Ecto-5′-nucleotidase and intestinal ion secretion by enteropathogenic Escherichia coli

Enteropathogenic Escherichia coli (EPEC) triggers a large release of adenosine triphosphate (ATP) from host intestinal cells and the extracellular ATP is broken down to adenosine diphosphate (ADP), AMP, and adenosine. Adenosine is a potent secretagogue in the small and large intestine. We suspected that ecto-5′-nucleotidase (CD73, an intestinal enzyme) was a critical enzyme involved in the conversion of AMP to adenosine and in the pathogenesis of EPEC diarrhea. We developed a nonradioactive method for measuring ecto-5′-nucleotidase in cultured T84 cell monolayers based on the detection of phosphate release from 5′-AMP. EPEC infection triggered a release of ecto-5′-nucleotidase from the cell surface into the supernatant medium. EPEC-induced 5′-nucleotidase release was not correlated with host cell death but instead with activation of phosphatidylinositol-specific phospholipase C (PI-PLC). Ecto-5′-nucleotidase was susceptible to inhibition by zinc acetate and by α,β-methylene-adenosine diphosphate (α,β-methylene-ADP). In the Ussing chamber, these inhibitors could reverse the chloride secretory responses triggered by 5′-AMP. In addition, α,β-methylene-ADP and zinc blocked the ability of 5′-AMP to stimulate EPEC growth under nutrient-limited conditions in vitro. Ecto-5′-nucleotidase appears to be the major enzyme responsible for generation of adenosine from adenine nucleotides in the T84 cell line, and inhibitors of ecto-5′-nucleotidase, such as α,β-methylene-ADP and zinc, might be useful for treatment of the watery diarrhea produced by EPEC infection.
Introduction
Ecto-5′-nucleotidase (CD73, EC 3.1.3.5) is a key extracellular enzyme which catalyzes the hydrolysis of extracellular nucleoside monophosphates to their corresponding nucleosides, with 5′-AMP being the preferred substrate. Ecto-5′-nucleotidase is highly expressed in endothelium, liver, and intestinal mucosa and at varying levels in other tissues including lymphocytes, kidney, and certain cancers. Ecto-5′-nucleotidase is tethered to the extracellular surface of mammalian cells by a glycosylphosphatidylinositol (GPI) lipid anchor. Most of the research on this enzyme has been in the cardiovascular system because of its role in the production of adenosine, which protects cells against ischemia by multiple mechanisms including vasodilation [1, 2].
Enteropathogenic Escherichia coli (EPEC) is a common cause of watery diarrhea in children in developing countries. While some aspects of EPEC infection, such as adherence, are well studied, the way that EPEC triggers watery diarrhea has been obscure, since EPEC produces no toxins. We recently proposed a theory that release of adenine nucleotides from host intestinal cells, followed by breakdown to adenosine, could trigger watery diarrhea by activation of adenosine receptors on intestinal cells [3].
In the gastrointestinal tract, ecto-5′-nucleotidase was found to be a key enzyme necessary for enterocyte responses to 5′-AMP, the neutrophil-derived secretagogue [4–6]. At that time the significance of ecto-5′-nucleotidase was felt to be in the setting of invasive, inflammatory pathogens because polymorphonuclear neutrophils (PMNs) release 5′-AMP during the process of chemotaxis into areas of infection or inflammation. Since that time, however, we have discovered that a noninvasive pathogen, EPEC, triggers a large release of ATP directly from host intestinal cells without any requirement for PMNs to be present [3]. Other diarrheal pathogens not considered classically invasive, such as Aeromonas hydrophila, also cause ATP release from enterocytes [7]. Extracellular ATP is broken down to ADP, AMP, and then adenosine, where it triggers a vigorous fluid secretory response in intestinal epithelium. We suspected that ecto-5′-nucleotidase also played a role in the diarrheal fluid secretion induced by EPEC and initiated this study to determine if it is the case, and, if so, whether inhibitors of ecto-5′-nucleotidase could block fluid secretion triggered by adenine nucleotides and EPEC infection. We used the T84 colon carcinoma cell line as a model because we have used these cells to study EPEC infection, they express ecto-5′-nucleotidase, and they can be studied in the Ussing chamber to measure chloride secretion. In order to facilitate our study we also developed a nonradioactive, non-high-pressure liquid chromatography (non-HPLC) method for measuring ecto-5′-nucleotidase in living T84 cell monolayers.
Materials and methods
Bacterial strains used E. coli strains used included laboratory E. coli strain HB101 (O: rough), commensal strain HS (O9: H4), and classic human EPEC strains E2348/69 (serotype O127: H6), B171-8 (O111: NM), and JCP88 (O119: B14) as described in several publications [8–12]. EPEC mutants included JPN15, an E2348 derivative which has lost the EPEC adherence factor (EAF) plasmid [13], UMD874, the espF mutant derived from E2348, which is deficient in host cell killing [3, 14], and SE1010, with a mutation in sepZ (also called espZ), which is defective in type III secretion [15]. Bacteria were added to yield a multiplicity of infection (MOI) of 100:1.
Materials The following reagents were obtained from Sigma-Aldrich Chemicals: α,β-methylene-ADP, adenosine, adenosine 5′-monophosphate (AMP), tetramisole (also called levamisole), polymyxin B, neomycin, purified phosphatidylinositol-specific phospholipase C (PI-PLC, from Bacillus cereus), and zinc acetate. BIOMOL (Plymouth Meeting, PA, USA) was the source of the BIOMOL GREEN reagent used in the phosphate release assay for nucleotidase activity and of U73122, a PI-PLC inhibitor. U73122 is 1-(6-[17 beta-3-methoxyestra-1,3,5- (10) triene-17-yl] amino/hexyl) 1H-pyrroledione. A cell permeant PI-PLC activator, 3M3-FBS, was from the Calbiochem Division of EMD Biosciences (La Jolla, CA, USA). 3M3-FBS is 2,4,6-trimethyl-N-(m-3-trifluoromethylphenyl)benzenesulfonamide. Phosphate-free DMEM medium was purchased from MP Biomedicals (formerly ICN Biomedicals, Aurora, OH, USA). UNIFILTER plates were from Whatman (Clifton, NJ, USA).
Bacterial culture E. coli strains were grown overnight in Luria-Bertani (LB) broth at 37°C with 300 rpm shaking, then subcultured for 2 h in serum-free DMEM/F12 medium supplemented with 18 mM NaHCO3, 25 mM hydroxyethylpiperazine ethanesulfonic acid (HEPES) buffer, pH 7.4, and 1% D-mannose as previously described [3]. For experiments with bacteria in minimal medium, bacteria were subcultured at a dilution of 1:2,000 into minimal medium (M9 salts plus casamino acids supplemented with 2 mM glucose). For convenience we used M9-CA liquid broth packets (E. coli Fast Media, MBI-Fermentas, Hanover, MD, USA) and added 2 mM glucose before use.
Cell culture T84 colon cancer cells were grown in DMEM/F12 medium supplemented with 7.5% fetal bovine serum (Gibco/Invitrogen, Grand Island, NY, USA), 18 mM NaHCO3, 20 μg/ml vancomycin, and 15 μg/ml gentamicin as previously described [16]. Ussing chamber studies of secretion were performed on T84 cell monolayers grown in Snapwell inserts (Corning Costar, Corning, NY, USA). The Snapwell inserts, which had a 0.4 μm pore size, were coated with 32 μg collagen per well by applying 0.16 ml of 0.2 mg/ml type III collagen (Sigma; dissolved in warm 0.2 M acetic acid) to the Snapwell and allowing it to dry in the tissue culture hood under UV light. T84 cells were seeded onto the Snapwell inserts at ~1.2 × 106 cells per well and allowed to grow to confluency for 7–9 days. At this time the monolayers had transepithelial electrical resistances (TER) of 400–1,000 Ω · cm2.
Assay for ecto-5′-nucleotidase by phosphate release An assay for ecto-5′-nucleotidase activity in living cells was developed based on the ability to detect inorganic phosphate (Pi) released from 5′-AMP. This method has been used to detect activity of protein phosphatases such as PTEN [17] and lipid phosphatases [18] and is based on sensitive detection of low levels of Pi using the BIOMOL GREEN reagent, an enhanced and stabilized formulation of malachite green. To carry out the assay a phosphate-free buffer was used consisting of (in mM): NaCl, 154; KCl, 2: MgCl2, 4; NaHCO3, 18: HEPES, pH 7.4, 25; and glucose, 10. This buffer is referred to as nucleotidase buffer. To measure ecto-5′-nucleotidase activity, the cell monolayer was rinsed once with sterile normal saline, then the medium was replaced with warm nucleotidase buffer. For cells in a 48-well plate, 0.25 ml of nucleotidase buffer were added per well, and the cells were allowed to rewarm to 37°C in the CO2 incubator. During pipetting the multiwell plate was kept warm using a metal heating block set at 37°, and a stopwatch was used to time the AMP addition and to terminate the assay. The procedure used for measuring monolayer activity was slightly different from that used to measure 5′-nucleotidase activity released into the supernatant, as described below.
Cell-bound or monolayer activity To measure cell-bound ecto-5′-nucleotidase activity in cell monolayers, 5′-AMP was added to yield a final concentration of 0.2 mM to quadruplicate wells. Two other wells were left without addition of AMP (the “no AMP blank”). After a 10-min incubation at 37° an aliquot (usually 50 μl) was removed and quickly transferred to a well of a 96-well plate to terminate the reaction.
E. coli-induced release of nucleotidase activity into the supernatant medium For nucleotidase release experiments, the cell monolayer was changed to warm, phosphate-free DMEM, then infected with an E. coli strain for 35 min to allow adherence, then the medium was changed to nucleotidase buffer and the infection was allowed to continue for 2 or 3 h. Note that in this procedure any nucleotidase activity that is released in the first 35 min is discarded and not detected by our method. However, this two-stage procedure with the medium change was necessary because EPEC bacteria did not adhere normally if they first encountered the host cell in nucleotidase buffer. After a period of infection, supernatant medium was collected with a multichannel pipettor and transferred to the wells of a Whatman UNIFILTER plate (a 96-well with 0.45-μm membrane for sterile filtration). Sterile filtrates were prepared by centrifugation with collection of the filtered medium into another 96-well plate placed beneath the UNIFILTER as previously described [19]. Once again, experimental conditions were usually done in groups of six, with two wells not receiving any AMP (no AMP blanks) and four wells receiving 0.2 mM AMP. Again, the usual assay condition was 10 min at 37° before the reaction was stopped by addition of 10 μl of 1 M HCl (“stop solution”).
