Facile synthesis of C5-azido derivatives of thiosialosides and 2,3-dehydro-5-N-acetylneuraminic acid (DANA).

Neuraminic acid (Neu5Ac, also known as sialic acid) is an important monosaccharide found in glycoproteins and glycolipids which plays a vital role in regulation of physiological functions and pathological conditions. The study of sialoglycans has benefitted from the development of glycomimetic probes and inhibitors for proteins and enzymes that interact with and modify neuraminic acid in glycan chains. Methods to access sialoside intermediates with high yield are needed to facilitate the design of new targets. Here, we report the synthesis of C5-azido thiosialosides using a mild method to deprotect the C5-acetamido functional group followed by the use of a diazo-transfer reagent. We examined two diazo-transfer strategies and compared their yields and tolerance of acetate protecting groups. The same methods and comparisons were also performed for the 2,3-dehydro-5-N-acetylneuraminic acid (DANA) scaffold which is commonly used to generate inhibitors of neuraminidase (sialidase) enzymes. We found that C5-azido derivatives of both thiosialosides and DANA could be produced in five or six steps with yields up to 76 % and 83 %, respectively. Diazo-transfer reagents compared in this study were trifluoromethanesulfonyl azide (TfN3) and imidazole-1-sulfonyl azide (ImzSO2N3). We found that both reagents were compatible with this method and showed comparable yields. Finally, we show that C5-azido derivatives can help to avoid O, N-acyl protecting group migration which was observed in C5-NHAc analogs.

Convergent synthesis of a hexadecavalent heterobifunctional ABO blood group glycoconjugate.

Naturally occurring glycans are often found in a multivalent presentation. Cell surface receptors that recognize these displays may form clusters, which can lead to signalling or endocytosis. One of the challenges in generating synthetic displays of multivalent carbohydrates is providing high valency as well as access to heterofunctional conjugates to allow attachment of multiple antigens or payloads. We designed a strategy based on a set of bifunctional linkers to generate a heterobifunctional multivalent display of two carbohydrate antigens to bind BCR and CD22 with four and twelve antigen copies, respectively. We confirmed that the conjugates were able to engage both CD22 and BCR on cells by observing receptor clustering. The strategy is modular and would allow for alternative carbohydrate antigens to be attached bearing amine and alkyne groups and should be of interest for the development of immunomodulators and vaccines.

Severe kidney dysfunction in sialidosis mice reveals an essential role for neuraminidase 1 in reabsorption.

Sialidosis is an ultra-rare multisystemic lysosomal disease caused by mutations in the neuraminidase 1 (NEU1) gene. The severe type II form of the disease manifests with a prenatal/infantile or juvenile onset, bone abnormalities, severe neuropathology, and visceromegaly. A subset of these patients present with nephrosialidosis, characterized by abrupt onset of fulminant glomerular nephropathy. We studied the pathophysiological mechanism of the disease in 2 NEU1-deficient mouse models, a constitutive Neu1-knockout, Neu1ΔEx3, and a conditional phagocyte-specific knockout, Neu1Cx3cr1ΔEx3. Mice of both strains exhibited terminal urinary retention and severe kidney damage with elevated urinary albumin levels, loss of nephrons, renal fibrosis, presence of storage vacuoles, and dysmorphic mitochondria in the intraglomerular and tubular cells. Glycoprotein sialylation in glomeruli, proximal distal tubules, and distal tubules was drastically increased, including that of an endocytic reabsorption receptor megalin. The pool of megalin bearing O-linked glycans with terminal galactose residues, essential for protein targeting and activity, was reduced to below detection levels. Megalin levels were severely reduced, and the protein was directed to lysosomes instead of the apical membrane. Together, our results demonstrated that desialylation by NEU1 plays a crucial role in processing and cellular trafficking of megalin and that NEU1 deficiency in sialidosis impairs megalin-mediated protein reabsorption.

New insights into the glycobiology of immune thrombocytopenia.

PURPOSE OF REVIEW: The platelet surface harbors a lush forest of glycans (carbohydrate polymers) attached to membrane proteins and lipids. Accumulating evidence suggests that these glycans may be relevant to the pathophysiology of immune thrombocytopenia (ITP). Here, we critically evaluate data that point to a possible role for loss of sialic acid in driving platelet clearance in ITP, comment on the potential use of neuraminidase inhibitors for treatment of ITP, and highlight open questions in this area. RECENT FINDINGS: Multiple lines of evidence suggest a role for loss of platelet sialic acid in the pathophysiology of thrombocytopenia. Recent work has tested the hypothesis that neuraminidase-mediated cleavage of platelet sialic acid may trigger clearance of platelets in ITP. Some clinical evidence supports efficacy of the viral neuraminidase inhibitor oseltamivir in ITP, which is surprising given its lack of activity against human neuraminidases. SUMMARY: Further study of platelet glycobiology in ITP is necessary to fill key knowledge gaps. A deeper understanding of the roles of platelet glycans in ITP pathophysiology will help to guide development of novel therapies.

