Genetic variations in human testosterone-estradiol binding globulin

The human testosterone-estradiol-binding globulin (hTeBG) is a plasma heterogeneous glycoprotein with high affinity for a number of circulating steroid hormones. The heterogeneity originates from differential glycosylation of a common protein precursor. Analysis of desialylated hTeBG by isoelectric focusing (IEF) has revealed that microheterogeneity could be partly attributed to variability in sialic acid content or rearrangement of amino acid composition. We have studied this possibility by the analysis of desialylated serum hTeBG by Western blotting of proteins previously separated on IEF-gels. Two distinct well-defined IEF patterns were identified. The most frequent consisted of two major IEF-bands of equal color intensity. The other pattern consisting of four IEF-bands was present in only 5.55% of the total serum samples analyzed. Family studies showed that these phenotypes were autosomally inherited with a simple Mendelian transmission and allele frequencies had an excellent agreement between the observed and expected phenotypes. Androgen affinity constants and serum concentrations of hTeBG variant were similar to those of normal hTeBG. Molecular analyses of each of the exons of hTeBG gene by denaturing gradient gel electrophoresis revealed the presence of a point mutation in exon 8. The studies presented herein confirm and extend previous reports on the existence of structural variants of hTeBG. In addition, the mutation reported in this study is probably the same as that recently identified within numerous ethnic groups throughout the world, thus further supporting the concept of a two allele gene worldwide concoding hTeBG.     

Analysis of the oligosaccharides on androgen-binding proteins: Implications concerning their role in structure/function relationships

Rat androgen-binding protein (rABP), human testosterone-binding globulin (hTeBG) and rabbit (rb) TeBG are heterodimeric proteins. The source of the heterogeneity arises from the differential glycosylation of a common protein core. This glycosylation results in a heavy subunit (more glycosylation) and a light subunit (less glycosylation). Glycosylation is one factor responsible for multiple charged species seen when rABP, hTeBG, and rbTeBG are analyzed by two-dimensional gel electrophoresis. Enzymatic digestion with the endoglycosidase, peptide: N-glycosidase F indicated that all three proteins have asparagine (Asn)-linked oligosaccharides as their major glycan substituent. Treatment with exoglycosidases provided evidence for terminal sialic acid, galactose and mannose and N-acetylglucosamine residues. About 16–22% of the mass of the heavy subunit and about 8–14% of the mass of the light subunit is contributed by carbohydrate.

Serial lectin chromatography indicated that rABP is glycosylated differently from hTeBG and rbTeBG. About 40% of the rABP contains tri and tetraantennary complex oligosaccharides, while only about 20% of the hTeBG and TeBG from pregnant rabbits contains these types of glycans. About 9% of the TeBG from male rabbits bears these types of oligosaccharides. All of the biantennary complex oligosaccharides on rABP are fucosylated on the chitobiose core, but only 8% of those on hTeBG and none of those on rbTeBG are fucosylated in this manner. All three proteins are glycosylated at more than one site. The data indicate that the proteins may have more than one type of oligosaccharide on them. It is likely that differences in glycosylation are responsible for different physiological roles of the proteins.     

There are two forms of androgen binding protein in human testes. Comparison of their protomeric variants with serum testosterone-estradiol binding globulin

