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The Journal of Clinical Endocrinology & Metabolism Vol. 85, No. 11 3996-3999
Copyright © 2000 by The Endocrine Society

Special Articles

A Characteristic Serpin Cleavage Product of Thyroxine-Binding Globulin Appears in Sepsis Sera

Benjaporn Jirasakuldech, George C. Schussler, Maria G. Yap, Hazel Drew, Alan Josephson and Josef Michl

Division of Endocrinology, Department of Medicine (B.J., G.C.S., M.G.Y.); Division of Immunology, Department of Medicine (H.D., A.J.); and Departments of Pathology, Anatomy, and Cell Biology and Microbiology and Immunology (J.M.), State University of New York Health Sciences Center, Brooklyn, New York 11203

Address all correspondence and requests for reprints to: Dr. George C. Schussler, Division of Endocrinology, Department of Medicine, State University of New York Health Sciences Center, Brooklyn, New York 11203. E-mail: george.c.schussler-new-york{at}worldnet.att.net


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
T4-binding globulin (TBG), the principal thyroid hormone-binding protein of serum, is a member of the serine protease inhibitor (serpin) superfamily. We report a characteristic serpin cleavage product of TBG in sepsis sera. At 49–50 kDa, the TBG remnant is 4–5 kDa smaller than the intact protein and is the same molecular mass as a TBG cleavage product produced by incubation with polymorphonuclear elastase. Incubation with polymorphonuclear leukocytes also produces the 49- to 50-kDa remnant, and this proteolysis is stimulated by zymosan activation. Polymorphonuclear cell cleavage of TBG increases the ratio of free/bound T4. As previously described, in vitro cleavage of TBG by elastase also increases free/bound T4. These findings are consistent with the hypothesis that serine proteases present at inflammatory sites cleave TBG, releasing its hormonal ligands.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
T4-BINDING GLOBULIN (TBG) and cortisol-binding globulin (CBG) are the principal serum binding proteins for their respective ligands. Sequence analysis reveals that although neither binding protein is a protease inhibitor, they are both members of the serine protease inhibitor (serpin) superfamily (1, 2). It is characteristic of serpins that upon interaction with a serine protease, an exposed loop or bait is cleaved, causing a conformational change from a stressed (thermodynamically unstable) to a relaxed (thermodynamically stable) form with the loss of a small C-terminal strand (4–7 kDa) (3, 4). This results in functional changes with important physiological effects in a variety of systems (5, 6). Pemberton et al. reported that both TBG and CBG undergo the characteristic serpin conformational changes on cleavage by neutrophil elastase and that the relaxed form of CBG has a markedly decreased affinity for cortisol (7). Subsequently, Hammond et al. showed that activated granulocytes from septic patients cleaved CBG, releasing cortisol, and suggested that activated granulocytes may release T4 from TBG by an analogous mechanism (8). Although Pemberton et al. found no decrease in the T4 affinity of the relaxed form of TBG, Janssen et al. reported that elastase cleavage of TBG increased the resin uptake of T4 (9), and our own studies with 125I-labeled TBG and T4 show that incubation of TBG with polymorphonuclear cells (PMN) cleaves TBG and increases the proportion of dialyzable T4 (10). Thus, these in vitro studies suggest that proteolytic cleavage of TBG occurs at inflammatory sites and releases T4. We therefore sought evidence of in vivo cleavage of TBG in sepsis.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Subjects

Sera from a recent study of the effects of sepsis on TBG concentration (11) were examined for the presence of the cleaved form of TBG. Normal subjects [two women and six men; age, 37 ± 11 (mean ± SD)] were volunteers from the hospital staff. Sepsis patients (two women and six men; age, 64 ± 13 yr) had been febrile for at least 48 h, with fever over 102 F, tachycardia, and a white count greater than 12,000. All had positive blood cultures. The diagnoses were pneumococcal pneumonia (n = 5), empyema (n = 1), and Gram-negative sepsis (n = 2). The sepsis patients had the characteristic thyroid function changes observed in the euthyroid sick syndrome, including weakened serum T4 binding as demonstrated by a markedly elevated T4 uptake [46 ± 7.1% (±sem)] compared with controls (32 ± 2.6%) and a decreased total T4 concentration (4.71 ± 1.4 µg/dL) compared with controls (8.4 ± 0.14 µg/dL). The TSH level was slightly, but not significantly lower, in sepsis patients than in controls (11). The study was approved by the institutional review board, and informed consent was obtained from each subject.

