Scan Science and Technology
Contact Leading Edge Experts & Companies
The Experts below are selected from a list of 315 Experts worldwide ranked by ideXlab platform
Peter Pahlsson – 1st expert on this subject based on the ideXlab platform
Determination of Fucose Concentration in a Lectin-Based Displacement Microfluidic Assay.Applied Biochemistry and Biotechnology, 2019Co-Authors: Per G. Erlandsson, Eva Astrom, Peter Pahlsson, Nathaniel D. RobinsonAbstract:
We compare three different methods to quantify the monosaccharide fucose in solutions using the displacement of a large glycoprotein, lactoferrin. Two microfluidic analysis methods, namely fluorescence detection of (labeled) lactoferrin as it is displaced by unlabeled fucose and the displacement of (unlabeled) lactoferrin in SPR, provide fast responses and continuous data during the experiment, theoretically providing significant information regarding the interaction kinetics between the saccharide groups and binding sites. For comparison, we also performed a static displacement ELISA. The stationary binding site in all cases was immobilized S2-AAL, a monovalent polypeptide based on Aleuria aurantia lectin. Although all three assays showed a similar dynamic range, the microfluidic assays with fluorescent or SPR detection show an advantage in short analysis times. Furthermore, the microfluidic displacement assays provide a possibility to develop a one-step analytical platform.
elucidating the selectivity of recombinant forms of Aleuria aurantia lectin using weak affinity chromatographyJournal of Chromatography B, 2012Co-Authors: Maria Bergstrom, Eva Astrom, Peter Pahlsson, Sten OhlsonAbstract:
Aberrant glycosylation is connected to several pathological conditions and lectins are useful tools to characterize glycosylated biomarkers. The Aleuria aurantia lectin (AAL) is of special interest …
Elucidating the selectivity of recombinant forms of Aleuria aurantia lectin using weak affinity chromatography.Journal of chromatography. B Analytical technologies in the biomedical and life sciences, 2011Co-Authors: Maria Bergstrom, Eva Astrom, Peter Pahlsson, Sten OhlsonAbstract:
Aberrant glycosylation is connected to several pathological conditions and lectins are useful tools to characterize glycosylated biomarkers. The Aleuria aurantia lectin (AAL) is of special interest since it interacts with all types of fucosylated saccharides. AAL has been expressed in Escherichia coli as a fully functional recombinant protein. Engineered variants of AAL have been developed with the aim of creating monovalent lectins with more homogenous binding characteristics. Four different forms of AAL were studied in the present work: native AAL purified from A. aurantia mushrooms, recombinant AAL dimer, recombinant AAL monomer and recombinant AAL site 2 (S2-AAL). The affinities of these AAL forms toward a number of saccharides were determined with weak affinity chromatography (WAC). Disaccharides with fucose linked α1-3 to GlcNAc interacted with higher affinity compared to fucose linked α1-6 or α1-4 and the obtained dissociation constants (K(d)) were in the range of 10μM for all AAL forms. Tetra- and pentasaccharides with fucose in α1-2, α1-3 or α1-4 had K(d) values ranging from 0.1 to 7mM while a large α1-6 fucosylated oligosaccharide had a K(d) of about 20μM. The recombinant multivalent AAL forms and native AAL exhibited similar affinities toward all saccharides, but S2-AAL had a lower affinity especially regarding a sialic acid containing fucosylated saccharide. It was demonstrated that WAC is a valuable technique in determining the detailed binding profile of the lectins. Specific advantages with WAC include a low consumption of non-labeled saccharides, possibility to analyze mixtures and a simple procedure using standard HPLC equipment.
Anand Mehta – 2nd expert on this subject based on the ideXlab platform
development and application of a novel recombinant Aleuria aurantia lectin with enhanced core fucose binding for identification of glycoprotein biomarkers of hepatocellular carcinomaProteomics, 2016Co-Authors: Pamela A Norton, Josef Houser, Michaela Wimmerova, Patrick R Romano, Mary Ann Comunale, Harmin Herrera, Mengjun Wang, Anand MehtaAbstract:
The Aleuria aurantia lectin (AAL) derived from orange peel
fungus contains five fucose-binding sites that recognizes
fucose bound in -1,2, -1,3, -1,4, and -1,6 linkages to
N-acetylglucosamine and galactose. Recently, we have created
several recombinant AAL (rAAL) proteins that had altered
binding affinity to fucose linkages. In this report, we further
characterize the binding specificity of one of the mutated
lectins, N224Q lectin. This lectin was characterized by lectin
Western blotting, surface plasmon resonance, and glycan
microarray and shown to have increased binding to fucosylated
glycan. Subsequently, we used this lectin to identify secreted
fucosylated glycoproteins from a fetal hepatic cell line.
