Characterization of a xylose-specific antiserum that reacts with the complex asparagine-linked glycans of extracellular and vacuolar glycoproteins

Antibodies were raised against carrot (Daucus carota) cell wall β-fructosidase that was either in a native configuration (this serum is called anti-βF₁) or chemically deglycosylated (anti-βF₂). The two antisera had completely different specificities when tested by immunoblotting. The anti-βF₁ serum reacted with β-fructosidase and many other carrot cell wall proteins as well as with many proteins in extracts of bean (Phaseolus vulgaris) cotyledons and tobacco (Nicotiana tabacum) seeds. It did not react with chemically deglycosylated β-fructosidase. The anti-βF₁ serum also reacted with the bean vacuolar protein, phytohemagglutinin, but not with deglycosylated phytohemagglutinin. The anti-βF₂ serum reacted with both normal and deglycosylated β-fructosidase but not with other proteins. These results indicate that the βF₂ antibodies recognize the β-fructosidase polypeptide, while the βF₁ antibodies recognize glycan sidechains common to many glycoproteins. We used immunoadsorption on glycoprotein-Sepharose columns and hapten inhibition of immunoblot reactions to characterize the nature of the antigenic site. Antibody binding activity was found to be associated with Man₃(Xyl)(GIcNAc)₂Fuc, Man₃(Xyl)(GIcNAc)₂, and Man(Xyl) (GIcNAc)₂ glycans, but not with Man₃(GIcNAc)₂. Treatment of phytohemagglutinin, a glycoprotein with a Man₃(Xyl)(GIcNAc)₂Fuc glycan, with Charonia lampas β-xylosidase (after treatment with jack-bean α-mannosidase) greatly diminished the binding between the antibodies and phytohemagglutinin. We conclude, therefore, that the antibodies bind primarily to the xyloseβ, 1→ 2mannose structure commonly found in the complex glycans of plant glycoproteins.     

Expression of Two Different Glutamine Synthetase Polypeptides during Root Development in Phaseolus vulgaris L

Immunoprecipitation and two-dimensional gel electrophoresis analysis of the glutamine synthetase (GS) polypeptides (α and β) during Phaseolus vulgaris root development shows that the α polypeptide is the main component of the enzyme in the embryo and in up to 5 day old roots. From 5 days on, the β polypeptide becomes the root predominant GS monomer. The α/β ratio of the in vitro translated GS polypeptides from the total polysomal RNA isolated at different root ages correlates with the α/β ratio observed in the root extracts. These results suggest that the two root GS polypeptides are encoded by different mRNA species in Phaseolus vulgaris.     

Regulation of Nodule Glutamine Synthetase by CO(2) Levels in Bean (Phaseolus vulgaris L.)

Nodulated bean (Phaseolus vulgaris) plants were grown for 17 days after infection in normal (0.02%) CO₂ and from day 8 to 17 in high (0.1%) CO₂ in order to increase nitrogen fixation and define how nodule glutamine synthetase (GS) isoforms are regulated by the ammonia derived from the bacteroid. Nitrogenase activity was detected by day 10, and by day 17 activity was over twofold higher in 0.1% of CO₂ compared with plants grown in 0.02% CO₂ and inoculated with Rhizobium wild-type strain CE3. Likewise, plant fresh weight increased in response to increased CO₂, particularly in plants inoculated with the Rhizobium phaseoli mutant strain CFN037. Glutamine synthetase specific activity increased 2.5- to 6.5-fold from day 11 to 17. However, increased CO₂ did not appear to have an effect on GS specific activity. Analysis of the nodule GS polypeptide composition revealed that the γ polypeptide was significantly reduced in response to high CO₂, whereas the β polypeptide was not affected. The significance of this result in relation to the regulation of GS isoforms and their role in the assimilation of ammonia in the nodule is discussed in this paper.     

