Periplasmically-exported lupanine hydroxylase undergoes transition from soluble to functional inclusion bodies in Escherichia coli

Archives of Biochemistry and Biophysics 484 (2009) 8–15

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Periplasmically-exported lupanine hydroxylase undergoes transition from soluble to functional inclusion bodies in Escherichia coli Pavlos Stampolidis, Naheed N. Kaderbhai, Mustak A. Kaderbhai * Institute of Biological Sciences, Cledwyn Building, Aberystwyth University, Aberystwyth, Ceredigion SY23 3DD, United Kingdom

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Article history: Received 3 December 2008 and in revised form 14 January 2009 Available online 23 January 2009 Keywords: Protein export Periplasmic space Post-translational modifications Amorphous protein aggregation Inclusion bodies Disulphide-bond formation LH Quinocytochrome c Pyrroloquinoline quinine

a b s t r a c t Pseudomonas lupanine hydroxylase is a periplasmic-localised, two domain quinocytochrome c enzyme. It requires numerous post-translocation modifications involving signal peptide processing, disulphide bridge formation and, heme linkage in the carboxy-terminal cytochrome c domain to eventually generate a Ca2+-bound quino-c hemoprotein that hydroxylates the plant alkaloid, lupanine. An exported, functional recombinant enzyme was generated in Escherichia coli by co-expression with cytochrome c maturation factors. Increased growth temperatures ranging from 18 to 30 °C gradually raised the enzyme production to a peak together with its concomitant aggregation as red solid particles, readily activatable in a fully functional form by mild chaotropic treatment. Here, we demonstrate that the exported lupanine hydroxylase undergoes a cascade transition from a soluble to ‘‘non-classical” inclusion body form when build-up in the periplasm exceeded a basal threshold concentration. These periplasmic aggregates were distinct from the non-secreted, signal-sequenceless counterpart that occurred as misfolded, non-functional concatamers in the form of classical inclusion bodies. We discuss our findings in the light of current models of how aggregation of lupanine hydroxylase arises in the periplasmic space. Ó 2009 Elsevier Inc. All rights reserved.

Introduction Many over-produced recombinant proteins in the artificial Escherichia coli factories accumulate as inclusion bodies. These aggregated proteins are misfolded or partially-folded intermediates clustered through inter- or intra-molecular interactions of solvent-exposed, hydrophobic polypeptide stretches [1]. Their formation is attributed to the inability of the bacterial cell factories to maintain protein quality control during over-expression or thermo-induction of heterologous proteins. Quality control can be as a result of (i) a lack of sufficient amount of the appropriate catalytic and molecular chaperoning machinery, e.g. trigger factor [2], DnaK-DnaJ-GrpE [3,4] and GroEL-ES or other heat-shock proteins [5], (ii) failure of the cognate co- or post-translational modifying enzymes [6] to operate in an orderly manner or to repair the misfolded structures [7]. Aggregation may also be potentiated by the high rate of thermally-driven vectorial protein discharge [8], coupled with the intensity of molecular crowding within the cells [9]. Failure to maintain precursors in an unfolded state for their localisation/translocation to extra-cytoplasmic compartments may also contribute to formation of protein clusters [10]. The ‘‘classical” inclusion bodies in the bacterial cytoplasm [11] are typically enriched in a single over-expressed species but, nevertheless, admixed with other common cellular components * Corresponding author. E-mail address: [email protected] (M.A. Kaderbhai). 0003-9861/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2009.01.017