To determine how the androgen binding protein in human testes (hABP) is related to the serum protein, testosterone-estradiol binding globulin (hTeBG), both proteins were isolated and compared. The hABP in extracts of human testes was composed of two molecular species based on concanavalin A (ConA)-Sepharose chromatography. Form I hABP did not interact with ConA while Form II hABP bound to ConA and eluted with alpha-methylmannoside. Form I and Form II hABP from five batches of testes were then purified approximately 30,500- and 30,000-fold to apparent homogeneity by high-performance liquid chromatography and compared with hTeBG isolated from human pregnancy serum. Fractionation of both forms of hABP and hTeBG by polyacrylamide gel electrophoresis in the absence of sodium dodecyl sulfate suggested that the native forms of these proteins were indistinguishable. However, analysis of the purified proteins on sodium dodecyl sulfate-containing polyacrylamide gels indicated that all three were dimers and that each was composed of monomers of at least two sizes which were not present in equimolar concentrations. Two distinctive monomers or protomers of each protein were designated as heavy (H) and light (L) according to their electrophoretic mobilities on sodium dodecyl sulfate-polyacrylamide gels. The H and L protomers of Form I hABP showed apparent molecular weights of 55,000 and 52,000, respectively, in all preparations and were usually present in a 4:5 ratio (H:L). The two components of Form II hABP had apparent molecular weights of 53,000 and 48,000, respectively, and existed in a ratio of approximately 20:1. These two components could not be distinguished in some preparations where Form II hABP migrated as a broad band rather than as distinct protomers. By contrast, hTeBG, which was similar to Form II hABP with respect to ConA binding, always exhibited discrete H and L protomers in a 10:1 ratio. Photolysis of these highly purified proteins with delta 6-[3H]testosterone resulted in specific covalent labeling of their binding sites, confirming that the products identified by silver staining and immunoblotting were indeed steroid binding proteins. The H and L protomers of Form I hABP and hTeBG were separated and examined by peptide mapping using Staphylococcus aureus protease V8 and chymotrypsin. The comparison of the respective fragmentation patterns of protomers indicated that Form I hABP and hTeBG contained distinctive peptides.(ABSTRACT TRUNCATED AT 400 WORDS)     

Hormonal regulation of rat androgen-binding protein (ABP) messenger ribonucleic acid and homology of human testosterone-estradiol-binding globulin and ABP complementary deoxyribonucleic acids

Complementary DNA clones coding for rat androgen-binding protein (rABP) were isolated from a rat testis cDNA library constructed in the bacteriophage lambda gt11. The library was screened immunochemically, using two different antibodies against rABP. The identity of the isolated clones was confirmed by epitope selection and DNA sequence analysis. The mRNA encoding rABP could be detected in the testes of 20- and 46-day-old-rats, but not in the 10-day-old rats by hybridization with 32P-labeled rABP cDNA in a Northern blot of poly(A)+-RNA fractioned by agarose gel electrophoresis. No hybridization signal was seen with poly(A)+-RNA isolated from kidney and liver. The rABP mRNA appeared as a single species with a size of 1.65 kilobase, sufficient to encode a protein of 42,000 daltons. The concentration of rABP mRNA in the testes of 37-day-old hypophysectomized rats increased after treatment with testosterone and FSH, given alone or in combination. Sequence and hybridization analysis of cDNAs for rABP, human testosterone-estradiol-binding globulin, and human ABP demonstrates that the cDNAs for human testosterone-estradiol binding globulin and human ABP have greater sequence similarity with each other than either has with rABP.     

A cDNA coding for human sex hormone binding globulin. Homology to vitamin K-dependent protein S

Affinity purified antibodies to human sex hormone binding globulin (SHBG) were used in screening a human liver cDNA library, constructed in the expression vector lambda gt11. One clone, identified as producing recombinant SHBG, carried a cDNA insert of 1.1 kb. The nucleotide sequence of the insert had an open reading frame coding for 356 amino acid residues. The coding sequence was followed by a short 3'-region of 19 non-translated nucleotides and a poly(A) tail. Confirmation that the cDNA clone represented human SHBG was obtained by the finding of a complete agreement in amino acid sequence with several peptide fragments generated from purified SHBG by proteolytic cleavage. The primary structure of SHBG shows a considerable homology to that of protein S, a vitamin K-dependent protein with functions in the coagulation system.     

