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Åke Lundwall - One of the best experts on this subject based on the ideXlab platform.
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the common marmoset callithrix jacchus has two very similar Semenogelin genes as the result of gene conversion
Biology of Reproduction, 2007Co-Authors: Camilla Valtonenandre, Yvonne A Olsson, P L Nayudu, Morgan Kullberg, Åke LundwallAbstract:The semen coagulum proteins have undergone substantial structural changes during evolution. In primates, these seminal vesicle-secreted proteins are known as Semenogelin I (SEMG1) and Semenogelin II (SEMG2). Previous studies on the common marmoset (Callithrix jacchus) showed that ejaculated semen from this New World monkey contains Semenogelin, but it remained unclear whether it carries both genes or only SEMG1 and no SEMG2, like the closely related cotton-top tamarin (Saguinus oedipus). In this study we show that there are two genes, both expressed in the seminal vesicles. Surprisingly, the genes show an almost perfect sequence identity in a region of 1.25 kb, encompassing nearly half of the genes and containing exon 1, intron 1, and the first 0.9 kb of exon 2. The underlying molecular mechanism is most likely gene conversion, and a phylogenetic analysis suggests that SEMG1 is the most probable donor gene. The marmoset SEMG1 in this report differs from a previously reported cDNA by a lack of nucleotides encoding one repeat of 60 amino acids, suggesting that marmoset SEMG1 displays allelic size variation. This is similar to what was recently demonstrated in humans, but in marmosets the polymorphism was generated by a repeat duplication, whereas in humans it was a deletion. Together, these studies shed new light on the evolution of Semenogelins and the mechanisms that have generated the structural diversity of semen coagulum proteins.
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truncated Semenogelin i binds zinc and is cleaved by prostate specific antigen
Journal of Andrology, 2006Co-Authors: Magnus P Jonsson, Åke Lundwall, Sara Linse, Birgitta Frohm, Johan MalmAbstract:Semenogelins I and II are major coagulum-forming proteins in semen, and they are secreted mainly by the seminal vesicles. These proteins bind Zn(2+) and act as substrates for prostate-specific antigen and transglutaminase. A variant Semenogelin I lacking 60 amino acids has been described that occurs in different populations with an allele frequency of 1%-3%. To better understand the function of the Semenogelins in vivo, our aim was to characterize the properties of the variant form and compare with the wild type. Recombinant proteins were synthesized in insect cells. Binding of Zn(2+) was studied by titration of metal ions in the presence of a zinc (II) fluorophore chelator. SDS-PAGE was used to visualize the results of cleavage by prostate-specific antigen and cross-linking with transglutaminase. We found that the truncated and wild-type Semenogelin molecules had similar Zn(2+)-binding properties (ie, a stoichiometry of at least 9-10 mol per mol of protein and an average dissociation constant of 5 mumol/L per site), and they showed also similar susceptibility for degradation by prostate-specific antigen. Furthermore, like the wild-type form, the truncated Semenogelin I was able to serve as a substrate for transglutaminase. These findings imply that the studied characteristics do not depend on a well-defined tertiary structure, or that the deletion has no major effect on the structure responsible for these features.
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characterization of Semenogelin proteins in the human retina
Experimental Eye Research, 2006Co-Authors: Vera L Bonilha, Åke Lundwall, Johan Malm, Mary E Rayborn, K G Shadrach, Sanjoy K Bhattacharya, John W Crabb, Joe G HollyfieldAbstract:Semenogelin I and II are the major proteins present in semen coagulum. In the present study, Semenogelin I and II were detected in human RPE lysates by proteomic analysis. We further analyzed the expression of these proteins in the retinal cells in vivo and in vitro. Western blots detected Semenogelin I and II in both RPE and neural retina while the vitreous contained only SgII. Cryo and paraffin sections of human retina were processed for both immunofluorescence and DAB reaction with an antibody that recognizes both forms of Semenogelin proteins. Retina and RPE total lysates were evaluated for the presence of these proteins and in a human RPE cell line (D407). Both proteins were detected by western blot in human RPE and in D407 cell lysates. Immunoreactivity was detected in the ganglion cell and photoreceptor layer of the retina. Our data support the expression of Semenogelin I and II in the human retina in several different compartments. Further studies towards addressing the function of these proteins in the retina are in progress.