αvβ3 Integrin Mediates the Cell-adhesive Capacity and Biological Activity of Basic Fibroblast Growth Factor (FGF-2) in Cultured Endothelial Cells

Fibroblast growth factor-2 (FGF-2) immobilized on non-tissue culture plastic promotes adhesion and spreading of bovine and human endothelial cells that are inhibited by anti-FGF-2 antibody. Heat-inactivated FGF-2 retains its cell-adhesive activity despite its incapacity to bind to tyrosine-kinase FGF receptors or to cell-surface heparan sulfate proteoglycans. Recombinant glutathione-S-transferase-FGF-2 chimeras and synthetic FGF-2 fragments identify two cell-adhesive domains in FGF-2 corresponding to amino acid sequences 38–61 and 82–101. Both regions are distinct from the FGF-receptor-binding domain of FGF-2 and contain a DGR sequence that is the inverse of the RGD cell-recognition sequence. Calcium deprivation, RGD-containing eptapeptides, soluble vitronectin (VN), but not fibronectin (FN), inhibit cell adhesion to FGF-2. Conversely, soluble FGF-2 prevents cell adhesion to VN but not FN, thus implicating VN receptor in the cell-adhesive activity of FGF-2. Accordingly, monoclonal and polyclonal anti-α_vβ₃ antibodies prevent cell adhesion to FGF-2. Also, purified human α_vβ₃ binds to immobilized FGF-2 in a cation-dependent manner, and this interaction is competed by soluble VN but not by soluble FN. Finally, anti-α_vβ₃ monoclonal and polyclonal antibodies specifically inhibit mitogenesis and urokinase-type plasminogen activator (uPA) up-regulation induced by free FGF-2 in endothelial cells adherent to tissue culture plastic. These data demonstrate that FGF-2 interacts with α_vβ₃ integrin and that this interaction mediates the capacity of the angiogenic growth factor to induce cell adhesion, mitogenesis, and uPA up-regulation in endothelial cells.     

Variability in Exposure of Epitope G40-R43 of Domain I in Commercial Anti-Beta2-Glycoprotein I IgG ELISAs

Background

A major problem for diagnosing the antiphospholipid syndrome (APS) is the high variability between commercial anti-β₂glycoprotein I (β₂GPI) assays. Predominantly antibodies reactive against cryptic epitope Glycine40-Arginine43 (G40-R43) in domain I are associated with an increased risk for thrombosis. Upon interaction with anionic surfaces β₂GPI opens up, thereby exposing G40-R43.

Objectives

To examine whether suboptimal exposure of epitope G40-R43 explains the variations in results observed between commercial assays.

Methods

Two patient-derived monoclonal antibodies were tested on neutral versus anionic plates. Antibody P1-117 reacts with G40-R43 in the open conformation while P2-6 recognizes β₂GPI irrespective of its conformation. These antibodies were tested in commercial anti-β₂GPI assays (A–E).

Results

In assay A, both antibodies showed equal reactivity towards β₂GPI, indicating that all the β₂GPI exposes G40-R43. In other assays P1-117 displayed lower reactivity than P2-6, demonstrating reduced G40-R43 availability. To exclude influences of other assay features, reactivity was re-examined on plates of assay A and B using the protocol/reagents from each assay. In all combinations, reactivity of both antibodies on a plate was comparable to results obtained with its own protocol/reagents, suggesting that the coating, rather than other assay components, accounts for the observed differences. In two patient cohorts we demonstrated that a number of domain I-reactive samples are missed in assays characterized by a decreased exposure of epitope G40-R43.

Conclusions

Exposure of epitope G40-R43 on β₂GPI is highly variable between commercial anti-β₂GPI assays. As a consequence, patients can be falsely assigned negative in assays characterized by a reduced exposure of G40-R43.     

Monoclonal anti-β1-adrenergic receptor antibodies activate G protein signaling in the absence of β-arrestin recruitment

Thermostabilized G protein-coupled receptors used as antigens for in vivo immunization have resulted in the generation of functional agonistic anti-β₁-adrenergic (β₁AR) receptor monoclonal antibodies (mAbs). The focus of this study was to examine the pharmacology of these antibodies to evaluate their mechanistic activity at β₁AR. Immunization with the β₁AR stabilized receptor yielded five stable hybridoma clones, four of which expressed functional IgG, as determined in cell-based assays used to evaluate cAMP stimulation. The antibodies bind diverse epitopes associated with low nanomolar agonist activity at β₁AR, and they appeared to show some degree of biased signaling as they were inactive in an assay measuring signaling through β-arrestin. In vitro characterization also verified different antibody-receptor interactions reflecting the different epitopes on the extracellular surface of β₁AR to which the mAbs bind. The anti-β₁AR mAbs only demonstrated agonist activity when in dimeric antibody format, but not as the monomeric Fab format, suggesting that agonist activation may be mediated through promoting receptor dimerization. Finally, we have also shown that at least one of these antibodies exhibits in vivo functional activity at a therapeutically-relevant dose producing an increase in heart rate consistent with β₁AR agonism.     

