Structural characterization and biological properties of human gastrokine 1

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.

Anti-amyloidogenic property of human gastrokine 1

“…To accomplish this deficiency, we have recently reported the characterization and secondary structural properties of human recombinant GKN1 (rGKN1). Using bio-informatics tools, we found that GKN1 BRICHOS domain has structural features similar to those endowed by BRI-CHOS domain-containing protein family [8]. In particular, predicted GKN1 3D models suggested a structural organization of its BRICHOS domain resembling that of the corresponding BRICHOS domain of prosurfactant protein C (proSP-C), a trans membrane (TM) protein expressed in epithelial type II cells.…”

“…Human GKN1 is made of 185 amino acids containing a 20 amino acid extracellular signal peptide localized in the N-terminal region [1]. The protein is characterized by the presence of a central structural BRICHOS domain [7] of about 100 amino acids containing two conservative cysteine residues most likely involved in disulfide bridges [8]. The association of GKN1 BRICHOS domain with biological functions has been proposed but not yet ascertained.…”

Human Gastrokine 1 and its anti-amyloidogenic properties

“…In addition, also the 3D model of GKN1 BRICHOS domain constructed at Swiss Model Server 11 shows a structural organization approaching that of the corresponding domain of proSP-C 13 . Both ProSP-C and BRI2 are membrane proteins containing a region with a tendency to form β-sheet structures that can be proteolytically cleaved with mechanisms still unclear.…”

“…The first two conserved cysteines (C 95 and C 156 ), are present in all BRICHOS domains, whereas the other two cysteines (C 179 and C 193 ), are conserved in all other mammalian gastrokines. In the case of human GKN1, the two latter cysteines are also involved in disulfide bridge as determined by mass spectrometry (MS) performed on rGKN1 11 ( Figure. 2).…”

“…Over-expression of GKN1 in gastric adenocarcinoma cell lines (AGS and MKN28) activates the expression of Fas receptor, while treatment with an antiFas antibody appreciably increases apoptosis 10 . Moreover, treatment of tumor cells with recombinant human GKN1 (rGKN1) reduceds the proliferation of AGS cells compared with human embryonic kidney cells (HEK 293) and nongastric cancer cells (H1355) 11 . These findings denotes that GKN1 is a tumor-suppressor and a modulator of apoptotic signals in GC.…”