Glycolipid Binding Research Articles

SC134-TCB Targeting Fucosyl-GM1, a T Cell-Engaging Antibody with Potent Antitumor Activity in Preclinical Small Cell Lung Cancer Models.

Small cell lung cancer (SCLC) is an aggressive disease with limited treatment options. Fucosyl-GM1 (FucGM1) is a glycolipid overexpressed in the majority of SCLC tumors but virtually absent from normal healthy tissues. In this study, we validate a FucGM1-targeting T cell-redirecting bispecific (TCB) antibody for the treatment of SCLC. More than 80% of patient-derived xenograft tissues of SCLC expressed FucGM1, whereas only three normal human tissues: pituitary, thymus, and skin expressed low and focal FucGM1. A FucGM1-targeting TCB (SC134-TCB), based on the Fc-silenced humanized SC134 antibody, exhibited nanomolar efficiency in FucGM1 glycolipid and SCLC cell surface binding. SC134-TCB showed potent ex vivo killing of SCLC cell lines with donor-dependent EC50 ranging from 7.2 pmol/L up to 211.0 pmol/L, effectively activating T cells, with picomolar efficiency, coinciding with target-dependent cytokine production such as IFNγ, IL2, and TNFα and robust proliferation of both CD4 and CD8 T cells. The ex vivo SC134-TCB tumor controlling activity translated into an effective in vivo anti-DMS79 tumor therapy, resulting in 100% tumor-free survival in a human peripheral blood mononuclear cell admixed setting and 40% overall survival (55% tumor growth inhibition) with systemically administered human peripheral blood mononuclear cells. Combination treatment with atezolizumab further enhanced survival and tumor growth inhibition (up to 73%). A 10-fold SC134-TCB dose reduction maintained the strong in vivo antitumor impact, translating into 70% overall survival (P < 0.0001). Whole-blood incubation with SC134-TCB, as well as healthy human primary cells analysis, revealed no target-independent cytokine production. SC134-TCB presents an attractive candidate to deliver an effective immunotherapy treatment option for patients with SCLC.

Abstract 4807: Gaucher disease treatment reduces the progression of smoldering myeloma

Abstract Background: Gaucher disease (GD), a hereditary condition characterized by glucocerebrosidase deficiency, results in the impaired degradation of glucocerebroside (GL1) and its deacetylated form, lyso-GL1 (LGL1). This leads to the accumulation of these substrates primarily in the bone marrow (BM), liver, and spleen. Notably, individuals with GD face an increased risk of plasma cell dyscrasias (PCD) by approximately nine-fold. We present two compelling cases of the regression of smoldering multiple myeloma (SMM) following GD treatment, substantiated by biochemical evidence of lipid-reactive clonal paraproteins (PP) in both patients. Methods: Peripheral blood samples were obtained with IRB approval from patients with concurrent SMM and GD at the Icahn School of Medicine at Mount Sinai. Utilizing C-18 beads coupled with gly-sphingosine-coated liposomes and Sphingosine-1 phosphate bead liposome-based pulldown, we established the binding of glycolipids to serum PP. Results: In a 66-year-old male, hepatosplenomegaly and pancytopenia revealed compound heterozygous N370S/V394L mutations and reduced β-glucosidase activity, confirming Type 1 GD. Laboratory findings included substantial pancytopenia (WBC 2.5 k/µL, Hgb 12.1 g/dL, platelet 44 k/µL) and myeloma markers (M spike 1.58 g/dL, monoclonal IgA λ). A BM biopsy indicated 30% λ-restricted plasma cells. PET-CT showed hepatosplenomegaly without FDG avid lytic lesions, consistent with SMM. Imiglucerase enzyme therapy improved GD biomarkers, decreased hepatosplenomegaly, and blood count improvement (WBC 4.7 k/µL, Hgb 13.6 g/dL, platelet 66 k/µL). Notably, myeloma burden significantly reduced (M-spike 0.94 g/dL, IgA 1373 g/dL and free lambda 26.1 mg/L). Similarly, a 41-year-old female with mild fatigue, Hgb levels of 10.9 g/dL, a platelet count of 169 k/µL, M spike of 0.9 g/dL, IgG κappa on immunofixation, and IgG levels at 1730 mg/dL, indicating SMM. GBA analysis showed homozygous N370S mutation and reduced beta-glucosidase activity consistent with Type 1 GD. Substrate reduction therapy with eliglustat alleviated fatigue, reduced GD biomarkers, normalized cytopenias, and gradually decreased myeloma markers. Laboratory results indicated an M spike of 0.5 g/dL, IgG levels at 1295 mg/dL, and free kappa at 31.3 mg/L. To explore the interaction between clonal immunoglobulins (Ig) and lyso-lipids, we utilized Gly-sphingolipids (see methods) and demonstrated a robust interaction with IgA (patient 1) and IgG (patient 2) paraprotein by western blotting. Conclusion: Our experimental findings confirm specific clonal Ig binding in SMM and GD patients and demonstrate potent and timely benefits from addressing GD in managing SMM patients. This holds significance amid ongoing clinical trials exploring aggressive interventions for SMM, including bispecific and CAR-T therapies. Citation Format: Ravi Prakash Shukla, Anshuman Parekh, Violetta V Leshchenko, Kevin Barley, Deepa Vatti, Daniel Verina, Manisha Balwani, Samir Parekh. Gaucher disease treatment reduces the progression of smoldering myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4807.

