Animal glycolipids as attachment sites for microbes

The abundance of carbohydrate at the animal cell surface may explain why microbes have selected primarily carbohydrates as essential attachment sites for colonization or infection. Of the various surface glycoconjugates of interest, primary attention has been given to glycolipids, due in part to an efficient binding assay based on a thin-layer chromatogram with separated glycolipids. In this way the general character of carbohydrate recognition by microbes is being mapped. Mainly two examples are briefly described to illustrate some generalizations: lactosylceramide-recognition by several bacteria, and Gal alpha l----4Gal-binding by Escherichia coli and the Shiga toxin. The unique recognition of internally placed sequences, the often low-affinity binding, and the preference of certain sequences before others are interpreted to be of decisive biological value. The binding to internal parts makes it technically possible to approximate the binding epitope on a receptor glycolipid. For this the binding preferences to glycolipids carrying the binding site in different saccharide environments (isoreceptors) are compared with the computer-calculated preferred conformations (definition of steric hindrances to epitope access). Several binding epitopes dissected with this approach have a common surface character: a nonpolar area of ring hydrogens over one or two sugars, surrounded by polar oxygens or amide. This is in agreement with the recent Lemieux concept for antibody-carbohydrate interaction. This information facilitates a rational synthesis of receptor analogues for potential applications. An outline is finally given of an improved general approach for receptor analysis.     

Host-specificity of uropathogenic Escherichia coli depends on differences in binding specificity to Gal alpha 1-4Gal-containing isoreceptors.

Four G adhesins, cloned from uropathogenic Escherichia coli strains, were examined for binding to glycolipids and various eukaryotic cells. PapGAD110 and PapGIA2 showed virtually identical binding patterns to Gal alpha 1-4Gal-containing glycolipids, while PapGJ96 differed slightly and PrsGJ96 markedly with respect to the effect of neighbouring groups on the binding. Their hemagglutination patterns confirmed the existence of three receptor-binding specificities. While the PapG adhesins bound to uroepithelial cells from man (T24) but not to those from the dog (MDCK II), the reverse was true of PrsG. These binding patterns were largely explained by the absence or presence of appropriate glycolipid isoreceptors, although the inability of the PapG adhesins to bind MDCK II cells was attributed to an inappropriate presentation of their receptor epitopes. The high prevalence of PrsG-like specificities observed among wild-type dog uropathogenic E. coli isolates, together with the determined isoreceptor composition of human and dog kidney target tissues, suggest variation in receptor specificity as a mechanism for shifting host specificity, and that this variation has evolved in response to the topography of the host cellular receptors. The receptor-binding half proposed for the predicted amino acid sequences of the four G adhesins and the corresponding adhesin of one of the dog E. coli isolates varied considerably among the three receptor-binding groups of adhesins, but only little within each group.     

Structure of human rhinovirus serotype 2 (HRV2)

Human rhinoviruses are classified into a major and a minor group based on their binding to ICAM-1 or to members of the LDL-receptor family, respectively. They can also be divided into groups A and B, according to their sensitivity towards a panel of antiviral compounds. The structure of human rhinovirus 2 (HRV2), which uses the LDL receptor for cell attachment and is included in antiviral group B, has been solved and refined at 2.6 A resolution by X-ray crystallography to gain information on the peculiarities of rhinoviruses, in particular from the minor receptor group. The main structural differences between HRV2 and other rhinoviruses, including the minor receptor group serotype HRV1A, are located at the internal protein shell surface and at the external antigenic sites. In the interior, the N termini of VP1 and VP4 form a three-stranded beta-sheet in an arrangement similar to that present in poliovirus, although myristate was not visible at the amino terminus of VP4 in the HRV2 structure. The betaE-betaF loop of VP2, a linear epitope within antigenic site B recognized by monoclonal antibody 8F5, adopts a conformation considerably different from that found in the complex of 8F5 with a synthetic peptide of the same sequence. This either points to considerable structural changes impinged on this loop upon antibody binding, or to the existence of more than one single conformation of the loop when the virus is in solution. The hydrophobic pocket of VP1 was found to be occupied by a pocket factor apparently identical with that present in the major receptor group virus HRV16. Electron density, consistent with the presence of a viral RNA fragment, is seen stacked against a conserved tryptophan residue.     

