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Background: The disturbed metabolism of ceramide (Cer) is supposed to evoke the autoimmune response, contributing to MS pathology., Objectives: To determine levels of anti-Cer immunoglobulins G (IgGs) in the CSF and serum of subjects with various phenotypes of MS, and to investigate relationships between levels of anti-Cer antibodies and MS-related variables., Methods: IgGs isolated from serum and the CSF of 68 MS patients and appropriate controls were examined for their reactivity to Cer subspecies. Their levels were compared between the studied groups and compartments, and analyzed with regard to clinical variables., Results: Increased levels of anti-C16:0-, C18:0-, C18:1-, C24:0- and C24:1-Cer IgGs were detected in the CSF and serum of MS patients in comparison with controls. For IgGs against particular Cer subspecies, correlations were found between their CSF and serum level, as well as with the Link index. Serum and the CSF anti-Cer IgGs differed between patients with clinically isolated syndrome (CIS) and relapsing-remitting MS from those with progressive MS. No correlations were found between anti-Cer IgGs and other MS-related clinical variables., Conclusion: Patients with MS have shown altered panels of anti-Cer IgGs in the CSF and serum, which might suggest a relevant, though limited role of Cer as a target for autoimmune humoral response. Utility of antibodies against Cer subspecies as potential markers for MS activity and progression deserves further investigations., Competing Interests: Declaration of Competing Interest The authors have no conflicting financial interests., (Copyright © 2023. Published by Elsevier B.V.)

Multiple sclerosis (MS) is a CNS disease characterized by immune-mediated demyelination and progressive axonal loss. MS-related CNS damage and its clinical course have two main phases: active and inactive/progressive. Reliable biomarkers are being sought to allow identification of MS pathomechanisms and prediction of its course. The purpose of this study was to identify sphingolipid (SL) species as candidate biomarkers of inflammatory and neurodegenerative processes underlying MS pathology. We performed sphingolipidomic analysis by HPLC-tandem mass spectrometry to determine the lipid profiles in post mortem specimens from the normal-appearing white matter (NAWM) of the normal CNS (nCNS) from subjects with chronic MS (active and inactive lesions) as well as from patients with other neurological diseases. Distinctive SL modification patterns occurred in specimens from MS patients with chronic inactive plaques with respect to NAWM from the nCNS and active MS (Ac-MS) lesions. Chronic inactive MS (In-MS) lesions were characterized by decreased levels of dihydroceramide (dhCer), ceramide (Cer), and SM subspecies, whereas levels of hexosylceramide and Cer 1-phosphate (C1P) subspecies were significantly increased in comparison to NAWM of the nCNS as well as Ac-MS plaques. In contrast, Ac-MS lesions were characterized by a significant increase of major dhCer subspecies in comparison to NAWM of the nCNS. These results suggest the existence of different SL metabolic pathways in the active versus inactive phase within progressive stages of MS. Moreover, they suggest that C1P could be a new biomarker of the In-MS progressive phase, and its detection may help to develop future prognostic and therapeutic strategies for the disease., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2020 Podbielska et al. Published by The American Society for Biochemistry and Molecular Biology, Inc.)

Glycophorins are heavily glycosylated sialoglycoproteins of human and animal erythrocytes. In humans, there are four glycophorins: A, B, C and D. Glycophorins play an important role in the invasion of red blood cells (RBCs) by malaria parasites, which involves several ligands binding to RBC receptors. Four Plasmodium falciparum merozoite EBL ligands have been identified: erythrocyte-binding antigen-175 (EBA-175), erythrocyte-binding antigen-181 (EBA-181), erythrocyte-binding ligand-1 (EBL-1) and erythrocyte-binding antigen-140 (EBA-140). It is generally accepted that glycophorin A (GPA) is the receptor for P. falciparum EBA-175 ligand. It has been shown that α(2,3) sialic acid residues of GPA O-glycans form conformation-dependent clusters on GPA polypeptide chain which facilitate binding. P. falciparum can also invade erythrocytes using glycophorin B (GPB), which is structurally similar to GPA. It has been shown that P. falciparum EBL-1 ligand binds to GPB. Interestingly, a hybrid GPB-GPA molecule called Dantu is associated with a reduced risk of severe malaria and ameliorates malaria-related morbidity. Glycophorin C (GPC) is a receptor for P. falciparum EBA-140 ligand. Likewise, successful binding of EBA-140 depends on sialic acid residues of N- and O-linked oligosaccharides of GPC, which form a cluster or a conformational structure depending on the presence of peptide fragment encompassing amino acids (aa) 36-63. Evaluation of the homologous P. reichenowi EBA-140 unexpectedly revealed that the chimpanzee homolog of human glycophorin D (GPD) is probably the receptor for this ligand. In this review, we concentrate on the role of glycophorins as erythrocyte receptors for Plasmodium parasites. The presented data support the long-lasting idea of high evolutionary pressure exerted by Plasmodium on the human glycophorins, which emerge as important receptors for these parasites.