Proteomic analysis revealed several glycoproteins secreted by
the fetal cell line that were bound by N224Q lectin. These
findings were confirmed by subsequent proteomic analysis of
human serum from control patients or patients with
hepatocellular carcinoma. These represent candidate oncofetal
markers for liver cancer.
Aleuria aurantia lectin aal reactive immunoglobulin g rapidly appears in sera of animals following antigen exposurePLOS ONE, 2012Co-Authors: Songming Chen, Anand Mehta, Chen Lu, Hongbo Gu, Jianwei Li, Patrick B Romano, David Horn, Craig D Hooper, Carthene R Bazemorewalker, Timothy M BlockAbstract:
We have discovered an Aleuria aurantia Lectin (AAL)-reactive immunoglobulin G (IgG) that naturally occurs in the circulation of rabbits and mice, following immune responses induced by various foreign antigens. AAL can specifically bind to fucose moieties on glycoproteins. However, most serum IgGs are poorly bound by AAL unless they are denatured or treated with glycosidase. In this study, using an immunogen-independent AAL-antibody microarray assay that we developed, we detected AAL-reactive IgG in the sera of all animals that had been immunized 1–2 weeks previously with various immunogens with and without adjuvants and developed immunogen-specific responses. All of these animals subsequently developed immunogen-specific immune responses. The kinetics of the production of AAL-reactive IgG in mice and rabbits were distinct from those of the immunogen-specific IgGs elicited in the same animals: they rose and fell within one to two weeks, and peaked between four to seven days after exposure, while immunogen-specific IgGs continued to rise during the same period. Mass spectrometric profiling of the Fc glycoforms of purified AAL-reactive IgGs indicates that these are mainly comprised of IgGs with core-fucosylated and either mono-or non-galactosylated Fc N-glycan structures. Our results suggest that AAL-reactive IgG could be a previously unrecognized IgG subset that is selectively produced at the onset of a humoral response.
development of recombinant Aleuria aurantia lectins with altered binding specificities to fucosylated glycansBiochemical and Biophysical Research Communications, 2011Co-Authors: Patrick R Romano, Andrew Mackay, Minh Vong, Johann Desa, Anne Lamontagne, Mary Ann Comunale, Julie Hafner, Timothy M Block, Anand MehtaAbstract:
Abstract Changes in glycosylation have long been associated with disease. While there are many methods to detect changes in glycosylation, plant derived lectins are often used to determine changes on specific proteins or molecules of interest. One change in glycosylation that has been observed by us and by others is a disease or antigen associated increase in fucosylation on N-linked glycans. To measure this change, the fucose binding Aleuria aurantia lectin (AAL) is often utilized in plate and solution based assays. AAL is a mushroom derived lectin that contains five fucose binding sites that preferentially bind fucose linked (α-1,3, α-1,2, α-,4, and α-1,6) to N -acetyllactosamine related structures. Recently, several reports by us and by others have indicated that specific fucose linkages found on certain serum biomarker glycoprotein’s are more associated with disease than others. Taking a site-directed mutagenesis approach, we have created a set of recombinant AAL proteins that display altered binding affinities to different analytes containing various fucose linkages.
Johan Olausson – 3rd expert on this subject based on the ideXlab platform
production and characterization of a monomeric form and a single site form of Aleuria aurantia lectinGlycobiology, 2011Co-Authors: Johan Olausson, Eva Astrom, Lena Tibell, Bengt-harald Jonsson, Peter PahlssonAbstract:
The presented work describes construction and analysis of recombinantly produced forms of Aleuria aurantia lectin (AAL). The binding properties of the produced AAL forms were studied using techniques such as tryptophan fluorescence, hemagglutination analysis, ELISA and surface plasmon resonance analysis. Lectins are proteins that are ubiquitous in nature with the ability to bind specifically to different types of carbohydrates. The physiological function of different lectins is not always known, but they are involved in many recognition events at molecular and cellular levels. In research, lectins are widely used for structural and functional studies of complex carbohydrates, and they are also used to detect changes in the carbohydrate pattern on glycoproteins in different diseases. With the use of recombinant technology it is now possible to refine properties of lectins such as decreasing the valency and alter specificity and affinity. This may be a way of constructing more suitable reagents for use in diagnostic glycosylation analysis assays. AAL has been extensively used in different types of research for its ability to bind the monosaccharide fucose and to fucose-containing oligosaccharides. It is composed of two identical subunits where each subunit contains five binding sites for fucose. AAL was expressed recombinantly (rAAL) and its properties was investigated. These studies reveled that one of the binding sites in rAAL had unusually high affinities towards fucose and fucosecontaining oligosaccharides with Kd-values in the nanomolar range. This binding site is not detected in AAL that have been exposed to fucose during its purification, and therefore we proposed that this site may be blocked with free fucose in commercial preparations of AAL. Normally lectin-oligosaccharide interactions are considered to be of weak affinity, so the finding of a high affinity site was interesting for the future study of recombinant forms of AAL. The next step was to produce recombinant AAL forms with decreased valency. This was done using site-directed mutagenesis. First a monomeric form of AAL (mAAL) was constructed and then a monovalent form of AAL, containing only one fucose-binding site (S2-AAL) was constructed. Both of these forms had retained ability to bind fucose. The binding characteristics of mAAL were similar to that of rAAL, but mAAL showed decreased hemagglutinating activity. S2-AAL showed a lower binding affinity to fucosylated oligosaccharides and did not bind to sialylated fuco-oligosaccharides such as sialyl-LewisX. This study shows that molecular engineering techniques could be important tools for development of reliable and specific diagnostic and biological assays for carbohydrate analysis.