Novel binding epitope for Helicobacter pylori found in neolacto carbohydrate chains: structure and cross-binding properties

Helicobacter pylori is a bacterium that colonizes the stomach of a majority of the global human population causing common gastric diseases like ulcers and cancer. It has an unusually complex pattern of binding to various host glycoconjugates including interaction with sialylated, sulfated, and fucosylated sequences. The present study describes an additional binding epitope comprising the neolacto internal sequence of GlcNAcbeta3-Galbeta4GlcNAcbeta. The binding was detected on TLC plates as an interaction with a seven-sugar ganglioside of rabbit thymus. The glycolipid was purified and characterized as Neu5Gcalpha3Galbeta4GlcNAcbeta3Galbeta4GlcNAcbeta3-Galbeta4Glcbeta1Cer with less than 10% of the fraction carrying a repeated lacto (type-1) core chain, Galbeta3Glc-NAcbeta3Galbeta3GlcNAcbeta. After stepwise chemical and enzymatic degradation and structural analysis of products the strongest binder was found to be the pentaglycosylceramide GlcNAcbeta3Galbeta4GlcNAcbeta3Galbeta4Glcbeta1-Cer, whereas the hexa- and tetraglycosylceramides were less active, and the trihexosylceramide was inactive. Further studies revealed that the terminal GlcNAcbeta of the pentaglycosylceramide may be exchanged for either GalNAcbeta3, GalNAcalpha3, or Galalpha3 without loss of the activity. Calculated minimum energy conformers of these four isoreceptors show a substantial topographical similarity suggesting that this binding is a result of a molecular mimicry. Although the glycoconjugate composition of human gastric epithelial cells is not known in detail it is proposed that repeating N-acetyllactosamine units of glycoconjugates may serve as bacterial attachment sites in the stomach.     

Novel oligodendrocyte transmembrane signaling systems

Antibodies are increasingly being used as tools to study the function of cell surface markers. Several types of responses may occur upon the selective binding of an antibody to an epitope on a receptor. Antibody binding may trigger signals that are normally transduced by endogenous ligands. Moreover, antibody binding may activate normal signals in a manner that disrupts a sequence of events that coordinates either differentiation, mitogenesis, or morphogenesis. Alternately, it is possible that binding elicits either a modified signal or no signal. This article focuses on the cascade of events that occur following specific antibody binding to myelin markers expressed by cultured murine oligodendrocytes. Binding of specific antibodies to the oligodendrocyte membrane surface markers myelin/oligodendrocyte glycoprotein (MOG), myelin/oligodendrocyte specific protein (MOSP), galactocerebroside (GalC), and sulfatide on cultured murine oligodendrocytes results in different effects with regard to phospholipid turnover, Ca²⁺ influxes, and antibody:marker distribution. The consequence of each antibody-elicited cascade of events appears to be the regulation of the cytoskeleton within the oligodendroglial membrane sheets. The antibody binding studies described in this article demonstrate that these myelin surface markers are capable of transducing signals. Since endogenous ligands for these myelin markers have yet to be identified, it is not known if these signals are normally transduced or are a modification of normally transduced signals.     

The use of monoclonal antibodies to localize the low density lipoprotein receptor-binding domain of apolipoprotein B