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ejaculates from the common marmoset callithrix jacchus contain Semenogelin and beta microseminoprotein but not prostate specific antigen
Molecular Reproduction and Development, 2005Co-Authors: Camilla Valtonenandre, Yvonne A Olsson, P L Nayudu, Åke LundwallAbstract:Human seminal plasma contains high concentrations of prostatic acid phosphatase (PAP), prostate-specific antigen (PSA), beta-microseminoprotein (MSP), Semenogelin I (SgI), and Semenogelin II (SgII), whereas only PAP and MSP are present in rodents. In order to gain a better understanding of the evolution and function of semen proteins, we have studied ejaculates from the common marmoset (Callithrix jacchus)—a New World monkey. Semen samples were analyzed with SDS–PAGE, Western blotting, and isoelectric focusing. Under reducing conditions the dominating protein components appear as heterogeneous material of 55–70 kDa and distinct protein bands of 85, 17, 16, and 15 kDa. The heterogeneous material contains glycosylated material detected by an antiserum recognizing both human SgI and SgII. Southern blotting indicates that the common marmoset has genes for both SgI and SgII. There are several marmoset MSP genes, but the strong immunoreactivity against one 15 kDa semen component with pI 7.3 suggests preferential expression of one gene in the prostate. Expression of two other genes cannot be excluded as indicated by weak reaction to isoforms with pI 6.6 and 4.9. Unexpectedly, PSA was not detected by either immunological methods or activity measurements. This is in agreement with results from Southern blotting suggesting that the common marmoset might not have a PSA gene. Thus, in this study we have shown that semen coagulum proteins are present in marmoset seminal plasma, but the lack of PSA precludes a similar liquefaction as of human semen. Mol. Reprod. Dev. 71: 247–255, 2005. © 2005 Wiley-Liss, Inc.
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a frequent allele codes for a truncated variant of Semenogelin i the major protein component of human semen coagulum
Molecular Human Reproduction, 2003Co-Authors: Åke Lundwall, Hans Lilja, Aleksander Giwercman, Yasir Ruhayel, Yvonne Lundberg Giwercman, Christer Hallden, Johan MalmAbstract:Human semen coagulum predominantly consists of high molecular mass complexes of the seminal vesicle secreted Semenogelin I (SgI) and Semenogelin II (SgII). Here we describe a previously unknown variant of the SgI gene that is present at an allele frequency of approximately 3% in the Swedish population. It gives rise to a protein with a molecular mass of 43 kDa, SgI(43), which compared with the 50 kDa variant, SgI(50), is lacking a tandem repeat of 60 amino acid residues that was probably deleted by homologous recombination. In spite of the size difference, SgI(43) has many properties in common with SgI(50), such as a very high iso-electric point and susceptibility to proteolytic degradation by prostate-specific antigen. Heterozygous carriers of the SgI(43) allele neither show impaired fertility nor do they significantly differ from individuals homozygous for SgI(50) with respect to sperm parameters such as semen volume, sperm count and fraction of motile spermatozoa.
Richard T. Richardson - One of the best experts on this subject based on the ideXlab platform.