Antigens of the Gerbich blood group system are expressed on glycophorin C (GPC) and glycophorin D (GPD), minor sialoglycoproteins of human erythrocytes. GPC and GPD help maintain erythrocyte shape of and contributes to the stability of its membrane. There are six high-prevalence Gerbich antigens: Ge2, Ge3, Ge4, GEPL (GE10), GEAT (GE11), GETI (GE12) and five low-prevalence Gerbich antigens: Wb (GE5), Ls a (GE6), An a (GE7), Dh a (GE8), GEIS (GE9). Some Gerbich antigens (Ge4, Wb, Dh a , GEAT) are expressed only on GPC, two (Ge2, An a ) are expressed only on GPD, while others (Ge3, Ls a , GEIS, GEPL, GETI) are expressed on both GPC and GPD. Antibodies recognizing GPC/GPD may arise naturally (so-called "naturally-occurring RBC antibodies") or as the result of alloimmunization, and some of them may be clinically relevant. Gerbich antibodies usually do not cause serious hemolytic transfusion reactions (HTR); autoantibodies of anti-Ge2- or anti-Ge3 specificity can cause autoimmune hemolytic anemia (AIHA)., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: All symptoms of malaria are caused by the intraerythrocytic proliferation of Plasmodium merozoites. Merozoites invade erythrocytes using multiple binding ligands that recognise specific surface receptors. It has been suggested that adaptation of Plasmodium parasites to infect specific hosts is driven by changes in genes encoding Plasmodium erythrocyte-binding ligands (EBL) and reticulocyte-binding ligands (RBL). Homologs of both EBL and RBL, including the EBA-140 merozoite ligand, have been identified in P. falciparum and P. reichenowi, which infect humans and chimpanzees, respectively. The P. falciparum EBA-140 was shown to bind human glycophorin C, a minor erythrocyte sialoglycoprotein. Until now, the erythrocyte receptor for the P. reichenowi EBA-140 remained unknown., Methods: The baculovirus expression vector system was used to obtain the recombinant EBA-140 Region II, and flow cytometry and immunoblotting methods were applied to characterise its specificity., Results: We showed that the chimpanzee glycophorin D is the receptor for the P. reichenowi EBA-140 ligand on chimpanzee red blood cells., Conclusions: We propose that the development of glycophorin C specificity is spurred by the P. falciparum lineage. We speculate that the P. falciparum EBA-140 evolved to hijack GPC on human erythrocytes during divergence from its ape ancestor.