Siglec-6 mediates the uptake of extracellular vesicles through a noncanonical glycolipid binding pocket

Immunomodulatory Siglecs are controlled by their glycoprotein and glycolipid ligands. Siglec-glycolipid interactions are often studied outside the context of a lipid bilayer, missing the complex behaviors of glycolipids in a membrane. Through optimizing a liposomal formulation to dissect Siglec–glycolipid interactions, it is shown that Siglec-6 can recognize glycolipids independent of its canonical binding pocket, suggesting that Siglec-6 possesses a secondary binding pocket tailored for recognizing glycolipids in a bilayer. A panel of synthetic neoglycolipids is used to probe the specificity of this glycolipid binding pocket on Siglec-6, leading to the development of a neoglycolipid with higher avidity for Siglec-6 compared to natural glycolipids. This neoglycolipid facilitates the delivery of liposomes to Siglec-6 on human mast cells, memory B-cells and placental syncytiotrophoblasts. A physiological relevance for glycolipid recognition by Siglec-6 is revealed for the binding and internalization of extracellular vesicles. These results demonstrate a unique and physiologically relevant ability of Siglec-6 to recognize glycolipids in a membrane.

Adaptive Binding of Alkyl Glycosides by Nonpeptidic Helix Bundles in Water: Toward Artificial Glycolipid Binding Proteins.

Amphipathic water-soluble helices formed from synthetic peptides or foldamers are promising building blocks for the creation of self-assembled architectures with non-natural shapes and functions. While rationally designed artificial quaternary structures such as helix bundles have been shown to contain preformed cavities suitable for guest binding, there are no examples of adaptive binding of guest molecules by such assemblies in aqueous conditions. We have previously reported a foldamer 6-helix bundle that contains an internal nonpolar cavity able to bind primary alcohols as guest molecules. Here, we show that this 6-helix bundle can also interact with larger, more complex guests such as n-alkyl glycosides. X-ray diffraction analysis of co-crystals using a diverse set of guests together with solution and gas-phase studies reveals an adaptive binding mode whereby the apo form of the 6-helix bundle undergoes substantial conformational change to accommodate the hydrocarbon chain in a manner reminiscent of glycolipid transfer proteins in which the cavity forms upon lipid uptake. The dynamic nature of the self-assembling and molecular recognition processes reported here marks a step forward in the design of functional proteomimetic molecular assemblies.