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1 Characterization of EPPIN’s Semenogelin I Binding Site: A Contraceptive Drug Target1
2016Co-Authors: Erick J. R. Silva, Katherine G. Hamil, Richard T. Richardson, Michael G. O’rAbstract:Summary sentence: Residues Cys102, Tyr107, and Phe117 within EPPIN’s Kunitz domain are critical for binding SEMG1 and as targets for contraceptive drug design
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Characterization of EPPIN’s Semenogelin I Binding Site: A Contraceptive Drug Target1
2016Co-Authors: Erick J. R. Silva, Katherine G. Hamil, Richard T. Richardson, Michael G. O’rAbstract:Epididymal protease inhibitor (EPPIN) is found on the surface of spermatozoa and works as a central hub for a sperm surface protein complex (EPPIN protein complex [EPC]) that inhibits sperm motility on the binding of Semenogelin I (SEMG1) during ejaculation. Here, we identify EPPIN’s amino acids involved in the interactions within the EPC and demonstrate that EPPIN’s sequence C102-P133 contains the major binding site for SEMG1. Within the same region, the sequence F117-P133 binds the EPC-associated protein lactotransferrin (LTF). We show that residues Cys102, Tyr107, and Phe117 in the EPPIN C-terminus are required for SEMG1 binding. Additionally, residues Tyr107 and Phe117 are critically involved in the interaction between EPPIN and LTF. Our findings demonstrate that EPPIN is a key player in the protein-protein interactions within the EPC. Target identifi-cation is an important step toward the development of a novel male contraceptive, and the functionality of EPPIN’s residues Cys102, Tyr107, and Phe117 offers novel opportunities for contraceptive compounds that inhibit sperm motility by targeting this region of the molecule. contraception, EPPIN, Semenogelin I, spermatozo
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1 Inhibition of Human Sperm Motility by Contraceptive Anti-Eppin Antibodies from Infertile Male Monkeys: Effect on Cyclic Adenosine Monophosphate
2016Co-Authors: Michael G. O’r, Esther E. Widgren, Stan Beyler, Richard T. RichardsonAbstract:Summary sentence: Anti-eppin antibodies and recombinant Semenogelin inhibit human sperm motility
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Inhibition of Human Sperm Motility by Contraceptive Anti-Eppin Antibodies from Infertile Male Monkeys: Effect on Cyclic Adenosine Monophosphate1
2016Co-Authors: Michael G. O’r, Esther E. Widgren, Stan Beyler, Richard T. RichardsonAbstract:Epididymal protease inhibitor (eppin [official symbol, SPINLW1]) is of interest as a male contraceptive target because of its specificity and location on the human sperm surface. We have examined the effect of anti-eppin antibodies from infertile male monkeys and the effect of recombinant human semenoge-lin on human sperm motility. Anti-eppin antibodies significantly decreased the progressive motility of human spermatozoa as measured by decreased total distance traveled, decreased straight-line distance, and decreased velocity. Anti-eppin treat-ment of spermatozoa significantly increased the amount of cAMP present in nonprogressive spermatozoa; however, ap-proximately 25 % of antibody-treated spermatozoa could be rescued by the addition of cAMP-acetoxymethyl ester, indicating that anti-eppin-treated spermatozoa have a compromised ability to utilize cAMP. Addition of recombinant human Semenogelin has a concentration-dependent inhibitory effect on progressive motility (increased tortuosity and decreased velocity). We tested the hypothesis that anti-eppin antibodies bound to eppin would subsequently block Semenogelin binding to eppin. Anti-eppin antibodies from infertile monkeys inhibited eppin from binding to Semenogelin. Addition of affinity-purified antibodies made to the dominant C-terminal epitope of eppin had an inhibitory effect on progressive motility (increased tortuosity, decreased velocity, and straight distance). Our results suggest that the eppin-Semenogelin binding site is critical for the removal of Semenogelin in vivo during semen liquefaction and for the initiation of progressive motility. We conclude that the eppin-Semenogelin binding site on the surface of human spermatozoa is an ideal target for a nonsteroidal male contraceptive. cAMP, contraception, eppin, gamete biology, Semenogelin, seminal plasma, sperm, spermatozo
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characterization of eppin s Semenogelin i binding site a contraceptive drug target
Biology of Reproduction, 2012Co-Authors: Erick J. R. Silva, Katherine G. Hamil, Richard T. Richardson, Michael G OrandAbstract:Epididymal protease inhibitor (EPPIN) is found on the surface of spermatozoa and works as a central hub for a sperm surface protein complex (EPPIN protein complex [EPC]) that inhibits sperm motility on the binding of Semenogelin I (SEMG1) during ejaculation. Here, we identify EPPIN's amino acids involved in the interactions within the EPC and demonstrate that EPPIN's sequence C102-P133 contains the major binding site for SEMG1. Within the same region, the sequence F117-P133 binds the EPC-associated protein lactotransferrin (LTF). We show that residues Cys102, Tyr107, and Phe117 in the EPPIN C-terminus are required for SEMG1 binding. Additionally, residues Tyr107 and Phe117 are critically involved in the interaction between EPPIN and LTF. Our findings demonstrate that EPPIN is a key player in the protein-protein interactions within the EPC. Target identification is an important step toward the development of a novel male contraceptive, and the functionality of EPPIN's residues Cys102, Tyr107, and Phe117 offers novel opportunities for contraceptive compounds that inhibit sperm motility by targeting this region of the molecule.
