Neutrophil elastase-cleaved corticosteroid-binding globulin is absent in human plasma

Corticosteroid-binding globulin (CBG) transports glucocorticoids in blood and is a serine protease inhibitor family member. Human CBG has a reactive center loop (RCL) which, when cleaved by neutrophil elastase (NE), disrupts its steroid-binding activity. Measurements of CBG levels are typically based on steroid-binding capacity or immunoassays. Discrepancies in ELISAs using monoclonal antibodies that discriminate between intact vs RCL-cleaved CBG have been interpreted as evidence that CBG with a cleaved RCL and low affinity for cortisol exists in the circulation. We examined the biochemical properties of plasma CBG in samples with discordant ELISA measurements and sought to identify RCL-cleaved CBG in human blood samples. Plasma CBG-binding capacity and ELISA values were consistent in arterial and venous blood draining skeletal muscle, liver and brain, as well as from a tissue (adipose) expected to contain activated neutrophils in obese individuals. Moreover, RCL-cleaved CBG was undetectable in plasma from critically ill patients, irrespective of whether their ELISA measurements were concordant or discordant. We found no evidence of RCL-cleaved CBG in plasma using a heat-dependent polymerization assay, and CBG that resists immunoprecipitation with a monoclonal antibody designed to specifically recognize an intact RCL, bound steroids with a high affinity. In addition, mass spectrometry confirmed the absence of NE-cleaved CBG in plasma in which ELISA values were highly discordant. Human CBG with a NE-cleaved RCL and low affinity for steroids is absent in blood samples, and CBG ELISA discrepancies likely reflect structural differences that alter epitopes recognized by specific monoclonal antibodies.

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N-Glycosylation influences human corticosteroid-binding globulin measurements

Endocrine Connections ◽

10.1530/ec-19-0242 ◽

2019 ◽

Vol 8 (8) ◽

pp. 1136-1148 ◽

Cited By ~ 2

Author(s):

Lesley A Hill ◽

Zeynep Sumer-Bayraktar ◽

John G Lewis ◽

Eva Morava ◽

Morten Thaysen-Andersen ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Liver Diseases ◽

Binding Capacity ◽

Congenital Disorders ◽

Congenital Disorders Of Glycosylation ◽

Blood Samples ◽

Reactive Center Loop ◽

Reactive Center ◽

Corticosteroid Binding Globulin ◽

Steroid Binding

Objective Discrepancies in ELISA measurements of human corticosteroid-binding globulin (CBG) using detection monoclonal antibodies that recognize an epitope (9G12) within its reactive center loop (RCL), versus an epitope (12G2) in a different location, have suggested that CBG with a proteolytically cleaved RCL exists in blood samples. We have previously been unable to verify this biochemically, and sought to determine if N-glycosylation differences account for discrepancies in ELISA measurements of CBG. Methods and subjects Molecular biological, biochemical and glycopeptide analyses were used to examine how N-glycosylation at specific sites, including at N347 within the RCL, affect CBG ELISA or steroid-binding capacity assay (BCA) measurements. Plasma from patients with congenital disorders of glycosylation (CDG) was also examined in these assays as examples of N-glycosylation defects. Results We demonstrate that an N-glycan at N347 within the CBG RCL limits the 9G12 antibody from recognizing its epitope, whereas the 12G2 antibody reactivity is unaffected, thereby contributing to discrepancies in ELISA measurements using these two antibodies. Qualitative differences in N-glycosylation at N238 also negatively affect the steroid-binding of CBG in the absence of an N-glycan at N347 caused by a T349A substitution. Desialylation increased both ELISA measurements relative to BCA values. Similarly, plasma CBG levels in both ELISAs were much higher than BCA values in several CDG patients. Conclusions Plasma CBG measurements are influenced by variations in N-glycosylation. This is important given the increasing number of CDG defects identified recently and because N-glycosylation abnormalities are common in patients with metabolic and liver diseases.

