Identification of calcium-binding proteins associated with the human sperm plasma membrane

<p>Abstract</p> <p>Background</p> <p>The precise composition of the human sperm plasma membrane, the molecular interactions that define domain specific functions, and the regulation of membrane associated proteins during the capacitation process, still remain to be fully understood. Here, we investigated the repertoire of calcium-regulated proteins associated with the human sperm plasma membrane.</p> <p>Methods</p> <p>Surface specific radioiodination was combined with two-dimensional gel electrophoresis, a 45Ca-overlay assay, computer assisted image analysis and mass spectrometry to identify calcium-binding proteins exposed on the human sperm surface.</p> <p>Results</p> <p>Nine acidic 45Ca-binding sperm proteins were excised from stained preparative 2D gels and identified by mass spectrometry. Five of the calcium binding proteins; HSPA2 (HSP70-1), HSPA5 (Bip), HYOU1 (ORP150), serum amyloid P-component (SAP) and protein kinase C substrate 80K-H (80K-H) were found to be accessible to Iodo-Bead catalyzed 125I-labelling on the surface of intact human sperm. Agglutination and immunofluorescence analysis confirmed that SAP is situated on the plasma membrane of intact, motile sperm as well as permeabilized cells. Western blot analysis showed increased phosphorylation of human sperm 80K-H protein following in vitro capacitation. This is the first demonstration of the 80K-H protein in a mammalian sperm.</p> <p>Conclusion</p> <p>The presence of SAP on the surface of mature sperm implies that SAP has a physiological role in reproduction, which is thought to be in the removal of spermatozoa from the female genital tract via phagocytosis. Since 80K-H is a Ca2+-sensor recently implicated in the regulation of both inositol 1,4,5-trisphosphate receptor and transient receptor potential (TRP) cation channel activities, its detection in sperm represents the first direct signaling link between PKC and store-operated calcium channels identified in human sperm.</p

Combined affinity labelling and mass spectrometry analysis of differential cell surface protein expression in normal and prostate cancer cells

Differences in the expression of cell surface proteins between a normal prostate epithelial (1542-NP2TX) and a prostate cancer cell line (1542-CP3TX) derived from the same patient were investigated. A combination of affinity chromatographic purification of biotin-tagged surface proteins with mass spectrometry analysis identified 26 integral membrane proteins and 14 peripheral surface proteins. The findings confirm earlier reports of altered expression in prostate cancer for several cell surface proteins, including ALCAM/CD166, the Ephrin type A receptor, EGFR and the prostaglandin F2 receptor regulatory protein. In addition, several novel findings of differential expression were made, including the voltage-dependent anion selective channel proteins Porin 1 and 2, ecto-5'-nucleotidase (CD73) and Scavenger receptor B1. Cell surface protein expression changed both qualitatively and quantitatively when the cells were grown in the presence of either or both interferon INFalpha and INFgamma. Costimulation with type I and II interferons had additive or synergistic effects on the membrane density of several, mainly peripherally attached surface proteins. Concerted upregulation of surface exposed antigens may be of benefit in immuno-adjuvant-based treatment of interferon-responsive prostate cancer. In conclusion, this study demonstrates that differences in the expression of membrane proteins between normal and prostate cancer cells are reproducibly detectable following vectorial labelling with biotin, and that detailed analysis of extracellular-induced surface changes can be achieved by combining surface-specific labelling with high-resolution two-dimensional gel electrophoresis and mass spectrometry

Proteomic approaches in the analysis of hypertension

The completion of the genomic sequence and the definition of the genes provide a wealth of data to interpret cellular protein expression patterns and relate them to protein function. Proteomics is the large-scale study of proteins in the post-genomic era, aimed at identifying and characterizing protein expression, function, posttranslational modification, regulation, trafficking, interaction and structure, and their perturbation by disease and drug action. The multigenetic background and essentially unknown etiology of hypertension, makes this main killer a prime candidate for proteomic analysis. The classical proteomic approaches are based on two-dimensional gel electrophoretic protein separation and their subsequent identification and characterization by mass spectrometry analysis. However, expression level analysis may not reflect the functional state of proteins and is biased towards long-lived abundant proteins. This review describes a variety of techniques that can be used to identify low-abundance proteins that may be of more functional interest. The modification of classical two-dimensional electrophoresis in order to study post-translational modifications, e.g., phosphorylation, is also discussed

Proteomic approaches in the analysis of hypertension

The completion of the genomic sequence and the definition of the genes provide a wealth of data to interpret cellular protein expression patterns and relate them to protein function. Proteomics is the large-scale study of proteins in the post-genomic era, aimed at identifying and characterizing protein expression, function, posttranslational modification, regulation, trafficking, interaction and structure, and their perturbation by disease and drug action. The multigenetic background and essentially unknown etiology of hypertension, makes this main killer a prime candidate for proteomic analysis. The classical proteomic approaches are based on two-dimensional gel electrophoretic protein separation and their subsequent identification and characterization by mass spectrometry analysis. However, expression level analysis may not reflect the functional state of proteins and is biased towards long-lived abundant proteins. This review describes a variety of techniques that can be used to identify low-abundance proteins that may be of more functional interest. The modification of classical two-dimensional electrophoresis in order to study post-translational modifications, e.g., phosphorylation, is also discussed