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Asn347 Glycosylation of Corticosteroid-binding Globulin Fine-tunes the Host Immune Response by Modulating Proteolysis by Pseudomonas aeruginosa and Neutrophil Elastase*

  • Zeynep Sumer-Bayraktar
    Footnotes
    Affiliations
    Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, New South Wales 2109, Australia
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  • Oliver C. Grant
    Affiliations
    Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia 30602
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  • Vignesh Venkatakrishnan
    Footnotes
    Affiliations
    Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, New South Wales 2109, Australia
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  • Robert J. Woods
    Footnotes
    Affiliations
    Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia 30602
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  • Nicolle H. Packer
    Footnotes
    Affiliations
    Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, New South Wales 2109, Australia
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  • Morten Thaysen-Andersen
    Correspondence
    Supported by an Early Career Fellowship from the Cancer Institute, New South Wales, Australia and a Macquarie University Research Development Grant (MQRDG). To whom correspondence should be addressed. Tel.: 61-2-9850-7487; Fax: 61-2-9850-6192
    Affiliations
    Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, New South Wales 2109, Australia
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  • Author Footnotes
    * The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
    This article contains supplemental Table S1.
    1 Recipient of an International Macquarie University postgraduate student research scholarship (iMQRES).
    2 Supported by National Institutes of Health Grant P41 GM103390.
    3 Supported by ARC Super Science Grant FS110200026 and ARC Centre of Excellence in Nanoscale Biophotonics Grant CE140100003.
Open AccessPublished:June 23, 2016DOI:https://doi.org/10.1074/jbc.M116.735258
      Corticosteroid-binding globulin (CBG) delivers anti-inflammatory cortisol to inflamed tissues upon elastase-based proteolysis of the exposed reactive center loop (RCL). However, the molecular mechanisms that regulate the RCL proteolysis by co-existing host and bacterial elastases in inflamed/infected tissues remain unknown. We document that RCL-localized Asn347 glycosylation fine-tunes the RCL cleavage rate by human neutrophil elastase (NE) and Pseudomonas aeruginosa elastase (PAE) by different mechanisms. NE- and PAE-generated fragments of native and exoglycosidase-treated blood-derived CBG of healthy individuals were monitored by gel electrophoresis and LC-MS/MS to determine the cleavage site(s) and Asn347 glycosylation as a function of digestion time. The site-specific (Val344-Thr345) and rapid (seconds to minutes) NE-based RCL proteolysis was significantly antagonized by several volume-enhancing Asn347 glycan features (i.e. occupancy, triantennary GlcNAc branching, and α1,6-fucosylation) and augmented by Asn347 NeuAc-type sialylation (all p < 0.05). In contrast, the inefficient (minutes to hours) PAE-based RCL cleavage, which occurred equally well at Thr345-Leu346 and Asn347-Leu348, was abolished by the presence of Asn347 glycosylation but was enhanced by sialoglycans on neighboring CBG N-sites. Molecular dynamics simulations of various Asn347 glycoforms of uncleaved CBG indicated that multiple Asn347 glycan features are modulating the RCL digestion efficiencies by NE/PAE. Finally, high concentrations of cortisol showed weak bacteriostatic effects toward virulent P. aeruginosa, which may explain the low RCL potency of the abundantly secreted PAE during host infection. In conclusion, site-specific CBG N-glycosylation regulates the bioavailability of cortisol in inflamed environments by fine-tuning the RCL proteolysis by endogenous and exogenous elastases. This study offers new molecular insight into host- and pathogen-based manipulation of the human immune system.

      Introduction

      Human corticosteroid-binding globulin (CBG)
      The abbreviations used are:
      CBG
      corticosteroid-binding globulin
      CBG-Ct
      corticosteroid-binding globulin C-terminal fragment
      CBG-Nt
      corticosteroid-binding globulin N-terminal fragment
      A1AT
      α1-antitrypsin
      CID
      collision-induced dissociation
      EIC
      extracted ion chromatogram
      GlcNAc
      N-acetylglucosamine
      MD
      molecular dynamics
      NE
      neutrophil elastase
      NeuAc
      N-acetylneuraminic acid
      PAE
      P. aeruginosa elastase
      PAO1
      wound-derived P. aeruginosa laboratory strain
      PASS1
      cystic fibrosis-derived P. aeruginosa strain
      RCL
      reactive center loop
      TBG
      thyroxine-binding globulin
      Fuc
      fucose
      Man
      mannose
      PDB
      Protein Data Bank.
      is the main carrier of cortisol in the blood circulatory system. CBG regulates the bioavailability of this anti-inflammatory steroid hormone by binding up to 90% of the entire cortisol pool with high affinity and specificity in a single binding pocket in a temperature-sensitive manner (
      • Siiteri P.K.
      • Murai J.T.
      • Hammond G.L.
      • Nisker J.A.
      • Raymoure W.J.
      • Kuhn R.W.
      The serum transport of steroid hormones.
      ,
      • Cameron A.
      • Henley D.
      • Carrell R.
      • Zhou A.
      • Clarke A.
      • Lightman S.
      Temperature-responsive release of cortisol from its binding globulin: a protein thermocouple.
      ). Although produced primarily by hepatocytes (
      • Khan M.S.
      • Aden D.
      • Rosner W.
      Human corticosteroid binding globulin is secreted by a hepatoma-derived cell line.
      ,
      • Hammond G.L.
      • Smith C.L.
      • Underhill D.A.
      Molecular studies of corticosteroid binding globulin structure, biosynthesis and function.
      ), human CBG mRNA and protein have been found in smaller amounts in other tissues, including lungs, kidneys, testes, placenta, endometrium, fallopian tube, heart, and some regions of the brain (
      • Hammond G.L.
      • Smith C.L.
      • Goping I.S.
      • Underhill D.A.
      • Harley M.J.
      • Reventos J.
      • Musto N.A.
      • Gunsalus G.L.
      • Bardin C.W.
      Primary structure of human corticosteroid binding globulin, deduced from hepatic and pulmonary cDNAs, exhibits homology with serine protease inhibitors.
      • Misao R.
      • Iwagaki S.
      • Sun W.S.
      • Fujimoto J.
      • Saio M.
      • Takami T.
      • Tamaya T.
      Evidence for the synthesis of corticosteroid-binding globulin in human placenta.
      • Misao R.
      • Hori M.
      • Ichigo S.
      • Fujimoto J.
      • Tamaya T.
      Corticosteroid-binding globulin mRNA levels in human uterine endometrium.
      • Miska W.
      • Peña P.
      • Villegas J.
      • Sánchez R.
      Detection of a CBG-like protein in human Fallopian tube tissue.
      • Schäfer H.H.
      • Gebhart V.M.
      • Hertel K.
      • Jirikowski G.F.
      Expression of corticosteroid-binding globulin CBG in the human heart.
      • Sivukhina E.V.
      • Jirikowski G.F.
      • Bernstein H.G.
      • Lewis J.G.
      • Herbert Z.
      Expression of corticosteroid-binding protein in the human hypothalamus, co-localization with oxytocin and vasopressin.
      • Perrot-Applanat M.
      • Racadot O.
      • Milgrom E.
      Specific localization of plasma corticosteroid-binding globulin immunoreactivity in pituitary corticotrophs.
      • Jirikowski G.F.
      • Pusch L.
      • Möpert B.
      • Herbert Z.
      • Caldwell J.D.
      Expression of corticosteroid binding globulin in the rat central nervous system.
      ). The varying circulatory levels of CBG in childhood, puberty, and adulthood and in different physiological states like pregnancy have been documented; for example, the CBG concentration in the blood of healthy individuals was measured to be ∼40 mg/l (
      • Heyns W.
      • Coolens J.L.
      Physiology of corticosteroid-binding globulin in humans.
      ). The circulatory half-life of human CBG has been established to be approximately 6 days, although some forms of CBG may be removed much faster (
      • Heyns W.
