Properties of an immunoglobulin binding factor from human seminal plasma.

A soluble factor (IBF) in human seminal plasma that binds serum immunoglobulins (Ig) of various species was purified to homogeneity by ammonium sulfate precipitation, preparative isoelectrofocusing, and gel filtration chromatography. The purified IBF interacted weakly with Fc and F(ab')2 fragments and not with Fab. It interacted with anti-Leu 11b and polyclonal anti-Fc gamma RIII antibodies, but not with other anti-Fc gamma R antibodies (32.2, IV.3 and 3G8). IBF is probably a non-glycosylated protein with isoelectric point ranging from 5.1 to 5.8. The estimated Mr of the purified native IBF is 27 kD, determined by sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE) under non-reducing condition. In its native form, IBF did not bind Ig or interact with anti-Fc gamma R antibodies. Following SDS-PAGE under reducing condition, IBF migrated as a single protein with an estimated Mr of 16 kD and interacted with Ig of various species and with anti-Leu 11b antibodies. When carboxymethylated, however, IBF did not bind IgG. The present results suggest that free sulfhydryl groups of IBF is required for Ig binding.     

Acid-activated insulin-like growth factor binding protein protease activity of Cathepsin D in normal and malignant prostatic epithelial cells and seminal plasma

In this study, we demonstrate insulin-like growth factor binding protein (IGFBP) acid proteolysis in conditioned media (CM) from normal and malignant primary cultures of prostatic epithelial cells, prostatic cell lines, and in seminal plasma. We further demonstrate the absence of such activity in CM from prostatic stromal cells. Radio-labeled IGFBPs (1–6) were incubated with various acidified CM and seminal plasma. None of these media showed IGFBP proteolytic activity at neutral pH, but all CM from prostatic epithelial cells (PC-E) demonstrated strong IGFBP proteolysis at acidic pH. No acid-activated proteolysis was observed in the CM from stromal cell cultures. In order to ascertain the role of cathepsin D, anti-cathepsin antibodies were used to immunodeplete the media of the selected enzymes prior to incubation with IGFBPs. Depletion of cathepsin D greatly reduced the proteolytic activity of the PC-E CM. Additionally, purified cathepsin D yielded a digestion pattern identical to that produced by prostatic cell CM and seminal plasma, following acidic incubation with IGFBP-3. Remarkably, the proteolytic pattern generated by seminal plasma, when incubated with IGFBP-3 at neutral pH, corresponded to that produced by prostate-specific antigen (PSA), demonstrating the interpolation of both neutral and acid proteases from prostate cells into seminal plasma. In conclusion, prostatic epithelial cells secrete acid-specific IGFBP protease(s) related to cathepsin D. Although no significant statistical difference was observed in the degree of acid-specific proteolysis in the media from normal versus malignant primary epithelial cell cultures, physiologicalcharacteristics of the malignant state might facilitate increased cathepsin D activity. We suspect this proteolysis may play a role in prostatic cell proliferationand invasive tumor growth. J. Cell. Physiol. 171:196–204, 1997. © 1997 Wiley-Liss, Inc.     

Zinc binding properties of human prostatic tissue, prostatic secretion and seminal fluid

When studied by gel filtration, zinc in prostatic cytosol (10(5) g, 1 h) was associated with high (greater than 80000), medium (3000-80000) and low molecular weight (less than 3000) molecules in approximately equal proportions. The molecules of high and medium Mr were not secreted into the prostatic fluid where all the zinc was associated with molecules of low Mr (probably citric acid). After ejaculation much of the zinc is redistributed and becomes bound to molecules of high and medium Mr of vesicular origin.     

Purification of prostatic acid phosphatase from human seminal plasma

Prostatic acid phosphatase has been isolated from human seminal plasma. The purification method utilizes gel filtration on Sephadex G100, ammonium sulfate precipitation and a series of chromatographical steps including concanavalin A Sepharose 4B, anion exchange and gel filtration chromatography. The final product appears homogenous when analyzed by gel filtration on Sephadex G100. It gives one major band on SDS polyacrylamide gels. The specific activity is similar to that obtained by other purification schemes. The yield of the method described above has allowed to set up a sensitive radioimmunoassay of prostatic acid phosphatase.     