[12,13]. Their formation is not attributed to the size of the expressed protein, usage of a fusion partner, subunit structure or degree of hydrophobicity [14,15]. Nevertheless, their significant resistance to proteolysis imparts an enhanced stability of the entrapped recombinant proteins [14,16]. The finding that some inclusion bodies contain enzymatically active recombinant proteins in various conformations [17] yet with preserved active sites has necessitated their differentiation into ‘‘classical” and ‘‘non-classical” types [18,1]. In the light of these developments, solubility has been discredited as the sole measure of biological activity since inclusion bodies are regarded as ‘‘dynamic reservoirs” consisting of a pool of both active and inactive enzyme [19] with a variety of conformations. While cytoplasmic inclusion bodies have been extensively characterised, there are comparatively few reports on the formation of recombinant protein aggregates in the periplasmic space [20]. The periplasmic space of E coli is a membrane-partitioned interface between the cell exterior and the cytoplasm. It comprises from 20% to 40% of the total cell volume [21,22] and holds significant biotechnological importance in the production of secreted recombinant proteins that require essential post-translocational modifications such as signal sequence processing [23], disulphide bond shuffling [24], covalent heme linkage [25,26], inter and intra-proteolysis [27,28] and prolyl cis–trans isomerisation [6]. Many of these post-translocational modifications and chaperoning functions have been attributed to Dsb-proteins, Skp, SurA, FkpA, DegP, Prc [27]. These activities maintain rigorous

P. Stampolidis et al. / Archives of Biochemistry and Biophysics 484 (2009) 8–15

quality control on the processing and refolding of the polypeptides threaded from the cytoplasm through the inner membrane into the periplasm [29]. A more controlled protein folding rate imposed by both the signal sequence and the polypeptide threading through the Sec-translocon [30] may be at least part of the reason why many periplasmically-targeted recombinant proteins are generally resistant to aggregation [31]. The fact that the periplasm has a lower protease activity than the cytoplasm makes it a more amenable location for secretion. The mode by which periplasmic inclusion bodies arise share some remarkable similarities to those of the cytoplasm in that their specific aggregation is induced in artificial cell factories programmed to operate at elevated growth temperatures, under environmental stress, at hyperosmolarity, in the presence of mutation-induced defective molecular folds and even insufficiency of modifying or chaperoning activities [32]; however their physical characteristics appear to be distinct from their cytoplasmic counterparts [33]. Fusion reporter and mutant protein chimera studies suggest that susceptible intermolecular interactions between the defective carrier are the driving force towards aggregation without affecting the activities of the adjacent functional passenger molecule within the aggregate [20]. Highly expressed or mutant proteins can accumulate as periplasmic inclusion bodies invoking stress-induced responses to macromolecular crowding, heat shock or insufficiency of chaperoning activities rendering the quality control system inoperative [34]. For example, thiol-containing recombinant proteins form concactamers by incorrect bridging of polymers [35]. Aggregation induced as a result of insufficiencies of the post-translocational processing activities has been shown to alleviated by over-expression of DegP, a protease chaperone that can clear the ‘dust-storm’ of aggregation [27]. The Cpx two-component system and the heat shock rE-mediated signalling are the two main distinct, but overlapping, signalling pathways that become operative during the periplasmic stress response by activating their selective proteases and foldase activities [29]. Presently, we have investigated the mode of aggregation of the periplasmically-secreted recombinant lupanine hydroxylase (LH) of a Pseudomonas species. This 72 kDa monomeric quino-hemoprotein is made of two domains and catalyses the first step of degradation of the alkaloid, lupanine [36]. The larger hydroxylase domain contains the cofactor pyrroloquinoline quinine (PQQ), a disulphide bond and Ca2+ in its active site. Initial dehydrogenation of lupanine results in the transfer of electrons to the adjacent Cterminal cytochrome c domain. Covalent heme linkage in the cytochrome c domain of the exported enzyme demands the constitutive co-expression of the entire complement of the cytochrome maturation factors (Ccm) A to H. Thus, successful periplasmic secretion of LH necessitates its translocation through the inner membrane along with a subset of complex post-translational modifications involving PQQ and covalent heme linkage along with disulphide bond formation. The need for these modifications in conjunction with the quantifiable export of a functional recombinant protein constituted a novel and valuable reporter system for investigating the mode of protein aggregation in the periplasmic space. Here we report that the fully post-translocationally modified, processed, and functional LH undergoes a concentration-dependent transition from a soluble to an aggregated state, readily recoverable by mild chaotropic in an active form. Our findings suggest a mechanism involving the formation of ‘‘non-classical” LH inclusion bodies which converges with the model proposed by GonzalezMontalban [19]. Moreover, in contrast to the periplasmic aggregates, the engineered signal-less, non-secreted counterpart, cytoplasmic-resident form was generated as an inactive, hemedeficient, misfolded, intermolecular disulphide-bonded concatamer in the form of ‘‘classical” inclusion bodies.