      • Coolens J.L.
      Physiology of corticosteroid-binding globulin in humans.
      ), as supported recently by investigation of the human CBG half-life in rabbits (
      • Lewis J.G.
      • Saunders K.
      • Dyer A.
      • Elder P.A.
      The half-lives of intact and elastase cleaved human corticosteroid-binding globulin (CBG) are identical in the rabbit.
      ). The CBG synthesis rate, which from the above can be estimated to be ∼0.1–0.2 mg/h under normal physiology, has been reported to be regulated by the glucocorticoids and IL-6 (
      • Perogamvros I.
      • Ray D.W.
      • Trainer P.J.
      Regulation of cortisol bioavailability: effects on hormone measurement and action.
      ). For instance, during inflammation, CBG acts as a negative acute-phase protein (
      • Verhoog N.
      • Allie-Reid F.
      • Vanden Berghe W.
      • Smith C.
      • Haegeman G.
      • Hapgood J.
      • Louw A.
      Inhibition of corticosteroid-binding globulin gene expression by glucocorticoids involves C/EBPβ.
      ), where the level of intact functionally active CBG in circulation is reduced due to the increased rate of proteolytic cleavage of this glucocorticoid carrier with a concomitant cortisol release (
      • Nenke M.A.
      • Rankin W.
      • Chapman M.J.
      • Stevens N.E.
      • Diener K.R.
      • Hayball J.D.
      • Lewis J.G.
      • Torpy D.J.
      Depletion of high-affinity corticosteroid-binding globulin corresponds to illness severity in sepsis and septic shock; clinical implications.
      ) and an IL-6-driven reduction in CBG synthesis (
      • Emptoz-Bonneton A.
      • Crave J.C.
      • LeJeune H.
      • Brébant C.
      • Pugeat M.
      Corticosteroid-binding globulin synthesis regulation by cytokines and glucocorticoids in human hepatoblastoma-derived (HepG2) cells.
      ,
      • Tsigos C.
      • Kyrou I.
      • Chrousos G.P.
      • Papanicolaou D.A.
      Prolonged suppression of corticosteroid-binding globulin by recombinant human interleukin-6 in man.
      ). The delivery mechanism of cortisol to target tissues, which is only partially understood, involves 1) interaction of the cortisol-bound CBG with a putative receptor on the membranes of some cortisol-sensitive tissues (i.e. liver, endometrium, placenta, and prostate) (
      • Strel'chyonok O.A.
      • Avvakumov G.V.
      Interaction of human CBG with cell membranes.
      ,
      • Hryb D.J.
      • Khan M.S.
      • Romas N.A.
      • Rosner W.
      Specific binding of human corticosteroid-binding globulin to cell membranes.
      ); 2) direct internalization of an intact cortisol-bound CBG complex in specific tissues (e.g. placental syncytiotrophoblasts) (
      • Strel'chyonok O.A.
      • Avvakumov G.V.
      Interaction of human CBG with cell membranes.
      ); and, the most documented, 3) extracellular elastase-based proteolytic cleavage of the exposed reactive center loop (RCL) of CBG, resulting in cortisol release and cellular uptake of cortisol upon a “stressed-to-relaxed” conformational change of CBG at the site of inflammation (
      • Pemberton P.A.
      • Stein P.E.
      • Pepys M.B.
      • Potter J.M.
      • Carrell R.W.
      Hormone binding globulins undergo serpin conformational change in inflammation.
      • Lin H.Y.
      • Underhill C.
      • Gardill B.R.
      • Muller Y.A.
      • Hammond G.L.
      Residues in the human corticosteroid-binding globulin reactive center loop that influence steroid binding before and after elastase cleavage.
      • Hammond G.L.
      • Smith C.L.
      • Underhill C.M.
      • Nguyen V.T.
      Interaction between corticosteroid binding globulin and activated leukocytes in vitro.
      • Klieber M.A.
      • Underhill C.
      • Hammond G.L.
      • Muller Y.A.
      Corticosteroid-binding globulin, a structural basis for steroid transport and proteinase-triggered release.
      • Qi X.
      • Loiseau F.
      • Chan W.L.
      • Yan Y.
      • Wei Z.
      • Milroy L.G.
      • Myers R.M.
      • Ley S.V.
      • Read R.J.
      • Carrell R.W.
      • Zhou A.
      Allosteric modulation of hormone release from thyroxine and corticosteroid-binding globulins.
      • Lewis J.G.
      • Elder P.A.
      Corticosteroid-binding globulin reactive centre loop antibodies recognise only the intact natured protein: elastase cleaved and uncleaved CBG may coexist in circulation.
      ). Readers are directed to a recent review as a source of more in depth coverage of the CBG biology (
      • Meyer E.J.
      • Nenke M.A.
      • Rankin W.
      • Lewis J.G.
      • Torpy D.J.
      Corticosteroid-binding globulin: a review of basic and clinical advances.
      ). The highly flexible RCL spanning the Glu333–Ile354 region of CBG is the target of human neutrophil elastase (NE), which cleaves Val344-Thr345 to form two complementary fragments: the large (50–55 kDa) N-terminal fragment (CBG-Nt) and the small (5–10 kDa) C-terminal fragment (CBG-Ct) (
      • Pemberton P.A.
      • Stein P.E.
      • Pepys M.B.
      • Potter J.M.
      • Carrell R.W.
      Hormone binding globulins undergo serpin conformational change in inflammation.
      ). The reduced cortisol affinity of cleaved CBG increases the local concentration of free cortisol, which is beneficial toward resolving inflammation in affected tissues by the anti-inflammatory effects of cortisol (
      • Nenke M.A.
      • Rankin W.
      • Chapman M.J.
      • Stevens N.E.
      • Diener K.R.
      • Hayball J.D.
      • Lewis J.G.
      • Torpy D.J.
      Depletion of high-affinity corticosteroid-binding globulin corresponds to illness severity in sepsis and septic shock; clinical implications.
      ,
      • Chan W.L.
      • Carrell R.W.
      • Zhou A.
      • Read R.J.
      How changes in affinity of corticosteroid-binding globulin modulate free cortisol concentration.
      ).
      It was recently suggested that in basal, low inflammatory conditions, proteases other than NE may be causing systemic CBG cleavage (
      • Nenke M.A.
      • Holmes M.
      • Rankin W.
      • Lewis J.G.
      • Torpy D.J.
      Corticosteroid-binding globulin cleavage is paradoxically reduced in α-1 antitrypsin deficiency: implications for cortisol homeostasis.
      ). Both endogenous and exogenous proteases were documented to cleave the RCL and reduce the cortisol affinity of CBG. It was also shown that chymotrypsin cleaves the RCL of CBG at Leu346-Asn347 and Leu348-Thr349, but the biological relevance of this pancreatic protease in the context of the host immune response in inflamed tissues remains unknown (
      • Lewis J.G.
      • Elder P.A.
      The reactive centre loop of corticosteroid-binding globulin (CBG) is a protease target for cortisol release.
      ). In addition, the Thr349-Ser350 and Ser350-Lys351 sites of E. coli-produced (non-glycosylated) recombinant human CBG were reported to be proteolytic target sites, but the responsible protease(s) remains elusive (
      • Gardill B.R.
      • Vogl M.R.
      • Lin H.Y.
      • Hammond G.L.
      • Muller Y.A.
      Corticosteroid-binding globulin: structure-function implications from species differences.
      ). Finally, P. aeruginosa elastase (PAE), the major virulence factor of this opportunistic Gram-negative pathogen and a zinc metalloprotease that is structurally unrelated to the serine proteases NE and chymotrypsin, was shown to cleave the RCL of CBG primarily between Asn347 and Leu348, leading to reduced cortisol affinity and release of the hormone from CBG (
      • Simard M.
      • Hill L.A.
      • Underhill C.M.
      • Keller B.O.
      • Villanueva I.
      • Hancock R.E.
