The physiological function of metallothionein

TIBS

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143

April 1982

first half (residues 1-30) of the sequence of metallothionein and 11 cysteines in the second half (residues 31-61) of the sequence. It might be expected that cluster B is associated with the first half of the molecule and cluster A with the second half. In a distantly related, crab metallothionein, two cadmium binding clusters are also present, each containing three bound metalsL

The physiological function of metallothionein Frank O. Brady Metallothionein seems to provide zinc and copper to newly synthesized apoenzymes in tissues which are undergoing rapid growth and development. High concentrations o f this protein are also induced in the liver by external physical and internal chemical stress and when the liver regenerates after partial hepatectomy. Metallothionein, thus, has a central role in macromolecular synthesis. Metallothionein, is a low molecular weight protein that is rich in cysteine and binds metals. It is chiefly found in eucaryotes although primitive, smaller metallothioneins also exist in some procaryotes. Metailothionein was originally thought to be a cadmium protein but it is now known to bind Zn, Cd, Cu, Hg, and Ag in increasing order of affinity. Under most conditions it can be purified with a mixture of bound metals, depending on the prior exposure of the animal to these metals. Several reviews t,2 are available on the structural, physical, and chemical aspects of metallothionein and over 200 papers on metallothionein have appeared since Kojima and K~igi3 published their review in T1BS. Much of the earlier work focused on metallothionein's role in heavy metal detoxification; this has been amply reviewed ~-3. However, recently attention has turned to its role in normal zinc and copper metabolism. This review focuses on recent advances in our understanding of the physiological function of metallothionein, the nature of its metal binding, and the regulation of its gene.

In vitro evidence fora physiological function We have shown that zinc thionein can transfer zinc to apoenzymes in vitro under mild conditions. Apoenzymes prepared from yeast aldolase, thermolysin, Escherichia coli alkaline phosphatase and bovine erythrocyte carbonic anhydrase were successfully reactivated by rat hepatic zinc thionein and this reactivation was as good as, or better than that obtained using simple zinc salts4. Petering's group5 has continued these studies, looking at the reactions of EDTA or apo-carbonic anhydrase with zinc thionein, (Cd, Zn)-thionein, and cadmium thionein. Apo-carbonic anhydrase is able to remove zinc from the first two proteins 100-1000 times more rapidly than EDTA (k2 = 0.44-1.50 × 103 Frank O. Brady is at the Department of Biochemistry, University of South Dakota School of Medk'ine, Vermillion, SD 57069, U.S.A.

Presence of zinc thionein in rapidly growing and developing tissues

Mechanism I : (Zn,Cu)-MT~---free

Zn2+ 7

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free Cu2+ apoenzy~s Mechanism 2:

(Zn,Cu)-MT--~--~hol0enzymes apoenzymes

Fig. 1. Metal donation by metallothionein. M - 1 s e c - 1 ) , a rate approaching that for the

binding of free, unligated zinc (k2 = 2.05 × 103 M-~sec 1). Winge's group6,7 has also studied the reactivation of apoenzymes in vitro, using copper thionein and other metallothioneins. Using this copper thionein (10 g-atoms Cu/mole), they were able to observe a transfer of copper to apocarbonic anhydrase ~. Fig. 1 shows two possible mechanisms for the transfer of zinc and copper from metallothionein to apoenzymes. Current world -7 is consistent with mechanism 2. A recent study of the metal binding by metallothionein, using mCd-NMR, suggested a structure for the bound metals. This structure has two metal clusters, the first 'A' has four metals and 11 cysteines, and the second 'B' has three metals and nine cysteiness.9 (Fig. 2). These clusters are intriguing since they involve bridging, twoand three-centered, thiolate ligands of cadmium, and, by inference, zinc. In mixed (Cd, Zn)-thionein most of the cadmium resides in cluster A, with most of the zinc in cluster B. There are nine cysteines in the