studies of recombinant forms of Aleuria aurantia lectin, 2009Co-Authors: Johan OlaussonAbstract:
Lectins have been widely used in structural and functional studies of complex carbohydrates. Lectins usually bind carbohydrates with relatively low affinity but compensate for this by multivalency. When using lectins in different biological and analytical assays the multivalent nature of lectins can sometimes produce unwanted reactions such as agglutination or precipitation of target glycoproteins. The mushroom lectin Aleuria aurantia binds to fucose-containing oligosaccharides. It is composed of two identical subunits where each subunit contains five binding sites for fucose. In the present study two forms of recombinant AAL were produced using site-directed mutagenesis. A monomeric form of AAL was produced by exchange of Tyr6 to Arg6, and a monovalent fragment of AAL was produced by insertion of a NdeI restriction enzyme cleavage site and a stop codon in the coding sequence. The AAL forms were expressed as His-tagged proteins in E.coli and purified by affinity chromatography. Binding properties of the two AAL forms were performed using hemagglutination assay, surface plasmon resonance and enzyme-linked lectin assay analyses. Both the monomeric AAL form (mAAL) and the monovalent AAL form (S2-AAL) retained their capacity to bind fucosylated oligosaccharides. However, both constructs exhibited properties that differed from the intact recombinant AAL (rAAL). Monomeric AAL showed similar binding affinities to fucosylated oligosaccharides compared to rAAL but had less hemagglutinating capacity. S2-AAL showed a lower binding affinity to fucosylated oligosaccharides and, in contrast to rAAL and mAAL, S2-AAL did not bind to sialylated fuco-oligosaccharides such as sialyl-Lex. The study shows that molecular engineering techniques may be a tool for producing lectins with more defined properties such as decreased valency and defined specificities and affinities. This may be very valuable for development of reliable diagnostic and biological assays for carbohydrate analysis.
Detection of a high affinity binding site in recombinant Aleuria aurantia lectinGlycoconjugate Journal, 2008Co-Authors: Johan Olausson, Lena Tibell, Bengt-harald Jonsson, Peter PahlssonAbstract:
Lectins are carbohydrate binding proteins that are involved in many recognition events at molecular and cellular levels. Lectin-oligosaccharide interactions are generally considered to be of weak affinity, however some mushroom lectins have unusually high binding affinity towards oligosaccharides with K _d values in the micromolar range. This would make mushroom lectins ideal candidates to study protein–carbohydrate interactions. In the present study we investigated the properties of a recombinant form of the mushroom lectin Aleuria aurantia (AAL). AAL is a fucose-binding lectin composed of two identical 312-amino acid subunits. Each subunit contains five binding sites for fucose. We found that one of the binding sites in rAAL had unusually high affinities towards fucose and fucose-containing oligosaccharides with K _d values in the nanomolar range. This site could bind to oligosaccharides with fucose linked α1-2, α1-3 or α1-4, but in contrast to the other binding sites in AAL it could not bind oligosaccharides with α1-6 linked fucose. This binding site is not detected in native AAL (nAAL) one possible explanation may be that this site is blocked with free fucose in nAAL. Recombinant AAL was produced in E. coli as a His-tagged protein, and purified in a one-step procedure. The resulting protein was analyzed by electrophoresis, enzyme-linked lectin assay and circular dichroism spectroscopy, and compared to nAAL. Binding properties were measured using tryptophan fluorescence and surface plasmon resonance. Removal of the His-tag did not alter the binding properties of recombinant AAL in the enzyme-linked lectin assay. Our study forms a basis for understanding the AAL-oligosaccharide interaction and for using molecular techniques to design lectins with novel specificities and high binding affinities towards oligosaccharides.