9

Experimental procedures Plasmids, culturing and sub-cellular fractionation Plasmid pEV-cLH, encoding a signal-less LH, was PCR-amplified using pEV-LH32 [26] as the template and the following primers: NdeI  Forward primer: 50 -CATATGTCGGCTAATAAGAATATATGGA EcoRV  Reverse primer: 50 -GATATCCTATTGCGCGCCCGTATCGCT The purified NdeI-EcoRV-cleaved, 2200 bp DNA was ligated downstream of the phoA promoter into the identically-cleaved pINKc vector. Of the several positive clones, an authentic cell line, verified by DNA sequencing, was used in this study. E. coli TB1 [F ara D(lac-proAB) rps /80d lacZDM15 thi hsdR17 rpsL (Strr)] harbouring pEV-LH32 and/or pEC86 were cultured in Luria broth (1% (w/v) Tryptone, 0.5% (w/v) yeast extract and 1% (w/v) NaCl containing 75 lg ampicillin/ml and/or 50 lg chloramphenicol/ml. Cultures grown to saturation (16 h at 37 °C), were applied as a 2% (v/v) inoculum for batch cultivation in the MOPS medium [37] with orbital agitation at 125 rev/min for 18 h. Periplasmic extracts were prepared by an osmotic shock procedure as described previously [30]. The residual cell pellet, resuspended in 50 mM Tris–HCl (pH 8) and 5 mM Na2EDTA, was lysed by soniciation and served as a source for cytoplasmic and membrane fractions. The membranes and the IAs1 were recovered as a sediment by centrifugation at 105,000g for 1 h. The IAs were separated from the membranes as a red solid sediment by centrifuging at 150,000g through 77% sucrose, 10 mM Tris–HCl (pH 8) and 1 mM EDTA (TE) for 18 h at 4 °C using a TST 41.14 Kontron swingout rotor. Isolation and reactivation of lupanine hydroxylase Aggregates, isolated from the membrane fraction of E. coli pEC86/pEV-LH32 or pEV-LH32 cells (1 L culture) were resuspended by gentle homogenization in 10 ml TE containing 8 M urea. After leaving on ice for 30 min they were centrifuged at 105,000g for 30 min at 22 °C. The supernatant was diluted with 90 ml TE and left to stand at room temperature (22 °C) for 30 min. Slurry of DEAE-cellulose (50 ml) in TE was added and the mixture was gently agitated for 30 min at 22 °C. The exchanger was removed by filtration and eluted with 250 ml TE containing 1 M NaCl. After overnight dialysis against TE (4 °C), the eluate was applied to a Q Sepharose FF column (6  2.5 cm) pre-equilibrated with TE. Following a 100 ml TE wash, a linear gradient of 0–0.5 M NaCl in TE eluted the LH. Enzyme activity was detected in column fractions by spectrophotometric assay after reactivating 100 ll portions pre-incubated with 50 lM PQQ and 5 mM CaCl2 for 30 min at 22 °C. Enriched-enzyme fractions were pooled and concentrated by pressure filtration (Amicon PM10 membrane). The pooled fractions were gel filtered by FPLC on a Superdex 75 16/60 column using 20 mM Tris–HCl (pH 8.0) at 4 °C. LH was finally purified by passage through a 10 ml bed volume of cellulose phosphate with a hemoprotein specific content of about 90%. The native enzyme was isolated from the Pseudomonas sp. as described previously [36].

1 Abbreviations used: IA, Insoluble aggregate; LH, Lupanine hydroxylase; PQQ, pyrroloquinoline quinine; TE, 10 mM Tris–HCl (pH 8), 1 mM EDTA.