BIOMOL GREEN detection of phosphate released from AMP Stopped samples in a 96-well plate were brought to 100 or 110 μl volume with water if necessary, then treated with 100 μl of BIOMOL GREEN reagent. A standard curve of inorganic phosphate was prepared and run with every experiment; standards and unknown samples were incubated at room temperature for 20 min to allow a green color to develop, then the 96-well plate was read on a multiwell plate spectrophotometer at 620 nm. Unknown values were calculated from the standard curve using a hyperbolic curve fit using GraphPad Prism software, version 4.0. Results of monolayer activity were expressed as nmol of Pi produced/min per 106 cells. For experiments showing nucleotidase release, the results were often expressed as nmol Pi released/ min per well since the assay was done on a cell-free filtrate and because we often noted some detachment of cells during the longer incubations of 2–3 h needed to observe release.Although we believed we were developing a new method for assay of ecto-5′-nucleotidase by phosphate release, during the course of this work another group reported using a virtually identical method, also based on detection of phosphate released from 5′-AMP [1].
Detection of CD73 by Western immunoblot To prove that the released 5′-nucleotidase activity we measured was of host cell rather than bacterial origin, we performed immunoblots on the supernatants of infected T84 cells with antibodies against CD73. Initial attempts at immunoblotting using a commercially available monoclonal anti-CD73 antibody (Abnova Corp., Taipei, Taiwan) were unsuccessful. Dr. Linda F. Thompson, Oklahoma Medical Research Foundation, kindly sent us mouse monoclonal antibodies against human CD73 which had been generated by Dr. Wolf Gutensohn several years earlier. Of these, the two antibodies that gave the best results were designated CD73.4 and CD73.6 by Dr. Gutensohn; both were of isotype IgG2b and were used at a concentration of 1 μg/ml. After washings, the secondary antibody was goat anti-mouse IgG2b conjugated to peroxidase at a dilution of 1:3,000 (Roche Molecular Biochemicals, Indianapolis, IN, USA). Blots were developed by chemiluminescence as previously described [20].
Ussing chamber studies A Snapwell insert containing a monolayer of T84 cells was placed in the plexiglass “slider” and inserted into the Ussing chamber (Physiologic Instruments, San Diego, CA, USA) at 37°C and continuously short-circuited by a four electrode, automatic voltage-clamp apparatus which measured short-circuit current (Isc) and transepithelial resistance (TER); chamber fluid resistance was automatically subtracted. Transepithelial resistance was determined by passing 10-s 10-mV current pulses through the tissues. Short-circuit current was measured by passing sufficient current through the tissues via Ag/AgCl electrodes to reduce the spontaneous transepithelial potential to zero. The composition of the tissue bathing solution was (in mM): 140 Na+, 124 Cl−, 21 HCO3-, 5.4 K+, , 1.2 Mg2+, 1.2 Ca2+, and 10 glucose. Raw short-circuit current (Isc) values were converted to μA per cm2 by dividing by the area of the Snapwell monolayer (1.13 cm2). Other details of the Ussing chamber methods were exactly as described [16].
Protein assay Protein assay was by the Coomassie blue dye binding assay method of Bradford, using a Bio-Rad kit [21].
Expression of ecto-5′-nucleotidase RNA by reverse transcription and real-time polymerase chain reaction (PCR) T84 cells grown in 24-well plates were infected with EPEC for 35 min, then the medium was changed to remove unbound bacteria. Three hours after the medium change, ciprofloxacin was added to 25 μg/ml to kill EPEC and the incubation was continued 1 more hour. Old medium was removed, and the cell monolayer was lysed in extraction buffer with 10% β-mercaptoethanol (RNeasy Kits, Qiagen, Valencia, CA, USA). RNA was subjected to reverse transcription using Invitrogen Superscript III reverse transcriptase; 5 μl of purified RNA was used per 50 μl reaction, and gene-specific primers at 0.2 μM were used. Reverse transcription reaction was at 55° for 1 h. Copy DNA from reverse transcription was diluted 100-fold, then analyzed by quantitative real-time PCR using the same oligonucleotide primers. For ecto-5′-nucleotidase the primers used were 5′-TTC CAC CCT GAA GAA GGC CTT TGA-3′ (forward) and 5′-ATA ACT GGG CAC TCG ACA CTT GGT-3′ (reverse). As a normalizing gene we used glyceraldehyde phosphate dehydrogenase (GAPDH) as described by Khan et al. [22] except that we redesigned longer primers which were 5′-TCG ACA GTC AGC CGC ATC TTC TTT-3′ and 5′-ACC AAA TCC GTT GAC TCC GACC CTT-3′. PCR was carried out using a MyiQ Single-Color qRT-PCR machine from Bio-Rad (Hercules, CA, USA) using SYBR Green as the dye to monitor the amplification. Relative expression was calculated by the ΔΔCt (“Livak”) method as described [23], where Ct is the number of cycles to threshold. SYBR Green PCR reagents were from Bio-Rad and to reduce the cost, the PCR reaction volume was reduced to 25 μl. PCR was performed using a two-step protocol with an annealing temperature of 58.7° and denaturation at 95° for 30 s each (i.e, no extension step) for 35 cycles. Thermal melt curve analysis was performed at the end of the PCR amplification and showed a single sharp peak for the genes analyzed.
Data analysis and presentation All error bars shown in graphs and error values reported in the text are standard deviations. Significance was tested by one-way analysis of variance (ANOVA) with the Tukey-Kramer post-test for multiple comparisons, using InStat software for the Macintosh from GraphPad software (San Diego, CA, USA). Graphs were prepared using Prism 4.0 software, also from GraphPad. Asterisks shown on graphs indicate a p value of < 0.05.
Results
Ecto-5′-nucleotidase activity was readily measurable in T84 cell monolayers using the phosphate release assay method developed in this study. Figure 1 shows the characteristics of the ectoenzyme in this system. Figure 1a shows a curve of activity vs substrate concentration, demonstrating that the enzyme shows Michaelis-Menten kinetics. In four experiments similar to that shown in Fig. 1a, the KM was 229 ± 30 μM and the Vmax was 2.7 ± 1.8 nmol/min per 106 cells (mean ± SD of 4 experiments). While the KM was very consistent over time and from experiment to experiment, the Vmax was more variable as we compared experiments separated by months or years. We noted that highly T84 passaged cells (passage number of 70 or greater) showed lower levels of ecto-5′-nucleotidase activity than less passaged cells from the same source (experiments not shown). The KM we observed in intact T84 cells was higher than that reported for the purified enzyme derived from a colon carcinoma cell line, BCS-TC2 cells [24]. Figure 1b shows the raw data of Pi generated over time in the presence and absence of added AMP substrate and demonstrates that the assay was linear for 10 min; therefore, 10 min was chosen as the standard assay duration.
Fig. 1Characterization of ecto-5′-nucleotidase activity in T84 cell monolayers. 5′-Nucleotidase activity was measured by the release of inorganic phosphate (Pi) from AMP in phosphate-free buffer using a colorimetric method as described in “Materials and methods.” For a, c, and d, the spectrophotometric reading in the absence of AMP (“no AMP blank”) was subtracted from the total reading. In b the “no AMP blank” was not subtracted and is shown in b as labeled. a Michaelis-Menten curve of enzyme velocity vs concentration of AMP substrate. b Time course of Pi release vs time. c Lack of inhibition by tetramisole, an inhibitor of alkaline phosphatase. d pH dependence of 5′-nucleotidase activity. e, f HPLC traces showing conversion of AMP (e) to adenosine (Ado, f) in T84 cell monolayers. g Sensitivity of the AMP-hydrolyzing activity to the inhibitor α,β-methylene-ADP, confirming it as ecto-5′-nucleotidase
Alkaline phosphatase is another abundant intestinal ectoenzyme which can hydrolyze 5′-AMP [25]. Alkaline phosphatase can be distinguished from 5′-nucleotidase by its alkaline pH optimum and sensitivity to inhibition by tetramisole. Figure 1c shows that the ectonucleotidase we measured on T84 cells was not inhibited by tetramisole (also known as levamisole). Furthermore, its activity did not increase with increasing pH (Fig. 1d) providing evidence that the activity being measured was not alkaline phosphatase. In addition, we used HPLC to confirm that 5′-AMP was being converted to adenosine in the supernatant medium of T84 cells (Fig. 1e and f). Morover, the 5′-nucleotidase activity we measured by phosphate release was sensitive to inhibition by α,β-methylene-ADP, a known inhibitor of ecto-5′-nucleotidase (Fig. 1g and see also Fig. 4). Lastly, after we initiated this study, other researchers also reported using phosphate release from 5′-AMP as their assay for ecto-5′-nucleotidase [1]. We have found that the increased throughput of the phosphate release assay increases its usefulness compared to the much slower HPLC methods for ecto-5′-nucleotidase.
Figure 2 shows the effect of EPEC infection on ecto-5′-nucleotidase activity released into the supernatant medium during an experimental EPEC infection of cultured T84 cells. Wild-type EPEC strains E2348/69 and JCP88 triggered a release of 5′-nucleotidase into the supernatant medium while nonpathogenic E. coli strains HB101 and HS did not. The supernatant medium was subjected to sterile filtration prior to assay to remove bacterial cells as well as any detached host cells, so that the activity reflects a soluble and not a cell-bound form. In experiments where we determined both the supernatant release and the monolayer activity in the same experiment, supernatant release by wild-type EPEC was 30–40% of monolayer activity 3 h after the medium change (Fig. 2a and data not shown). To determine if the 5′-nucleotidase could possibly be derived from the E. coli bacteria rather than from the host we also assayed sterile filtrates of E. coli for 5′-nucleotidase activity and it was virtually absent from the culture filtrates of all E. coli strains tested, including EPEC (Fig. 2b, light gray bars). Even after treatment with a concentration of polymyxin B sufficient to cause > 99% bacterial cell lysis the amount of bacterial nucleotidase-like activity was far less than the amounts we observed in the supernatant in Fig. 2a. In addition, our protocol included a medium change step in order to remove unbound bacteria. Therefore we concluded that the 5′-nucleotidase activity being released in Fig. 2a was of host cell origin.
