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Philip H. Petra - One of the best experts on this subject based on the ideXlab platform.
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arginine 140 and isoleucine 141 determine the 17β estradiol Binding specificity of the sex Steroid Binding Protein sbp or shbg of human plasma
Protein Science, 2001Co-Authors: Philip H. Petra, Katherine T. Woodcock, William R. Orr, Elinor T Adman, Christine Groff, Li-ming SuiAbstract:Human sex-Steroid-Binding Protein (SBP, also referred to as SHBG, sex-hormone-Binding globulin) is a 93.4 kD homodimeric plasma glycoProtein, present in most species tested, that specifically binds one mole of testosterone (T), 5α-dihydrotestosterone (DHT), or 17β-estradiol (E2) per mole dimer (Petra 1979, 1991; Westphal 1986; Joseph 1994). All three Steroid hormones compete for the same Binding site. The human SBP monomer contains 373 amino acid residues and three oligosaccharide side chains, one O-linked to T7 and two N-linked to N351 and N367 (Walsh et al. 1986). Removal of these side chains either enzymatically or genetically does not affect Steroid-Binding activity (Petra et al. 1992; Sui et al. 1999). Although SBP binds 2 moles of calcium per monomer (Ross et al. 1985), the metal does not appear to play a role in Steroid Binding or dimerization (Sui et al. 1996); however, it could function in stabilizing the folding of certain regions of the Protein, as indicated by the protection by calcium against heat denaturation (Rosner et al. 1974). Electron microscopy reveals that SBP has a rodlike structure, and calculations from circular dichroism measurements indicate that it contains 15% helix, 43% β-sheet, and 10–16% β-turns (Beck et al., 1997). The physiological role of SBP is to regulate the bioavailability of sex Steroid hormones to target tissues by controlling their metabolic clearance rates in plasma (Bardin and Lipsett 1967; Vermeulen et al. 1969: Petra et al. 1985; Plymate et al. 1990). The Protein has also been proposed to play two additional roles, one in assisting the uptake of hormone by cells (Strel'chyonok et al. 1984; Pardridge 1988) and the other in signaling (Nakhla et al. 1990; Fissore et al. 1994). Four amino acid side chains have been identified as functional in Steroid Binding, Y57 (Petra et al. 2000), M107 (Petra et al. 2000), M139 (Grenot et al. 1992; Sui et al. 1992), and K134 (Namkung et al. 1990); the first two residues only play a role in DHT Binding (Petra et al. 2000). A recently published crystal structure of the N-terminal domain of human SBP (residues 13–188; Grishkovskaya et al. 2000) confirms the presence of M107 and M139 in the Steroid-Binding site; however, Y57 does not make direct contact with the ligand and the peptide region containing K134 did not exhibit electron density and could not be modeled. Because the calcium atoms are located ∼20 Å from the Steroid-Binding site and ∼18 Å from the subunit interface, they do not contribute directly to ligand Binding and dimerization, as previously found (Sui et al. 1996). A most interesting finding is the presence of two Steroid-Binding sites located one in each truncated subunit resulting in a ligand-Binding stoichiometry of 2 moles per dimer; this result is in contrast to that found for the native Protein, which binds 1 mole ligand per mole dimer (Petra et al. 1986 and references cited therein). The amino acid sequences of human and rabbit SBP4 are 80% identical (Walsh et al. 1986; Griffin et al. 1989; Lee et al. 1997). The Steroid-Binding site of the latter contains M133 (Kassab et al. 1998), as well as I101 (Petra et al. 2000), the homologs of M139 and M107 in human SBP (Walsh et al. 1986). The two Proteins bind DHT with similar affinities but rabbit SBP has a markedly reduced affinity for E2 (Rosner and Darmstadt 1973) with a Kd equal to 80 nM, compared to 4 nM for human SBP (Mickelson and Petra 1978). Because both E2 and DHT bind at the same site and the only major difference between the two Steroids lies in the saturation and orientation of ring A, these data suggest that the structural motif recognizing ring A of E2 in human SBP is significantly altered in rabbit SBP. We reasoned that this motif must be specified by residues that are among the 77 residues that are different in the two sequences (excluding the six residues at the N terminus of human SBP that are missing in the rabbit sequence). The experiments described here led to the identification of R140 and I141 as key determinants of E2-Binding specificity in human SBP.
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The sex Steroid Binding Protein (SBP or SHBG) of human plasma: identification of Tyr-57 and Met-107 in the Steroid Binding site.
