Mass spectrometry and non-covalent protein-ligand complexes: confirmation of binding sites and changes in tertiary structure

An experimental approach is described for determining protein-small molecule non-covalent ligand binding sites and protein conformational changes induced by ligand binding. The methodology utilizes time resolved limited proteolysis and the high throughput analysis capability of MALDI TOF MS to determine the binding site in a tetanus toxin C-fragment (51 kDa)-doxorubicin (543 Da) non-covalent complex. Comparing relative ion abundances of peptides released from the time resolved limited proteolysis of tetanus toxin C-fragment (TetC) and the TetC-doxorubicin complex every 10 min from 10 to 120 min of digestion revealed that the binding of doxorubicin induced a significant change in surface topology of TetC. Four of the twenty-nine peptides observed by MALDI MS, including amino acids 351-360, 299-304, 305-311 and 312-316, had a lower abundance in the TetC-doxorubicin complex relative to TetC from 10 to 100 min of digestion. A decrease in ion abundance suggests doxorubicin obstructs the access of the protease to one or both termini of these peptides, identifying doxorubicin binding site(s). Conversely, five peptide ions, including amino acids 335-350, 364-375, 364-376, 281-298, and 316-328, all had a greater abundance in the digest of the complex, indicating an increase in accessibility to these sites. These five peptides flank regions of decreased ion abundance, suggesting that doxorubicin not only binds to the surface, but also induces a conformational change in TetC.     

Gangliosides and sulphatide in human cerebrospinal fluid: quantitation with immunoaffinity techniques

A sensitive micromethod involving extraction, purification and thin-layer chromatography (TLC)-enzyme immunostaining was developed for the quantation of gangliosides and sulphatide, as markers for neuronal disorders and myelin disturbances, in individual samples of less than 5 ml of cerebrospinal fluid. The gangliosides of the gangliotetraose series were individually determined with cholera toxin subunit B by TLC-enzyme-linked immunosorbent assay (ELISA) after chromatography and subsequent sialidase hydrolysis to II3NeuAc-GgOse4Cer (GM1). Other gangliosides and sulphatide were determined with specific monoclonal antibodies by TLC-ELISA. The total ganglioside content varied between 100 and 230 nmol/l in ten normal cerebrospinal fluid samples from adults. The major gangliosides were of the gangliotetraose series, represented by GM1, IV3NeuAc,II3NeuAc-GgOse4Cer, (GD1a), II3(NeuAc)2-GgOse4Cer (GD1b) and IV3NeuAc,II3 (NeuAc)2-GgOse4Cer (GT1b) of which the b-series gangliosides dominated, i.e., GD1b and GT1b.     

A Novel Method for the Production of Glycosphingolipids

Neolacto‐series ganglioside sialylparagloboside (SPG) is a ganglioside species present in various human tissues, and used in many important studies. In this study, four ganglioside analogs, GM3, GD3, SPG, and NeuAc‐Gal‐GlcNAc‐Gal‐GlcNAc‐Gal‐Glc‐Cer, were synthesized by the saccharide‐primer method using MDCK cells and β‐lactoside primer with different aglycons. As compared to former methods for producing SPG, the primer method was rapid and convenient. Moreover, the yield of SPG was much higher than that obtained by former methods. The production of gangliosides with an azido group in the aglycon moiety was also achieved by using MDCK cells.     

Determination of the absolute configuration of sialic acids in gangliosides from the sea cucumber Cucumaria echinata

Enantiomeric pairs of sialic acid, D- and L-NeuAc (N-acetylneuraminic acid), were converted to D- and L-arabinose, respectively, by chemical degradation. Using this method, the absolute configuration of the sialic acid residues, NeuAc and NeuGc (N-glycolylneuraminic acid), in the gangliosides from the sea cucumber Cucumaria echinata was determined to be the D-form. Although naturally occurring sialic acids have been believed to be the D-form on the basis of biosynthetic evidence, this is the first report of the determination of the absolute configuration of the sialic acid residues in gangliosides using chemical methods.     