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Protein analyses

Results and discussion

The protein content of biological samples was determined using the Coomassie Blue dye-binding procedure [38] employing bovine serum albumin as the standard. Unless stated otherwise, the proteins were separated using 12% polyacrylamide gel electrophoresis in the presence of SDS, using a discontinuous buffer system [39] with a loading of 10 lg protein in each lane. Samples were pretreated with equal volumes of SDS sample buffer containing 0.125 M Tris–HCl (pH 6.8 at 25 °C), 4% (w/v) SDS, 0.04% (w/v) bromophenol blue, 40% (w/v) glycerol and 50 mM dithiothreitol. After leaving for 30 min at room temperature, the samples were subjected to electrophoretic separation. The resolved proteins were detected by heme-staining [40] followed by Coomassie Blue R250 binding. LH activity was measured by spectrophotometric assay using horse heart cytochrome c as the electron acceptor [26]. A unit of enzyme activity is defined as an amount that converts 1 lmol of lupanine to hydroxylupanine under the standard assay conditions at 25 °C. Cytochrome c was estimated from the difference in a-peak absorption (at 550 nm) of the Na dithionite-reduced and oxidized protein using an absorption coefficient of 21.1 mM1 cm1 [41]. Alkaline phosphatase activity was assayed using the substrate p-nitrophenylphosphate (1 mM) as previously described [37]. LH solubility was estimated using polyethylene glycol excluded volume interaction to enforce protein precipitation by crowding, essentially as described by Stevenson and Hageman [42]. Purified enzyme at 12.5 mg in 0.5 ml 50 mM Tris–HCl, was vigorously mixed with polyethylene glycol 4000 (PEG 4000) ranging from 7.5% to 20% in a final 1 ml volume in 50 mM Tris–HCl, pH 8 (l = 50). After a 30 min incubation at room temperature, the mixture was centrifuged at 5000g for 45 min at 22 °C. The hemoprotein content in the aqueous phase was spectrophotometrically gauged by scanning the absorbance at 280 nm and 415 nm (oxidised Soret peak) against appropriate blanks as described below. The potential maximum solubility of LH at zero PEG 4000 concentration was determined by graphical extrapolation. The second conventional approach to determine the solubility involved stirring an excess of solid enzyme (25 °C) in 50 mM Tris–HCl (pH 8) until the LH monitored in the aqueous phase had reached a constant value over a period of three days. N-terminal sequence analysis of the purified LH was performed by automated Edman degradation using an Applied Biosystems, Inc. Model 473A sequencer.

Aggregated, periplasmically-localised, signal sequence-processed LH is functionally active

Electron microscopy E. coli TB1 pEC86/pEV-LH32 cells were subjected to cold fixation (0.1 M sodium cacodylate buffer at pH 7.4 (containing 2.5% (v/v) glutaraldehyde, 2% (v/v) formaldehyde and 2.5 mM calcium chloride) and then embedded in low melting agarose. They were then fixed with 1% (w/v) osmium tetroxide and 1.5% (w/v) potassium ferricyanide in 0.1 M sodium cacodylate buffer pH 7.4 and tertiary fixed with 1% (w/v) uranyl acetate. Following dehydration through a graded series of ethanol concentration and infiltration with LR WhiteTM resin, the preparations were transferred to BEEMTM capsules and the resin cured at 60 °C under N2. Ultrathin cut-sections with silver/gold interference colours were post-stained with 5% (w/v) uranyl acetate [43] and lead citrate [44] using microwave irradiation (800 W, 10% power for 1 min). Sections were examined in a JEOL JEM1010 electron microscope (JEOL, Tokyo) at 80 kV and images recorded on KodakTM electron microscope film (type 4489, Agar Scientific).