Human apolipoprotein (apo) B-100 is composed of 4536 amino acids. It is thought that the binding of apoB to the low density lipoprotein (LDL) receptor involves an interaction between basic amino acids of the ligand and acidic residues of the receptor. Three alternative models have been proposed to describe this interaction: 1) a single region of apoB is involved in receptor binding; 2) groups of basic amino acids from throughout the apoB primary structure act in concert in apoB receptor binding; and 3) apoB contains multiple independent binding regions. We have found that monoclonal antibodies (Mabs) specific for a region that spans a thrombin cleavage site at apoB residue 3249 (T2/T3 junction) totally blocked LDL binding to the LDL receptor. Mabs specific for epitopes outside this region had either no or partial ability to block LDL binding. In order to define the region of apoB directly involved in the interaction with the LDL receptor we have tested 22 different Mabs for their ability to bind to LDL already fixed to the receptor. A Mab specific for an epitope situated between residues 2835 and 2922 could bind to its epitope on LDL fixed to its receptor whereas a second epitope between residues 2980 and 3084 is inaccessible on receptor-bound LDL. A series of epitopes near residue 3500 of apoB is totally inaccessible, and another situated between residues 4027 and 4081 is poorly accessible on receptor-bound LDL. In contrast, an epitope that is situated between residues 4154 and 4189 is fully exposed. Mabs specific for epitopes upstream and downstream of the region 3000-4000 can bind to receptor-bound LDL with a stoichiometry close to unity. Our results strongly suggest that the unique region of apoB directly involved in the LDL-receptor interaction is that of the T2/T3 junction.     

The constraints protein-protein interactions place on sequence divergence

IL4-BP, the extracellular binding domain of the IL-4 receptor alpha chain, contains a high-affinity binding epitope for IL-4 (K(D) 150 pM). Previous results on the crystal structure of the IL-4/IL4-BP complex and on the functional epitope of IL-4 suggested that this contact comprises a mosaic of two binding clusters. The present mutational analysis of IL4-BP supports this view and demonstrates that the energetically most important group is the receptor carboxylate group of D72 forming an ion pair with IL-4 R88 in cluster II. The second main receptor determinant is the hydroxyl group of Y183 forming a hydrogen bond with IL-4 E9 in cluster I. The latter is engaged in additional hydrogen bonds with Y13 and also in van der Waals contacts with Y127. Receptor residue D72 as well as Y183 are each surrounded by a shell of hydrophobic groups from residues that upon mutation lead to smaller decreases in binding affinity. Analysis of IL4-BP double mutants showed that receptor side-chains within one cluster but not those of different clusters cooperate. Interaction analysis of IL-4 and IL4-BP single mutants also revealed additivity in binding of side-chains between clusters and cooperativity within each cluster I or II.These results show that the high-affinity IL-4/IL4-BP contact is constituted by two independent binding units, each containing a central polar or charged side-chain surrounded by hydrophobic groups (avocado cluster).     

Mapping the functional domains of human transcobalamin using monoclonal antibodies

Recombinant human transcobalamin (TC) was probed with 17 monoclonal antibodies (mAbs), using surface plasmon resonance measurements. These experiments identified five distinct epitope clusters on the surface of holo-TC. Western blot analysis of the CNBr cleavage fragments of TC allowed us to distribute the epitopes between two regions, which spanned either the second quarter of the TC sequence GQLA...TAAM(103-198) or the C-terminal peptide LEPA...LVSW(316-427). Proteolytic fragments of TC and the synthetic peptides were used to further specify the epitope map and define the functional domains of TC. Only one antibody showed some interference with cobalamin (Cbl) binding to TC, and the corresponding epitope was situated at the C-terminal stretch TQAS...QLLR(372-399). We explored the receptor-blocking effect of several mAbs and heparin to identify TC domains essential for the interaction between holo-TC and the receptor. The receptor-related epitopes were located within the TC sequence GQLA...HHSV(103-159). The putative heparin-binding site corresponded to a positively charged segment KRSN...RTVR(207-227), which also seemed to be necessary for receptor binding. We conclude that conformational changes in TC upon Cbl binding are accompanied by the convergence of multiple domains, and only the assembled conformation of the protein (i.e. holo-TC) has high affinity for the receptor.     

