scholarly journals Characterization of mini-protein S, a recombinant variant of protein S that lacks the sex hormone binding globulin-like domain

1998 ◽  
Vol 330 (1) ◽  
pp. 389-396 ◽  
Author(s):  
Merel VAN WIJNEN ◽  
G. Jeanette STAM ◽  
T. G. Glenn CHANG ◽  
C. M. Joost MEIJERS ◽  
H. Pieter REITSMA ◽  
...  
Keyword(s):  
Recombinant Protein ◽  
Solid Phase ◽  
Anticoagulant Activity ◽  
Protein S ◽  
Sex Hormone ◽  
Sex Hormone Binding Globulin ◽  
Hormone Binding ◽  
Wild Type ◽  
Cofactor Activity

Protein S is a vitamin K-dependent glycoprotein involved in the regulation of the anticoagulant activity of activated protein C (APC). Also, an anticoagulant role for protein S, independent of APC, has been described. Protein S has a unique C-terminal sex hormone binding globulin (SHBG)-like domain that represents about half of the molecule. To define the role of this domain in APC cofactor activity and in binding to C4b-binding protein (C4BP), we have constructed a recombinant protein S molecule of N-terminal residues 1-242 that lacks the SHBG domain (mini-protein S). A panel of monoclonal antibodies directed against the N-terminal region of protein S recognized plasma-derived protein S, wild-type recombinant protein S and mini-protein S with similar affinities, whereas a monoclonal antibody that recognizes an epitope in the SHBG domain did not detect mini-protein S. Mini-protein S did not bind to C4BP in a solid-phase binding assay, and the cofactor activity of mini-protein S was not inhibited by preincubation with C4BP. In a plasma coagulation assay, the cofactor activity of mini-protein S was lower than wild-type or plasma-derived preparations. In contrast, no difference in APC cofactor activities was observed when the preparations were tested in purified systems that monitor the APC-mediated degradation of factors Va or VIIIa. In conclusion, we constructed a protein S molecule that fails to bind C4BP and still displays cofactor activity for APC. This confirms the role of the C-terminal SHBG region in C4BP binding and demonstrates that N-terminal residues 1-242 are sufficient for the expression of APC cofactor activity in a system using purified components. In plasma, however, the C-terminal SHBG region plays a role in the expression of optimal APC cofactor activity.

scholarly journals Dietary Intake of 17α-Ethinylestradiol Promotes HCC Progression in Humanized Male Mice Expressing Sex Hormone-Binding Globulin

2021 ◽  
Vol 22 (22) ◽  
pp. 12557
Author(s):  
Sang R. Lee ◽  
Su Hee Jeong ◽  
Jun H. Heo ◽  
Seong Lae Jo ◽  
Je-Won Ko ◽  
...  
Keyword(s):  
High Plasma ◽  
Sex Hormone ◽  
Sex Hormone Binding Globulin ◽  
Hormone Action ◽  
Male Mice ◽  
Hormone Binding ◽  
Wild Type ◽  
Synthetic Estrogen ◽  
Androgen Binding ◽  
Old Mice

Hepatocellular carcinoma (HCC) is a male-oriented malignancy; its progression is affected by sex hormones. 17α-ethinylestradiol (EE2) is a synthetic estrogen widely used as an oral contraceptive; however, it is unknown whether EE2 regulates sex hormone action in HCC. We investigated whether EE2 influences HCC risk in male androgenic environments, using mice expressing human sex hormone-binding globulin (SHBG). Two-week-old male mice were injected with diethyl-nitrosamine (DEN, 25 mg/kg) and fed an EE2 diet for 10 weeks from 30 weeks of age. Development and characteristics of liver cancer were evaluated in 40-week-old mice via molecular and histological analyses. Although EE2 did not increase HCC progression in wild-type mice, SHBG mice exhibited remarkably higher HCC risk when fed EE2. The livers of EE2-treated SHBG mice exhibited substantially increased pro-inflammatory necrosis with high plasma levels of ALT and HMGB1, and intrahepatic injury and fibers. Additionally, increased androgen response and androgen-mediated proliferation in the livers of EE2-treated SHBG mice and EE2-exposed hepatocytes under SHBG conditions were observed. As a competitor of SHBG-androgen binding, EE2 could bind with SHBG and increase the bioavailability of androgen. Our results revealed that EE2 is a novel risk factor in androgen-dominant men, predisposing them to HCC risk.

