scholarly journals Pyrexia and acidosis act independently of neutrophil elastase reactive center loop cleavage to effect cortisol release from corticosteroid‐binding globulin

2020 ◽  
Vol 29 (12) ◽  
pp. 2495-2509
Author(s):  
Emily J. Meyer ◽  
David J. Torpy ◽  
Anastasia Chernykh ◽  
Morten Thaysen‐Andersen ◽  
Marni A. Nenke ◽  
...  
Keyword(s):  
Neutrophil Elastase ◽  
Reactive Center Loop ◽  
Reactive Center ◽  
Corticosteroid Binding Globulin ◽  
Cortisol Release

scholarly journals N-Glycosylation influences human corticosteroid-binding globulin measurements

2019 ◽  
Vol 8 (8) ◽  
pp. 1136-1148 ◽  
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.

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

Endocrinology ◽  
2014 ◽  
Vol 155 (8) ◽  
pp. 2900-2908 ◽  
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.

scholarly journals Residues in the Human Corticosteroid-binding Globulin Reactive Center Loop That Influence Steroid Binding before and after Elastase Cleavage

2008 ◽  
Vol 284 (2) ◽  
pp. 884-896 ◽  
Author(s):  
Hai-Yan Lin ◽  
Caroline Underhill ◽  
Bernd R. Gardill ◽  
Yves A. Muller ◽  
Geoffrey L. Hammond
Keyword(s):  
Reactive Center Loop ◽  
Reactive Center ◽  
Corticosteroid Binding Globulin ◽  
Before And After ◽  
Steroid Binding

A protein structural approach to the solution of biological problems: alpha 1-antitrypsin as a recent example

1993 ◽  
Vol 265 (3) ◽  
pp. L211-L219 ◽  
Author(s):  
D. A. Lomas ◽  
R. W. Carrell
Keyword(s):  
Endoplasmic Reticulum ◽  
Neutrophil Elastase ◽  
Serine Proteinase ◽  
Reactive Center Loop ◽  
Hepatocellular Damage ◽  
Reactive Center ◽  
Normal Gene ◽  
Alpha 1 Antitrypsin ◽  
And Function ◽  
Positively Charged

alpha 1-Antitrypsin is a circulating serine proteinase inhibitor that protects the lungs against proteolysis by the enzyme neutrophil elastase. Most northern Europeans have only the normal M form, but some 4% are heterozygotes for the Z deficiency mutant. This mutant is characterized by the substitution of a positively charged lysine residue for a negatively charged glutamic acid at position 342 and results in normal gene translation but reduced protein secretion into the plasma. The plasma levels of antitrypsin in homozygotes are only 15% of normal, the other 85% being retained in the endoplasmic reticulum of the hepatocyte. This review describes the effect of the Z mutation on the structure and function of antitrypsin and illustrates the importance of understanding protein structure in solving the mechanism of Z antitrypsin retention within the liver. We demonstrate that antitrypsin accumulation in the liver results from a unique interaction between antitrypsin molecules. The Z mutation perturbs the gap between the third and fifth strands of the A sheet, allowing the reactive center loop of one molecule to insert into the A sheet of a second. This loop-sheet polymerization results in the formation of chains of protein which form insoluble inclusions in the endoplasmic reticulum, resulting in hepatocellular damage and cirrhosis. In addition, the Z mutation results in a distortion of the circular dichroic spectrum, a rearrangement of the reactive center loop with respect to the A sheet, and a reduction in association rate constant with the cognate proteinase neutrophil elastase.

scholarly journals Functional implications of corticosteroid-binding globulin N-glycosylation

2018 ◽  
Vol 60 (2) ◽  
pp. 71-84 ◽  
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.

scholarly journals Serpin reactive center loop mobility is required for inhibitor function but not for enzyme recognition.

1994 ◽  
Vol 269 (44) ◽  
pp. 27657-27662 ◽  
Author(s):  
D A Lawrence ◽  
S T Olson ◽  
S Palaniappan ◽  
D Ginsburg
Keyword(s):  
Reactive Center Loop ◽  
Reactive Center ◽  
Enzyme Recognition

A reactive center loop–based prediction platform to enhance the design of therapeutic SERPINs

2021 ◽  
Vol 118 (45) ◽  
pp. e2108458118
Author(s):  
Wariya Sanrattana ◽  
Thibaud Sefiane ◽  
Simone Smits ◽  
Nadine D. van Kleef ◽  
Marcel H. Fens ◽  
...  
Keyword(s):  
Cleavage Site ◽  
Serine Proteases ◽  
Protein C ◽  
Activated Protein C ◽  
Reactive Center Loop ◽  
Physiological Processes ◽  
Naturally Occurring ◽  
Reactive Center ◽  
Insight Into

Serine proteases are essential for many physiological processes and require tight regulation by serine protease inhibitors (SERPINs). A disturbed SERPIN–protease balance may result in disease. The reactive center loop (RCL) contains an enzymatic cleavage site between the P1 through P1’ residues that controls SERPIN specificity. This RCL can be modified to improve SERPIN function; however, a lack of insight into sequence–function relationships limits SERPIN development. This is complicated by more than 25 billion mutants needed to screen the entire P4 to P4’ region. Here, we developed a platform to predict the effects of RCL mutagenesis by using α1-antitrypsin as a model SERPIN. We generated variants for each of the residues in P4 to P4’ region, mutating them into each of the 20 naturally occurring amino acids. Subsequently, we profiled the reactivity of the resulting 160 variants against seven proteases involved in coagulation. These profiles formed the basis of an in silico prediction platform for SERPIN inhibitory behavior with combined P4 to P4’ RCL mutations, which were validated experimentally. This prediction platform accurately predicted SERPIN behavior against five out of the seven screened proteases, one of which was activated protein C (APC). Using these findings, a next-generation APC-inhibiting α1-antitrypsin variant was designed (KMPR/RIRA; / indicates the cleavage site). This variant attenuates blood loss in an in vivo hemophilia A model at a lower dosage than the previously developed variant AIKR/KIPP because of improved potency and specificity. We propose that this SERPIN-based RCL mutagenesis approach improves our understanding of SERPIN behavior and will facilitate the design of therapeutic SERPINs.

scholarly journals Crystal Structure of Monomeric Native Antithrombin Reveals a Novel Reactive Center Loop Conformation

2006 ◽  
Vol 281 (46) ◽  
pp. 35478-35486 ◽  
Author(s):  
Daniel J. D. Johnson ◽  
Jonathan Langdown ◽  
Wei Li ◽  
Stephan A. Luis ◽  
Trevor P. Baglin ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Reactive Center Loop ◽  
Reactive Center ◽  
Loop Conformation

The Reactive-center Loop of Active PAI-1 is Folded Close to the Protein Core and can be Partially Inserted

2004 ◽  
Vol 335 (3) ◽  
pp. 823-832 ◽  
Author(s):  
Peter Hägglöf ◽  
Fredrik Bergström ◽  
Malgorzata Wilczynska ◽  
Lennart B.-Å Johansson ◽  
Tor Ny
Keyword(s):  
Protein Core ◽  
Reactive Center Loop ◽  
Reactive Center ◽  
Pai 1
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