Plasmodium reichenowi, an ape malaria parasite is morphologically identical and genetically similar to Plasmodium falciparum, infects chimpanzees but not humans. Genomic studies revealed that all primate malaria parasites belong to Laverania subgenus. Laverania parasites exhibit strict host specificity, but the molecular mechanisms underlying these host restrictions remain unexplained. Plasmodium merozoites express multiple binding ligands that recognize specific receptors on erythrocytes, including micronemal proteins belonging to P. falciparum EBL family. It was shown that erythrocyte binding antigen-175 (EBA-175), erythrocyte binding ligand-1 (EBL-1), erythrocyte binding antigen-140 (EBA-140) recognize erythrocyte surface sialoglycoproteins - glycophorins A, B, C, respectively. EBA-140 merozoite ligand hijacks glycophorin C (GPC), a minor erythrocyte sialoglycoprotein, to invade the erythrocyte through an alternative invasion pathway. A homolog of P. falciparum EBA-140 protein was identified in P. reichenowi. The amino acid sequences of both EBA-140 ligands are very similar, especially in the conservative erythrocyte binding region (Region II). It has been suggested that evolutionary changes in the sequence of EBL proteins may be associated with Plasmodium host restriction. In this study we obtained, for the first time, the recombinant P. reichenowi EBA-140 ligand Region II using baculovirus expression vector system. We show that the ape EBA-140 Region II is host specific and binds to chimpanzee erythrocytes in the dose and sialic acid dependent manner. Further identification of the erythrocyte receptor for this ape ligand is of great interests, since it may reveal the molecular basis of host restriction of both P. reichenowi and its deadliest human counterpart, P. falciparum., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

The erythrocyte binding ligand 140 (EBA-140) is a member of the Plasmodium falciparum erythrocyte binding antigens (EBA) family, which are considered as prospective candidates for malaria vaccine development. EBA proteins were identified as important targets for naturally acquired inhibitory antibodies. Natural antibody response against EBA-140 ligand was found in individuals living in malaria-endemic areas. The EBA-140 ligand is a paralogue of the well-characterized P. falciparum EBA-175 protein. They both share homology of domain structure, including the binding region (Region II), which consists of two homologous F1 and F2 domains and is responsible for ligand-erythrocyte receptor interaction during merozoite invasion. It was shown that the erythrocyte receptor for EBA-140 ligand is glycophorin C-a minor human erythrocyte sialoglycoprotein. In studies on the immunogenicity of P. falciparum EBA ligands, the recombinant proteins are of great importance. In this report, we have demonstrated that the recombinant baculovirus-obtained EBA-140 Region II is immunogenic and antigenic. It can raise specific antibodies in rabbits, and it is recognized by natural antibodies present in sera of patients with malaria, and thus, it may be considered for inclusion in multicomponent blood-stage vaccines.