High-affinity binding of seminal plasma PSP94 to human immunoglobulin is through the Fab domain

Prostate secretory protein of 94 amino acids (PSP94) is one of the major proteins present in human seminal plasma. We had earlier reported that PSP94 has the ability to bind to human IgG. The aims of the present study were to further delineate the PSP94–IgG interaction and to understand whether this could have any significance in sperm function. Direct binding of IgG fragments to PSP94 showed maximal binding with F(ab′)₂ followed by Fab, while Fc displayed least binding in ELISA. Binding kinetics of PSP94–IgG interaction using surface plasmon resonance (SPR) revealed high-affinity binding of IgG to PSP94 with a dissociation constant (K_D) of 8.8 × 10⁻¹¹ M. PSP94–IgG interaction was found to be through the Fab domains of IgG. Real-time interaction kinetics revealed association constants for binding of IgG, Fab, and F(ab′)₂ towards PSP94 to be of the same order but with altered dissociation constants. IgG and its F(ab′)₂ fragment once complexed to PSP94 demonstrated negligible dissociation, while dissociation rate of Fab fragment was 6.6 × 10⁻⁴. In silico molecular modeling of PSP94–IgG complex identified N- and C-terminal β-strands of PSP94 to be the most plausible region involved in IgG interaction. Immunofluorescence studies revealed that IgG bound to human spermatozoa predominantly in the tail region, which could be prevented when IgG was preincubated with PSP94. This study reports for the first time that IgG forms a high-affinity complex with PSP94 through its F(ab′)₂ domain and reveals the ability of PSP94 to prevent binding of IgG to spermatozoa.     

Lactoferrin binding molecules in human seminal plasma

During ejaculation, the iron binding protein lactoferrin binds to sperm and forms a major component of sperm-coating antigens. Physicochemical properties of lactoferrin in seminal plasma (SP) and on sperm differ from those of purified lactoferrin. These differences have been attributed to the binding of unknown seminal macromolecules to lactoferrin. We have studied lactoferrin binding molecules in SP. The SP samples were coated onto microtiter plates and tested for binding of biotinylated lactoferrin. SP was found to specifically bind biotinylated lactoferrin. This binding was competitively inhibited by coincubation with unlabeled lactoferrin but was not affected by control incubations done with human IgG or transferrin. Lactoferrin binding molecules in SP were biochemically characterized by using SDS-PAGE and ligand blotting. Biotinylated lactoferrin bound to SP molecules of approximately 120, 60 and 30 kDa. No binding was observed with biotinylated transferrin. The presence of molecules that associate with lactoferrin in SP was further studied by using crossed immunoelectrophoresis. Lactoferrin in SP immunoprecipitated as two peaks, one of which corresponded to purified lactoferrin. These results suggest that some lactoferrin molecules in SP are free and that others are associated with lactoferrin binding molecules. Binding of lactoferrin to lactoferrin binding molecules appears to change its physicochemical properties and thus could influence its biologic activity and its affinity to sperm.     

The major protein of bull seminal plasma is a secretory product of seminal vesicle

We isolated the major protein with apparent molecular weight, Mr, 15,000-16,000 from seminal plasma as well as from seminal vesicle secretion of bull and proved by amino acid analysis and tryptic peptide mapping that the two proteins were identical. An antiserum against this major protein was employed to quantitate and identify the major protein in seminal plasma as well as in seminal vesicle secretion. The antiserum did not cross-react with proteins from bovine or human plasma or follicular fluid, respectively. Cell-free translation of poly(A+)RNA isolated from seminal vesicle tissue resulted in formation of one major species with apparent Mr 18,000. Using the anti-major protein antiserum, this major species was specifically immuno absorbed. We thus provided evidence that the major protein component of bull seminal plasma is a secretory protein of seminal vesicles. Furthermore, it appeared that the isolated major protein may be closely related to the protein PDC109, purified from bull seminal plasma and sequenced by Esch et al. (Biochem. Biophys. Res. Commun. 113, 861-867 (1983).     

