Interaction between corticosteroid binding globulin and activated leukocytes in vitro

The interaction between human corticosteroid binding globulin and activated leukocytes is restricted to the granulocyte population, and is characterized by specific proteolytic cleavage of corticosteroid binding globulin which markedly reduces its steroid binding activity. A direct interaction between corticosteroid binding globulin and the activated cells appears to enhance this event, and does not involve cellular internalization of corticosteroid binding globulin or its proteolytic degradation products, which resemble those obtained after incubation of corticosteroid binding globulin with neutrophil elastase. These data suggest that corticosteroid binding globulin interacts with elastase on the surface of activated neutrophils, and may promote glucocorticoid delivery to these cells during inflammation.     

Measurement of human corticosteroid binding globulin by enzyme-linked immunoassay

Thirteen monoclonal antibodies have been raised against corticosteroid binding globulin (CBG). From four of those with highest affinity for the antigen, two were selected for development of a sandwich enzyme-linked immunoassay (ELISA). The sensitivity of the assay was such that 0.7 fmol CBG could be detected. Levels of the binding protein in men (740 +/- 67 nmol/l) and women (690 +/- 103 nmol/l) were not significantly different, while those found during the third trimester of pregnancy (1500 +/- 423 nmol/l) were approximately twice these levels. CBG denatured by heating to 60 degrees C could not be detected by the ELISA.     

Adrenal glucocorticoids regulate adipsin gene expression in genetically obese mice

Adipsin expression at the protein and mRNA levels is greatly reduced in several distinct syndromes of obesity in the mouse: genetic obesity due to the db/db and ob/ob genes, and a chemically induced model secondary to neonatal exposure to monosodium glutamate. We considered first the possibility that the adipsin gene might be identical to the db or ob locus and the lowered expression of this protein might result from a mutation in this gene. We show here that the adipsin structural gene is located on chromosome 10 and hence is physically distinct from any obesity genes so far identified in the mouse. A major role for the adrenal gland and adrenal glucocorticoids in the aberrant regulation of adipsin in these models of obesity is indicated by several experiments. Adrenalectomy of the ob/ob mouse raises the circulating levels of adipsin protein and the amount of this mRNA in epididymal fat pads (5-fold), although neither is increased to the levels seen in lean controls. Exogenous administration of corticosterone completely blocks the effects of adrenalectomy on adipsin, suggesting that the effect of this endocrine ablation is through reduction of adrenal glucocorticoids. Corticosterone administration also causes suppression in the levels of adipsin mRNA and protein in lean mice, although this decrease is never as severe as that seen in obese mice. The effect of exogenous corticosterone in lean mice occurs within 2 days and hence is not secondary to the obesity which these hormones eventually elicit. These results indicate that glucocorticoids can regulate adipsin expression in vivo and strongly suggest that the hyperglucocorticoid state seen in certain obese models plays a significant role in lowering adipsin mRNA and protein levels. Quantitative analysis of these experiments suggests that other as yet unknown neuroendocrine factors also function to suppress adipsin in obesity.     

Progesterone measurement using stripped corticosteroid binding globulin (CBG)

Modifications in an existing competitive protein binding assay for progesterone have been made which provide a readily available and rich source of binding sites on the corticosteroid binding globulin (CBG). The primary and most important modification is the rapid removal of endogenous steroids from plasma by gel filtration at an elevated temperature. The ‘stripped’ protein retains full CBG activity, but is cleared of 95% of its endogenous steroids. This stripping procedure provides not only increased number of binding sites, but in conjunction with the other modifications also eliminates some of the variability in the assay.     

Human corticosteroid binding globulin is secreted by a hepatoma-derived cell line

We used a sensitive radioimmunoassay for corticosteroid-binding-globulin (CBG) to demonstrate that a human hepatoma-derived cell line secretes a protein which: (a) the radioimmunoassay cannot distinguish from CBG; (b) has the same Stokes' radius as CBG; (c) is absorbed by a steroid affinity column which is specific for CBG.     

An amino acid substitution in biobreeding rat corticosteroid binding globulin results in reduced steroid binding affinity

