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Misfolding and aggregation of tau protein are closely associated with the onset and progression of Alzheimer's Disease (AD). By interrogating IgG + memory B cells from asymptomatic donors with tau peptides, we have identified two somatically mutated V H 5-51/V L 4-1 antibodies. One of these, CBTAU-27.1, binds to the aggregation motif in the R3 repeat domain and blocks the aggregation of tau into paired helical filaments (PHFs) by sequestering monomeric tau. The other, CBTAU-28.1, binds to the N-terminal insert region and inhibits the spreading of tau seeds and mediates the uptake of tau aggregates into microglia by binding PHFs. Crystal structures revealed that the combination of V H 5-51 and V L 4-1 recognizes a common Pro-X n -Lys motif driven by germline-encoded hotspot interactions while the specificity and thereby functionality of the antibodies are defined by the CDR3 regions. Affinity improvement led to improvement in functionality, identifying their epitopes as new targets for therapy and prevention of AD.

Unlabelled: The extraordinary diversity of the human immunodeficiency virus type 1 (HIV-1) envelope (Env) glycoprotein poses a major challenge for the development of an HIV-1 vaccine. One strategy to circumvent this problem utilizes bioinformatically optimized mosaic antigens. However, mosaic Env proteins expressed as trimers have not been previously evaluated for their stability, antigenicity, and immunogenicity. Here, we report the production and characterization of a stable HIV-1 mosaic M gp140 Env trimer. The mosaic M trimer bound CD4 as well as multiple broadly neutralizing monoclonal antibodies, and biophysical characterization suggested substantial stability. The mosaic M trimer elicited higher neutralizing antibody (nAb) titers against clade B viruses than a previously described clade C (C97ZA.012) gp140 trimer in guinea pigs, whereas the clade C trimer elicited higher nAb titers than the mosaic M trimer against clade A and C viruses. A mixture of the clade C and mosaic M trimers elicited nAb responses that were comparable to the better component of the mixture for each virus tested. These data suggest that combinations of relatively small numbers of immunologically complementary Env trimers may improve nAb responses., Importance: The development of an HIV-1 vaccine remains a formidable challenge due to multiple circulating strains of HIV-1 worldwide. This study describes a candidate HIV-1 Env protein vaccine whose sequence has been designed by computational methods to address HIV-1 diversity. The characteristics and immunogenicity of this Env protein, both alone and mixed together with a clade C Env protein vaccine, are described., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

The discovery and characterization of broadly neutralizing antibodies (bnAbs) against influenza viruses have raised hopes for the development of monoclonal antibody (mAb)-based immunotherapy and the design of universal influenza vaccines. Only one human bnAb (CR8020) specifically recognizing group 2 influenza A viruses has been previously characterized that binds to a highly conserved epitope at the base of the hemagglutinin (HA) stem and has neutralizing activity against H3, H7, and H10 viruses. Here, we report a second group 2 bnAb, CR8043, which was derived from a different germ-line gene encoding a highly divergent amino acid sequence. CR8043 has in vitro neutralizing activity against H3 and H10 viruses and protects mice against challenge with a lethal dose of H3N2 and H7N7 viruses. The crystal structure and EM reconstructions of the CR8043-H3 HA complex revealed that CR8043 binds to a site similar to the CR8020 epitope but uses an alternative angle of approach and a distinct set of interactions. The identification of another antibody against the group 2 stem epitope suggests that this conserved site of vulnerability has great potential for design of therapeutics and vaccines.

