Identification of a nuclear localization signal in activin/inhibin betaA subunit; intranuclear betaA in rat spermatogenic cells.
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Activin is a dimeric glycoprotein hormone that was initially characterized by its ability to stimulate pituitary FSH secretion and was subsequently recognized as a growth factor with diverse biological functions in a large variety of tissues. In the testis, activin has been implicated in the auto/paracrine regulation of spermatogenesis through its cognate cell membrane receptors on Sertoli and germ cells. In this study we provide evidence for intranuclear activin/inhibin betaA subunit and show its distribution in the rat seminiferous epithelium. We have shown by transient expression in HeLa cells of beta-galactosidase fusion proteins that the betaA subunit precursor contains a functional nuclear localization signal within the lysine-rich sequence corresponding to amino acids 231-244. In all stages of the rat seminiferous epithelial cycle, an intense immunohistochemical staining of nuclear betaA was demonstrated in intermediate or type B spermatogonia or primary spermatocytes in their initial stages of the first meiotic prophase, as well as in pachytene spermatocytes and elongating spermatids primarily in stages IX-XII. In some pachytene spermatocytes, the pattern of betaA immunoreactivity was consistent with the characteristic distribution of pachytene chromosomes. In the nuclei of round spermatids, betaA immunoreactivity was less intense, and in late spermatids it was localized in the residual cytoplasm, suggesting disposal of betaA before spermatozoal maturation. Immunoblot analysis of a protein extract from isolated testicular nuclei revealed a nuclear betaA species with a molecular mass of approximately 24 kDa, which is more than 1.5 times that of the mature activin betaA subunit present in activin dimers. These results suggest that activin/inhibin betaA may elicit its biological functions through two parallel signal transduction pathways, one involving the dimeric molecule and cell surface receptors and the other an alternately processed betaA sequence acting directly within the nucleus. According to our immunohistochemical data, betaA may play a significant role in the regulation of nuclear functions during meiosis and spermiogenesis. (+info)
Identification of low density lipoprotein receptor-related protein-2/megalin as an endocytic receptor for seminal vesicle secretory protein II.
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The low density lipoprotein receptor-related protein-2/megalin (LRP-2) is an endocytic receptor that is expressed on the apical surfaces of epithelial cells lining specific regions of the male and female reproductive tracts. In the present study, immunohistochemical staining revealed that LRP-2 is also expressed by epithelial cells lining the ductal region and the ampulla of the rat seminal vesicle. To identify LRP-2 ligands in the seminal vesicle, we probed seminal vesicle fluid with 125I-labeled LRP-2 in a gel-blot overlay assay. A 100-kDa protein (under non-reducing conditions) was found to bind the radiolabeled receptor. The protein was isolated and subjected to protease digestion, and the proteolytic fragments were subjected to mass spectroscopic sequence analysis. As a result, the 100-kDa protein was identified as the seminal vesicle secretory protein II (SVS-II), a major constituent of the seminal coagulum. Using purified preparations of SVS-II and LRP-2, solid-phase binding assays were used to show that the SVS-II bound to the receptor with high affinity (Kd = 5.6 nM). The binding of SVS-II to LRP-2 was inhibited using a known antagonist of LRP-2 function, the 39-kDa receptor-associated protein RAP. Using a series of recombinant subfragments of SVS-II, the LRP-2 binding site was mapped to a stretch of repeated 13-residue modules located in the central portion of the SVS-II polypeptide. To evaluate the ability of LRP-2 to mediate 125I-SVS-II endocytosis and lysosomal degradation, ligand clearance assays were performed using differentiated mouse F9 cells, which express high levels of LRP-2. Radiolabeled SVS-II was internalized and degraded by the cells, and both processes were inhibited by antibodies to LRP-2 or by RAP. The results indicate that LRP-2 binds SVS-II and can mediate its endocytosis leading to lysosomal degradation. (+info)
Characterisation of the conformational and quaternary structure-dependent heparin-binding region of bovine seminal plasma protein PDC-109.