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Webb's group 1°-12 has systematically studied the levels of (Zn, Cu)-thionein in various tissues of the neonatal and developing rat. The liver 1°, kidneys 12, testes12, and gastrointestinal tract" have been studied in detail. The working hypothesis is that high levels of metallothionein are present in tissues undergoing rapid growth and development, to supply zinc, and possibly copper, for nucleic acid metabolism, protein synthesis and other metabolic processes. In fetal rat liver just before birth the concentrations of zinc and copper rise dramatically to 120 and 35 /xg/g wet weight, respectively. 50% of the zinc and 25% of the copper is associated with metallothionein. The concentration of total zinc and zinc in metallothionein declines steadily until adult concentrations are reached at day 26, but the organ content (/xg/liver) of zinc in metaUothionein remains stable until day 15 before declining. Hepatic metallothionein may provide a reservoir of zinc suitable for use by other components of the liver during this rapid growth phase. Rat kidney metallothionein does not undergo these rapid and dramatic changes in concentration during neonatal growth and development. The concentration of zinc in renal metallothionein remains stable at 4---6 /zg/g wet wt from birth to weaning at day 21. The Zn:Cu ratio is > 4 during this period. After weaning, the zinc concentration drops and the copper concentration rises until, at day 70, the Zn:Cu ratio is <0.65 though the total amount of metal is about the same (4-5/xg/g wet wt). Testis is the most rapidly dividing tissue in young rats

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144 and 35-50 day old male rats have more zinc intracellular protein TM. thionein in their testes than in their livers The induction process for metallothioand kidneys combined. Concentrations of nein appears to occur in the following manzinc in metallothionein of 6-9 /zg/g wet ner: (a) the target cell is exposed to metals weight are observed after 35 days of age. or glucocorticoids; (b) an induction of synVery little copper is associated with testicu- thesis of thionein mRNA begins, reaching a lar metallothionein (Zn:Cu ratio > 10). steady state at about 4 h (this process is senNeonatal rat gastrointestinal tract was sitive to actinomycin D but not to found to be extremely rich in copper cycloheximide); (c) metallothionein pro(10(O150/xg Cu/g wet wt) (Zn:Cu ratio tein synthesis is stimulated with detectable <0.3) from birth to 13 days of age. Most of increases observable at 2-4 h; (d) maximal this copper was present in the cytosol, levels of metallothionein accumulate at associated with a low molecular weight pro- 18-24 h; (e) if the inducers have been tein. This protein was difficult to purify and removed, thionein levels decrease with a t, did not behave like a classical metallothio- of 12-22 h (longer ifCd 2+ is the inducer); nein, but this may have been due to aerobic and (f) basal levels are attained after 48-72 instability owing to a high Cu:Zn ratio h. The ability to induce metallothionein is (> 10). This protein disappeared before day much greater for metals (20-50-fold 21, as the gastrointestinal tract matured and increases) than for glucocorticoids underwent closure. A concomitant increase (2-4-fold increases). in hepatic copper occurred during this period. These data are consistent with the Stress-related induction of hepatic zinc hypothesis that high levels of metallothio- thionein nein are required in tissues which rapidly Whanger's group TM was the fh'st to report synthesize macromolecules. that stress could increase the concentration of zinc thionein in rat liver. Rats were subInduction of zinc thionein by hormones jected to various acute physical and and metals chemical stresses, and the increase in hepaExposure of an animal to metals (e.g. tic zinc thionein was measured. The followzinc, cadmium, mercury, etc.), induces the ing results were obtained: cold environment synthesis of thionein in the liver, and, to a (322% increase), hot environment (56%), lesser extent, in the kidney, induction heat burn (17%), strenuous exercise proceeds until most, if not all, of the (218%), and CC14 intoxication (580%). exogenous metal has been sequestered by Sobocinski et al. ~° presented evidence to metallothionein. These early observations show that the increase in hepatic zinc thiosuggested that metallothioneins were part nein is involved in the zinc redistribution of a detoxification mechanism for protec- which occurs during acute bacterial infecttion against heavy metals. More recently, ions. Sham operation for adrenalectomy glucocorticoid hormones have been shown produced the largest stress-related increase to act as independent inducers of metal- in hepatic zinc thionein yet seen (30-fold), lothionein. Several research groups have when followed over a 2 day period 2~. studied thionein induction in vivo and in Regenerating liver after partial hepateccell culture. tomy also requires a high concentration of Using HeLa cells grown in serum-free zinc thionein (0.5/xg increases to 18/zg Zn medium, Herschman's group 13 has demon- in MT/g wet wt22). strated that dexamethasone and Zn 2+ are Two things are apparent from these and primary inducers of metallothionein and similar studies. First, when a rat is subthat they operate by independent mechan- jected to external or internal physical or isms. The Los Alamos group '4,15 has chemical stress, the liver plays a central role developed a series of CHO cells in culture, in the animal's response. Zinc is mobilized which have increased resistance to the toxic from depots, migrates to the liver, the zinc effects of Cd 2~ in the culture media. thionein concentration increases and hepaIncreased resistance correlates well with the tic metabolism is stimulated. Second, stresability of these mutants to take up Cd 2~ and ses which are hepatotoxic result in an Zn 2~ from the medium, to induce thionein increase in zinc thionein concentrations synthesis, and to bind these metals to metal- concomitant with or consequent to the onset Iothionein. Cousins' group '6.~7 has studied of liver regeneration. The involvement of the induction process, as stimulated by glucocorticoids in these processes seems dexamethasone, in rat liver in vivo and in clear, but the possible involvement of primary hepatocyte culture. Using in vitro catecholamines requires further study21. translation with a wheat germ system, they have also shown that thionein mRNA is Molecular biology of metallothionein present in free polysomes (>90%), and not genes membrane bound polysomes. Hence, The most recent and exciting area of metallothionein should be considered as an metallothionein research concerns the