The Journal of Steroid Biochemistry and Molecular Biology, 2000Co-Authors: Philip H. Petra, Katherine T. Woodcock, William R. Orr, Dat Nguyen, Li-ming SuiAbstract:Abstract Tyrosine-57 (Y57) and methionine-107 (M107) have been identified in the Binding site of the sex Steroid Binding Protein (SBP) (or sex hormone Binding globulin) of human plasma by replacing the two amino acids with a number of residues of varying structure. Replacement of Y57 with phenylalanine resulted in a fourfold increase in the K d of 5α-dihydrotestosterone but left the K d of 17β-estradiol unchanged. Except in two cases, no further loss in Binding took place when replacing Y57 with other residues, suggesting that the phenolic group of Y57 may form a hydrogen bond with the ligand. Replacement of M107 with isoleucine increased the 5α-dihydrotestosterone K d fourfold to a value equal to that of rabbit SBP, which contains isoleucine at the corresponding position; however, the K d of 17β-estradiol remained unchanged. Replacement of M107 with threonine resulted in a tenfold decrease in 5α-dihydrotestosterone Binding affinity, whereas replacement with leucine left the K d unchanged. These data indicate that substitutions on the β-carbon of the amino acid side-chain at position 107 causes significant loss of Binding affinity but, as in the case of Y57, the activity was not totally eliminated. We conclude that Y57 and M107 form part of a structural motif within the Steroid Binding site and specifically contribute Binding energy to ring A of 5α-dihydrotestosterone but not to ring A of 17β-estradiol. We also propose that the integrated contribution of several side chains may be required to optimize the ligand affinity of the Steroid Binding site. This proposal may fit a ‘lock and key’ model where little movement of the side chains occurs during Binding as might be expected for a rigid structure like the Steroid nucleus.
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heterologous expression of wild type and deglycosylated human sex Steroid Binding Protein sbp or shbg in the yeast pichia pastoris characterization of the recombinant Proteins
The Journal of Steroid Biochemistry and Molecular Biology, 1999Co-Authors: Li-ming Sui, I Woo, J Lennon, I Mccann, Philip H. PetraAbstract:Abstract Wild type, partially and fully-deglycosylated human sex Steroid-Binding Protein (SBP or SHBG) cDNAs lacking the native eucaryotic signal sequence were cloned into a yeast expression vector containing the Saccharomyces cerevisiae α-factor for extracellular secretion. Following transformation into Pichia pastoris, the wild type and all constructed mutants were successfully expressed. The levels were lower for the deglycosylated mutants indicating that oligosaccharide side chains may play a role in SBP secretion. Under fermentation conditions, the wild type Protein was expressed at a level of 4 mg/l while the fully-deglycosylated mutant T7A/N351Q/N367Q was expressed at about 1.5 mg/l. The latter was purified from several fermentation runs and was found to be completely deglycosylated, electrophoretically homogeneous and fully active. The aminoterminus was found to have the sequence NH2QSAHDPPAV- indicating that cleavage of the α-factor occurred at the A+7–Q+8 peptide bond. The molecular mass of the subunit was determined to be 39,717.8 Da, which is in complete agreement with the amino acid sequence of the T7A/N351Q/N367/Q mutant. The equilibrium constants for the dissociation of 5α-dihydrotestosterone and Steroid Binding specificity were found to be identical to that of the human plasma Protein indicating that the missing N-terminal segment NH2-LRPVLPT and the removal of oligosaccharide side chains do not affect the stability and active conformation of the Protein. In conclusion, the data presented reveal that the SBP mutant T7A/N351Q/N367/Q is the Protein of choice for solving the three-dimensional structure.
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Secondary structure and shape of plasma sex Steroid-Binding Protein - comparison with domain G of laminin results in a structural model of plasma sex Steroid-Binding Protein
European Journal of Biochemistry, 1997Co-Authors: Konrad Beck, Li-ming Sui, Tanja M. Gruber, Catherine C. Ridgway, William Hughes, Philip H. PetraAbstract:We have analyzed the secondary structure, shape and dimensions of plasma sex Steroid-Binding Protein (SBP) by CD, size-exclusion chromatography and electron microscopy. CD spectra show extrema at 186 nm and 216 nm characteristic for beta-sheet structures. Analysis with different algorithms indicates 15% alpha-helix, 43% beta-sheet and 10-16% beta-turn structures. An irreversible structural change is observed upon heating above 60 degrees C, which correlates with the loss of Steroid-Binding activity. As the SBP sequence shows similarity with domains of several multidomain Proteins, including laminins, we evaluated the structure of domain G of laminin-1. The CD spectrum shows extrema at 200 nm and 216 nm. Deconvolution results in 13% alpha-helix, 32% beta-sheet and 15% beta-turn structures. Steroid-Binding assays indicate that laminin and fragments thereof have no activity. Size-exclusion chromatography reveals that SBP has an extended shape and can be modeled as a cylinder with a length and diameter of 23 nm and 3 nm, respectively. This shape and the dimensions are in agreement with the appearance on electron micrographs. We propose a model for the structure of SBP in which two monomers assemble head to head with the Steroid-Binding site located in the center of the rod-like particle.
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Direct evidence for the localization of the Steroid-Binding site of the plasma sex Steroid-Binding Protein (SBP or SHBG) at the interface between the subunits.
