-glycans, which we confirmed by both antibody staining and mass spectrometric O-glycomic analysis. Unexpectedly, ST3Gal1-induced changes in O-glycan length also correlated with altered binding of two glycosylation-sensitive CD45 antibodies, RA3-6B2 (more commonly called B220) and MEM55, which (in humans) have previously been reported to favor binding to naïve/GC subsets and memory/plasmablast subsets, respectively. Analysis of primary B cell binding to B220, MEM55, and several plant lectins suggested that B cell differentiation is accompanied by significant loss of O-glycan complexity, including loss of extended Core 2 O-glycans. To our surprise, decreased O-glycan length from naïve to post-GC fates best correlated not with ST3Gal1, but rather downregulation of the Core 2 branching enzyme GCNT1. Thus, our data suggest that O-glycan remodeling is a feature of B cell differentiation, dually regulated by ST3Gal1 and GCNT1, that ultimately results in expression of distinct O-glycosylation states/CD45 glycoforms at each stage of B cell differentiation.
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http://dx.doi.org/10.3389/fimmu.2018.02857DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6302748PMC
October 2019

Publisher Correction: Host and viral determinants of influenza A virus species specificity.

Nat Rev Microbiol 2019 Jan;17(2):124

Department of Medicine, Imperial College London, London, UK.

In Figure 4, seasonal influenza virus was erroneously indicated as having "HA α2-3 SA preference" instead of "HA drift from population immunity" to represent ongoing evolution of seasonal influenza virus. This has now been corrected in all versions of the Review. The publisher apologizes to the authors and to readers for this error.
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http://dx.doi.org/10.1038/s41579-018-0140-yDOI Listing
January 2019

Host and viral determinants of influenza A virus species specificity.

Nat Rev Microbiol 2019 01;17(2):67-81

Department of Medicine, Imperial College London, London, UK.

Influenza A viruses cause pandemics when they cross between species and an antigenically novel virus acquires the ability to infect and transmit between these new hosts. The timing of pandemics is currently unpredictable but depends on ecological and virological factors. The host range of an influenza A virus is determined by species-specific interactions between virus and host cell factors. These include the ability to bind and enter cells, to replicate the viral RNA genome within the host cell nucleus, to evade host restriction factors and innate immune responses and to transmit between individuals. In this Review, we examine the host barriers that influenza A viruses of animals, especially birds, must overcome to initiate a pandemic in humans and describe how, on crossing the species barrier, the virus mutates to establish new interactions with the human host. This knowledge is used to inform risk assessments for future pandemics and to identify virus-host interactions that could be targeted by novel intervention strategies.
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http://dx.doi.org/10.1038/s41579-018-0115-zDOI Listing
January 2019

Thioglycosides Are Efficient Metabolic Decoys of Glycosylation that Reduce Selectin Dependent Leukocyte Adhesion.

Cell Chem Biol 2018 12 18;25(12):1519-1532.e5. Epub 2018 Oct 18.

Department of Chemical and Biological Engineering, State University of New York, 906 Furnas Hall, Buffalo, NY 14260, USA; Clinical & Translational Research Center and State University of New York, Buffalo, NY 14260, USA. Electronic address:

Metabolic decoys are synthetic analogs of naturally occurring biosynthetic acceptors. These compounds divert cellular biosynthetic pathways by acting as artificial substrates that usurp the activity of natural enzymes. While O-linked glycosides are common, they are only partially effective even at millimolar concentrations. In contrast, we report that N-acetylglucosamine (GlcNAc) incorporated into various thioglycosides robustly truncate cell surface N- and O-linked glycan biosynthesis at 10-100 μM concentrations. The >10-fold greater inhibition is in part due to the resistance of thioglycosides to hydrolysis by intracellular hexosaminidases. The thioglycosides reduce β-galactose incorporation into lactosamine chains, cell surface sialyl Lewis-X expression, and leukocyte rolling on selectin substrates including inflamed endothelial cells under fluid shear. Treatment of granulocytes with thioglycosides prior to infusion into mouse inhibited neutrophil homing to sites of acute inflammation and bone marrow by ∼80%-90%. Overall, thioglycosides represent an easy to synthesize class of efficient metabolic inhibitors or decoys. They reduce N-/O-linked glycan biosynthesis and inflammatory leukocyte accumulation.
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http://dx.doi.org/10.1016/j.chembiol.2018.09.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6474417PMC
December 2018

The mucinous domain of pancreatic carboxyl-ester lipase (CEL) contains core 1/core 2 glycans that can be modified by ABO blood group determinants.