      • Hammond G.L.
      Pseudomonas aeruginosa elastase disrupts the cortisol-binding activity of corticosteroid-binding globulin.
      ). It was also reported that PAE cleaves several RCL sites (i.e. Thr345-Leu346, Leu346-Asn347, Asn347-Leu348 (main cleavage site), and Leu348-Thr349) (
      • Simard M.
      • Hill L.A.
      • Underhill C.M.
      • Keller B.O.
      • Villanueva I.
      • Hancock R.E.
      • Hammond G.L.
      Pseudomonas aeruginosa elastase disrupts the cortisol-binding activity of corticosteroid-binding globulin.
      ). Interestingly, PAE and NE are known to co-exist in the inflamed and bacteria-infected respiratory tract of individuals with cystic fibrosis and chronic obstructive pulmonary disease (
      • Henke M.O.
      • John G.
      • Rheineck C.
      • Chillappagari S.
      • Naehrlich L.
      • Rubin B.K.
      Serine proteases degrade airway mucins in cystic fibrosis.
      ). Although growing evidence supports an elastase-driven release of cortisol from CBG as a mechanism for cortisol delivery to inflamed tissues, the molecular basis for the regulation of such a delivery mechanism by endogenous and exogenous elastases remains poorly understood.
      The six occupied N-glycosylation sites of mature CBG (383 amino acid residues) create extensive glycoform heterogeneity (
      • Hammond G.L.
      • Smith C.L.
      • Underhill D.A.
      Molecular studies of corticosteroid binding globulin structure, biosynthesis and function.
      ,
      • Hammond G.L.
      • Smith C.L.
      • Goping I.S.
      • Underhill D.A.
      • Harley M.J.
      • Reventos J.
      • Musto N.A.
      • Gunsalus G.L.
      • Bardin C.W.
      Primary structure of human corticosteroid binding globulin, deduced from hepatic and pulmonary cDNAs, exhibits homology with serine protease inhibitors.
      ,
      • Avvakumov G.V.
      • Hammond G.L.
      Glycosylation of human corticosteroid-binding globulin. Differential processing and significance of carbohydrate chains at individual sites.
      ). We recently performed a detailed site-specific glycoprofiling of all six glycosylation sites of blood-derived CBG from healthy individuals (
      • Sumer-Bayraktar Z.
      • Kolarich D.
      • Campbell M.P.
      • Ali S.
      • Packer N.H.
      • Thaysen-Andersen M.
      N-Glycans modulate the function of human corticosteroid-binding globulin.
      ). The sites displayed different heterogeneous populations of branched, primarily bi- and triantennary, complex glycans displaying α1,6-linked (core) fucose (Fuc) and terminal N-acetylneuraminic acid (NeuAc) residues. It was suggested that CBG carries predominantly α2,3-sialylation, as assessed by LC-MS/MS-based glycomics of free reduced N-glycans at the global (site-nonspecific) level of CBG with and without α2,3-linkage-specific sialidase treatment. In addition, sialylation was found to be an inhibitory feature for the CBG-receptor complex interaction (
      • Sumer-Bayraktar Z.
      • Kolarich D.
      • Campbell M.P.
      • Ali S.
      • Packer N.H.
      • Thaysen-Andersen M.
      N-Glycans modulate the function of human corticosteroid-binding globulin.
      ). Other lines of evidence suggest that N-glycosylation is a key modulator of CBG functions, including cortisol binding and delivery (e.g. glycosylated CBG was found to bind cortisol with significantly higher affinity and was more temperature-sensitive relative to non-glycosylated CBG) (
      • Chan W.L.
      • Carrell R.W.
      • Zhou A.
      • Read R.J.
      How changes in affinity of corticosteroid-binding globulin modulate free cortisol concentration.
      ). Moreover, upon NE-based RCL cleavage, the cortisol affinity of glycosylated CBG was reduced more than non-glycosylated forms, indicating that CBG glycosylation may yield a more rapid surge of free cortisol at target tissues to thereby facilitate a quicker resolution of inflammation (
      • Chan W.L.
      • Carrell R.W.
      • Zhou A.
      • Read R.J.
      How changes in affinity of corticosteroid-binding globulin modulate free cortisol concentration.
      ). Intriguingly, the C-terminally located Asn347, which is 84.7% occupied by primarily triantennary sialoglycans, is located on the RCL in close proximity to the reported cleavage sites (
      • Sumer-Bayraktar Z.
      • Kolarich D.
      • Campbell M.P.
      • Ali S.
      • Packer N.H.
      • Thaysen-Andersen M.
      N-Glycans modulate the function of human corticosteroid-binding globulin.
      ). Similarly, other highly occupied glycosylation sites of CBG displaying mainly bi- and triantennary sialoglycans appear to be in relatively close spatial vicinity of the RCL when evaluated on the three-dimensional structure of human CBG (
      • Gardill B.R.
      • Vogl M.R.
      • Lin H.Y.
      • Hammond G.L.
      • Muller Y.A.
      Corticosteroid-binding globulin: structure-function implications from species differences.
      ,
      • Zhou A.
      • Wei Z.
      • Stanley P.L.
      • Read R.J.
      • Stein P.E.
      • Carrell R.W.
      The S-to-R transition of corticosteroid-binding globulin and the mechanism of hormone release.
      ). However, the site-specific effects of CBG N-glycosylation on the endogenous and exogenous elastase-based RCL proteolysis and cortisol release remain undocumented.
      We establish here that CBG uses specific glycan features, in particular at the Asn347 site, to fine-tune the RCL proteolysis by two unrelated and co-existing elastases of host and pathogen origin that may be found in the respiratory tract of bacteria-infected individuals; the endogenous NE and P. aeruginosa-derived PAE. The site-specific modulatory functions of CBG N-glycosylation contribute to advancing our understanding of the complex molecular mechanisms underpinning the competing interests of the host and pathogen that facilitate subtle yet effective manipulation of the human immune response during infection and inflammation.

      Discussion

      CBG is a central protein in inflammation due to its transport of corticosteroids in circulation and its ability to deliver such anti-inflammatory stress hormones in an accurate and timely way to the required tissues. Although not fully resolved, one aspect of the delivery mechanism that is rather well established is the NE-driven proteolysis of the RCL leading to cortisol release upon a so-called stress-to-relaxed conformational change of CBG at the neutrophil-rich inflammatory site (
      • Pemberton P.A.
      • Stein P.E.
      • Pepys M.B.
      • Potter J.M.
      • Carrell R.W.
      Hormone binding globulins undergo serpin conformational change in inflammation.
      ,
      • Hammond G.L.
      • Smith C.L.
      • Underhill C.M.
      • Nguyen V.T.
      Interaction between corticosteroid binding globulin and activated leukocytes in vitro.
      ). As demonstrated here by time-based digestion assays and structural analysis of the resulting glycoform fragments and supported by MD simulations, the digestion efficiency of the highly potent and cleavage site-specific (Val344-Thr345) endogenous NE is modulated in an intriguingly complex manner by the Asn347 glycosylation of CBG. The predominant bi- and trivalent NeuAc-type sialylation of the Asn347 glycans was found to be highly beneficial for NE digestion, possibly by enabling electrostatic attraction to the arginine-rich surface of NE (
      • Hansen G.
      • Gielen-Haertwig H.
      • Reinemer P.
      • Schomburg D.
      • Harrenga A.
      • Niefind K.
      Unexpected active-site flexibility in the structure of human neutrophil elastase in complex with a new dihydropyrimidone inhibitor.
      ) upon docking onto the RCL. With respect to the RCL cleavage rate, no additional benefits of trivalent over bivalent sialylation of the Asn347 glycan were observed, indicating that the orientation and the position of the anionic charges around the RCL are more important than the sheer number of negative charges. The MD simulations indicated that some Asn347 sialoglycoforms form interactions with the CBG protein surface (data not shown). However, further work is warranted to investigate whether these interactions promote RCL digestion by NE.