Mechanisms of ligand binding and efficacy at the human D2(short) dopamine receptor

Mechanisms of ligand binding and receptor activation for the human D2(short) dopamine receptor have been probed using two homologous series of monohydroxylated and dihydroxylated agonists (phenylethylamines and 2-dipropylaminotetralins). In ligand binding studies, the majority of compounds exhibited competition curves versus [3H]spiperone that were best fitted using a two site binding model. The compounds had different abilities (potencies and maximal effects) to stimulate [35S]GTPgammaS binding and to inhibit forskolin-stimulated cAMP accumulation. From the data it can be concluded that: (i) the ability of an agonist to stabilize receptor/G protein coupling can be used to predict agonist efficacy for some groups of compounds (2-dipropylaminotetralins) but not for others (phenylethylamines); (ii) the receptor may be activated by unhydroxylated compounds; (iii) single hydroxyl groups or pairs of hydroxyl groups on the agonist may contribute to binding affinity, potency and efficacy; and (iv) for the 2-dipropylaminotetralin series two modes of agonist/receptor interaction have been identified associated with different relative efficacy.     

Identification of the beta-dystroglycan binding epitope within the C-terminal region of alpha-dystroglycan.

Dystroglycan is a receptor for extracellular matrix proteins that plays a crucial role during embryogenesis in addition to adult tissue stabilization. A precursor product of a single gene is post-translationally cleaved to form two different subunits, alpha and beta. The extracellular alpha-dystroglycan is a membrane-associated, highly glycosylated protein that binds to various extracellular matrix molecules, whereas the transmembrane beta-dystroglycan binds, via its cytosolic domain, to dystrophin and many other proteins. alpha- and beta-Dystroglycan interact tightly but noncovalently. We have previously shown that the N-terminal region of beta-dystroglycan, beta-DG(654-750), binds to the C-terminal region of murine alpha-dystroglycan independently from glycosylation. Preparing a series of deleted recombinant fragments and using solid-phase binding assays, the C-terminal sequence of alpha-dystroglycan containing the binding epitope for beta-dystroglycan has been defined more precisely. We found that a region of 36 amino acids, from position 550-585, is required for binding the extracellular region, amino acids 654-750 of beta-dystroglycan. Recently, a dystroglycan-like gene was identified in Drosophila that showed a moderate degree of conservation with vertebrate dystroglycan (31% identity, 48% similarity). Surprisingly, the Drosophila sequence contains a region showing a higher degree of identity and conservation (45% and 66%) that coincides with the 550-585 sequence of vertebrate alpha-dystroglycan. We have expressed this Drosophila dystroglycan fragment and measured its binding to the extracellular region of vertebrate (murine) beta-dystroglycan (Kd = 6 +/- 1 microM). These data confirm the proper identification of the beta-dystroglycan binding epitope and stress the importance of this region during evolution. This finding might help the rational design of dystroglycan-specific binding drugs, that could have important biomedical applications.     

A monoclonal anti-IgE antibody against an epitope (amino acids 367-376) in the CH3 domain inhibits IgE binding to the low affinity IgE receptor (CD23)

We have produced three different mAb specific for human IgE-Fc. Their binding pattern to either heat-denatured IgE or a family of overlapping IgE-derived recombinant peptides and their ability to affect interaction of IgE with its low affinity receptor Fc epsilon R2/CD23 demonstrate that they recognize distinct epitopes on the IgE molecule. All three mAb were able to induce basophil degranulation as measured by the induction of histamine release. mAb 173 recognizes a thermolabile epitope in the CH4 domain. It does not affect the binding of IgE to Fc epsilon R2/CD23. mAb 272 recognizes a thermostable epitope that maps to a sequence of 36 amino acids (AA) spanning part of the CH2 and CH3 domain and it does not affect the binding of IgE to Fc epsilon R2/CD23. mAb 27 recognizes a thermolabile epitope located on a 10 AA stretch (AA 367-376) in the CH3 domain. This area contains one N-linked oligosaccharide (Asn-371), but the antibody is not directed against carbohydrate because it binds to Escherichia coli-derived IgE peptides. mAb 27 inhibits the binding of IgE to Fc epsilon R2/CD23 but is still capable of reacting with IgE already bound to Fc epsilon R2/CD23. These data suggest that upon binding to Fc epsilon R2/CD23, the IgE molecule engages one of two equivalent-binding sites close to the glycosylated area of the CH3 domain.     