J Biol Chem 2018 12 12;293(50):19476-19491. Epub 2018 Oct 12.

From the Gade Laboratory for Pathology, Department of Clinical Medicine, University of Bergen, N-5020 Bergen, Norway,

Carboxyl-ester lipase (CEL) is a pancreatic fat-digesting enzyme associated with human disease. Rare mutations in the gene cause a syndrome of pancreatic exocrine and endocrine dysfunction denoted MODY8, whereas a recombined allele increases the risk for chronic pancreatitis. Moreover, CEL has been linked to pancreatic ductal adenocarcinoma (PDAC) through a postulated oncofetal CEL variant termed feto-acinar pancreatic protein (FAPP). The monoclonal antibody mAb16D10 was previously reported to detect a glycotope in the highly glycosylated, mucin-like C terminus of CEL/FAPP. We here assessed the expression of human CEL in malignant pancreatic lesions and cell lines. CEL was not detectably expressed in neoplastic cells, implying that FAPP is unlikely to be a glycoisoform of CEL in pancreatic cancer. Testing of the mAb16D10 antibody in glycan microarrays then demonstrated that it recognized structures containing terminal GalNAc-α1,3(Fuc-α1,2)Gal (blood group A antigen) and also repeated protein sequences containing GalNAc residues linked to Ser/Thr (Tn antigen), findings that were supported by immunostainings of human pancreatic tissue. To examine whether the CEL glycoprotein might be modified by blood group antigens, we used high-sensitivity MALDI-TOF MS to characterize the released glycan pool of CEL immunoprecipitated from human pancreatic juice. We found that the glycome of CEL consisted mainly of core 1/core 2 structures with a composition depending on the subject's and gene polymorphisms. Thus, among digestive enzymes secreted by the pancreas, CEL is a glycoprotein with some unique characteristics, supporting the view that it could serve additional biological functions to its cholesteryl esterase activity in the duodenum.
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http://dx.doi.org/10.1074/jbc.RA118.001934DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6302156PMC
December 2018

XBP1s activation can globally remodel N-glycan structure distribution patterns.

Proc Natl Acad Sci U S A 2018 10 10;115(43):E10089-E10098. Epub 2018 Oct 10.

Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139;

Classically, the unfolded protein response (UPR) safeguards secretory pathway proteostasis. The most ancient arm of the UPR, the IRE1-activated spliced X-box binding protein 1 (XBP1s)-mediated response, has roles in secretory pathway maturation beyond resolving proteostatic stress. Understanding the consequences of XBP1s activation for cellular processes is critical for elucidating mechanistic connections between XBP1s and development, immunity, and disease. Here, we show that a key functional output of XBP1s activation is a cell type-dependent shift in the distribution of N-glycan structures on endogenous membrane and secreted proteomes. For example, XBP1s activity decreased levels of sialylation and bisecting GlcNAc in the HEK293 membrane proteome and secretome, while substantially increasing the population of oligomannose N-glycans only in the secretome. In HeLa cell membranes, stress-independent XBP1s activation increased the population of high-mannose and tetraantennary N-glycans, and also enhanced core fucosylation. mRNA profiling experiments suggest that XBP1s-mediated remodeling of the N-glycome is, at least in part, a consequence of coordinated transcriptional resculpting of N-glycan maturation pathways by XBP1s. The discovery of XBP1s-induced N-glycan structural remodeling on a glycome-wide scale suggests that XBP1s can act as a master regulator of N-glycan maturation. Moreover, because the sugars on cell-surface proteins or on proteins secreted from an XBP1s-activated cell can be molecularly distinct from those of an unactivated cell, these findings reveal a potential new mechanism for translating intracellular stress signaling into altered interactions with the extracellular environment.
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http://dx.doi.org/10.1073/pnas.1805425115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6205500PMC
October 2018

Loss of GCNT2/I-branched glycans enhances melanoma growth and survival.