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Functional implications of corticosteroid-binding globulin N-glycosylation

Journal of Molecular Endocrinology ◽

10.1530/jme-17-0234 ◽

2018 ◽

Vol 60 (2) ◽

pp. 71-84 ◽

Cited By ~ 4

Author(s):

Marc Simard ◽

Caroline Underhill ◽

Geoffrey L Hammond

Keyword(s):

Neutrophil Elastase ◽

Binding Capacity ◽

Binding Activity ◽

Glycosylation Site ◽

Binding Affinities ◽

Glycosylation Sites ◽

Reactive Center ◽

Corticosteroid Binding Globulin ◽

Pngase F ◽

Steroid Binding

Corticosteroid-binding globulin (CBG) is a plasma carrier of glucocorticoids. Human and rat CBGs have six N-glycosylation sites. Glycosylation of human CBG influences its steroid-binding activity, and there are N-glycosylation sites in the reactive center loops (RCLs) of human and rat CBGs. Proteolysis of the RCL of human CBG causes a structural change that disrupts steroid binding. We now show that mutations of conserved N-glycosylation sites at N238 in human CBG and N230 in rat CBG disrupt steroid binding. Inhibiting glycosylation by tunicamycin also markedly reduced human and rat CBG steroid-binding activities. Deglycosylation of fully glycosylated human CBG or human CBG with only one N-glycan at N238 with Endo H-reduced steroid-binding affinity, while PNGase F-mediated deglycosylation does not, indicating that steroid binding is preserved by deamidation of N238 when its N-glycan is removed. When expressed in N-acetylglucosaminyltransferase-I-deficient Lec1 cells, human and rat CBGs, and a human CBG mutant with only one glycosylation site at N238, have higher (2–4 fold) steroid-binding affinities than when produced by sialylation-deficient Lec2 cells or glycosylation-competent CHO-S cells. Thus, the presence and composition of an N-glycan in this conserved position both appear to influence the steroid binding of CBG. We also demonstrate that neutrophil elastase cleaves the RCL of human CBG and reduces its steroid-binding capacity more efficiently than does chymotrypsin or the Pseudomonas aeruginosa protease LasB. Moreover, while glycosylation of N347 in the RCL limits these activities, N-glycans at other sites also appear to protect CBG from neutrophil elastase or chymotrypsin.

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Pseudomonas Aeruginosa Elastase Disrupts the Cortisol-Binding Activity of Corticosteroid-Binding Globulin

Endocrinology ◽

10.1210/en.2014-1055 ◽

2014 ◽

Vol 155 (8) ◽

pp. 2900-2908 ◽

Cited By ~ 22

Author(s):

Marc Simard ◽

Lesley A. Hill ◽

Caroline M. Underhill ◽

Bernd O. Keller ◽

Ivan Villanueva ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Neutrophil Elastase ◽

Binding Capacity ◽

Binding Activity ◽

Spectrometric Analysis ◽

Reactive Center ◽

Corticosteroid Binding Globulin ◽

Active Protease ◽

Humoral Responses ◽

Pseudomonas Aeruginosa Elastase

The serine protease inhibitor (SERPIN) family member corticosteroid-binding globulin (CBG) is the main carrier of glucocorticoids in plasma. Human CBG mediates the targeted release of cortisol at sites of inflammation through cleavage of its reactive center loop (RCL) by neutrophil elastase. The RCLs of SERPIN family members are targeted by diverse endogenous and exogenous proteases, including several bacterial proteases. We tested different bacteria for their ability to secrete proteases that disrupt CBG cortisol-binding activity, and characterized the responsible protease and site of CBG cleavage. Serum CBG integrity was assessed by Western blotting and cortisol-binding capacity assay. Effects of time, pH, temperature, and protease inhibitors were tested. Proteolytically active proteins from bacterial media were purified by fast protein liquid chromatography, and the active protease and CBG cleavage sites were identified by mass spectrometry. Among the bacteria tested, medium from Pseudomonas aeruginosa actively disrupted the cortisol-binding activity of CBG. This proteolytic activity was inhibited by zinc chelators and occurred most efficiently at pH 7 and elevated physiological temperature (ie, 41°C). Mass spectrometric analysis of a semi-purified fraction of P. aeruginosa media identified the virulence factor LasB as the responsible protease, and this was confirmed by assaying media from LasB-deficient P. aeruginosa. This metalloprotease cleaves the CBG RCL at a major site, distinct from that targeted by neutrophil elastase. Our results suggest that humoral responses to P. aeruginosa infection are influenced by this pathogen's ability to secrete a protease that promotes the release of the anti-inflammatory steroid, cortisol, from its plasma transport protein.

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Novel Corticosteroid-Binding Globulin Variant That Lacks Steroid Binding Activity

Endocrine Reviews ◽

10.1210/edrv.31.4.9978 ◽

2010 ◽

Vol 31 (4) ◽

pp. 607-608

Author(s):

I. Perogamvros ◽

C. Underhill ◽

D. E. Henley ◽

K. D. Hadfield ◽

W. G. Newman ◽

...