Human Gb3/CD77 synthase (α1,4-galactosyltransferase, P(k) synthase), encoded by A4GALT gene, is known for synthesis of Gal(α1-4)Gal moiety in globotriaosylceramide (Gb3Cer, CD77, P(k) blood group antigen), a glycosphingolipid of the globo series. Recently, it was shown that c.631C > G mutation in A4GALT, which causes p.Q211E substitution in the open reading frame of the enzyme, broadens the enzyme specificity, making it able also to synthesize Gal(α1-4)GalNAc moiety, which constitutes the defining terminal disaccharide of the NOR antigen (carried by two glycosphingolipids: NOR1 and NOR2). Terminal Gal(α1-4)Gal disaccharide is also present in another glycosphingolipid blood group antigen, called P1, which together with P(k) and NOR comprises the P1PK blood group system. Despite several attempts, it was never clearly shown that P1 antigen is synthesized by Gb3/CD77 synthase, leaving open an alternative hypothesis that there are two homologous α1,4-galactosyltransferases in humans. In this study, using recombinant Gb3/CD77 synthase produced in insect cells, we show that the consensus enzyme synthesizes both the P(k) and P1 antigens, while its p.Q211E variant additionally synthesizes the NOR antigen. This is the first direct biochemical evidence that Gb3/CD77 synthase is able to synthesize two different glycosphingolipid antigens: P(k) and P1, and when p.Q211E substitution is present, the NOR antigen is also synthesized., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Glycophorins C and D are highly glycosylated integral sialoglycoproteins of human red blood cell membranes carrying the Gerbich blood group antigens. The O- and N-glycosidic chains of the major erythrocyte glycoprotein (Lisowska E. 2001, Antigenic properties of human glycophorins - an update. Adv Exp Med Biol, 491:155-169; Tomita M and Marchesi VT. 1975, Amino-acid sequence and oligosaccharide attachment sites of human erythrocyte glycophorin. Proc Natl Acad Sci USA, 72:2964-2968.) are well characterized but the structure of GPC N-glycans has remained unknown. This problem became important since it was reported that GPC N-glycans play an essential role in the interaction with Plasmodium falciparum EBA-140 merozoite ligand. The elucidation of these structures seems essential for full characterization of the GPC binding site for the EBA-140 ligand. We have employed detailed structural analysis using sequential mass spectrometry to show that many GPC N-glycans contain H2 antigen structures and several contain polylactosamine structures capped with fucose. The results obtained indicate structural heterogeneity of the GPC N-glycans and show the existence of structural elements not found in glycophorin A N-glycans. Our results also open a possibility of new interpretation of the data concerning the binding of P. falciparum EBA-140 ligand to GPC. We hypothesize that preferable terminal fucosylation of N-glycosidic chains containing repeating lactosamine units of the GPC Gerbich variant could be an explanation for why the EBA-140 ligand does not react with GPC Gerbich and an indication that the EBA-140 interaction with GPC is distinctly dependent on the GPC N-glycan structure., (© The Author 2014. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The erythrocyte binding ligand 140 (EBA-140) is a member of the Plasmodium falciparum DBL family of erythrocyte binding proteins, which are considered as prospective candidates for malaria vaccine development. The EBA-140 ligand is a paralogue of the well-characterized P. falciparum EBA-175 protein. They share homology of domain structure, including Region II, which consists of two homologous F1 and F2 domains and is responsible for ligand-erythrocyte receptor interaction during invasion. In this report we describe, for the first time, the glycophorin C specificity of the recombinant, baculovirus-expressed binding region (Region II) of P. falciparum EBA-140 ligand. It was found that the recombinant EBA-140 Region II binds to the endogenous and recombinant glycophorin C, but does not bind to Gerbich-type glycophorin C, neither normal nor recombinant, which lacks amino acid residues 36-63 of its polypeptide chain. Our results emphasize the crucial role of this glycophorin C region in EBA-140 ligand binding. Moreover, the EBA-140 Region II did not bind either to glycophorin D, the truncated form of glycophorin C lacking the N-glycan or to desialylated GPC. These results draw attention to the role of glycophorin C glycans in EBA-140 binding. The full identification of the EBA-140 binding site on glycophorin C molecule, consisting most likely of its glycans and peptide backbone, may help to design therapeutics or vaccines that target the erythrocyte binding merozoite ligands.

Rare polyagglutinable NOR erythrocytes contain three unique globoside (Gb4Cer) derivatives, NOR1, NOR(int), and NOR2, in which Gal(α1-4), GalNAc(β1-3)Gal(α1-4), and Gal(α1-4)GalNAc(β1-3)Gal(α1-4), respectively, are linked to the terminal GalNAc residue of Gb4Cer. NOR1 and NOR2, which both terminate with a Gal(α1-4)GalNAc- sequence, react with anti-NOR antibodies commonly present in human sera. While searching for an enzyme responsible for the biosynthesis of Gal(α1-4)GalNAc, we identified a mutation in the A4GALT gene encoding Gb3/CD77 synthase (α1,4-galactosyltransferase). Fourteen NOR-positive donors were heterozygous for the C>G mutation at position 631 of the open reading frame of the A4GALT gene, whereas 495 NOR-negative donors were homozygous for C at this position. The enzyme encoded by the mutated gene contains glutamic acid instead of glutamine at position 211 (substitution Q211E). To determine whether this mutation could change the enzyme specificity, we transfected a teratocarcinoma cell line (2102Ep) with vectors encoding the consensus Gb3/CD77 synthase and Gb3/CD77 synthase with Glu at position 211. The cellular glycolipids produced by these cells were analyzed by flow cytometry, high-performance thin-layer chromatography, enzymatic degradation, and MALDI-TOF mass spectrometry. Cells transfected with either vector expressed the P1 blood group antigen, which was absent from untransfected cells. Cells transfected with the vector encoding the Gb3/CD77 synthase with Glu at position 211 expressed both P1 and NOR antigens. Collectively, these results suggest that the C631G mutation alters the acceptor specificity of Gb3/CD77 synthase, rendering it able to catalyze synthesis of the Gal(α1-4)Gal and Gal(α1-4)GalNAc moieties.

