Régis Daniel

Degradation of algal (Ascophyllum nodosum) fucoidan by an enzymatic activity contained in digestive glands of the marine mollusc Pecten maximus

Abstract A protein extract from digestive glands of the marine mollusc Pecten maximus was shown to possess fucoidan-degrading activity. This activity was able to release l -fucose from fucoidan derived from the brown algae Ascophyllum nodosum, and markedly reduce the molecular size of the polysaccharide. An enzymatically degraded fucoidan was produced and analysed by NMR spectroscopy. The 2D 1H NMR data obtained for the first time on low-molecular-weight fractions of algal fucoidan provided new insight into the structure of the polysaccharide. The latter has a randomly organised structure, involving (1→3)- and (1→4)-linked unsulfated and 2-sulfated-α- l -fucose residues.

Interaction of fucoidan with the proteins of the complement classical pathway.

Fucoidan inhibits complement by mechanisms that so far remain to be unraveled, and the objective of this work was to delineate the mode of inhibition by this sulfated polysaccharide. For that purpose, low molecular weight fractions of algal (Ascophyllum nodosum) fucoidan containing the disaccharide unit [-->3)-alpha-L-Fuc(2SO3(-))-(1-->4)-alpha-L-Fuc(2,3diSO3(-))-(1-->](n) have been studied. Gel co-affinity electrophoresis and a new affinity capillary electrophoresis (ACE) method have been implemented to characterize fucoidan-complement protein complexes. Fucoidan binds C1q, likely to its collagen-like region through interactions involving lysine residues, and then prevents the association of the C1r(2)-C1s(2) subunit, required to form the fully active C1. In addition to C1q, fucoidan forms a complex with the protein C4 as observed by ACE. The fucoidan inhibits the first steps of the classical pathway activation that is of relevance in view of the proinflammatory effects of the subsequent products of the cascade. This study shows that a high level of inhibitory activity can be achieved with low molecular weight carbohydrate molecules and that the potential applicability of fucoidan oligosaccharides for therapeutic complement inhibition is worthy of consideration.

Sulfated oligosaccharides (heparin and fucoidan) binding and dimerization of stromal cell-derived factor-1 (SDF-1/CXCL 12) are coupled as evidenced by affinity CE-MS analysis.

Chemokine stromal cell-derived factor-1 (SDF-1) is a potent chemoattractant involved in leukocyte trafficking and metastasis. Heparan sulfate on the cell surface binds SDF-1 and may modulate its function as a coreceptor of this chemokine. A major effect of the glycosaminoglycan binding may be on the quaternary structure of SDF-1, which has been controversially reported as a monomer or a dimer. We have investigated the effect of sulfated oligosaccharides on the oligomerization of SDF-1 and of its mutated form SDF-1 (3/6), using affinity capillary electrophoresis (ACE) hyphenated to mass spectrometry (MS). Coupled to MS, ACE allowed the study for the first time of the effect of size-defined oligosaccharides on the quaternary organization of SDF-1 in muM range concentrations, i.e., lower values than the mM values previously reported in NMR, light scattering, and ultracentrifugation experiments. Our results showed that in the absence of sulfated oligosaccharides, SDF-1 is mostly monomeric in solution. However, dimer formation was observed upon interaction with heparin-sulfated oligosaccharides despite the mM Kd values for dimerization. A SDF-1/oligosaccharide 2/1 complex was detected, indicating that oligosaccharide binding promoted the dimerization of SDF-1. Heparin tetrasaccharide but not disaccharide promoted dimer formation, suggesting that the dimer required to be stabilized by a long enough bound oligosaccharide. The SDF-1/oligosaccharide 1/1 complex was only observed with heparin disaccharide and fucoidan pentasaccharide, pointing out the role of specific structural determinants in promoting dimer formation. These results underline the importance of dimerization induced by glycosaminoglycans for chemokine functionality.

