ORIGINAL PAPER Purification and characterization of a mucin-binding mycelial lectin from Aspergillus nidulans with potent mitogenic activity Ram Sarup Singh • Ranjeeta Bhari • Jatinder Singh • Ashok Kumar Tiwary Received: 18 March 2010 / Accepted: 17 June 2010 / Published online: 26 June 2010 Ó Springer Science+Business Media B.V. 2010 Abstract Mucin-specific lectin from mycelium of Aspergillus nidulans was purified using anion exchange and gel filtration chromatographic techniques with an overall recovery of 32% and 21.97-fold purification. The purified lectin migrated as a single band in SDS–PAGE with an apparent molecular mass of 34 kDa. Carbohydrate analysis revealed that it is a glycoprotein with total sugar content of 2.54%. Optimal agglutination was observed when serially diluted lectin was incubated with human type O erythrocyte suspension at pH 7.0–8.0 and temperature 20–30°C. Lectin was found to be completely stable within pH 5.0–8.0 and temperature at or below 40°C. Demetalli- zation by extensive dialysis against EDTA did not alter its haemagglutination activity. Lectin activity was reduced to half after 24 h incubation with urea and thiourea, with no such effect of guanidine HCl. The lectin showed potent mitogenic response towards mouse splenocytes, attaining a maximum at 200 lg/ml as compared to untreated control cells. Mitogenic lectins are invaluable tools to assess the functioning of immune cells. None of the microfungal lectin has yet been investigated for mitogenic activity. This is the first report on mitogenic activity of lectin from Aspergillus sp. Keywords Aspergillus nidulans Á Lectin Á Mucin-binding Á Purification Á Haemagglutination Á Mitogenic potential Introduction Cell surfaces of organisms bear abundant carbohydrates which have potential roles in biological recognition mechanisms, involving cell–cell interactions and cell-sub- strate interactions. Lectins are ubiquitous carbohydrate binding proteins that have been isolated from a wide range of organisms including plants, animals and microbes (Singh et al. 1999). Owing to their defined carbohydrate specificity, these molecules have gained recognition as specific probes for investigating the distribution, structure and biological functions of carbohydrate chains on cell surfaces. Lectins have been reported as chemotherapeutic agents, as mitogens and as site-specific drug delivery agents (Tiwary and Singh 1998, 1999). Earlier, most of the research had been focused on plant and animal lectins while during the past few years, there had been mounting interest in fungal lectins due to their poten- tially exploitable therapeutic applications. Biomedical applications of lectins from higher fungi (Singh et al. 2010b) and current trends in lectins from microfungi (Singh et al. 2010a) have been reviewed recently by our group. Lower fungi displaying lectin activity include Rhizoctonia solani (Candy et al. 2001), Arthrobotrys oligospora (Rosen et al. 1992), Sclerotium rolfsii (Wu et al. 2001), Sclerotinia sclerotiorum (Candy et al. 2003), Penicillium marneffei (Hamilton et al. 1998), P. thomii, P. griseofulvum (Singh et al. 2009a), Fusarium solani (Khan et al. 2007), Aspergillus oryzae (Ishimaru et al. 1996) and A. fumigatus (Tronchin et al. 2002). Matsumura et al. (2007) have demonstrated R. S. Singh (&) Á R. Bhari Carbohydrate and Protein Biotechnology Laboratory, Department of Biotechnology, Punjabi University, Patiala, Punjab 147 002, India e-mail: rssingh11@lycos.com J. Singh Department of Molecular Biology and Biochemistry, Guru Nanak Dev University, Amritsar, Punjab 143 005, India A. K. Tiwary Department of Pharmaceutical Sciences and Drug Research, Punjabi University, Patiala, Punjab 147 002, India 123 World J Microbiol Biotechnol (2011) 27:547–554 DOI 10.1007/s11274-010-0488-2