A Role for Uric Acid and the Nalp3 Inflammasome in Antiphospholipid Antibody-Induced IL-1β Production by Human First Trimester Trophoblast

Women with antiphospholipid syndrome (APS) are at risk of recurrent pregnancy loss and obstetrical disorders, such as preeclampsia and intrauterine growth restriction (IUGR). Antiphospholipid antibodies (aPL) directly target the placenta by binding beta₂-glycoprotein I (β₂GPI) expressed on the trophoblast. We recently demonstrated in human first trimester trophoblast cells that anti-β₂GPI antibodies (Abs) induce the secretion of IL-1β in a Toll-like receptor 4 (TLR4)-dependent manner. IL-1β secretion requires processing of pro-IL-1β and this is mediated by the inflammasome, a complex of Nalp3, apoptosis-associated speck-like protein containing a CARD (ASC) and caspase-1. The objective of this study was to determine if aPL induce IL-1β production in trophoblast via the inflammasome. Using a human first trimester trophoblast cell line, we demonstrated that a mouse anti-β₂GPI mAb and human polyclonal aPL-IgG induce IL-1β processing and secretion, which was partially blocked upon caspase-1 inhibition. Nalp3 and ASC knockdown also attenuated anti-β₂GPI Ab-induced IL-1β secretion. Furthermore, aPL stimulated the production of uric acid in a TLR4-dependent manner; and inhibition of uric acid prevented aPL-induced IL-1β production by the trophoblast. These findings demonstrate that aPL, via TLR4 activation, induce a uric acid response in human trophoblast, which in turn activates the Nalp3/ASC inflammasome leading to IL-1β processing and secretion. This novel mechanism may account for the inflammation at the maternal-fetal interface, which causes placental dysfunction and increases the risk of adverse pregnancy outcome in patients with APS.     

Structure and expression of class II defective herpes simplex virus genomes encoding infected cell polypeptide number 8

Defective genomes present in serially passaged virus stocks derived from the tsLB2 mutant of herpes simplex virus type 1 were found to consist of repeat units in which sequences from the U_L region, within map coordinates 0.356 and 0.429 of standard herpes simplex virus DNA, were covalently linked to sequences from the end of the S component. The major defective genome species consisted of repeat units which were 4.9 × 10⁶ in molecular weight and contained a specific deletion within the U_L segment. These tsLB2 defective genomes were stable through more than 35 sequential virus passages. The ratios of defective virus genomes to helper virus genomes present in different passages fluctuated in synchrony with the capacity of the passages to interfere with standard virus replication. Cells infected with passages enriched for defective genomes overproduced the infected cell polypeptide number 8, which had previously been mapped within the U_L sequences present in the tsLB2 defective genomes. In contrast, the synthesis of most other infected cell polypeptides was delayed and reduced. The abundant synthesis of infected cell polypeptide number 8 followed the β regulatory pattern, as evident from kinetic studies and from experiments in which cycloheximide, canavanine, and phosphonoacetate were used. However, in contrast to many β (early) and γ (late) viral polypeptides, the synthesis of infected cell polypeptide number 8 was only minimally reduced when cells infected with serially passaged tsLB2 were incubated at 39°C. The tsLB2 mutation had previously been mapped within the domains of the gene encoding infected cell polypeptide number 4, the function of which was shown to be required for β and γ viral gene expression. It is thus possible that the tsLB2 mutation affects the synthesis of only a subset of the β and γ viral polypeptides. An additional polypeptide, 74.5 × 10³ in molecular weight, was abundantly produced in cells infected with a number of tsLB2 passages. This polypeptide was most likely expressed from truncated gene templates within the most abundant, deleted repeats of tsLB2 defective virus DNA.     