BioBreeding (BB) rats are derived from an outbred colony of Wistar rats and are used as a model of autoimmune diabetes mellitus. A corticosteroid binding globulin (CBG) variant with reduced affinity for glucocorticoids has now been found in the blood of these animals. The dissociation rate constants of BB CBG for cortisol (4.42 nM) and corticosterone (1.43 nM) are both about 50% higher than those associated with Wistar CBG, but no obvious difference in the steroid binding specificity of BB and Wistar CBGs was detected. Purified BB and Wistar CBGs exhibit the same size heterogeneity when examined by polyacrylamide gel electrophoresis under denaturing conditions, and the sizes of their respective hepatic mRNAs are identical. The genetic basis for this abnormality was therefore determined by comparing the cDNA sequences for BB and Wistar CBG, and this revealed a point mutation that results in a single amino acid substitution at residue 276 (Ile in BB CBG and Met in Wistar CBG). To confirm that this mutation is responsible for the reduced steroid binding affinity associated with BB CBG, the cDNAs for rat CBG-Ile276 and CBG-Met276 were expressed in Chinese hamster ovary cells. The steroid binding affinities of the CBGs secreted by these cells were essentially identical with those observed in the corresponding serum samples from these two rat strains. The amino acid substitution identified in BB rat CBG therefore clearly accounts for the reduction in its steroid binding affinity, and further analysis of this and other natural CBG variants may reveal important information about the CBG steroid binding site. It is also possible that this mutation may contribute to the etiology of pathological abnormalities that are characteristic of the BB rat.     

Enhancement by LPS of B-cell function in genetically deficient mice.

CBA/N mice are apparently deficient in mature B cells, but not in immature B cells. Experiments reported here were designed to test the ability of the adjuvant and B-cell mitogen, bacterial lipopolysaccharide (LPS), to induce B-cell function in CBA/N mice. CBA/N spleen cells, which respond poorly to erythrocyte antigens in vitro, developed high numbers of hemolytic antibody-forming cells when cultured with LPS. When injected along with erythrocyte antigens, LPS enhanced in vivo immune responses, as shown by increases in direct and indirect hemolytic antibody-forming cells. These data demonstrate that both in vivo and in vitro, in the presence of LPS, CBA/N B cells can become antibody-forming cells.     

A new role for corticosteroid binding globulin (CBG), member of SERPIN superfamily]

Recent advances in molecular endocrinology have shed a new light on the role and mode of action of CBG. It is now not only demonstrated that this plasma glycoprotein, a steroid carrier, can be internalized by glucocorticoid target tissues, but it is also certain that CBG mRNA is synthesized by extra-hepatic tissues. Moreover, some authors have reported a modulation of CBG properties by free fatty acids. The existence of CBG receptors (or high affinity membrane-binding sites), and even a positive effect of CBG on adenylate cyclase activity, have also been reported. To progress in the understanding of these diverse results, one must first integrate them in a general scheme where it is considered that CBG is a member of the SERPIN (SERine Protease INhibitors) superfamily. In the case of CBG, that means a protein which functions as a substrate for elastase at the surface of neutrophils, for instance at sites of inflammation. CBG is specifically cleaved by this protease at a precise site close to its carboxy-terminus. This induces a conformation change and disrupts the binding between glucocorticoids and CBG, and promotes a significant and local release of glucocorticoids (over 90% of them are bound to CBG in human plasma). In this context, CBG directs glucocorticoids to sites of inflammation, and plays in consequence a crucial role in efficient glucocorticoid action in physiology. The elucidation of the CBG sequence, the knowledge of its gene structure, and the discovery of its chromosomal localization near two other SERPIN genes, are three sets of data in concordance to demonstrate that CBG is a SERPIN; and this has allowed the understanding of a new role for CBG, possibly with important consequences in pathology. Moreover, it could be more appropriate to say that CBG is a member of the SERine Protease INhibitors and Substrates superfamily (SERPINS).     

Purification and properties of squirrel monkey (Saimiri sciureus) corticosteroid binding globulin

Corticosteroid binding globulin (CBG), a serum glycoprotein which binds glucocorticoids and progestins with high affinity, is widely distributed throughout the animal world. Although its charge and size characteristics have largely been conserved across species, we found the behavior of CBG in squirrel monkey (Saimiri sciureus) serum during fractionation by polyacrylamide gel electrophoresis or Sephadex chromatography was consistent with a molecule about twice the size of that found in most species. To more fully understand the basis for this difference, we purified the protein by sequential affinity and DEAE-Sepharose chromatographies. The final product was obtained in greater than 60% yield and was found to migrate as a single homogeneous band when examined by electrophoresis at pH 8.3 in polyacrylamide gels varying total acrylamide concentration or under conditions of severe protein overload. The steroid binding specificity of the purified protein was identical with that of the protein in the starting serum. The ultraviolet absorption spectrum of the isolated CBG-steroid complexes revealed that the protein had no pyridine nucleotide cofactor or nucleic acid. Amino acid analyses showed that the composition of the squirrel monkey protein is quite similar to that of CBG molecules from other species but distinct from albumins, hemoglobin, or rabbit progesterone receptor. In contrast to the single protein band observed following electrophoresis under normal conditions, separations in the presence of sodium dodecyl sulfate (SDS) resolved the pure protein into two bands: one at 54,000 daltons and one at 57,000 daltons.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of dexamethasone on the levels of plasma corticosteroid binding globulin in rats and monkeys.

