Localization of the steroid-binding site of the human sex steroid-binding protein of plasma (SBP or SHBG) by site-directed mutagenesis.

The amino-terminal region of the human sex steroid-binding protein of plasma (SBP or SHBG) containing K134 and M139 was found to represent part of the steroid-binding site. This was accomplished by constructing and expressing site-directed mutants having the following replacements: M139L, M139K, M139S, K134A, H235S, and Y57F. The results indicated that M139L and H235S were fully-active, K134A and Y57F were 50 and 67% active, M139K was 7% active, and M139S was inactive. These results support affinity-labeling data indicating that both K134 and M139 are located in or near the site, and suggest that Y57 may play a role in steroid binding. The fully active H235S mutant reveals that H235 is not involved in the steroid-binding process.

Identification of lysine 134 in the steroid-binding site of the sex steroid-binding protein of human plasma.

The sex steroid-binding protein of human plasma SBP (or sex hormone-binding globulin, SHBG) was specifically inhibited with the alkylating affinity label, 17 beta-[[( 2-14C]bromoacetyl)oxy]-5 alpha-androstan-3-one. The natural ligand, 5 alpha-dihydrotestosterone, was shown to protect against inactivation and labeling. The steroid-binding activity of the protein was abolished when approximately 1 mol of label was incorporated into 1 mol of dimeric SBP. In order to identify and locate the labeled amino acid in the steroid-binding site, the steroidal portion of the bound label was first removed and the protein was digested with Achromobacter protease and subdigested with trypsin. Seven radioactive peptides were isolated, sequenced, and found to contain the common sequence QVSGPLTSXR. Residue X was identified as lysine-134 from the SBP amino acid sequence (Walsh, K. A., Titani, K., Kumar, S., Hayes, R., and Petra, P. H. (1986) Biochemistry 25, 7584-7590). The results indicate that only 1 of the 2 lysine-134 residues in the homodimer was labeled. This suggests that the steroid-binding site is constructed from an association of the two subunits in an AB to BA "sandwich" configuration with lysine-134 residue of one subunit on one surface near the D-ring and the lysine-134 of the other subunit at the opposite end of the steroid, or well away from the steroid-binding site. Although the nature of the data does not allow description of a specific role for lysine-134, its proximity to the 17 beta-OH of the steroid nucleus suggests participation in the binding process through direct or indirect hydrogen bonding.

Direct effect of plasma sex hormone binding globulin (SHBG) on the metabolic clearance rate of 17 beta-estradiol in the primate.

Sex hormone binding globulin (SHBG) has been shown to be a major determinant of testosterone clearance in the primate. It has also been suggested that SHBG would also be a determinant of estradiol clearance (MCR-E2). However, published studies have suggested that the MCR-E2 do not always vary with changes in the level of SHBG. Therefore, the present study was undertaken to address this issue. The baseline MCR-E2 was determined in adult male pigtail macaques (Macaca nemestrina). Following the baseline determination of MCR-E2 the animals were infused with either purified human (h) SHBG or antibody against hSHBG, which also has a high degree of cross-reactivity with primate SHBG. Following the infusions of either hSHBG or anti-SHBG, MCR-E2 was again determined. In addition, luteinizing hormone (LH) was measured using a mouse Leydig cell bioassay. Following the infusion of hSHBG, a marked increase in serum SHBG was noted and the MCR-E2 decreased. Associated with the increase in SHBG, the SHBG bound T levels decreased and LH increased. Following the infusion of antibody, serum SHBG decreased, and the MCR-E2 also decreased. With the decrease in SHBG following the antibody infusion, non-SHBG bound T increased and serum LH fell. This study demonstrates that an increase in the serum SHBG levels is associated with a decrease in MCR-E2, however, an acute decrease in serum SHBG also decreases the MCR-E2. This later result demonstrates that factors in addition to serum SHBG binding may be important in determining the MCR-E2.

The amino acid sequence of the sex steroid-binding protein of rabbit serum.

The amino acid sequence of the sex steroid-binding protein (SBP or SHBG) of rabbit serum, specific for binding testosterone and 5 alpha-dihydrotestosterone, was determined using a complementary combination of mass spectrometric and Edman degradation techniques. The monomeric unit of the homodimeric protein is a single chain glycopeptide of 367 amino acid residues, with N-linked oligosaccharide side chains at Asn-345 and Asn-361 and disulfide bonds connecting Cys-158 to Cys-182 and Cys-327 to Cys-355. The polypeptide molecular weight of the monomer calculated from the sequence is 39,769. The molecular weight of the homodimer including 9% carbohydrate is 87,404. The sequence contains a relatively hydrophobic segment between Trp-241 and Leu-282, which includes many leucine residues in an alternating pattern. An amino acid sequence repeat is also located within that segment. Both of these patterns are present in human SBP and in the androgen-binding protein of rat epididymis. The sequence data indicate that the previously reported microheterogeneity of rabbit SBP in sodium dodecyl sulfate-polyacrylamide gel electrophoresis reflects variants generated by differential glycosylation of the monomer rather than different gene products. Seventy-nine percent of the amino acids of rabbit SBP are identical to those of human SBP; rabbit SBP thus joins human SBP and rat androgen-binding protein in one gene family that is distinct from the steroid hormone receptor superfamily. It appears that the problem of binding sex steroid hormones has been solved independently in two different gene families that contain completely different steroid-binding domains. Since the nonhomologous steroid-binding domains of both families of proteins recognize essentially the same steroid structure, it will be interesting to determine the structural basis of the two different protein designs that lead to similar steroid-binding specificity.