Multiscale Molecular Dynamics Studies Reveal Different Modes of Receptor Clustering by Gb3-Binding Lectins

The recognition of carbohydrate receptors on host cell membranes by pathogenic lectins is a crucial step in the microbial invasion. Two bacterial lectins, the B-subunit of Shiga toxin from Shigella dysenteria (StxB) and lectin I from Pseudomonas aeruginosa (LecA), are specific to the same galactolipid-globotriaosylceramide (Gb3). In this study we present a coarse-grained (cg) model of Gb3, which we further apply to unravel the molecular details of glycolipid binding by two lectins on the surface of a DOPC/cholesterol/Gb3 bilayer. In cg molecular dynamics simulations with time scales of dozens of microseconds, Gb3 was randomly distributed. The binding of both StxB or LecA is accompanied by Gb3 clustering in a cholesterol environment and with exclusion of DOPC in protein vicinity. StxB being bound by all 15 binding sites induced membrane bending, while LecA interacted with two out of four binding sites for most of the time causing a smaller inward curvature of the model membrane. Stable interactions occurred preferably when LecA was normal to the membrane surface. Furthermore, all-atom simulations revealed that LecA bound Gb3's headgroup at only one out of two possible conformations of the carbohydrate moiety observed at protein-free conditions. The results shed light on the mechanism of interactions between two lectins and Gb3 on the membrane surface and offer a coarse-grained model to study more complex systems at large spatiotemporal scales.

Efficient preparation of human and mouse CD1d proteins using silkworm baculovirus expression system

CD1d is a major histocompatibility complex (MHC) class I-like glycoprotein and binds to glycolipid antigens that are recognized by natural killer T (NKT) cells. To date, our understanding of the structural basis for glycolipid binding and receptor recognition of CD1d is still limited. Here, we established a preparation method for the ectodomain of human and mouse CD1d using a silkworm-baculovirus expression system. The co-expression of human and mouse CD1d and β2-microglobulin (β2m) in the silkworm-baculovirus system was successful, but the yield of human CD1d was low. A construct of human CD1d fused with β2m via a flexible GS linker as a single polypeptide was prepared to improve protein yield. The production of this single-chained complex was higher (50 μg/larva) than that of the co-expression complex. Furthermore, differential scanning calorimetry revealed that the linker made the CD1d complex more stable and homogenous. These results suggest that the silkworm-baculovirus expression system is useful for structural and biophysical studies of CD1d in several aspects including low cost, easy handling, biohazard-free, rapid, and high yielding.

Rescue from Stx2-Producing E.coli-Associated Encephalopathy by Intravenous Injection of Muse Cells in NOD-SCID Mice.

Shiga toxin-producing Escherichia coli (STEC) causes hemorrhagic colitis, hemolytic uremic syndrome, and acute encephalopathies that may lead to sudden death or severe neurologic sequelae. Current treatments, including immunoglobulin G (IgG) immunoadsorption, plasma exchange, steroid pulse therapy, and the monoclonal antibody eculizumab, have limited effects against the severe neurologic sequelae. Multilineage-differentiating stress-enduring (Muse) cells are endogenous reparative non-tumorigenic stem cells that naturally reside in the body and are currently under clinical trials for regenerative medicine. When administered intravenously, Musecells accumulate to the damaged tissue, where they exert anti-inflammatory, anti-apoptotic, anti-fibrotic, and immunomodulatory effects, and replace damaged cells by differentiating into tissue-constituent cells. Here, severely immunocompromised non-obese diabetic/severe combined immunodeficiency (NOD-SCID) mice orally inoculated with 9 × 109 colony-forming units of STEC O111 and treated 48 h later with intravenous injection of 5 × 104 Muse cells exhibited 100% survival and no severe after-effects of infection. Suppression of granulocyte-colony-stimulating factor (G-CSF) by RNAi abolished the beneficial effects of Muse cells, leading to a 40% death and significant body weight loss, suggesting the involvement of G-CSF in the beneficial effects of Muse cells in STEC-infected mice. Thus, intravenous administration of Muse cells could be a candidate therapeutic approach for preventing fatal encephalopathy after STEC infection.

Biomedical Applications of Glycoconjugates.

Glycans expressed on the forefront surface of cells participate in many key biological processes via forming various glycoconjugates. The recognition of complex glycoconjugates as mediators of important biological processes has stimulated investigation into their therapeutic potential. As the increasing accessibility of glycoconjugates, it has been widely applied in the field of drug delivery. This review particularly refers to the constitutive glycoconjugates of receptor-mediated binding of glycoprotein, glycolipids and glycopeptides for cell-selective drug delivery, in order to broaden the future therapeutic scope of drug delivery system and effective cancer therapy.

