Porphobilinogen deaminase is unstable in the absence of its substrate

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Biochimica et Biophysica A cta 882 (1986) 384-388 Elsevier

BBA 22364

P o r p h o b i l i n o g e n d e a m i n a s e is u n s t a b l e in the a b s e n c e o f its substrate Carole Beaumont, Bernard Grandchamp, Marc Bogard, Hubert de Verneuil and Yves Nordmann Department of Biochemistry, Hospital Louis Mourier, 92701 Colombes (France)

(Received January 21st, 1986)

Key words: Porphobilinogen deaminase; Succinylacetone; Enzyme stability

Porphobilinogen deaminase is induced during the dimethyl sulfoxide-mediated differentiation of Friend erythroleukemia cells. We have previously shown that when succinylacetone, a potent inhibitor of porphobiiinogen formation, is present during the differentiation process, the induction of the enzyme is apparently suppressed. Here, we provide evidence that, in this condition, porphobilinogen deaminase is synthesized normally but does not accumulate as a consequence of an accelerated turnover. The normal half-life of the protein is 24 h but decreases to 10 h when the formation of its substrate is impaired by succinylacetone. We propose that when the enzyme is covalently bound to its substrate, a normal step in this enzymatic reaction, it is protected from proteolytic degradation, and we show that this new finding is relevant to the human disorder acute intermittent porphyria.

Introduction Porphobilinogen deaminase (EC 4.3.1.8), the third enzyme of the heme biosynthetic pathway, catalyses the sequential addition of four molecules of porphobilinogen to form the linear tetrapyrrole hydroxymethylbilane, which is then rapidly converted into uroporphyrinogen III by uroporphyrinogen-II! cosynthase [1]. Multiple electrophoretic forms of red blood cell porphobilinogen deaminase have been described and have been shown to correspond to stable enzyme-substrate covalent intermediates [2,3]. More recently, we were able to demonstrate that there is a tissuespecific expression of two different molecular forms of porphobilinogen deaminase, in mouse [4] as well as in man (unpublished data). In mouse liver, porphobilinogen deaminase is present in a heavier form (M r 44 000) than in mouse red blood

Abbreviation: DMSO, dimethyl sulfoxide.

cells (M r 42000) and from cell-free synthesis experiments, it clearly appears that these two forms do not derive from one another by post-translational processing. In Friend cells, which are virustransformed murine erythroid precursor cells, both the erythroid and the non-erythroid forms are present in the undifferentiated state, whereas only the erythroid species accumulates in fully differentiated cells. Recently, we assessed the effect of succinylacetone on the dimethyl sulfoxide(DMSO-) mediated induction of heme pathway enzyme activities in cultured Friend erythroleukemia cells [5]. Succinylacetone is a potent irreversible inhibitor of aminolevulinate dehydratase [6], the second enzyme of the heme biosynthetic pathway, which catalyses the formation of porphobilinogen. Succinylacetone has been shown to markedly reduce the intracellular heme content of various cell types when added to the culture medium [7,8]. In Friend cells, aminolevulinate dehydratase was actually inhibited by succinylacetone, but determination of the catalytically in-

0304-4165/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)

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active protein by an enzyme immunoassay revealed that the enzyme synthesis was induced by DMSO to the same extent as in the absence of succinylacetone [5,18]. Unexpectedly, the DMSOmediated increase in porphobilinogen deaminase activity was suppressed by the presence of succinylacetone in the culture medium during the induction experiment. This effect could not be related to an inhibition of the enzyme by succinylacetone or its derivatives and could not be overcome by the addition of exogenous hemin. In this paper, we explore the possibility that conditions which impair porphobilinogen formation, i.e., the inhibition of aminolevulinate dehydratase by succinylacetone, might alter the stability or the translational efficiency of porphobilinogen deaminase mRNA or the stability of either forms of the protein itself. Material and Methods

All reagents were analytical grade. Succinylacetone (4,6-dioxoheptanoic acid) was purchased from Calbiochem (La Jolla, CA). [35S]Methionine (specific activity 1475 C i / m m o l ) and Amplify were from Amersham, Zeta-Probe membranes were from Bio-Rad Laboratories (Richmond, CA).