Half-life of plasma sex steroid-binding protein (SBP) in the primate.

We have recently shown that the metabolic clearance of testosterone in plasma is directly dependent on sex steroid-binding protein (SBP or SHBG) levels [J. steroid. Biochem. 22, 739 (1986)]. In order to further understand the relationship between these two parameters, we have measured the half-life of SBP in plasma of female rhesus monkeys. SBP was purified to homogeneity from pooled Macaca nemestrina serum, and iodinated with 125I. The labeled protein ([125I]nSBP) was purified by chromatography on DEAE-agarose and fractions identified as immunologically reactive against anti-human SBP were collected. Protein purity of [125I]nSBP was established by SDS gel electrophoresis using an unlabelled Macaca nemestrina SBP as standard. The labeled protein was infused intravenously into two different adult female Macaca mulatta (rhesus) monkeys. Plasma samples were collected at short intervals during the first 24 h after infusion, and then daily for 7-9 days. The clearance profile of labeled SBP in plasma was quantitated by radioactivity measurement and immunoprecipitation. Analysis of the results indicate that the rate of SBP clearance in plasma has two components, the t1/2 (app) of the first component is 7.5 h (r = 0.94), and the t1/2 (app) of the second component is 3.95 days (r = 0.95). Over 90% of the injected 125I-nSBP was removed from plasma within 24 h at a rate corresponding to the t1/2 (app) of the first component. The data indicate that most of the SBP rapidly distributes into extravascular spaces during the first 24 h following infusion, and are consistent with the hypothesis that SBP may be directly involved in sex steroid hormone transport into tissues.

Molecular characterization of the sex steroid binding protein (SBP) of plasma. Re-examination of rabbit SBP and comparison with the human, macaque and baboon proteins.

Physico-chemical characterization of the sex steroid-binding protein, SBP, of rabbit plasma reveals that it is a dimer of mol. wt 85,800 composed of similar subunits of mol. wt 43,000. These data confirm our original proposal for a dimeric structure. The protein contains 9% carbohydrate, comprised of mannose, galactose, N-acetylglucosamine and sialic acid. It is devoid of N-acetylgalactosamine and fucose. The protein binds one molecule of 5 alpha-dihydrotestosterone per dimer with a Kd of 0.89 nM (12 degrees C). Comparison with the human, monkey and baboon SBPs indicates that all these proteins have the same dimeric molecular organization and exhibit microheterogeneity in SDS-PAGE and isoelectricfocusing. Rabbit SBP, however, contains less carbohydrate and has a higher polypeptide molecular weight than all the other SBPs. Spectrophotometric data also indicate that some tryptophan residues are in a different chemical environment than those in other SBPs. The observed microheterogeneity in all four SBP species is due for the most part to variable glycosylation of the subunit and variability at the amino-terminal region of the subunit. Combination of these and other phenomena will generate a significant number of isomeric forms of the SBP subunit which will then interact stoichiometrically to yield active dimeric SBP molecules. These differ slightly from each other depending upon the charge and size of the subunit comprising the dimeric structure, and will result in the observed microheterogeneity of pure SBP preparations. Based on these results along with more recent amino acid sequence data, we conclude that all four SBPs are dimers composed of identical polypeptide chains.

Alterations in sex steroid-binding protein (SBP), corticosteroid-binding globulin (CBG), and steroid hormone concentrations during pregnancy in rhesus macaques.

Temporal relationships between concentrations of sex steroid-binding protein (SBP), corticosteroid-binding globulin (CBG), total and free estradiol, total and free testosterone, cortisol, and progesterone were studied in plasma obtained at 1- to 3-day intervals throughout gestation in six rhesus macaques. Concentrations of SBP and CBG were measured by diethylaminoethyl cellulose filter assays. Total and free steroids were estimated by radioimmunoassay and ultrafiltration dialysis, respectively. We found that SBP was elevated between days 30 and 50 and CBG between days 60 and 140; both then declined until term (167 days). Estradiol increased gradually throughout gestation. Testosterone was elevated between days 10 and 40, then declined, and rose slightly in late gestation until approximately 15 days before delivery, when it increased markedly. Free estradiol and testosterone increased dramatically before parturition. Progesterone was elevated between days 25 and 45 and declined to relatively constant levels thereafter. Cortisol was essentially unchanged throughout gestation. Our data show that in the pregnant rhesus, levels of SBP and CBG vary independently of one another, but both decline before term; concentrations of both total and free estradiol and testosterone increase markedly before parturition; in late gestation, elevated estrogen is not associated with increased levels of SBP or CBG (as it is in human females).