Protein Science, 1996Co-Authors: Li-ming Sui, William Hughes, Agnes J. Hoppe, Philip H. PetraAbstract:Complete dissociation of dimeric plasma sex Steroid-Binding Protein (SBP or SHBG) was obtained in 6 M urea at 10 degrees C. Removal of urea resulted in the refolding of monomers, followed by reformation of dimeric SBP, which migrates with the same mobility as the native Protein. Dimerization does not require Ca+2 or Steroid. Renatured monomers yield dimers with dissociation constants for 5 alpha-dihydrotesterone (DHT) and 17 beta-estradiol (E2) indistinguishable from those of native human SBP. This phenomenon was also demonstrated by mixing human and rabbit SBPs that, upon renaturation, form a hybrid dimer composed of one human subunit and one rabbit subunit. The hybrid binds both DHT and E2 in contrast to rSBP, which only binds the androgen. Therefore, we conclude that (1) docking of the two subunits creates an asymmetric Steroid-Binding site located at the interface between the subunits, and (2) only one face of the dimer defines the specificity for Binding E2 by encompassing portion of a structural motif that recognizes the flat ring A of E2. The remaining portion, which recognizes the saturated ring A of DHT, is shared by both faces of the dimer. Because native monomers do not exist alone, the often-asked question of whether the SBP monomer binds Steroid can be considered meaningless; Steroid-Binding activity is expressed only in the dimeric state. Finally, formation of the hybrid indicates that SBP dimerization represents a conserved event during the molecular evolution of SBP, suggesting that the structural elements responsible for dimerization will be homologous in SBPs from other species.
Knut Erik Tollefsen - One of the best experts on this subject based on the ideXlab platform.
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Binding of alkylphenols and alkylated non phenolics to the rainbow trout oncorhynchus mykiss plasma sex Steroid Binding Protein
Ecotoxicology and Environmental Safety, 2007Co-Authors: Knut Erik TollefsenAbstract:Alkylphenols are well-known endocrine disrupters, mediating effects through the estrogen receptor (ER). Although the estrogenic properties of the alkylphenols are well documented, alternative mechanisms of action are poorly described. In the present work, the interaction of a range of alkyl-substituted phenols and alkyl-substituted non-phenolics with the rainbow trout (Oncorhynchus mykiss) sex Steroid-Binding Protein (rtSBP) were determined by competitive ligand-Binding studies. The role of alkyl chain length and branching, substituent position, number of alkylated groups, and the requirement of a phenolic ring structure were assessed. The results showed that the rtSBP binds to most chemical structures tested, although the highest affinity was obtained for mono-substituted alkylphenols with a chain length of four to eight methyl groups. Interestingly, rtSBP Binding was also observed for non-phenolic compounds such as 4-t-butylcyclohexanol and 4-t-butylnitrobenzene suggesting that the rtSBP has a broad Binding specificity for alkylphenols and alkylated non-phenolics.
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Binding of xenoestrogens to the sex Steroid Binding Protein in plasma from arctic charr salvelinus alpinus l
Comparative Biochemistry and Physiology C-toxicology & Pharmacology, 2004Co-Authors: Knut Erik Tollefsen, J Ovrevik, J StenersenAbstract:Abstract A specific sex Steroid-Binding Protein (SBP) is believed to be involved in regulation of circulating sex Steroids, Steroid delivery to target cells and intracellular signalling in sex Steroid-sensitive tissues. In the present work, interactions between xenoestrogens and the plasma SBP in Arctic charr (Salvelinus alpinus L.) were determined using ligand–Protein Binding studies. The test compounds were all able to displace tritiated 17β-estradiol (E2) from the Arctic charr SBP (acSBP) in a competitive and dose-dependent manner. The acSBP affinities for the xenoestrogens ranged over several orders of magnitude (17β-estradiol≫ethynylestradiol (EE2)>zearalenone (ZEA)>diethylstilbestrol (DES)>genistein (GEN)>bisphenol A (BPA), 4-t-octylphenol (OP)≫o,p′-DDT, and dieldrin (DIN)), but were consistently lower than that of 17β-estradiol (about 4×102–106-fold less potent). The relative Binding affinity (RBA) for selected chemicals were independent of both gender, age and maturation status, as well as variations of acSBP Binding affinity. The affinity of endogenous Steroids and estrogen mimics for the acSBP shows a high correlation to the affinity for the rainbow trout SBP, thus suggesting a phylogenetically conserved ligand-Binding site between closely related species. Furthermore, it is argued that interaction with the acSBP- and SBP-mediated processes may introduce novel pathways for endocrine disruption, which may work in concert with the classical receptor-mediated effects.