HABA-based ionic liquid matrices for UV-MALDI-MS analysis of heparin and heparan sulfate oligosaccharides

Polysulfated carbohydrates such as heparin (HP) and heparan sulfate (HS) are not easily amenable to usual ultraviolet matrix-assisted laser desorption/ionization-mass spectrometry (UV-MALDI)-MS analysis due to the thermal lability of their O- and N-SO(3) moieties, and their poor ionization efficiency with common crystalline matrices. Recently, ionic liquid matrices showed considerable advantages over conventional matrices for MALDI-MS of acidic compounds. Two new ionic liquid matrices (ILMs) based on the combination of 2-(4-hydroxyphenylazo)benzoic acid (HABA) with 1,1,3,3-tetramethylguanidine and spermine were evaluated in the study herein. Both ILMs were successfully applied to the analysis of synthetic heparin oligosaccharides of well-characterized structures as well as to heparan sulfate-derived oligosaccharides from enzymatic depolymerization. HABA-based ILMs showed improved signal-to-noise ratio as well as a decrease of fragmentation/desulfation processes and cation exchange. Sulfated oligosaccharides were detected with higher sensitivity than usual crystalline matrices, and their intact fully O- and N-sulfated species [M-Na](-) were easily observed on mass spectra. MALDI-MS characterization of challenging analytes such as heparin octasaccharide carrying 8-O and 4 N-sulfo groups, and heparin octadecasulfated dodecasaccharide was successfully achieved.

Capillary electrophoresis determination of the binding affinity of bioactive sulfated polysaccharides to proteins: study of the binding properties of fucoidan to antithrombin

The interaction of proteins with polysaccharides represents a major and challenging topic in glycobiology, since such complexes mediate fundamental biological mechanisms. An affinity capillary electrophoresis method has been developed to evidence the complex formation and to determine the binding properties between an anticoagulant polysaccharide of marine origin, fucoidan, and a potential target protein, antithrombin. This method is a variant of zonal electrophoresis in the mobility shift format. A fixed amount of protein was injected into a capillary filled with a background electrolyte containing the polysaccharide in varying concentrations. The effective mobility data of the protein were processed according to classical linearization treatments to obtain the binding constant for the polysaccharide/antithrombin complex. The results indicate that fucoidan binds to antithrombin in a 1:1 stoichiometry and with an affinity depending on the molecular weight of the polysaccharide. For heparin, the binding constant obtained similarly is in accordance with the literature. This is the first report showing the implementation of a capillary electrophoresis method contributing to the mechanistic understanding of the biological activities of fucoidan and providing evidence for the complex formation between fucoidan and the protein inhibitor of the coagulation antithrombin.

Hyphenation of surface plasmon resonance imaging to matrix-assisted laser desorption ionization mass spectrometry by on-chip mass spectrometry and tandem mass spectrometry analysis.

Most of the recent developments aiming to the coupling between surface plasmon resonance (SPR) and mass spectrometry (MS) are based on the use of a biochip with a limited number of flow cells requiring elution steps for the recovery of the captured biomolecules. In this work, a direct on-chip MALDI-MS detection is presented using a SPRi-sensor biochip in a microarray format that allows a multiplex SPR-MS analysis. The biochip gold surface was functionalized by a self-assembled monolayer (SAM) of short polyoxyethylene (POE) chains carrying a N-hydroxysuccinimide (NHS) group for the immobilization of biomolecules. The SPR measurement of the interaction of grafted antibodies anti-beta-lactoglobulin and anti-ovalbumin with their corresponding antigens indicated that the POE-NHS SAM preserved the binding activity of the antibodies immobilized on the biochips surface. SPR-MS experiments were carried out through MALDI-MS detection of the retained antigens (beta-lactoglobulin and ovalbumin) directly from the biochip surface. Mass spectra were obtained from each distinct spot on the arrayed biochips. Femtomole amounts of specifically retained antigen proteins as determined by SPR were sufficient to obtain good quality mass spectra. These mass spectra showed protein ions corresponding to the specific antigen, without any trace of nonspecific binding. The underivatized portion of the chip was also devoid of nonspecifically bound proteins, indicating that the functionalization of the biochips surface by short polyoxyethylene chains greatly minimizes the unspecific binding. In addition, it allowed on-chip digestion of the specifically bound analyte and coupling with MS/MS experiments, opening numerous applications in the proteomic field.

NMR characterization and molecular modeling of fucoidan showing the importance of oligosaccharide branching in its anticomplementary activity.

Fucoidan is a potent inhibitor of the human complement system whose activity is mediated through interactions with certain proteins belonging to the classical pathway, particularly the protein C4. Branched fucoidan oligosaccharides displayed a higher anticomplementary activity as compared to linear structures. Nuclear magnetic resonance (NMR) characterization of the branched oligosaccharides and saturation transfer difference-NMR experiment of the interaction with the protein C4 allowed the identification of the glycan residues in close contact with the target protein. Transferred nuclear Overhauser effect spectroscopy experiment and molecular modeling of fucoidan oligosaccharides indicated that the presence of side chains reduces the flexibility of the oligosaccharide backbone, which thus adopts a conformation which is very close to the one recognized by the protein C4. Together, these results suggest that branching of fucoidan oligosaccharides, determining their conformational state, has a major impact on their anticomplementary activity.