      In contrast, other volume-enhancing neutral features of the Asn347 glycans, including the site occupancy, core fucosylation, outer antennary β-GlcNAc branching, and β-galactosylation, were found to antagonize the RCL digestion efficiency of NE. It is anticipated that these inhibitory glycan features are exerting their action by steric hindrance by forming hydrophilic (hydrogen bonding from hydroxyl groups) and/or hydrophobic (from N-acetyl groups of GlcNAc and NeuAc) interactions with the CBG surface, thereby masking the digestion sites of the RCL. The presence of core fucosylation of N-glycans has previously been shown to inhibit the proximal proteolysis of polypeptides by serine proteases (
      • Deshpande N.
      • Jensen P.H.
      • Packer N.H.
      • Kolarich D.
      GlycoSpectrumScan: fishing glycopeptides from MS spectra of protease digests of human colostrum sIgA.
      ). None of the five other CBG N-glycans (at sites Asn9, Asn74, Asn154, Asn238, and Asn308) displayed any detectable modulation of the NE digestion efficiency of the RCL, which can be rationalized from the relative small size of NE (mature polypeptide, 25.5 kDa) and the significant distance of the digestion site to the “neighboring” Asn154 (∼44 Å) and Asn238 (∼34 Å) of uncleaved CBG (data not shown, but see FIGURE 4., FIGURE 5. for model). Despite the presence of these multiple inhibitory features of the Asn347 glycans, NE yielded significant CBG fragments in the second-to-minute time scale. Thus, the Asn347 glycosylation does not prevent the NE-based cleavage of CBG but instead fine-tunes the digestion rate and, thus, is able to manipulate the speed at which cortisol is released into inflamed tissues. Consequently, the Asn347 glycosylation may be viewed as an anti-inflammatory regulator.
      Unlike NeuAc-type α-sialylation and β-galactosylation, which forms the terminal and penultimate glycoepitopes, respectively, of all native CBG glycoforms on Asn347, it is interesting to note that the other regulatory Asn347 glycan features are partial modifications (i.e. the Asn347 site occupancy is ∼85%, core fucosylation is ∼35%, and triantennary GlcNAc branching is ∼70% as evaluated from an “average” CBG population derived from a pool of healthy donors) (
      • Sumer-Bayraktar Z.
      • Kolarich D.
      • Campbell M.P.
      • Ali S.
      • Packer N.H.
      • Thaysen-Andersen M.
      N-Glycans modulate the function of human corticosteroid-binding globulin.
      ). The degree of expression of these glycan features on CBG may be manipulated by hepatic cells or extrahepatic tissues as required to alter the NE-based RCL digestion rate and the cortisol release from CBG (e.g. during inflammation or bacterial infection). Both cellular factors (e.g. levels of glycosylation enzymes and nucleotide sugar donors) (
      • Rudd P.M.
      • Dwek R.A.
      Glycosylation: heterogeneity and the 3D structure of proteins.
      ) and protein features (e.g. glycosylation site accessibility (
      • Thaysen-Andersen M.
      • Packer N.H.
      Site-specific glycoproteomics confirms that protein structure dictates formation of N-glycan type, core fucosylation and branching.
      ) and subcellular localization/trafficking rate) (
      • Rudd P.M.
      • Dwek R.A.
      Glycosylation: heterogeneity and the 3D structure of proteins.
      ,
      • Lee L.Y.
      • Lin C.H.
      • Fanayan S.
      • Packer N.H.
      • Thaysen-Andersen M.
      Differential site accessibility mechanistically explains subcellular-specific N-glycosylation determinants.
      ) may contribute to the regulation of the N-glycosylation of a protein. Specifically, the global glycosylation pattern of hepatocyte-derived CBG has indeed been shown, in vitro, to be regulated by glucocorticoids (
      • Mihrshahi R.
      • Lewis J.G.
      • Ali S.O.
      Hormonal effects on the secretion and glycoform profile of corticosteroid-binding globulin.
      ); however, it still needs to be assessed whether altered cortisol levels affect the secretion and glycosylation pattern of hepatocyte-derived CBG and/or potentially the expression of extrahepatic CBG displaying different glycoforms and activities in a feedback mechanism. In relation to this, it would be interesting to map the natural variation of the Asn347 glycosylation of blood- and tissue-derived CBG in healthy donors and to compare these profiles with those of individuals undergoing local/systemic inflammation and bacterial infection to further test the observations described herein. Understanding the molecular basis for rapid tissue-specific cortisol delivery may have great utility in the rational design of CBG glycoforms with enhanced cleavage characteristics as a new type of therapeutics that can be directed to inflammatory tissues (e.g. in septic shock, prostatic hyperplasia, and rheumatoid arthritis) (
      • Sprung C.L.
      • Annane D.
      • Keh D.
      • Moreno R.
      • Singer M.
      • Freivogel K.
      • Weiss Y.G.
      • Benbenishty J.
      • Kalenka A.
      • Forst H.
      • Laterre P.F.
      • Reinhart K.
      • Cuthbertson B.H.
      • Payen D.
      • Briegel J.
      • CORTICUS Study Group
      Hydrocortisone therapy for patients with septic shock.
      • Chan W.L.
      • Zhou A.
      • Read R.J.
      Towards engineering hormone-binding globulins as drug delivery agents.
      • Nenke M.A.
      • Lewis J.G.
      • Rankin W.
      • McWilliams L.
      • Metcalf R.G.
      • Proudman S.M.
      • Torpy D.J.
      Reduced corticosteroid-binding globulin cleavage in active rheumatoid arthritis.
      ).
      The immune modulation caused by other endogenous and exogenous proteases upon CBG cleavage is even less studied. PAE produced by virulent P. aeruginosa was shown to cleave the RCL of CBG, causing a diminished cortisol binding capacity (
      • Simard M.
      • Hill L.A.
      • Underhill C.M.
      • Keller B.O.
      • Villanueva I.
      • Hancock R.E.
      • Hammond G.L.
      Pseudomonas aeruginosa elastase disrupts the cortisol-binding activity of corticosteroid-binding globulin.
      ). Importantly, other pathogens (i.e. Burkholderia cenocepacia and Staphylococcus aureus) did not produce RCL-cleaving proteases, suggesting that this ability may be unique to P. aeruginosa (
      • Simard M.
      • Hill L.A.
      • Underhill C.M.
      • Keller B.O.
      • Villanueva I.
      • Hancock R.E.
      • Hammond G.L.
      Pseudomonas aeruginosa elastase disrupts the cortisol-binding activity of corticosteroid-binding globulin.
      ). Supporting these observations, the RCL of CBG was here shown to be a proteolytic target of PAE; two equally preferred cleavage sites (Thr345-Leu346 and Asn347-Leu348) were identified. PAE is the most secreted protein of P. aeruginosa, and despite its poor proteolytic activity (acting on a minutes-to-hours time scale) relative to NE, the total proteolytic capacity of PAE may still be significant, considering its abundance. In contrast to NE, the action of PAE was affected by the glycans on multiple glycosylation sites of CBG. Specifically, PAE digestion was shown to benefit from the sialylation of glycans on sites other than Asn347. Further investigation needs to specify whether these promoting effects are generated directly by electrostatic attractions of the neighboring Asn154 and Asn238 to the larger (relative to NE) PAE molecule (mature polypeptide, 33.1 kDa) upon RCL docking or whether the sialic acid residues are indirectly facilitating a more cleavage-susceptible conformation of CBG by engaging in charge-based interactions with its polypeptide backbone. Despite the lack of any direct evidence, we cannot rule out the possibility that negative charges from intra- or intermolecular sources other than sialylation of CBG, including glycosaminoglycans and phosphorylation, may similarly play a yet to be explored role in RCL modulation. It is also of interest to note that although the CBG sialylation appears to be a promoting feature of both NE and PAE digestion, we have previously shown that these negatively charged terminal glycoepitopes are antagonistic features reducing the interaction of CBG to yet to be thoroughly characterized putative cell surface receptors (
      • Sumer-Bayraktar Z.