Fig. 2Effect of E. coli infection on 5′-nucleotidase activity in the supernatant medium. a Nonpathogenic E. coli strains (HB101 and HS) or EPEC strains E2348/69 and JCP88 were subcultured in DMEM medium for 2 h, then used to infect T84 cells at a multiplicity of infection of 100:1 in phosphate-free DMEM. After 35 min to allow adherence, the medium was changed to nucleotidase buffer and aliquots were collected at various times after the medium change, filtered through a 0.45-μm filter to remove bacterial cells, then assayed for 5′-nucleotidase activity. Since the activity was measured in the cell-free sterile filtrates the activity is expressed as nmol/min per well. bE. coli suspensions were subjected to sterile filtration without treatment (light gray bars) or following treatment with 50 μg/ml polymyxin B, a lytic antibiotic (dark gray bars). Then the nucleotidase activity of a 100-μl aliquot was measured. The 100 μl volume was chosen because this was the volume of inoculum needed to achieve an MOI of 100:1 for the slowest growing strain (E2348/69) in a typical 48-well plate of T84 cells
To determine if this hypothesis was correct and to try to determine the mechanism of EPEC-induced 5′-nucleotidase release, we compared the 5′-nucleotidase release triggered by wild-type EPEC strain E2348/69 with the plasmid-cured derivative, JPN15, and its espF mutant, UMD874 (Fig. 3a). The espF mutant is defective in host cell killing and in inducing damage to monolayer integrity [14, 26]. Interestingly, the espF mutant was not attenuated in its ability to trigger 5′-nucleotidase release, but instead it consistently outperformed the wild-type strain in this regard. In six experiments 5′-nucleotidase release induced by the espF mutant exceeded that of the wild-type E2348/69 by an average of 2.3 ± 0.14 fold (p = 0.03 by paired t-test), suggesting that host cell death or damage is not the mechanism of EPEC-induced nucleotidase release.
Fig. 3a Effect of EPEC mutants and PI-PLC inhibitors and activators on induced 5′-nucleotidase release into supernatant. Release of 5′-nucleotidase activity into supernatant medium was measured as described in “Materials and methods” and in the legend to Fig. 2. Once again, activity in the cell-free sterile filtrates is expressed as nmol/min per well. However, for purposes of comparison 48-well plates contain ~0.25 × 106 T84 cells per well at confluency. b U73122, an inhibitor of PI-PLC, was used at a concentration of 2 μm and was re-added after the medium change; *significantly decreased compared to the EPEC strain alone, p < 0.05. cm-3M3-FBS, a cell-permeant sulfonamide activator of PI-PLC, was added at the concentrations and for the times indicated. d Inhibitory effect of neomycin on PI-PLC-induced 5′-nucleotidase release; this experiment was performed on cells grown in a 24-well plate, with ~0.8 × 106 cells per well; *significantly decreased compared to PI-PLC alone. e PI-PLC was again added to a final concentration of 0.1 U/ml for the times shown; *significantly different from the corresponding control. This figure is a composite of experiments that were separated in time and with cells of different passage number; therefore the absolute amount of activity varies among the figure parts. f Effect of EPEC infection on expression of RNA encoding ecto-5′-nucleotidase in T84 cells, by reverse transcription and real-time PCR. T84 cells were infected for 35 min, then the supernatant medium was changed to remove unattached bacteria, then the T84 cell monolayer harvested for RNA extraction 4 h after the medium change. Reverse transcription and PCR conditions were as described in “Materials and methods.” Expression of ecto-5′-nucleotidase was normalized to that of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). f Normalized expression of a single representative experiment (mean ± SD of 6 PCR wells). g Immunoblot analysis of the proteins released into the supernatant after 2 h of infection with EPEC or an EPEC mutant (SE1010, sepZ/espZ), using monoclonal antibody against CD73. Lane 1, supernatant from uninfected, control T84 cells. Lanes 2 and 3, supernatant from cells infected with wild-type E2348/69. Lanes 4 and 5, supernatants from cells infected with the sepZ mutant. GPI-linked CD73 has an apparent molecular size of 72 kDa (not seen in this blot) and the GPI-cleaved soluble portion runs at 51 kDa (heavy band in lanes 2 and 3)
Ecto-5′-nucleotidase is tethered to the extracellular surface of the cell by a glycosylphosphatidylinositol (GPI) lipid anchor [24, 27]. GPI lipid anchors are cleaved by phosphatidylinositol-specific phospholipases C (PI-PLC), and EPEC has been shown to activate at least one type of PI-PLC in the host cell, PI-PLC-γ [28]. Therefore we considered whether PI-PLC activation by EPEC could be a nonlethal mechanism by which EPEC triggered 5′-nucleotidase release from host intestinal cells.
Figure 3b shows an experiment in which we tested whether a PI-PLC inhibitor could block EPEC-induced nucleotidase release. Indeed, 2 μM U73122 did reverse the nucleotidase release triggered by wild-type EPEC strains. Conversely, a newly described cell-permeable activator of PI-PLC, called m-3M3-FBS [29], triggered nucleotidase release from uninfected cells in a time- and concentration-dependent manner (Fig. 3c). Experiments to test if m-3M3-FBS would enhance EPEC-induced release were inconsistent, with some experiments showing a mild enhancement or additive effect and others showing no enhancement or mild antagonism (results not shown).
In addition we also tested whether purified PI-PLC added to the apical surface of the T84 cell monolayer induced 5′-nucleotidase release and it did (Fig. 3d and e). Figure 3d also shows that 50 μg/ml neomycin could partially block 5′-nucleotidase release by purified PI-PLC. In addition to its antimicrobial effects, neomycin is an inhibitor of several types of PLC enzymes including PI-PLC. Figure 3e shows that after a 2-h treatment with PI-PLC not only was 5′-nucleotidase activity increased in the supernatant, but that monolayer activity was depleted as well (right portion of Fig. 3e). In experiments similar to Fig. 3e done with EPEC infection, we could not detect any decrease in monolayer activity 2–3 h after infection, despite the liberation of 5′-nucleotidase activity into the supernatant. We considered the possibility that increased host cell synthesis of ecto-5′-nucleotidase might be occurring in response to the cleavage of the enzyme from the cell surface. We analyzed the abundance of RNA encoding ecto-5′-nucleotidase by reverse transcription and real-time quantitative PCR in response to EPEC infection. Figure 3f and Table 1 show that infection with wild-type EPEC and the EPEC espF mutant (UMD874) increased the abundance of ecto-5′-nucleotidase RNA compared to uninfected control cells. The increase in expression of ecto-5′-nucleotidase RNA by wild-type EPEC was 33% above control. While this is not a large increase, it does explain the maintenance of normal levels of ecto-5′-nucleotidase on the surface of EPEC-infected cells, because under the most optimum conditions the release of enzyme activity into the supernatant is about 30–40% of monolayer activity (e.g., Fig. 3e).
Table 1Effect of EPEC infection on expression of RNA encoding ecto-5′-nucleotidase in T84 cells, by reverse transcription and real-time PCRConditionNormalized expression ratioaUninfected control1.0, by definition+1 U/ml PI-PLC1.23 ± 0.26HS-infectedb0.66 ± 0.22E2348/69-infectedb1.33 ± 0.21cUMD874 (espF)-infectedb1.40 ± 0.19caCells were infected for 35 min, then the supernatant medium was changed to remove unattached bacteria, then the T84 cell monolayer harvested for RNA extraction 4 h after the medium change. Expression of ecto-5′-nucleotidase was normalized to that of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Data shown are the means ± SD of 5 similar experimentsbBacteria were added with the intent of achieving a multiplicity of infection (MOI) of 100:1; actual MOIs in the 5 experiments ranged from 71 to 144cSignificantly increased compared to uninfected control by paired t-test (p = 0.04)
The 5′-nucleotidase released into the supernatant appeared to be of host cell origin, because it could be triggered by chemical stimuli in the absence of EPEC infection (Fig. 3c–e). The host origin was also proven using a mouse monoclonal antibody against human CD73. Wild-type EPEC infection of T84 cells triggered the release into the supernatant of a 51 kDa protein recognized by a mAb against CD73 (Fig. 3g, lanes 2 and 3). In contrast, the EPEC sepZ/espZ mutant, which is defective in type III secretion, triggered the appearance of a fainter band which was of ~55 kDa in size. The origin of this faint band is unknown, but it is possible it is generated by proteolysis rather than PI-PLC activation. For example, E2348/69 expresses EspC, an autotransporter protease whose export is independent of type III secretion.
Figure 4 shows that 5′-nucleotidase activity in living T84 cells was susceptible to the same inhibitors as the purified enzyme studied by others. Figure 4a shows that α,β-methylene-ADP inhibited ecto-5′-nucleotidase activity and that its inhibition has the characteristics of a competitive inhibitor. In the presence of α,β-methylene-ADP the enzyme shows an apparent increase in KM but no increase in the Vmax, and the inhibition can be overcome at high concentrations of AMP substrate. In contrast in Fig. 4b one observes that zinc acetate behaves as a noncompetitive inhibitor of the enzyme, with a decrease in the Vmax but no increase in the KM (in fact there is a small, paradoxical decrease in KM in the presence of zinc). Figures 4c and d show the inhibitory dose-response curves at a single 0.2 mM concentration of AMP substrate. For both inhibitors, when concentration is expressed on a logarithmic scale, the curves are well fit by a sigmoidal, single-site model, allowing determination of the inhibitory concentration 50% (IC50) as shown in Figs. 4c and d. In addition to α,β-methylene-ADP and zinc acetate, ecto-5′-nucleotidase activity was also inhibited by 3–10 mM sodium fluoride as reported for the purified enzyme (data not shown). 5′-Nucleotidase activity released into the supernatant was also sensitive to inhibition by α,β-methylene-ADP and zinc acetate with similar potencies (data not shown).
Fig. 4Effect of the 5′-nucleotidase inhibitors on enzyme activity in T84 cell monolayers. a, b Michaelis-Menten curves of enzyme activity vs substrate concentration in the absence (open symbols) and presence (closed symbols) of the inhibitor shown. Enzyme parameters were calculated by nonlinear curve fitting using GraphPad Prism and the fitted parameters of Vmax and KM are shown below the graphs. c, d Dose-response curves for inhibition of ecto-5′-nucleotidase activity by α,β-methylene-ADP (c) and zinc acetate (d) using a 0.2 mM concentration of AMP substrate. In all parts of Fig. 4 the inhibitor was added before the AMP, and in c and d the curves were fitted to a sigmoidal inhibitory dose-response curve and the inhibitory concentration 50% (IC50) value derived from the curve fit is shown
In Fig. 4 the inhibitor was always added to the cell monolayer prior to the AMP substrate. Using the Ussing chamber apparatus to measure chloride secretion, we realized we could monitor the effects of inhibition of 5′-nucleotidase activity dynamically and assess the ability of inhibitors to reverse a secretory response already underway. In preliminary experiments we found that to reverse an ongoing secretory stimulus we had to add inhibitors at concentrations considerably above their IC50 values. The requirement for addition of inhibitors well above the IC50 is due to the fact that adenosine is a very potent secretagogue in intestinal tissues and a very high degree of inhibition of ecto-5′-nucleotidase is needed to reduce the production of adenosine below the secretory threshold.