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PDC-109, the major heparin-binding protein of bull seminal plasma, binds to sperm choline lipids at ejaculation and modulates capacitation mediated by heparin. Affinity chromatography on heparin-Sepharose showed that polydisperse, but not monomeric, PDC-109 displayed heparin-binding capability. We sought to characterise the surface topology of the quaternary structure-dependent heparin-binding region of PDC-109 by comparing the arginine- and lysine-selective chemical modification patterns of the free and the heparin-bound protein. A combination of reversed-phase peptide mapping of endoproteinase Lys-C-digested PDC-109 derivatives and mass spectrometry was employed to identify modified and heparin-protected residues. PDC-109 contains two tandemly arranged fibronectin type II domains (a, Cys24-Cys61; b, Cys69-Cys109). The results show that six basic residues (Lys34, Arg57, Lys59, Arg64, Lys68, and Arg104) were shielded from reaction with acetic anhydride and 1,2-cyclohexanedione in heparin-bound PDC-109 oligomers. In the 1H-NMR solution structures of single fibronectin type II domains, residues topologically equivalent to PDC-109 Arg57 (Arg104) and Lys59 lay around beta-strand D on the same face of the domain. In full-length PDC-109, Arg64 and Lys68 are both located in the intervening polypeptide between domains a and b. Our data suggest possible quaternary structure arrangements of PDC-109 molecules to form a heparin-binding oligomer. (+info)
Presentation of antigen in immune complexes is boosted by soluble bacterial immunoglobulin binding proteins.
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Using a snake toxin as a proteic antigen (Ag), two murine toxin-specific monoclonal antibodies (mAbs), splenocytes, and two murine Ag-specific T cell hybridomas, we showed that soluble protein A (SpA) from Staphylococcus aureus and protein G from Streptococcus subspecies, two Ig binding proteins (IBPs), not only abolish the capacity of the mAbs to decrease Ag presentation but also increase Ag presentation 20-100-fold. Five lines of evidence suggest that this phenomenon results from binding of an IBP-Ab-Ag complex to B cells possessing IBP receptors. First, we showed that SpA is likely to boost presentation of a free mAb, suggesting that the IBP-boosted presentation of an Ag in an immune complex results from the binding of IBP to the mAb. Second, FACS analyses showed that an Ag-Ab complex is preferentially targeted by SpA to a subpopulation of splenocytes mainly composed of B cells. Third, SpA-dependent boosted presentation of an Ag-Ab complex is further enhanced when splenocytes are enriched in cells containing SpA receptors. Fourth, the boosting effect largely diminishes when splenocytes are depleted of cells containing SpA receptors. Fifth, the boosting effect occurs only when IBP simultaneously contains a Fab and an Fc binding site. Altogether, our data suggest that soluble IBPs can bridge immune complexes to APCs containing IBP receptors, raising the possibility that during an infection process by bacteria secreting these IBPs, Ag-specific T cells may activate IBP receptor-containing B cells by a mechanism of intermolecular help, thus leading to a nonspecific immune response. (+info)
Bovine seminal plasma (BSP) contains a family of phospholipid-binding proteins (BSP-A1/-A2, BSP-A3 and BSP-30-kDa, collectively called BSP proteins) that potentiate sperm capacitation induced by high-density lipoproteins. We showed recently that BSP proteins stimulate cholesterol efflux from epididymal spermatozoa and play a role in capacitation. Here, we investigated whether or not BSP proteins could stimulate cholesterol and phospholipid efflux from fibroblasts. Cells were radiolabeled ([3H]cholesterol or [3H]choline) and the appearance of radioactivity in the medium was determined in the presence of BSP proteins. Alcohol precipitates of bovine seminal plasma (designated crude BSP, cBSP), purified BSP-A1/-A2, BSP-A3 and BSP-30-kDa proteins stimulated cellular cholesterol and choline phospholipid efflux from fibroblasts. Efflux mechanistic differences were observed between BSP proteins and other cholesterol acceptors. Preincubation of BSP-A1/-A2 proteins with choline prevented cholesterol efflux, an effect not observed with apolipoprotein A-I. Also, the rate of BSP-induced efflux was rapid during the first 20 min, but leveled off thereafter in contrast to a relatively slow, but constant, rate of cholesterol efflux mediated by apolipoprotein A-I, apolipoprotein A-I-containing reconstituted lipoproteins (LpA-I) and high-density lipoproteins. These results indicate that fibroblasts are a good cell model to study the mechanism of lipid efflux mediated by BSP proteins. (+info)
Characterization of lipid efflux particles generated by seminal phospholipid-binding proteins.