      • Kolarich D.
      • Campbell M.P.
      • Ali S.
      • Packer N.H.
      • Thaysen-Andersen M.
      N-Glycans modulate the function of human corticosteroid-binding globulin.
      ). Thus, the same glycosylation features may display multiple, diverse, and even opposite modulatory functions.
      Interestingly, PAE-induced RCL cleavage was restricted to the subpopulation of CBG molecules lacking Asn347 glycosylation, amounting to only ∼15% of native CBG molecules (
      • Sumer-Bayraktar Z.
      • Kolarich D.
      • Campbell M.P.
      • Ali S.
      • Packer N.H.
      • Thaysen-Andersen M.
      N-Glycans modulate the function of human corticosteroid-binding globulin.
      ). This suggests that PAE can only access a small proportion of the CBG-bound cortisol in a biological context, which may, however, be sufficient for promoting long term inhibition of the host immune response and thus allow the bacteria to gain a growth advantage by initiating and sustaining colonization. P. aeruginosa is an opportunistic pathogen that is known to establish chronic infection of immunocompromised individuals (e.g. in the respiratory tract of cystic fibrosis and chronic obstructive pulmonary disease patients) by having evolved refined strategies for long term infection, such as quorum sensing and biofilm formation (
      • Kamath S.
      • Kapatral V.
      • Chakrabarty A.M.
      Cellular function of elastase in Pseudomonas aeruginosa: role in the cleavage of nucleoside diphosphate kinase and in alginate synthesis.
      • Lee J.
      • Zhang L.
      The hierarchy quorum sensing network in Pseudomonas aeruginosa.
      • Ciofu O.
      • Hansen C.R.
      • Høiby N.
      Respiratory bacterial infections in cystic fibrosis.
      ). Because the release of free cortisol delays the production of pro-inflammatory factors designed to combat bacterial invasion, the PAE-induced RCL cleavage of CBG may also be viewed as one such infection strategy that enhances the chances of this opportunistic pathogen to evade the host immune system. The low potency of PAE may be crucial to avoid triggering a more complete release of cortisol, which appears to be detrimental to the growth of virulent P. aeruginosa, as demonstrated here. The weak bacteriostatic effect of low micromolar cortisol concentrations and the underlying suppressive mechanisms on the growth of P. aeruginosa need to be further investigated by orthogonal approaches.
      The RCL of human α1-antitrypsin (A1AT, UniProtKB, P01009), another member of the serpin superfamily, is also a target of PAE (
      • Rapala-Kozik M.
      • Potempa J.
      • Nelson D.
      • Kozik A.
      • Travis J.
      Comparative cleavage sites within the reactive-site loop of native and oxidized α1-proteinase inhibitor by selected bacterial proteinases.
      ). This is important because A1AT, unlike CBG, shows serpin-inhibitory activity by irreversibly binding and inactivating NE (and other serine proteases) and thereby reducing its CBG-cleaving abilities (
      • Beatty K.
      • Bieth J.
      • Travis J.
      Kinetics of association of serine proteinases with native and oxidized α-1-proteinase inhibitor and α-1-antichymotrypsin.
      ). Thus, the PAE-induced cleavage of A1AT may result in reduced NE inactivation and consequently an increased NE-based CBG digestion and cortisol release. Similar to NE, A1AT is present at the site of inflammation due to its localized production and secretion from leukocytes (
      • Brantly M.
      α1-Antitrypsin: not just an antiprotease: extending the half-life of a natural anti-inflammatory molecule by conjugation with polyethylene glycol.
      ). Future work will reveal whether glycosylation is involved in the A1AT-based inhibition of NE and whether this inhibition is as potent as the well described NE inhibition displayed by a number of chemical inhibitors (e.g. N-benzoylindazole derivatives (
      • Crocetti L.
      • Schepetkin I.A.
      • Cilibrizzi A.
      • Graziano A.
      • Vergelli C.
      • Giomi D.
      • Khlebnikov A.I.
      • Quinn M.T.
      • Giovannoni M.P.
      Optimization of N-benzoylindazole derivatives as inhibitors of human neutrophil elastase.
      ) as well as acylating and carbamylating agents and inhibitors based on other mechanisms (
      • Groutas W.C.
      • Dou D.
      • Alliston K.R.
      Neutrophil elastase inhibitors.
      )) and other protein/peptide-based inhibitors, such as cyclotides (
      • Craik D.J.
      • Cemazar M.
      • Daly N.L.
      The cyclotides and related macrocyclic peptides as scaffolds in drug design.
      ). Interestingly, the exposed RCLs of serpins generally do not share a common amino acid sequence and harbor no conserved N-glycosylation sequon motifs (NX(S/T), where X is not proline) (
      • Hill R.E.
      • Hastie N.D.
      Accelerated evolution in the reactive centre regions of serine protease inhibitors.
      ). For example, no RCL-localized glycosylation sites similar to Asn347 of CBG were identified for A1AT and the structurally related thyroxine-binding globulin (TBG). It could be speculated that the RCL of especially inhibitory serpins encompassing the majority of the members within this superfamily lacks glycosylation sites because the binding and inactivation of proteases through the RCL may be compromised by the presence of a glycan in this region. However, it remains to be investigated whether any of the more than 70 serpin members, in addition to CBG, harbor an RCL glycosylation site, which may similarly modulate their inhibitory/non-inhibitory functions (
      • Law R.H.
      • Zhang Q.
      • McGowan S.
      • Buckle A.M.
      • Silverman G.A.
      • Wong W.
      • Rosado C.J.
      • Langendorf C.G.
      • Pike R.N.
      • Bird P.I.
      • Whisstock J.C.
      An overview of the serpin superfamily.
      ). The lack of serpin sequence conservation between species is attributed to the adaptive changes necessary to escape pathogenic species-specific elastases (
      • Hill R.E.
      • Hastie N.D.
      Accelerated evolution in the reactive centre regions of serine protease inhibitors.
      ) and to accommodate for species-specific hormonal and physiological differences (
      • Vashchenko G.
      • Das S.
      • Moon K.M.
      • Rogalski J.C.
      • Taves M.D.
      • Soma K.K.
      • Van Petegem F.
      • Foster L.J.
      • Hammond G.L.
      Identification of avian corticosteroid-binding globulin (SerpinA6) reveals the molecular basis of evolutionary adaptations in SerpinA6 structure and function as a steroid-binding protein.
      ). In this context, the Asn347 glycosylation of the RCL of human CBG may have evolved as an additional level of fine control of and/or protection from digestion by exogenous and endogenous elastases, considering the diverse environments of CBG (i.e. produced in liver, circulates in blood, and migrates to inflammatory and steroid-sensitive tissues). The RCL glycosylation of CBG may regulate its spatial and temporal cleavage and thus be crucial to ensure accurate cortisol delivery.
      Although the ability of glycans to protect their carrier proteins from proteolytic degradation has been known for many years (
      • Bernard B.A.
      • Yamada K.M.
      • Olden K.
      Carbohydrates selectively protect a specific domain of fibronectin against proteases.
      ,
      • Rutledge E.A.
      • Enns C.A.
      Cleavage of the transferrin receptor is influenced by the composition of the O-linked carbohydrate at position 104.
      ), still very few examples have been reported, in particular, in biological systems where the regulatory role of the individual glycan features have been documented (
      • Hane M.
      • Matsuoka S.
      • Ono S.
      • Miyata S.
      • Kitajima K.
      • Sato C.
      Protective effects of polysialic acid on proteolytic cleavage of FGF2 and ProBDNF/BDNF.