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interaction of estrogen mimics singly and in combination with plasma sex Steroid Binding Proteins in rainbow trout oncorhynchus mykiss
Aquatic Toxicology, 2002Co-Authors: Knut Erik TollefsenAbstract:The plasma sex Steroid-Binding Protein (SBP) is believed to be involved in regulating circulating endogenous sex Steroids as well as cellular signal transduction to nuclear Steroid receptors in sex Steroid-sensitive tissues. In this study, a variety of estrogen mimics (EMs), which may contribute to the endocrine disrupting effects observed in fish, were tested for the ability to interact with the rainbow trout plasma sex Steroid-Binding Protein (rtSBP) either singly or in binary combinations. The EMs ethynylestradiol, diethylstilbestrol, 4-hydroxytamoxifen, genistein, zearalenone, 4-t-octylphenol, bisphenol A and o,p′-DDT were all able to displace 1,2,4,6,7-[3H]estradiol from the sex Steroid-Binding site at the rtSBP (Kd=2.1±0.5 nM, Bmax=2963±303 fmol estradiol/mg Protein) in a dose-dependent and competitive manner. The plastizicer n-butyl benzyl phthalate only displayed weak Binding affinity for the rtSBP, whereas the pesticide dieldrin was not able to compete for the high affinity estradiol-Binding sites in plasma. None of the compounds tested was able to clearly promote the Binding of the others when given in combination, indicating that synergy did not occur at the ligand-SBP Binding level. The rtSBP Binding affinity for EMs ranged over several orders of magnitude, but were consistently lower than those for the endogenous sex Steroids (about 102–106 less potent). The results suggest that the presence of rtSBP may potentially modulate the bioavailability of EMs to estrogen receptors relative to each other and to the endogenous sex Steroids themselves. Since the Binding of the endogenous ligands is reversible, SBP-bound estrogens (or androgens) potentially may also become displaced by potent EMs.
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partial characterization of a sex Steroid Binding Protein in plasma from arctic charr salvelinus alpinus l
General and Comparative Endocrinology, 2001Co-Authors: J Ovrevik, J Stenersen, Kjell J Nilssen, Knut Erik TollefsenAbstract:Abstract A sex Steroid-Binding Protein (SBP) that binds 17β-estradiol with high affinity and moderate capacity was identified in the plasma from Arctic charr (Salvelinus alpinus L.) sampled during the early stage of gonadal maturation in June and prior to spawning in October. Maximum specific Binding (Bmax) and equilibrium dissociation constant (Kd) of males (Bmax = 2122 fmol E2/mg Protein, Kd = 1.9 nM), females (Bmax = 4115 fmol E2/mg Protein, Kd = 3.0 nM), and juveniles (Bmax = 4355 fmol E2/mg Protein, Kd = 1.8 nM) resembled Binding characteristics of SBP from related species. The early-maturing females displayed both Bmax and Kd values significantly higher than those of males (June samples). No significant differences in Binding characteristics between fully matured males or females and immature juveniles were observed in the October samples. Interestingly, despite large individual variations there was a strong correlation between SBP levels and affinity. The association rate for 17β-estradiol was rapid (t1/2 ≈ 1–2 min) compared with the dissociation rate (t1/2 ≈ 3 h). Several native hormones (estrogens, androgens, and progesterone) were able to compete with tritiated 17β-estradiol for the Binding site. Gel filtration chromatography demonstrated a peak of estradiol Binding at approximately 60 kDa, when eluted on a Sephadex S-200 HR column.
Li-ming Sui - One of the best experts on this subject based on the ideXlab platform.
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arginine 140 and isoleucine 141 determine the 17β estradiol Binding specificity of the sex Steroid Binding Protein sbp or shbg of human plasma
Protein Science, 2001Co-Authors: Philip H. Petra, Katherine T. Woodcock, William R. Orr, Elinor T Adman, Christine Groff, Li-ming SuiAbstract:Human sex-Steroid-Binding Protein (SBP, also referred to as SHBG, sex-hormone-Binding globulin) is a 93.4 kD homodimeric plasma glycoProtein, present in most species tested, that specifically binds one mole of testosterone (T), 5α-dihydrotestosterone (DHT), or 17β-estradiol (E2) per mole dimer (Petra 1979, 1991; Westphal 1986; Joseph 1994). All three Steroid hormones compete for the same Binding site. The human SBP monomer contains 373 amino acid residues and three oligosaccharide side chains, one O-linked to T7 and two N-linked to N351 and N367 (Walsh et al. 1986). Removal of these side chains either enzymatically or genetically does not affect Steroid-Binding activity (Petra et al. 1992; Sui et al. 1999). Although SBP binds 2 moles of calcium per monomer (Ross et al. 1985), the metal does not appear to play a role in Steroid Binding or dimerization (Sui et al. 1996); however, it could function in stabilizing the folding of certain regions of the Protein, as indicated by the protection by calcium against heat denaturation (Rosner et al. 1974). Electron