Homology model of human corticosteroid binding globulin: a study of its steroid binding ability and a plausible mechanism of steroid hormone release at the site of inflammation

Corticosteroid binding globulin (CBG) and thyroxin binding globulin (TBG) both belong to the same SERPIN superfamily of serine-proteinase inhibitors but in the course of evolution CBG has adapted to its new role as a transport agent of insoluble hormones. CBG binds corticosteroids in plasma, delivering them to sites of inflammation to modify the inflammatory response. CBG is an effective drug carrier for genetic manipulation, and hence there is immense biological interest in the location of the hormone binding site. The crystal structure of human CBG (hCBG) has not been determined, but sequence alignment with other SERPINs suggests that it conforms as a whole to the tertiary structure shared by the superfamily. Human CBG shares 52.15% and 55.50% sequence similarity with alpha1-antitrypsin and alpha1-antichymotrypsin, respectively. Multiple sequence alignment among the three sequences shows 73 conserved regions. The molecular structures of alpha1-antitrypsin and alpha1-antichymotrypsin, the archetype of the SERPIN superfamily, obtained by X-ray diffraction methods are used to develop a homology model of hCBG. Energy minimization was applied to the model to refine the structure further. The homology model of hCBG contains 371 residues (His13 to Val383 ). The secondary structure comprises 11 helices, 15 turns and 11 sheets. The putative corticosteroid binding region is found to exist in a pocket between beta-sheets S4, S10, S11 and alpha helix H10. Both cortisol and aldosterone are docked to the elongated hydrophobic ligand binding pocket with the polar residues at the two extremities. A difference accessible surface area (DASA) study revealed that cortisol binds with the native hCBG more tightly than aldosterone. Cleavage at the Val379-Met380 peptide bond causes a deformation of hCBG (also revealed through a DASA study). This deformation could probably trigger the release of the bound hormone. Figure Stereoscopic view of the ribbon diagram of hCBG complexed with cortisol. The bound cortisol is shown in space filling model in blue. Helices and sheets are shown in red and magenta respectively. Turns are shown in yellow.     

Cloning, expression and sequencing the molybdenum-pterin binding protein (mop) gene of Clostridium pasteurianum in Escherichia coli

mop is the structural gene for the molybdenum-pterin binding protein, which is the major molybdenum binding protein in Clostridium pastuerianum. The mop gene was detected by immunoscreening genomic libraries of C. pastuerianum and identified by determining the nucleotide sequence of the cloned insert of clostridial DNA. The deduced amino acid sequence of an open reading frame proved to be identical to the first twelve residues of purified Mop. The DNA sequence flanking the mop gene contains promoter-like consensus sequences which are probably responsible for the expression of Mop in Escherichia coli. The deduced amino acid composition shows that the protein is hydrophobic, lacks aromatic and cysteine residues and has a calculated molecular weight of 7,038. The N-terminal amino acid sequence of Mop has sequence homology with DNA binding proteins. The pattern and type of residues in the N-terminal region suggest it forms the helix-turn-helix structure observed in DNA binding proteins. We propose that Mop may be a regulatory protein binding the anabolic source of molybdenum.     

Sequences of Escherichia coli uvrA gene and protein reveal two potential ATP binding sites

We have determined the nucleotide sequence of the uvrA gene of Escherichia coli. The coding region of the gene is 2820 base pairs which specifies a protein of 940 amino acids and Mr = 103,874. The polypeptide sequence predicted from the DNA sequence was confirmed by analyzing the UvrA protein: the sequence of the first 7 NH2-terminal amino acids as well as the amino acid composition of the pure protein agreed with those predicted from the nucleotide sequence. By comparing the sequence of UvrA protein to the amino acid sequences of other ATPases, we found that two regions in the UvrA protein, separated from one another by about 600 amino acids, have the highly conserved G-X4-GKT(S)-X6-I(V) sequence found at the active sites of many, but not all, ATPases. Our findings suggest that UvrA protein may have two ATP binding sites.     

Rabbit FKBP59-heat shock protein binding immunophillin (HBI) is a calmodulin binding protein.