Identification of the carbohydrate receptor for Shiga toxin produced by Shigella dysenteriae type 1

The binding of Shiga toxin isolated from the bacterium Shigella dysenteriae type 1 to a series of glycolipids and to cells or cell homogenates has been studied. Bound toxin was detected using either 125I-labeled toxin or specific monoclonal antibody and 125I-labeled anti-antibody. Overlay of toxin on thin-layer chromatograms with separated glycolipids and binding to glycolipids coated in microtiter wells established that the toxin specifically bound to Gal alpha 1-4Gal beta (galabiose) placed terminally or internally in the oligosaccharide chain. No glycolipid shown to lack this sequence binds the toxin. Most of the glycolipids with internally placed galabiose were not active, indicating a sterical hindrance for toxin access to the binding epitope. Binding of toxin to HeLa cells in monolayers could be inhibited by preincubation of the toxin with galabiose covalently linked to bovine serum albumin (BSA), but not with free oligosaccharides containing galabiose or with lactose coupled to BSA. This demonstrated that the inhibition is specifically dependent on galabiose and requires multivalency of the disaccharide to be efficient. The inhibitory effect was successively enhanced by increasing the substitution on BSA (7, 18, and 25 mol of galabiose/mol of BSA). The BSA-coupled galabiose could also prevent the cytotoxic effect on HeLa cells (detachment of killed cells). There are cell lines with a dense number of receptor sites, but which are resistant to toxin action (uptake and inhibition of protein synthesis) which may suggest two types of receptor substances which are functionally different and unevenly expressed. In analogy with the mechanism earlier formulated for cholera toxin, we propose glycolipid-bound, bilayer-close galabiose as the functional receptor for membrane penetration of the toxin, while galabiose bound in glycoproteins affords binding sites but is not able to mediate penetration.     

The role of the CPNKEKEC sequence in the beta(2) subunit I domain in regulation of integrin alpha(L)beta(2) (LFA-1)

The alpha(L) I (inserted or interactive) domain of integrin alpha(L)beta(2) undergoes conformational changes upon activation. Recent studies show that the isolated, activated alpha(L) I domain is sufficient for strong ligand binding, suggesting the beta(2) subunit to be only indirectly involved. It has been unclear whether the activity of the alpha(L) I domain is regulated by the beta(2) subunit. In this study, we demonstrate that swapping the disulfide-linked CPNKEKEC sequence (residues 169-176) in the beta(2) I domain with a corresponding beta(3) sequence, or mutating Lys(174) to Thr, constitutively activates alpha(L)beta(2) binding to ICAM-1. These mutants do not require Mn(2+) for ICAM-1 binding and are insensitive to the inhibitory effect of Ca(2+). We have also localized a component of the mAb 24 epitope (a reporter of beta(2) integrin activation) in the CPNKEKEC sequence. Glu(173) and Glu(175) of the beta(2) I domain are identified as critical for mAb 24 binding. Because the epitope is highly expressed upon beta(2) integrin activation, it is likely that the CPNKEKEC sequence is exposed or undergoes conformational changes upon activation. Deletion of the alpha(L) I domain did not eliminate the mAb 24 epitope. This confirms that the alpha(L) I domain is not critical for mAb 24 binding, and indicates that mAb 24 detects a change expressed in part in the beta(2) subunit I domain. These results suggest that the CPNKEKEC sequence of the beta(2) I domain is involved in regulating the alpha(L) I domain.     

Epitope Mapping by Label-Free Biomolecular Interaction Analysis

The diversity of B-cell response to a large immunogen gives rise to a series of antibodies that can be used for epitope mapping of an antigen. This is based on the relative reaction pattern for all antibodies in relation to each other and other ligands to the studied protein. With the introduction of an instrument system, BIAcore, label-free real-time biomolecular interaction analysis (BIA) was made possible. It is based on biosensor technology, with a carboxymethyl-dextran-coated gold surface and an integrated fluidics for transport of liquid. The basic idea is to measure label-free binding of an analyte from a continuous flow to an immobilized ligand in real time. With an automatic approach, quantitative analysis and sequential injection characteristic biospecific binding parameters such as affinity and kinetic constants can be measured. The instrument system was adopted at an early stage for epitope mapping. With label-free detection, antibodies from tissue culture media can be analyzed without purification. Binding of both antigen and a series of antibodies can be individually determined in molar ratio by sequential injections. The quantitative aspects of BIA offer the possibility of further refined epitope mapping. The relative binding pattern for 30 monoclonal antibodies against HIV-1 p24 core protein has been analyzed. Multideterminant analysis and peptide identification of binding sites were performed. Verification of the binding pattern has also been performed in relation to mapping with ELISA as well as the binding to peptides derived from the antigen sequence. Functional domains of proteins in relation to an epitope map have been identified forTaqpolymerase.     