Globo-A Binds to the Recombinant Natural Cytotoxicity Receptor NKp44.

Natural killer (NK) cells play an important role in tumor immunity and infection control. The natural cytotoxicity receptors (NCRs) NKp46, NKp44 and NKp30 are involved in the control of the activation of NK cells. Few reports have investigated the ligands of NCRs. We previously reported the NCRs binding affinity to heparin and glycosaminoglycans. We also showed that multimeric sialyl Lewis X-expressing transferrin, secreted by human hepatoma HepG2 cells, binds to NKp46 and NKp44, but not to NKp30. In this study, we investigated the binding between NCRs and glycolipids. The possible binding of glycolipids to NCRs was screened by microarray, using the recombinant extracellular domain of NKp46, NKp44 and NKp30 tagged with 6×His (rNKp46, rNKp44 and rNKp30). We found that rNKp44 binds to Globo-A. However, we did not detect the interaction between rNKp46 or rNKp30 and any of the glycolipids investigated. Direct binding assays supported the results of the microarray screening. Therefore, we concluded that Globo-A is a novel ligand for NKp44 but not NKp46 and NKp30, and showed differences in the ligand selectivity of NCRs.

Functional evaluation of tryptophans in glycolipid binding and membrane interaction by HET-C2, a fungal glycolipid transfer protein

HET-C2 is a fungal glycolipid transfer protein (GLTP) that uses an evolutionarily-modified GLTP-fold to achieve more focused transfer specificity for simple neutral glycosphingolipids than mammalian GLTPs. Only one of HET-C2's two Trp residues is topologically identical to the three Trp residues of mammalian GLTP. Here, we provide the first assessment of the functional roles of HET-C2 Trp residues in glycolipid binding and membrane interaction. Point mutants HET-C2W208F, HET-C2W208A and HET-C2F149Y all retained >90% activity and 80–90% intrinsic Trp fluorescence intensity; whereas HET-C2F149A transfer activity decreased to ~55% but displayed ~120% intrinsic Trp emission intensity. Thus, neither W208 nor F149 is absolutely essential for activity and most Trp emission intensity (~85–90%) originates from Trp109. This conclusion was supported by HET-C2W109Y/F149Y which displayed ~8% intrinsic Trp intensity and was nearly inactive. Incubation of the HET-C2 mutants with 1-palmitoyl-2-oleoyl-phosphatidylcholine vesicles containing different monoglycosylceramides or presented by lipid ethanol-injection decreased Trp fluorescence intensity and blue-shifted the Trp λmax by differing amounts compared to wtHET-C2. With HET-C2 mutants for Trp208, the emission intensity decreases (~30–40%) and λmax blue-shifts (~12nm) were more dramatic than for wtHET-C2 or F149 mutants and closely resembled human GLTP. When Trp109 was mutated, the glycolipid induced changes in HET-C2 emission intensity and λmax blue-shift were nearly nonexistent. Our findings indicate that the HET-C2 Trp λmax blue-shift is diagnostic for glycolipid binding; whereas the emission intensity decrease reflects higher environmental polarity encountered upon nonspecific interaction with phosphocholine headgroups comprising the membrane interface and specific interaction with the hydrated glycolipid sugar.

A Peptide-Glycolipid Interaction Probed by Retroinverso Peptide Analogues.

Cell surface glycolipids are implicated in the formation of lipid rafts and membrane microdomains, where they interact with protein receptors to mediate a variety of cellular processes such as cell-cell recognition, cell adhesion, and membrane signaling. Studies of glycolipid function at the local membrane structures have not been straightforward to date, because the locally clustered structures are labile and their protein binding affinities tend to be weak. While specific glycolipid-binding proteins have been employed as molecular probes for detecting lipid rafts, small peptides may be more suitable for probing glycolipids at the cell surface due to their small size as well as their ease of synthetic preparation and functionalization. Here we report an application of the retroinverso approach as a rapid method to obtain novel glycolipid-binding D-peptide sequences. We have prepared analogues of two known GM1-binding peptides by replacing L-amino acids with D-amino acids, followed by inverting the sequences and characterized their conformational propensity and glycolipid binding properties. Circular dichroism (CD) spectroscopic analysis indicated that all the peptide sequences interacted with GM1 under a micellar condition. We found, by a microplate-based competitive glycolipid binding assay, that one of the retroinverso D-peptide analogues, peptide 3, also binds GM1 as the parent L-peptide 1. These results suggested that in this glycolipid-peptide interaction, the positioning of the side chain functionalities of the peptide is important, while the peptide backbone polarity is not. Glycolipid binding retroinverso D-peptides should be useful for the design of new peptide-based probes for investigating the biological role of cell surface glycolipids.

