Markiv, Anatoliy and Peiris, Diluka and Curley, G. Paul and Odell, Mark and Dwek, Miriam (2011) Identification, cloning and characterization of two N-acetylgalactosamine binding lectins from the albumen gland of Helix pomatia. Journal of Biological Chemistry, 286 (23). pp. 20260-20266. ISSN 0021-9258
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Official URL: http://dx.doi.org/10.1074/jbc.M110.184515
Helix pomatia agglutinin (HPA), the lectin from the albumen gland of the Roman snail, has been used in histochemical studies relating glycosylation changes to the metastatic potential of solid tumors. To facilitate the use of HPA in a clinical (diagnostic) setting, detailed analysis of the lectin, including cloning and recombinant production of HPA is required. A combination of isoelectric focusing, amino acid sequence analysis and cloning revealed two polypeptides in native HPA preparations (HPAI and HPAII) both consistent with GalNAc binding lectins of the H-type family. Pairwise sequence alignment showed that HPAI and HPAII share 54% sequence identity while molecular modelling using SWISS-MODEL suggest they are likely to adopt similar tertiary structure. The inherent heterogeneity of native HPA highlighted the need for production of functional recombinant protein; this was addressed by preparing His-Trx tagged fusion products in Escherichia coli Rosetta-gami B (DE3) cells. The recombinant lectins agglutinated human blood group A erythrocytes while their oligosaccharide specificity, evaluated using glycan microarrays, showed they predominantly bind glycans with terminal alpha-GalNAc residues. Surface plasmon resonance with immobilized GalNAc-BSA confirmed that recombinant HPAI and HPAII bind strongly with this ligand (Kd = 0.60 nM and 2.00 nM, respectively) with a somewhat higher affinity to native HPA (Kd = 7.67 nM). Recombinant HPAII also bound the breast cancer cells of breast cancer tissue specimens in a similar manner to native lectin. The recombinant HPA described here shows important potential for future studies of cancer cell glycosylation and as a reagent for cancer prognostication.
|Research Community:||University of Westminster > Life Sciences, School of|
|Deposited On:||14 Mar 2011 10:29|
|Last Modified:||18 Oct 2012 11:39|
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