Background: Humans are genetically defective in synthesizing the common mammalian sialic acid N-glycolylneuraminic acid (Neu5Gc), but can metabolically incorporate it from dietary sources (particularly red meat and milk) into glycoproteins and glycolipids of human tumors, fetuses and some normal tissues. Metabolic incorporation of Neu5Gc from animal-derived cells and medium components also results in variable contamination of molecules and cells intended for human therapies. These Neu5Gc-incorporation phenomena are practically significant, because normal humans can have high levels of circulating anti-Neu5Gc antibodies. Thus, there is need for the sensitive and specific detection of Neu5Gc in human tissues and biotherapeutic products. Unlike monoclonal antibodies that recognize Neu5Gc only in the context of underlying structures, chicken immunoglobulin Y (IgY) polyclonal antibodies can recognize Neu5Gc in broader contexts. However, prior preparations of such antibodies (including our own) suffered from some non-specificity, as well as some cross-reactivity with the human sialic acid N-acetylneuraminic acid (Neu5Ac)., Methodology/principal Findings: We have developed a novel affinity method utilizing sequential columns of immobilized human and chimpanzee serum sialoglycoproteins, followed by specific elution from the latter column by free Neu5Gc. The resulting mono-specific antibody shows no staining in tissues or cells from mice with a human-like defect in Neu5Gc production. It allows sensitive and specific detection of Neu5Gc in all underlying glycan structural contexts studied, and is applicable to immunohistochemical, enzyme-linked immunosorbent assay (ELISA), Western blot and flow cytometry analyses. Non-immune chicken IgY is used as a reliable negative control. We show that these approaches allow sensitive detection of Neu5Gc in human tissue samples and in some biotherapeutic products, and finally show an example of how Neu5Gc might be eliminated from such products, by using a human cell line grown under defined conditions., Conclusions: We report a reliable antibody-based method for highly sensitive and specific detection of the non-human sialic acid Neu5Gc in human tissues and biotherapeutic products that has not been previously described.

CD33-related-Siglecs are lectins on immune cells that recognize sialic acids via extracellular domains, and deliver negative signals via cytosolic tyrosine-based regulatory motifs. We report that while Siglec-6/OB-BP1 (which can also bind leptin) is expressed on immune cells of both humans and the closely related great apes, placental trophoblast expression is human-specific, with little or no expression in ape placentae. Human-specific transcription factor recognition site changes in the Siglec-6 promoter region can help explain the human-specific expression. Human placenta also expresses natural ligands for Siglec-6 (a mixture of glycoproteins carrying cognate sialylated targets), in areas adjacent to Siglec-6 expression. Ligands were also found in uterine endometrium and on cell lines of trophoblastic or endometrial origin. Thus, Siglec-6 was recruited to placental expression during human evolution, presumably to interact with sialylated ligands for specific negative signaling functions and/or to regulate leptin availability. The control of human labor is poorly understood, but involves multiple cues, including placental signaling. Human birthing is also prolonged in comparison to that in our closest evolutionary relatives, the great apes. We found that Siglec-6 levels are generally low in placentae from elective surgical deliveries without known labor and the highest following completion of labor. We therefore speculate that the negative signaling potential of Siglec-6 was recruited to human-specific placental expression, to slow the tempo of the human birth process. The leptin-binding ability of Siglec-6 is also consistent with this hypothesis, as leptin-deficient mice have increased parturition times.

Covalent attachment of polyethylene glycol, PEGylation, has been shown to prolong the half-life and enhance the pharmacodynamics of therapeutic proteins. Current methods for PEGylation, which rely on chemical conjugation through reactive groups on amino acids, often generate isoforms in which PEG is attached at sites that interfere with bioactivity. Here, we present a novel strategy for site-directed PEGylation using glycosyltransferases to attach PEG to O-glycans. The process involves enzymatic GalNAc glycosylation at specific serine and threonine residues in proteins expressed without glycosylation in Escherichia coli, followed by enzymatic transfer of sialic acid conjugated with PEG to the introduced GalNAc residues. The strategy was applied to three therapeutic polypeptides, granulocyte colony stimulating factor (G-CSF), interferon-alpha2b (IFN-alpha2b), and granulocyte/macrophage colony stimulating factor (GM-CSF), which are currently in clinical use.