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We reported recently that the choline phospholipid-binding proteins (BSP-A1/-A2, BSP-A3 and BSP-30-kDa) of bovine seminal plasma (BSP) stimulate cholesterol and choline phospholipid efflux from fibroblasts. In this study, we characterized the lipid efflux particles generated by BSP proteins. The density gradient ultracentrifugation of the efflux medium from radiolabeled fibroblasts incubated with BSP proteins showed a single peak of [3H]cholesterol between density (d) 1.12 and 1.14 g/ml, which is in the range of high-density lipoproteins. Size-exclusion chromatographic and immunoblot analysis revealed that the efflux particles have a large size equal to or bigger than very low-density lipoproteins and contained BSP proteins. Lipid analysis of density gradient and gel filtration fractions from efflux medium of simultaneously labeled fibroblasts ([3H]cholesterol and [3H]choline) incubated with BSP proteins showed that the efflux particles were homogeneous and composed of cholesterol and choline phospholipids. The lipid particles contained BSP proteins, cholesterol and choline phospholipids in molar ratio of 0.05:1.21:1, respectively. Agarose gel electrophoresis showed that the BSP-generated lipid particles had a gamma migration pattern which is slower than low-density lipoproteins. The sonication of cholesterol and BSP proteins followed by gel filtration chromatographic analysis indicated no direct binding of cholesterol to BSP proteins. These results taken together indicate that BSP proteins induce a concomitant cholesterol and choline phospholipid efflux and generate large protein-lipid particles. (+info)
Complementary deoxyribonucleic acid cloning and tissue expression of BSP-A3 and BSP-30-kDa: phosphatidylcholine and heparin-binding proteins of bovine seminal plasma.
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BSP-A1, BSP-A2, BSP-A3, and BSP-30-kDa are four major proteins of bovine seminal plasma (BSP protein family). These heparin- and phosphatidylcholine-binding proteins potentiate the capacitation of spermatozoa. Here we determined the complete sequences of the two cDNAs coding for the BSP-A3 and BSP-30-kDa proteins. Degenerate oligonucleotides designed on the basis of the primary sequences of the proteins were used as primers in reverse transcription-polymerase chain reaction, with cDNA preparations of bovine seminal vesicles as templates, to amplify an internal fragment of each BSP cDNA. Specific oligonucleotides designed on the basis of these partial cDNA sequences were used to clone the two complete cDNAs by using the 3' rapid amplification of cDNA ends (RACE) and 5' RACE methods. We also verified the expression of all members of the bovine BSP protein family in several adult bovine tissues by RNase protection assays. The results indicated that each BSP protein mRNA is expressed only in seminal vesicles and in the ampullae. Homologous genes were detected in human, rat, hamster, and rabbit genomic DNA, using high-stringency Southern hybridization with a specific BSP-30-kDa cDNA probe. (+info)
Target cells for an immunosuppressive cytokine, glycosylation-inhibiting factor.
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Receptors for bioactive glycosylation-inhibiting factor (GIF) were demonstrated using a bioactive mutant of recombinant human (rh) GIF, which is comparable to the suppressor T (Ts) cell-derived bioactive GIF in its affinity for the receptors on helper T (Th) hybridoma cells. Both naive T and B cells in normal mouse spleen lacked GIF receptors. However, presentation of specific antigen to naive T cells resulted in the expression of the receptors on activated T cells. Furthermore, activation of small resting B cells with F(ab')2 fragments of anti-mouse IgM plus IL-4, lipopolysaccharide (LPS) plus IL-4 or LPS plus dextran sulfate induced the expression of the receptors within 48 h of B cell stimulation. It was also found that NK T cells freshly isolated from mouse spleen, but not conventional NK cells, expressed receptors for GIF. CD4(+) and CD4(-) subpopulations of NK T cells showed a similar binding capability. Mature dendritic cells derived from bone marrow did not bear the receptors. The dissociation constant (Kd) of the interaction between the bioactive rhGIF mutant and the high-affinity receptors was 10-100 pM, whereas inactive wild-type rhGIF failed to bind to the receptors. A bioactive derivative of rhGIF suppressed both IgG1 and IgE synthesis by purified B cells activated by LPS and IL-4, indicating that the binding of bioactive GIF to its receptors on activated B cells results in suppression of their differentiation. (+info)