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Enzyme replacement therapy
95
TIBS - April 1981
Future Trends Enzyme replacement therapy Ernest Beutler Enzyme replacement therapy, treatment of genetic diseases accomplished by administering the missing enzyme, is often regarded as a futuristic medical tour de force. In a certain sense it is a promising therapeutic manoeuvre which has not as yet been achieved. In reality, however, enzyme replacement therapy has been practiced by at least two generations of physicians who transfused fresh whole blood into patients with bleeding disorders. The missing enzyme that they replaced was a plasma protein. Similarly, the oral administration of digestive enzymes to patients with cystic fibrosis may be considered a form of enzyme replacement therapy. But what has not yet been accomplished is the targeting of enzymes to their proper specific intracellular site where they are able to perform their normal function and thereby reverse the effects of a disease caused by enzyme deficiency.
Diseases suitable for treatment by enzyme replacement
metabolizes it, accumulates to thousands of times the normal concentration. Even a relatively brief restoration of normal enzyme concentrations in the tissues of such patients could result in catabolism of most of the accumulated material, giving longer lasting benefit than might be expected in other types of metabolic disorders. It is partly for this reason that most initial attempts at enzyme replacement therapy have focused on this group of diseases. Not all storage diseases are equally suitable targets for enzyme replacement therapy. In some disorders, such as GM2 ganglioside storage disease, Tay-Sachs' disease and Sandhoff's disease, considerable damage to the nervous system has occurred very early in life. Since damage to the central nervous system is usually not reversible removal of the GM= ganglioside from tissues of such a patient would probably accomplish little. Moreover, since one of the principal difficulties in implementing enzyme replacement therapy is the accessibility of the target cell, patients with storage diseases which affect the nervous system are relatively unsuitable subjects. In selecting a disorder to be treated by enzyme replacement therapy, it is important to consider the frequency with which the disorder occurs. The selection of excessively rare diseases would be imprudent because it would be difficult to find suitable test subjects, and because the number of people benefiting would be very small. Because of these considerations Gaucher's disease is clearly one of the best candidates for enzyme replacement therapy. Storage of the glycolipid which accumulates, glucocerebroside, is to a large degree limited to the macrophage-monocyte system. Lipid-swollen macrophages accumulate in the liver, spleen and bones. It is easy to see why the disease was regarded as a neoplasm when it was first described by Phillipe Gaucher a century ago.
Perusal of any standard work on modern biochemical genetics reveals that an enormous variety of enzyme defects have been described in man. Most of these defects produce distorted patterns in the intermediate metabolic pathways which they serve. Altered amounts of metabolite directly or indirectly produce the disease process associated with the enzyme deficiency. However. in most defects of intermediary metabolism sequestration of unmetabolized substrate does not occur, and restoration of a normal enzyme level for a short period of time would therefore produce only brief clinical benefits. The so-called storage diseases are an exception to this rule. These disorders, most of which are caused by a deficiency of a lysosomal enzyme, result in accumulation of a relatively insoluble substrate in tissues. The clinical manifestations of such disorders are caused by engorgement of cells by a metabolic intermediate which is normally present in very small amounts but which, in the absence of the enzyme that normally Techniques of replacement therapy
Administration o f exogenous enzyme Ernest Beutler is at the Department of Clinical Research, Scripps Clinic and Research Foundation, 10666 North Torrey Pines Road, La Jolla, California 92037. U,S.A.