Rudolf Dernick - One of the best experts on this subject based on the ideXlab platform.
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biological activity of prostate specific antigen isolated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and electroelution
Electrophoresis, 1995Co-Authors: Uwe Tessmer, Folkert Donn, Astrid Leuner, Thomas Quack, Rudolf DernickAbstract:: Human prostate-specific antigen (PSA), a 33 kDa kallikrein-like serine protease, occurring in the prostate, in seminal plasma and in blood, was prepared under nonreducing conditions in an enzymatically active form from seminal plasma by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by fast copper staining, electroelution from gel slices and dialysis against isotonic phosphate-buffered saline (PBS). Enzymatic activity was demonstrated for the first time directly by cleavage of Semenogelin, one of the biological substrates of PSA, isolated by the same procedure, i.e. SDS-PAGE and electroelution, but from seminal vesicle fluid. The purified PSA formed SDS-stable complexes with the two major extracellular protease inhibitors in blood, alpha 1-antichymotrypsin (alpha 1-ACH) and alpha 2-macroglobulin (alpha 2-M). PSA isolated under reducing conditions was enzymatically inactive and could not bind to the protease inhibitors alpha 1-ACH and alpha 2-M.
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biological activity of prostate specific antigen isolated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and electroelution
Electrophoresis, 1995Co-Authors: Uwe Tessmer, Folkert Donn, Astrid Leuner, Thomas Quack, Rudolf DernickAbstract:: Human prostate-specific antigen (PSA), a 33 kDa kallikrein-like serine protease, occurring in the prostate, in seminal plasma and in blood, was prepared under nonreducing conditions in an enzymatically active form from seminal plasma by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by fast copper staining, electroelution from gel slices and dialysis against isotonic phosphate-buffered saline (PBS). Enzymatic activity was demonstrated for the first time directly by cleavage of Semenogelin, one of the biological substrates of PSA, isolated by the same procedure, i.e. SDS-PAGE and electroelution, but from seminal vesicle fluid. The purified PSA formed SDS-stable complexes with the two major extracellular protease inhibitors in blood, alpha 1-antichymotrypsin (alpha 1-ACH) and alpha 2-macroglobulin (alpha 2-M). PSA isolated under reducing conditions was enzymatically inactive and could not bind to the protease inhibitors alpha 1-ACH and alpha 2-M.
Michael G Orand - One of the best experts on this subject based on the ideXlab platform.
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characterization of eppin s Semenogelin i binding site a contraceptive drug target
Biology of Reproduction, 2012Co-Authors: Erick J. R. Silva, Katherine G. Hamil, Richard T. Richardson, Michael G OrandAbstract:Epididymal protease inhibitor (EPPIN) is found on the surface of spermatozoa and works as a central hub for a sperm surface protein complex (EPPIN protein complex [EPC]) that inhibits sperm motility on the binding of Semenogelin I (SEMG1) during ejaculation. Here, we identify EPPIN's amino acids involved in the interactions within the EPC and demonstrate that EPPIN's sequence C102-P133 contains the major binding site for SEMG1. Within the same region, the sequence F117-P133 binds the EPC-associated protein lactotransferrin (LTF). We show that residues Cys102, Tyr107, and Phe117 in the EPPIN C-terminus are required for SEMG1 binding. Additionally, residues Tyr107 and Phe117 are critically involved in the interaction between EPPIN and LTF. Our findings demonstrate that EPPIN is a key player in the protein-protein interactions within the EPC. Target identification is an important step toward the development of a novel male contraceptive, and the functionality of EPPIN's residues Cys102, Tyr107, and Phe117 offers novel opportunities for contraceptive compounds that inhibit sperm motility by targeting this region of the molecule.