Rat marrow cells were preincubated for 45 hours with 5.5 × 10^?4M sodium hexachloroiridate. This treatment abolished DNA synthesis whilst improving cell survival over that of controls. The synthesis of RNA, protein and glycoprotein continued and could be further increased by the addition of erythropoietin for up to 44 more hours. Heme synthesis also continued in the absence of DNA synthesis but could not be stimulated by erythropoietin.     

A Leu----His substitution at residue 93 in human corticosteroid binding globulin results in reduced affinity for cortisol.

A steroid binding capacity assay and a radioimmunoassay were both used to measure corticosteroid binding globulin (CBG) in serum samples from 22 patients with sepsis. An approximately 50% discordancy between the two values in one patient suggested the presence of a CBG variant with reduced affinity for cortisol, and this was confirmed by Scatchard analysis. We therefore used the polymerase chain reaction to amplify exons that encode for human CBG from the genomic DNA of this patient. This revealed two mutations within the coding sequences: one of which results in a Leu----His substitution at residue 93 and another which encodes a Ser----Ala substitution at residue 224 of the human CBG polypeptide. To assess the impact of each substitution on the steroid binding affinity of CBG, each mutation was introduced separately into a normal human CBG cDNA, and the normal and mutated cDNAs were expressed in Chinese hamster ovary cells. Scatchard analysis of the CBG produced in culture indicated that the His93 mutation (Kd = 2.24 +/- 1.75 nM) reduced the cortisol binding affinity of CBG (mean +/- SD) significantly (P less than 0.024) when compared to normal CBG (Kd = 0.64 +/- 0.31 nM), while the Ala224 mutation (Kd = 0.63 +/- 0.33 nM) did not influence cortisol binding affinity. We therefore conclude that residue 93 may play an important role in determining the structure of the CBG steroid binding site.     

Serum corticosteroid binding globulin expression is modulated by fasting in polar bears (Ursus maritimus)

Polar bears (Ursus maritimus) from several subpopulations undergo extended fasting during the ice-free season. However, the animals appear to conserve protein despite the prolonged fasting, though the mechanisms involved are poorly understood. We hypothesized that elevated concentrations of corticosteroid binding globulin (CBG), the primary cortisol binding protein in circulation, lead to cortisol resistance and provide a mechanism for protein conservation during extended fasting. The metabolic state (feeding vs. fasting) of 16 field sampled male polar bears was determined based on their serum urea to creatinine ratio (>25 for feeding vs. <5 for fasting). There were no significant differences in serum cortisol levels between all male and female polar bears sampled. Serum CBG expression was greater in lactating females relative to non-lactating females and males. CBG expression was significantly higher in fasting males when compared to non-fasting males. This leads us to suggest that CBG expression may serve as a mechanism to conserve protein during extended fasting in polar bears by reducing systemic free cortisol concentrations. This was further supported by a lower serum glucose concentration in the fasting bears. As well, a lack of an enhanced adrenocortical response to acute capture stress supports our hypothesis that chronic hunger is not a stressor in this species. Overall, our results suggest that elevated serum CBG expression may be an important adaptation to spare proteins by limiting cortisol bioavailability during extended fasting in polar bears.     

Sperm from mice genetically deficient for the PCSK4 proteinase exhibit accelerated capacitation, precocious acrosome reaction, reduced binding to egg zona pellucida, and impaired fertilizing ability

The gene for proprotein convertase subtilisin/kexin-like 4 (PCSK4, previously known as PC4) is primarily transcribed in testicular spermatogenic cells. Its inactivation in mouse causes severe male subfertility. To better understand the reproductive function of PCSK4, we examined its subcellular localization in the testicular epithelium via immunohistochemistry, and on intact sperm via indirect immunofluorescence and immunoelectron microscopy. PCSK4 was detected in the acrosomal granules of round spermatids, in the acrosomal ridges of elongated spermatids, and on the sperm plasma membrane overlying the acrosome. We also investigated PCSK4 relevance for sperm acquisition of fertilizing ability by comparing wild-type and PCSK4-null sperm for their abilities in capacitation, acrosome reaction, and egg binding in vitro. PCSK4-null sperm underwent capacitation at a faster rate; they were induced to acrosome react by lower concentrations of zona pellucida; and their egg-binding ability was only half that of wild-type sperm. These sperm physiologic anomalies likely contribute to the severe subfertility of PCSK4-deficient male mice.     

