Abstract
BackgroundS100A4 is a metastasis-associated protein which has been linked to multiple cellular events, and has been identified extracellularly, in the cytoplasm and in the nucleus of tumor cells; however, the biological implications of subcellular location are unknown. Associations between a variety of posttranslational protein modifications and altered biological functions of proteins are becoming increasingly evident. Identification and characterization of posttranslationally modified S100A4 variants could thus contribute to elucidating the mechanisms for the many cellular functions that have been reported for this protein, and might eventually lead to the identification of novel drugable targets.MethodsS100A4 was immuoprecipitated from a panel of in vitro and in vivo sources using a monoclonal antibody and the samples were separated by 2D-PAGE. Gels were analyzed by western blot and silver staining, and subsequently, several of the observed spots were identified as S100A4 by the use of MALDI-TOF and MALDI-TOF/TOF.ResultsA characteristic pattern of spots was observed when S100A4 was separated by 2D-PAGE suggesting the presence of at least three charge variants. These charge variants were verified as S100A4 both by western immunoblotting and mass spectrometry, and almost identical patterns were observed in samples from different tissues and subcellular compartments. Interestingly, recombinant S100A4 displayed a similar pattern on 2D-PAGE, but with different quantitative distribution between the observed spots.ConclusionEndogenously expressed S100A4 were shown to exist in several charge variants, which indicates the presence of posttranslational modifications altering the net charge of the protein. The different variants were present in all subcellular compartments and tissues/cell lines examined, suggesting that the described charge variants is a universal phenomenon, and cannot explain the localization of S100A4 in different subcellular compartments. However, the identity of the specific posttranslational modification and its potential contribution to the many reported biological events induced by S100A4, are subject to further studies.
Highlights
S100A4 is a metastasis-associated protein which has been linked to multiple cellular events, and has been identified extracellularly, in the cytoplasm and in the nucleus of tumor cells; the biological implications of subcellular location are unknown
Characteristic distribution pattern of human S100A4 when separated by 2D-PAGE A characteristic pattern of spots was observed when S100A4 immunoprecipitated from SW620 cell lysate was separated by 2D-PAGE, blotted and stained with antiS100A4 (Fig. 1)
In the present work we have demonstrated that endogenous human S100A4 distributes in a characteristic pattern when separated by 2D-PAGE, and irrespective of source or subcellular compartment used for immunoisolation of the protein, the observed pattern was almost identical
Summary
S100A4 is a metastasis-associated protein which has been linked to multiple cellular events, and has been identified extracellularly, in the cytoplasm and in the nucleus of tumor cells; the biological implications of subcellular location are unknown. S100A4 is a small (approximately 12 kDa) acidic calciumbinding protein that has been associated with a range of biological functions, such as cell migration, invasion and angiogenesis, potentially contributing to higher metastatic capacity of tumor cells [1,2,3,4]. The human variant of S100A4 consists of 101 amino acids and is characterized by two calcium-binding EF-hands connected by an intermediate region referred to as the hinge region, and a distinct C-terminal extension. The S100 protein family shows a high degree of sequence homology, especially in the calcium binding EF-hand regions, while the composition of the C-terminal extension and the hinge region is more diversified and characterize each member [6,7]. Upon calcium binding the homodimer undergo a conformational change that leads to exposure of the hydrophobic regions in the C-terminal end, initially buried in the complex [8]
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