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Detection of a high affinity binding site in recombinantAleuria aurantia lectin
Linköping University, Department of Clinical and Experimental Medicine. Linköping University, Faculty of Health Sciences.
Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Linköping University, The Institute of Technology.
Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.ORCID iD: 0000-0002-4694-5611
Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
2008 (English)In: Glycoconjugate Journal, ISSN 0282-0080, E-ISSN 1573-4986, Vol. 25, no 8, 753-762 p.Article in journal (Refereed) Published
Abstract [en]

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 Kd 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 fucosecontaining oligosaccharides with Kd 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.

Place, publisher, year, edition, pages
Springer Netherlands , 2008. Vol. 25, no 8, 753-762 p.
Keyword [en]
Aleuria aurantia lectin, Fucose, Surface plasmon resonance, Tryptophan fluorescence, Oligosaccharides
National Category
Cell and Molecular Biology
Identifiers
URN: urn:nbn:se:liu:diva-20306DOI: 10.1007/s10719-008-9135-7OAI: oai:DiVA.org:liu-20306DiVA: diva2:233889
Projects
Studies of recombinant forms of Aleuria aurantia lectin
Available from: 2009-09-03 Created: 2009-09-03 Last updated: 2017-12-13Bibliographically approved
In thesis
1. Studies of recombinant forms of Aleuria aurantia lectin
Open this publication in new window or tab >>Studies of recombinant forms of Aleuria aurantia lectin
2009 (English)Licentiate thesis, comprehensive summary (Other academic)
Abstract [en]

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.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2009. 38 p.
Series
Linköping Studies in Health Sciences. Thesis, ISSN 1100-6013 ; 105
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:liu:diva-20311 (URN)978-91-7393-558-6 (ISBN)
Presentation
2009-09-01, Elsa Bränströmssalen, Hälsouniversitetet, Campus US, Linköpings Universitet, Linköping, 09:00 (English)
Opponent
Supervisors
Available from: 2009-09-18 Created: 2009-09-03 Last updated: 2009-09-18Bibliographically approved

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Olausson, JohanJonsson, Bengt-HaraldTibell, LenaPåhlsson, Peter

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