FKBP59-HBI, a heat shock protein hsp90-binding immunophilin that was originally detected in heterooligomer forms of steroid receptors, is retained on Calmodulin (CAM)-Sepharose 4B in the presence of 2 mM Ca2+ and is eluted by EGTA, demonstrating a specific p59-CAM interaction. The p59 amino acid sequence reveals the presence of two putative CAM binding sites in a helix regions of the protein, as well as PEST sequences which are generally present in CAM-binding proteins. In vitro proteolysis by calpain II (a Ca(2+)-activated neutral protease), another feature of CAM-binding proteins, generates shorter peptides revealed by the mAb EC1, but not by the pAb 173 which recognizes the C-terminal of the protein. The potential function of CAM binding by the hsp90-binding immunophilin is discussed.     

Nucleotide sequence and expression of a cDNA clone encoding a fetal rat binding protein for insulin-like growth factors

The insulin-like growth factors (IGFs), IGF-I and IGF-II, occur in plasma and tissue fluids complexed to specific binding proteins. Although the role of the binding proteins is not completely defined, they are capable of modulating the biological activity of the IGFs. In order to better understand the function of these proteins, we have isolated a clone from the BRL-3A rat liver cell line that encodes a protein corresponding to the IGF binding protein in fetal rat serum. The cDNA clone encodes a precursor protein of 304 amino acids (32,886 daltons), comprised of a 34-residue hydrophobic prepeptide and a 270-residue mature protein (29,564 daltons). The deduced amino acid sequence agrees with the sequence of 173 amino acid residues determined by Edman degradation. The mature protein contains 18 cysteines and no N-glycosylation sites. It contains an Arg-Gly-Asp (RGD) sequence near the carboxyl terminus. A similar sequence is present on many extracellular matrix proteins and contributes to their recognition by cellular adhesion receptors. The cloned cDNA has been transcribed in vitro and the resulting RNA expressed in Xenopus oocytes. Injected oocytes secrete a 33-kDa protein that is immunoprecipitated by polyclonal antibodies to the BRL-3A binding protein and binds IGF-I and IGF-II with the same affinity and specificity as does purified BRL-3A binding protein. The binding protein cDNA probe hybridizes to an approximately 2-kilobase mRNA in BRL-3A cells and in multiple fetal rat tissues including liver, kidney, intestine, and lung. Levels of this mRNA are greatly reduced in the corresponding adult tissues. The rat IGF binding protein is closely related to the partial amino acid sequences reported for a bovine IGF binding protein and more distantly related to a human IGF binding protein that recently has been cloned. No significant homologies were identified to other proteins. Thus, the rat IGF binding protein that we have cloned appears to be a distinct member of a family of related IGF binding proteins. We postulate that the structurally distinct IGF binding proteins may have different biological functions.     

Interaction of carbohydrate and protein in thyroxine binding globulin

The fluorescence properties of human thyroxine binding globulin were evaluated during enzymatic deglycosylation by using both neuraminidase and a mixture of glycosidases. Three fluorescent chromophores, one intrinsic and two extrinsic, were monitored, and all showed changes in fluorescent parameters that have been interpreted in terms of a loss of interactions between the carbohydrate and amino acid residues during deglycosylation. The loss of carbohydrates also results in a decrease in stability of the protein to both acid and guanidinium chloride inactivation. Since deglycosylation decreases the frictional ratio of thyroxine binding globulin, it is concluded that, although sialic acid and other sugar residues are in contact with the protein surface, the hydrated carbohydrate chains protrude partially into the solvent.     