Figure 5a shows that secretion triggered by 10 μM AMP is reversed by theophylline, an adenosine receptor antagonist. Figure 5a emphasizes the fact that 5′-AMP has no secretory ability unless it is converted into adenosine. Adenosine then acts via apical adenosine A2b receptors to trigger chloride secretion toward the apical side of the monolayer. Figure 5b shows that chloride secretion triggered by 1 μM AMP is partially antagonized by 2 mM zinc acetate even when the inhibitor is added after secretion has started. Addition of 1 μM adenosine restored the short-circuit current (Isc) to its previous trend, showing that zinc does not block adenosine receptors or “poison” the secretory machinery. Under the same experimental conditions, 200 μM α,β-methylene-ADP produced a much larger degree of reversal of Isc than did zinc (Fig. 5c). As with zinc, α,β-methylene-ADP did not prevent a subsequent, normal Isc response to addition of adenosine (Fig. 5c, right-hand portion), showing that it is acting as a 5′-nucleotidase inhibitor and not an adenosine antagonist. The efficacy of inhibition by α,β-methylene-ADP in this paradigm, however, was strongly dependent on the concentration of AMP used to trigger secretion. At higher concentrations of AMP (e.g., 20 μM), the degree of inhibition by α,β-methylene-ADP was markedly less (Fig. 5d) than when 1 μM AMP was used as the stimulus. In contrast, zinc acetate produced the same, modest inhibition of Isc regardless of the concentration of AMP used. Figure 5e summarizes the effectiveness of the two inhibitors, zinc and α,β-methylene-ADP, when tested against various different concentrations of AMP as the initial stimulus. Figure 5e shows that the effectiveness of α,β-methylene-ADP declines significantly with increasing concentrations of AMP used, whereas zinc’s inhibitory effects are fairly constant over a range of AMP concentrations. The results of Fig. 5 (b–e) vividly illustrate, at the level of of an electrophysiologic response, the difference between a competitive and a noncompetitive mode of action of an enzyme inhibitor (compare with Fig. 4a and b).
Fig. 5Effect of 5′-nucleotidase inhibitors on the chloride secretory response to AMP in T84 cells in the Ussing chamber. Short-circuit current (Isc) was measured in T84 cells grown in Snapwell inserts as described in “Materials and methods”; in this cell line and electrode configuration, a positive Isc (upward deflection on the graphs) represents chloride secretion in the basolateral to apical direction. All of the additions of agonists and inhibitors in these parts of the figure was on the apical (mucosal) side of the monolayers only. a Reversal of AMP-induced secretory response by theophylline, an adenosine receptor antagonist. b Partial inhibition of AMP-induced secretion by 2 mM zinc acetate, with restoration of the tracing to its previous trend by adenosine, demonstrating that adenosine receptors are not blocked by zinc. c Substantial reversal of Isc by α,β-methylene-ADP when a low concentration of AMP (1 μM) is used as the stimulus. Note that adenosine fully restores secretion to its previous level. d Lesser degree of reversal of Isc by α,β-methylene-ADP when a higher concentration of AMP (20 μM) is used as stimulus. e Summary of many experiments in which fixed concentrations of zinc and α,β-methylene-ADP were tested for inhibitory efficacy against varying concentrations of AMP as test stimulus. In Fig. 5e the slope of the α,β-methylene-ADP curve was −1.73 ± 0.5 by linear regression (95% confidence limits of the slope value −3.27 to −0.2, significantly different from zero, p < 0.04). In contrast the slope of the zinc curve was −0.04 ± 0.23 and did not differ significantly from zero
Since the discovery of EPEC-induced ATP release we have hypothesized that EPEC bacteria may gain a nutritional advantage by this inflicting kind of damage on the host cell [3]. The lumen of the gastrointestinal tract is a purine-limited environment, as demonstrated by in vivo gene expression screens such as in vivo expression technology (IVET) [30] and signature-tagged mutagenesis (STM) [31]. Nucleotides and nucleosides enhance growth of EPEC strains in vitro, and these growth-promoting effects are most obvious when adenosine is added to minimal medium (Fig. 6). Compared to minimal medium, addition of 30 μM adenosine markedly accelerated growth of EPEC strains such as E2348/69 (Fig. 6). Addition of 5′-AMP also increased EPEC growth, although not as much as adenosine. Alpha-β-methylene-ADP alone has no effect, stimulatory or inhibitory, on EPEC growth, but it did completely reverse the growth-promoting effects of 5′-AMP (Fig. 6a and b, right-hand bars). Alpha-β-methylene-ADP did not block the enhanced growth seen with adenosine (not shown). Similarly, 100 μM zinc acetate did not affect growth in minimal medium (Fig. 6c, left-hand bars) or the enhanced growth seen with adenosine (Fig. 6c, two middle bars), but zinc did abolish the enhancement of growth by AMP. E. coli 5′-nucleotidase is susceptible to inhibition by α,β-methylene-ADP and zinc; in fact, the crystal structure of the E. coli enzyme was solved with α,β-methylene-ADP bound in the catalytic pocket [32]. Bacterial utilization of extracellular nucleotides is usually by dephosphorylation followed by uptake of the nucleoside (in this case adenosine) and inorganic phosphate [33]. Although we began studying α,β-methylene-ADP and zinc as inhibitors of the host cell enzyme, CD73, a side effect (or side benefit) of these compounds may be inhibition of bacterial 5′-nucleotidase activity as well, an effect that is able to block the nutritional benefits that E. coli bacteria derive from extracellular AMP (Fig. 6).
Fig. 6Effect of 5′-nucleotidase inhibitors on growth of EPEC strain E2348/69 in minimal medium supplemented with 30 μM adenosine or AMP. Minimal medium here refers to M9 salts, 2% casamino acids, and 2 mM glucose. Growth was at 37°C with 300 rpm shaking after a 1: 2,000 dilution from an overnight culture in LB medium. a, c Culture turbidity by spectrophotometric absorbance at 600 nm. b Bacterial protein content. a, b Growth-enhancing effects of AMP are blocked by α,β-methylene-ADP. c Growth-enhancing effects of AMP are blocked by zinc acetate; *significantly decreased compared to AMP alone (p < 0.05)
Discussion
We became interested in ecto-5′-nucleotidase (CD73) because of its likely role in the generation of adenosine from adenine nucleotides released during EPEC infection. This host enzyme has not received a great deal of scrutiny in the context of microbial pathogenesis, and when it was studied its role was felt to be in the setting of a polymorphonuclear neutrophil (PMN) response, since activated PMNs release 5′-AMP during migration into an infected area [4, 34]. EPEC behaves clinically as a noninvasive pathogen and does not trigger an influx of fecal leukocytes, but nevertheless triggers ATP release from the host. Therefore our results suggest that ecto-5′-nucleotidase plays a role in the response to a broader range of pathogens, including noninvasive enteric pathogens, than was previously thought.
One important finding of our study is that EPEC infection triggers a release of ecto-5′-nucleotidase from a cell-bound to a soluble, free form (Figs. 2 and 3). The significance of this finding is not clear at present but there are several possibilities to be considered. First, studies by other investigators have shown that cleavage of ecto-5′-nucleotidase from its GPI lipid anchor increases its activity significantly via an increase in the Vmax [27]. This could result in an increase in the activity of 5′-nucleotidase present in an EPEC-infected portion of the intestine, which could be an advantage to EPEC (see Fig. 6 and additional discussion below). The observed increase in transcription of ecto-5′-nucleotidase in response to EPEC infection (Fig. 3f and Table 1), if accompanied by a similar increase in protein synthesis and surface expression of the enzyme, would replace ecto-5′-nucleotidase lost due to cleavage of the lipid anchor and also contribute to an increase in total activity. Third, release of nucleotidase from its lipid anchor would change its distribution from a mucosal location to a soluble form in the intestinal lumen, where it could possibly be more accessible to substrate. This could be important in the human or animal EPEC infection because expression of ecto-5′-nucleotidase is highest in the intestinal crypts [35], whereas EPEC adheres to villus tips as well as crypts. Last, after many days of infection the mucosa could be stripped of ecto-5′-nucleotidase activity, leading to malabsorption of nucleotides in the diet. Indeed, malabsorption of electrolytes has been demonstrated after 7 days of infection in a rabbit model of EPEC infection [36]. In addition to ecto-5′-nucleotidase, several other important digestive enzymes have GPI lipid anchors, including alkaline phosphatase and aminopeptidase N (CD13) and these also could be liberated by EPEC-induced activation of PI-PLC. Another GPI-linked protein of interest in the intestinal tract is decay-accelerating factor (CD55), the cellular receptor for adhesion by diffuse adherent E. coli [37, 38] and which is also involved in internalization of Salmonella into vacuoles.
Several virulent pathogens have been found to secrete their own microbial ATPases or nucleotidases into the extracellular medium, including Entamoeba histolytica [39], Trichinella spiralis [40], and Vibrio cholerae [41]. Researchers have speculated that secreted microbial nucleotidases may serve in protection against killing by host immune cells, acquisition of nutrients from the host, or dampening of host immune responses. EPEC does not appear to secrete nucleotidase activity under any conditions we have encountered, but may achieve the same goal by triggering the release of 5′-nucleotidase from the host cell.
Ecto-5′-nucleotidase acts on nucleoside monophosphates, with 5′-AMP as the preferred substrate, and is inactive toward ATP. Therefore, the generation of AMP from ATP requires another enzyme or enzymes. CD39, or ectonucleoside triphosphate diphosphohydrolase, is the major enzyme responsible for this conversion in the cardiovascular system and probably in the gastrointestinal tract as well [42, 43]. CD39 is also deserving of further study in the context of microbial pathogenesis.
One surprising finding of our study was that the espF EPEC mutant was not attenuated but in fact better than the wild-type EPEC in release of ecto-5′-nucleotidase (Fig. 3a). Although this mutant is defective in host cell killing, its phenotype has been instructive in many ways. For example, the ability of the espF mutant to trigger a robust ATP release led to us the discovery of a new pathway for ATP release from the host cell; this “second pathway” is not dependent on cell death but is dependent on the cystic fibrosis transmembrane regulator (CFTR) for ATP efflux [19]. The competence of the espF mutant in release of ATP and of ecto-5′-nucleotidase from the host cell may help explain why this mutant is only modestly attenuated compared to wild-type in some animal models of EPEC infection, such as Citrobacter rodentium in mice [44]. On the other hand, the sepZ/espZ mutant is attenuated in its ability to trigger ecto-5′-nucleotidase release (Fig. 3f), showing that type III secretion is necessary for PI-PLC activation and release of the enzyme from its lipid anchor.