Nat Commun 2018 08 22;9(1):3368. Epub 2018 Aug 22.

Department of Dermatology, Brigham and Women's Hospital, Boston, MA, 02115, USA.

Cancer cells often display altered cell-surface glycans compared to their nontransformed counterparts. However, functional contributions of glycans to cancer initiation and progression remain poorly understood. Here, from expression-based analyses across cancer lineages, we found that melanomas exhibit significant transcriptional changes in glycosylation-related genes. This gene signature revealed that, compared to normal melanocytes, melanomas downregulate I-branching glycosyltransferase, GCNT2, leading to a loss of cell-surface I-branched glycans. We found that GCNT2 inversely correlated with clinical progression and that loss of GCNT2 increased melanoma xenograft growth, promoted colony formation, and enhanced cell survival. Conversely, overexpression of GCNT2 decreased melanoma xenograft growth, inhibited colony formation, and increased cell death. More focused analyses revealed reduced signaling responses of two representative glycoprotein families modified by GCNT2, insulin-like growth factor receptor and integrins. Overall, these studies reveal how subtle changes in glycan structure can regulate several malignancy-associated pathways and alter melanoma signaling, growth, and survival.
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http://dx.doi.org/10.1038/s41467-018-05795-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6105653PMC
August 2018

Photoactivable Glycolipid Antigens Generate Stable Conjugates with CD1d for Invariant Natural Killer T Cell Activation.

Bioconjug Chem 2018 09 23;29(9):3161-3173. Epub 2018 Aug 23.

Vaccinex Inc. , 1895 Mount Hope Avenue , Rochester , New York 14620 , United States.

Activation of invariant natural killer T lymphocytes (iNKT cells) by α-galactosylceramide (α-GC) elicits a range of pro-inflammatory or anti-inflammatory immune responses. We report the synthesis and characterization of a series of α-GC analogues with acyl chains of varying length and a terminal benzophenone. These bound efficiently to the glycolipid antigen presenting protein CD1d, and upon photoactivation formed stable CD1d-glycolipid covalent conjugates. Conjugates of benzophenone α-GCs with soluble or cell-bound CD1d proteins retained potent iNKT cell activating properties, with biologic effects that were modulated by acyl chain length and the resulting affinities of conjugates for iNKT cell antigen receptors. Analysis by mass spectrometry identified a unique covalent attachment site for the glycolipid ligands in the hydrophobic ligand binding pocket of CD1d. The creation of covalent conjugates of CD1d with α-GC provides a new tool for probing the biology of glycolipid antigen presentation, as well as opportunities for developing effective immunotherapeutics.
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http://dx.doi.org/10.1021/acs.bioconjchem.8b00484DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6586470PMC
September 2018

Towards automation of glycomic profiling of complex biological materials.

Glycoconj J 2018 06 16;35(3):311-321. Epub 2018 Jun 16.

Department of Life Sciences, Imperial College London, London, SW7 2AZ, UK.

Glycosylation is considered one of the most complex and structurally diverse post-translational modifications of proteins. Glycans play important roles in many biological processes such as protein folding, regulation of protein stability, solubility and serum half-life. One of the ways to study glycosylation is systematic structural characterizations of protein glycosylation utilizing glycomics methodology based around mass spectrometry (MS). The most prevalent bottleneck stages for glycomic analyses is laborious sample preparation steps. Therefore, in this study, we aim to improve sample preparations by automation. We recently demonstrated the successful application of an automated high-throughput (HT), glycan permethylation protocol based on 96-well microplates, in the analysis of purified glycoproteins. Therefore, we wanted to test if these developed HT methodologies could be applied to more complex biological starting materials. Our automated 96-well-plate based permethylation method showed very comparable results with established glycomic methodology. Very similar glycomic profiles were obtained for complex glycoprotein/protein mixtures derived from heterogeneous mouse tissues. Automated N-glycan release, enrichment and automated permethylation of samples proved to be convenient, robust and reliable. Therefore we conclude that these automated procedures are a step forward towards the development of a fully automated, fast and reliable glycomic profiling system for analysis of complex biological materials.
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http://dx.doi.org/10.1007/s10719-018-9825-8DOI Listing
June 2018