Structural characterization and biological properties of human gastrokine 1

Luigi Michele Pavone; Pompea Del Vecchio; Paolo Mallardo; Filomena Altieri; Valeria De Pasquale; Silviana Rea; Nicola M. Martucci; Chiara Stella Di Stadio; Pietro Pucci; Angela Flagiello; Mariorosario Masullo; Paolo Arcari; Emilia Rippa

doi:10.1039/C2MB25308A

Structural characterization and biological properties of human gastrokine 1†

Luigi Michele Pavone,^ab Pompea Del Vecchio,*^c Paolo Mallardo,^a Filomena Altieri,^a Valeria De Pasquale,^a Silviana Rea,^a Nicola M. Martucci,^ad Chiara Stella Di Stadio,^a Pietro Pucci,^bc Angela Flagiello,^bc Mariorosario Masullo,^ad Paolo Arcari*^ab and Emilia Rippa^a

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* Corresponding authors

^a Department of Biochemistry and Medical Biotechnology, University of Naples Federico II, Via S. Pansini 5, 80131 Naples, Italy
E-mail: arcari@unina.it
Tel: +39-081-746-3120

^b CEINGE Advanced Biotechnology scarl, Via Gaetano Salvatore 486, 80145 Naples, Italy

^c Department of Chemical Sciences, University of Naples Federico II, Via Cintia, 80126 Naples, Italy
E-mail: pompea.delvecchio@unina.it
Tel: +39-081-674-254

^d Department of DiSIST, University of Naples “Parthenope”, Via Medina 40, Naples, Italy

Abstract

Gastrokine-1 (GKN1), a protein expressed in normal gastric tissue, but absent in gastric cancer tissues and derived cell lines, has recently emerged as a potential biomarker for gastric cancer. To better establish the molecular properties of GKN1, the first protocol for the production of mature human GKN1 in the expression system of Pichia pastoris was settled. The recombinant protein showed anti-proliferative properties specifically on gastric cancer cell lines thus indicating that it was properly folded. Characterization of structural and biochemical properties of recombinant GKN1 was achieved by limited proteolysis analysis, circular dichroism and fluorescence spectroscopy. The analysis of GKN1 primary structure coupled to proteolytic experiments highlighted that GKN1 was essentially resistant to proteolytic enzymes and showed the presence of at least a disulphide bond between Cys61 and one of the other three Cys (Cys122, Cys145 and Cys159) of the molecule. The secondary structure analysis revealed a prevailing β-structure. Spectroscopic and calorimetric investigations on GKN1 thermal denaturation pointed out its high thermal stability and suggested a more complex than a two-state unfolding process. The resulting protein was endowed with a globular structure characterized by domains showing different stabilities toward chemical and physical denaturants. These results are in agreement with the prediction of GKN1 secondary structure and a three-dimensional structure model. Our findings provide the basis for the development of new pharmaceutical compounds of potential use for gastric cancer therapy.

Molecular BioSystems

Structural characterization and biological properties of human gastrokine 1†

Abstract

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