Laboratory methods

SDS-PAGE and immunoblotting of sera. Control and sepsis sera were subjected to SDS-PAGE with immunoblotting for TBG. Although purified TBG (Sigma, St. Louis, MO) was demonstrable by immunoblotting after SDS-PAGE, serum TBG could not be identified even when the serum was enriched with TBG. Serum albumin has previously been shown to interfere with immunoblotting of heat shock proteins (12). It seemed likely that high concentrations of albumin overlapping the TBG zone after SDS-PAGE similarly interfered with immunoblotting of TBG. Removal of albumin by a modification of the method of Rengarajan et al. (12) revealed endogenous TBG. Affi-Gel Blue beads (Bio-Rad Laboratories, Inc., Hercules, CA) were washed with 20 mmol/L potassium phosphate buffer, pH 7.1. Twenty microliters of serum diluted with 230 µL potassium phosphate buffer were added to the beads, and the mixture was gently shaken for 3 h at room temperature and then centrifuged. An equal volume of Tris-glycine-SDS sample buffer containing 5% 2-mercaptoethanol was added to an aliquot of each supernatant and incubated at 100 C for 5 min. A 25-µL aliquot of the incubated samples was subjected to SDS-PAGE (4% stacking gel and 12% separating gel) and electrotransferred to a nitrocellulose membrane (Immobilon-p, Millipore Corp., Bedford, MA) that was blocked with 2% non fat dry milk. The membrane was incubated with goat antiserum against human TBG (INCSTAR Corp., Stillwater, MN) diluted 1:500 in 2% nonfat dry milk overnight, washed twice with 2% nonfat dry milk and twice with TBS (20 mmol/L Tris and 150 mmol/L NaCl, pH 7.4), then incubated with affinity-purified alkaline phosphatase-conjugated rabbit antigoat IgG (Sigma) diluted 1:5000 in TBS for 2 h and washed twice with TBS and twice with distilled water. The membrane was then incubated with alkaline phosphatase substrate solution containing 5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium (Sigma) in 10 mL distilled water until the intensity of the bands became adequate.

Elastase cleavage of TBG. For Western blot analysis, 0.05 µg TBG was incubated with 0.05–0.4 µg PMN elastase (Sigma) in 25 µL saline for 5 min at 37 C, diluted to 50 µL in Tris-glycine-SDS sample buffer containing 5% mercaptoethanol, and incubated at 100 C for 5 min. Twenty-five-microliter aliquots were subjected to SDS-PAGE with immunoblotting as described above.

In a separate series of experiments, previously reported in abstract (10), the effect of elastase cleavage on T4 binding by TBG was examined. Purified TBG (5.5 µg) in 1.1 mL 0.05 mol/L potassium phosphate buffer, pH 7.4, was incubated with 0.01–0.5 mg porcine pancreatic elastase (Sigma) for 1 h at 37 C. Porcine pancreatic elastase has the same substrate specificity as PMN elastase. T4 binding was then analyzed by dialysis (see below).

Incubation of purified TBG with PMN. Normal human PMN were isolated to more than 95% purity from heparinized blood by dextran sedimentation and Ficoll-Hypaque gradient centrifugation (13). For Western blot analysis, 10 x 106 PMN in 1 mL HBSS were incubated with 7 µg purified TBG for 15 min at 37 C in the absence or presence of 1000 µg zymosan. After incubation, the supernatant and cells were separated, lyophilized, and dissolved in HBSS and Tris-glycine-SDS sample buffer containing 5% 2-mercaptoethanol and then subjected to SDS-PAGE and immunoblotting as described above.

For determining the effect of PMN on T4 binding, the cells were incubated with 7.5 µg TBG for 1 h at 37 C in the absence or presence of 1000 µg zymosan. The supernatant was taken for determination of free/bound T4 by dialysis (see below).

Dialysis for determination of free/bound T4. [125I]T4 prepared by iodination of 3,5-diiodothyronine was chromatographically isolated, extracted, and purified by predialysis (14, 15, 16). The tracer was added to supernatants of PMN that had been incubated with TBG and to TBG that had been incubated with elastase. One-milliliter aliquots of supernatant were then placed in dialysis bags (Spectra/Por dialysis tubing, 25-mm diameter, Spectrum, Laguna Hills, CA) and dialyzed overnight against 5 mL 0.05 mol/L potassium phosphate buffer, pH 7.4. After dialysis, 200 µL pooled serum were added to 1-mL aliquots of the dialysate, allowed to stand for 30 min, then precipitated with 1 mL 20% cold trichloroacetic acid (Sigma), washed three times with 3 mL 5% cold trichloroacetic acid, dissolved in 200 µL 2 N NaOH, and counted in a well scintillation counter.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Serpin cleavage product of TBG in sepsis sera