Extending the in vivo persistence of synthetic glycoconjugates using a serum-protein binder.

Synthetic glycoconjugates are used in the development of vaccines and the design of inhibitors for glycan-protein interactions. The in vivo persistence of synthetic glycoconjugates is an important factor in their efficacy, especially when prolonged interactions with specific cell types may be required. In this study, we applied a strategy for non-covalent association of an active compound with serum proteins for extension of glycoconjugate half-life in serum. The small molecule, AG10, has previously been used to extend the half-life of small molecules through its high affinity for transthyretin (TTR), a serum protein. Using a tetravalent polyethylene glycol (PEG)-based scaffold we developed a synthetic strategy for glycoconjugates that allowed for controlled addition of multiple tags, such as a TTR affinity tag or fluorophore. We designed a version of AG10 modified at the pyrazole core, named GD10, amenable to our conjugation strategy and introduced to glycoconjugates using a tri-functional linker. This approach allowed for attachment of GD10 and fluorophore tags, as well as carbohydrate antigens. We then tested the influence of the GD10 tag on glycoconjugate half-life in vivo using a mouse model. Our results suggest that the combination of the GD10 tag and the PEG scaffold extended the half-life of glycoconjugates by as much as 10-fold when compared to proteins of similar molecular weight. The GD10 tag was able to extend the half-life of similar glycoconjugates by as much as 2-fold. We observed a role for the terminal saccharide residue of the carbohydrate antigen and confirmed that conjugates were able to penetrate multiple compartments in vivo including bone marrow, lymph nodes, and other organs. The introduction of the GD10 tag did not obstruct the ability of conjugates to interact with lectin receptors. We conclude that serum protein binders can be used to extend the persistence of glycoconjugates in vivo.

NEU1 and NEU3 enzymes alter CD22 organization on B cells.

The B cell membrane expresses sialic-acid-binding immunoglobulin-like lectins, also called Siglecs, that are important for modulating immune response. Siglecs have interactions with sialoglycoproteins found on the same membrane (cis-ligands) that result in homotypic and heterotypic receptor clusters. The regulation and organization of these clusters, and their effect on cell activation, is not clearly understood. We investigated the role of human neuraminidase enzymes NEU1 and NEU3 on the clustering of CD22 on B cells using confocal microscopy. We observed that native NEU1 and NEU3 activity influence the cluster size of CD22. Using single-particle tracking, we observed that NEU3 activity increased the lateral mobility of CD22, which was in contrast to the effect of exogenous bacterial NEU enzymes. Moreover, we show that native NEU1 and NEU3 activity influenced cellular Ca2+ levels, supporting a role for these enzymes in regulating B cell activation. Our results establish a role for native NEU activity in modulating CD22 organization and function on B cells.

The Janus-like role of neuraminidase isoenzymes in inflammation.

The processes of activation, extravasation, and migration of immune cells to a site are early and essential steps in the induction of an acute inflammatory response. These events are an essential part of the inflammatory cascade, which involves multiple regulatory steps. Using a murine air pouch model of inflammation with LPS as an inflammation inducer, we demonstrate that isoenzymes of the neuraminidase family (NEU1, 3, and 4) play essential roles in these processes by acting as positive or negative regulators of leukocyte infiltration. In genetically knocked-out (KO) mice for different NEU genes (Neu1 KO, Neu3 KO, Neu4 KO, and Neu3/4 double KO mice) with LPS-induced air pouch inflammation, leukocytes at the site of inflammation were counted, and the inflamed tissue was analyzed using immunohistochemistry. Our data show that leukocyte recruitment was decreased in NEU1- and NEU3-deficient mice, while it was increased in NEU4-deficient animals. Consistent with these results, systemic as well as pouch exudate levels of pro-inflammatory cytokines were reduced in Neu1 and increased in Neu4 KO mice. Pharmacological inhibitors specific for NEU1, NEU3, and NEU4 isoforms also affected leukocyte recruitment. Together our data demonstrate that NEU isoenzymes have distinct-and even opposing-effects on leukocyte recruitment, and therefore warrant further investigation to determine their mechanisms and importance as regulators of the inflammatory cascade.