Batch induction of E. coli pEV-LH32/pEC86 (Fig. 1a) at 32 °C produced LH that was devoid of enzymatic activity in the periplasmic or whole cell extracts. The pEC86 plasmid coded for the entire complement of the ccm operon (Fig. 1a). However, an active, homogeneous enzyme preparation was isolatable by urea solubilisation of the IAs separated from the membranes by ultracentrifugation (Fig. 1b, lane 4) with an activity of 170 units/mg protein [36]. The amino acid sequence of the first ten N-terminal residues confirmed that the signal sequence was processed at the bond position predicted by the SigPep program [45]. The fact that the aggregated, matured form was devoid of the signal sequence suggested that it must have been fully translocated to the periplasmic space to be processed by the signal peptidase-I which has an exo-cytoplasmic-facing active site [23]. Generation of heme-assembled LH necessitated the constitutive co-expression of ccm [46] (Fig. 1b, lanes 3 and 4). However, in a ccm background, heme-deficient, inactive LH protein prevailed (Fig. 1b, lane 2). Nevertheless, in both the backgrounds LH was produced in aggregated forms; an active LH was only recoverable by urea solubilisation when ccm was co-expressed. Clearly, maturation of LH to holo c-type hemoprotein necessitated an obligatory co-expression of the entire complement of eight cytochrome c maturation factors. A signal-less LH, also produced as inclusion bodies, lacked the bound heme in both ccm+ or ccm backgrounds (see later section). In accordance with the known model of periplasmic cytochrome c biogenesis in prokaryotes [47], ccm-mediated periplasmic heme import and its covalent coupling to the signal peptide-processed apo-LH must occur post-translocationally, requiring a conformationally-competent C-terminal cytochrome c domain localized in the more oxidising periplasmic space [25]. Hence, LH protein must have been translocated, processed, folded and heme-associated prior to its aggregation. Aggregated recombinant LH is intramolecularly disulphide bonded Of the four Cys residues in LH, Cys586,590 are in the putative cytochrome c heme-binding sequence CGACH [26]. The two remaining Cys residues, Cys124,143, are significantly displaced in comparison to the homologous motifs in the related quinoproteins [48]; these vicinal Cys103,104 in methanol dehydrogenase and Cys116,117 in ethanol dehydrogenase form the disulphide bridge floor of the active site [49]. Free thiols were neither detectable by Ellman reaction [50], nor were they susceptible to modification by iodomethane alkylation unless the enzyme was pre-reduced with dithiothreitol (data not shown). This suggested that as with the enzyme in the native Pseudomonas sp., the two Cys124 and Cys143 sulphydryl residues in the recombinant LH domain, derived from the isolated aggregates, were intramolecularly bonded. In contrast, a signal-less version of LH was produced in an inactive form as both monomer and dimer (see later section). The findings verify that the translocated LH must have been correctly folded to have gained intra-molecular disulphide-bonding in the envelope zone [24] prior to its aggregation. Thermoinduced transition of LH from soluble to aggregated state As shown in Figs. 2a and 3, E. coli pEV-LH32/pEC86 batch cultured at 18° to 630 °C efficiently exported soluble, enzymatically active, heme-assembled LH to the periplasmic space, accounting for 40% (30 °C) of total protein, as analysed by Phoretix 1D

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PphoA

C

H

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68

45

30

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LH3 2 pEV -

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M proarker tein s

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IA

Fig. 1. (a) Linear maps of expression plasmid maps (i) pEV-LH32 encoding LH gene (lh) fused to signal sequence of alkaline phosphatase (ss) under the transcriptional control of alkaline phosphatase promoter (PphoA); (ii) pEC86 containing the ccm operon encoding the full complement of cytochrome c maturation factors constitutively expressed via tetracycline resistance promoter (Ptetr). S/D, Shine/Dalgarno sequence, Ori, origin of replication, ampr, b-lactamase gene, cmr, chloramphenicol resistance gene. (b) Ccmdependency on the formation of heme-assembled LH. SDS–PAGE analyses of recombinant LH in the IA derived from E. coli harbouring either pEV-LH32 (lane 2) or pEV-LH32/ pEC86 (lanes 3); isolated LH from IA of E. coli pEV-LH32/pEC86 (lane 4); marker proteins (lane 1). The gel was first heme-stained (H) followed by staining with Coomassie blue R250 (C).