Tobacco Plants Transformed with the Bean alphaai Gene Express an Inhibitor of Insect alpha-Amylase in Their Seeds

Bean (Phaseolus vulgaris L.) seeds contain a putative plant defense protein that inhibits insect and mammalian but not plant α-amylases. We recently (J Moreno, MJ Chrispeels [1989] Proc Natl Acad Sci USA 86:7885-7889) presented strong circumstantial evidence that this α-amylase inhibitor (αAI) is encoded by an already-identified lectin gene whose product is referred to as lectin-like-protein (LLP). We have now made a chimeric gene consisting of the coding sequence of the lectin gene that encodes LLP and the 5′ and 3′ flanking sequences of the lectin gene that encodes phytohemagglutinin-L. When this chimeric gene was expressed in transgenic tobacco (Nicotiana tabacum), we observed in the seeds a series of polypeptides (M_r 10,000-18,000) that cross-react with antibodies to the bean α-amylase inhibitor. Most of these polypeptides bind to a pig pancreas α-amylase affinity column. An extract of the seeds of the transformed tobacco plants inhibits pig pancreas α-amylase activity as well as the α-amylase present in the midgut of Tenebrio molitor. We suggest that introduction of this lectin gene (to be called αai) into other leguminous plants may be a strategy to protect the seeds from the seed-eating larvae of Coleoptera.     

Intractable Headaches,Ischemic Stroke,and Seizures Are Linked to the Presence of Anti-β2GPI Antibodies in Patients with Systemic Lupus Erythematosus

Background

Neuropsychiatric systemic lupus erythematosus (NPSLE) is a common and potentially fatal manifestation of SLE. Antiphospholipid antibodies (aPL) such as lupus anticoagulant (LA), anticardiolipin (aCL) and antibodies to β2glycoprotein I (anti-β2GPI), the most important aPL antigen, are thought to play a role in some forms of NPSLE. As of yet, their specific roles in NPSLE manifestations remain to be elucidated.

Methodology/Principal Findings

57 SLE patients (53 women) were assessed for LA, aCL and anti-β₂GPI twice, to determine persistent positivity. All patients were examined by neurology and psychiatry specialists. 69 healthy subjects were assessed as controls. NPSLE was diagnosed in 74% of patients. Headaches were the most prevalent manifestation of NPSLE (39%), followed by cerebrovascular disease (CVD) (23%), depressive disorders (19.0%), and seizures (14%). NPSLE and non-NPSLE patients showed comparable SLE activity and corticosteroid use. In 65% of patients neuropsychiatric manifestations preceded SLE diagnosis. aPL profiles of NPSLE patients and non-NPSLE patients were similar. Headaches and ischemic stroke were independently associated with anti-β₂GPI-IgM (OR=5.6; p<0.05), and seizures were linked to anti-β₂GPI-IgG (OR=11.3; p=0.01).

Conclusions

In SLE patients, neuropsychiatric manifestations occur frequently and early, often before the disease is diagnosed. Autoantibodies to β2GPI are linked to non-specific headaches, ischemic stroke and seizures, and show a better predictive value than aCL and LA. These findings may help to improve the diagnosis of NPSLE and should prompt further studies to characterize the role of anti-β2GPI in the pathogenesis of this condition.     

The Effects of NF-κB and c-Jun/AP-1 on the Expression of Prothrombotic and Proinflammatory Molecules Induced by Anti-β2GPI in Mouse