Identification of the physiological antigen presenting cell for glycolipid antigens

Abstract A major subset of T cells that recognize complexes formed by the binding of glycolipids to CD1d are called invariant natural killer T (iNKT) cells. Unlike conventional T cells that need to proliferate and differentiate to become activated effector T cells, iNKT cells exist as an effector population and start responding rapidly after glycolipid recognition. The prototypical iNKT stimulatory antigen is synthetic α-galactosylceramide (αGC), initially designated as KRN7000. Administration of this antigen in vivo results in the production of cytokines associated with both T helper type 1 (Th1) or type 2 (Th2) responses. Several variants of this antigen having different fatty acyl- and phytosphinganine chains have been synthesized and polarize the response towards a predominance of either Th1 or Th2 cytokines, and are therefore described as Th1- or Th2-biasing analogues. It is not clear how the Th1- or Th2-bias in iNKT mediated response is induced, although selective presentation by distinct cell types has been proposed as a possible mechanism. By visualizing glycolipid antigen presentation directly with monoclonal antibodies specific for complexes of αGC bound to CD1d, we showed that the CD8αPosDEC-205Pos dendritic cells were the major APCs in the spleen for a range of αGC analogues, irrespective of their chemical structures and cytokine biasing activities. We found that DCs presenting Th1-biasing agonists upregulated ligands for stimulatory NK receptor molecules, while Th2-agonist presenting cells showed a marked upregulation of ligands for inhibitory receptors. Furthermore, modulation of accessory immune proteins was dependent on CD1d, suggesting direct cell-cell interactions between DC and iNKT cells.

Phosphatidylserine Stimulates Ceramide 1-Phosphate (C1P) Intermembrane Transfer by C1P Transfer Proteins

Genetic models for studying localized cell suicide that halt the spread of pathogen infection and immune response activation in plants include Arabidopsis accelerated-cell-death 11 mutant (acd11). In this mutant, sphingolipid homeostasis is disrupted via depletion of ACD11, a lipid transfer protein that is specific for ceramide 1-phosphate (C1P) and phyto-C1P. The C1P binding site in ACD11 and in human ceramide-1-phosphate transfer protein (CPTP) is surrounded by cationic residues. Here, we investigated the functional regulation of ACD11 and CPTP by anionic phosphoglycerides and found that 1-palmitoyl-2-oleoyl-phosphatidic acid or 1-palmitoyl-2-oleoyl-phosphatidylglycerol (≤15 mol %) in C1P source vesicles depressed C1P intermembrane transfer. By contrast, replacement with 1-palmitoyl-2-oleoyl-phosphatidylserine stimulated C1P transfer by ACD11 and CPTP. Notably, "soluble" phosphatidylserine (dihexanoyl-phosphatidylserine) failed to stimulate C1P transfer. Also, none of the anionic phosphoglycerides affected transfer action by human glycolipid lipid transfer protein (GLTP), which is glycolipid-specific and has few cationic residues near its glycolipid binding site. These findings provide the first evidence for a potential phosphoglyceride headgroup-specific regulatory interaction site(s) existing on the surface of any GLTP-fold and delineate new differences between GLTP superfamily members that are specific for C1P versus glycolipid.