Mouse CD33/Siglec-3 (mCD33) is the apparent ortholog of human CD33/Siglec-3 (hCD33), a member of the Siglec (sialic acid-binding Ig superfamily lectin) family of sialic acid-recognizing cell-surface lectins. We examined the binding specificity and expression pattern of mCD33 and explored its functions by generating mice deficient in this molecule. Like hCD33, mCD33 is expressed on myeloid precursors in the bone marrow, albeit mostly in the more mature stages of the granulocytic lineage. Moreover, unlike hCD33, mCD33 in peripheral blood is primarily expressed on granulocytes. Also, unlike hCD33, mCD33 did not bind to alpha2-3- or alpha2-6-linked sialic acids on lactosamine units. Instead, it showed distinctive sialic acid-dependent binding only to the short O-linked glycans of certain mucins and weak binding to the sialyl-Tn epitope. Binding was enhanced by removal of 9-O-acetyl groups and attenuated by truncation of the glycerol-like side chain of sialic acids. Mice deficient in CD33 were viable and fertile in a controlled-access specific-pathogen-free vivarium, showed no major morphological or histological abnormalities, had no changes in bone marrow or peripheral leukocyte subpopulations, and had very minor differences in biochemical and erythrocyte parameters. Cellular responses to intraperitoneally injected proinflammatory stimulants, as well as subsequent interleukin-6 secretion, were also apparently unaffected. These results indicate substantial species differences in CD33 expression patterns and ligand recognition and suggest functional degeneracy between mCD33 and the other CD33-related Siglec proteins expressed on cells of the myeloid lineage.

The siglecs (sialic acid-binding immunoglobulin superfamily lectins) are immunoglobulin superfamily members recognizing sialylated ligands. Most prior studies of siglec specificities focused on alpha2-3- and alpha2-6-sialyllactos(amin)es and on one or two of the siglecs at a time. Here, we explore several new aspects of specificities of the first six reported siglecs, using sialylated glycans presented in multivalent form, on synthetic polyacrylamide backbones, or on mucin polypeptides. First, we report that binding of siglec-1 (sialoadhesin), siglec-3 (CD33), siglec-4a (myelin-associated glycoprotein), and siglec-5 to alpha2-3 sialyllactosamine is affected markedly by the presence of an alpha1-3-linked fucose. Thus, while siglecs may not interfere with selectin-mediated recognition, fucosylation could negatively regulate siglec binding. Second, in contrast to earlier studies, we find that siglec-3 prefers alpha2-6-sialyllactose. Third, siglec-5 binds alpha2-8-linked sialic acid, making it the siglec least specific for linkage recognition. Fourth, siglecs-2 (CD22), -3, -5, and -6 (obesity-binding protein 1) showed significant binding to sialyl-Tn (Neu5Acalpha2-6-GalNAc), a tumor marker associated with poor prognosis. Fifth, siglec-6 is an exception among siglecs in not requiring the glycerol side chain of sialic acid for recognition. Sixth, all siglecs require the carboxyl group of sialic acid for binding. Finally, the presentation of the sialyl-Tn epitope and/or more extended structures that include this motif may be important for optimal recognition by the siglecs. This was concluded from studies using ovine, bovine, and porcine submaxillary mucins and Chinese hamster ovary cells transfected with ST6GalNAc-I and/or the mucin polypeptide MUC1.

The common sialic acids of mammalian cells are N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc). Humans are an exception, because of a mutation in CMP-sialic acid hydroxylase, which occurred after our common ancestor with great apes. We asked if the resulting loss of Neu5Gc and increase in Neu5Ac in humans alters the biology of the siglecs, which are Ig superfamily members that recognize sialic acids. Human siglec-1 (sialoadhesin) strongly prefers Neu5Ac over Neu5Gc. Thus, humans have a higher density of siglec-1 ligands than great apes. Siglec-1-positive macrophages in humans are found primarily in the perifollicular zone, whereas in chimpanzees they also occur in the marginal zone and surrounding the periarteriolar lymphocyte sheaths. Although only a subset of chimpanzee macrophages express siglec-1, most human macrophages are positive. A known evolutionary difference is the strong preference of mouse siglec-2 (CD22) for Neu5Gc, contrasting with human siglec-2, which binds Neu5Ac equally well. To ask when the preference for Neu5Gc was adjusted in the human lineage, we cloned the first three extracellular domains of siglec-2 from all of the great apes and examined their preference. In fact, siglec-2 had evolved a higher degree of recognition flexibility before Neu5Gc was lost in humans. Human siglec-3 (CD33) and siglec-6 (obesity-binding protein 1) also recognize both Neu5Ac and Neu5Gc, and siglec-5 may have some preference for Neu5Gc. Others showed that siglec-4a (myelin-associated glycoprotein) prefers Neu5Ac over Neu5Gc. Thus, the human loss of Neu5Gc may alter biological processes involving siglec-1, and possibly, siglec-4a or -5.