CD36 peptides enhance or inhibit CD36-thrombospondin binding. A two-step process of ligand-receptor interaction.

CD36 (glycoprotein IV or IIIB) is an integral plasma membrane protein of wide cellular distribution and functions as a receptor site for thrombospondin (TSP), an adhesive protein important in cell-cell and cell-matrix interactions. OKM5, a monoclonal anti-CD36 antibody, has been reported to block CD36 cell adhesive functions suggesting that the OKM5 epitope on CD36 is functionally important. A panel of 10 synthetic CD36 peptides was made. One peptide, P139-155, specifically inhibited the immunoadsorption of CD36 by OKM5, and P139-155 was directly immunoadsorbed by OKM5, indicating that CD36 sequence 139-155 represents part of the OKM5 epitope. TSP bound to immobilized P139-155 in a dose-dependent and saturable manner. Surprisingly, P139-155 significantly augmented, instead of inhibited, binding of CD36 to TSP. This peptide did not induce platelet aggregation but augmented ADP- and collagen-induced aggregation in platelet-rich plasma. Another CD36 peptide, P93-110, which had no effect on OKM5 immunoadsorption, blocked binding of CD36 to immobilized TSP and partially inhibited collagen-induced platelet aggregation. P93-110 by itself did not bind to TSP; however, in the presence of P139-155, there was a marked enhancement of P93-110 binding to TSP, with a stoichiometry consistent with the trimeric nature of TSP. The data suggest that CD36-TSP interaction is a two-step process; the sequence 139-155 region of CD36 binds first to TSP, triggering a change in TSP to reveal a second site, which binds the 93-110 region of CD36 with high affinity. CD36 peptides can be used as stimulators or inhibitors in cellular adhesive events involving TSP-CD36 interaction. Conformational changes leading to the exposure or activation of high affinity binding sites may occur in both the receptor and the ligand upon cell-cell and cell-matrix adhesion.     

Identification of a novel epitope in the thyroid-stimulating hormone receptor ectodomain acting as intramolecular signaling interface

Glycoprotein hormone receptors (GPHRs) differ from the other seven transmembrane receptors mainly through a complex activation mechanism that requires the binding of a large hormone toward a large N-terminal ectodomain. The intramolecular mechanism of the signal transduction to the serpentine domain upon hormone binding at the ectodomain is not understood. To identify determinants at the GPHR ectodomain that may be involved in signal transduction, we first searched for homologous structural features. Based on high sequence similarity to the determined structures of the Nogo-receptor ectodomain and the intermolecular complex of the Interleukin-8 ligand (IL8) and the N-terminal peptide of the IL8 receptor (IL8RA), the hypothesis was developed that portions of the intramolecular components, Cysteine-box-2 and Cysteine-box-3, of the GPHR ectodomain interact and localize at the interface between ectodomain and serpentine domain. Indeed, point mutations within the D403EFN406 motif at Cysteine-box-3 of the thyrotropin receptor resulted in increased basal cAMP levels, suggesting that this motif may be important for transduction of the signal from the ectodomain to the transmembrane domain. New indications are provided about the tight spatial cooperation and relative location of the new epitope and other determinants at the thyrotropin receptor ectodomain, such as the leucine-rich repeat motif Ser281 and the cysteine boxes. According to the high sequence conservation, the results are of general relevance for the signal transduction mechanism of other glycoprotein hormone receptors such as choriogonadotrophic/luteinizing hormone receptor and follicle-stimulating hormone receptor.     