Dissecting the cholera toxin-ganglioside GM1 interaction by isothermal titration calorimetry

The complex of cholera toxin and ganglioside GM1 is one of the highest affinity protein-carbohydrate interactions known. Herein, the GM1 pentasaccharide is dissected into smaller fragments to determine the contribution of each of the key monosaccharide residues to the overall binding affinity. Displacement isothermal titration calorimetry (ITC) has allowed the measurement of all of the key thermodynamic parameters for even the lowest affinity fragment ligands. Analysis of the standard free energy changes using Jencks' concept of intrinsic free energies reveals that the terminal galactose and sialic acid residues contribute 54% and 44% of the intrinsic binding energy, respectively, despite the latter ligand having little appreciable affinity for the toxin. This analysis also provides an estimate of 25.8 kJ mol(-1) for the loss of independent translational and rotational degrees of freedom on complexation and presents evidence for an alternative binding mode for ganglioside GM2. The high affinity and selectivity of the GM1-cholera toxin interaction originates principally from the conformational preorganization of the branched pentasaccharide rather than through the effect of cooperativity, which is also reinvestigated by ITC.     

Deciphering the molecular details for the binding of the prion protein to main ganglioside GM1 of neuronal membranes

The prion protein (PrP) resides in lipid rafts in?vivo, and lipids modulate misfolding of the protein to infectious isoforms. Here we demonstrate that binding of recombinant PrP to model raft membranes requires the presence of ganglioside GM1. A combination of liquid- and solid-state NMR revealed the binding sites of PrP to the saccharide head group of GM1. The binding epitope for GM1 was mapped to the folded C-terminal domain of PrP, and docking simulations identified key residues in the C-terminal region of helix C and the loop between strand S2 and helix B. Crucially, this region of PrP is linked to prion resistance in?vivo, and structural changes caused by lipid binding in this region may explain the requirement for lipids in the generation of infectious prions in?vitro.     

Liposomal glyco-microarray for studying glycolipid–protein interactions

A microarray enables high-throughput interaction screening of numerous biomolecules; however, fabrication of a microarray composed of cellular membrane components has proven difficult. We report fabrication of a liposomal glyco-microarray by using an azide-reactive liposome that carries synthetic and natural glycolipids via chemically selective and biocompatible liposome immobilization chemistry. Briefly, liposomes carrying anchor lipid dipalmitoylphosphatidylethanolamine (DPPE)-PEG(2000)-triphenylphosphine and ganglioside (GM1 or GM3) were prepared first and were then printed onto an azide-modified glass slide so as to afford a liposomal glyco-microarray via Staudinger ligation. Fluorescent dye release kinetics and fluorescence imaging confirmed successful liposome immobilization and specific protein binding to the intact arrayed glycoliposomes. The liposomal glyco-microarray with different gangliosides showed their specific lectin and toxin binding with different binding affinity. The azide-reactive liposome provides a facile strategy for fabrication of either a natural or a synthetic glycolipid-based membrane-mimetic glycoarray. This liposomal glyco-microarray is simple and broadly applicable and thus will find important biomedical applications, such as studying glycolipid-protein interactions and toxin screening applications.     

Ganglioside patterns of metastatic and non-metastatic transplantable hepatocellular carcinomas of the rat

In previous investigations, we correlated levels of sialic acid, gangliosides, and ganglioside glycosyltransferases with tumorigenesis over a 24-week continuum of growth of hepatocellular neoplasms of the rat induced by the carcinogen N-2-fluorenylacetamide. However, metastatic tumors developed only rarely and were not analyzed. To investigate surface changes associated with metastasis, well-differentiated and poorly differentiated hepatocellular carcinomas were transplanted to syngeneic recipient rats. From those, several metastatic and nonmetastatic isolates were obtained and compared. Both total and ganglioside sialic acid amounts in transplantable hepatomas were elevated above control liver values but were significantly lower for metastatic lines than for nonmetastatic lines. The nonmetastatic lines were characterized by ganglioside patterns depleted in the precursor ganglioside GM3 (sialic acid-galactose-glucose-ceramide) and elevated in the products of the monosialoganglioside pathway. In contrast, metastatic isolates exhibited a restoration of GM3 and nearer normal amounts of other gangliosides. The findings point to differences in sialic acid-containing glycolipids, comparing metastatic and nonmetastatic hepatocellular carcinomas, and further extend the concept that ganglioside alterations do not cause tumorigenesis but are the end result of a cascade of events which apparently continue beyond the onset of metastasis.     