      ). The fine control provided by the Asn347 glycosylation of CBG regulating the RCL proteolysis by important endogenous and exogenous proteases as reported here represents a fascinating example of the extremely complex molecular regulation underpinning the immune system. This work has demonstrated the central role of CBG glycosylation in providing an efficient immune response upon challenge and has brought us closer to understanding the molecular basis underpinning the regulation of these processes in the context of inflammation and bacterial infection.

      Author Contributions

      Z. S.-B. and M. T.-A. conceived the experimental design and hypotheses. Z. S.-B., V. V., and O. C. G. performed the experiments. Z. S.-B., O. C. G., V. V., R. J. W., and M. T.-A. analyzed data. R. J. W., N. H. P., and M. T.-A. supplied reagents/analytical tools/expertise. Z. S.-B., O. C. G., V. V., R. J. W., N. H. P., and M. T.-A. wrote the paper.

      Acknowledgments

      We thank Dr. Karthik Kamath and Dr. Jodie Abrahams for assistance with LC-MS/MS data collection and interpretation. This research was facilitated through access to the Australian Proteomics Analysis Facility (APAF).

      References

        • Siiteri P.K.
        • Murai J.T.
        • Hammond G.L.
        • Nisker J.A.
        • Raymoure W.J.
        • Kuhn R.W.
        The serum transport of steroid hormones.
        Recent Prog. Horm. Res. 1982; 38: 457-510
        • Cameron A.
        • Henley D.
        • Carrell R.
        • Zhou A.
        • Clarke A.
        • Lightman S.
        Temperature-responsive release of cortisol from its binding globulin: a protein thermocouple.
        J. Clin. Endocrinol. Metab. 2010; 95: 4689-4695
        • Khan M.S.
        • Aden D.
        • Rosner W.
        Human corticosteroid binding globulin is secreted by a hepatoma-derived cell line.
        J. Steroid Biochem. 1984; 20: 677-678
        • Hammond G.L.
        • Smith C.L.
        • Underhill D.A.
        Molecular studies of corticosteroid binding globulin structure, biosynthesis and function.
        J. Steroid Biochem. Mol. Biol. 1991; 40: 755-762
        • Hammond G.L.
        • Smith C.L.
        • Goping I.S.
        • Underhill D.A.
        • Harley M.J.
        • Reventos J.
        • Musto N.A.
        • Gunsalus G.L.
        • Bardin C.W.
        Primary structure of human corticosteroid binding globulin, deduced from hepatic and pulmonary cDNAs, exhibits homology with serine protease inhibitors.
        Proc. Natl. Acad. Sci. U.S.A. 1987; 84: 5153-5157
        • Misao R.
        • Iwagaki S.
        • Sun W.S.
        • Fujimoto J.
        • Saio M.
        • Takami T.
        • Tamaya T.
        Evidence for the synthesis of corticosteroid-binding globulin in human placenta.
        Horm. Res. 1999; 51: 162-167
        • Misao R.
        • Hori M.
        • Ichigo S.
        • Fujimoto J.
        • Tamaya T.
        Corticosteroid-binding globulin mRNA levels in human uterine endometrium.
        Steroids. 1994; 59: 603-607
        • Miska W.
        • Peña P.
        • Villegas J.
        • Sánchez R.
        Detection of a CBG-like protein in human Fallopian tube tissue.
        Andrologia. 2004; 36: 41-46
        • Schäfer H.H.
        • Gebhart V.M.
        • Hertel K.
        • Jirikowski G.F.
        Expression of corticosteroid-binding globulin CBG in the human heart.
        Horm. Metab. Res. 2015; 47: 596-599
        • Sivukhina E.V.
        • Jirikowski G.F.
        • Bernstein H.G.
        • Lewis J.G.
        • Herbert Z.
        Expression of corticosteroid-binding protein in the human hypothalamus, co-localization with oxytocin and vasopressin.
        Horm. Metab. Res. 2006; 38: 253-259
        • Perrot-Applanat M.
        • Racadot O.
        • Milgrom E.
        Specific localization of plasma corticosteroid-binding globulin immunoreactivity in pituitary corticotrophs.
        Endocrinology. 1984; 115: 559-569
        • Jirikowski G.F.
        • Pusch L.
        • Möpert B.
        • Herbert Z.
        • Caldwell J.D.
        Expression of corticosteroid binding globulin in the rat central nervous system.
        J. Chem. Neuroanat. 2007; 34: 22-28
        • Heyns W.
        • Coolens J.L.
        Physiology of corticosteroid-binding globulin in humans.
        Ann. N.Y. Acad. Sci. 1988; 538: 122-129
        • Lewis J.G.
        • Saunders K.
        • Dyer A.
        • Elder P.A.
        The half-lives of intact and elastase cleaved human corticosteroid-binding globulin (CBG) are identical in the rabbit.
        J. Steroid Biochem. Mol. Biol. 2015; 149: 53-57
        • Perogamvros I.
        • Ray D.W.
        • Trainer P.J.
        Regulation of cortisol bioavailability: effects on hormone measurement and action.
        Nat. Rev. Endocrinol. 2012; 8: 717-727
        • Verhoog N.
        • Allie-Reid F.
        • Vanden Berghe W.
        • Smith C.
        • Haegeman G.
        • Hapgood J.
        • Louw A.
        Inhibition of corticosteroid-binding globulin gene expression by glucocorticoids involves C/EBPβ.
        PLoS One. 2014; 9: e110702
        • Nenke M.A.
        • Rankin W.
        • Chapman M.J.
        • Stevens N.E.
        • Diener K.R.
        • Hayball J.D.
        • Lewis J.G.
        • Torpy D.J.
        Depletion of high-affinity corticosteroid-binding globulin corresponds to illness severity in sepsis and septic shock; clinical implications.
        Clin. Endocrinol. 2015; 82: 801-807
        • Emptoz-Bonneton A.
        • Crave J.C.
        • LeJeune H.
        • Brébant C.
        • Pugeat M.
        Corticosteroid-binding globulin synthesis regulation by cytokines and glucocorticoids in human hepatoblastoma-derived (HepG2) cells.
        J. Clin. Endocrinol. Metab. 1997; 82: 3758-3762
        • Tsigos C.
        • Kyrou I.
        • Chrousos G.P.
        • Papanicolaou D.A.
        Prolonged suppression of corticosteroid-binding globulin by recombinant human interleukin-6 in man.
        J. Clin. Endocrinol. Metab. 1998; 83: 3379
        • Strel'chyonok O.A.
        • Avvakumov G.V.
        Interaction of human CBG with cell membranes.
        J. Steroid Biochem. Mol. Biol. 1991; 40: 795-803
        • Hryb D.J.
        • Khan M.S.
        • Romas N.A.
        • Rosner W.
        Specific binding of human corticosteroid-binding globulin to cell membranes.
        Proc. Natl. Acad. Sci. U.S.A. 1986; 83: 3253-3256
        • Pemberton P.A.
        • Stein P.E.
        • Pepys M.B.
        • Potter J.M.
        • Carrell R.W.
        Hormone binding globulins undergo serpin conformational change in inflammation.
        Nature. 1988; 336: 257-258
        • Lin H.Y.
        • Underhill C.
        • Gardill B.R.
        • Muller Y.A.
        • Hammond G.L.
        Residues in the human corticosteroid-binding globulin reactive center loop that influence steroid binding before and after elastase cleavage.
        J. Biol. Chem. 2009; 284: 884-896
        • Hammond G.L.
        • Smith C.L.
        • Underhill C.M.
        • Nguyen V.T.
        Interaction between corticosteroid binding globulin and activated leukocytes in vitro.
        Biochem. Biophys. Res. Commun. 1990; 172: 172-177
        • Klieber M.A.
        • Underhill C.
        • Hammond G.L.
        • Muller Y.A.
        Corticosteroid-binding globulin, a structural basis for steroid transport and proteinase-triggered release.
        J. Biol. Chem. 2007; 282: 29594-29603
        • Qi X.
        • Loiseau F.
        • Chan W.L.
        • Yan Y.