The role of ecto-5′-nucleotidase in triggering EPEC-induced fluid secretion is highlighted in the Ussing chamber studies shown in Fig. 5. Even after AMP has induced a chloride secretory response, addition of the nucleotidase inhibitor α,β-methylene-ADP is able to reverse the short-circuit current in a manner similar to that of an adenosine receptor antagonist, theophylline (compare Fig. 5a and c). Tracings such as those in Fig. 5 seem to indicate that to sustain a secretory response there must be on ongoing production of adenosine from AMP. In other words, there seems to be a “sink” for depletion of adenosine from the extracellular medium, either by cellular reuptake or by adenosine deaminase. The rapid turnover of adenosine implied in these experiments suggested that inhibitors of ecto-5′-nucleotidase might be able to block EPEC-induced fluid secretion in animal models of infection.
In discussions of zinc on ecto-5′-nucleotidase it is worthwhile to note that zinc supplements have been shown to reduce the duration and severity of watery diarrhea in children in developing countries around the world [45–48]. In most of these trials the microbial etiology of the diarrhea was not carefully investigated, so it is unknown if zinc is particularly efficacious in diarrhea due to certain pathogens, such as EPEC, or if it beneficial “across the board” for multiple agents. Likewise, the mechanism by which zinc exerts its beneficial effects has not been carefully studied in the field trials. In earlier reports it was assumed that zinc was acting by correcting a zinc deficiency, but this assumption has been called into question by the finding that zinc supplements have a beneficial effect on diarrhea even in children with normal serum zinc levels at the outset [49]. Kelleher et al. showed that zinc provided some added benefit beyond a probiotic (Lactobacillus rhamnosus) in rhesus monkeys infected with the human EPEC strain E2348/69; the monkeys studied were not zinc deficient [50]. Inhibition of ecto-5′-nucleotidase or K+ channels [51] by zinc could slow diarrhea but this should be considered a pharmacological effect of the mineral rather than repletion of a deficiency. Inhibition of 5′-nucleotidase also blocks the growth-enhancing effects of AMP on EPEC bacteria (Fig. 6); since this is a direct effect on bacterial cells this action of zinc would not depend on the nutritional status of the host. The effects of zinc on EPEC infection and on diarrheal diseases in non-zinc-deficient children deserve further study.

adenosine
zinc
extracellular nucleotides
diarrheal disease


Plasticity Within the Obligatory Folding Nucleus of an Immunoglobulin-like Domain

A number of β-sandwich immunoglobulin-like domains have been shown to fold using a set of structurally equivalent residues that form a folding nucleus deep within the core of the protein. Formation of this nucleus is sufficient to establish the complex Greek key topology of the native state. These nucleating residues are highly conserved within the immunoglobulin superfamily, but are less well conserved in the fibronectin type III (fnIII) superfamily, where the requirement is simply to have four interacting hydrophobic residues. However, there are rare examples where this nucleation pattern is absent. In this study, we have investigated the folding of a novel member of the fnIII superfamily whose nucleus appears to lack one of the four buried hydrophobic residues. We show that the folding mechanism is unaltered, but the folding nucleus has moved within the hydrophobic core.
Introduction
Studies of structurally related proteins have clearly indicated that for
many proteins the folding mechanism is determined primarily by the native state
topology.1,2 This is evident from comparative Φ-value analyses of proteins
that share similar folds but are very different in sequence. Such studies
include representatives from all protein classes; all-α proteins,3–9 all-β proteins,10–22 and mixed α/β proteins.23–28 The folding mechanisms of these proteins range from purely
hierarchical, where secondary structural elements form before any tertiary
structure, to pure nucleation-condensation, where secondary and tertiary
structure form concomitantly.29 A study of representative members of the homeodomain superfamily
family has suggested that their folding mechanisms are dependent on inherent
secondary structural propensity, and the authors propose that all folding
mechanisms are in fact variations of the same theme:3 as the propensity for forming secondary structures decreases,
the folding mechanism shifts from pure hierarchical to polarized transition
states, and ultimately to the classical nucleation-condensation mechanism first
shown for CI2.30 Separate investigations have suggested that a folding nucleus
consists of obligatory and critical components: the specific interactions
necessary to establish the correct topology form first, followed by a subset of
surrounding residues that provide the critical stabilising
interactions.31–33
As part of the “fold approach” we have studied the folding of a number of
proteins with an Ig-like fold,16–18,34–36 all of which are composed of two anti-parallel β-sheets packed
against each other. The deep hydrophobic core is always formed from the packing
of the four central B, C, E and F β-strands,37 but the number and position of the edge strands varies between
the superfamilies.38 Although proteins in different superfamilies share the same
fold, they are apparently unrelated in sequence and are found in proteins with a
wide variety of functions. The stabilities of the Ig-like proteins studied to
date range from about 1 kcal mol− 1 to 9 kcal
mol− 1, and the folding rate
constants vary by six orders of magnitude. However, there is a correlation
between folding rate and thermodynamic stability, which suggests that the
interactions that are critical in stabilising the fold also govern the folding
process.34
All members of the fold studied to date fold via a
nucleation-condensation mechanism where the obligatory folding nucleus comprises
a set of structurally equivalent buried hydrophobic residues in the B, C, E and
F-strands that form a “ring” of interactions in the core (Figure 1). Early
packing of the residues, which are distant in sequence, ensures formation of the
correct native state topology.35,36 The critical nucleus surrounds this obligatory nucleus, but the
degree of structure formation varies between different proteins.
The residues that form the obligatory folding nucleus are highly conserved
within immunoglobulin domains but are conserved only in terms of residue type in
fibronectin type III (fnIII) domains. However, there are rare examples of
proteins that appear to have a disparate nucleation pattern. Here, we have
identified a fnIII domain in which one of the hydrophobic residues in the
conserved folding nucleus has been replaced by a surface polar residue, and we
ask how the folding mechanism has been affected. An extensive protein
engineering Φ-value analysis reveals that the folding mechanism is unaltered,
but that a spatially different set of core residues is used to form the
obligatory folding nucleus, where interactions within each sheet establish the
correct hydrogen bond registry between the core β-strands. Subsequent
interactions between two such pairs are able to bring the β-sheets together and
set up the complex Greek key topology.
Results
Residue conservation within the folding nucleus of fnIII
domains
A non-redundant multiple sequence alignment from Pfam was used to
analyse the residue conservation in the putative folding nucleus of fnIII
domains.39 These nucleation positions were identified through
comparison with the third fnIII domain from human tenascin (TNfn3), which
has been studied extensively in our laboratory. In most fnIII domains (73%),
all four residues in the proposed folding nucleus positions are hydrophobic.
Further analysis reveals that where there is a polar residue in one of these
folding positions, it is almost invariably in the C or E strand, and these
hydrophilic residues are usually arginine or lysine. These can act as
hydrophobic residues, since the long aliphatic side-chains can traverse the
core and allow the charged terminus to reside on the surface of the
protein.40 Small hydrophilic residues, such as asparagine or aspartate,
are found very rarely (only in ∼ 3% of all cases).
Most fnIII domains (65%) have a single aromatic residue in the proposed
folding nucleus. Analysis of the distribution of aromatic residues in the
four obligatory folding nucleus positions shows clearly that aromatic amino
acids are located preferably within the C–F sheet (Figure 2).
Furthermore, the type of aromatic residue present is affected by the solvent
accessibility of the β-strand: the C-strand position is partly
solvent-accessible, and hence the majority of aromatic residues occurring
within this strand are tyrosine (thereby allowing hydrogen bonding of the
hydroxyl group with solvent molecules). In contrast, the F-strand position
is deep within the core and phenylalanine is almost always the aromatic
residue of choice. Approximately 20% of the sequences have more than one
aromatic residue in the folding nucleus, and again these residues are almost
exclusively located in the C–F sheet (86% of all such sequences). This
asymmetry is likely caused by the presence of an adjacent conserved
tryptophan in the B-strand that is essential for stability but not involved
in the folding nucleus.16,18 Interestingly, about 15% of the fnIII domains appear to
fold without any aromatic residues at the supposed folding positions,
suggesting that a large side-chain is not crucial for the formation of the
obligatory nucleus.
Selection of CAfn2 as a candidate
All known fnIII structures were surveyed to find a candidate protein
that was missing a hydrophobic residue at one of the four putative folding
positions. Only one candidate protein was identified, the second fnIII
domain in chitin A1 from Bacillus circulans (CAfn2),
which has a surface-exposed asparagine in the putative nucleus position in
the C-strand. It has an aromatic residue in the F-strand folding position
(Phe66).41
In this work we intended to compare the folding of CAfn2 with TNfn3, as
this is the most extensively studied “typical” fnIII domain. The structure
of CAfn2 was superimposed on the structure of TNfn3 to reveal a RMSD of only
1.7 Å over all structurally equivalent positions (69 residues), even though
the two proteins have just 13% sequence identity (Supplementary Data Figure 1). The major differences are
restricted to the loops and turns, which are different in length in the two
proteins. CAfn2 has the same Greek key topology as all other fnIII domains,
(Figure 1), and possesses both
the highly conserved Trp residue in the B-strand and the conserved tyrosine
corner motif (Supplementary Figure
1).42 A comparison of the structures of CAfn2 and TNfn3 reveals
that the lengths of the highly conserved EF-loop and the AB-turn are
identical in these proteins, whereas the rest of the loops show some
variation. Most notably the C and C′-strands are shorter in CAfn2 and are
joined by a short tight turn (Figures 1 and
3). The packing
interactions are almost identical in the two proteins, with the majority of
the contacts being made within the same sheet, and the interactions between
sheets occurring in “layers”.18 To help visualize the interactions that occur within the
proteins, all residue positions are described according to both the β-strand
and the core layer in which they reside (Figure
3).
Importantly, however, inspection of the supposed obligatory folding
nucleus of CAfn2 clearly shows that the C-strand residue, N40, does not pack
against the other putative nucleus residues (Figure 1). How, therefore, does this domain
fold?
Characterization of wild-type CAfn2
The equilibrium stabilities of wild-type CAfn2 and all its mutant
proteins were determined through the use of standard denaturation curves fit
to a two-state equation.43 From a number of repeated measurements of wild-type CAfn2
the free energy of unfolding (ΔGD–N)
was estimated to be 6.7( ± 0.3) kcal
mol− 1 at pH 5.0 and 25 °C.
Kinetic studies reveal a single unfolding phase, but two refolding
phases. Since the slower phase accounts for less than 15% of the total
amplitude, and is apparently independent of the concentration of denaturant,
it was attributed to proline isomerisation. Both arms of the chevron plot
are linear (Figure
4), and the
βT for wild-type CAfn2 is estimated to be 0.56. This is
similar to that of TNfn3,44 indicating that the two transition state structures are of a
similar compactness.