microscopy reveals that SBP has a rodlike structure, and calculations from circular dichroism measurements indicate that it contains 15% helix, 43% β-sheet, and 10–16% β-turns (Beck et al., 1997). The physiological role of SBP is to regulate the bioavailability of sex Steroid hormones to target tissues by controlling their metabolic clearance rates in plasma (Bardin and Lipsett 1967; Vermeulen et al. 1969: Petra et al. 1985; Plymate et al. 1990). The Protein has also been proposed to play two additional roles, one in assisting the uptake of hormone by cells (Strel'chyonok et al. 1984; Pardridge 1988) and the other in signaling (Nakhla et al. 1990; Fissore et al. 1994). Four amino acid side chains have been identified as functional in Steroid Binding, Y57 (Petra et al. 2000), M107 (Petra et al. 2000), M139 (Grenot et al. 1992; Sui et al. 1992), and K134 (Namkung et al. 1990); the first two residues only play a role in DHT Binding (Petra et al. 2000). A recently published crystal structure of the N-terminal domain of human SBP (residues 13–188; Grishkovskaya et al. 2000) confirms the presence of M107 and M139 in the Steroid-Binding site; however, Y57 does not make direct contact with the ligand and the peptide region containing K134 did not exhibit electron density and could not be modeled. Because the calcium atoms are located ∼20 Å from the Steroid-Binding site and ∼18 Å from the subunit interface, they do not contribute directly to ligand Binding and dimerization, as previously found (Sui et al. 1996). A most interesting finding is the presence of two Steroid-Binding sites located one in each truncated subunit resulting in a ligand-Binding stoichiometry of 2 moles per dimer; this result is in contrast to that found for the native Protein, which binds 1 mole ligand per mole dimer (Petra et al. 1986 and references cited therein). The amino acid sequences of human and rabbit SBP4 are 80% identical (Walsh et al. 1986; Griffin et al. 1989; Lee et al. 1997). The Steroid-Binding site of the latter contains M133 (Kassab et al. 1998), as well as I101 (Petra et al. 2000), the homologs of M139 and M107 in human SBP (Walsh et al. 1986). The two Proteins bind DHT with similar affinities but rabbit SBP has a markedly reduced affinity for E2 (Rosner and Darmstadt 1973) with a Kd equal to 80 nM, compared to 4 nM for human SBP (Mickelson and Petra 1978). Because both E2 and DHT bind at the same site and the only major difference between the two Steroids lies in the saturation and orientation of ring A, these data suggest that the structural motif recognizing ring A of E2 in human SBP is significantly altered in rabbit SBP. We reasoned that this motif must be specified by residues that are among the 77 residues that are different in the two sequences (excluding the six residues at the N terminus of human SBP that are missing in the rabbit sequence). The experiments described here led to the identification of R140 and I141 as key determinants of E2-Binding specificity in human SBP.
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The sex Steroid Binding Protein (SBP or SHBG) of human plasma: identification of Tyr-57 and Met-107 in the Steroid Binding site.
The Journal of Steroid Biochemistry and Molecular Biology, 2000Co-Authors: Philip H. Petra, Katherine T. Woodcock, William R. Orr, Dat Nguyen, Li-ming SuiAbstract:Abstract Tyrosine-57 (Y57) and methionine-107 (M107) have been identified in the Binding site of the sex Steroid Binding Protein (SBP) (or sex hormone Binding globulin) of human plasma by replacing the two amino acids with a number of residues of varying structure. Replacement of Y57 with phenylalanine resulted in a fourfold increase in the K d of 5α-dihydrotestosterone but left the K d of 17β-estradiol unchanged. Except in two cases, no further loss in Binding took place when replacing Y57 with other residues, suggesting that the phenolic group of Y57 may form a hydrogen bond with the ligand. Replacement of M107 with isoleucine increased the 5α-dihydrotestosterone K d fourfold to a value equal to that of rabbit SBP, which contains isoleucine at the corresponding position; however, the K d of 17β-estradiol remained unchanged. Replacement of M107 with threonine resulted in a tenfold decrease in 5α-dihydrotestosterone Binding affinity, whereas replacement with leucine left the K d unchanged. These data indicate that substitutions on the β-carbon of the amino acid side-chain at position 107 causes significant loss of Binding affinity but, as in the case of Y57, the activity was not totally eliminated. We conclude that Y57 and M107 form part of a structural motif within the Steroid Binding site and specifically contribute Binding energy to ring A of 5α-dihydrotestosterone but not to ring A of 17β-estradiol. We also propose that the integrated contribution of several side chains may be required to optimize the ligand affinity of the Steroid Binding site. This proposal may fit a ‘lock and key’ model where little movement of the side chains occurs during Binding as might be expected for a rigid structure like the Steroid nucleus.
