Comparative proteomic analysis of spermatozoa isolated by swim-up or density gradient centrifugation.

BACKGROUND: Reports about the morphologic and functional characteristics of spermatozoa prepared by density gradient centrifugation (DC) or swim-up (SU) have produced discordant results. We have performed a proteomic comparison of cells prepared by DC and SU providing a molecular insight into the differences between these two methods of sperm cell isolation. METHODS: Protein maps were obtained by 2-dimensional (2-D) separations consisting of isoelectrofocusing (IEF) from pI 3 to 11 followed by SDS-polyacrylamide gel electrophoresis. 2-D gels were stained with Sypro Ruby. Map images of DC and SU spermatozoa were compared using dedicated software. Intensities of a given spot were considered different between DC and SU when their group mean differed by >1.5-fold (p<0.05, Anova). RESULTS: No differences were observed for 853 spots, indicating a 98.7% similarity between DC and SU. Five spots were DC>SU and 1 was SU>DC. Proteins present in 3 of the differential spots could be identified. One DC>SU spot contained lactate dehydrogenase C and gamma-glutamylhydrolase, a second DC>SU spot contained fumarate hydratase and glyceraldehyde-3-phosphate dehydrogenase-2, and a SU>DC spot contained pyruvate kinase M1/M2. CONCLUSIONS: The differences in protein levels found on comparison of DC with SU spermatozoa indicate possible dissimilarities in their glycolytic metabolism and DNA methylation and suggest that DC cells may have a better capacitation potential.

Comparative analysis of the seminal plasma proteomes of oligoasthenozoospermic and normozoospermic men.

A comparative proteomic study of oligoasthenozoospermic and normozoospermic seminal plasmas was conducted to establish differences in protein expression. Oligoasthenozoospermia (when semen presents with a low concentration and reduced motility of spermatozoa) is common in male infertility. Two-dimensional protein maps from seminal plasma samples from 10 men with normozoospermia and 10 men with idiopathic oligoasthenozoospermia were obtained by isoelectric focusing followed by sodium dodecyl-sulphate polyacrylamide electrophoresis. Map images were analysed using dedicated software involving normalization, spot-to-spot volume comparison and statistical treatment of the results to establish the significance of differences between normal and oligoasthenozoospermic samples. Six out of 1028 spots showed over 1.5-fold relative intensity differences (P < 0.05, analysis of variance). Four proteins were identified by nano liquid chromatography-electrospray ionization-mass spectrometry/mass spectrometry of their tryptic peptides and database searches. Two proteins were more than three-fold under-expressed in oligoasthenozoospermia, namely epididymal secretory protein E1 and galectin-3-binding protein; the other (lipocalin-1 and a prolactin-inducible protein form) were over-expressed. The identity and differential expression of epididymal secretory protein E1 was verified by Western-blotting. The statistically significant differential expression of these four proteins in oligoasthenozoospermia compared with normozoospermia provides a molecular basis for further investigations into the pathogenic mechanisms underlying idiopathic oligoasthenozoospermia.

Extracellular Vesicles in Mycobacterial Infections: Their Potential as Molecule Transfer Vectors.

Extracellular vesicles are membrane-bound structures released by living cells and present in body fluids. Their composition includes proteins, lipids, carbohydrates, and nucleic acids and are involved in transfers between cells. Extracellular vesicles can deliver molecules to cells and tissues even if distant. As a consequence, they have a role in information transmission and in the modulation of the biological function of recipient cells. Among other things, they are involved in antigen presentation and the induction of secretion events by immune cells. Thus, extracellular vesicles participate in the regulation of immune responses during infections. We will discuss their potential as effectors and disease biomarkers concerning only mycobacterial infections.

Mycobacteria-containing phagosomes associate less annexins I, VI, VII and XI, but not II, concomitantly with a diminished phagolysosomal fusion.

We have studied the intracellular localization of annexins I,II, VI, VII, and XI in cells containing latex beads or Mycobacterium avium at different times after ingestion in order to establish whether a correlation existed between the association of annexins to phagosomes and phagolysosomal fusion, since the intracellular survival of mycobacteria is linked to an impairment of phagosome maturation. We demonstrate an important decrease in the levels of association of annexins I, VI, VII and XI, but not II to phagosomes containing either live or killed mycobacteria compared with phagosomes containing inert latex particles. The reduced association of annexins observed was detected only on M. avium-containing phagosomes and not in other cell membrane nor in cytosolic fractions from infected cells, and was apparent from 8 hours through to 4 days after phagocytosis. These findings add elements to the present knowledge of the phagosomal modifications that accompany the survival of intracellular pathogens, suggesting that annexins I, VI, VII, and XI play a secondary role in phagosomal fusion events while annexin II does not seem to be related to the mechanism of regulation of endolysosomal fusion.

An extragranular compartment of blood eosinophils contains eosinophil protein X/eosinophil-derived neurotoxin (EPX/EDN).