T1BS - A p r i l 1 982

molecular biology and inducibility of the genes coding for metallothionein. Palmiter's group has isolated and characterized a metallothionein gene (the mouse MT-I gene 23) using a eDNA probe inserted into pBR322 to transform E. coli. The mouse gene was partially characterized and shown to include three exons and two introns. This gene has now been sequenced~. The eDNA probe has subsequently been used to monitor the glucorticoid induction of mouse liver metallothionein in vivo, confirming that regulation occurs at the level of transcription ~. The characteristics of induction of metallothionein have been studied using a variety of mammalian cell lines in culture. Usually a cadmium-resistant cell line is selected by growing a wild-type cell in the presence of increasing amounts of cadmium. Cadmit,n-resistant cell lines, derived from CHO cells2~, murine hepatoma cells (Hepa IA) 27, murine sarcoma cells (S180) ~7, and Friend erythroleukemia cells28, all exhibit an increased induction of metallothionein to which cadmium binds. Amplified thionein genes have also been observed. A murine thymoma cell line (W7), which does not normally express the metallothionein gene in the presence of cadmium or dexamethasone, was also found to be inducible, when DNA methylation was prevented using 5-azacytidine~. Hypomethylation of the metallothionein genes in these cells was correlated with increased metallothionein mRNA synthesis in cells selected for cadmium resistance. Metallothionein has proved to be a useful tool for the study of its own and generalized gene regulation.

Prospects for the future Research on metallothionein is currently varied and active. Areas to be studied in the near future include: elucidation of the three dimensional structure of metallothionein; the molecular basis of metal induction; metal transfer in vivo; the chromosomal location of metallothionein genes; and the role of hormones other than glucocorticoids in induction. References 1 K~igi, J. H. R. and Nordberg, M. (eds) (1979) Metallothionein, Birkh~user Verlag, Basel 2 Webb, M. (ed.) (1979) The Chemistry, Biochemistry, and Biology of Cadmium, Elsevier North-Holland, New York 3 Kojima, Y. and Khgi, J. H. R. (1978) Trends Biochem. Sci. 3, 90-93 4 Udorn, A. O. and Brady, F. O. (1980)Biochem. J. 187,329-335 5 Li, T.-Y., Kraker, A. J., Shaw HI, C. F. and Petering, D. H. (1980) Proc. Natl Acad. Sci. U.S.A. 77, 6334-6338