In order to successfully treat an enzyme deficiency disease by administration of enzyme, four goals must be achieved: (1) a
source of enzyme must be developed, (2) the enzyme must be delivered to the cell in which it is to function, (3) the enzyme must be sufficiently stable in the cell to perform its function, and (4) it must interact with its substrate long enough to produce clinical benefits. The source of enzyme is clearly of major importance, and whenever possible should be human in origin. Although enzymes from non-human sources, even those derived from bacteria or fungi, have been used in human therapy, it is virtually impossible to prevent antibodies developing against them. Therapy with agents such as L-asparaginase is usually associated with a high incidence of unpleasant and even life-threatening reactions, although generally given to an immuno-compromised host with leukemia. Even the administration of human enzyme is not entirely free of the risk. However, most enzyme deficient individuals do produce CRM (cross-reacting material) which is immunologically nearly identical to the normal enzyme. In these patients, therefore, it is unlikely that enzyme derived from human sources would be treated as a foreign substance. Purity of the infused material is desirable, but by no means absolutely essential. Enzyme replacement with fresh frozen plasma or with the more modern cryoprecipitate as a source of human factor VIII is quite effective in the management of hemophilia. Yet, the active principle represents only a minute fraction of the material injected. In general, however, the greater the number of extraneous substances infused, the greater the probability of an untoward reaction. The source of the human material requires careful consideration. Those accustomed to the preparation of mammalian enzymes from other species customarily begin their purification process with large quantities of liver, kidney, muscle or other appropriate organs. This approach is impractical when using human material, since only tissue from healthy humans should be used to avoid transmitting disease. Tissues from a victim of an accident may serve as an occasional source of starting material, but a combination of legal constraints and of a lack of predictability as to the availability of such material seriously limits its usefulness. Human sources are therefore limited to samples of blood, urine and placenta. Fortunately, these sources provide access to many enzymes which might be used for replaceElsevier/Notlh-Holland
Biomedical Press 1981
96 ment therapy. In the case of Gaucher's disease it has been possible to purify the missing enzyme, glucocerebrosidase, from h u m a n placenta 1.2. Enzymes do not diffuse freely across cell membranes. The delivery of enzymes to an intracellular location requires development of a strategy for the uptake of the enzyme by the target cell. If the enzyme is attached to a carrier which is normally ingested by the target cell this goal might, in some instances, be accomplished. Such an approach is particularly feasible in attempting to deliver enzymes to phagocytic cells. These cells normally take up materials from their environment by invaginating their exterior membrane and enclosing them within the cell. This process is specifically triggered by a number of substances including the Fc fragment of g a m m a globulins. But there are other mechanisms which may be used by cells for enzyme uptake. A classical example of such a mechanism is the galactose receptor on liver parenchymal cells which removes glycoproteins with terminal galactosyl residues from the plasmaL Recently, it has been found that cultured fibroblasts have a membrane receptor which appears to recognize mannose-6-phosphate. Fibroblasts rapidly accumulate exogenous lysosomal enzymes which contain the mannose-6-phosphate signal and such uptake is inhibited by addition of free mannose-6-phosphate. The physiologic significance of such signalreceptor systems is not altogether clear, although it has been proposed that they may represent mechanisms which guide an enzyme made in the Golgi apparatus to its appropriate intracellular location 4. Breaking the code of such signals may make it possible, in the future, to modify enzymes in such a way that they are directed to the desired location. A variety of means which might be employed to direct enzymes to target cells may be envisaged. For