Friend cell cultures Friend cells, clone 745, were grown in McCoy 5A medium supplemented with 10% fetal calf serum. Cells for induction were diluted to 105 cells/ml to maintain a logarithmic growth rate. 12-16 h later, DMSO a n d / o r succinylacetone were added in an equal volume of fresh medium at twice the final desired concentration. DMSO was added to 1.5% (v/v) and succinylacetone to 0.5 mM. For each time-point, cells were harvested by centrifugation at 800 × g and washed with sterile phosphate-buffered saline. The cell pellet was homogenized for isolation of total cellular RNA [10]. Cell-free translation of porphobilinogen deaminase message Total cellular RNA was extracted from cells harvested 48 h after DMSO a n d / o r succinylacetone addition and was translated in a nucleasetreated reticulocyte lysate according to Pelham and Jackson [11]. Neosynthesized porphobilinogen

deaminase was immunoprecipitated from the same amount of trichloroacetic acid-precipitable radioactivity and analysed by SDS-polyacrylamide gel electrophoresis. The gel was then treated with Amplify and subjected to fluorography.

Metabolic labefing of cells with [35S]methionine Cells (6-10 s cells/ml) previously cultured in the presence of DMSO with or without succinylacetone for 48 h were pulse-labeled with [35S]methionine for 2 h using 175 #Ci in 10 ml total volume of methionine-free Eagle's medium supplemented with 10% fetal calf serum. Labeled cells were washed twice using McCoy medium and then incubated in the same medium for various chase periods. [ 35S]Methionine-labeled porphobilinogen deaminase was immunoprecipitated from cell lysates and analysed by SDS-polyacrylamide gel electrophoresis, followed by fluorography of the gel and scanning of the film. Results

In vitro translation of porphobilinogen deaminase We have already shown by hybridization with specific cDNA probes that maximum accumulation of the porphobilinogen deaminase mRNA is reached 48 h after DMSO addition and is not

Mr ( xlO-:3] 67 .4.__NE

43 30 a

b

c

d

Fig. 1. Porphobilinogen deaminase synthesized in vitro by total mRNA extracted from Friend cells after 48 h of culture in the presence of standard medium (lane a), DMSO (lane b) and DMSO plus succinylacetone (lane c). NE, non-erythroid form; E, erythroid form. Lane d: 14C-labeled purified erythrocyte porphobilinogen deaminase (M r 42000). Exposure was for 3 days.

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affected by the presence of succinylacetone in the medium during the induction experiment [12]. We now investigate a possible effect of succinylacetone on the translational efficiency of the porphobilinogen deaminase mRNA. The fluorogram in Fig. 1 shows the porphobilinogen deaminase synthesized in vitro under the direction of total RNA from Friend cells cultured

Chase ( h )

0

18

24

48

DMSO

.,,-.-- E

C)MSO + SA

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for 48 h in various conditions. Immunoprecipitation of products translated from RNA extracted from non-induced cells produced two bands (Fig. 1, lane a). These bands correspond to the two erythroid and non-erythroid forms of porphobilinogen deaminase that we recently described [4]. The lower band (M~ 42000) migrates in the same position as purified red blood cell porphobilinogen deaminase (Fig. 1, lane d) and is synthesized in greater amounts from RNA extracted from DMSO-treated cells (Fig. 1, lane b). The in vitro synthesis of this erythroid form is apparently not affected by the presence of succinylacetone during the DMSO treatment (Fig. 1, lane c). The minor non-erythroid species (Mr 44000), which is also specifically immunoprecipitated from non-induced cells, is only synthesized in trace amounts in DMSO- and DMSO plus succinylacetonetreated cells. It is therefore difficult to assess any possible effect of succinylacetone on the translational efficiency of the non-erythroid porphobiiinogen deaminase mRNA.

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18 24 HOURS CHASE

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Fig. 2. Turnover rate of [35S]methionine-labeled porphobilinogen deaminase. The figure shows fluorograms of porphobilinogen deaminase immunoprecipitates obtained after labeling Friend cells with [35S]methionine (175/~Ci/10 ml, 2 h), washing, then incubating in chase medium for 0, 18, 24 and 48 h. The fluorograms were scanned and, for the erythroid form, a semi-log plot is shown for the percentage of integrated values (relative to the 0-h chase time-point) vs. hours of chase in DMSO- (zx) or DMSO plus succinylacetone-treated cells (A). The plot is drawn from the mean of three separate experiments. NE, non-erythroid form; E, erythroid form.