Structure-based design and confirmation of peptide ligands for neuronal polo-like kinase to promote neuroregeneration

Neuronal polo-like kinase (nPLK) is an essential regular of cell cycle and differentiation in nervous system, and targeting nPLK has been established as a promising therapeutic strategy to treat neurological disorders and to promote neuroregeneration. The protein contains an N-terminal kinase domain (KD) and a C-terminal Polo-box domain (PBD) that are mutually inhibited by each other. Here, the intramolecular KD–PBD complex in nPLK was investigated at structural level via bioinformatics analysis, molecular dynamics (MD) simulation and binding affinity scoring. From the complex interface two regions representing separately two continuous peptide fragments in PBD domain were identified as the hot spots of KD–PBD interaction. Structural and energetic analysis suggested that one (PBD peptide 1) of the two peptides can bind tightly to a pocket nearby the active site of KD domain, which is thus potential as self-inhibitory peptide to target and suppress nPLK kinase activity. The knowledge harvesting from computational studies were then used to guide the structural optimization and mutation of PBD peptide 1. Consequently, two of three peptide mutants separately exhibited moderately and considerably increased affinity as compared to the native peptide. The computationally modeled complex structures of KD domain with these self-inhibitory peptides were also examined in detail to unravel the structural basis and energetic property of nPLK-peptide recognition and interaction.     

Glycopolymers Mimicking GM1 Gangliosides: Cooperativity of Galactose and Neuraminic Acid for Cholera Toxin Recognition

Glycopolymers mimicking GM1 gangliosides were synthesized by incorporating multiple types of carbohydrates into the polymer backbone. The glycopolymers were immobilized onto gold surfaces, and the interactions with the cholera toxin B subunit (CTB) were analyzed using surface plasmon resonance imaging. The glycopolymer containing both galactose and neuraminic acid showed enhanced recognition of CTB. The interaction was enhanced mainly because of an improvement in the dissociation process by the binding of the neuraminic acid group in the GM1 binding pocket. This cooperativity of galactose and neuraminic acid was achieved by incorporation into the same flexible polymer backbone, and the importance of the close placement of galactose and neuraminic acid groups was revealed. These results will be valuable in medical fields and also for the development of biofunctional materials.     

Affinity capillary electrophoresis for the assessment of binding affinity of carbohydrate‐based cholera toxin inhibitors

Developing tools for the study of protein carbohydrate interactions is an important goal in glycobiology. Cholera toxin inhibition is an interesting target in this context, as its inhibition may help to fight against cholera. For the study of novel ligands an affinity capillary electrophoresis (ACE) method was optimized and applied. The method uses unlabeled cholera toxin B‐subunit (CTB) and unlabeled carbohydrate ligands based on ganglioside GM1‐oligosaccharides (GM1os). In an optimized method at pH 4, adsorption of the protein to the capillary walls was prevented by a polybrene‐dextran sulfate‐polybrene coating. Different concentrations of the ligands were added to the BGE. CTB binding was observed by a mobility shift that could be used for dissociation constant (K_d) determination. The K_d values of two GM1 derivatives differed by close to an order of magnitude (600 ± 20 nM and 90 ± 50 nM) which was in good agreement with the differences in their reported nanomolar IC₅₀ values of an ELISA‐type assay. Moreover, the selectivity of GM1os towards CTB was demonstrated using Influenza hemagglutinin (H5) as a binding competitor. The developed method can be an important platform for preclinical development of drugs targeting pathogen‐induced secretory diarrhea.     

Constituents of Crinoidea. 5. Isolation and structure of a new glycosyl inositolphosphoceramide-type ganglioside from the feather star Comanthina schlegeli

A new glycosyl inositolphosphoceramide-type ganglioside, CSP2, was obtained from the polar lipid fraction of the chloroform/methanol extract of the feather star Comanthina schlegeli together with a known same type of ganglioside CJP2. The structure of this ganglioside has been determined on the basis of chemical and spectroscopic evidence to be 9-O-methyl-(N-acetyl-alpha-D-neuraminosyl)-(2-->3)-inositolphosphoceramide, which contains C(16)-sphingosine and C(22:0)-, C(24:0)-fatty acid as major component. This is the first report on the isolation and structural elucidation of a glycosyl inositolphosphoceramide-type ganglioside possessing N-acetyl-neuraminic acid (NeuAc) residue.     

白喉毒素活性中心的量子化学计算与149位突变体的酶学动力学

通过量子化学计算确定白喉毒素分子催化区活性中心的关键氨基酸残基, 评价其取代后的酶活性的改变, 为导向性抗癌药物研究提供高效杀伤细胞工具. 结合目前关于白喉毒素结构与功能的研究状况和量子化学计算结果, 将白喉毒素催化区的第149位酪氨酸突变为苯丙氨酸, 对其酶活性和与底物的结合能力进行评价. Y149位酪氨酸位于正电中心, 起受电子作用, 与野生白喉毒素相比, 苯丙氨酸突变体的酶催化活性增加约一倍, 而与底物结合能力没有变化. Y149是酶活性中心的关键氨基酸残基, 对其取代能够影响蛋白质的生物活性.     