Keyword(s):

Binding Capacity ◽

Serum Cortisol ◽

Pulse Frequency ◽

Binding Activity ◽

Glucocorticoid Signaling ◽

Free Cortisol ◽

Corticosteroid Binding Globulin ◽

Undetectable Serum ◽

Steroid Binding ◽

Cortisol Levels

Abstract Background: Corticosteroid-binding globulin (CBG) is the principal carrier for glucocorticoids in the circulation and a regulator of their bioavailability. Inherited CBG deficiencies are rarely reported, and only three causative mutations in four families have been described. Patients, Methods, and Results: In a 26-yr-old female with hypotension, fatigue, and undetectable serum cortisol at presentation, we have identified a novel homozygous c.776g>t transversion in exon 3 of the CBG (SERPINA6) gene. This results in a p.Gly237Val substitution that is predicted to influence the positioning of two β-sheets that constitute part of the CBG steroid-binding site. Two siblings were also homozygous for the variant, whereas her mother and an unaffected sibling were heterozygous. No other symptomatic family members were identified apart from the proband. Individuals homozygous for the variant had serum CBG levels below the reference range when measured by RIA, but CBG was unmeasurable in cortisol-binding capacity assays. In the same individuals, we observed very low baseline and stimulated serum cortisol levels but normal free serum and salivary cortisol and plasma ACTH. In a study of ultradian cortisol pulsatility, increased pulse frequency was only observed in the proband. Conclusion: We describe a novel CBG variant that lacks steroid binding. All mutant homozygotes have very low serum cortisol, but normal free cortisol levels. The only biochemical feature to distinguish the symptomatic subject was increased cortisol pulsatility, and we suggest that this may influence glucocorticoid signaling and contribute to symptoms previously associated with CBG deficiency.

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A comparison of HER2 tumor accumulations of Ga-67 labeled anti-HER2 antibody with chemically and site-specifically conjugated bifunctional chelators

10.21203/rs.3.rs-27077/v1 ◽

2020 ◽

Author(s):

Yumiko Kono ◽

Keita Utsunomiya ◽

Yuta Ohira ◽

Hirokazu Satoh ◽

Naoki Kan ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Binding Capacity ◽

Binding Activity ◽

Pharmacokinetic Analysis ◽

Pharmacokinetic Studies ◽

Her2 Positive ◽

Site Specific ◽

Planar Images

Abstract Background Monoclonal antibodies (mAb) developed to target specific cancers have achieved considerable success to-date. To further enhance therapeutic efficacy monoclonal antibodies may be conjugated with a cytotoxic drug or radioisotope. We present the development of new method based on site-specific conjugation (SSC) for targeting HER2. The study design involves a comparison of the accumulation of Ga-67 labeled anti-HER2 antibodies with SSC versus conventional chemical conjugation in HER2-positive tumors. Anti-HER2 antibodies were chemically conjugated (Chem) with the bifunctional chelator deferoxamine (Chem-mAb). The resulting chemical conjugate was radiolabeled with Ga-67 yielding Ga-67-Chem -mAb. The SSC anti-HER2 antibodies enzymatically conjugated with deferoxamine using transglutaminase (SSC-mAb) and radiolabeled with Ga-67 yielding Ga-67-SSC-mAb. In vitro, the binding activity of HER2 to both conjugated antibodies was measured using surface Plasmon resonance. In vivo, a xenograft mouse model consisting of transplanted CHO/HER2 were divided into two groups, the Chem and the SSC group. Planar images were acquired over three days after each mAb injection, respectively. Pharmacokinetic analysis was used to compare the Chem group to the SSC group, for Ga-67 accumulation. Result The SSC and Chem groups were found to have similar HER2 binding capacity, however the tumor accumulation ratio gradually increased in the SSC group. The pharmacokinetic studies also found that radiolabeled mAb accumulation was significantly higher in the SSC group than the Chem group in not only the tumors, but also in blood and in other organs. Conclusion The new site-specific conjugation may improve targeted antigen-specific cancer radioimmunotherapy and may, due to higher retention, require a lower dose.