Inhibitors of Human Neuraminidase Enzymes Block Transmigration in vitro.

Cell migration to a site of inflammation is an important step of the immune response. This process is coordinated by cytokines, receptors, and the signal processing machinery of the cell. Many cellular receptors are glycosylated, and their activity can be modulated through changes in glycan structure. Furthermore, glycosylation can be critical to the folding and trafficking of receptors. In this work, we investigated the role of native human neuraminidase enzymes (NEU) in transmigration. We used a cultured T cell line (Jurkat) and a transwell assay with fibronectin (FN) coated wells and cytokines (IL-4 and TNF-α) as chemoattractants in the bottom chamber. We observed that NEU1, NEU3, and NEU4 were positive regulators of transmigration using an siRNA knockdown. Furthermore, we found that pharmacological inhibition of these enzymes inhibited transmigration. We conclude that human NEU isoenzymes NEU1, NEU3, and NEU4 can act as positive regulators of transmigration and should be investigated as targets for anti-inflammatory strategies.

Profiling of glycosphingolipids with SCDase digestion and HPLC-FLD-MS.

Lipid components of cells and tissues feature a large diversity of structures that present a challenging problem for molecular analysis. Glycolipids from mammalian cells contain glycosphingolipids (GSLs) as their major glycolipid component, and these structures vary in the identity of the glycan headgroup as well as the structure of the fatty acid and sphingosine (Sph) tails. Analysis of intact GSLs is challenging due to the low abundance of these species. Here, we develop a new strategy for the analysis of lyso-GSL (l-GSL), GSL that retain linkage of the glycan headgroup with the Sph base. The analysis begins with digestion of a GSL sample with sphingolipid ceramide N-deacylase (SCDase), followed by labelling with an amine-reactive fluorophore. The sample was then analyzed by HPLC-FLD-MS and quantitated by addition of an external standard. This method was compared to analysis of GSL glycans after cleavage by an Endoglycoceramidase (EGCase) enzyme and labeling with a fluorophore (2-anthranilic acid, 2AA). The two methods are complementary, with EGCase providing improved signal (due to fewer species) and SCDase providing analysis of lyso-GSL. Importantly the SCDase method provides Sph composition of GSL species. We demonstrate the method on cultured human cells (Jurkat T cells) and tissue homogenate (porcine brain).

Characterization of ABH-subtype donor-specific antibodies in ABO-A-incompatible kidney transplantation.

ABO-incompatible (ABOi) transplantation requires preemptive antibody reduction; however, the relationship between antibody-mediated rejection (AMR) and ABO-antibodies, quantified by hemagglutination (HA), is inconsistent, possibly reflecting variable graft resistance to AMR or HA assay limitations. Using an ABH-glycan microarray, we quantified ABO-A antigen-subtype (A-subtype)-specific IgM and IgG in 53 ABO-O recipients of ABO-A kidneys, before and after antibody removal (therapeutic plasma exchange [TPE] or ABO-A-trisaccharide immunoadsorption [IA]) and 1-year posttransplant. IgM binding to all A-subtypes correlated highly (R2  ≥ .90) and A-subtype antibody specificities was reduced equally by IA versus TPE. IgG binding to the A-subtypes (II-IV) expressed in kidney correlated poorly (.27 ≤ R2  ≤ .69). Reduction of IgG specific to A-subtype-II was equivalent for IA and TPE, whereas IgG specific to A-subtypes-III/IV was not as greatly reduced by IA (p < .005). One-year posttransplant, IgG specific to A-II remained the most reduced antibody. Immunostaining revealed only A-II on vascular endothelium but A-subtypes II-III/IV on tubular epithelium. These results show that ABO-A-trisaccharide is sufficient for IgM binding to all A-subtypes; this is true for IgG binding to A-II, but not subtypes-III/IV, which exhibits varying degrees of specificity. We identify A-II as the major, but importantly not the sole, antigen relevant to treatment and immune modulation in adult ABO-A-incompatible kidney transplantation.

Neuraminidases 1 and 3 Trigger Atherosclerosis by Desialylating Low-Density Lipoproteins and Increasing Their Uptake by Macrophages.