Carbohydrate chain of ganglioside GM1 as a ligand: identification of the binding strategies of three 15 mer peptides and their divergence from the binding modes of growth-regulatory galectin-1 and cholera toxin

The branched pentasaccharide chain of ganglioside GM1 is a prominent cell surface ligand, for example, for cholera toxin or tumor growth-regulatory homodimeric galectins. This activity profile via protein recognition prompted us to examine the binding properties of peptides with this specificity. Our study provides insights into the mechanism of molecular interaction of this thus far unexplored size limit of the protein part. We used three pentadecapeptides in a combined approach of mass spectrometry, NMR spectroscopy and molecular modelling to analyze the ligand binding in solution. Availability of charged and hydrophobic functionalities affected the intramolecular flexibility of the peptides differently. Backfolding led to restrictions in two cases; the flexibility was not reduced significantly by association of the ligand in its energetically privileged conformations. Major contributions to the interaction energy arise from the sialic acid moiety contacting Arg/Lys residues and the N-terminal charge. Considerable involvement of stacking between the monovalent ligand and aromatic rings could not be detected. This carbohydrate binding strategy is similar to how an adenoviral fiber knob targets sialylated glycans. Rational manipulation for an affinity enhancement can now be directed to reduce the flexibility, exploit the potential for stacking and acquire the cross-linking capacity of the natural lectins by peptide attachment to a suitable scaffold.     

Synthesis,Biological Evaluation,WAC and NMR Studies of S‐Galactosides and Non‐Carbohydrate Ligands of Cholera Toxin Based on Polyhydroxyalkylfuroate Moieties

The synthesis of several non‐carbohydrate ligands of cholera toxin based on polyhydroxyalkylfuroate moieties is reported. Some of them have been linked to D ‐galactose through a stable and well‐tolerated S‐glycosidic bond. They represent a novel type of non‐hydrolyzable bidentate ligand featuring galactose and polyhydroxyalkylfuroic esters as pharmacophoric residues, thus mimicking the GM1 ganglioside. The affinity of the new compounds towards cholera toxin was measured by weak affinity chromatography (WAC). The interaction of the best candidates with this toxin was also studied by saturation transfer difference NMR experiments, which allowed identification of the binding epitopes of the ligands interacting with the protein. Interestingly, the highest affinity was shown by non‐carbohydrate mimics based on a polyhydroxyalkylfuroic ester structure.     

Interaction between a Novel Oligopeptide Fragment of the Human Neurotrophin Receptor TrkB Ectodomain D5 and the C-Terminal Fragment of Tetanus Neurotoxin

This article presents experimental evidence and computed molecular models of a potential interaction between receptor domain D5 of TrkB with the carboxyl-terminal domain of tetanus neurotoxin (Hc-TeNT). Computational simulations of a novel small cyclic oligopeptide are designed, synthesized, and tested for possible tetanus neurotoxin-D5 interaction. A hot spot of this protein-protein interaction is identified in analogy to the hitherto known crystal structures of the complex between neurotrophin and D5. Hc-TeNT activates the neurotrophin receptors, as well as its downstream signaling pathways, inducing neuroprotection in different stress cellular models. Based on these premises, we propose the Trk receptor family as potential proteic affinity receptors for TeNT. In vitro, Hc-TeNT binds to a synthetic TrkB-derived peptide and acts similar to an agonist ligand for TrkB, resulting in phosphorylation of the receptor. These properties are weakened by the mutagenesis of three residues of the predicted interaction region in Hc-TeNT. It also competes with Brain-derived neurotrophic factor, a native binder to human TrkB, for the binding to neural membranes, and for uptake in TrkB-positive vesicles. In addition, both molecules are located together in vivo at neuromuscular junctions and in motor neurons.     

Rapid Profiling of Bovine and Human Milk Gangliosides by Matrix-Assisted Laser Desorption/Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