example, the enzyme may be entrapped in erythrocytes or in liposomes which are then coated with a material which would stimulate uptake by the target cell. Alternatively, the enzyme may be attached covalently to various carrier molecules, or sugars may be attached to it. In our thus-far-unsuccessful attempt to treat Gaucher's disease by administration of exogenous h u m a n enzyme, we have encapsulated the purified glucocerebrosidase in resealed h u m a n red cells which were then coated with g a m m a globulin 5'e. Such red cells are avidly taken up by cultured monocytes in vitro ~ and they are cleared rapidly from the blood with subsequent accumulation in the liver and spleen 6.8. Other investigators have injected large
T I B S - A p r i l 1981
amounts of enzymes directly intravenously into patients with Gaucher's disease. Although initially the results were interpreted as biochemically encouraging 9, it is quite clear that little or nothing has been achieved clinically using such an approach TM. The putative decrease in storage material has not been convincingly demonstrated. The results obtained by infusion of enzyme-laden liposomes too have been of doubtful benefit". Organ transplantation
Transplantation of the affected organ or organ system from a genetically normal, immunologically compatible donor is one means of replacing the missing enzyme. Attempts to achieve this through kidney transplantation in the treatment of ~-galactosidase A deficiency (Fabry's disease) n.13 and to transplant a spleen TMinto a patient with Gaucher's disease have been unsuccessful. However, the technology of bone marrow transplantation has markedly advanced in the past few years 15,18,and the possibility of treating a disorder such as Gaucher's disease which affects the macrophage-monocyte system, by transplanting bone marrow does not appear to be hopelessly remote. Administration o f genes to replace missing enzymes
The striking advances which have occurred in the past few years in the technology of isolating genes and cloning them has made it possible to produce vast numbers of copies which code for needed proteins. To use such a technique in the treatment of disease, it is necessary not only to isolate and clone the required gene, but also to introduce it into the target cell relatively efficiently. Theoretically possible though it may be, there are many major technological hurdles to overcome before this type of enzyme replacement can be implemented. The isolation of the gene for a missing enzyme is, in itself, a major problem. Those genes which have been isolated in the past are generally ones which direct production of a vast a m o u n t of proteins. Thus, most of the synthetic activity of the developing erythroid cell is directed to the production of globin chains and plasma cells produce primarily immunoglobulin subunits. Genes which code for these proteins have been isolated. In contrast, the enzymes which are deficient in storage diseases exist in only minute quantities in any cell type. Once a gene has been cloned, it must be introduced into the target cell and inserted into its genome in such a way that regulation of its production is not seriously
impaired. D N A can be introduced into a few million cells by exposing them to large amounts of the D N A , particularly under conditions which facilitate D N A uptake, such as in the presence of calcium phosphate. The D N A may also be incorporated into a viral vector to allow its integration into the genome of a target cell. In either case, the number of cells receiving functional units is likely to be small, especially since introduction of D N A would probably have to be carried out in vitro. An interesting way in which this problem may be approached is to introduce the required gene into a cell with high proliferative potential together with a gene for drug resistance 17. If this is accomplished, then cells which have not received the exogenous D N A may be eliminated by suitable drug treatment.
Conclusions In spite of a n u m b e r of attempts, enzyme replacement therapy has not yet been successful in the treatment of disease. With advancing knowledge of factors which direct the uptake and incorporation of enzyme molecules by various cell types, with advances in immunology permitting organ transplantation, and with the development of sophisticated technology for the purification and implanting of DNA, it is to be hoped that diseases which are essentially untreatable today may yield to enzyme replacement therapy tomorrow.