Performance evaluation on a wide set of matrix‐assisted laser desorption ionization matrices for the detection of oligosaccharides in a high‐throughput mass spectrometric screening of carbohydrate depolymerizing enzymes

Compared to other analytical methods, matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) presents several unique advantages for the structural characterization of degradation products of carbohydrates. Our final goal is to implement this technique as a high-throughput platform, with the aim of exploring natural bio-diversity to discover new carbohydrate depolymerizing enzymes. In this approach, a variety of carbohydrates will be used as enzymes substrates and MALDI-MS will be employed to monitor the oligosaccharides produced. One drawback of MALDI, however, is that the choice of the matrix is largely dependent on the chemical properties of the analyte. In this context, our objective in the present work was to find the smallest set of MALDI matrices able to detect chemically heterogeneous oligosaccharides. This was done through the performance evaluation of more than 40 MALDI matrices preparations. Homogeneity of analyte-matrix deposits was considered as a critical feature, especially since the final objective is to fully automate the analyses. Evaluation of the matrices was done by means of a rigorous statistical approach. Amongst all tested compounds, our work proposes the use of the DHB/DMA ionic matrix as the most generic matrix, for rapid detection of a variety of polysaccharides including neutral, anionic, methylated, sulfated, and acetylated compounds. The selected matrices were then used to screen crude bacterial incubation media for the detection of enzymatic degradation products.

Expression of recombinant human complement C1q allows identification of the C1r/C1s-binding sites

Complement C1q is a hexameric molecule assembled from 18 polypeptide chains of three different types encoded by three genes. This versatile recognition protein senses a wide variety of immune and nonimmune ligands, including pathogens and altered self components, and triggers the classical complement pathway through activation of its associated proteases C1r and C1s. We report a method for expression of recombinant full-length human C1q involving stable transfection of HEK 293-F mammalian cells and fusion of an affinity tag to the C-terminal end of the C chain. The resulting recombinant (r) C1q molecule is similar to serum C1q as judged from biochemical and structural analyses and exhibits the characteristic shape of a bunch of flowers. Analysis of its interaction properties by surface plasmon resonance shows that rC1q retains the ability of serum C1q to associate with the C1s-C1r-C1r-C1s tetramer, to recognize physiological C1q ligands such as IgG and pentraxin 3, and to trigger C1r and C1s activation. Functional analysis of rC1q variants carrying mutations of LysA59, LysB61, and/or LysC58, in the collagen-like stems, demonstrates that LysB61 and LysC58 each play a key role in the interaction with C1s-C1r-C1r-C1s, with LysA59 being involved to a lesser degree. We propose that LysB61 and LysC58 both form salt bridges with outer acidic Ca2+ ligands of the C1r and C1s CUB (complement C1r/C1s, Uegf, bone morphogenetic protein) domains. The expression method reported here opens the way for deciphering the molecular basis of the unusual binding versatility of C1q by mapping the residues involved in the sensing of its targets and the binding of its receptors.

Single molecule detection of glycosaminoglycan hyaluronic acid oligosaccharides and depolymerization enzyme activity using a protein nanopore.

Glycosaminoglycans are biologically active anionic carbohydrates that are among the most challenging biopolymers with regards to their structural analysis and functional assessment. The potential of newly introduced biosensors using protein nanopores that have been mainly described for nucleic acids and protein analysis to date, has been here applied to this polysaccharide-based third class of bioactive biopolymer. This nanopore approach has been harnessed in this study to analyze the hyaluronic acid glycosamiglycan and its depolymerization-derived oligosaccharides. The translocation of a glycosaminoglycan is reported using aerolysin protein nanopore. Nanopore translocation of hyaluronic acid oligosaccharides was evidenced by the direct detection of translocated molecules accumulated into the arrival compartment using high-resolution mass spectrometry. Anionic oligosaccharides of various polymerization degrees were discriminated through measurement of the dwelling time and translocation frequency. This molecular sizing capability of the protein nanopore device allowed the real-time recording of the enzymatic cleavage of hyaluronic acid polysaccharide. The time-resolved detection of enzymatically produced oligosaccharides was carried out to monitor the depolymerization enzyme reaction at the single-molecule level.