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loss of calcium in human spermatozoa via eppin the Semenogelin receptor
Biology of Reproduction, 2012Co-Authors: Michael G Orand, Esther E. WidgrenAbstract:The development of a new male contraceptive requires a transition from animal model to human and an understanding of the mechanisms involved in the target's inhibition of human spermatozoan fertility. We now report that Semenogelin (SEMG1) and anti-EPPIN antibodies to a defined target site of 21 amino acids on the C terminal of EPPIN cause the loss of intracellular calcium, as measured by Fluo-4. The loss of intracellular calcium explains our previous observations of an initial loss of progressive motility and eventually the complete loss of motility when spermatozoa are treated with SEMG1 or anti-EPPIN antibodies. Thimerosal can rescue the effects of SEMG1 on motility, implying that internal stores of calcium are not depleted. Additionally, SEMG1 treatment of spermatozoa decreases the intracellular pH, and motility can be rescued by ammonium chloride. The results of this study demonstrate that EPPIN controls sperm motility in the ejaculate by binding SEMG1, resulting in the loss of calcium, most likely through a disturbance of internal pH and an inhibition of uptake mechanisms. However, the exact steps through which the EPPIN-SEMG1 complex exerts its effect on internal calcium levels are unknown. Anti-EPPIN antibodies can substitute for SEMG1, and, therefore, small-molecular weight compounds that mimic anti-EPPIN binding should be able to substitute for SEMG1, providing the basis for a nonantibody, nonhormonal male contraceptive.
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analysis of recombinant human Semenogelin as an inhibitor of human sperm motility
Biology of Reproduction, 2010Co-Authors: Anurag Mitra, Richard T. Richardson, Michael G OrandAbstract:Eppin (epididymal protease inhibitor [SPINLW1]) is present in a protein complex on the human sperm surface that contains lactotransferrin, clusterin, and Semenogelin (SEMG1). During ejaculation the presence of Semenogelin inhibits sperm progressive motility until Semenogelin is hydrolyzed by prostate-specific antigen (PSA). Although eppin binds all three components in its protein complex, the binding of Semenogelin to eppin appears to be critical for the inhibition of progressive motility. The effect of the originally identified seminal plasma motility inhibitor fragment has not been clearly defined on live spermatozoa. Therefore, we have used recombinant Semenogelin (rSEMG1) and its fragments, including a Semenogelin mutant in which cysteine 239 was changed to glycine, coupled with a computer assisted sperm analysis assay to study the motility inhibitory properties of Semenogelin. Each fragment and the mutant were tested for their effects on motility. Recombinant Semenogelin significantly inhibited sperm progressive motility in a dose- and time-dependent manner. The C-terminal Semenogelin fragment (amino acids 164-283) containing cysteine 239 significantly inhibited sperm progressive motility, whereas the N-terminal fragment (amino acids 24-163), a short C-terminal fragment (amino acids 172-215) without cysteine 239, and the mutant fragment (amino acids 24-283 with glycine 239) did not inhibit motility. After treatment with recombinant Semenogelin, spermatozoa could be washed and treated with PSA, partially reversing the inhibition of progressive motility. Cysteine 239 of rSEMG1 appears to be the critical amino acid for both binding to eppin and inhibiting sperm motility.
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inhibition of human sperm motility by contraceptive anti eppin antibodies from infertile male monkeys effect on cyclic adenosine monophosphate
Biology of Reproduction, 2009Co-Authors: Michael G Orand, Stan Beyler, Esther E. Widgren, Richard T. RichardsonAbstract:Epididymal protease inhibitor (eppin [official symbol, SPINLW1]) is of interest as a male contraceptive target because of its specificity and location on the human sperm surface. We have examined the effect of anti-eppin antibodies from infertile male monkeys and the effect of recombinant human Semenogelin on human sperm motility. Anti-eppin antibodies significantly decreased the progressive motility of human spermatozoa as measured by decreased total distance traveled, decreased straight-line distance, and decreased velocity. Anti-eppin treatment of spermatozoa significantly increased the amount of cAMP present in nonprogressive spermatozoa; however, approximately 25% of antibody-treated spermatozoa could be rescued by the addition of cAMP-acetoxymethyl ester, indicating that anti-eppin-treated spermatozoa have a compromised ability to utilize cAMP. Addition of recombinant human Semenogelin has a concentration-dependent inhibitory effect on progressive motility (increased tortuosity and decreased velocity). We tested the hypothesis that anti-eppin antibodies bound to eppin would subsequently block Semenogelin binding to eppin. Anti-eppin antibodies from infertile monkeys inhibited eppin from binding to Semenogelin. Addition of affinity-purified antibodies made to the dominant C-terminal epitope of eppin had an inhibitory effect on progressive motility (increased tortuosity, decreased velocity, and straight distance). Our results suggest that the eppin-Semenogelin binding site is critical for the removal of Semenogelin in vivo during semen liquefaction and for the initiation of progressive motility. We conclude that the eppin-Semenogelin binding site on the surface of human spermatozoa is an ideal target for a nonsteroidal male contraceptive.