        • Wei Z.
        • Milroy L.G.
        • Myers R.M.
        • Ley S.V.
        • Read R.J.
        • Carrell R.W.
        • Zhou A.
        Allosteric modulation of hormone release from thyroxine and corticosteroid-binding globulins.
        J. Biol. Chem. 2011; 286: 16163-16173
        • Lewis J.G.
        • Elder P.A.
        Corticosteroid-binding globulin reactive centre loop antibodies recognise only the intact natured protein: elastase cleaved and uncleaved CBG may coexist in circulation.
        J. Steroid Biochem. Mol. Biol. 2011; 127: 289-294
        • Meyer E.J.
        • Nenke M.A.
        • Rankin W.
        • Lewis J.G.
        • Torpy D.J.
        Corticosteroid-binding globulin: a review of basic and clinical advances.
        Horm. Metab. Res. 2016; 48: 359-371
        • Chan W.L.
        • Carrell R.W.
        • Zhou A.
        • Read R.J.
        How changes in affinity of corticosteroid-binding globulin modulate free cortisol concentration.
        J. Clin. Endocrinol. Metab. 2013; 98: 3315-3322
        • Nenke M.A.
        • Holmes M.
        • Rankin W.
        • Lewis J.G.
        • Torpy D.J.
        Corticosteroid-binding globulin cleavage is paradoxically reduced in α-1 antitrypsin deficiency: implications for cortisol homeostasis.
        Clin. Chim. Acta. 2016; 452: 27-31
        • Lewis J.G.
        • Elder P.A.
        The reactive centre loop of corticosteroid-binding globulin (CBG) is a protease target for cortisol release.
        Mol. Cell. Endocrinol. 2014; 384: 96-101
        • Gardill B.R.
        • Vogl M.R.
        • Lin H.Y.
        • Hammond G.L.
        • Muller Y.A.
        Corticosteroid-binding globulin: structure-function implications from species differences.
        PLoS One. 2012; 7: e52759
        • Simard M.
        • Hill L.A.
        • Underhill C.M.
        • Keller B.O.
        • Villanueva I.
        • Hancock R.E.
        • Hammond G.L.
        Pseudomonas aeruginosa elastase disrupts the cortisol-binding activity of corticosteroid-binding globulin.
        Endocrinology. 2014; 155: 2900-2908
        • Henke M.O.
        • John G.
        • Rheineck C.
        • Chillappagari S.
        • Naehrlich L.
        • Rubin B.K.
        Serine proteases degrade airway mucins in cystic fibrosis.
        Infect. Immun. 2011; 79: 3438-3444
        • Avvakumov G.V.
        • Hammond G.L.
        Glycosylation of human corticosteroid-binding globulin. Differential processing and significance of carbohydrate chains at individual sites.
        Biochemistry. 1994; 33: 5759-5765
        • Sumer-Bayraktar Z.
        • Kolarich D.
        • Campbell M.P.
        • Ali S.
        • Packer N.H.
        • Thaysen-Andersen M.
        N-Glycans modulate the function of human corticosteroid-binding globulin.
        Mol. Cell. Proteomics. 2011; (10.1074/mcp.M111.009100)
        • Zhou A.
        • Wei Z.
        • Stanley P.L.
        • Read R.J.
        • Stein P.E.
        • Carrell R.W.
        The S-to-R transition of corticosteroid-binding globulin and the mechanism of hormone release.
        J. Mol. Biol. 2008; 380: 244-251
        • Melby J.C.
        • Spink W.W.
        Comparative studies on adrenal cortical function and cortisol metabolism in healthy adults and in patients with shock due to infection.
        J. Clin. Invest. 1958; 37: 1791-1798
        • Hansen G.
        • Gielen-Haertwig H.
        • Reinemer P.
        • Schomburg D.
        • Harrenga A.
        • Niefind K.
        Unexpected active-site flexibility in the structure of human neutrophil elastase in complex with a new dihydropyrimidone inhibitor.
        J. Mol. Biol. 2011; 409: 681-691
        • Deshpande N.
        • Jensen P.H.
        • Packer N.H.
        • Kolarich D.
        GlycoSpectrumScan: fishing glycopeptides from MS spectra of protease digests of human colostrum sIgA.
        J. Proteome Res. 2010; 9: 1063-1075
        • Rudd P.M.
        • Dwek R.A.
        Glycosylation: heterogeneity and the 3D structure of proteins.
        Crit. Rev. Biochem. Mol. Biol. 1997; 32: 1-100
        • Thaysen-Andersen M.
        • Packer N.H.
        Site-specific glycoproteomics confirms that protein structure dictates formation of N-glycan type, core fucosylation and branching.
        Glycobiology. 2012; 22: 1440-1452
        • Lee L.Y.
        • Lin C.H.
        • Fanayan S.
        • Packer N.H.
        • Thaysen-Andersen M.
        Differential site accessibility mechanistically explains subcellular-specific N-glycosylation determinants.
        Front. Immunol. 2014; 5: 404
        • Mihrshahi R.
        • Lewis J.G.
        • Ali S.O.
        Hormonal effects on the secretion and glycoform profile of corticosteroid-binding globulin.
        J. Steroid Biochem. Mol. Biol. 2006; 101: 275-285
        • Sprung C.L.
        • Annane D.
        • Keh D.
        • Moreno R.
        • Singer M.
        • Freivogel K.
        • Weiss Y.G.
        • Benbenishty J.
        • Kalenka A.
        • Forst H.
        • Laterre P.F.
        • Reinhart K.
        • Cuthbertson B.H.
        • Payen D.
        • Briegel J.
        • CORTICUS Study Group
        Hydrocortisone therapy for patients with septic shock.
        N. Engl. J. Med. 2008; 358: 111-124
        • Chan W.L.
        • Zhou A.
        • Read R.J.
        Towards engineering hormone-binding globulins as drug delivery agents.
        PLoS One. 2014; 9: e113402
        • Nenke M.A.
        • Lewis J.G.
        • Rankin W.
        • McWilliams L.
        • Metcalf R.G.
        • Proudman S.M.
        • Torpy D.J.
        Reduced corticosteroid-binding globulin cleavage in active rheumatoid arthritis.
        Clin. Endocrinol. 2016; 48: 359-371
        • Kamath S.
        • Kapatral V.
        • Chakrabarty A.M.
        Cellular function of elastase in Pseudomonas aeruginosa: role in the cleavage of nucleoside diphosphate kinase and in alginate synthesis.
        Mol. Microbiol. 1998; 30: 933-941
        • Lee J.
        • Zhang L.
        The hierarchy quorum sensing network in Pseudomonas aeruginosa.
        Protein Cell. 2015; 6: 26-41
        • Ciofu O.
        • Hansen C.R.
        • Høiby N.
        Respiratory bacterial infections in cystic fibrosis.
        Curr. Opin. Pulm. Med. 2013; 19: 251-258
        • Rapala-Kozik M.
        • Potempa J.
        • Nelson D.
        • Kozik A.
        • Travis J.
        Comparative cleavage sites within the reactive-site loop of native and oxidized α1-proteinase inhibitor by selected bacterial proteinases.
        Biol. Chem. 1999; 380: 1211-1216
        • Beatty K.
        • Bieth J.
        • Travis J.
        Kinetics of association of serine proteinases with native and oxidized α-1-proteinase inhibitor and α-1-antichymotrypsin.
        J. Biol. Chem. 1980; 255: 3931-3934
        • Brantly M.
        α1-Antitrypsin: not just an antiprotease: extending the half-life of a natural anti-inflammatory molecule by conjugation with polyethylene glycol.
        Am. J. Respir. Cell Mol. Biol. 2002; 27: 652-654
        • Crocetti L.
        • Schepetkin I.A.
        • Cilibrizzi A.
        • Graziano A.
        • Vergelli C.
        • Giomi D.
        • Khlebnikov A.I.
        • Quinn M.T.