Figure 1Go shows purified TBG and TBG in eight normal and eight sepsis sera at 54 kDa. An additional band at 49–50 kDa was present in the sepsis sera. This is shown most clearly in Fig. 1AGo, lanes 4, 6, and 8, and Fig. 1BGo, lanes 6 and 8. The 49- to 50-kDa band was not present in the lanes containing normal sera. The major band at approximately 27 kDa was probably apolipoprotein A-I, which has been identified as a monomeric component of a 68-kDa high density lipoprotein that binds T4 (17). Grimaldi et al. (18) showed that reaction of apparently homogenous TBG antibody with the 27-kDa component was not due to the TBG antibody itself, but, rather, to an antibody produced by contamination of the TBG used as an antigen. This contamination probably occurs during isolation of TBG by T4 agarose affinity chromatography (19).



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  		Figure 1. A—C, SDS-PAGE and immunoblotting of TBG from eight normal and eight sepsis sera. Lane 1, Molecular mass standards (Bio-Rad Laboratories, Inc.); lane 2, purified human TBG; lanes 3, 5, and 7, normal sera; lanes 4, 6, and 8, sepsis sera. C has only lanes 1–6. Intact TBG is seen at 54 kDa, and the cleaved TBG is at 49–50 kDa. The band at approximately 27 kDa corresponds to apolipoprotein A1 (17 ).

 
Serpin cleavage of TBG by elastase in vitro

To confirm that the 49- to 50-kDa band corresponds to the expected serine protease cleavage product of TBG, purified TBG (0.025 µg) was incubated with PMN elastase. As shown in Fig. 2Go, this resulted in the rapid appearance of the 49- to 50-kDa band as well as a number of lower molecular mass bands. Similar in vitro proteolysis of TBG was reported previously by Hammond et al. (8).



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  		Figure 2. SDS-PAGE and immunoblotting of purified TBG after 5-min incubation with PMN elastase. Lane 1, Molecular mass standards; lane 2, purified human TBG (0.025 µg); lane 3, TBG incubated in buffer without elastase; lane 4, TBG plus elastase (0.2 µg); lane 5, TBG plus elastase (0.1 µg); lane 6, TBG plus elastase (0.05 µg); lane 7, TBG plus elastase (0.025 µg); lane 8, molecular mass standards. A major band of cleaved TBG appears at 49–50 kDa.

 
PMN cleavage of TBG

To determine whether sepsis-induced cleavage of TBG could be attributed to proteolysis by activated PMN, as has been shown in vitro by Hammond et al. for cleavage of CBG (8), TBG was incubated with PMN. This resulted in the appearance of the 49- to 50-kDa band. Activation of PMN by zymosan increased the intensity of this band (Fig. 3Go).



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  		Figure 3. SDS-PAGE and immunoblotting of purified TBG after incubation with PMN in the absence or presence of zymosan. Lane 1, Molecular mass standards; lane 2, purified TBG; lane 3, supernatant from TBG incubation with PMN; lane 4, supernatant from TBG incubation with PMN and zymosan; lane 5, cell extract from TBG incubation with PMN; lane 6, cell extract from TBG incubation with PMN and zymosan; lane 7, purified TBG. The 49- to 50-kDa cleavage product appears in the supernatant after TBG incubation with PMN. The density of the 49- to 50-kDa band is increased after activation of PMN by zymosan.

 
Effects of elastase and PMN incubation on TBG binding of T4

Figure 4Go shows that incubation of TBG with increasing amounts of elastase resulted in weakened binding, as demonstrated by an increased free/bound T4 ratio. As shown in Fig. 5Go, incubation of TBG with PMN caused a small, but significant, increase in free/bound T4. A much larger increase was observed when PMN were stimulated with zymosan.



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  		Figure 4. Effect of incubation with elastase on T4 binding by TBG. Purified human TBG was incubated with increasing amounts of pancreatic elastase at 37 C for 1 h. Pancreatic elastase has the same substrate specificity as PMN elastase. Free/bound T4 was determined by overnight dialysis in the presence of tracer, [125I]T4.