Sequence of membrane-associated thyroid hormone binding protein from bovine liver: its identity with protein disulphide isomerase

The complementary DNAs of the bovine liver membrane-associated 3,5,3'-triiodo-L-thyronine binding protein with 55 k-dalton (T3BP) were cloned and the nucleotide sequences were determined. Monospecific antibodies against T3BP were used to screen a bovine liver cDNA library in lambda gtll. We analyzed the sequences of two cloned T3BP cDNAs. The clones encoded a polypeptide of 510 amino acid residues, including a signal peptide of 20 amino acid. Northern blot analysis of bovine and human RNA showed that the mRNAs encoding T3BP are 2.7 kilobase in length. Amino acid sequence of N-terminal and other three peptides isolated from purified T3BP were found in the predicted amino acid sequence from the cDNA sequence. The predicted amino acid sequence is closely homologous (93%) with that of rat protein disulphide isomerase (EC 5.3.4.1), which catalyzes the isomerization of the protein disulphide bonds and has been shown to play an important role in post-translational regulation. The results suggest that T3BP and protein disulphide isomerase should be the same protein.     

Insulin-like growth factor (IGF) binding protein complementary deoxyribonucleic acid from human HEP G2 hepatoma cells: predicted protein sequence suggests an IGF binding domain different from those of the IGF-I and IGF-II receptors

The primary structure of an insulin-like growth factor (IGF) binding protein produced by human HEP G2 hepatoma cells has been deduced from the cDNA sequence. The 234 amino acid protein has a predicted molecular mass of 25,274 and contains a single, distinctive cysteine-rich region. The N-terminal sequence of this protein is quite similar to the limited sequence data available for a rat IGF binding protein produced by BRL-3A cells and suggests a common ancestral origin. In contrast, the HEP G2 IGF binding protein sequence bears no similarity to the N-terminal 15 amino acids of a 53 kilodalton binding protein purified from human plasma. Comparison of full-length protein sequences for the IGF-I and IGF-II receptors with that of the HEP G2 IGF binding protein also fails to demonstrate any significant similarities among these three proteins, and suggests that each contains a unique binding domain for the IGF peptides.     

cDNA cloning of a human 25 kDa FK506 and rapamycin binding protein.

The abilities of FK506 and rapamycin to block distinct signal transduction pathways are mediated by soluble binding proteins. Previously, a family of these receptors has been recognized that includes a 25 kDa protein, FKBP25. We now report the isolation of a cDNA for FKBP25 from a human hippocampal cDNA library by oligonucleotide screening. The nucleotide sequence reveals an open reading frame that encodes a 224 amino acid polypeptide. Human FKBP25 shows 97% amino acid identity with bovine FKBP25 and 62% homology with human FKBP12.     

Identification and characterization of an IgG binding protein in the secretion of the rat coagulating gland

A merocrine released protein (named 115k protein) was highly enriched from the secretion of the rat coagulating gland. The protein has a molecular mass of 115 kDa as calculated by SDS-PAGE under reducing conditions. Furthermore, the 115 kDa protein is glycosylated, and carries Man, GlcNAc, Gal, Fuc and sialic acid residues. For identification, N-terminal amino acid and nucleotide sequence analyses were performed. The sequences obtained showed 86 to 100% identity with human and mouse IgGFc binding proteins. The functional capacity of IgG binding of the 115 kDa protein was shown by overlay experiments, indicating its membership in the IgG binding protein family.     

Novel structure of a high molecular weight FK506 binding protein fromArabidopsis thaliana

We have isolated clones of an Arabidopsis gene (ROF1, forrotamaseFKBP) encoding a high molecular weight member of the FK506 binding protein (FKBP) family. The deduced amino acid sequence of ROF1 predicts a 551-amino acid, 62 kDa polypeptide which is 44% identical to human FKBP59 — a 59 kDa FKBP which binds to the 90 kDa heat shock protein and is associated with inactive steroid hormone receptors. ROF1 contains three FKBP12-like domains in the N-terminal portion of the protein (in contrast to two domains in mammalian FKBP59), an internal repeat structure associated with protein-protein interactions (tetratricopeptide repeats), and a putative calmodulin binding domain near the C-terminal region of the protein. No sequences associated with protein translocation out of the cytosol were found in ROF1.ROF1 mRNA was found at equivalent low levels in light-grown roots, stems, and flowers and at slightly higher levels in leaves. The abundance ofROF1 mRNA increased several-fold under stress conditions such as wounding or exposure to elevated NaCl levels.The nucleotide sequences in this paper have been submitted to the GenBank/EMBL Data bank with accession numbers U49453 and U57838     