        • Giovannoni M.P.
        Optimization of N-benzoylindazole derivatives as inhibitors of human neutrophil elastase.
        J. Med. Chem. 2013; 56: 6259-6272
        • Groutas W.C.
        • Dou D.
        • Alliston K.R.
        Neutrophil elastase inhibitors.
        Expert Opin. Ther. Pat. 2011; 21: 339-354
        • Craik D.J.
        • Cemazar M.
        • Daly N.L.
        The cyclotides and related macrocyclic peptides as scaffolds in drug design.
        Curr. Opin. Drug Discov. Devel. 2006; 9: 251-260
        • Hill R.E.
        • Hastie N.D.
        Accelerated evolution in the reactive centre regions of serine protease inhibitors.
        Nature. 1987; 326: 96-99
        • Law R.H.
        • Zhang Q.
        • McGowan S.
        • Buckle A.M.
        • Silverman G.A.
        • Wong W.
        • Rosado C.J.
        • Langendorf C.G.
        • Pike R.N.
        • Bird P.I.
        • Whisstock J.C.
        An overview of the serpin superfamily.
        Genome Biol. 2006; 7: 216
        • Vashchenko G.
        • Das S.
        • Moon K.M.
        • Rogalski J.C.
        • Taves M.D.
        • Soma K.K.
        • Van Petegem F.
        • Foster L.J.
        • Hammond G.L.
        Identification of avian corticosteroid-binding globulin (SerpinA6) reveals the molecular basis of evolutionary adaptations in SerpinA6 structure and function as a steroid-binding protein.
        J. Biol. Chem. 2016; 291: 11300-11312
        • Bernard B.A.
        • Yamada K.M.
        • Olden K.
        Carbohydrates selectively protect a specific domain of fibronectin against proteases.
        J. Biol. Chem. 1982; 257: 8549-8554
        • Rutledge E.A.
        • Enns C.A.
        Cleavage of the transferrin receptor is influenced by the composition of the O-linked carbohydrate at position 104.
        J. Cell. Physiol. 1996; 168: 284-293
        • Hane M.
        • Matsuoka S.
        • Ono S.
        • Miyata S.
        • Kitajima K.
        • Sato C.
        Protective effects of polysialic acid on proteolytic cleavage of FGF2 and ProBDNF/BDNF.
        Glycobiology. 2015; 25: 1112-1124
        • Penesyan A.
        • Kumar S.S.
        • Kamath K.
        • Shathili A.M.
        • Venkatakrishnan V.
        • Krisp C.
        • Packer N.H.
        • Molloy M.P.
        • Paulsen I.T.
        Genetically and phenotypically distinct Pseudomonas aeruginosa cystic fibrosis isolates share a core proteomic signature.
        PLoS One. 2015; 10: e0138527
        • Luria S.E.
        • Burrous J.W.
        Hybridization between Escherichia coli and Shigella.
        J. Bacteriol. 1957; 74: 461-476
        • Schneider C.A.
        • Rasband W.S.
        • Eliceiri K.W.
        NIH Image to ImageJ: 25 years of image analysis.
        Nat. Methods. 2012; 9: 671-675
        • Sumer-Bayraktar Z.
        • Nguyen-Khuong T.
        • Jayo R.
        • Chen D.D.
        • Ali S.
        • Packer N.H.
        • Thaysen-Andersen M.
        Micro- and macroheterogeneity of N-glycosylation yields size and charge isoforms of human sex hormone binding globulin circulating in serum.
        Proteomics. 2012; 12: 3315-3327
        • Thaysen-Andersen M.
        • Mysling S.
        • Højrup P.
        Site-specific glycoprofiling of N-linked glycopeptides using MALDI-TOF MS: strong correlation between signal strength and glycoform quantities.
        Anal. Chem. 2009; 81: 3933-3943
        • Stavenhagen K.
        • Hinneburg H.
        • Thaysen-Andersen M.
        • Hartmann L.
        • Varón Silva D.
        • Fuchser J.
        • Kaspar S.
        • Rapp E.
        • Seeberger P.H.
        • Kolarich D.
        Quantitative mapping of glycoprotein micro-heterogeneity and macro-heterogeneity: an evaluation of mass spectrometry signal strengths using synthetic peptides and glycopeptides.
        J. Mass Spectrom. 2013; 48: 627-639
        • Eswar N.
        • Webb B.
        • Marti-Renom M.A.
        • Madhusudhan M.S.
        • Eramian D.
        • Shen M.Y.
        • Pieper U.
        • Sali A.
        Comparative protein structure modeling using Modeller.
        Curr. Protoc. Bioinformatics. 2006; (10.1002/0471250953.bi0506s15)
        • Stanley P.
        • Schachter H.
        • Taniguchi N.
        N-Glycans.
        in: Varki A. Cummings R.D. Esko J.D. Freeze H.H. Stanley P. Bertozzi C.R. Hart G.W. Etzler M.E. Essentials of Glycobiology. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY2009: 101-114
        • Petrescu A.J.
        • Milac A.L.
        • Petrescu S.M.
        • Dwek R.A.
        • Wormald M.R.
        Statistical analysis of the protein environment of N-glycosylation sites: implications for occupancy, structure, and folding.
        Glycobiology. 2004; 14: 103-114
        • Salomon-Ferrer R.
        • Götz A.W.
        • Poole D.
        • Le Grand S.
        • Walker R.C.
        Routine microsecond molecular dynamics simulations with AMBER on GPUs. 2. Explicit solvent particle mesh Ewald.
        J. Chem. Theory Comput. 2013; 9: 3878-3888
        • Götz A.W.
        • Williamson M.J.
        • Xu D.
        • Poole D.
        • Le Grand S.
        • Walker R.C.
        Routine microsecond molecular dynamics simulations with AMBER on GPUs. 1. Generalized Born.
        J. Chem. Theory Comput. 2012; 8: 1542-1555
        • Kirschner K.N.
        • Yongye A.B.
        • Tschampel S.M.
        • González-Outeiriño J.
        • Daniels C.R.
        • Foley B.L.
        • Woods R.J.
        GLYCAM06: a generalizable biomolecular force field: carbohydrates.
        J. Comput. Chem. 2008; 29: 622-655
        • Case D.A.
        • Babin V.
        • Berryman J.T.
        • Betz R.M.
        • Cai Q.
        • Cerutti D.S.
        • Cheatham III, T.E.
        • Darden T.A.
        • Duke R.E.
        • Gohlke H.
        • Goetz A.W.
        • Gusarov S.
        • Homeyer N.
        • Janowski P.
        • Kaus J.
        • et al.
        The FF14SB force field.
        in: AMBER 14 Reference Manual. AMBER 14, University of California, San Francisco2014
        • Darden T.
        • York D.
        • Pedersen L.
        Particle mesh Ewald: an N·log(N) method for Ewald sums in large systems.
        J. Chem. Phys. 1993; 98: 10089
        • Ryckaert J.-P.
        • Ciccotti G.
        • Berendsen H.J.C.
        Numerical integration of the Cartesian equations of motion of a system with constraints: molecular dynamics of n-alkanes.
        J. Comput. Phys. 1977; 23: 327-341
        • Mahoney M.W.
        • Jorgensen W.L.
        A five-site model for liquid water and the reproduction of the density anomaly by rigid, nonpolarizable potential functions.
        J. Chem. Phys. 2000; 112: 8910-8922
        • Varki A.
        • Cummings R.D.
        • Aebi M.
        • Packer N.H.
        • Seeberger P.H.
        • Esko J.D.
        • Stanley P.
        • Hart G.
        • Darvill A.
        • Kinoshita T.
        • Prestegard J.J.
        • Schnaar R.L.
        • Freeze H.H.
        • Marth J.D.
        • Bertozzi C.R.
        • et al.
        Symbol nomenclature for graphical representations of glycans.
        Glycobiology. 2015; 25: 1323-1324