Our previous data demonstrated that nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) are involved in the process of anti-β₂GPI/β₂GPI-induced tissue factor (TF) expression in monocytes. However, the role of NF-κB and AP-1 in pathogenic mechanisms of antiphospholipid syndrome (APS) in vivo has been rarely studied. This study aimed to investigate whether NF-κB and c-Jun/AP-1 are involved in anti-β₂GPI-induced expression of prothrombotic and proinflammatory molecules in mouse. IgG-APS or anti-β₂GPI antibodies were injected into BALB/c mice in the presence or absence of PDTC (a specific inhibitor of NF-κB) and Curcumin (a potent inhibitor of AP-1) treatment. Our data showed that both IgG-APS and anti-β₂GPI could induce the activation of NF-κB and c-Jun/AP-1 in mouse peritoneal macrophages. The anti-β₂GPI-induced TF activity in homogenates of carotid arteries and peritoneal macrophages from mice could significantly decrease after PDTC and/or Curcumin treatment, in which PDTC showed the strongest inhibitory effect, but combination of two inhibitors had no synergistic effect. Furthermore, anti-β₂GPI-induced expression of TF, VCAM-1, ICAM-1 and E-selectin in the aorta and expression of TF, IL-1β, IL-6 and TNF-α in peritoneal macrophages of mice were also significantly attenuated by PDTC and/or Curcumin treatment. These results indicate that both NF-κB and c-Jun/AP-1 are involved in regulating anti-β₂GPI-induced expression of prothrombotic and proinflammatory molecules in vivo. Inhibition of NF-κB and c-Jun/AP-1 pathways may be beneficial for the prevention and treatment of thrombosis and inflammation in patients with APS.     

Characterization of alpha-Amylase-Inhibitor, a Lectin-Like Protein in the Seeds of Phaseolus vulgaris

The common bean, Phaseolus vulgaris, contains a glycoprotein that inhibits the activity of mammalian and insect α-amylases, but not of plant α-amylases. It is therefore classified as an antifeedant or seed defense protein. In P. vulgaris cv Greensleeves, α-amylase inhibitor (αAl) is present in embryonic axes and cotyledons, but not in other organs of the plant. The protein is synthesized during the same time period that phaseolin and phytohemagglutinin are made and also accumulates in the protein storage vacuoles (protein bodies). Purified αAl can be resolved by SDS-PAGE into five bands (M_r 15,000-19,000), four of which have covalently attached glycans. These bands represent glycoforms of two different polypeptides. All the glycoforms have complex glycans that are resistant to removal by endoglycosidase H, indicating transport of the protein through the Golgi apparatus. The two different polypeptides correspond to the N-terminal and C-terminal halves of a lectin-like protein encoded by an already identified gene or a gene closely related to it (LM Hoffman [1984] J Mol Appl Genet 2: 447-453; J Moreno, MJ Chrispeels [1989] Proc Natl Acad Sci USA 86:7885-7889). The primary translation product of αAl is a polypeptide of M_r 28,000. Immunologically cross-reacting glycopolypeptides of M_r 30,000 to 35,000 are present in the endoplasmic reticulum, while the smaller polypeptides (M_r 15,000-19,000) accumulate in protein storage vacuoles (protein bodies). Together these data indicate that αAl is a typical bean lectin-type protein that is synthesized on the rough endoplasmlc reticulum, modified in the Golgi, and transported to the protein storage vacuoles.     

Synthesis of Oat Globulin Precursors : Analogy to Legume 11S Storage Protein Synthesisa

The oat (Avena sativa L.) seed globulin was found to be synthesized in vitro as 60,000 to 64,000 dalton precursors. In vivo protein labeling yielded polypeptides of 58,000 to 62,000 daltons, suggesting cleavage of signal sequences from the precursors. Further cleavage is apparently required to separate the α and β polypeptide sequences which are known to form disulfide-linked 53,000 to 58,000 dalton species in the (αβ)₆ holoprotein. The data are discussed with respect to analogous synthesis and processing of some legume 11S storage proteins.     

Relationship between the Autoantibody and Expression of β3-Adrenoceptor in Lung and Heart

Background

Evidences suggest that β₃ -adrenoceptor (β₃-AR) plays an important role in heart failure (HF), although no data is reported indicating how these effects may change with the increasing age. Pulmonary congestion and edema are the major life-threatening complications associated with HF. The purpose of this study is to explore the relationship between the anti-β₃-AR autoantibody and the expression of β₃-AR in the lungs and heart for both aged patients and rats with HF.