Acknowledgement This work was supported by Grant A M 2 5 6 9 6 from the National Institute of Arthritis, Metabolic and Digestive Diseases.
References I Dale, G. L. and Beutler, E. (1976) Proc. Natl. Acad, Sci. U.S.A. 73, 46724674 2 Furbish, F. S., Blair, H. E., Shiloach,J., Pentchev, P. G. and Brady, R. O. 11977) Proc. Natl. Acad. Sci. U.S.A. 74, 3560-3563 3 Ashwell, G. and Morell, A, G. (1974) Adv. Enzymol. 41.99-128 4 Sly, W. S. and Stahl, P. (1978) (Silverstein,S. C., ed.), pp. 229-244, Dahlem Konferenzen,Berlin 5 Beutler, E., Dale, G. L., Guinto, E. and Kuhl, W, (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 46204623 6 Beutler, E., Dale, G. L. and Kuhl, W. 11980) in Enzyme Therapy in Genetic Diseases:2. (Desnick, R. J, ed.), pp. 369-381, Alan R. Liss,New York 7 Dale, G. L., Kuhl, W. and Beutler, E. (1979) Proc. Natl. Acad. Sei. U.S.A. 76, 473475 8 Beutler, E., Dale, G. L. and Kuhl, W. (1977) New Engl. J. Med. 296, 942-943 9 Brady, R. O., Pentchev, P. G., Gal, A. E., Hibbert, S. R. and Dekaban. A. S. (1974) New Engl. J. Med. 291,989 993 I0 Brady, R. O., Barranger, J. A., Gal, A. E., Pentchev. P. G. and Furbish, F. S. (1980) Enzyme
97
TIBS - April 1981 Therapy in Gaucher's Diseases:2. (Desnick, R. J., 13 Van den Bergh, F., Rietra, P., Kolk-Vegter,A. G., Bosch, E. and Tager, J. M. (1976) Acta. Med. ed.), pp. 361-368, Alan R. Liss,New York Scand. 200, 249-256 11 Belchetz,P. E., Crawley,J. C. W., Braidman, 1. P. and Gregoriadis, G. (1977) Lancet ii, 116-- 14 Groth, C. G., Dreborg, S., Ockerman, P. A., Svennerholm, L., Hagenfeldt, L., L6fstroem, B., 117 Samuelsson, K., Werner, B. and Westberg, G. 12 Spence, M. W., MacKinnon,K. E., Burgess,J. K., ( 1971) Lanceti, 1260-1264 D'Entremont, D. M., Belitsky, P., Lannon, S. G. and MacDonald, A. S. (1976)Ann. Intern. Med. 15 Thomas, E. D. (1979) Am. J. Clin. PathoL 72, 887-892 84, 13~16
16 Blume, K. G., Beutler, E., Bross, K. J., Chillar, R. K., Ellington, O. B., Fahey, J. L., Farbstein, M. J., Forman, S. J., Schmidt,G. M., Scott, E. P., Spruce, W. E., Turner, M. A. and Wolf, J. L. (1980) New EngLJ. Med. 302, 1041-1046 17 Cline, M. J., Stang, H., Mercola, K., Morse, L., Ruprecht, R., Browne. J. and Salser. W. (1980) Nature (London) 284, 422-425
Reviews Glycoproteins as cell-surface structural components Matthew F. Mescher The major cell-surface component o f Halobacterium salinarium is a glycoprotein having structural and biosynthetic features in common with eukaryotic glycoproteins. The occurrence o f glycoproteins in eukaryotes and archaebacteria suggests that they may have originated in primitive cells, which existed before the divergence o f the three major lines o f descent, possibly as structural components to stabilize the cell membrane.
All cells have limiting m e m b r a n e s of the same basic structure, ie. a lipid bilayer and associated integral m e m b r a n e proteins. In addition, peripheral components are present which interact with the membrane to stabilize it, providing greater rigidity than is possible with a bilayer alone. In many cases, these components provide the cell with a distinctive morphology. Organisms of each of the three major lines of evolutionary descent - eukaryotes, eubacteria and archaebacteria 1'2 - appear to use fundamentally different peripheral components to accomplish this stabilization. Eukaryotes have internal cytoskeletal systems of microtubules and microfilaments which are thought to interact with the inner surface of the membrane affecting cell shape, motility and influencing the mobility of the membrane proteins3; erythrocytes have a cytoskeletal matrix at the inner membrane surface'; and eubacteria have external components, in particular peptidoglycan, which maintain cell shape and rigidity 5. Organisms of the kingdom Archaebacteriae have neither a cytoskeletal system nor peptidoglycan 1.2. At least one m e m b e r of this group, Halobacterium salinarium, has a cell surface glycoprotein which appears to play a structural role in stabilizing the membrane and maintaining cell morphology. The similarities in structure and biosynthesis between this glycoprotein and those of eukaryotic cell surfaces have interesting evolutionary implications. Matthew F. Mescher is at the Department o f Pathology, Harvard Medical School, Boston, Massachusetts
02115, U.S.A.