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heterologous expression of wild type and deglycosylated human sex Steroid Binding Protein sbp or shbg in the yeast pichia pastoris characterization of the recombinant Proteins
The Journal of Steroid Biochemistry and Molecular Biology, 1999Co-Authors: Li-ming Sui, I Woo, J Lennon, I Mccann, Philip H. PetraAbstract:Abstract Wild type, partially and fully-deglycosylated human sex Steroid-Binding Protein (SBP or SHBG) cDNAs lacking the native eucaryotic signal sequence were cloned into a yeast expression vector containing the Saccharomyces cerevisiae α-factor for extracellular secretion. Following transformation into Pichia pastoris, the wild type and all constructed mutants were successfully expressed. The levels were lower for the deglycosylated mutants indicating that oligosaccharide side chains may play a role in SBP secretion. Under fermentation conditions, the wild type Protein was expressed at a level of 4 mg/l while the fully-deglycosylated mutant T7A/N351Q/N367Q was expressed at about 1.5 mg/l. The latter was purified from several fermentation runs and was found to be completely deglycosylated, electrophoretically homogeneous and fully active. The aminoterminus was found to have the sequence NH2QSAHDPPAV- indicating that cleavage of the α-factor occurred at the A+7–Q+8 peptide bond. The molecular mass of the subunit was determined to be 39,717.8 Da, which is in complete agreement with the amino acid sequence of the T7A/N351Q/N367/Q mutant. The equilibrium constants for the dissociation of 5α-dihydrotestosterone and Steroid Binding specificity were found to be identical to that of the human plasma Protein indicating that the missing N-terminal segment NH2-LRPVLPT and the removal of oligosaccharide side chains do not affect the stability and active conformation of the Protein. In conclusion, the data presented reveal that the SBP mutant T7A/N351Q/N367/Q is the Protein of choice for solving the three-dimensional structure.
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Secondary structure and shape of plasma sex Steroid-Binding Protein - comparison with domain G of laminin results in a structural model of plasma sex Steroid-Binding Protein
European Journal of Biochemistry, 1997Co-Authors: Konrad Beck, Li-ming Sui, Tanja M. Gruber, Catherine C. Ridgway, William Hughes, Philip H. PetraAbstract:We have analyzed the secondary structure, shape and dimensions of plasma sex Steroid-Binding Protein (SBP) by CD, size-exclusion chromatography and electron microscopy. CD spectra show extrema at 186 nm and 216 nm characteristic for beta-sheet structures. Analysis with different algorithms indicates 15% alpha-helix, 43% beta-sheet and 10-16% beta-turn structures. An irreversible structural change is observed upon heating above 60 degrees C, which correlates with the loss of Steroid-Binding activity. As the SBP sequence shows similarity with domains of several multidomain Proteins, including laminins, we evaluated the structure of domain G of laminin-1. The CD spectrum shows extrema at 200 nm and 216 nm. Deconvolution results in 13% alpha-helix, 32% beta-sheet and 15% beta-turn structures. Steroid-Binding assays indicate that laminin and fragments thereof have no activity. Size-exclusion chromatography reveals that SBP has an extended shape and can be modeled as a cylinder with a length and diameter of 23 nm and 3 nm, respectively. This shape and the dimensions are in agreement with the appearance on electron micrographs. We propose a model for the structure of SBP in which two monomers assemble head to head with the Steroid-Binding site located in the center of the rod-like particle.
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Direct evidence for the localization of the Steroid-Binding site of the plasma sex Steroid-Binding Protein (SBP or SHBG) at the interface between the subunits.
Protein Science, 1996Co-Authors: Li-ming Sui, William Hughes, Agnes J. Hoppe, Philip H. PetraAbstract:Complete dissociation of dimeric plasma sex Steroid-Binding Protein (SBP or SHBG) was obtained in 6 M urea at 10 degrees C. Removal of urea resulted in the refolding of monomers, followed by reformation of dimeric SBP, which migrates with the same mobility as the native Protein. Dimerization does not require Ca+2 or Steroid. Renatured monomers yield dimers with dissociation constants for 5 alpha-dihydrotesterone (DHT) and 17 beta-estradiol (E2) indistinguishable from those of native human SBP. This phenomenon was also demonstrated by mixing human and rabbit SBPs that, upon renaturation, form a hybrid dimer composed of one human subunit and one rabbit subunit. The hybrid binds both DHT and E2 in contrast to rSBP, which only binds the androgen. Therefore, we conclude that (1) docking of the two subunits creates an asymmetric Steroid-Binding site located at the interface between the subunits, and (2) only one face of the dimer defines the specificity for Binding E2 by encompassing portion of a structural motif that recognizes the flat ring A of E2. The remaining portion, which recognizes the saturated ring A of DHT, is shared by both faces of the dimer. Because native monomers do not exist alone, the often-asked question of whether the SBP monomer binds Steroid can be considered meaningless; Steroid-Binding activity is expressed only in the dimeric state. Finally, formation of the hybrid indicates that SBP dimerization represents a conserved event during the molecular evolution of SBP, suggesting that the structural elements responsible for dimerization will be homologous in SBPs from other species.
Clelia M Riera - One of the best experts on this subject based on the ideXlab platform.