Sphingolipid Transfer by GLTP-fold Proteins is Differentially Regulated by Phosphatidylinositol Derivatives

Immune response activation and localized cell suicide that halt the spread of pathogen infection in plants benefit from genetic models such as Arabidopsis accelerated-cell-death11 (acd11). In this mutant, depletion of ACD11, a lipid transfer protein that is specific for ceramide-1-phosphate (C1P) and phyto-C1P, disrupts sphingolipid homeo-stasis triggering programmed cell death. The C1P binding site in ACD11 and in human ceramide-1-phosphate transfer protein (CPTP) is surrounded by cationic residues. Here, we studied the regulation of C1P intermembrane transfer by ACD11 and CPTP when C1P-source vesicles contain various phosphatidylinositol (PI) derivatives (≤10 mol%) using Forster resonance energy transfer (FRET) involving anthrylvinyl and perylenoyl lipid probes. The presence and location of phosphate on the PI head group (PI-4P, PI-3P, PI-4,5P2) exerted differing effects on C1P transfer by ACD11 and CPTP. In contrast, none of the PI derivatives affected transfer action by human glycolipid lipid transfer protein (GLTP) which forms a glycolipid-specific GLTP-fold and has no cationic residue cluster near its glycolipid binding site. The findings provide evidence for the existence of a potential regulatory site for certain PI-derivatives on the surface C1P-specific, but not glycolipid-specific GLTP superfamily members. The results are discussed within the context of recent studies involving other anionic phosphoglycerides (phosphatidylglycerol, phosphatidylserine, phosphatidic acid) and these same sphingolipid transfer proteins. (Support: NIGMS GM45928; NHLBI HL125353; NCI CA121493; Russian Foundation for Basic Research 015-04-07415; Danish Strategic Research Council 09‑067148; Abby Rockefeller Mauze Trust; and the Maloris & Hormel Foundations.

Design and synthesis of fluorescent glycolipid photoaffinity probes and their photoreactivity

Glycolipid–protein interactions at the cell surface are implicated in various biological processes. Toward the investigation of glycolipid binding proteins, we designed and synthesized trifunctional photoaffinity probes, which present a sugar head group with a triazole linkage to the lipid tail unit containing a photoreactive group and a fluorescent tag. The glycolipid photoaffinity probes bearing benzophenone group or diazirine group were evaluated for their photocrosslinking reactivity toward a carbohydrate head group specific protein. The diazirine based glycolipid photoaffinity probe was found to be more effective than the benzophenone-based probe in a comparative analysis involving a competitive ligand to distinguish a specific binding protein.

Aβ1-25-Derived Sphingolipid-Domain Tracer Peptide SBD Interacts with Membrane Ganglioside Clusters via a Coil-Helix-Coil Motif.

The Amyloid-β (Aβ)-derived, sphingolipid binding domain (SBD) peptide is a fluorescently tagged probe used to trace the diffusion behavior of sphingolipid-containing microdomains in cell membranes through binding to a constellation of glycosphingolipids, sphingomyelin, and cholesterol. However, the molecular details of the binding mechanism between SBD and plasma membrane domains remain unclear. Here, to investigate how the peptide recognizes the lipid surface at an atomically detailed level, SBD peptides in the environment of raft-like bilayers were examined in micro-seconds-long molecular dynamics simulations. We found that SBD adopted a coil-helix-coil structural motif, which binds to multiple GT1b gangliosides via salt bridges and CH–π interactions. Our simulation results demonstrate that the CH–π and electrostatic forces between SBD monomers and GT1b gangliosides clusters are the main driving forces in the binding process. The presence of the fluorescent dye and linker molecules do not change the binding mechanism of SBD probes with gangliosides, which involves the helix-turn-helix structural motif that was suggested to constitute a glycolipid binding domain common to some sphingolipid interacting proteins, including HIV gp120, prion, and Aβ.

