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Production and characterization of a monomeric form and a single-site form of Aleuria aurantia lectin
Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. 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 Science and Technology, Media and Information Technology. Linköping University, The Institute of Technology.ORCID iD: 0000-0002-4694-5611
Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
2011 (English)In: Glycobiology, ISSN 0959-6658, E-ISSN 1460-2423, Vol. 21, no 1, 34-44 p.Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
Oxford University Press , 2011. Vol. 21, no 1, 34-44 p.
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:liu:diva-20310DOI: 10.1093/glycob/cwq129ISI: 000285193100005OAI: oai:DiVA.org:liu-20310DiVA: diva2:233891
Available from: 2009-09-03 Created: 2009-09-03 Last updated: 2016-05-04
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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