The cell surface glycoprotein of H. salinarium About 52% of the neutral hexose present in the H. salinarium cell envelope is in the form of glycolipid. The remaining hexose and all of the amino sugar is attached to a high molecular weight glycoprotein which accounts for about half of the total envelope protein and is exposed at the cell surface 8.7. The carbohydrate units consist of di- and trisaccharides attached via O-glycosidic linkages between galactose and threonine, and a relatively large heterosaccharide attached via an N-glycosidic linkage to asparagine 7 (Fig. 1). N- and O-glycosidic linkages to amino and hydroxyl groups are the major means of attachment of carbohydrate to eukaryotic glycoproteins. The H. salinarium glycoprotein also resembles those of eukaryotes with respect to the number, size and composition of the carbohydrate units 7. Attachment of the N-linked carbohydrate, and possibly the O-linked units, of the H. salinarium glycoprotein occurs via isoprenol lipid-linked intermediates 8.~(Fig. 2). Similar pathways involving isoprenol lipid-linked intermediates were first demonstrated in peptidoglycan and lipopolysaccharide synthesis in eubacteria. They are now known to be involved in the glycosylation of eukaryotic glycoproteins 1° as well. The H. salinarium glycoprotein forms a rigid matrix at the cell surface and is responsible for maintenance of cell shape 6. These organisms are rod-shaped under normal growth conditions, despite the
absence of the peptidoglycan layer necessary for the maintenance of shape in most other bacteria. Lactoperoxidase-catalyzed iodination of surface proteins and treatment of intact cells with proteolytic enzymes show that the glycoprotein is the major cell surface component. It is visible in thin-section electron micrographs as a densely staining layer approximately 15 nm thick lying external to the plasma membrane. Removal of the glycoprotein from the surface of intact cells with proteolytic enzymes results in the rod-shaped cells becoming spherical. The spherical ceils remain viable, will grow and will regain the normal rod-shape on removal of the protease and the addition of fresh growth medium. When the glycoprotein content of the cells was examined during treatment with protease and subsequent regrowth, it was found that the glycoprotein disappeared concomitantly with the conversion from rods to spherical cells and reappeared at the same time as the cells returned to rod shape. Further evidence for the structural role of the glycoprotein was provided by experiments using bacitracin, an antibiotic which blocks the lipidintermediate pathway for glycosylation 200,000
5~ooo GOI ic
GOl GIc',XU
2-14
Fig. 1. Structureof the Halobacteriumsalinariumcell surface glycoprotein=. XUA is an unidentified hexuronic acid and XNAc an unidentified amino sugar. ~ Elsevier/North-Holland
Biomedical Press 1981
TIBS - April 1981
Future Trends Enzyme replacement therapy Ernest Beutler Enzyme replacement therapy, treatment of genetic diseases accomplished by administering the missing enzyme, is often regarded as a futuristic medical tour de force. In a certain sense it is a promising therapeutic manoeuvre which has not as yet been achieved. In reality, however, enzyme replacement therapy has been practiced by at least two generations of physicians who transfused fresh whole blood into patients with bleeding disorders. The missing enzyme that they replaced was a plasma protein. Similarly, the oral administration of digestive enzymes to patients with cystic fibrosis may be considered a form of enzyme replacement therapy. But what has not yet been accomplished is the targeting of enzymes to their proper specific intracellular site where they are able to perform their normal function and thereby reverse the effects of a disease caused by enzyme deficiency.
Diseases suitable for treatment by enzyme replacement
metabolizes it, accumulates to thousands of times the normal concentration. Even a relatively brief restoration of normal enzyme concentrations in the tissues of such patients could result in catabolism of most of the accumulated material, giving longer lasting benefit than might be expected in other types of metabolic disorders. It is partly for this reason that most initial attempts at enzyme replacement therapy have focused on this group of diseases. Not all storage diseases are equally suitable targets for enzyme replacement therapy. In some disorders, such as GM2 ganglioside storage disease, Tay-Sachs' disease and Sandhoff's disease, considerable damage to the nervous system has occurred very early in life. Since damage to the central nervous system is usually not reversible removal of the GM= ganglioside from tissues of such a patient would probably accomplish little. Moreover, since one of the principal difficulties in implementing enzyme replacement therapy is the accessibility of the target cell, patients with storage diseases which affect the nervous system are relatively unsuitable subjects. In selecting a disorder to be treated by enzyme replacement therapy, it is important to consider the frequency with which the disorder occurs. The selection of excessively rare diseases would be imprudent because it would be difficult to find suitable test subjects, and because the number of people benefiting would be very small. Because of these considerations Gaucher's disease is clearly one of the best candidates for enzyme replacement therapy. Storage of the glycolipid which accumulates, glucocerebroside, is to a large degree limited to the macrophage-monocyte system. Lipid-swollen macrophages accumulate in the liver, spleen and bones. It is easy to see why the disease was regarded as a neoplasm when it was first described by Phillipe Gaucher a century ago.