Growth factor receptor signalling: location, location, location

Ligand binding to plasma membrane receptors initiates a series of events culminating in a variety of changes in cellular phenotypes. Although numerous publications have documented the activation/inactivation of signalling molecules following receptor binding, relatively few investigations have focused on the cellular compartment responsible for either initiating or selecting the particular pathway that mediates the response. Specifically, does receptor signalling occur only at the plasma membrane; is signalling dependent upon the location of defined endosome populations; or are components of both plasma membrane and endosomal activity operative depending upon the particular signalling pathway or cell type? This review addresses aspects of these questions by discussing the evidence supporting or contrasting the interplay between the endocytic and signalling systems for a subset of tyrosine kinase, serine/threonine kinase and G-protein-coupled receptors.     

Mu opiate isoreceptors: differentiation with kappa agonists

In vivo pharmacological data support the concept of mu (mu) isoreceptors in the rat CNS. The mu 1 receptor mediates analgesia and regulation of cholinergic neurons. Naloxazone may be a specific antagonist of this receptor type. In contrast, the mu 2 receptor is responsible for respiratory depression and regulation of nigrostriatal dopaminergic neurons. In this case, the kappa agonists EKC and MR-2034 appear to be specific mu 2 antagonists.     

Influenza Human Monoclonal Antibody 1F1 Interacts with Three Major Antigenic Sites and Residues Mediating Human Receptor Specificity in H1N1 Viruses

Most monoclonal antibodies (mAbs) to the influenza A virus hemagglutinin (HA) head domain exhibit very limited breadth of inhibitory activity due to antigenic drift in field strains. However, mAb 1F1, isolated from a 1918 influenza pandemic survivor, inhibits select human H1 viruses (1918, 1943, 1947, and 1977 isolates). The crystal structure of 1F1 in complex with the 1918 HA shows that 1F1 contacts residues that are classically defined as belonging to three distinct antigenic sites, Sa, Sb and Ca₂. The 1F1 heavy chain also reaches into the receptor binding site (RBS) and interacts with residues that contact sialoglycan receptors and determine HA receptor specificity. The 1F1 epitope is remarkably similar to the previously described murine HC63 H3 epitope, despite significant sequence differences between H1 and H3 HAs. Both antibodies potently inhibit receptor binding, but only HC63 can block the pH-induced conformational changes in HA that drive membrane fusion. Contacts within the RBS suggested that 1F1 may be sensitive to changes that alter HA receptor binding activity. Affinity assays confirmed that sequence changes that switch the HA to avian receptor specificity affect binding of 1F1 and a mAb possessing a closely related heavy chain, 1I20. To characterize 1F1 cross-reactivity, additional escape mutant selection and site-directed mutagenesis were performed. Residues 190 and 227 in the 1F1 epitope were found to be critical for 1F1 reactivity towards 1918, 1943 and 1977 HAs, as well as for 1I20 reactivity towards the 1918 HA. Therefore, 1F1 heavy-chain interactions with conserved RBS residues likely contribute to its ability to inhibit divergent HAs.     

Dimerization of the immunosuppressive peptide fragment of HLA-DR molecule enhances its potency

Our previous studies revealed that the nonapeptide fragment of HLA-DR molecule, located in the beta chain 164-172 with the VPRSGEVYT sequence, suppresses the immune responses. The sequence is located on the exposed molecule loop, therefore it may be involved in the interactions with other proteins. We suggested that the loop may serve as a functional epitope on the HLA class II surface for intermolecular binding, and that possible mechanism of biological action of the synthesized peptides is associated with interfering of adhesion of HLA class II molecules to their coreceptors. It has been postulated that oligomerization of the coreceptors is required for stable binding to class II HLA. Based on the crystal dimeric structure of HLA-DR molecules, we designed, and synthesized molecules able to induce the putative coreceptors dimerization. The synthesized series of compounds consisted of two VPRSGEVYT sequences linked through their C-termini by spacers of different length: (VPRSGEVYTGn)2K-NH2 ( n = 4-6). The results demonstrate that the dimerization of the nonapeptide fragment of HLA-DR results in enhanced immunosuppressory properties.