Book of abstract 2008

Antiproliferative and adhesion-stimulating effects on human cancer cell
lines elicited by lectins from mushroom Clitocybe nebularis
Jure Pohleven1 ,2, Nataša Obermajer2, Jerica Sabotič1, Bogdan Kralj3, Janko Kos2, Borut
Štrukelj1 ,2, Jože Brzin1
1Dept. of Biotechnology, Jožef Stefan Institute, Jamova 39, SI-1000 Ljubljana, Slovenia; 2Dept. of
Pharmaceutical Biology, Faculty of Pharmacy, University of Ljubljana, Aškerčeva 7, SI-1000 Ljubljana,
Slovenia; 3Mass Spectrometry Centre, Dept. of Environmental Chemistry, Jožef Stefan Institute, Jamova
39, SI-1000 Ljubljana, Slovenia
Fungi are well known for their medicinal values and numerous bioactive compounds with
pharmacological properties have been isolated from mushrooms. Lectins are a diverse
and ubiquitous group of non-homologous proteins that specifically bind different types
of carbohydrates. Most lectins are multivalent, containing more than one carbohydratebinding
site; therefore they can cross-link cell surface carbohydrates and agglutinate cells.
Their biological activity is exerted through binding to glycoconjugates on cell surfaces,
extracellular matrix and cytoplasmic or nuclear glycoproteins. Lectins elicit a variety of
effects in different organisms and are suggested to have many biological functions which
are still not fully understood.
In this study, several lectins were isolated from fruiting bodies of mycorrhizal basidiomycete
fungus Clouded agaric (Clitocybe nebularis) by affinity chromatography using carbohydrates
(glucose, galactose, sucrose, lactose) as ligands. Lectins were purified using HPLC and
their molecular masses were determined by mass spectrometry. They were shown to
be oligomeric by gel filtration and native gel electrophoresis. Hemagglutination and
hemagglutination inhibition assays with human type A, B, 0, AB and bovine erythrocytes
were performed to determine lectins specificity and they were shown to be pH and heat
stable. Their N-terminal and internal amino acid sequences were determined by protein
sequencing and degenerative PCR primers were designed to obtain genomic and cDNA
p38
nucleotide sequences using genome walking and reverse-transcription PCR as well as
rapid amplification of cDNA ends methods. Deduced amino acid sequence was compared
to protein sequences in databases. Effects on proliferation and adhesion were studied on
human breast epithelial tumour cell line (MCF-10A NeoT), human T leukaemia cell
line (Mo-T), differentiated and undifferentiated human monocytic lymphoma cell line
(U-937).
From among isolated proteins, lactose-specific and galactose-specific lectins showed the
most potent effects on human cancer cell lines. Galactose-specific lectins stimulated plastic
adherence of differentiated U-937 cells, suggesting that they interact with integrins and
activate integrin-mediated adhesion. Lactose-specific lectin exerted antiproliferative effect
on human T leukaemia cell line. This lectin has a potential use in treating haematopoietic
malignances so complete genomic and cDNA sequences were obtained. The gene consists
of five exons and four introns. Comparing a deduced amino acid sequence to protein
databases using BLASTP, SMART and Pfam tools revealed that lactose-specific lectin
belongs to a ricin B-like lectin protein family.
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