Advanced software (Nonlinear Dynamics, UK). However, a dramatic decline in the periplasmic recovery of the soluble enzyme sets in at P30 °C (Figs. 2a and 3). Similarly, a qualitative electrophoretic survey of the heme-assembled LH protein (Fig. 2b) and urea (8 M)-solubilised (Fig. 3) enzymatic activity from the isolated IA demonstrated that active LH protein gradually accumulates in an insoluble, yet active form, reaching a maximum at 30 °C. However, at subsequent higher growth temperatures a rapid decline in the recovery of both the LH protein and the enzymatic activity was apparent; almost complete loss of the recombinant LH occurred at 37 °C (Figs. 2b and 3). These observations suggested that the properties of the aggregated LH formed within a growth temperature range of 18–30 °C differed from those at 30 °C or above. In the former the temperature range the active aggregated enzyme is almost quantitatively extractable with urea whereas in the latter a similar recovery was not found (Fig. 2b) despite the presence of a substantial amount of the LH protein in the residual material (Fig. 2c). Notably, a substantial amount of LH in the non-recoverable urea extract was heme bound but, nevertheless, enzymatically active (Fig. 2c and 3). Interestingly, the kinetics of the formation of IAs of functional LH, even at the earliest induction temperature of 20 °C, suggested a temperature-dependent transition of the soluble- or correctly folded, soluble periplasmic pool of LH to IA, recoverable in fully active form (Fig. 2). However, LH IAs generated at

lower temperatures differed from those formed at higher temperatures. For a further insight into the qualitative trend of production and sub-cellular localisation of LH, morphological examination of cross-sections of E. coli pEV-LH32/pEC86 cells cultured at different temperatures were undertaken (Fig. 4). At 18 °C, loose folds of the inner or cytoplasmic membranes were perceptible with light grey deposits in an apical islandic forms (Fig. 4a and b). Clearly the build-up of the periplasmic dark zones corresponded with the accumulating LH in the periplasmic space zones. At 28 °C, the interposed amorphous patches become enlarged, darker and extend over a wider periplasmic zone (Fig. 4c and d), reflecting increased localisation and denser packing of the recombinant protein. At 35 °C the enlarged, distended periplasmic patches further intensified and appeared more densely packed with the protein cargo evenly distributed around the entire cellular periplasmic zone (Fig. 4e and f). Our findings are consistent with previous observations that, in contrast to cytoplasmic inclusion bodies which appear as large, dark, spherical bodies, periplasmic inclusion bodies are rod shaped structures and are visibly separated from the cytoplasm as a lightly stained boundary [33]. The co-induced endogenous periplasmic alkaline phosphatase, expressed through the same promoter in the absence (E. coli

P. Stampolidis et al. / Archives of Biochemistry and Biophysics 484 (2009) 8–15

10

30

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ke r 18 s o C 22 o C 25 o C 27 o C 30 o C 32 o C 35 o C 37 o C

ar M

M

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ke rs 18 o C 22 o C 25 o C 27 o C 30 o C 32 o C 35 o C 37 o C

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24 26 28 30 32 Growth temperature ( oC)

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Fig. 3. The effect of varying the temperature of cultivation on the production of active lupanine hydroxylase in the isolated sub-cellular fractions of E. coli cells harbouring plasmids pEV-LH32 and pEC86.

s

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20

Fig. 2. Localisation of soluble and insoluble LH (a) Periplasmic extract, (b) 8 M urea extracted LH from isolated IA, (c) residue left over following extraction in (b). The gels were first heme-stained (H) followed by staining with Coomassie blue R250 (C).

pEC86) or presence (E. coli pEV-LH32/pEC86) of LH, showed no significant change at different growth temperatures (data not shown). An insignificant amount of alkaline phosphatase activity was detected in the solubilised membrane fraction derived from both the clone lines, implying that the temperature-dependent formation of IAs is an event specifically related to expression of the recombinant LH. Transition of correctly modified and active LH to the aggregated form could be attributed to thermal denaturation at higher growth temperatures. However, a one hour pre-incubation of the isolated LH at 35, 55, and 70 °C (isolated from periplasmic extract of E. coli cultured at 22 °C) gave full, half maximal and complete loss of enzymatic activity, respectively (Supplementary Fig. 1); 80% of the activity was, however, retained following a 16 h incubation at 35 °C. Thus the considerable in vitro stability of the enzyme over a wide temperature zone makes thermal denaturation of the pre-

