Studies of recombinant forms of Aleuria aurantia lectin
2009 (English)Licentiate thesis, comprehensive summary (Other academic)
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.
Linköping Studies in Health Sciences. Thesis, ISSN 1100-6013 ; 105
Cell and Molecular Biology
IdentifiersURN: urn:nbn:se:liu:diva-20311ISBN: 978-91-7393-558-6OAI: oai:DiVA.org:liu-20311DiVA: diva2:233897
2009-09-01, Elsa Bränströmssalen, Hälsouniversitetet, Campus US, Linköpings Universitet, Linköping, 09:00 (English)
Konradsson, Peter, ProfessorLarsson, Anders, Professor
Påhlsson, Peter, Professor
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