Turnover rate of porphobilinogen deaminase The porphobilinogen deaminase mRNA content is not modified by succinylacetone, nor is its translational efficiency, thus raising the possibility that the enzyme itself is unstable in this condition. To explore this hypothesis, we carried out a pulse-chase experiment of 35S-labeled porphobilinogen deaminase in Friend cells cultured for 48 h in the presence of DMSO with or without succinylacetone (Fig. 2). Synthesis and degradation rates of total cellular proteins are not affected by the presence of succinylacetone in the culture medium, and 50% of the total labeled proteins are degraded after 48 h of chase. After 2 h of labeling, the same amount of [35S]methionine is incorpo-

TABLE I Changes in the relative amount of the non-erythroid form of porphobilinogen deaminase, expressed as a percentage of the total enzyme, during the chase period. SA, succinylacetone. Chase period (h)

DMSO D M S O + SA

0

18

24

48

5 5

10 30

15 40

20 50

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rated into porphobilinogen deaminase in thg two conditions. The half-life of the erythroid form of porphobilinogen deaminase in DMSO-treated cells is approximately 24 h but the protein seems to turnover more rapidly when succinylacetone is also present in the medium; 50% of the labeled enzyme is lost within 10 h. There is some evidence that the non-erythroid form of the enzyme (Fig. 2), which represents a minor fraction of the total porphobilinogen deaminase remaining at the end of the labeling period, is less affected by the presence of succinylacetone. After 48 h of chase, the minor species represents 50% of the total porphobilinogen deaminase immunoprecipitated from DMSO plus succinylacetone-treated cells (Table I), whereas it only represents 20% of the total enzyme in the absence of succinylacetone. Discussion

In this paper, we provide evidence that porphobilinogen deaminase is unstable in the absence of its substrate. We previously reported that when porphobilinogen synthesis is inhibited by succinylacetone in cultured Friend cells, a progressive decline in porphobilinogen deaminase protein is observed [5], which cannot be accounted for by an effect of succinylacetone on the accumulation of the porphobilinogen deaminase mRNA [4]. From this paper, it also appears that the amount of porphobilinogen deaminase synthesized both in vivo and in vitro is not affected by the presence of succinylacetone. However, investigation of the turnover rate of the erythroid form of porphobilinogen deaminase in cultured Friend cells clearly shows that the enzyme is degraded more rapidly when succinylacetone is present in the culture medium ( q / 2 "~ 10 h) than in normal conditions of culture ( t l / 2 ~ 24 h). This difference in turnover rate can easily account for the low porphobilinogen deaminase activity previously observed in Friend cells cultured in the presence of succinylacetone [5]. Determination of the exact turnover rate of the non-erythroid form is more difficult, since this form represents only a minor fraction of the total porphobilinogen deaminase present in Friend cells after 48 h of induction by DMSO. However, the relative proportion of the M r 44 000 form seems to increase at the end of the chase

period, suggesting that it is less affected by the absence of porphobilinogen than the M r 42000 form. This differential turnover rate between the two species of porphobilinogen deaminase might be part of possible functional differences between the two isoforms of the enzyme. Porphobilinogen deaminase has been shown to form covalent enzyme-substrate complexes [13] arising from the sequential addition of four molecules of porphobilinogen while the initially bound porphobilinogen remains covalently linked to the enzyme, probably through a lysine residue [14]. We can speculate that when porphobilinogen synthesis is prevented by inhibition of aminolevulinate dehydratase with succinylacetone, more porphobilinogen deaminase will accumulate as free enzyme, which may be more susceptible to degradation than the mono-, di-, tri- and tetrapyrrole-bound intermediates. This is consistent with the finding that the enzyme-substrate intermediates are more resistant to in vitro heat inactivation than the native enzyme [3]. In humans, the clinical disorder acute intermittent porphyria is a dominant inherited defect in heme biosynthesis characterized by a 50% deficiency in porphobilinogen deaminase activity [1]. Anderson et al. [15] have shown that the erythrocyte enzyme from heterozygotes for acute intermittent porphyria has the same pattern of DEAE-cellulose chromatography as that from normal individuals. However, during an acute attack, which is characterized by an overproduction of 3-aminolevulinic acid and porphobilinogen, they observed an increase in one of the intermediate enzyme-substrate complexes. Furthermore, it has been observed in some patients with clinically expressed acute intermittent porphyria that the activity of erythrocyte porphobilinogen deaminase was almost normal [16] although some relatives of the patient (asymptomatic carriers) displayed a diminished activity. These findings could be explained in view of our present results, assuming that during an attack of acute intermittent porphyria more of the normal erythrocyte enzyme is bound to porphobilinogen and is therefore protected from degradation. Recently, Desnick et al. [17] characterized a new mutation in some acute intermittent porphyria patients who exhibited a highly elevated level of porphobilinogen deamin-

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ase-cross-reacting immunological material. They speculate that an altered release of the end product protect the enzyme from degradation, an explanation which is consistent with the results reported here.

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Acknowledgements

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This work was supported in part by a research grant from the Caisse Nationale de l'Assurance Maladie des Travailleurs Salari6s and grants from the University of Paris VII. The authors thank Mrs C. Guyomard for typing the manuscript.

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