Perusal of any standard work on modern biochemical genetics reveals that an enormous variety of enzyme defects have been described in man. Most of these defects produce distorted patterns in the intermediate metabolic pathways which they serve. Altered amounts of metabolite directly or indirectly produce the disease process associated with the enzyme deficiency. However. in most defects of intermediary metabolism sequestration of unmetabolized substrate does not occur, and restoration of a normal enzyme level for a short period of time would therefore produce only brief clinical benefits. The so-called storage diseases are an exception to this rule. These disorders, most of which are caused by a deficiency of a lysosomal enzyme, result in accumulation of a relatively insoluble substrate in tissues. The clinical manifestations of such disorders are caused by engorgement of cells by a metabolic intermediate which is normally present in very small amounts but which, in the absence of the enzyme that normally Techniques of replacement therapy
Administration o f exogenous enzyme Ernest Beutler is at the Department of Clinical Research, Scripps Clinic and Research Foundation, 10666 North Torrey Pines Road, La Jolla, California 92037. U,S.A.
In order to successfully treat an enzyme deficiency disease by administration of enzyme, four goals must be achieved: (1) a
source of enzyme must be developed, (2) the enzyme must be delivered to the cell in which it is to function, (3) the enzyme must be sufficiently stable in the cell to perform its function, and (4) it must interact with its substrate long enough to produce clinical benefits. The source of enzyme is clearly of major importance, and whenever possible should be human in origin. Although enzymes from non-human sources, even those derived from bacteria or fungi, have been used in human therapy, it is virtually impossible to prevent antibodies developing against them. Therapy with agents such as L-asparaginase is usually associated with a high incidence of unpleasant and even life-threatening reactions, although generally given to an immuno-compromised host with leukemia. Even the administration of human enzyme is not entirely free of the risk. However, most enzyme deficient individuals do produce CRM (cross-reacting material) which is immunologically nearly identical to the normal enzyme. In these patients, therefore, it is unlikely that enzyme derived from human sources would be treated as a foreign substance. Purity of the infused material is desirable, but by no means absolutely essential. Enzyme replacement with fresh frozen plasma or with the more modern cryoprecipitate as a source of human factor VIII is quite effective in the management of hemophilia. Yet, the active principle represents only a minute fraction of the material injected. In general, however, the greater the number of extraneous substances infused, the greater the probability of an untoward reaction. The source of the human material requires careful consideration. Those accustomed to the preparation of mammalian enzymes from other species customarily begin their purification process with large quantities of liver, kidney, muscle or other appropriate organs. This approach is impractical when using human material, since only tissue from healthy humans should be used to avoid transmitting disease. Tissues from a victim of an accident may serve as an occasional source of starting material, but a combination of legal constraints and of a lack of predictability as to the availability of such material seriously limits its usefulness. Human sources are therefore limited to samples of blood, urine and placenta. Fortunately, these sources provide access to many enzymes which might be used for replaceElsevier/Notlh-Holland
Biomedical Press 1981
96 ment therapy. In the case of Gaucher's disease it has been possible to purify the missing enzyme, glucocerebrosidase, from h u m a n placenta 1.2. Enzymes do not diffuse freely across cell membranes. The delivery of enzymes to an intracellular location requires development of a strategy for the uptake of the enzyme by the target cell. If the enzyme is attached to a carrier which is normally ingested by the target cell this goal might, in some instances, be accomplished. Such an approach is particularly feasible in attempting to deliver enzymes to phagocytic cells. These cells normally take up materials from their environment by invaginating their exterior membrane and enclosing them within the cell. This process is specifically triggered by a number of substances including the Fc fragment of g a m m a globulins. But there are other mechanisms which may be used by cells for enzyme uptake. A classical example of such a mechanism is the galactose receptor on liver parenchymal cells which removes glycoproteins with terminal galactosyl residues from the plasmaL Recently, it has been found that cultured fibroblasts have a membrane receptor which appears to recognize mannose-6-phosphate. Fibroblasts rapidly accumulate exogenous lysosomal enzymes which contain the mannose-6-phosphate signal and such uptake is inhibited by addition of free mannose-6-phosphate. The