 


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  		Figure 5. Effect of incubation with PMN on T4 binding by TBG. TBG (7.5 µg) in 1 mL buffer with and without PMN (10 x 106 cells) was incubated for 1 h at 37 C. Free/bound T4 in the supernatant was determined as described in Fig. 4Go. Incubation with cells produced only a small, but significant, increase in free/bound T4. When zymosan (Z; 1 mg), a stimulant of phagocytic activity, was added, there was a further 50% increase in the free/bound T4.

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
Although characteristic protease cleavage of serpins including TBG is demonstrable in vitro (4, 5, 7, 8), we have not found a report of the direct identification of typical serpin cleavage products in serum. This could be due to interference with immunoblotting by serum proteins (12). We were encouraged to search for the presumptive TBG cleavage product in sepsis sera by the report that the cleavage product of another serpin, plasma C1 inhibitor, had been identified in the sera of sepsis patients by SDS-PAGE and immunoblotting after preliminary purification and concentration (20). By removing albumin, it was possible to identify TBG and the TBG cleavage product without preliminary processing. This relatively simple method may be applicable to the direct demonstration of other serpin cleavage products in serum.

The appearance of the 49- to 50-kDa TBG cleavage product in septic sera confirms that inflammation causes the characteristic serine protease cleavage of TBG that was predicted by in vitro studies with PMN elastase (7, 8) and by incubation with PMN as shown here. The cleavage of TBG by elastase decreases its affinity for T4 as originally shown by Janssen et al. (9) and confirmed here. As would be expected, PMN cleavage of TBG, presumably by the action of elastase, was also associated with the release of T4. Cleavage of TBG and release of T4 by PMN were markedly increased after activation of PMN with zymosan. These findings suggest that activation of PMN in the inflammatory response cleaves TBG and releases T4. Because serine proteases are neutralized by the {alpha}1-protease inhibitor in plasma, the proteolysis of TBG and the associated release of T4 are probably restricted to the locus of cellular proteases. This is consistent with accumulation of T4 iodine at inflammatory sites (21) and with the rapid decrease in serum T4 during acute inflammation (22). TBG has been considered a passive carrier for T4 with a distributive function and a storage/buffering capacity that maintains a stable free T4 concentration during cellular uptake (23). It seems possible that noninflammatory serine protease activity cleaves TBG at a lower rate and releases T4, contributing to local transfer of T4 from plasma to tissue sites. In contrast to the continuous transfer of T4 to tissues via the picomolar serum free T4 concentration, proteolytic cleavage of TBG with release of T4 has the potential for intermittent site-specific access to the much higher total serum T4 concentration. More sensitive methods may reveal the lower TBG remnant concentrations to be expected in the absence of sepsis. The hypothesis that proteolytic cleavage of TBG releases T4 to sites at which it is metabolized implies that the concentration of T4-TBG complexes contributes to T4 metabolism. This is consistent with reports by Arafah that requirements for replacement T4 in hypothyroidism decrease during treatment with androgens, which decrease TBG, and increase during estrogen replacement, which increases TBG (24, 25).

The weakening of T4 binding by TBG cleavage would be expected to contribute to the increased free/bound T4 ratio and the consequent decrease in total T4 that are characteristic of the euthyroid sick syndrome. By analogy to other serpin cleavage products, the possibility that the cleaved TBG has functions distinct from the loss of binding affinity should be considered (6, 27). Persistence of the TBG remnant in the circulation also raises the question of whether it contributes to the immunoassayable TBG and the reported discrepancy between weakened serum T4 binding affinity and relatively small or absent decreases of TBG in nonthyroid illness (28). In our recent study of sepsis sera (11), assays that depended on tracer T4 binding by immobilized TBG antibody and immunodiffusion both showed decreases in TBG concentration in sepsis sera. Together with decreases in albumin and transthyretin concentrations, these were sufficient to account for the observed increase in the ratio of free/bound T4. However, these determinations of serum TBG may have been predominantly measurements of the native TBG with high affinity for T4. Recent studies in our laboratory (unpublished data) indicate that elastase cleavage as well as heat inactivation decrease the TBG measurable by tracer T4 binding to TBG immobilized by antibody (GammaDab TBG RIA Kit, INCSTAR Corp.). Surprisingly, exposure to elastase and heat inactivation also decreased the measurable TBG by radial immunodiffusion (Bind A RID, The Binding Site Ltd., Birmingham, UK), suggesting that binding to the TBG antibody used for radial immunodiffusion can be weakened by conformational changes in TBG.

Received March 1, 2000.

Revised June 23, 2000.

Revised August 2, 2000.

Accepted August 9, 2000.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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