Methods

Synthetic β₃-AR peptides served as the target antigens in ELISA were used to screen the anti-β₃-AR autoantibody in aged patients and rats. Two aged rat models were constructed based on aortic banding and sham-operation. The expression of β₃-AR mRNA and protein in the lung and heart was measured in intervention and non-intervention groups by Western blot analysis at the baseline, 5^th, 7^th, 9^th and 11^th week, respectively.

Results

The frequency and titer of anti-β₃-AR autoantibody in aged patients and rats with HF were higher than those in the control group (p<0.05). The expression of β₃-AR mRNA and protein in pulmonary tissues decreased continually from the 7^th week (p<0.05), followed by HF observed during the 9^th week. The expression of β₃-AR in myocardial tissues continued to increase after the 9^th week (p<0.05), and the expression of both β₃-AR mRNA and protein in the BRL group [HF group with BRL37344 (4-[-[2-hydroxy-(3-chlorophenyl)ethyl-amino] phenoxyacetic acid) (a β₃-AR agonist) injection] was positively correlated with BRL37344 when compared with non-BRL group (HF group without BRL37344 injection) (p<0.05).

Conclusion

Anti-β₃-AR autoantibody was detected in aged patients and rats with HF. The expression of β₃-AR mRNA and protein in pulmonary tissues decreased continually, and began earlier than in the heart, but its expression in myocardial tissues increased continually and could be further promoted by β₃-AR agonist.     

Ero1α Is Expressed on Blood Platelets in Association with Protein-disulfide Isomerase and Contributes to Redox-controlled Remodeling of αIIbβ3

Recent evidence supports a role of protein-disulfide isomerase (PDI) in redox-controlled remodeling of the exofacial domains of α_IIbβ₃ in blood platelets. The aim of this study was to explain whether Ero1α can be responsible for extracellular reoxidation of the PDI active site. We showed that Ero1α can be found on platelets and is rapidly recruited to the cell surface in response to platelet agonists. It is physically associated with PDI and α_IIbβ₃, as suggested by colocalization analysis in confocal microscopy and confirmed by immunoprecipitation experiments. Apart from monomeric oxidized Ero1α, anti-α_IIbβ₃ immunoprecipitates showed the presence of several Ero1α-positive bands that corresponded to the complexes α_IIbβ₃-PDI-Ero1α, PDI-Ero1α, and Ero1α-Ero1α dimers. It binds more efficiently to the activated α_IIbβ₃ conformer, and its interaction is inhibited by RGD peptides. Ero1α appears to be involved in the regulation of α_IIbβ₃ receptor activity because of the following: (a) blocking the cell surface Ero1α by antibodies leads to a decrease in platelet aggregation in response to agonists and a decrease in fibrinogen and PAC-1 binding, and (b) transfection of MEG01 with Ero1α increases α_IIbβ₃ receptor activity, as indicated by increased binding of fibrinogen.     

pH responsiveness of fibrous assemblies of repeat-sequence amphipathic α-helix polypeptides

We reported previously that our designed polypeptide α3 (21 residues), which has three repeats of a seven-amino-acid sequence (LETLAKA)₃, forms not only an amphipathic α-helix structure but also long fibrous assemblies in aqueous solution. To address the relationship between the electrical states of the polypeptide and its α-helix and fibrous assembly formation, we characterized mutated polypeptides in which charged amino acid residues of α3 were replaced with Ser. We prepared the following polypeptides: 2Sα3 (LSTLAKA)₃, in which all Glu residues were replaced with Ser residues; 6Sα3 (LETLASA)₃, in which all Lys residues were replaced with Ser; and 2S6Sα3 (LSTLASA)₃; in which all Glu and Lys residues were replaced with Ser. In 0.1M KCl, 2Sα3 formed an α-helix under basic conditions and 6Sα3 formed an α-helix under acid conditions. In 1M KCl, they both formed α-helices under a wide pH range. In addition, 2Sα3 and 6Sα3 formed fibrous assemblies under the same buffer conditions in which they formed α-helices. α-Helix and fibrous assembly formation by these polypeptides was reversible in a pH-dependent manner. In contrast, 2S6Sα3 formed an α-helix under basic conditions in 1M KCl. Taken together, these findings reveal that the charge states of the charged amino acid residues and the charge state of the Leu residue located at the terminus play an important role in α-helix formation.     