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prostatein or Steroid Binding Protein psbp induces experimental autoimmune prostatitis eap in nod mice
Clinical Immunology, 2002Co-Authors: Virginia E Rivero, Claude Carnaud, Clelia M RieraAbstract:Abstract In a previous study, we showed that nonobese diabetic (NOD) mice, a strain that present an inherited predisposition to develop both spontaneous and induced autoimmune lesions, are susceptible to the induction of experimental autoimmune prostatitis (EAP), developing a severe inflammatory reaction in the prostate, accompanied by humoral and T-cell-mediated responses. In this study we asked whether the Protein Steroid Binding Protein (PSBP) or prostatein (a major autoantigen in the rat model of EAP) is a potential autoantigen in the NOD mouse model and examined the ability of purified PSBP to induce EAP in this strain. Our results indicate clearly that NOD male mice react immunologically to PSBP by developing lymphocytic inflammatory lesions in prostatic tissue and producing both a cellular- and humoral-specific autoimmune response. But our results suggest also the existence of other prostatic autoantigens present only in total prostate extract. Such additional antigens could enhance the autoimmune response and result in more severe forms of inflammation. We also analyzed the respective contributions of MHC antigens and CD4/CD8 T-cell subsets in NOD mice lacking expression of β 2-microglobulin (NOD.β2m°/°) or MHC class II β chain (NOD.Aβ°/°) and demonstrate an essential role for CD4 + T cells in the development of EAP in the NOD model. In conclusion, we demonstrate that PSBP is an autoantigen recognized by the NOD immune system, capable of generating humoral and cellular autoimmune responses and of inducing EAP. Moreover, using selected knock-out NOD mice we demonstrate an essential role for CD4 + T cells in the development of EAP.
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identification of rat prostatic Steroid Binding Protein psbp as an immunosuppressive factor
Journal of Reproductive Immunology, 2001Co-Authors: Mariana Maccioni, Clelia M Riera, Virginia E RiveroAbstract:Abstract Prostatic Steroid Binding Protein (PSBP) is the major Protein produced (∼20% of the total cytosolic Protein) and secreted into the seminal fluid by the rat ventral prostate but its physiological function has not been elucidated yet. Since PSBP is secreted into the seminal fluid (which is itself a potent immunosuppressor) and has strong homology with uteroglobin (which possess an important anti-inflammatory function) our aim was to determine what effect, if any, PSBP would have on the immune system. With that purpose in mind we performed mononuclear cell cultures in the presence or absence of purified PSBP and analysed the effect of this Protein on different functional parameters. PSBP inhibits the mitogen-induced proliferation of normal rat spleen mononuclear cells (MNC) specifically and in a dose-dependent manner. It reduces the production of IL-2 and the expression of its receptor (analysed by flow cytometry) which are important events for lymphocyte proliferation. Also, PSBP was able to inhibit OVA-specific proliferation of lymph node cells from previously primed animals. The immunosuppressive effect of PSBP is not due to an inherent toxic effect to the cells, since the cell viability was kept intact at the different times of culture studied. We also analysed the effect of rat PSBP on mitogen-induced proliferation of mouse spleen and human blood MNC. The proliferation was strongly abolished in a dose-dependent and non-species specific fashion. Moreover, PSBP strongly inhibits the human mixed lymphocyte reaction. Taken together, the present data support evidence for a new type of function for PSBP. We report that PSBP is a potent immunosuppressor factor and we describe its effect on the immune function in vitro. Here, we discuss the possible implications of these findings in the protection of sperm from immunologic damage in the feminine reproductive tract.
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prostatein or rat prostatic Steroid Binding Protein is a major autoantigen in experimental autoimmune prostatitis
Clinical and Experimental Immunology, 1998Co-Authors: Mariana Maccioni, Virginia E Rivero, Clelia M RieraAbstract:Experimental autoimmune prostatitis (EAP) is a disease that could be considered an experimental model of human non-bacterial prostatitis. In this experimental model, male rats are intradermally immunized with a saline extract of male sex accessory glands (RAG) in an adequate adjuvant. The prostatitis observed in the immunized animals develops as a consequence of the immune response against RAG antigens, and the histological lesion is strikingly similar to the pattern of prostatic inflammation observed in the human disease. In this study, we purified one of the prostatic autoantigens recognized by the autoantibodies in our model. Amino acid sequence analysis identified the purified Protein as prostatein or rat prostatic Steroid Binding Protein, a member of the uteroglobin superfamily. Prostatein was recognized not only by the humoral autoimmune response, but also by the cellular autoimmune response. Certainly, the DTH response and lymph node cell proliferative assays against prostatein in immunized animals yielded positive results. Prostatein is not only the target of the autoimmune response in animals immunized with the whole extract, but also an inducing antigen of the disease. Purified prostatein, when incorporated to an adequate adjuvant, elicited cellular and humoral autoimmune response and lesion in the prostate gland. The identification of one of the target antigens in autoimmune prostatitis has provided a further refinement and characterization of our model, which could serve for a better understanding of the aetiology, pathogenesis and pathophysiology of non-bacterial prostatitis.
Virginia E Rivero - One of the best experts on this subject based on the ideXlab platform.