Role of the Ovary in the Regulation of Sex Hormone Binding Globulin and Its Contribution to Peripheral Levels of Androstenedione

2009 ◽  
Vol 82 (04) ◽  
pp. 29-34 ◽  
Author(s):  
P. Bolufer ◽  
P. Antonio ◽  
R. Garcia ◽  
J. Munoz ◽  
A. Rodriguez ◽  
...  
Keyword(s):  
Sex Hormone ◽  
Sex Hormone Binding Globulin ◽  
Hormone Binding

The Ser460Pro mutation in recombinant protein S Heerlen does not affect its APC-cofactor and APC-independent anticoagulant activities

2004 ◽  
Vol 91 (06) ◽  
pp. 1105-1114 ◽  
Author(s):  
Rory Koenen ◽  
Lucio Gomes ◽  
Guido Tans ◽  
Jan Rosing ◽  
Tilman Hackeng
Keyword(s):  
Recombinant Protein ◽  
Vitamin K ◽  
Dissociation Constants ◽  
Anticoagulant Activity ◽  
Protein S ◽  
Wild Type ◽  
Free Protein ◽  
Increased Risk ◽  
Plasma Factor ◽  
Thrombotic Complications

SummaryProtein S is a vitamin K-dependent plasma protein that functions as an APC-cofactor, but also exhibits anticoagulant activity in the absence of APC. The Heerlen polymorphism of protein S is characterized by a Ser460Pro substitution and lacks glycosylation at Asn458. It is associated with decreased protein S levels due to selective deficiency of free protein S Heerlen.To understand the lack of thrombotic complications associated with the protein S Heerlen mutation, we compared recombinant protein S Heerlen, wild type (wt) protein S and plasmaderived protein S. wt-Protein S and protein S Heerlen each bound 1:1 to C4BP with dissociation constants of 0.27 and 0.33 nM, respectively. Both wt-protein S and protein S Heerlen, either free or in complex with C4BP, were equally active as prothrombinase inhibitors in the absence of APC. All three protein S preparations stimulated APC-catalyzed inactivation of normal FVa, FVa Leiden and FVIIIa to the same extent. If extrapolated to plasma, it is not likely that the decreased free protein S levels in carriers of the protein S Heerlen mutation are compensated by an increased anticoagulant activity of protein S Heerlen-C4BP complexes. It is possible that an unrecognized plasma factor selectively enhances the anticoagulant activity of protein S Heerlen. If not, the reduction of free protein S levels in heterozygous protein S Heerlen-carriers combined with (low) normal total protein S levels apparently minimally affects the total anticoagulant activity of protein S (APC-cofactor and APC-independent activity) and hence is not associated with increased risk of venous thrombosis.

A Method for the Determination of Sex Hormone Binding Globulin Using Concanavalin A-Sepharose

1992 ◽  
Vol 29 (2) ◽  
pp. 168-171 ◽  
Author(s):  
J A Whittaker ◽  
M L Cawood ◽  
R E Oakey
Keyword(s):  
Concanavalin A ◽  
Binding Assay ◽  
Solid Phase ◽  
Sex Hormone ◽  
Sex Hormone Binding Globulin ◽  
Research Use ◽  
Hormone Binding

A binding assay for sex hormone binding globulin (SHBG) has been developed in which SHBG is saturated with tritiated dihydrotestosterone (DHT). Separation of bound and free DHT is achieved by using Concanavalin A-Sepharose as a solid phase matrix. The method is described and its performance, including linearity, imprecision and comparison with other methods, is assessed. The assay is simple and robust and is suitable for analysis of samples of plasma or serum for clinical or research use.

scholarly journals Human sex hormone-binding globulin does not provide metabolic protection against diet-induced obesity and dysglycemia in mice

2018 ◽  
Vol 7 (1) ◽  
pp. 91-96 ◽  
Author(s):  
Yael Sofer ◽  
Nava Nevo ◽  
Michal Vechoropoulos ◽  
Gabi Shefer ◽  
Etty Osher ◽  
...  
Keyword(s):  
Metabolic Syndrome ◽  
Fasting Blood Glucose ◽  
Serum Triglyceride ◽  
Tolerance Test ◽  
Sex Hormone ◽  
Sex Hormone Binding Globulin ◽  
Hormone Binding ◽  
Wild Type ◽  
Diet Induced Obesity ◽  
Increased Risk

