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Molecules 2015, 20(6), 10848-10865; doi:10.3390/molecules200610848

Molecular Cloning, Carbohydrate Specificity and the Crystal Structure of Two Sclerotium rolfsii Lectin Variants

1
Department of Biochemistry and Biotechnology, University of Thessaly, 26 Ploutonos Street, Larissa 41221, Greece
2
Department of Biochemistry, Kuvempu University, Shimoga, Karnataka 577451, India
3
Department of Post Graduate Studies in Biochemistry, Karnatak University, Dharwad 580003, India
4
Institute of Biology, Medicinal Chemistry & Biotechnology, National Hellenic Research Foundation, 48 Vas. Constantinou Ave, Athens 11635, Greece
These authors contributed equally to this work.
*
Authors to whom correspondence should be addressed.
Academic Editor: Els Van Damme
Received: 31 March 2015 / Revised: 3 June 2015 / Accepted: 5 June 2015 / Published: 12 June 2015
(This article belongs to the Special Issue Protein-Carbohydrate Interactions, and Beyond)
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Abstract

SRL is a cell wall associated developmental-stage specific lectin secreted by Sclerotium rolfsii, a soil-born pathogenic fungus. SRL displays specificity for TF antigen (Galβ1→3GalNAc-α-Ser//Thr) expressed in all cancer types and has tumour suppressing effects in vivo. Considering the immense potential of SRL in cancer research, we have generated two variant gene constructs of SRL and expressed in E. coli to refine the sugar specificity and solubility by altering the surface charge. SSR1 and SSR2 are two different recombinant variants of SRL, both of which recognize TF antigen but only SSR1 binds to Tn antigen (GalNAcα-Ser/Thr). The glycan array analysis of the variants demonstrated that SSR1 recognizes TF antigen and their derivative with high affinity similar to SRL but showed highest affinity towards the sialylated Tn antigen, unlike SRL. The carbohydrate binding property of SSR2 remains unaltered compared to SRL. The crystal structures of the two variants were determined in free form and in complex with N-acetylglucosamine at 1.7 Å and 1.6 Å resolution, respectively. Structural analysis highlighted the structural basis of the fine carbohydrate specificity of the two SRL variants and results are in agreement with glycan array analysis. View Full-Text
Keywords: Sclerotium rolfsii lectin; variant forms; gene constructs; glycan array; carbohydrate binding; X-ray crystallography Sclerotium rolfsii lectin; variant forms; gene constructs; glycan array; carbohydrate binding; X-ray crystallography
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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Peppa, V.I.; Venkat, H.; Kantsadi, A.L.; Inamdar, S.R.; Bhat, G.G.; Eligar, S.; Shivanand, A.; Chachadi, V.B.; Satisha, G.J.; Swamy, B.M.; Skamnaki, V.T.; Zographos, S.E.; Leonidas, D.D. Molecular Cloning, Carbohydrate Specificity and the Crystal Structure of Two Sclerotium rolfsii Lectin Variants. Molecules 2015, 20, 10848-10865.

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