title: Molecular Cloning, Carbohydrate Specificity and the Crystal Structure of Two Sclerotium rolfsii Lectin Variants.
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.
authors.affiliation: Department of Biochemistry and Biotechnology, University of Thessaly, 26 Ploutonos Street, Larissa 41221, Greece. vikpeppa@gmail.com.
authors.forename: Vassiliki I
authors.initials: VI
authors.lastname: Peppa
authors.affiliation: Department of Biochemistry, Kuvempu University, Shimoga, Karnataka 577451, India. hemalatha.venkat@enzene.com.
authors.forename: Hemalatha
authors.initials: H
authors.lastname: Venkat
authors.affiliation: Department of Biochemistry and Biotechnology, University of Thessaly, 26 Ploutonos Street, Larissa 41221, Greece. natassakan@windowslive.com.
authors.forename: Anastassia L
authors.initials: AL
authors.lastname: Kantsadi
authors.affiliation: Department of Post Graduate Studies in Biochemistry, Karnatak University, Dharwad 580003, India. srinamdar2009@gmail.com.
authors.forename: Shashikala R
authors.initials: SR
authors.lastname: Inamdar
authors.affiliation: Department of Post Graduate Studies in Biochemistry, Karnatak University, Dharwad 580003, India. ganapati.bd@gmail.com.
authors.forename: Ganapati G
authors.initials: GG
authors.lastname: Bhat
authors.affiliation: Department of Post Graduate Studies in Biochemistry, Karnatak University, Dharwad 580003, India. sachinbchem@gmail.com.
authors.forename: Sachin
authors.initials: S
authors.lastname: Eligar
authors.affiliation: Department of Post Graduate Studies in Biochemistry, Karnatak University, Dharwad 580003, India. anupamas.biochem@gmail.com.
authors.forename: Anupama
authors.initials: A
authors.lastname: Shivanand
authors.affiliation: Department of Post Graduate Studies in Biochemistry, Karnatak University, Dharwad 580003, India. vishwanath1014@yahoo.co.in.
authors.forename: Vishwanath B
authors.initials: VB
authors.lastname: Chachadi
authors.affiliation: Department of Biochemistry, Kuvempu University, Shimoga, Karnataka 577451, India. satishlec@gmail.com.
authors.forename: Gonchigar J
authors.initials: GJ
authors.lastname: Satisha
authors.affiliation: Department of Post Graduate Studies in Biochemistry, Karnatak University, Dharwad 580003, India. sawmy_bm@yahoo.co.in.
authors.forename: Bale M
authors.initials: BM
authors.lastname: Swamy
authors.affiliation: Department of Biochemistry and Biotechnology, University of Thessaly, 26 Ploutonos Street, Larissa 41221, Greece. vskamnaki@bio.uth.gr.
authors.forename: Vassiliki T
authors.initials: VT
authors.lastname: Skamnaki
authors.affiliation: Institute of Biology, Medicinal Chemistry & Biotechnology, National Hellenic Research Foundation, 48 Vas. Constantinou Ave, Athens 11635, Greece. sez@eie.gr.
authors.forename: Spyridon E
authors.initials: SE
authors.lastname: Zographos
authors.affiliation: Department of Biochemistry and Biotechnology, University of Thessaly, 26 Ploutonos Street, Larissa 41221, Greece. ddleonidas@bio.uth.gr.
authors.forename: Demetres D
authors.initials: DD
authors.lastname: Leonidas
mesh_terms: D000595:Amino Acid Sequence; D001487:Basidiomycota / Q000235:genetics* / Q000378:metabolism; D050260:Carbohydrate Metabolism; D002241:Carbohydrates / Q000737:chemistry; D003001:Cloning, Molecular; D014644:Genetic Variation; D006860:Hydrogen Bonding; D037102:Lectins / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011134:Polysaccharides / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity
chemical_list: D002241:Carbohydrates; D037102:Lectins; D011134:Polysaccharides
keywords: Sclerotium rolfsii lectin; X-ray crystallography; carbohydrate binding; gene constructs; glycan array; variant forms