Gangliosides are anionic glycosphingolipids widely distributed in vertebrate tissues and fluids. Their structural and quantitative expression patterns depend on phylogeny and are distinct down to the species level. In milk, gangliosides are exclusively associated with the milk fat globule membrane. They may participate in diverse biological processes but more specifically to host-pathogen interactions. However, due to the molecular complexities, the analysis needs extensive sample preparation, chromatographic separation, and even chemical reaction, which makes the process very complex and time-consuming. Here, we describe a rapid profiling method for bovine and human milk gangliosides employing matrix-assisted desorption/ionization (MALDI) Fourier transform ion cyclotron resonance (FTICR) mass spectrometry (MS). Prior to the analyses of biological samples, milk ganglioside standards GM3 and GD3 fractions were first analyzed in order to validate this method. High mass accuracy and high resolution obtained from MALDI FTICR MS allow for the confident assignment of chain length and degree of unsaturation of the ceramide. For the structural elucidation, tandem mass spectrometry (MS/MS), specifically as collision-induced dissociation (CID) and infrared multiphoton dissociation (IRMPD) were employed. Complex ganglioside mixtures from bovine and human milk were further analyzed with this method. The samples were prepared by two consecutive chloroform/methanol extraction and solid phase extraction. We observed a number of differences between bovine milk and human milk. The common gangliosides in bovine and human milk are NeuAc-NeuAc-Hex-Hex-Cer (GD3) and NeuAc-Hex-Hex-Cer (GM3); whereas, the ion intensities of ganglioside species are different between two milk samples. Kendrick mass defect plot yields grouping of ganglioside peaks according to their structural similarities. Gangliosides were further probed by tandem MS to confirm the compositional and structural assignments. We found that only in human milk gangliosides was the ceramide carbon always even numbered, which is consistent with the notion that differences in the oligosaccharide and the ceramide moieties confer to their physiological distinctions.     

Carbohydrate Chain of Ganglioside GM1 as a Ligand: Identification of the Binding Strategies of Three 15 mer Peptides and Their Divergence from the Binding Modes of Growth‐Regulatory Galectin‐1 and Cholera Toxin

The branched pentasaccharide chain of ganglioside GM₁ is a prominent cell surface ligand, for example, for cholera toxin or tumor growth‐regulatory homodimeric galectins. This activity profile via protein recognition prompted us to examine the binding properties of peptides with this specificity. Our study provides insights into the mechanism of molecular interaction of this thus far unexplored size limit of the protein part. We used three pentadecapeptides in a combined approach of mass spectrometry, NMR spectroscopy and molecular modelling to analyze the ligand binding in solution. Availability of charged and hydrophobic functionalities affected the intramolecular flexibility of the peptides differently. Backfolding led to restrictions in two cases; the flexibility was not reduced significantly by association of the ligand in its energetically privileged conformations. Major contributions to the interaction energy arise from the sialic acid moiety contacting Arg/Lys residues and the N‐terminal charge. Considerable involvement of stacking between the monovalent ligand and aromatic rings could not be detected. This carbohydrate binding strategy is similar to how an adenoviral fiber knob targets sialylated glycans. Rational manipulation for an affinity enhancement can now be directed to reduce the flexibility, exploit the potential for stacking and acquire the cross‐linking capacity of the natural lectins by peptide attachment to a suitable scaffold.     

Beyond carbohydrate binding: new directions in plant lectin research

Although for a long time carbohydrate binding property has been used as the defining feature of lectins, studies carried out mostly during the last two decades or so demonstrate that many plant lectins exhibit specific interactions with small molecules that are predominantly hydrophobic in nature. Such interactions, in most cases, appear to be at specific sites that do not interfere with the ability of the lectins to recognise and bind carbohydrates. Further, several of these ligands have binding affinities comparable to those for the binding of specific carbohydrates to the lectins. Given the ability of lectins to specifically recognise the glycocode (carbohydrate code) on different cell surfaces and distinguish between diseased and normal tissues, these additional sites may be viewed as potential drug carrying sites that could be exploited for targeted delivery to sites of choice. Porphyrin-lectin complexes are especially suited for such targeting since porphyrins are already under investigation in photodynamic therapy for cancer. This review will provide an update on the interactions of plant lectins with non-carbohydrate ligands, with particular emphasis on porphyrin ligands. The implications and potential applications of such studies will also be discussed.     

Isolation and structure of a monomethylated ganglioside possessing neuritogenic activity from the ovary of the sea urchin Diadema setosum

A new monomethylated ganglioside, DSG-A (3), was obtained, together with four known gangliosides, compounds (1, 2, 4, 5), from the lipid fraction of the chloroform/methanol extract of the ovary of the sea urchin Diadema setosum. The structures of the new ganglioside was determined on the basis of chemical and spectroscopic evidence to be 1-O-[9-O-methyl-(N-acetyl-alpha-D-neuraminosyl)-(2-->6)-beta-D-glucopyranosyl]-ceramide (3). The ceramide moiety of 3 was composed of C18-phytosphingosine base, and 2-hydroxy and nonhydroxylated fatty acid units. These gangliosides showed neuritogenic activity toward the rat pheochromocytoma cell line PC-12 in the presence of nerve growth factor, in which compound 3 showed the most potent activity.