Background Chronic vascular disease atherosclerosis starts with an uptake of atherogenic modified low-density lipoproteins (LDLs) by resident macrophages, resulting in formation of arterial fatty streaks and eventually atheromatous plaques. Increased plasma sialic acid levels, increased neuraminidase activity, and reduced sialic acid LDL content have been previously associated with atherosclerosis and coronary artery disease in human patients, but the mechanism underlying this association has not been explored. Methods and Results We tested the hypothesis that neuraminidases contribute to development of atherosclerosis by removing sialic acid residues from glycan chains of the LDL glycoprotein and glycolipids. Atherosclerosis progression was investigated in apolipoprotein E and LDL receptor knockout mice with genetic deficiency of neuraminidases 1, 3, and 4 or those treated with specific neuraminidase inhibitors. We show that desialylation of the LDL glycoprotein, apolipoprotein B 100, by human neuraminidases 1 and 3 increases the uptake of human LDL by human cultured macrophages and by macrophages in aortic root lesions in Apoe-/- mice via asialoglycoprotein receptor 1. Genetic inactivation or pharmacological inhibition of neuraminidases 1 and 3 significantly delays formation of fatty streaks in the aortic root without affecting the plasma cholesterol and LDL levels in Apoe-/- and Ldlr-/- mouse models of atherosclerosis. Conclusions Together, our results suggest that neuraminidases 1 and 3 trigger the initial phase of atherosclerosis and formation of aortic fatty streaks by desialylating LDL and increasing their uptake by resident macrophages.

Therapeutic Effect of Neuraminidase-1-Selective Inhibition in Mouse Models of Bleomycin-Induced Pulmonary Inflammation and Fibrosis.

Pulmonary fibrosis remains a serious biomedical problem with no cure and an urgent need for better therapies. Neuraminidases (NEUs), including NEU1, have been recently implicated in the mechanism of pulmonary fibrosis by us and others. We now have tested the ability of a broad-spectrum neuraminidase inhibitor, 2,3-dehydro-2-deoxy-N-acetylneuraminic acid (DANA), to modulate the in vivo response to acute intratracheal bleomycin challenge as an experimental model of pulmonary fibrosis. A marked alleviation of bleomycin-induced body weight loss and notable declines in accumulation of pulmonary lymphocytes and collagen deposition were observed. Real-time polymerase chain reaction analyses of human and mouse lung tissues and primary human lung fibroblast cultures were also performed. A predominant expression and pronounced elevation in the levels of NEU1 mRNA were observed in patients with idiopathic pulmonary fibrosis and bleomycin-challenged mice compared with their corresponding controls, whereas NEU2, NEU3, and NEU4 were expressed at far lower levels. The levels of mRNA for the NEU1 chaperone, protective protein/cathepsin A (PPCA), were also elevated by bleomycin. Western blotting analyses demonstrated bleomycin-induced elevations in protein expression of both NEU1 and PPCA in mouse lungs. Two known selective NEU1 inhibitors, C9-pentyl-amide-DANA (C9-BA-DANA) and C5-hexanamido-C9-acetamido-DANA, dramatically reduced bleomycin-induced loss of body weight, accumulation of pulmonary lymphocytes, and deposition of collagen. Importantly, C9-BA-DANA was therapeutic in the chronic bleomycin exposure model with no toxic effects observed within the experimental timeframe. Moreover, in the acute bleomycin model, C9-BA-DANA attenuated NEU1-mediated desialylation and shedding of the mucin-1 ectodomain. These data indicate that NEU1-selective inhibition offers a potential therapeutic intervention for pulmonary fibrotic diseases. SIGNIFICANCE STATEMENT: Neuraminidase-1-selective therapeutic targeting in the acute and chronic bleomycin models of pulmonary fibrosis reverses pulmonary collagen deposition, accumulation of lymphocytes in the lungs, and the disease-associated loss of body weight-all without observable toxic effects. Such therapy is as efficacious as nonspecific inhibition of all neuraminidases in these models, thus indicating the central role of neuraminidase-1 as well as offering a potential innovative, specifically targeted, and safe approach to treating human patients with a severe malady: pulmonary fibrosis.

Human neuraminidases have reduced activity towards modified sialic acids on glycoproteins.