Effect of mutations
Using the Φ-value analysis of TNfn3 as a basis, a total of 23
non-disruptive mutations were made throughout the CAfn2 protein. Where
possible, all mutations were conservative deletions.45 Contacts lost upon mutation are given in Table 1. The
difference in free energy between wild-type and mutant
(ΔΔGD–N) was calculated using an
average m-value,
<m>, of 1.15(± 0.02)
kcal mol− 1 M− 1. It has been shown that m-values are
hard to determine with accuracy (the range of
m-values observed is typical for that observed in
most large-scale protein engineering studies) so that use of an average
m-value reduces the error in
ΔΔG.46 Most mutations are destabilizing but
ΔΔGD–N values range between
− 1.5 kcal mol− 1
and + 6.0 kcal mol− 1.
Chevron plots for all mutants are shown in Figure 4, with the kinetic data given in Table 2.
Using these data, Φ-values were calculated from refolding data at 0 M
denaturant. Note that for three highly destabilized proteins (L22A, Y36L and
L58A) there were too few data points in the refolding arm to determine the
gradient (mkf) accurately. In these
cases an average mkf (1.06
M− 1) was used to fit the data.
Note that this has no effect on the final Φ-value determined at 0 M
denaturant. Only one mutant, V38A, has a folding
m-value that is significantly different from this
mean value.
Several mutant proteins exhibit roll-over in the unfolding arm, and
some mutants have increased mku
values. Such behavior in unfolding has several possible explanations. It has
been ascribed to “Hammond” behaviour, where there is a broad transition
state barrier,47 or to population of a high-energy intermediate.48 We do not have sufficient data to distinguish these two
possibilities and, since there is no roll-over in the wild-type protein, it
is not possible to determine Φ-values for the “late” transition state using
unfolding data. However, the model used has no effect on analysis of the
“early” transition state, at 0 M denaturant.49
Structure of the transition state
A number of positions in each β-strand of CAfn2 were probed using
Φ-value analysis. Φ is a measure of the extent of structure at a given
residue in the transition state (‡). A Φ-value of 1 indicates that the
interactions are fully formed in ‡, whereas a Φ-value of 0 indicates that
the structure is as unfolded in ‡ as in the denatured state. The precise
interpretation of fractional Φ-values is ambiguous but is usually taken to
mean that the residue is partly structured in ‡.50 However, it is generally accepted that, particularly when
comparing homologous proteins, the best approach is to look at patterns of
Φ-values rather than considering the absolute values of individual
residues.2
The CAfn2 Φ-values range from 0 to 0.5, indicating that none of the
positions analyzed is completely structured at the transition state
(Table 2). In general, the
Φ-values in the A and G-strands are close to 0, while those in the central
B, C, C′, E and F β-strands are higher, and the Φ-values in these central
strands are higher in the central layers of the core than at the extremes
(Figure 3), as observed in
TNfn3,18 the tenth fnIII domain of fibronectin (FNfn10)16 and the titin immunoglobulin domain TI I27.17 The Φ-values were classified into low (Φ ≤ 0.2), medium (0.2 < Φ < 0.4) and high (Φ ≥ 0.4)
classes. These Φ-values are mapped onto the CAfn2 structure in Figure 5.
A and G-strands
All mutations in the A and G-strands gave low Φ-values, indicating
very little structure formation in these strands in the transition state
(Table 2; Figures 3 and
4). Both mutated sites in the
A-strand, L10A(A3) and S12A(A2), pack onto the neighbouring B-strand,
whilst inter-sheet interactions are formed with residues from the F and
G-strands (Table 1). The
ΔΔGD–N for the S12A(A2)
mutation is too low for a reliable Φ-value to be determined. The three
residues probed in the G-strand, S81(G4), V84(G3) and V86(G2), make
interactions mainly with residues from the A and F-strands
(Table 1).
Strands B, C, E and F
All core positions in CAfn2 were mutated, with the exception of W24
in the B-strand. The distribution of Φ-values reveals that the
hydrophobic core is only partially formed in the transition state
(Table 2; Figures 3 and
4). The highest values occur
at positions V38(C4) and I55(E3), with moderate Φ-values at four other
positions: I20(B2), L22(B3), V68(F4) and A70(F5).
C′-strand
The C′-strand is connected by two short loops to the central C and
E-strands. Three of the four mutations within this strand, L44A(C′3),
T46A(C′4), and V48A(C′5), have high Φ-values (Table 2; Figures 3 and
4). T46 and V48 interact
mainly with the buried residues from the C and E-strands, whereas the
L44 contacts are limited to residues within the C and C′-strands
(Table 1).
Discussion
CAfn2 folds by a nucleation-condensation mechanism
There has been much discussion of folding mechanisms in recent years.
The two “extremes” are represented by the framework model, where local
secondary structure forms before tertiary structure, and
nucleation-condensation, where secondary structure and tertiary structure
form concomitantly. Such extremes have distinct patterns of Φ-values. In the
framework model, Φ-values will fall into two groups, one set close to 1 and
the other close to 0. This has been termed a polarised transition state. In
a nucleation-condensation mechanism, the transition state structure will be
more diffuse, involving most of the protein, and Φ-values will all be
generally between 1 and zero. Furthermore, in the nucleation condensation
mechanism, the pattern of Φ-values is generally distinctive, with a subset
of residues having slightly higher Φ-values, with Φ-values gradually
becoming lower as structure condenses around the early “nucleus”. The
pattern of Φ-values shows CAfn2 to have a diffuse nucleus, with two-thirds
of the residues having Φ-values between 0.10 and 0.45. Moreover, these are
arranged in the structure as one would predict from a
nucleation-condensation pattern, with higher Φ-values at the centre of the
core, becoming lower towards the edges of the molecule. This suggests
strongly that CAfn2 folds, as do other Ig-like proteins,
via a nucleation-condensation folding mechanism.
However, again like other Ig-like proteins, there is a significant number of
residues, in the peripheral A and G-strands, and in loops that have Φ-values
close to 0, suggesting that in the final stage of folding these peripheral
strands and loops pack onto the central region of the protein.
Identification of the obligate (embryonic) folding nucleus
Oliveberg and co-workers have suggested that residues that constitute
the transition state for folding in a nucleation-condensation mechanism
might be divided into two sets.31,32 The first set of residues make up the “embryonic” or
obligate folding nucleus, defined as the set of primary contacts that are
obliged to form to establish the topology of the protein. The second set is
the residues that pack onto this embryonic nucleus forming the “critical
contact layer”, providing sufficient interactions to drive the folding
process downhill. Note that residues that form the embryonic nucleus have to
form a network of contacts that establish the topology of the protein, but
that residues in the critical contact layer may contribute significantly
towards stabilising the transition state for folding. Identifying the most
likely obligate folding nucleus from a pattern of Φ-values is non-trivial,
especially for complex Greek key structures. This has been discussed in
detail.18 In summary, it is not possible simply to “pick” residues
with the highest Φ-values as being those that form the folding nucleus: one
also has to consider the packing of the residues. In TNfn3, as is observed
here for CAfn2, residues in the B-strand have generally low Φ-values
compared to the Φ-values in the other central β-strands. This does not
necessarily mean that the residues in the B-strand are less important for
folding. Residue L22(B3) in CAfn2, for example, forms about half of its
inter-strand contacts with residues in the A and G-strands, which have
Φ-values of ∼ 0. Thus, the contacts with the C (V38), E
(A53 and I55) and F (F66 and V68) strands must be more formed than the
moderate Φ-value would indicate. Similarly, the high Φ-values in the
C′-strand probably reflect the fact that the residues in this strand make
the vast majority of their tertiary contacts with residues in the C and
E-strands, which are themselves partially formed. In TNfn3 it was suggested
that this C′-strand is “obliged” to fold when the adjoining C and E-strands
pack together. Thus, we would now, following the nomenclature suggested by
the Oliveberg model, assign residues in the C′-strand to the critical
contact layer and not to the obligatory embryonic nucleus.
For TNfn3, the residues with the highest Φ-value in the B, C, E and
F-strands were initially chosen as putative nucleus residues. Examination of
the structure showed that these residues are all found in the same core
layer, and that they pack to form a “ring” of interactions in the core of
the protein. It was suggested that this “obligatory” nucleus alone was
sufficient to establish the topology of the native protein. This picture of
the folding transition state was confirmed by subsequent restrained
molecular dynamics simulations.36 A similar method has been used to identify the folding
nucleus in a structurally related immunoglobulin domain.17,35
Using the same strategy, the Φ-value pattern of CAfn2 was investigated
to identify the putative obligate folding nucleus, a set of residues with
significant Φ-values that interact such that these interactions are
sufficient to establish the topology of the protein. The layer that contains
residues with consistently high Φ-values in the B and E-strands is layer 3
(I55 and L22), as in TNfn3. However, for the C and F-strands, the layer with
the highest Φ-values is layer 4 (V38 and V68). Examination of the structure
of CAfn2 shows that although residues L22 and I55, and V38 and V68 sit in
different core layers, these four residues are still able to pack together
in the centre of the core to form a ring of contacts (Figure 6). We
suggest that the residues surrounding the obligate nucleus, residues in the
C′-strand and more peripheral residues in the B, C E and F-strands, pack
onto these obligate nucleus residues and, together, form the critical
contact layer required to stabilise the transition state structure
sufficiently to drive folding.
There is a caveat we should make. We note that in CAfn2 it is more
difficult to select which residues are likely to form part of the obligate
nucleus than it was in TNfn3. Consider the B-strand. I20(B2) exhibits a
Φ-value that is slightly lower, but is within error of L22(B3). However, I20
forms no contacts with the nucleating residues from the opposite sheet,
(V38(C4) and V68(E4)), suggesting that it does not form part of the
obligatory nucleus that establishes the topology of the molecule.
Furthermore, as was the case in TNfn3, we were unable to determine a Φ-value
for the highly conserved Trp in the position B4. Simulations confirmed for
TNfn3 that this Trp residue had a low Φ-value (as we had inferred from the
pattern of Φ-values surrounding the Trp residue). Trp 24 makes 150
side-chain–side-chain contacts in CAfn2, and 65% of these contacts are with
residues that have Φ-values that are (or are predicted to be) low (Φ ∼ 0.15, 52 contacts) or zero (46 contacts).
Less than one-third of the contacts made by Trp24 are with residues in the
putative obligatory nucleus (with L22, V38 and V68) and no contact is made
with I55. Thus, we tentatively propose that if Trp24 does have a role in the
folding nucleus, it is more likely to be in the critical layer than in the
topology-defining obligate nucleus. Also consider residue A70 in the
F-strand in position F5. The Φ-value for this residue is very slightly
higher than that for V68 in layer F4. However, Ala to Gly mutation must be
considered to be non-conservative and, furthermore, Ala70 makes no contact
within the proposed obligate nucleus i.e. it cannot have a role in
establishing the Greek key topology.