Serum and plasma profiles of eosinophil protein X (EPX/EDN) and those of other eosinophil proteins differ in various conditions, suggesting a different mobilisation from storage granules. This work studied the subcellular localisation of EPX/EDN in non-primed and in vivo primed blood eosinophils from healthy and allergic subjects, during and out of the pollen season. Primed eosinophils contain easily mobilisable secretory proteins. By fractionation on sucrose density gradients, EPX/EDN localised in the specific granules as well as in a cytoplasmic extra-granular compartment of low equilibrium density that partially overlapped with vesicular structures, cytosolic proteins and plasma membranes. This compartment was clearly separate from the low density peak of ECP that increases during the pollen season. There were no significant differences in the amounts of EPX/EDN present in the low density peak of healthy and allergic subjects. Immuno-gold labelling electron microscopy showed EPX/EDN in specific granules, cytoplasm and associated to plasma membranes. In conclusion, substantial amounts of EPX/EDN localise in an extra-granular, low equilibrium density compartment of human eosinophils.

A proteomic insight into the effects of the immunomodulatory hydroxynaphthoquinone lapachol on activated macrophages.

We report the effect of an immunomodulatory and anti-mycobacterial naphthoquinone, lapachol, on the bi-dimensional patterns of protein expression of toll-like receptor 2 (TLR2)-agonised and IFN-γ-treated THP-1 macrophages. This non-hypothesis driven proteomic analysis intends to shed light on the cellular functions lapachol may be affecting. Proteins of both cytosol and membrane fractions were analysed. After quantification of the protein spots, the protein levels corresponding to macrophages activated in the absence or presence of lapachol were compared. A number of proteins were identified, the levels of which were appreciably and significantly increased or decreased as a result of the action of lapachol on the activated macrophages: cofilin-1, fascin, plastin-2, glucose-6-P-dehydrogenase, adenylyl cyclase-associated protein 1, pyruvate kinase, sentrin-specific protease 6, cathepsin B, cathepsin D, cytosolic aminopeptidase, proteasome ß type-4 protease, tryptophan-tRNA ligase, DnaJ homolog and protein disulphide isomerase. Altogether, the comparative analysis performed indicates that lapachol could be hypothetically causing an impairment of cell migration and/or phagocytic capacity, an increase in NADPH availability, a decrease in pyruvate concentration, protection from proteosomal protein degradation, a decrease in lysosomal protein degradation, an impairment of cytosolic peptide generation, and an interference with NOS2 activation and grp78 function. The present proteomic results suggest issues that should be experimentally addressed ex- and in-vivo, to establish more accurately the potential of lapachol as an anti-infective drug. This study also constitutes a model for the pre-in-vivo evaluation of drug actions.

The hydroxy-naphthoquinone lapachol arrests mycobacterial growth and immunomodulates host macrophages.

The present study reports the anti-mycobacterial activity of 2-hydroxy-3-(3-methyl-2-butenyl)-1,4-naphthoquinone (lapachol) as well as its influence on macrophage functions. Lapachol (L) did not induce apoptosis/necrosis of THP-1 macrophages at ≤32 µg/mL. Mycobacterium avium liquid growth was arrested by ≥32 µg/mL and intra-macrophage proliferation by ≥16 µg/mL lapachol. The main immuno-modulatory effects of lapachol observed were an up-regulation of interferon-γ-receptor 1 (IFN-γR1) and major histocompatibility complex class II (MHCII) surface expression, and a marked inhibition of IL-10 secretion. Lapachol did not affect resting, IFN-γ- or toll-like receptor 2 (TLR2)-induced levels of oxygen and nitrogen metabolism key proteins nor the TLR2-mediated secretion of TNF-α, nor induced either oxidative or endoplasmic reticulum (ER) stress. Lapachol inhibited the surface expression of the co-stimulatory molecule CD86 but not that of CD80 and CD83. The results obtained indicate that the substituted naphthoquinone lapachol exhibits an anti-mycobacterial activity that is more efficient intra- than extra-cellularly, and exerts immuno-modulatory effects some of which may enhance the capacity of the host cell to control mycobacterial growth. The immune-modulatory action of lapachol could contribute to its more efficient intra-macrophage anti-mycobacterial activity.

The human eosinophil proteome. Changes induced by birch pollen allergy.

Proteins from human eosinophils were separated bidimensionally and identified by mass spectrometry (336 spots/bands, 98 different proteins). Of these, 24.7% belonged to the cytoskeleton/migration group. Highly basic proteins (11.3%) were concentrated in the granule-containing cell fraction. We detected novel hyperacidic forms of cofilin-1, profilin-1 and adenylyl cyclase-associated protein, and hyperbasic forms of eosinophil-derived neurotoxin/eosinophil protein X and major basic protein homologue. We also found evidence of the triglycosylation of the heavy chain of eosinophil peroxidase. In addition, through comparative 2D image analysis, spot quantification and MS, it was found that hsc70, actin-capping protein and hyperacidic forms of eosinophil peroxidase heavy chain are overexpressed in cells from birch pollen allergic subjects, at the peak of a season. The link between these findings and an increased cellular antigen-presenting capacity and motility are discussed.