Characterization of a synthetic peptide that inhibits the interaction between protein S and C4b-binding protein

Protein S is unique among the vitamin K-dependent proteins found in blood plasma because it is a cofactor rather than a zymogen of a serine protease. Instead of a trypsin-like domain, protein S contains a domain that has sequence homology with steroid binding proteins. In order to understand the function of this structural domain, peptides have been synthesized with amino acid sequences that are homologous between human protein S and rat androgen binding protein. Two peptides, corresponding to amino acids 400-407 (PINPRLDG) and 605-614 (GVQLDLDEAI) of the protein S sequence have been tested for their effects on protein S function. Neither peptide altered the clotting of bovine or human plasma. The peptide GVQLDLDEAI enhanced the anticoagulant activity of human-activated protein C in human plasma while the peptide PINPRLDG had no effect. The peptide GVQLDLDEAI was observed to inhibit the binding of protein S to C4b-binding protein in plasma, resulting in increased concentrations of free protein S. GVQLDLDEAI was also observed to enhance the disassociation of the protein S.C4b-binding protein complex when purified complex was used. Finally, C4b-binding protein was observed to bind to GVQLDLDEAI. These results suggest that the carboxyl-terminal region of protein S, which contains the sequence GVQLDLDEAI, is involved in the interaction between protein S and C4b-binding protein.     

The Ch21 protein, developmentally regulated in chick embryo, belongs to the superfamily of lipophilic molecule carrier proteins

Ch21, a developmentally regulated low molecular weight protein observed in chick embryo skeletal tissues, is expressed "in vitro" by differentiating chondrocytes at a late stage of development. Here we report the complete amino acid sequence of the protein. 86% of the total amino acid sequence was deduced by sequences of 17 high performance liquid chromatography-separated proteolytic fragments and 33 amino acid residues at the amino-terminal end of protein purified from spent culture medium of hypertrophic chondrocytes. Furthermore we isolated by molecular cloning the corresponding cDNA and determined its nucleotide sequence. By combining protein and nucleotide sequence data we determined the primary structure of the entire Ch21. It consists of 158 amino acids and has a molecular mass of 18.065 kDa. Computer-assisted analysis showed that the Ch21 belongs to the superfamily of low molecular weight proteins sharing a basic framework for binding and transport of small hydrophobic molecules.     

Molecular cloning of a 25-kDa high affinity rapamycin binding protein, FKBP25.

Two FK506 binding proteins of molecular mass 12 kDa (FKBP12) and 13 kDa (FKBP13) have been identified as common cellular receptors of the immunosuppressants FK506 and rapamycin. Here we report the molecular cloning and overexpression of a 25-kDa rapamycin and FK506 binding protein (termed FKBP25) with peptidylprolyl cis-trans-isomerase (PPIase) activity. The amino acid sequence, predicted from the FKBP25 cDNA, shares identity with FKBP12 (44%) and FKBP13 (47%) in the C-terminal 97 amino acids. Unlike either FKBP12 or FKBP13, the nucleotide sequence of FKBP25 contains a number of putative nuclear localization sequences. The PPIase activity of recombinant FKBP25 was comparable with that of FKBP12. The PPIase activity of FKBP25 was far more sensitive to inhibition by rapamycin (IC50 = 50 nM) than FK506 (IC50 = 400 nM). PPIase activity of 100 nM FKBP25 was almost completely inhibited by 150 nM rapamycin while only 90% inhibition was achieved by 4 microM FK506. These data demonstrate that FKBP25 has a higher affinity for rapamycin than for FK506 and suggest that this cellular receptor may be an important target molecule for immunosuppression by rapamycin.