We report the expression cloning of a novel leptin-binding protein of the immunoglobulin superfamily (OB-BP1) and a cross-hybridizing clone (OB-BP2) that is identical to a recently described sialic acid-binding I-type lectin called Siglec-5. Comparisons to other known Siglec family members (CD22, CD33, myelin-associated glycoprotein, and sialoadhesin) show that OB-BP1, OB-BP2/Siglec-5, and CD33/Siglec-3 constitute a unique related subgroup with a high level of overall amino acid identity: OB-BP1 versus Siglec-5 (59%), OB-BP1 versus CD33 (63%), and OB-BP2/Siglec-5 versus CD33 (56%). The cytoplasmic domains are not as highly conserved, but display novel motifs which are putative sites of tyrosine phosphorylation, including an immunoreceptor tyrosine kinase inhibitory motif and a motif found in SLAM and SLAM-like proteins. Human tissues showed high levels of OB-BP1 mRNA in placenta and moderate expression in spleen, peripheral blood leukocytes, and small intestine. OB-BP2/Siglec-5 mRNA was detected in peripheral blood leukocytes, lung, spleen, and placenta. A monoclonal antibody specific for OB-BP1 confirmed high expression in the cyto- and syncytiotrophoblasts of the placenta. Using this antibody on peripheral blood leukocytes showed an almost exclusive expression pattern on B cells. Recombinant forms of the extracellular domains of OB-BP1, OB-BP2/Siglec-5, and CD33/Siglec-3 were assayed for specific binding of leptin. While OB-BP1 exhibited tight binding (K(d) 91 nM), the other two showed weak binding with K(d) values in the 1-2 microM range. Studies with sialylated ligands indicated that OB-BP1 selectively bound Neu5Acalpha2-6GalNAcalpha (sialyl-Tn) allowing its formal designation as Siglec-6. The identification of OB-BP1/Siglec-6 as a Siglec family member, coupled with its restricted expression pattern, suggests that it may mediate cell-cell recognition events by interacting with sialylated glycoprotein ligands expressed on specific cell populations. We also propose a role for OB-BP1 in leptin physiology, as a molecular sink to regulate leptin serum levels.

Patients suffering from rheumatoid arthritis (RA) may experience a temporary reduction of disease symptoms during pregnancy. As indicated by the occurrence of RA-disease symptoms during pregnancy, three categories of patients were defined, namely, remission, relapse and unchanged. In all three categories changes in the plasma level and glycosylation of alpha1-acid glycoprotein (AGP) were determined longitudinally in comparison to those occurring in pregnancy of healthy women. In healthy pregnancy, we observed: (i) a peak in the plasma concentration at week 18 and a minimum at week 30; (ii) a continuous increase in the degree of branching of the glycans during the entire pregnancy period, and (iii) a decrease in the degree of alpha3-fucosylation of AGP-glycans with a minimum occurring at week 25. Comparable pregnancy-induced changes in glycosylation were found for two other acute-phase proteins alpha1-protease inhibitor (PI) and alpha1-antichymotrypsin (ACT). Increased oestrogen levels, known to occur during pregnancy, may be one of the factors that induce these changes, because the increased branching and decreased alpha3-fucosylation is in agreement with our earlier findings regarding an involvement of this hormone in the regulation of acute phase protein glycosylation in oestrogen-treated males as well as females. In all three clinical categories in RA, pregnancy also induced a continuous increase in the degree of branching of the glycans of AGP. However, similar changes in concentration and fucosylation were only found during remission of the disease symptoms. In the relapse and unchanged categories in RA, the degree of fucosylation and the plasma concentration of AGP remained constant throughout pregnancy. This indicates a relationship between changes in alpha3-fucosylation of AGP and RA disease activity.