Multiple levels of diversity in sialic acid presentation can influence the substrate activity of sialosides for glycoside hydrolases. Few reports have investigated the specificity of human neuraminidase (hNEU) activity towards modified sialic acid residues that can occur on glycoproteins. Previously, we evaluated hNEU activity towards 9-O-acetylated sialic acid in glycolipid substrates and found that hNEU generally discriminated against 9-O-acetylated sialic acid over Neu5Ac. Here, we have investigated the substrate specificity of hNEU enzymes for a glycoprotein substrate (bovine submaxillary mucin) containing 9-O-acetylated and Neu5Gc residues. Using this model substrate, we observe a general trend for hNEU tolerance of Neu5Ac > Neu5Gc ≫ Neu5,9Ac2, consistent with our previous results with glycolipid substrates. These results expand our understanding of hNEU enzyme specificity and suggest that naturally occurring modifications of sialic acids can play a role in regulating hNEU activity.

Isoenzyme-Selective Inhibitors of Human Neuraminidases Reveal Distinct Effects on Cell Migration.

The human neuraminidase enzymes (NEU1, NEU2, NEU3, and NEU4) are a class of enzymes implicated in pathologies including cancer and diabetes. Several reports have linked neuraminidase activity to the regulation of cell migration in cancer cells. Using an in vitro cell migration assay on fibronectin (FN) coated surfaces, we have investigated the role of these enzymes in integrin-mediated cell migration. We observed that neuraminidase inhibition caused significant retardation of cell migration in breast cancer (MDA-MB-231) and prostate cancer (PC-3) cell lines when using inhibitors of NEU3 and NEU4. In contrast, inhibition of NEU1 caused a significant increase in cell migration for the same cell lines. We concluded that the blockade of human neuraminidase enzymes with isoenzyme-selective inhibitors can lead to disparate results and has significant potential in the development of anticancer or wound healing therapeutics.

Neuraminidase-3 Is a Negative Regulator of LFA-1 Adhesion.

Within the plasma membrane environment, glycoconjugate-receptor interactions play an important role in the regulation of cell-cell interactions. We have investigated the mechanism and activity of the human neuraminidase (NEU) isoenzyme, NEU3, on T cell adhesion receptors. The enzyme is known to prefer glycolipid substrates, and we confirmed that exogenous enzyme altered the glycolipid composition of cells. NEU3 was able to modify the sialic acid content of purified LFA-1 in vitro. Enzymatic activity of NEU3 resulted in re-organization of LFA-1 into large clusters on the membrane. This change was facilitated by an increase in the lateral mobility of LFA-1 upon NEU3 treatment. Changes to the lateral mobility of LFA-1 were specific for NEU3 activity, and we observed no significant change in diffusion when cells were treated with a bacterial NEU (NanI). Furthermore, we found that NEU3 treatment of cells increased surface expression levels of LFA-1. We observed that NEU3-treated cells had suppressed LFA-1 adhesion to an ICAM-1 coated surface using an in vitro static adhesion assay. These results establish that NEU3 can modulate glycoconjugate composition and contribute to the regulation of integrin activity. We propose that NEU3 should be investigated to determine its role on LFA-1 within the inflammatory cascade.

A quantitative, high-throughput method identifies protein-glycan interactions via mass spectrometry.

Glycan binding by glycan-binding proteins and processing by carbohydrate-active enzymes is implicated in physiological and pathophysiological processes. Comprehensive mapping of glycan interactions is essential to understanding of glycan-mediated biology and can guide the development of new diagnostics and therapeutics. Here, we introduce the competitive universal proxy receptor assay (CUPRA), which combines electrospray ionization mass spectrometry, competitive binding and heterobifunctional glycan-based ligands to give a quantitative high-throughput method for screening glycan libraries against glycan-binding and glycan-processing proteins. Application of the assay to human (siglec-2), plant (Sambucus nigra and Maackia amurensis lectins) and bacterial (cholera toxin, and family 51 carbohydrate binding module) proteins allowed for the identification of ligands with affinities (Kd) ≤ 1 mM. The assay is unprecedentedly versatile and can be applied to natural libraries and, when implemented in a time-resolved manner, provides a quantitative measure of the activities and substrate specificity of carbohydrate-active enzymes.

Crystal structures of human lysosomal EPDR1 reveal homology with the superfamily of bacterial lipoprotein transporters.

EPDR1, a member of the ependymin-related protein family, is a relatively uncharacterized protein found in the lysosomes and secretomes of most vertebrates. Despite having roles in human disease and health, the molecular functions of EPDR1 remain unknown. Here, we present crystal structures of human EPDR1 and reveal that the protein adopts a fold previously seen only in bacterial proteins related to the LolA lipoprotein transporter. EPDR1 forms a homodimer with an overall shape resembling a half-shell with two non-overlapping hydrophobic grooves on the flat side of the hemisphere. EPDR1 can interact with membranes that contain negatively charged lipids, including BMP and GM1, and we suggest that EPDR1 may function as a lysosomal activator protein or a lipid transporter. A phylogenetic analysis reveals that the fold is more widely distributed than previously suspected, with representatives identified in all branches of cellular life.