physiologic significance of such signalreceptor systems is not altogether clear, although it has been proposed that they may represent mechanisms which guide an enzyme made in the Golgi apparatus to its appropriate intracellular location 4. Breaking the code of such signals may make it possible, in the future, to modify enzymes in such a way that they are directed to the desired location. A variety of means which might be employed to direct enzymes to target cells may be envisaged. For example, the enzyme may be entrapped in erythrocytes or in liposomes which are then coated with a material which would stimulate uptake by the target cell. Alternatively, the enzyme may be attached covalently to various carrier molecules, or sugars may be attached to it. In our thus-far-unsuccessful attempt to treat Gaucher's disease by administration of exogenous h u m a n enzyme, we have encapsulated the purified glucocerebrosidase in resealed h u m a n red cells which were then coated with g a m m a globulin 5'e. Such red cells are avidly taken up by cultured monocytes in vitro ~ and they are cleared rapidly from the blood with subsequent accumulation in the liver and spleen 6.8. Other investigators have injected large
T I B S - A p r i l 1981
amounts of enzymes directly intravenously into patients with Gaucher's disease. Although initially the results were interpreted as biochemically encouraging 9, it is quite clear that little or nothing has been achieved clinically using such an approach TM. The putative decrease in storage material has not been convincingly demonstrated. The results obtained by infusion of enzyme-laden liposomes too have been of doubtful benefit". Organ transplantation
Transplantation of the affected organ or organ system from a genetically normal, immunologically compatible donor is one means of replacing the missing enzyme. Attempts to achieve this through kidney transplantation in the treatment of ~-galactosidase A deficiency (Fabry's disease) n.13 and to transplant a spleen TMinto a patient with Gaucher's disease have been unsuccessful. However, the technology of bone marrow transplantation has markedly advanced in the past few years 15,18,and the possibility of treating a disorder such as Gaucher's disease which affects the macrophage-monocyte system, by transplanting bone marrow does not appear to be hopelessly remote. Administration o f genes to replace missing enzymes
The striking advances which have occurred in the past few years in the technology of isolating genes and cloning them has made it possible to produce vast numbers of copies which code for needed proteins. To use such a technique in the treatment of disease, it is necessary not only to isolate and clone the required gene, but also to introduce it into the target cell relatively efficiently. Theoretically possible though it may be, there are many major technological hurdles to overcome before this type of enzyme replacement can be implemented. The isolation of the gene for a missing enzyme is, in itself, a major problem. Those genes which have been isolated in the past are generally ones which direct production of a vast a m o u n t of proteins. Thus, most of the synthetic activity of the developing erythroid cell is directed to the production of globin chains and plasma cells produce primarily immunoglobulin subunits. Genes which code for these proteins have been isolated. In contrast, the enzymes which are deficient in storage diseases exist in only minute quantities in any cell type. Once a gene has been cloned, it must be introduced into the target cell and inserted into its genome in such a way that regulation of its production is not seriously
impaired. D N A can be introduced into a few million cells by exposing them to large amounts of the D N A , particularly under conditions which facilitate D N A uptake, such as in the presence of calcium phosphate. The D N A may also be incorporated into a viral vector to allow its integration into the genome of a target cell. In either case, the number of cells receiving functional units is likely to be small, especially since introduction of D N A would probably have to be carried out in vitro. An interesting way in which this problem may be approached is to introduce the required gene into a cell with high proliferative potential together with a gene for drug resistance 17. If this is accomplished, then cells which have not received the exogenous D N A may be eliminated by suitable drug treatment.
Conclusions In spite of a n u m b e r of attempts, enzyme replacement therapy has not yet been successful in the treatment of disease. With advancing knowledge of factors which direct the uptake and incorporation of enzyme molecules by various cell types, with advances in immunology permitting organ transplantation, and with the development of sophisticated technology for the purification and implanting of DNA, it is to be hoped that diseases which are essentially untreatable today may yield to enzyme replacement therapy tomorrow.
Acknowledgement This work was supported by Grant A M 2 5 6 9 6 from the National Institute of Arthritis, Metabolic and Digestive Diseases.