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T I B S - A p r i l 1 982 6 Geller, B. L. and Winge, D. R. Areh. Bioehem. Biophys. 213, 109-117 7 Winge, D. R. and Miktossy, K.-A. Arch. Biochem. Biophys. (in press) 80tvos, J. D. and Armitage, I, M (1980) Proe. Natl Acad. Sci. U.S.A. 77, 7094-7098 9 Otvos, J. D. and Armitage, 1. M. in Biochemical Structure Determination by NMR (Bothner-By, A. A., Sykes, B. D. and Glickson, J., eds), Marcel Dekker, New York (in press) 10 Mason, R.. Bakka, A., Samawickrama, G. P. and Webb, M. (1981) Br. J. Nutr. 45. 375-389 II Mason, R., Brady, F. O. and Webb, M. (1981) Br. J. Nutr. 45,391-399 12 Brady, F. O. and Webb, M. (1981) J. Bh~l. Chem. 256, 3931-3935 13 Karin, M. and Herschman, H. R. (198l) Eur. J. Biochem. 113,267-272 14 Hildebrand, C. E. and Enger, M. D. (1980) Biochemistry 19, 5850-5857

15 Hildebrand, C. E., Enger, M. D. andTobey, R. A. (1980) Biol. Trace Element Res. 2,235-246 16 Failla, M. A. and Cousins, R. J. ( 1978) Bioehim. Biophys. Acta 543,293-304 17 Etzel, K. R. and Cousins, R. J. (1981)Proc. Soe. Exp. Biol. Med. 167,233-236 18 Shapiro. S. G and Cousins, R. J. (1980) Biochem. J. 190, 755-764 19 Oh, S. H.. Deagen, J. T., Whanger, P. D. and Weswig, P. H. (1978)Amer. J. Physiol. 234, E282-E285 20 Sobocinski, P. Z.. Canterbury, Jr.. W. J., Mapes, C. A. and Dinterman. R. E. (1978) Amer. J. Physiol. 234, E399-E406 21 Brady, F. O. (1981) Life Sci. 28. 1647-1654 22 Ohtake, H . Hasegawa, K. and Koga, M. (1978) Biochem. J. 174, 999-1005 23 Durnam. D. M., Perrin. F., Gannon, F. and Paimiter. R. D. (1980) Proe. Natl Aead. Sci. U.S.A. 77. 6511~515

24 Glanville, N., Durham, D. M and Palmiter, R. D ( 1981) Nature (London) (in press) 25 Hager, L. J. and Paimiter, R. D. (1981)Nature (London) 291,3443-342 26 Waiters, R. A., Enger, M. D., Hildebrand. C. E. and Griffith, J. K. in Developmental Biology Using Purified Genes, ICN-UCLA Symposia on Molecular and Cellular Biology (Brown, D. andFox, C. F.. eds), Vol. XXIII, Academic Press. New York (in press) 27 Beach, L. F.. Mayo, D. E., Durnam. D. M. and Palmiter, R. D. in Developmental Biology using Purified Genes, ICN-UCLA Symposia on Molecular and Cellular Biology (Brown. D. and Fox, C. F.. eds). Vol. XXIII. Academic Press, New York (in press) 28 Beach. L. R. and Palmiter, R. D. (1981) Proc. Natl Acad. Sci. U.S.A. 78, 2110-2114 29 Compere, S. J. and Palmiter. R. D. (1981) ('ell 25,233-240

What do ectoenzymes do ?

measured is derived from a small percentage of leaky cells in the preparation.