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association of eppin with Semenogelin on human spermatozoa
Biology of Reproduction, 2005Co-Authors: Zengjun Wang, Michael G Orand, Esther E. Widgren, Perumal Sivashanmugam, Richard T. RichardsonAbstract:Eppin (SPINLW1; GeneID, 57119) is a single-copy gene encoding a cysteine-rich protein found only in the testis and epididymis, which contains both Kunitz-type and WAP-type four disulfide core protease inhibitor consensus sequences. This study demonstrates that, in seminal plasma and on human spermatozoa following ejaculation, Eppin is bound to Semenogelin I (Sg). Six different experimental approaches: 1) immunoprecipitation from spermatozoa and seminal plasma with anti-Eppin, 2) colocalization in semen and spermatozoa, 3) incubation of recombinant Eppin (rEppin) and rSg and immunoprecipitation with either anti-Eppin or anti-Sg, 4) far-Western blotting of Eppin and Sg, 5) Saturation binding of 125 I-Sg to Eppin, which is competed by unlabeled Sg, and 6) direct binding of 125 I-Sg to Eppin on a blot, all demonstrate that Eppin and Sg bind to each other. To study the specificity of binding, recombinant fragments of Eppin and Sg were made and demonstrate that the Eppin75‐133 C-terminal fragment binds the Sg164‐283 fragment containing the only cysteine in human Sg I (Cys-239). Reduction and carboxymethylation of Cys239 blocks binding of 125 I-rEppin, indicating that a disulfide bond may be necessary for Eppin binding. The physiological significance of the Eppin-Semenogelin complex bound on the surface of ejaculate spermatozoa lies in its ability to provide antimicrobial activity for spermatozoa, which has been reported for both Eppin and Semenogelin-derived peptides, and in its ability to provide for the survival and preparation of spermatozoa for fertility in the female reproductive tract.
Johan Malm - One of the best experts on this subject based on the ideXlab platform.
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a peptide from human Semenogelin i self assembles into a ph responsive hydrogel
Soft Matter, 2015Co-Authors: Birgitta Frohm, Johan Malm, Luigi Gentile, Ulf Olsson, Karin S Akerfeldt, J E Denizio, David Lee, Sara LinseAbstract:The peptide GSFSIQYTYHV derived from human Semenogelin I forms a transparent hydrogel through spontaneous self-assembly in water at neutral pH. Linear rheology measurements demonstrate that the gel shows a dominating elastic response over a large frequency interval. CD, fluorescence and FTIR spectroscopy and cryo-TEM studies imply long fibrillar aggregates of extended β-sheet. Dynamic light scattering data indicate that the fibril lengths are of the order of micrometers. Time-dependent thioflavin T fluorescence shows that fibril formation by GSFSIQYTYHV is a nucleated reaction. The peptide may serve as basis for development of smart biomaterials of low immunogenicity suitable for biomedical applications, including drug delivery and wound healing.
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Cleavage of protein substrates by PSA.
2014Co-Authors: Johanna M. Mattsson, Johan Malm, Magnus Jonsson, Suvi Ravela, Can Hekim, Ale Närvänen, Ulf-håkan Stenman, Hannu KoistinenAbstract:(A) Characterization of the proteolytic activity of PSA (0.2 µM) during 22 h incubation towards different protein substrates (1 µM each, except 0.5 µM Semenogelin I) by SDS-PAGE and silver staining. Approximate molecular weights of the proteins are: PSA (28 kDa), Semenogelin I (50 kDa), Semenogelin II (63 kDa), fibronectin (220 kDa), galectin-3 (26 kDa), IGFBP-3 (30 kDa) and nidogen-1 (130 kDa). The lanes in which ∼50% of the proteins were cleaved are bordered. (B) 1 µM MMP-3 (22 kDa), but not PSA, cleaved 1 µM plasminogen (88 kDa). Also 0.5 M fibronectin was incubated with 1 µM PSA as a control (SDS-PAGE with silver staining).