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The Effect of Human Serum on the Binding Activity of Radiolabelled Monoclonal Antibodies

Tumori Journal ◽

10.1177/030089168707300602 ◽

1987 ◽

Vol 73 (6) ◽

pp. 547-554

Author(s):

Silvia Camagni ◽

Silvana Canevari ◽

Marina Ripamonti ◽

Delia Mezzanzanica ◽

Rosaria Orlandi ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Human Blood ◽

Solid Phase ◽

Binding Capacity ◽

Binding Activity ◽

Ovarian Carcinomas ◽

Radiolabelled Monoclonal Antibodies ◽

In Vitro Stability ◽

Murine Monoclonal Antibodies

Three murine monoclonal antibodies (MoAbs), MBrl and MOv2 of IgM isotype and MOv8 of IgG isotype, with restricted reactivity for breast or ovarian carcinomas, were labelled with 125I in the perspective of obtaining specific and stable radioimmunopharmaceutical reagents. The radiolabeled MoAbs were analyzed for their « in vitro » stability in human blood. They were incubated at 37 °C for various lengths of time in human or, as a control, in murine blood and their binding capacity was evaluated by solid-phase RIA and compared with that obtained after incubation with buffer. In human blood, serum and plasma, but not with other components such as erythrocytes, leukocytes, HSA and IgG, the MoAbs revealed a loss of binding reactivity which was marked and constant for the IgM MoAbs, and only occasional for the IgG MoAb. In murine serum the decrease was not so rapid. The same change in the binding capacity was observed when the MoAbs were labelled with 3H or 35S, excluding the involvement of dehalogenating mechanisms. In the perspective of using MoAbs for intracavity therapy the effect of ascitic or pleural fluids on their binding activity was also evaluated. The inhibition of the binding reactivity was not as evident and was not related to the MoAb isotype.

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Novel Corticosteroid-Binding Globulin Variant That Lacks Steroid Binding Activity

Endocrinology ◽

10.1210/endo.151.8.9995 ◽

2010 ◽

Vol 151 (8) ◽

pp. 4067-4067

Author(s):

I. Perogamvros ◽

C. Underhill ◽

D. E. Henley ◽

K. D. Hadfield ◽

W. G. Newman ◽

...

Keyword(s):

Binding Activity ◽

Corticosteroid Binding Globulin ◽

Steroid Binding

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Identification and characterization of a human corticosteroid binding globulin variant with a reduced affinity for cortisol

Journal of Endocrinology ◽

10.1677/joe.0.1040269 ◽

1985 ◽

Vol 104 (2) ◽

pp. 269-277 ◽

Cited By ~ 17

Author(s):

P. A. Robinson ◽

G. L. Hammond

Keyword(s):

Binding Capacity ◽

Affinity Purification ◽

Gel Filtration ◽

Healthy Woman ◽

Late Pregnancy ◽

Physiological Significance ◽

Binding Characteristics ◽

Corticosteroid Binding Globulin ◽

Steroid Binding

ABSTRACT A corticosteroid binding globulin variant (CBGv) has been identified in a serum sample taken from an apparently healthy woman during late pregnancy. Identification was based on the observation that it exhibited approximately half the cortisol binding capacity expected when compared to its concentration measured by radioimmunoassay (RIA). Affinity purification of CBGv excluded the possibility that this anomaly was caused by assay interference, and demonstrated that immunoreactive CBGv was capable of binding cortisol. The CBGv had a molecular weight (63 800) similar to normal CBG, and no evidence of molecular aggregation was found by gel filtration. Although the electrophoretic mobility, isoelectric profile and immunochemical identity of CBGv appeared to be similar to normal CBG, it focussed as two distinct bands (pI 5·48 and pI 5·53) after desialylation with neuraminidase, unlike normal CBG which focusses only at pI 5·48. Investigation of the steroid binding characteristics of CBGv revealed a reduced association-rate constant (Ka = 1·05 × 109 1/mol) and dissociation half-time (12·5 min) when compared with normal CBG (Ka = 1·39 × 109l/mol and 25 min at 0 °C) but an apparently normal steroid binding specificity. Although the physiological significance of this variant is not known, the cortisol concentration in the variant serum was within the normal range of women during late pregnancy. No other CBG variants were identified among other normal controls (n = 66) or nine patients with Cushing's syndrome. It is suggested that comparisons between cortisol binding capacity and RIA will reveal other variants of CBG, and lead to greater understanding of their physiological significance. J. Endocr. (1985) 104, 269–277

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Decreased immunoreactivity and binding activity of corticosteroid-binding globulin in serum in septic shock.