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prostatein or Steroid Binding Protein psbp induces experimental autoimmune prostatitis eap in nod mice
Clinical Immunology, 2002Co-Authors: Virginia E Rivero, Claude Carnaud, Clelia M RieraAbstract:Abstract In a previous study, we showed that nonobese diabetic (NOD) mice, a strain that present an inherited predisposition to develop both spontaneous and induced autoimmune lesions, are susceptible to the induction of experimental autoimmune prostatitis (EAP), developing a severe inflammatory reaction in the prostate, accompanied by humoral and T-cell-mediated responses. In this study we asked whether the Protein Steroid Binding Protein (PSBP) or prostatein (a major autoantigen in the rat model of EAP) is a potential autoantigen in the NOD mouse model and examined the ability of purified PSBP to induce EAP in this strain. Our results indicate clearly that NOD male mice react immunologically to PSBP by developing lymphocytic inflammatory lesions in prostatic tissue and producing both a cellular- and humoral-specific autoimmune response. But our results suggest also the existence of other prostatic autoantigens present only in total prostate extract. Such additional antigens could enhance the autoimmune response and result in more severe forms of inflammation. We also analyzed the respective contributions of MHC antigens and CD4/CD8 T-cell subsets in NOD mice lacking expression of β 2-microglobulin (NOD.β2m°/°) or MHC class II β chain (NOD.Aβ°/°) and demonstrate an essential role for CD4 + T cells in the development of EAP in the NOD model. In conclusion, we demonstrate that PSBP is an autoantigen recognized by the NOD immune system, capable of generating humoral and cellular autoimmune responses and of inducing EAP. Moreover, using selected knock-out NOD mice we demonstrate an essential role for CD4 + T cells in the development of EAP.
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identification of rat prostatic Steroid Binding Protein psbp as an immunosuppressive factor
Journal of Reproductive Immunology, 2001Co-Authors: Mariana Maccioni, Clelia M Riera, Virginia E RiveroAbstract:Abstract Prostatic Steroid Binding Protein (PSBP) is the major Protein produced (∼20% of the total cytosolic Protein) and secreted into the seminal fluid by the rat ventral prostate but its physiological function has not been elucidated yet. Since PSBP is secreted into the seminal fluid (which is itself a potent immunosuppressor) and has strong homology with uteroglobin (which possess an important anti-inflammatory function) our aim was to determine what effect, if any, PSBP would have on the immune system. With that purpose in mind we performed mononuclear cell cultures in the presence or absence of purified PSBP and analysed the effect of this Protein on different functional parameters. PSBP inhibits the mitogen-induced proliferation of normal rat spleen mononuclear cells (MNC) specifically and in a dose-dependent manner. It reduces the production of IL-2 and the expression of its receptor (analysed by flow cytometry) which are important events for lymphocyte proliferation. Also, PSBP was able to inhibit OVA-specific proliferation of lymph node cells from previously primed animals. The immunosuppressive effect of PSBP is not due to an inherent toxic effect to the cells, since the cell viability was kept intact at the different times of culture studied. We also analysed the effect of rat PSBP on mitogen-induced proliferation of mouse spleen and human blood MNC. The proliferation was strongly abolished in a dose-dependent and non-species specific fashion. Moreover, PSBP strongly inhibits the human mixed lymphocyte reaction. Taken together, the present data support evidence for a new type of function for PSBP. We report that PSBP is a potent immunosuppressor factor and we describe its effect on the immune function in vitro. Here, we discuss the possible implications of these findings in the protection of sperm from immunologic damage in the feminine reproductive tract.
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prostatein or rat prostatic Steroid Binding Protein is a major autoantigen in experimental autoimmune prostatitis
Clinical and Experimental Immunology, 1998Co-Authors: Mariana Maccioni, Virginia E Rivero, Clelia M RieraAbstract:Experimental autoimmune prostatitis (EAP) is a disease that could be considered an experimental model of human non-bacterial prostatitis. In this experimental model, male rats are intradermally immunized with a saline extract of male sex accessory glands (RAG) in an adequate adjuvant. The prostatitis observed in the immunized animals develops as a consequence of the immune response against RAG antigens, and the histological lesion is strikingly similar to the pattern of prostatic inflammation observed in the human disease. In this study, we purified one of the prostatic autoantigens recognized by the autoantibodies in our model. Amino acid sequence analysis identified the purified Protein as prostatein or rat prostatic Steroid Binding Protein, a member of the uteroglobin superfamily. Prostatein was recognized not only by the humoral autoimmune response, but also by the cellular autoimmune response. Certainly, the DTH response and lymph node cell proliferative assays against prostatein in immunized animals yielded positive results. Prostatein is not only the target of the autoimmune response in animals immunized with the whole extract, but also an inducing antigen of the disease. Purified prostatein, when incorporated to an adequate adjuvant, elicited cellular and humoral autoimmune response and lesion in the prostate gland. The identification of one of the target antigens in autoimmune prostatitis has provided a further refinement and characterization of our model, which could serve for a better understanding of the aetiology, pathogenesis and pathophysiology of non-bacterial prostatitis.