The Dramatic Modulatory Role of the 2'N Substitution of the Terminal Amino Hexose of Globotetraosylceramide in Determining Binding by Members of the Verotoxin Family

Although globotetraosylceramide (Gb4) is only recognized by a single member of the verotoxin family namely, the pig edema disease toxin (VT2e), removal of the acetyl group from the terminal N-acetyl hexosamine of Gb4 to generate the free amino sugar containing species (aminoGb4) results in the generation of a glycolipid preferentially recognized by all members of the verotoxin family (i.e., VT1, VT2, VT2c, and VT2e). GT3, a site-specific mutant of VT2e, in which Gb4 recognition is lost but Gb3 binding is retained, also binds aminoGb4. We have now compared the binding of VT1, VT2, VT2e, and GT3 to a series of aminoGb4 derivatives using a TLC overlay technique. DimethylaminoGb4 is bound by VT1 and VT2 but not VT2e or GT3; formylaminoGb4 binds all toxins but poorly to VT2 and preferentially VT2e; trifluoroacetylaminoGb4 binds only VT2e and GT3; isopropylaminoGb4 binds VT1 and poorly to VT2; benzylaminoGb4 binds all four toxins. Thus, there is a marked distinction between the permissible amino substitutions for VT1 and VT2e binding. GT3 is a hybrid between these in that, according to the substitution, it behaves similarly either to VT1 or to VT2e. For each species, GT3 does not however, show a hybrid binding between that of VT1 and VT2e. Analysis of the binding as a function of pH shows opposite effects for VT1 and VT2e: decreased pH increases VT1, but decreases VT2e receptor glycolipid binding.

Different effects of identical symmetry-related mutations near the bacteriochlorophyll dimer in the photosynthetic reaction center of Rhodobacter sphaeroides.

In the bacterial photosynthetic reaction center (RC), asymmetric protein environment of the bacteriochlorophyll (BChl) dimer largely determines the photophysical and photochemical properties of the primary electron donor. Previously, we noticed significant differences in properties of Rhodobacter sphaeroides RCs with identical mutations in symmetry-related positions- I(M206)H and I(L177)H. The substitution I(L177)H resulted in covalent binding of BChl PA with the L-subunit, as well as in 6-coordination of BChl BB, whereas in RC I(M206)H no such changes of pigment-protein interactions were found. In addition, the yield of RC I(M206)H after its isolation from membranes was significantly lower than the yield of RC I(L177)H. This study shows that replacement of amino acid residues in the M203-M206 positions near BChls PB and BA by symmetry-related residues from the L-subunit near BChls PA and BB leads to further decrease in RC amount in the membranes associated obviously with poor assembly of the complex. Introduction of a new hydrogen bond between BChl PB and its protein environment by means of the F(M197)H mutation stabilized the mutant RC but did not affect its low yield. We suggest that the mutation I(M206)H and substitution of amino acid residues in M203-M205 positions could disturb glycolipid binding on the RC surface near BChl BA that is important for stable assembly of the complex in the membrane.

Membrane deformation by neolectins with engineered glycolipid binding sites.

Lectins are glycan-binding proteins that are involved in the recognition of glycoconjugates at the cell surface. When binding to glycolipids, multivalent lectins can affect their distribution and alter membrane shapes. Neolectins have now been designed with controlled number and position of binding sites to decipher the role of multivalency on avidity to a glycosylated surface and on membrane dynamics of glycolipids. A monomeric hexavalent neolectin has been first engineered from a trimeric hexavalent bacterial lectin, From this neolectin template, 13 different neolectins with a valency ranging from 0 to 6 were designed, produced, and analyzed for their ability to bind fucose in solution, to attach to a glycosylated surface and to invaginate glycolipid-containing giant liposomes. Whereas the avidity only depends on the presence of at least two binding sites, the ability to bend and invaginate membranes critically depends on the distance between two adjacent binding sites.

Membrane Deformation by Neolectins with Engineered Glycolipid Binding Sites

Lectins are glycan-binding proteins that are involved in the recognition of glycoconjugates at the cell surface. When binding to glycolipids, multivalent lectins can affect their distribution and alter membrane shapes. Neolectins have now been designed with controlled number and position of binding sites to decipher the role of multivalency on avidity to a glycosylated surface and on membrane dynamics of glycolipids. A monomeric hexavalent neolectin has been first engineered from a trimeric hexavalent bacterial lectin, From this neolectin template, 13 different neolectins with a valency ranging from 0 to 6 were designed, produced, and analyzed for their ability to bind fucose in solution, to attach to a glycosylated surface and to invaginate glycolipid-containing giant liposomes. Whereas the avidity only depends on the presence of at least two binding sites, the ability to bend and invaginate membranes critically depends on the distance between two adjacent binding sites.