Comparison of the transition states of CAfn2 and TNfn3
The Φ-values for TNfn3 are generally higher than those in CAfn2,
ranging from 0 to 0.6;18 however, the pattern of Φ-values is similar. For both
proteins, the mutational results can be separated into two classes. The
first group consists of residues in the central β-strands, which show
significant formation of structure in the transition state. The second group
consists of mutations probing the terminal A and G-strands, and residues
from the extremities of the central strands. These two populations are
clearly observed in a Brønsted plot (Figure
7).
Nevertheless, there are important differences between the two domains,
which are apparent in the pattern of Φ-values (Figure 8). In the
C–F-sheet, the highest Φ-values in TNfn3 are found in core layer 3 (V70,
0.54; Y36, 0.53), with the Φ-values in layer 4 being significantly lower
(L72, 0.29; L34, 0.35). However, in CAfn2 the Φ-values in layer 3 are very
low (F66, 0.07; N40, 0.01), whereas the Φ-values in layer 4 (V68, 0.25; V38,
0.40) are significantly higher (Figures 3
and 8). This indicates that the absence of a buried
hydrophobic residue in position C3 has forced the obligate folding nucleus
of CAfn2 to “migrate down” one layer within the core (Figure 6). Perhaps unexpectedly, a
corresponding “downwards migration” has not occurred in the B–E-sheet; (even
if Trp24 was important, the Φ-value for A53(E4) is unambiguously low
(0.14)). Such migration is not necessary; analysis of the CAfn2 structure
shows clearly that residues L22(B3) and I55(E3) form significant
interactions with V68(F4) and V38(C4) in the opposite sheet. Thus, these
inter-sheet interactions would be sufficient to establish the Greek key
topology.
Further support for this migration hypothesis comes from Φ-values in
the EF-loop. TNfn3 exhibits moderate Φ-values in this loop, (Y68(F2), 0.42;
L62(E2), 0.33), which indicates that it is significantly structured in the
transition state. It was argued that this loop is “obliged” to be structured
in the transition state of TNfn3 to allow for formation of the adjacent
folding nucleus: the more distant BC-loop exhibits lower values. However, in
CAfn2 the folding nucleus has shifted away from the EF-loop (Figure 6) and consequently it is less
restrained within the transition state (Φ-values for Y64(F2) and L58(E2) are
0.04 and 0.16, respectively).
Both TNfn3 and CAfn2 display high Φ-values in the C′-strand. We suggest
that these residues are not involved in the obligatory folding nucleus, but
result from short CE-loops that force the C′-strand to pack as the nucleus
forms;18 thus, these residues form part of the critical contact
layer. In CD2d1, an immunoglobulin Ig variable domain, the nucleating C and
E-strands are joined by a much longer loop comprising three β-strands, C′,
Cʺ and D. In this case these strands do not pack until late in
folding.18,19
In summary, for both proteins we observe the formation of a specific
nucleus in the core of the protein involving formation of long-range
tertiary contacts between a single residue from each of the B, C, E and
F-strands. Formation of this “obligate” nucleus establishes the topology of
the protein. Other residues pack around this obligate nucleus to form the
critical contact layer until sufficient contacts have formed to surmount the
free-energy barrier. This is typical of a nucleation-condensation folding
mechanism. The peripheral strands and the loops pack late, mainly after the
rate-limiting step for folding.
Conclusion: plasticity within the obligatory folding nucleus
in Ig-like domains
Unlike other classes of proteins, such as the homeodomain proteins, all
Ig-like proteins appear to fold by the same, nucleation condensation
mechanism. The obligate nucleus is defined by the interactions that are
necessary to establish the complex Greek key β-sheet topology of the native
state. Previous biophysical studies of members of the Ig-like fold have
shown that this folding nucleus always comprises a ring of interacting
residues within the hydrophobic core: one residue from each of the B, C, E
and F-strands. Whereas the obligatory nucleus in the immunoglobulin
superfamily proteins is highly conserved and is based around the invariant
tryptophan located within the C-strand, members of the fnIII superfamily
show more variability. Instead of restricting a particular structural
position to a specific amino acid, each position simply needs to possess a
hydrophobic residue. Here, we have shown that the fnIII nucleus is more
flexible still, and that when this pattern of residue conservation is lost
upon mutation, fnIII proteins can “migrate” the folding nucleus, thereby
revealing plasticity in the early stages of the folding process, while
retaining the same folding mechanism.
Such plasticity in the folding of Ig-like proteins has been observed
previously; the Ig domain TI I27 has been shown to fold by alternative,
parallel pathways.51 Although the wild-type protein folds only through one
pathway under physiological conditions, extremes of temperature and
denaturant, or mutations within its obligate folding nucleus also result in
a switch of folding pathway. Lindberg and Oliveberg have suggested recently
that a “malleable” protein folding energy landscape will allow proteins to
retain efficient folding during the course of evolution, even though the
finer details of the folding pathway are dependent on individual
sequence.52 It is possible that the ability of these Ig-like domains to
alter their folding pathway on mutation has contributed to the success of
this fold, and has contributed to its abundance in the proteome (over
40,000 Ig-like domains are listed in the current Pfam database).
Materials and Methods
Protein expression and purification
The fibronectin type III domain used in this work consists of 88
residues (SwissProt P20533, residues 559–646, PDB 1K85) of the
Bacillus circulans chitinase A1. The synthetic
gene was produced using overlapping primers and standard PCR techniques, and
was inserted into a modified version of pRSETA vector (Invitrogen)
containing an N-terminal His-tag followed by a thrombin cleavage site.
Site-directed mutagenesis was performed using the QuikChange Kit
(Stratagene). The identity of wild-type and mutants was confirmed by DNA
sequencing.
Protein expression was carried out in Escherichia
coli C41 cells.53 Transformed cells were grown to an absorbance at 600 nm of
0.6 at 37 °C before induction with IPTG and growth overnight at 28 °C. The
cells were harvested and lysed by sonication. The soluble fraction was bound
to Ni2+-agarose resin, washed several times to remove
weakly bound proteins, and eluted from the Ni2+-agarose
resin in a high concentration of imidazole. After dialysis to remove the
imidazole, the proteins were cleaved overnight with thrombin. Uncleaved
protein and remaining His-tag were removed by using small amounts of
Ni2+-resin before further purification by
gel-filtration chromatography using a Pharmacia Biotech Superdex 75 column.
When not used immediately, proteins were flash-frozen and stored at − 80 °C.
Equilibrium measurements
The stability of the CAfn2 wild-type and mutant proteins was determined
by equilibrium urea denaturation in 50 mM sodium acetate buffer, pH 5.0
(15 mM HOAc, 35 mM NaOAc) at 25 °C. The solutions were left to equilibrate
at 25 °C for at least 2 h before measurements were recorded. All experiments
were carried out in thermostatted cuvettes at 25 °C. The experiments used an
excitation wavelength of 280 nm, and an emission wavelength of 360 nm. Data
were fit to an equation describing a two-state transition.43
Change of free energy on mutation
The change of free energy on mutation,
ΔΔGD–N, was determined using
equation (1):54Where [urea]50% is the concentration of urea
at which 50% of the protein is unfolded for wild-type (wt) and mutant (mut)
proteins, and <m> is the mean
m-value determined from all measurements on
wild-type and mutant proteins.
Kinetic measurements
All kinetic experiments were done using an Applied Photophysics
stopped-flow fluorimeter. The excitation wavelength was 280 nm and the
emission was monitored at wavelengths > 320 nm. All
experiments were carried out in 50 mM sodium acetate buffer (pH 5.0) at
25 °C. The final concentration of all proteins was 1 μM. Refolding rates at
0 M denaturant were determined using CAfn2 unfolded at pH 12.4 as
described.18 Between three and five traces were averaged for each
concentration of denaturant. The refolding data were fit to an equation
using a single-exponential term. An average refolding
m-value of 1.06 M− 1 was used for mutations L22A, Y36L and L58A. Fitting data to an
equation with two exponentials did not improve the residuals. The unfolding
data were fit to an equation describing a single-exponential process with
curvature.
Φ-Value analysis
The Φ-value for folding was determined using equation (2):54.where ΔΔGD–‡ is the
change in the difference in free energy between D and the transition state
(‡) upon mutation and calculated from refolding data as follows:where kf and
kf′ are refolding rate constants
for wild-type and mutant proteins (at 0 M urea), respectively.

cafn2, the second fniii domain of chitin a1 from bacillus circulans

tnfn3, the third fniii domain of human fibronectin


Thoracic cord compression caused by disk herniation in Scheuermann’s disease

We present the case of a 14-year-old male with Scheuermann’s disease and significant neurological deficit due to thoracic disk herniation at the apex of kyphosis. He was treated with an anterior decompression, anterior and posterior fusion in the same setting using plate, cage and a segmental instrumentation system. The patient had an excellent outcome with complete neurological recovery.
Introduction
Scheuermann’s disease (juvenile kyphosis dorsalis) is a structural kyphosis of the thoracic spine initially described by Scheuermann in 1921 [19]. It occurs commonly in adolescents (0.4–8.3% of the general population) and in most cases is characterized by minimal deformity and few clinical symptoms. Scheuermann’s disease is rarely associated with neurological complications [5, 20].
The purpose of the present paper is to present the case of a patient with Scheuermann’s disease and severe neurological deficit due to a thoracic disc hernia at the level of kyphos, to review the literature and compare with previously published similar cases.
Case report
A 14-year-old male was admitted with a severe spastic paraparesis. He had gradually developed a gait disorder and bilateral numbness of lower limbs in the past 5 months. During that period his symptoms deteriorated rapidly. He was unable to walk and a week before his admission he developed spastic paraplegia. There was no history of trauma.
Clinical examination revealed spastic paraparesis below T9. The superficial sensation was diminished below the T9 dermatome. He had a muscle strength of grade II according to the Medical Research Council (MRC) scale. A barely detectable contraction could be seen below the knees by palpation over the muscles (M1 on the MRC scale). Plantar responses (Babinski sign) were present on both sides. Knee and ankle reflexes were brisk bilaterally, clonus was present in both ankles as well. Patient had hypalgesia and decreased temperature sensation. Light touch and pin prick sensation were diminished over both lower limbs and absent from the knees downward. Rectal tone and sensation while urinating were decreased.