folded protein an unlikely mechanism as the causation of its in vivo aggregation through denaturation. Signal sequence-appended and signal sequence – less expressed LH protein are generated in distinct aggregated forms To shed light on the nature of LH protein clustering, isolated aggregates from cells cultured at 30 °C were titrated with urea (Fig. 5). Recovery of the soluble, renatured, active LH following removal of urea showed that most of the active, renatured enzyme was quantitatively recovered at 4 M urea. However, recovery, measured as specific enzyme activity, occurred at a significantly lower urea concentration of about 1 M (Fig. 5). In this and other respects, discussed above, the periplasmic LH aggregate behaviour was different from that of classical inclusion bodies [51]. However, aggregates isolated at 35 °C were almost completely resistant to this pattern of LH solubilisation. This prompted us to construct by inverse PCR the plasmid pEVcLH, a progenitor of pEV-LH32, encoding the signal-less LH protein. E. coli/pEV-cLH strain cultured at 30 °C induced a four-fold excess LH protein in comparison with the secreted counterpart (Fig. 6). N-terminus sequencing and Western blotting of the eluted protein identified it as the LH protein (data not presented). In contrast to swift reactivation, under relatively mild chaotropic conditions, of the secretory aggregates, only 40% of the signal-less LH protein was extractable with 8 M urea (Supplementary Fig. 3). The signal-less LH occurred as a monomer and a prominent disulphideinterlinked dimer (Fig. 6). Of the 4 potential thiols, 1.2 was accessible in the non-reduced, isolated signal-less LH. The isolated, signal-less LH was neither heme bound nor enzymatically active. Thus the periplasmic aggregated LH was post-translocationally modified to give an authentic active enzyme, whereas, the comparably-expressed, signal-less LH, enzymatically was generated inactive misfolded state. Mode of aggregation of periplasmic LH LH is a valuable reporter of post-translocational events in the periplasmic space as its functional folding has obligatory require-

P. Stampolidis et al. / Archives of Biochemistry and Biophysics 484 (2009) 8–15

a (x15k)

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Fig. 4. Electron micrographs of cross-sections of E. coli/pEC86/pEV-LH32 cultured at the indicated temperatures showing potential deposits of LH at the cell periphery. The values in the bracket indicates the extent of magnification. The localised formation of LH and its amorphous aggregates are indicated by the arrow heads.

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Fig. 5. Urea concentration-dependent solubilisation of LH in IA. Cells of E. coli pEVLH32/pEC86 cultured at 30 °C were lysed by sonication and centrifuged at 105,000g for 1.5 h at 4 °C. The IA pellets were homogenised with solutions of urea at the indicated concentrations. After leaving at room temperature for 10 min and centrifuging at 200,000g for 1.5 h at 4 °C, the supernatants were diluted with TE to 0.8 M final urea concentration and assayed for LH activity.

ments for numerous biochemical processing and molecular chaperoning activities. Electron microscopic observations (Fig. 4), together with the sub-cellular localization of the enzyme activities (Fig. 2), suggest that the functional LH was efficiently localised to the periplasm. These observations suggest that LH must have been exported to the periplasmic space and correctly post-translationally modified and folded, thus eliminating the possibility that the IAs arise solely through misfolding. In this respect, the aggregates resemble the non-classical type (cf Fig. 2a, b and c) in that they are readily recoverable into functional form and yet are distinct from the classical type of the signal-less counterpart generated in the cytoplasm (Fig. 2c).