Background Sex hormone-binding globulin (SHBG) is the main transporter of sex hormones in most vertebrates. Low SHBG levels have been linked to increased risk for diabetes and metabolic syndrome. Polymorphisms of the SHBG gene linked to low SHBG protein levels also strongly predicted increased risk of type 2 diabetes, thus raising the possibility that SHBG may play a role in the pathogenesis of insulin resistance and diabetes. Aim To examine whether expression of human SHBG in mice may ameliorate the development of diabetes and metabolic syndrome in response to a high-fat diet (HFD). Methods Transgene mice expressing a human SHBG transgene (SHBG+) (N = 10/11; males/females) and their wild type littermates (N = 12/8; males/females) were fed HFD for 4.5 months. Results HFD induced comparable obesity in control and SHBG+ mice. Male transgenes had higher muscle mass after 2–3.5 months HFD (0.43 ± 0.028 (n = 4) vs 0.38 ± 0.053 g (n = 7), P = 0.05). Fasting blood glucose, as well as insulin or HOMA-IR, was not different in transgenic vs wild-type males after 4–5 months HFD. Female transgenes had higher fasting glucose (152 ± 29 (n = 7) vs 115 ± 27 mg/dL, P = 0.01 (n = 8)), but mean insulin and HOMA-IR were not different. Likewise, insulin tolerance test and intra-peritoneal glucose tolerance test (GTT) were not different. Finally, SHBG+ mice were not different from controls in terms of liver enzymes, serum triglyceride levels and blood pressure. Conclusion In mice with diet-induced obesity, human SHBG did not protect against development of obesity or dysglycemia.

Cleavage of Factor V at Arg 506 by Activated Protein C and the Expression of Anticoagulant Activity of Factor V

Blood ◽  
1999 ◽  
Vol 93 (8) ◽  
pp. 2552-2558 ◽  
Author(s):  
Elisabeth Thorelli ◽  
Randal J. Kaufman ◽  
Björn Dahlbäck
Keyword(s):  
Protein C ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Protein S ◽  
Factor V ◽  
Wild Type ◽  
Apc Resistance ◽  
S 100 ◽  
Negative Effect ◽  
Cofactor Activity

Activated protein C (APC) inhibits coagulation by cleaving and inactivating procoagulant factor Va (FVa) and factor VIIIa (FVIIIa). FV, in addition to being the precursor of FVa, has anticoagulant properties; functioning in synergy with protein S as a cofactor of APC in the inhibition of the FVIIIa-factor IXa (FIXa) complex. FV:Q506 isolated from an individual homozygous for APC-resistance is less efficient as an APC-cofactor than normal FV (FV:R506). To investigate the importance of the three APC cleavage sites in FV (Arg-306, Arg-506, and Arg-679) for expression of its APC-cofactor activity, four recombinant FV mutants (FV:Q306, FV:Q306/Q506, FV:Q506, and FV:Q679) were tested. FV mutants with Gln (Q) at position 506 instead of Arg (R) were found to be poor APC-cofactors, whereas Arg to Gln mutations at positions 306 or 679 had no negative effect on the APC-cofactor activity of FV. The loss of APC-cofactor activity as a result of the Arg-506 to Gln mutation suggested that APC-cleavage at Arg-506 in FV is important for the ability of FV to function as an APC-cofactor. Using Western blotting, it was shown that both wild-type FV and mutant FV was cleaved by APC during the FVIIIa inhibition. At optimum concentrations of wild-type FV (11 nmol/L) and protein S (100 nmol/L), FVIIIa was found to be highly sensitive to APC with maximum inhibition occurring at less than 1 nmol/L APC. FV:Q506 was inactive as an APC-cofactor at APC-concentrations ≤ 1 nmol/L and only partially active at higher APC concentrations. Our results show that increased expression of FV anticoagulant activity correlates with APC-mediated cleavage at Arg-506 in FV, but not with cleavage at Arg-306 nor at Arg-679.

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