All laboratory findings were normal including an investigation for systemic diseases. Plain radiographs of the spine in standing position revealed anterior wedging of more than 5° of several adjacent vertebrae (T7:12°, T8:14°, T9:17°, T10:15°) at the apex of the kyphosis and vertebral endplate irregularities. The thoracic kyphosis from T5 to T12 measured 66° (Fig. 1). These findings are in agreement with the criteria for diagnosis of Scheuermann’s disease. A magnetic resonance imaging scan revealed thoracic disc heniation with spinal cord compression at T8–T9 level (apex of the kyphosis). The intervertebral spaces from T6 to T10 were also very narrow and the vertebral bodies had an anterior wedging (Figs. 2, 3).
Fig. 1a, b Anteroposterior and lateral radiographs of the thoracic spine in standing position, at the time of admission. The thoracic kyphosis from T5 to T12 is 66°Fig. 2a, b Magnetic resonance imaging scan revealed thoracic disc herniation with spinal cord compression at T8–T9 level (apex of the kyphosis)Fig. 3Three-dimensional reconstruction computed tomography of the thoracic spine preoperatively
Surgical treatment was decided based on the severity of the neurological deficit. A right seventh rib transthoracic approach to the spinal column was performed followed by decompression at T8–T9 level. A disc fragment was found and removed behind the thoracic body of T9. We performed an anterior fusion using plate, screws, an interbody titanium cage and also a posterior fusion from T6 to T12 using a double-rod multihook and transpedicular screws segmental instrumentation system. Bone grafts were applied between T8 and T9 and intertransversaly from T7 to T10 (Fig. 4).Fig. 4a, b Postoperative anteroposterior and lateral radiographs of the thoracic spine. Note the anterior and posterior spinal fusion
Postoperatively the patient had a surprisingly rapid improvement of the neurological deficit. Four weeks after the operation he could walk steadily and 2 months later his neurological examination was entirely normal.
At 2 years follow-up the patient had a normal gait, he was symptoms free and there was no increase of the spinal deformity.
Discussion
Scheuermann in 1921 [19] described a condition, which he called juvenile dorsal kyphosis, distinguishing it from the more common postural kyphosis. The etiology of Scheuermann’s disease is unknown. Scheuermann proposed that the kyphosis resulted from avascular necrosis of the ring apophysis of the vertebral body [19]. Schmorl suggested that the vertebral wedging was caused by herniation of disc material into the vertebral body (Schmorl’s nodes). According to Ippolito and Ponseti [10] a biochemical abnormality of the collagen and matrix of the vertebral endplate cartilage may be another contributing factor. Bradford et al. [6] claimed that osteoporosis may be responsible for the development of Scheuermann’s disease. Mechanical factors and repetitive trauma have been also considered to play a significant role in the appearance of the disease [20]. Case reports in monozygotic twins support the theory that there is also a genetic contribution [9]. In conclusion, the etiology of Scheuermann’s disease remains unclear and probably is multifactorial.
The criteria for the diagnosis of Scheuermann’s disease are: (1) more than 5° of wedging of at least three adjacent vertebrae at the apex of the kyphosis, (2) endplates irregularities and (3) a thoracic kyphosis of more than 45° [5]. Our patient had typical roentgenografic features of Scheuermann’s disease and his kyphosis was 66° from T5 to T12.
Neurological complications in Scheuermann’s disease are rare [2–4, 7, 11, 15, 18, 21, 24]. Three different types of neural compression have been reported: (1) extradural spinal cyst, (2) compression of the cord at the apex of the kyphos and (3) disk hernia at the apex of the kyphos [4]. To the best of our knowledge only 20 cases fall in the last category [2–4, 8, 12, 14, 17, 21–24]. Data of all these cases are presented in Table 1.
Table 1Cases of Scheuermann’s disease causing spinal cord compression as the result of thoracic disc herniationReferencesLevelAge/sexFindingsAppro achProcedureResults (follow up)Muller [14]T1040 MParaplegiaNot reportedNot complete recoveryVan Landingham [23]T7–T8–T917 MSpastic paraparesis, no sphincter dysfunctionPT7–T9 laminectomyComplete recovery (4 months)Roth et al. [17]T9–T1061 MSpastic paraparesisPT9–T10 laminectomyParaplegiaTurinese and Raven [22]T5–T7–T816 MSpastic paraparesis, sphincter dysfunctionBed rest, Minerva jacketComplete recovery Bradford and Garcia [4]T7–T816 MSpastic paraparesisPT7–T9 laminectomyResidual hypereflexia(120 months) Ryan and Taylor [18]T8–T918 MSpastic paraparesisTT8–T10 partial vertebrectomy, anterior spinal fusionSpastic paraparesis(36 months)Yablon et al. [24]T7–T829 MSpastic paraparesis, sphincter dysfunctionCT/TT7–T8 discectomy and fusionComplete recovery (14 months)Lesoin et al. [12] (six cases) T9–T1049 FMonoparesis (left)PT8–T10 laminectomyNot complete recovery (12 months)T8–T955 FSpastic paraparesisTT8–T9 discectomySmall motor deficit (8 months)T11–T1261 MMonoparesis (right)PPosterior spinal fusionNot complete recovery (10 months)T8–T927 MLower back pain, Babinski’s sign (right)PLT8–T9 discectomy, T7–T10 posterior spinal fusionComplete recovery (3 months)T8–T930 MLower back pain, slight motor deficit (left)PLT8–T9 discectomy, T8–T9 posterior spinal fusionComplete recovery (3 months)T8–T935 MSpastic paraparesisPLT8–T9 discectomy, T8–T9 posterior spinal fusionSmall motor deficit (3 months)Bohlman and Zdeblick [3] ( two cases)T11–T12T12–L138 FMonoparesis. Back and leg pain (for 16 years)CTT11–L1 discectomy and fusionComplete recovery (36 months)T8–T925 FBack and leg pain (for 7 years)TT8–T9 discectomy and fusionSignificant pain relief (26 months)Stambough et al. [21]T6–T7–T821 MSpastic paraparesisTT6–T8 discectomy, anterior spinal fusion Complete recovery (24 months)Bhojraj and Dandawate [2] (three cases)T11–T1216 MSpastic paraparesis, urinary hesitancyPT10–T12 laminectomy, posterior interbody fusion Complete recovery (36 months)T12–L124 MSpastic paraparesis, urinary hesitancyPT12–L1 laminectomy, posterior interbody fusionComplete recovery (24 months)T11–T1216 FSpastic paraparesisPT11–T12 laminectomy, posterior interbody fusionTransient worsening postoperatively, subsequently complete recovery (15 months)Chiu and Luk [8]T11–L235 FSpastic paraparesisTT11–L2 discectomy, spinal fusion Small motor deficit (24 months)This report (2005)T8–T914 MRapidly progressive spastic paraparesis, finally spastic paraplegiaTT8–T9 discectomy, anterior fusion, T6–T12 posterior fusionComplete recovery (25 months)M Male, F female, P posterior, T transthoracic, CT costotransversectomy, PL posterolateral
Despite the fact that this condition has a uniform sex distribution [15, 20], only 6 of all 20 cases were female. Males with Scheuermann’s disease are at greater risk for the development of spinal cord compression in the second decade of life [1, 15]. This is in agreement with our case (male, 14 years old). This difference between the sexes is probably due to the fact that males have more longitudinal spinal growth than females and their maximum growth in trunk length, as well as the maximum deformity of the kyphosis, occurs about 2 years later.
In all cases in which the degree of deformity was reported, the average angle was only 56.3°. The severity of the neurological complications had no obvious correlation with the magnitude of the deformity [11, 18]. In our case the deformity was 66°. Lonstein et al. [13] found that in 43 cases with neurological deficits secondary to spinal deformity (what ever the etiology) the average angle of kyphosis was 95°. He suggested that the greater the angle of kyphosis, the greater the risk of neurological impairment. Neurological complications are less likely to occur when the deformity is present over a large number of segments, instead of sharply angular deformities.
Disc ruptures tend to occur at the apex of the thoracic kyphosis [13]. The majority of them have occurred at T7–T8 or at T8–T9. This is also confirmed by our case (at T8–T9).
It is interesting that a relatively small disc herniation such as in our case produces such a major neurological deficit. Factors that may influence the onset of cord compromise are the tenuous vascularization and the relatively small canal diameter of the thoracic cord [16]. Additionally, in young patients the disc material is not degenerated and can act as a solid mass. Bradford and Garcia [4] suggested that the high incidence of Schmorl’s nodes in Scheuermann’s disease can prevent disc herniation by decompressing the disc space. In our case there were no Schmorl’s nodes either at this level or at any other level.
Surgical treatment is indicated in the cases of Scheuermann’s disease with acute cord compression and generally combines anterior release with posterior instrumentation and fusion. A posterior decompression is ineffective because the compressive force is acting from the front [11, 13, 15, 18]. Although some authors have advocated posterior laminectomy and decompression, Patterson and Arbit [16] showed that 45% of patients undergoing laminectomies for thoracic disc herniations either had no relief or deteriorated.
For the 20 reported cases with thoracic disc herniation and Scheuermann’s disease six patients were treated with anterior spinal fusion, four patients with laminectomy, four with posterior spinal fusion, three patients with interbody fusion and pedicular screw plate fixation and one patient was treated with discectomy without spinal fusion. Only one patient was treated conservatively with bed rest and Minerva jacket without any decompressive surgery [22]. In one case [14] the treatment was not reported. If a significant or progressive kyphosis, i.e. 55° or more exists, a combined posterior spinal fusion and instrumentation should be considered [18, 21]. Due to the age of the described patient and to the degree of kyphosis we performed discectomy, decompression, anterior and also posterior fusion using a double-rod multihook and transpedicular screws segmental instrumentation system.
The majority of the reported cases had good results. In 10 out of 20 cases the patients had complete recovery while only in one case the final outcome was paraplegia [17]. Our patient had an excellent result with complete recovery in 3 months. At the final follow-up 2 years postoperatively, he is neurologically entirely normal and the spinal fusion is solid.
This case is interesting because of the severity of the symptoms. The patient had a progressive bilateral spastic paraparesis and finally a spastic paraplegia. Spastic paraparesis was the most common symptom in all cases but neurological symptoms were not so severe to result in paraplegia. Only one case had paraplegia, as the described patient [14]. It is impressive that despite the significant neurological deficit there was a complete recovery. It seems that when symptomatic compression of the spinal cord occurs, surgery is the best option.
Conclusion
Thoracic disc herniation is an extremely rare complication of Scheuermann’s disease, resulting in cord compression and marked neurological deficit, most commonly in young patients. This rare form of spinal cord compression once demonstrated, should be treated as soon as possible with surgical decompression and stabilization of the spine anteroposteriorly.

scheuermann’s disease
spastic paraparesis
spinal cord compression
thoracic disc herniation
acute myelopathy