Proteomic analysis of human cervical-vaginal fluids.

The pathophysiology of vaginal conditions is still ill-defined at a molecular level. Because the proteome of the human cervical-vaginal fluid (CVF) has not been reported to date, we undertook the identification of proteins present in the cell-free fraction of these fluids. Proteins were separated bidimensionally (2-D) by isoelectrofocusing (pH 3-11) followed by SDS-polyacrylamide electrophoresis. The proteins of 147 spots were identified by matrix-assisted laser desorption/ ionization-time-of-flight-mass spectrometry (MALDI-TOF/TOF). This approach was supplemented by immunoassays for markers of neutrophils (myeloperoxidase, MPO; neutrophil gelatinase-associated lipocalin, NGAL/HNL) and eosinophils (eosinophil cationic protein: ECP) and by immunoblotting (lactoferrin, calgranulins A and B and annexins A1 and A3. Nearly half of the proteins (69/147) and protein fragments detected were found to be plasma components, on the basis of which the human CVF can be broadly considered a plasma transudate. Although the pattern of protein spots was very similar for all fluids analyzed, a relative overabundance of major plasma proteins such as albumin, transferrin, immunoglobulins, apolipoproteins, alpha-1-acid glycoprotein 1, and calgranulins was associated with the presence of a high number of polymorphonuclear leukocytes in the lavages from which those cell-free fluids had been obtained. Instead, fluids from women experiencing vulvovaginal candidiasis did not show differences in the protein maps compared with asymptomatic individuals. Neutrophil and eosinophil granule secretion proteins were also detected in variable amounts in the lavage fluids by both immunoassay and immunoblotting, indicating polymorphonuclear cell activation.

Complement receptor 3 binds the Borrelia burgdorferi outer surface proteins OspA and OspB in an iC3b-independent manner.

Persistence of borreliae within the vertebrate host depends on the fate of interactions between the spirochetes and target cells. The present work demonstrates the direct binding of the Borrelia burgdorferi outer surface proteins OspA and OspB to CR3 and that this binding is independent of iC3b.

Determination of a large number of kinase activities using peptide substrates, P81 phosphocellulose paper arrays and phosphor imaging.

To perform phosphoproteomics and signal transduction studies, a number of protein kinase activities and levels must be simultaneously analyzed in different cell samples and correlated with phosphoprotein patterns to obtain conclusions with regard to the regulation of kinase networks. We describe here a miniaturized format of the classical phosphocellulose (P81) paper binding assay with which up to 594 kinase reactions can be simultaneously analyzed. Kinase peptide substrates possessing a minimum of three consecutive basic residues were subjected to phosphorylation in 96-well plates and aliquots of the phosphorylation reactions were spotted on arrays printed on P81 papers. Phosphorylation levels were quantified using a storage phosphor system imager. The versatility of the procedure was validated by analyzing casein kinase 2, protein kinase C, and p34cdc2/cyclin B in cell extracts and testing the effect of known inhibitors and activators on kinase activities. This improved, miniaturized version of the classical P81 paper method combines simplicity, high sensitivity, high reproducibility, high reliability, and optimal Z factors and takes into account possible sources of background signals. We discuss the possibility of automation and the advantages over other methods.

Priming of eosinophil migration across lung epithelial cell monolayers and upregulation of CD11b/CD18 are elicited by extracellular Ca2+.

In patients with asthma, eosinophils are primed and massively infiltrate lung tissues and migrate across epithelia into airways. Using blocking monoclonal antibodies, we found that eosinophil transmigration across a lung epithelial cell monolayer depended on the functions of alphaMbeta2 integrin CD11b/CD18. To study the role of Ca2+ in eosinophil priming and transepithelial migration, we treated eosinophils with eotaxin or thapsigargin (TG), reagents that increase cytoplasmic free Ca2+ concentrations by receptor- or nonreceptor-mediated mechanisms, respectively. Pretreatment of eosinophils with TG enhanced CD11b/CD18-dependent transmigration across lung epithelium. Within minutes, TG time- and dose-dependently upregulated the expression of CD11b/CD18 but did not upregulate the expression of alphaL (CD11a) or beta1 (CD29) integrin. The upregulation of CD11b/CD18 expression by eotaxin or TG was prevented when Ca2+ entry was blocked. The priming of eosinophil transmigration by TG was also abrogated by the blockade of Ca2+ entry. Our results indicate that induction of Ca2+ entry by the depletion of Ca2+ from intracellular stores upregulates CD11b/CD18 expression on eosinophils and primes eosinophil transmigration across lung epithelium. Both responses are therefore elicited by extracellular Ca2+. We suggest that, as an important priming signal for human eosinophil functional responses, store-operated Ca2+ entry may be one of the underlying mechanisms of eosinophilic inflammation in asthma.