Epidermal growth factor in human seminal plasma

In the present study, we have partially purified a characterized epidermal growth factor (EGF)-like substance(s) from human seminal plasma, and determined the concentrations of immunoreactive (IR)-hEGF in seminal plasma from normal and infertile males. Competitive binding curves of seminal plasma extracts were parallel to those of standard hEGF in both radioimmunoassay and receptor assay. Seminal IR-hEGF was similar to standard hEGF by gel exclusion chromatography, isoelectric focusing and sodium dodecyl sulfate polyacrylamide gel electrophoresis. The concentrations of IR-hEGF in normal seminal plasma (48 +/- 9 ng/ml) did not differ from those of infertile males (41 +/- 3 ng/ml); the concentrations of seminal plasma IR-hEGF did not correlate with density, motility or morphology of sperm. These data clearly demonstrate the presence of hEGF in human seminal plasma indistinguishable from hEGF of urinary origin, and suggest that it may not play an important role in the sperm function. The tissue(s) of its origin and its physiological function in the male reproductive organs remain undetermined.     

Detection of prostatic tissue and seminal plasma peptides reacting with human seminal fluid acid phosphatase antibodies

IgG1 monoclonal antibody to purified seminal fluid phosphatase was raised by fusion of spleen cells from immunized mice with cell line Sp2/O-Ag 14 using simple method of screening for antiphosphatase antibody secreting clones. All molecular forms of catalytically active seminal fluid phosphatase and prostatic tissue phosphatase, resolved by chromatofocusing in pH gradient, react with this monoclonal antibody and with rabbit antiserum to purified seminal fluid phosphatase. Peptides of Mr 25,000 to 76,000 and of Mr 13,000 to 76,000 were adsorbed from the prostatic tissue extract and from seminal plasma on the monoclonal antibody-Sepharose column.     

Seminalplasmin,the major basic protein of bull seminal plasma,is a secretory protein of the seminal vesicles

From the experimental results of three independent methods: (1) indirect immunofluorescence employing monospecific anti-seminalplasmin-IgGs, (2) cell-free translation of poly(A)⁺ RNA from seminal vesicle and testicular tissue, as well as (3) Northern analysis of poly(A)⁺ RNA of the latter tissues with a synthetic seminalplasmin-specific antisense DNA probe, it is concluded that the biosynthesis of seminalplasmin occurs in seminal vesicles but not in testis.     

Characterization of a seminal plasma-associated inhibitor of human seminal plasma protein kinase

Human sperm-free seminal plasma (HSP) contains inhibitors (I) of the seminal plasma histone kinase activity (HK). One I is dialyzable and the other I is nondialyzable and precipitable by dialysis of HSP against a hypotonic buffer. When the nondialyzable, precipitable I fraction is resolubilized, it inhibits HK in a concentration-dependent manner. Sephadex G-25 column chromatography of whole HSP resolves I in both the void (Vo) and inclusion (Vi) volumes. Rechromatography of the VoI resolves I solely in the Vo. These and other data suggest that the ViI does not originate from the VoI, and that both I activities represent separate molecular entities. VoI was further characterized and found to be heat labile, trypsin and neuraminidase insensitive, and alpha-chymotrypsin sensitive. VoI is not soluble in CHCl3 or CHCl3:CH3OH (2:1) and is not adsorbed by charcoal. Chromatography of VoI on Sephadex G-100 yields a broad peak of I that migrates just past the Vo. VoI has no detectable cyclic AMP (cAMP) binding activity and VoI activity is not affected by coincubation of VoI and HK with cAMP. VoI also does not bind to zinc-chelate or phenothiazine affinity columns. These data suggest that VoI is protein in nature with properties distinct from the class of previously described protein kinase inhibitors. Although the identity of VoI is not known, it does not appear to be the regulatory subunit of a cAMP-dependent protein kinase, calsemin or a zinc binding protein.     