GM2 synthase is a homodimer in which the subunits are joined by lumenal domain disulfide bond(s). To define the disulfide bond pattern of this enzyme, we analyzed a soluble form by chemical fragmentation, enzymatic digestion, and mass spectrometry and a full-length form by site-directed mutagenesis. All Cys residues of the lumenal domain of GM2 synthase are disulfide bonded with Cys(429) and Cys(476) forming a disulfide-bonded pair while Cys(80) and Cys(82) are disulfide bonded in combination with Cys(412) and Cys(529). Partial reduction to produce monomers converted Cys(80) and Cys(82) to free thiols while the Cys(429) to Cys(476) disulfide remained intact. CNBr cleavage at amino acid 330 produced a monomer-sized band under nonreducing conditions which was converted upon reduction to a 40-kDa fragment and a 24-kDa myc-positive fragment. Double mutation of Cys(80) and Cys(82) to Ser produced monomers but not dimers. In summary these results demonstrate that Cys(429) and Cys(476) form an intrasubunit disulfide while the intersubunit disulfides formed by both Cys(80) and Cys(82) with Cys(412) and Cys(529) are responsible for formation of the homodimer. This disulfide bond arrangement results in an antiparallel orientation of the catalytic domains of the GM2 synthase homodimer.

Many Golgi membrane-bound glycosyltransferases exist as intermolecular disulfide bonded species, some of which have been demonstrated to be homodimers. Evidence for homodimer formation has come primarily from radiation inactivation experiments. We utilized an alternative strategy to test for homodimer formation of the cloned beta 1,4 N-acetylgalactosaminyltransferase (GalNAcT) responsible for synthesis of the glycosphingolipids GM2, GD2, and GA2. We stably transfected CHO cells with myc epitopetagged GalNAcT, which localizes primarily to the Golgi, and a hemagglutinin (HA) epitope-tagged GalNAcT fusion protein in which the cytoplasmic domain of GalNAcT was replaced by an ER retention signal. We then sought evidence for dimer formation between the two forms of GalNAcT. Immunoprecipitation with anti-myc or anti-HA co-immunoprecipitated the HA-tagged form or the myc-tagged form, respectively, providing evidence for the physical association of the two forms of GalNAcT. As a result of this association, GalNAcT/myc increased in the ER as demonstrated by Western blots and immunofluorescence. The rapid formation of dimers provided further evidence for dimer formation occurring in the ER. In summary, these results demonstrate that GalNAcT forms homodimers as a result of intermolecular disulfide bond formation in the ER. Furthermore, this ER motif strategy is potentially useful for demonstrating homodimer formation of other Golgi enzymes.

Cloned human beta 1,4N-acetylgalactosaminyltransferase (GalNAcT) catalyzes the synthesis of the glycosphingolipids GM2, GD2, and gangliotriosylceramide. To determine the subcellular location of this enzyme and whether it exists in intermolecular disulfide bonded species, we stably transfected Chinese hamster ovary (CHO) cells with three myc epitope-tagged forms of the GalNAcT gene: the native enzyme; the lumenal domain of GalNAcT fused to the cytoplasmic and transmembrane domains of N-acetylglucosaminyltransferase I (GNT); and the transmembrane and lumenal domains of GalNAcT fused to the cytoplasmic domain of the Iip33 form of human invariant chain in order to retain the enzyme in the endoplasmic reticulum (ER). Immunoelectron microscopic analysis with anti-myc revealed that GalNAcT/myc was present throughout the Golgi stack, the GNT/GalNAcT/myc form was restricted primarily to the medial Golgi cisternae, and the Iip33/GalNAcT/myc form was restricted to the ER. Cells transfected with each of the three constructs contained high levels of GM2 synthase activity in vitro, but only the GalNAcT/myc form and the GNT/GalNAcT/myc forms were able to synthesize the GM2 product in vivo. The enzyme produced by all three constructs was present in the transfected cells in a disulfide bonded form having a molecular size consistent with that of a homodimer or higher aggregate.