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The effect of peptides on the proteolytic cleavage of different protein substrates by PSA.
2014Co-Authors: Johanna M. Mattsson, Johan Malm, Magnus Jonsson, Suvi Ravela, Can Hekim, Ale Närvänen, Ulf-håkan Stenman, Hannu KoistinenAbstract:(A) Peptide B2 enhanced the activity of PSA towards all protein substrates more strongly than C4, except for fibronectin this could not be detected. The peptides were preincubated with 0.2 µM PSA for 30 min prior to addition of 0.3–2.5 µM protein substrates and the cleavage of the proteins was detected after 10 min (Semenogelin I), 40 min (Semenogelin II), 4 h (fibronectin and nidogen-1) and 20–22 h (galectin-3 and IGFBP-3) incubation at 37°C. The notation 5* indicates that 5 µg of either peptide (with Semenogelins I and II peptide C4, and with the other proteins B2) was added to the control sample. (B) Concentration of intact IGFBP-3 after incubation with PSA and peptides shown in relation to IGFBP-3 control without added PSA as measured by immunofluorometric assay that recognizes only intact, not cleaved IGFBP-3 (n = 2, mean ± SD).
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Copyright E American Society of Andrology Truncated Semenogelin I Binds Zinc and Is Cleaved by Prostate-Specific Antigen
2013Co-Authors: Magnus Jonsson, Birgitta Frohm, Sara Linse, Ke A Lundwall, Johan MalmAbstract:ABSTRACT: Semenogelins I and II are major coagulum-forming proteins in semen, and they are secreted mainly by the seminal vesicles. These proteins bind Zn 2+ and act as substrates for prostate-specific antigen and transglutaminase. A variant Semenogelin I lacking 60 amino acids has been described that occurs in different populations with an allele frequency of 1%–3%. To better understand the function of the Semenogelins in vivo, our aim was to characterize the properties of the variant form and compare with the wild type. Recombinant proteins were synthesized in insect cells. Binding of Zn 2+ was studied by titration of metal ions in the presence of a zinc (II) fluorophore chelator. SDS-PAGE was used to visualize the results of cleavage by prostate-specific antigen and cross-linking with transglutaminase. We found that the truncated and wild-type Semenogelin molecules had similar Zn 2+-binding properties (ie, a stoichiometry of at least 9–10 mol per mol of protein and an average dissociation constant of 5 mmol/L per site), and they showed also similar susceptibility for degradation by prostate-specific antigen. Furthermore, like the wild-type form, the truncated Semenogelin I was able to serve as a substrate for transglutaminase. These findings imply that the studied characteristics do not depend on a well-defined tertiary structure, or that the deletion has no major effect on the structure responsible for these features
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the major bactericidal activity of human seminal plasma is zinc dependent and derived from fragmentation of the Semenogelins
Journal of Immunology, 2008Co-Authors: Anneli M L Edstrom, Johan Malm, Birgitta Frohm, Julie A Martellini, Aleksander Giwercman, Matthias Morgelin, Alexander M Cole, Ole E SorensenAbstract:One of the major roles of seminal plasma is to provide antimicrobial protection for the spermatozoa in the female reproductive tract. We found that the bactericidal activity of seminal plasma was highest after resolution of the seminal clot and that this antibacterial activity subsequently became greatly diminished. The antibacterial activity was derived from peptides generated by fragmentation of the Semenogelins while the Semenogelin holoproteins displayed no antibacterial activity. After ejaculation the Semenogelin-derived peptides were fragmented to smaller and smaller fragments over time and thereby lost antibacterial activity. This paralleled the loss of antibacterial activity of whole seminal plasma both in vitro and after sexual intercourse. Moreover, the antibacterial activity of the Semenogelin-derived peptides generated in seminal plasma was strictly zinc-dependent both at neutral and low pH. These data provide novel roles for the resolution of seminal clots and for the high zinc concentration in human seminal plasma.