Direct effect of sex steroid-binding protein (SBP) of plasma on the metabolic clearance rate of testosterone in the rhesus macaque.

We report direct evidence for the effect of the sex steroid-binding protein (SBP) on the metabolic clearance rate of testosterone (MCRT). Pure rhesus SBP or human SBP was infused intravenously into three different cycling female rhesus monkeys. MCRT was measured before and after SBP had reached 150-300% of basal levels. A decrease in MCRT was observed in all cases. The effect of SBP on MCRT was tested further in four additional cycling females by infusing immunoaffinity-purified monospecific human SBP antibodies known to cross-react with rhesus SBP. SBP dropped to 54, 40, 4 and 2% of basal levels with a concomitant increase of 118, 190, 320 and 640% of basal MCRT. In one of these animals, pure rabbit SBP was administered after the anti-human SBP infusion resulting in a decrease in MCRT. The magnitude of the SBP effect on MCRT is related to the distribution of testosterone (T) bound to SBP and albumin in the plasma. Calculations show that as long as the percent of T bound to SBP is equal or higher than the percent of T bound to albumin, the influence on MCRT is small. However, if SBP is reduced to the extent that T is bound mostly to albumin, the redistribution of T is associated with a dramatic increase in MCRT. We conclude that under normal conditions each animal has an optimum concentration of plasma SBP which binds a maximum amount of T. If SBP increases above this level, little effect on MCRT will result. However, a drop below the optimum level, as is the case in certain physiological or clinical conditions, will produce a large increase in the clearance of T.

Purification and characterization of the sex steroid binding protein from macaque serum. Comparison with the human protein.

The sex steroid binding protein (SBP) of Macaca mulatta and Macaca nemestrina sera has been purified to homogeneity and chemically characterized. The native protein is a glycoprotein having a molecular weight of approximately 88 000 and is composed of two similar subunits of molecular weight 47 000 as estimated by sodium dodecyl sulfate gel electrophoresis. One molecule of 5 alpha-dihydrotestosterone is bound per dimer with a KD equal to 1.6 nM at 11 degrees C. Isoelectric focusing patterns reveal the presence of at least 12 different forms of dimeric SBP molecules probably resulting from the presence of different amounts or types of carbohydrate side chains. The data indicate a very close similarity in molecular and steroid-binding properties to human SBP and establish the macaque monkey as a valuable animal model to study the physiological role of SBP in humans.

Current status of the molecular structure and function of the plasma sex steroid-binding protein (SBP).

Purification and characterization of the sex steroid-binding protein (SBP) from human, macaque, baboon, and rabbit sera indicate that the protein is composed of two polypeptide chains which associate noncovalently to yield a native structure having molecular weight distributions of about 88,000 for primate SBPs, and 80,000 for rabbit SBP. The subunit molecular weight distributions are 44,000 for human SBP, 47,000 for macaque and baboon SBP's, and 40,000 for rabbit SBP. Isoelectric focusing show extensive microheterogeneity for all four SBPs. The patterns appear to be unique for each species and reveal the presence of at least twelve bands of different colour intensity reflecting a specific spectrum of active SBP molecules. The existence of the large number of dimeric forms of SBP arises through the combination of many variants of the same two subunits containing different amounts and types of carbohydrate sidechains. Physiological studies on the intravenous infusion of pure rhesus SBP, human SBP, and purified monospecific SBP-antibodies into the rhesus reveal an inverse relationship between SBP and the metabolic clearance rate of testosterone. The effect is complex and depends on the concentration of SBP, albumin, and testosterone which in turn influences the distribution of testosterone between albumin and SBP.

Stability of estrogen receptors in frozen human breast tumor tissue.

The effect of freezing on the stability of the estrogen receptors in human breast tumors was measured. Portions of three tumors kept frozen for 3 days after removal from the patients were analyzed by the sucrose density gradient method. The initial measurements demonstrated positive receptor values. The remaining portions were kept frozen for 30 days, and the receptor values were reevaluated. The 8S receptor was 80% destroyed under these freezing conditions. The 4S receptor was found to be more stable. The inactivation reflects a loss of specific binding since the protein concentration was similar in the analyses. These results strongly indicate that human breast tumor specimens should be analyzed as soon as possible or within 3 weeks after freezing.