Design and Optimization of Anti-amyloid Domain Antibodies Specific for β-Amyloid and Islet Amyloid Polypeptide

Antibodies with conformational specificity are important for detecting and interfering with polypeptide aggregation linked to several human disorders. We are developing a motif-grafting approach for designing lead antibody candidates specific for amyloid-forming polypeptides such as the Alzheimer peptide (Aβ). This approach involves grafting amyloidogenic peptide segments into the complementarity-determining regions (CDRs) of single-domain (V_H) antibodies. Here we have investigated the impact of polar mutations inserted at the edges of a large hydrophobic Aβ42 peptide segment (Aβ residues 17–42) in CDR3 on the solubility and conformational specificity of the corresponding V_H domains. We find that V_H expression and solubility are strongly enhanced by introducing multiple negatively charged or asparagine residues at the edges of CDR3, whereas other polar mutations are less effective (glutamine and serine) or ineffective (threonine, lysine, and arginine). Moreover, Aβ V_H domains with negatively charged CDR3 mutations show significant preference for recognizing Aβ fibrils relative to Aβ monomers, whereas the same V_H domains with other polar CDR3 mutations recognize both Aβ conformers. We observe similar behavior for a V_H domain grafted with a large hydrophobic peptide from islet amyloid polypeptide (residues 8–37) that contains negatively charged mutations at the edges of CDR3. These findings highlight the sensitivity of antibody binding and solubility to residues at the edges of CDRs, and provide guidelines for designing other grafted antibody fragments with hydrophobic binding loops.     

Protection by Anti-β-Glucan Antibodies Is Associated with Restricted β-1,3 Glucan Binding Specificity and Inhibition of Fungal Growth and Adherence

Anti-β-glucan antibodies elicited by a laminarin-conjugate vaccine confer cross-protection to mice challenged with major fungal pathogens such as Candida albicans, Aspergillus fumigatus and Cryptococcus neoformans. To gain insights into protective β-glucan epitope(s) and protection mechanisms, we studied two anti-β-glucan monoclonal antibodies (mAb) with identical complementarity-determining regions but different isotypes (mAb 2G8, IgG2b and mAb 1E12, IgM). C. albicans, the most relevant fungal pathogen for humans, was used as a model.Both mAbs bound to fungal cell surface and to the β1,3-β1,6 glucan of the fungal cell wall skeleton, as shown by immunofluorescence, electron-microscopy and ELISA. They were also equally unable to opsonize fungal cells in a J774 macrophage phagocytosis and killing assay. However, only the IgG2b conferred substantial protection against mucosal and systemic candidiasis in passive vaccination experiments in rodents. Competition ELISA and microarray analyses using sequence-defined glucan oligosaccharides showed that the protective IgG2b selectively bound to β1,3-linked (laminarin-like) glucose sequences whereas the non-protective IgM bound to β1,6- and β1,4-linked glucose sequences in addition to β1,3-linked ones. Only the protective IgG2b recognized heterogeneous, polydisperse high molecular weight cell wall and secretory components of the fungus, two of which were identified as the GPI-anchored cell wall proteins Als3 and Hyr1. In addition, only the IgG2b inhibited in vitro two critical virulence attributes of the fungus, hyphal growth and adherence to human epithelial cells.Our study demonstrates that the isotype of anti-β-glucan antibodies may affect details of the β-glucan epitopes recognized, and this may be associated with a differing ability to inhibit virulence attributes of the fungus and confer protection in vivo. Our data also suggest that the anti-virulence properties of the IgG2b mAb may be linked to its capacity to recognize β-glucan epitope(s) on some cell wall components that exert critical functions in fungal cell wall structure and adherence to host cells.     

Urea-Elicited Changes in Relative Electrophoretic Mobility of Certain Glycinin and beta-Conglycinin Subunits

Six molar urea in sodium dodecyl sulfate-polyacrylamide gels altered the relative electrophoretic mobility of several soybean protein subunits. Glycinin acidic polypeptide components A₃ and A₄ could be resolved from the other acidic polypeptides. A variant of the δ′ subunit of β-conglycinin was identified.