Acute and chronic inflammation-induced expression of sialyl LewisX has already been shown to occur on alpha1-acid glycoprotein. We now demonstrate that this phenomenon is not restricted to alpha1-acid glycoprotein but also occurs on two other acute-phase proteins. ie on alpha1-antichymotrypsin and on haptoglobin. The level of expression of sialyl LewisX on these proteins was lower than on alpha1-acid glycoprotein, in all likelihood because alpha1-acid glycoprotein is the only acute-phase protein containing tetraantennary glycans. No expression of sialyl LewisX was detectable on alpha1-protease inhibitor, a protein with a high diantennary glycan content. Non-sialylated LewisX was not detectable on these major acute-phase proteins in any of the conditions studied. This indicates that the majority of the a3-linked fucose residues are present as sialyl LewisX on alpha1-acid glycoprotein, alpha1-antichymotrypsin and haptoglobin. The absolute contribution to the total phenotype in plasma of protein containing this determinant in a multivalent form was highest for alpha1-acid glycoprotein. This leads us to propose that alpha1-acid glycoprotein is, among the acute-phase proteins studied, the one with the highest potential for interference with the extravasation of leukocytes by binding to the selectins.

The major alpha3-fucosyltransferase activity in human plasma is encoded by the gene for fucosyltransferase VI (FUT6). A missense mutation (Gly-739 --> Ala) in this gene is responsible for deficiency of enzyme activity in plasma. To examine whether this fucosyltransferase is the sole enzyme responsible for the alpha3-fucosylation of serum glycoproteins in the liver, we studied the fucosylation of three glycoproteins in sera of individuals with or without inactivated FUT3 and/or FUT6 gene(s) but with a functional FUT5 gene. alpha1-Acid glycoprotein was used as the principal reporter protein for liver alpha3-fucosyltransferase activity, because of its high fucose content. In all individuals with the FUT6 missense mutation Gly-739 --> Ala in double dose, no fucosylation of alpha1-acid glycoprotein was found. This alpha1-acid glycoprotein was not intrinsically resistant to fucosylation, since it was susceptible to in vitro fucosylation using an alpha3/4-fucosyltransferase isolated from human milk. The same result was found for alpha1-antichymotrypsin and alpha1-protease inhibitor. On the other hand in all individuals with alpha3-fucosyltransferase activity in plasma, alpha3-fucosylated glycoforms of the glycoproteins studied were found. The degree of fucosylation of alpha1-acid glycoprotein was correlated with alpha3-fucosyltransferase activity (Rs = 0.82). These data indicate that the product of FUT6, but not of FUT3 or of FUT5, is responsible for the alpha3-fucosylation of glycoproteins produced in liver and suggest that this organ is a major source of alpha3-fucosyltransferase activity in plasma.

The occurrence of differences in acute-phase response, with respect to concentration and glycosylation of alpha 1-acid glycoprotein (AGP) was studied in the sera of patients, surviving or not from septic shock. Crossed affino-immunoelectrophoresis was used with concanavalin A and Aleuria aurantia lectin for the detection of the degree of branching and fucosylation, respectively, and the monoclonal CSLEX-1 for the detection of sialyl Lewisx (SLeX) groups on AGP. Septic shock apparently induced an acute-phase response as indicated by the increased serum levels and changed glycosylation of AGP. In the survivor group a transient increase in diantennary glycan content was accompanied by a gradually increasing fucosylation and SLeX expression, comparable to those observed in the early phase of an acute-inflammatory response. Remarkably, in the non-survivor group a modest increase in diantennary glycan content was accompanied by a strong elevation of the fucosylation of AGP and the expression of SLeX groups on AGP, typical for the late phase of an acute-phase response. Our results suggest that these changes in glycosylation of AGP can have a prognostic value for the outcome of septic shock.

The aim of this review is to summarize the research efforts of the last two decades with respect to (i) the determination and characterization of the changes in glycosylation of AGP under various physiological and pathological states; and (ii) the effects of such changes on its possible anti-inflammatory functions. It will become clear that the heterogeneity observed in the glycosylation of AGP in serum, represents various so-called glycoforms of AGP, of which the relative amounts are strictly determined by the (patho) physiological conditions.