It should be mentioned that we initially used an 8 M urea-based protocol to isolate aggregates and recover LH from periplasmic IAs, which were shown to be active aggregates. Urea is a denaturing agent used to solubilize protein aggregates and thus the solubilized LH protein could have been (i) recovered in the unfolded state and the subsequent filtration, dialysis and purification allowed LH to attain its native state, or ii) unaffeacted by 8 M urea to give an overall fully folded structure to yield active enzyme. Thus, it can be argued that LH solubilized from IAs maintained the structure that was embedded in IAs, either unfolded, partially folded or native. Indeed, the dynamics of thermo-induction of the periplasmically-localised lupanine hydroxylase can be divided into two temperature growth zones of 18–30 °C and 30–37 °C involving (i) accumulation, (ii) precipitation/aggregation into non-classical inclusion bodies and (iii) eventual clustering as classical inclusion bodies. In the lower temperature zones, production of soluble, functional LH was enhanced by increases in growth temperature up to 25 °C where the quality control of the chaperoning activities and post-translocational machinery would be expected to be functional in the periplasmic space [34]. Nevertheless, even within this temperature zone, the kinetics of the accumulation of active LH associated with IAs prevailed concomitantly with the increases in growth temperature (Fig. 2a and b), implying a transition from soluble to insoluble form. This may have been triggered by phase-induced precipitation of the over-produced protein exceeding its solubility in the periplasmic space (Fig. 3). Based on published estimates of the periplasmic size as 30% of the whole cell volume [21], at peak induction the soluble enzyme, which amounts to 10 mg/Lit of culture (Fig. 3), would translate to a concentration of 45 mg / ml in the periplasmic space. This is also consistent with the in vitro solubility of purified LH as determined by (i) PEG 4000 enforced precipitation (55 mg protein /ml (Supplementary Fig. 2) and (ii) maximum solubility (45 mg protein/ml) [52] in 50 mM Tris–HCl (pH 8). Moreover, compared with the native Pseudomonas sp., estimates of the recombinant LH production in E. coli periplasmic space was at least 2 orders of magnitude higher [36]. Thus, it is possible that LH production in the periplasmic space may have exceeded its solubility thus causing precipitation through non-co-operative aggregation.

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kDa 175 83 62 47

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n es Cy top l as m Per ipla sm Me mb ra n es Cy top l as m Per ipla sm Lu hyd pani rox ne yla se Me mb ra n es Cy top l as m Per ipla sm Me mb ra n es Cy top la s m Per ipla sm Per ipla sm Ma rke rs

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E. coli/ pEV-LH32/pEC86

Fig. 6. Sub-cellular localisation in E. coli of signal-less LH (pEV-cLH/pEC86) and signal-appended LH (pEV-LH32/pEC86) expressed at 30 °C. The reduced samples were treated with 50 mM dithiothreitol included in the SDS sample buffer. The positions of monomeric and dimeric forms of LH is indicated by the circles and squares, respectively.

However, an alternative possibility is that the temperature-induced hyper-produced soluble LH may have been triggered into aggregation not only by its limited solubility but also through the partially misfolded LH which may have been trapped in conformations close to that attained by the native state, the so-called non-classical inclusion bodies [19]. This model is also reinforced by recent findings reporting the successful detection of enzymatic activity within isolated inclusion bodies of expressed enzymes [20,53]. In the light of these developments, solubility may be discredited as a measure of biological activity since inclusion bodies are regarded as ‘‘dynamic reservoirs” consisting of a pool of both active and inactive enzyme located in both soluble and insoluble cell fractions [5]. Furthermore, expression above 30–35 °C, though yet with gradual loss of the soluble or non-recoverable functional LH from the IA (Fig. 2 and 3), suggested transition from non-classical to classical inclusion bodies (Fig. 4). Over-expressed recombinant proteins in the confined periplasmic space can cause crowding with considerable build-up of both soluble and insoluble macromolecules [54–56]. Macromolecularinduced crowding or ‘excluded volume effect’ can constrain space and diffusion limits [57] which in turn may induce hydrophobicinduced aggregation of the unfolded intermediates in the folding pathway. Such increased viscosity induced [55] by molecular crowding in the periplasm may render initially a mild but subsequently a stronger stress-induced response causing the formation of severe and/or extensive misfolded aggregated LH molecules. Such a phenomenon is reported to affect not only protein assembly, protein folding and protein aggregation [58] but also cell volume regulation and the detection of intercellular component concentration [59]. It is a function of aggregation control mechanisms to determine whether the IAs are to be later degraded or processed into a biologically active form [60]. Thus, the diminished LH activity at higher growth temperatures (Figs. 2b and 3) may be attributed to degradation by components of the CpxR and/or rE regulons which are known to be inducible during periplasmic stress response [60], although this proteolytic activity is likely to be restrained to an extent by macromolecular crowding, which is known to increase protein stability [61]. Electrophoretic

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