K. K. Stanley, A. C. Newby and J. P. Luzio

Movement of ectoenzymes during membrane circulation While early studies concentrated on proving that ectoenzymes were present on the surface of cells, it became apparent that some activity must also be present inside the cell, since plasma membrane proteins are k n o w n to be assembled in the endoplasmic reticulum. In experiments 6,7 designed to measure this intracellular pool it was found that a surprisingly large proportion of the ectoenzyme, 5'-nucleotidase, was present inside the cell (more than 50% in hepatocytes and about 20% in adipocytes, lymphocytes and polymorphonuclear leukocytes from rat). The intracellular 5'-nucleotidase in the hepatocyte was similar to the cell surface enzyme in its kinetics and topography, being situated with its active site on the extracytoplasmic side o f an intracellular membrane compartment. The possibility that this intracellular pool was derived from the cell surface by the endocytosis of the plasma membrane was checked using an antibody which inhibited the enzyme. When this antibody, or a monovalent Fab fragment derived from it, was incubated with intact cells at 37°C it was tbund that both pools of enzyme were inhibited, showing that there was a relationship between them. When the experiment was repeated at 4°C only the cell surface 5'-nucleotidase was inhibited. In a second experiment cells in which the surface 5'-nucleotidase had been inhibited at 4°C with antibody were washed and incubated at 37°C. Now enzyme activity reappeared at the cell surface suggesting that a complete cycle of membrane circulation through the cell was possible. Similar results have been obtained in other cell types, both for 5'-nucleotidase and for

The first convincing biochemical description o f cell m e m b r a n e enzymes with their catalytic sites directed towards the extracellular space occurred in the 1950s 1.2. Whilst their p r o b a b l e role in nutrient absorption in the gut was recognized immediately, the)" were also present in other cells. Engelhardt 2, who coined the term ectoenzyme, f o u n d an ecto-A TPase at the surface o f avian erythrocytes and stated that 'at present the role o f the ecto-A TPase . . . remains completely obscure'. M a n y other ectoenzymes have since been described but their physiological functions remain equally obscure. Here, we review s o m e possible functions f o r ectoenzymes which arise out o f recent experimental data. Membrane proteins have an absolute asymmetry in the bilayer, that is, all copies o f a particular type o f protein are oriented in the same manner. Integral membrane proteins which contain domains with either enzyme activity or ligand-binding sites consequently exhibit asymmetry o f these functions. A unique subset of these membrane proteins are those which have their functional groups facing the outside o f the cell. These are o f particular interest because of the wide range of biological processes (e.g. transport, hormone action, neurotransmission, chemotaxis, and cell protection) for which they are responsible. While the presence of receptor proteins on the cell surface was anticipated before their biological characterization, the presence of enzymes facing the outside of the cell came as a surprise. Reports o f ectoenzymes were therefore treated with scepticism until adequate criteria had been proposed.

K. K. Stanley is at the European Molecular Biology Laboratory, Meyerho~trasse I, 6900 Heidelberg, F.R. G. and A. C. Newby and J. P. Luzio are at the Department of Clinical Biochemistry, Universityof Cambridge, Addenbrooke's Hospital. Hills Road, Cambridge CB2 2QR, U.K.

Definition of an ectoenzyme T w o criteria must be established to prove that an enzyme is an ectoenzyme :' ~: ( 1) The enzyme must be an integral protein of the plasma m e m b r a n e and; (2) Its active site must be situated on the outside surface of the cell. The first of these two statements is necessary to distinguish ectoenzymes from secreted enzymes, e.g. exoenzymes in the gut, lipoprotein lipase in adipose tissue and periplasmic enzymes in bacteria. Ectoenzymes are integral membrane proteins, and, in the case of aminopeptidase N and 5'-nucleotidase, have been shown to span the bilayer. Proof of the second statement requires that non-penetrating substrates have access to the enzyme in whole cells. It is also desirable to show that the product formed is extracellular and that the enzyme activity can be inhibited by non-penetrating inhibitors (e.g. diazosulphanilic acid or antibodies). Due to the fragile nature of m a n y isolated cell preparations, especially those prepared by collagenase digestion ~''~, many controls are necessary to exclude the possibility that the enzyme activity being

ElsevierBit~medica]PressIO82 (}376 q)67/82/(l~KIOO(X~'$o275