The identity of the acid and alkaline phosphatases of human seminal plasma.

Examination of human seminal plasma showed that there was no p-nitrophenylphosphatase activity maximum at alkaline pH values. A constant ratio of enzyme activities in 8 split ejaculates, identical temperature dependence and copurification through a three-step purification procedure led us to conclude that the alkaline phosphatase in human semen is identical to acid phosphatase.     

Seminalplasmin, the major basic protein of bull seminal plasma, is a secretory protein of the seminal vesicles.

From the experimental results of three independent methods: (1) indirect immunofluorescence employing monospecific anti-seminalplasmin-IgGs, (2) cell-free translation of poly(A)+ RNA from seminal vesicle and testicular tissue, as well as (3) Northern analysis of poly(A)+ RNA of the latter tissues with a synthetic seminalplasmin-specific antisense DNA probe, it is concluded that the biosynthesis of seminalplasmin occurs in seminal vesicles but not in testis.     

Ovulation-inducing factor: a protein component of llama seminal plasma

Background  

Previously, we documented the presence of ovulation-inducing factor (OIF) in the seminal plasma of llamas and alpacas. The purpose of the study was to define the biochemical characteristics of the molecule(s) in seminal plasma responsible for inducing ovulation.     

Immunologically reactive albumin-like protein in human testis and seminal plasma

An immunologically reactive albumin-like protein (albumin) was localized, by an immunostaining technique, in the testis of infertile men (normal spermatogenesis, obstructive azoospermia) at the level of the Sertoli cells and in some cells of the germinal epithelium (secondary spermatocytes and early spermatids). No positive reaction was detectable in prepubertal testis. In vasectomized men, mean seminal albumin values were drastically reduced (by about 80%) in comparison to fertile controls, indicating a probable testicular origin. Mean seminal albumin values were also decreased in patients affected by azoospermia due to a seminiferous tubular lesion (about 40%) and in oligozoospermic patients (about 30%). In the same seminal samples transferrin, an index of Sertoli cell function, was also measured. Albumin and transferrin results were well correlated in the seminal plasma of each group (with the exception of vasectomized subjects), including a group of men with abnormally high concentrations of seminal transferrin. A weak correlation was found between seminal albumin and sperm count. We suggest that the presence of albumin in the human adult testis and in seminal plasma could be related to its ability to transport androgens.     

Computational analysis of Concanavalin A binding glycoproteins of human seminal plasma

Glycoproteins have immense clinical importance and comparative glycoproteomics has become a powerful tool for biomarker discovery and disease diagnosis. Seminal plasma glycoproteins participate in fertility related processes including sperm-egg recognition, modulation of capacitation and acrosome reaction inhibition. Affinity chromatography using broad specificity lectin such as Con A is widely applied for glycoproteins enrichment. More notably, Con A-interacting fraction of human seminal plasma has decapacitating activity which makes this fraction critically important. In our previous study, we isolated Con A-interacting glycoproteins from human seminal plasma and subsequently identified them by mass spectrometry. Here, we report the computational analysis of these proteins using bioinformatics tools. The analysis includes: prediction of glycosylation sites using sequence information (NetNGlyc 1.0), functional annotations to cluster these proteins into various functional groups (InterProScan and Blast2GO) and identification of protein interaction networks (STRING database). The results indicate that these proteins are involved in various biological processes including transport, morphogenesis, metabolic processes, cell differentiation and homeostasis. The clusters illustrate two major molecular functions - hydrolase activity (6) and protein (4)/carbohydrate (1)/lipid binding (1). The large interactomes of proteins point towards their versatile roles in wide range of biological processes.