Persistent reduction in sialylation of cerebral glycoproteins following postnatal inflammatory exposure.

BACKGROUND: The extension of sepsis encompassing the preterm newborn's brain is often overlooked due to technical challenges in this highly vulnerable population, yet it leads to substantial long-term neurodevelopmental disabilities. In this study, we demonstrate how neonatal neuroinflammation following postnatal E. coli lipopolysaccharide (LPS) exposure in rat pups results in persistent reduction in sialylation of cerebral glycoproteins. METHODS: Male Sprague-Dawley rat pups at postnatal day 3 (P3) were injected in the corpus callosum with saline or LPS. Twenty-four hours (P4) or 21 days (P24) following injection, brains were extracted and analyzed for neuraminidase activity and expression as well as for sialylation of cerebral glycoproteins and glycolipids. RESULTS: At both P4 and P24, we detected a significant increase of the acidic neuraminidase activity in LPS-exposed rats. It correlated with significantly increased neuraminidase 1 (Neu1) mRNA in LPS-treated brains at P4 and with neuraminidases 1 and 4 at P24 suggesting that these enzymes were responsible for the rise of neuraminidase activity. At both P4 and P24, sialylation of N-glycans on brain glycoproteins decreased according to both mass-spectrometry analysis and lectin blotting, but the ganglioside composition remained intact. Finally, at P24, analysis of brain tissues by immunohistochemistry showed that neurons in the upper layers (II-III) of somatosensory cortex had a reduced surface content of polysialic acid. CONCLUSIONS: Together, our data demonstrate that neonatal LPS exposure results in specific and sustained induction of Neu1 and Neu4, causing long-lasting negative changes in sialylation of glycoproteins on brain cells. Considering the important roles played by sialoglycoproteins in CNS function, we speculate that observed re-programming of the brain sialome constitutes an important part of pathophysiological consequences in perinatal infectious exposure.

Selective Inhibitors of Human Neuraminidase 1 (NEU1).

Inhibitors of human neuraminidase enzymes (NEU) are recognized as important tools for the study of the biological functions of NEU and will be potent tools for elucidating the role of these enzymes in regulating the repertoire of cellular glycans. Here we report the discovery of selective inhibitors of the human neuraminidase 1 (NEU1) and neuraminidase 2 (NEU2) enzymes with exceptional potency. A library of modified 2-deoxy-2,3-didehydro- N-acetylneuraminic acid (DANA) analogues, with variability in the C5- or C9-position, were synthesized and evaluated against four human neuraminidase isoenyzmes (NEU1-4). Hydrophobic groups with an amide linker at the C5 and C9 positions were well accommodated by NEU1, and a hexanamido group was found to give the best potency at both positions. While the C5-hexanamido-C9-hexanamido-DANA analogue did not show synergistic improvements for combined modification, an extended alkylamide at an individual position combined with a smaller group at the second gave increased potency. The best NEU1 inhibitor identified was a C5-hexanamido-C9-acetamido-DANA that had a Ki of 53 ± 5 nM and 340-fold selectivity over other isoenzymes. Additionally, we demonstrated that C5-modifications combined with a C4-guandino group provided the most potent NEU2 inhibitor reported, with a Ki of 1.3 ± 0.2 µM and 7-fold selectivity over other NEU isoenzymes.

A tyrosine sulfation-dependent HLA-I modification identifies memory B cells and plasma cells.

Memory B cells and plasma cells are antigen-experienced cells tasked with the maintenance of humoral protection. Despite these prominent functions, definitive cell surface markers have not been identified for these cells. We report here the isolation and characterization of the monoclonal variable lymphocyte receptor B (VLRB) N8 antibody from the evolutionarily distant sea lamprey that specifically recognizes memory B cells and plasma cells in humans. Unexpectedly, we determined that VLRB N8 recognizes the human leukocyte antigen-I (HLA-I) antigen in a tyrosine sulfation-dependent manner. Furthermore, we observed increased binding of VLRB N8 to memory B cells in individuals with autoimmune disorders multiple sclerosis and systemic lupus erythematosus. Our study indicates that lamprey VLR antibodies uniquely recognize a memory B cell- and plasma cell-specific posttranslational modification of HLA-I, the expression of which is up-regulated during B cell activation.