Clinical Chemistry ◽

10.1093/clinchem/35.8.1675 ◽

1989 ◽

Vol 35 (8) ◽

pp. 1675-1679 ◽

Cited By ~ 79

Author(s):

M Pugeat ◽

A Bonneton ◽

D Perrot ◽

B Rocle-Nicolas ◽

H Lejeune ◽

...

Keyword(s):

Septic Shock ◽

Ion Exchange ◽

Binding Capacity ◽

Normal Subjects ◽

Binding Activity ◽

Healthy Controls ◽

Corticosteroid Binding Globulin ◽

Ion Exchange Column ◽

Monospecific Antiserum ◽

Final Purification

Abstract To investigate the mechanism(s) responsible for the depletion of corticosteroid-binding globulin (CBG) activity in serum in septic shock, we developed a radioimmunoassay (RIA) for human CBG, using a monospecific antiserum to human CBG raised in rabbits. CBG was purified from pooled human serum by precipitation with ammonium sulfate and successive affinity chromatography treatments on corticosterone-Sepharose and concanavalin A-Sepharose. Final purification was achieved by HPLC on a diethylaminoethyl-PW (polymer matrix) ion-exchange column. Typical standard curves established for the CBG immunoassay showed parallelism for pure CBG and serial dilutions of sera from patients with septic or nonseptic shock and from healthy controls. Measurements of CBG by RIA showed a significantly (P less than 0.001) lower CBG concentration in patients with septic shock (22.9 +/- 5.9 mg/L, mean +/- SD; n = 23) than in controls (39.9 +/- 6.5 mg/L, n = 21) or in patients with nonseptic shock (33.3 +/- 6.5 mg/L, n = 12). The correlation between the concentrations determined by RIA and the CBG binding capacity was significant (r = 0.619, P less than 0.001, n = 33). The electrophoretic mobility of CBG was similar in sera from septic shock patients and normal subjects (Rf = 0.52-0.56). This suggests that the depletion of the corticosteroid-binding activity in serum during septic shock is associated with a decreased amount of CBG.

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A phylogenetic study of the structural and functional characteristics of corticosteroid binding globulin in primates

Journal of Endocrinology ◽

10.1677/joe.0.1040251 ◽

1985 ◽

Vol 104 (2) ◽

pp. 251-257 ◽

Cited By ~ 27

Author(s):

P. A. Robinson ◽

C. Hawkey ◽

G. L. Hammond

Keyword(s):

Plasma Cortisol ◽

World Monkey ◽

Binding Activity ◽

Rate Of Change ◽

Phylogenetic Study ◽

New World Monkeys ◽

Old World ◽

Old World Monkey ◽

Corticosteroid Binding Globulin ◽

Steroid Binding

ABSTRACT A monospecific antiserum against human corticosteroid binding globulin (hCBG) has been used to identify structural similarities between hCBG and CBG in the blood of other primates and representative species of different vertebrate classes. Double immunodiffusion analysis indicated that only CBG in Old World monkeys and apes cross-react with the hCBG antiserum. This was confirmed by a solid-phase radioimmunoassay for hCBG which also demonstrated that CBG in apes is immunologically identical to hCBG and that Old World monkey CBG comprises most, but not all, of the hCBG epitopes. The electrophoretic mobilities of human, gorilla and gibbon CBG were similar (RF 0·50–0·51), but differed from Old World monkey CBG (RF 0·44–0·49) and chimpanzee CBG (RF 0·47). Although serum/plasma cortisol binding capacities were similar in Old World primates, the dissociation half-times (t½) of cortisol were higher from human and ape CBG (18–25 min) than from Old World monkey CBG (14–18 min). The steroid binding specificities of human and ape (CBG corticosterone > cortisol > progesterone ≥ testosterone) were also different from those of Old World monkey CBG (corticosterone >> cortisol ≃ progesterone > testosterone). Lemur plasma cortisol binding capacity and CBG dissociation t½ of cortisol were similar to hCBG, but its steroid binding specificity was different (cortisol > corticosterone > progesterone ≥ testosterone) and it did not cross-react with the hCBG antiserum. We could not detect high affinity cortisol binding activity in blood samples from New World monkeys, and they did not cross-react with the hCBG antiserum. These results suggest that considerable modification in the steroid binding activity and structure of CBG has occurred since the evolutionary appearance of the primates, but that the rate of change decreased after the cladogenesis of Catarrhine primates. J. Endocr. (1985) 104, 251–257

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