Severe hypoalphalipoproteinemia in mice expressing human hepatic lipase deficient in binding to heparan sulfate proteoglycan

Unlike human hepatic lipase (hHL) that is mainly cell surface-anchored via binding to heparan sulfate proteoglycans (HSPG), mouse HL (mHL) has a low affinity to HSPG and thus is largely blood-borne. The reduced HSPG binding of mHL is attributable to the C-terminal amino acids. To determine the functions of HSPG binding of hHL in vivo, we created adenovirus vectors encoding hHL or a chimeric protein (designated hHLmt) in which the C-terminal HSPG-binding sequences were replaced with the corresponding mouse sequences. Injecting hHLmt-expressing virus into C57BL/6J mice (1.8 x 10(10) virus particles/mouse) resulted in a 3-fold increase in pre-heparin HL activity, whereas infection with an identical dose of hHL virus did not change pre-heparin HL activity. In hHLmt-expressing mice, the concentration of total cholesterol and phospholipids was inversely related to the hHL activity in pre-heparin plasma in a dose- and time-dependent manner, and the decrease was mainly attributable to high density lipoproteins (HDL) cholesterol and HDL phospholipids. The expression of hHL exhibited no change in plasma total cholesterol or phospholipid levels as compared with control mice infected with luciferase or injected with saline. The reduced HDL lipids in the hHLmt-expressing mice were accompanied by markedly decreased plasma and hepatic apolipoprotein (apo) A-I. In primary hepatocytes isolated from hHLmt-expressing mice, the concentration of cell-associated and secreted apoA-I was decreased by 2-3-fold as compared with hepatocytes isolated from control mice, whereas the levels of apoB and apoE were unaltered. Infection of primary hepatocytes with hHLmt virus ex vivo also resulted in reduced apoA-I secretion but had no effect on cell-associated apoA-I. These results suggest that expression of HSPG binding-deficient hHL has a profound HDL-lowering effect.     

Methotrexate-resistant Leishmania donovani genetically deficient in the folate-methotrexate transporter

From a mutagenized population of wild type Leishmania donovani promastigotes, a clone was isolated in a single step by virtue of its resistance to 1 mM methotrexate, a potent inhibitor of dihydrofolate reductase. This methotrexate-selected cell line, MTXA5, was cross-resistant to aminopterin but just as sensitive to growth inhibition caused by pyrimethamine, trimethoprim, and cytotoxic purine and pyrimidine analogs. Unlike previously characterized methotrexate-resistant Leishmania (Coderre, J. A., Beverley, S. M., Schimke, R., and Santi, D. V. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 2132-2136), resistance to the antimetabolite was not due to gene amplification or increased dihydrofolate reductase activity. The genetic defect in MTXA5 cells appeared to be in the methotrexate-folate transport system. The rate of uptake and transport of [3H]methotrexate and [3H]folate into MTXA5 cells was less than 1% of that of wild type parental cells. Neither wild type nor MTXA5 cells could multiply in folate-deficient medium, and thymine and thymidine at concentrations which circumvented methotrexate toxicity, did not restore the ability of Leishmania to grow. The concentration of exogenous folate that restored growth of wild type and mutant cells, however, was virtually identical, although MTXA5 cells, unlike parental cells, could not proliferate in folate-deficient medium supplemented with 10 microM biopterin. Interestingly, methotrexate and aminopterin could stimulate the growth of both leishmanial strains in folate-deficient medium, suggesting that these antifolate analogs were serving as a pteridine source for the parasite. These somatic cell genetic studies of folate transport in Leishmania provide genetic evidence for a specific folate permease in L. donovani promastigotes and have important implications concerning the mechanisms by which these parasites utilize exogenous pteridines and folates and by which they might become resistant to parasite-directed chemotherapeutic regimens.     

Rat corticosteroid binding globulin: primary structure and messenger ribonucleic acid levels in the liver under different physiological conditions

Rat corticosteroid binding globulin (CBG) cDNAs were isolated from a lambda gt11 liver cDNA library. When rat hepatic mRNA was hybrid selected and translated in vitro, a major product reacted with antibodies against rat CBG and its Mr (approximately 43,000) was consistent with a nonglycosylated, CBG precursor polypeptide. Two overlapping cDNAs produced a 1,432 nucleotide sequence with an open reading frame comprising 396 amino acids. This includes a potential signal peptide of 22 residues followed by the amino terminus of purified rat CBG. Rat CBG therefore contains 374 amino acids (Mr = 42,196), and has six consensus sites for N-glycosylation. There is 60% identity in the primary structures of rat and human CBG over 383 residues that comprise the human sequence. Furthermore, the single cysteine in rat CBG corresponds to one of two cysteines in human CBG, and this may be significant because a cysteine is located in the human CBG steroid binding site. Northern analysis of RNA from various rat tissues revealed an approximate 1.8 kilobase CBG mRNA only in the liver. Its relative abundance in a pregnant rat was only 30% higher than in an adult female; approximately 3-fold higher than in an adult male, and 25-fold higher than in the fetuses from the same animal. Southern analysis of rat genomic DNA suggests the presence of a single gene for CBG.