References I Dale, G. L. and Beutler, E. (1976) Proc. Natl. Acad, Sci. U.S.A. 73, 46724674 2 Furbish, F. S., Blair, H. E., Shiloach,J., Pentchev, P. G. and Brady, R. O. 11977) Proc. Natl. Acad. Sci. U.S.A. 74, 3560-3563 3 Ashwell, G. and Morell, A, G. (1974) Adv. Enzymol. 41.99-128 4 Sly, W. S. and Stahl, P. (1978) (Silverstein,S. C., ed.), pp. 229-244, Dahlem Konferenzen,Berlin 5 Beutler, E., Dale, G. L., Guinto, E. and Kuhl, W, (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 46204623 6 Beutler, E., Dale, G. L. and Kuhl, W. 11980) in Enzyme Therapy in Genetic Diseases:2. (Desnick, R. J, ed.), pp. 369-381, Alan R. Liss,New York 7 Dale, G. L., Kuhl, W. and Beutler, E. (1979) Proc. Natl. Acad. Sei. U.S.A. 76, 473475 8 Beutler, E., Dale, G. L. and Kuhl, W. (1977) New Engl. J. Med. 296, 942-943 9 Brady, R. O., Pentchev, P. G., Gal, A. E., Hibbert, S. R. and Dekaban. A. S. (1974) New Engl. J. Med. 291,989 993 I0 Brady, R. O., Barranger, J. A., Gal, A. E., Pentchev. P. G. and Furbish, F. S. (1980) Enzyme
97
TIBS - April 1981 Therapy in Gaucher's Diseases:2. (Desnick, R. J., 13 Van den Bergh, F., Rietra, P., Kolk-Vegter,A. G., Bosch, E. and Tager, J. M. (1976) Acta. Med. ed.), pp. 361-368, Alan R. Liss,New York Scand. 200, 249-256 11 Belchetz,P. E., Crawley,J. C. W., Braidman, 1. P. and Gregoriadis, G. (1977) Lancet ii, 116-- 14 Groth, C. G., Dreborg, S., Ockerman, P. A., Svennerholm, L., Hagenfeldt, L., L6fstroem, B., 117 Samuelsson, K., Werner, B. and Westberg, G. 12 Spence, M. W., MacKinnon,K. E., Burgess,J. K., ( 1971) Lanceti, 1260-1264 D'Entremont, D. M., Belitsky, P., Lannon, S. G. and MacDonald, A. S. (1976)Ann. Intern. Med. 15 Thomas, E. D. (1979) Am. J. Clin. PathoL 72, 887-892 84, 13~16
16 Blume, K. G., Beutler, E., Bross, K. J., Chillar, R. K., Ellington, O. B., Fahey, J. L., Farbstein, M. J., Forman, S. J., Schmidt,G. M., Scott, E. P., Spruce, W. E., Turner, M. A. and Wolf, J. L. (1980) New EngLJ. Med. 302, 1041-1046 17 Cline, M. J., Stang, H., Mercola, K., Morse, L., Ruprecht, R., Browne. J. and Salser. W. (1980) Nature (London) 284, 422-425
Reviews Glycoproteins as cell-surface structural components Matthew F. Mescher The major cell-surface component o f Halobacterium salinarium is a glycoprotein having structural and biosynthetic features in common with eukaryotic glycoproteins. The occurrence o f glycoproteins in eukaryotes and archaebacteria suggests that they may have originated in primitive cells, which existed before the divergence o f the three major lines o f descent, possibly as structural components to stabilize the cell membrane.
All cells have limiting m e m b r a n e s of the same basic structure, ie. a lipid bilayer and associated integral m e m b r a n e proteins. In addition, peripheral components are present which interact with the membrane to stabilize it, providing greater rigidity than is possible with a bilayer alone. In many cases, these components provide the cell with a distinctive morphology. Organisms of each of the three major lines of evolutionary descent - eukaryotes, eubacteria and archaebacteria 1'2 - appear to use fundamentally different peripheral components to accomplish this stabilization. Eukaryotes have internal cytoskeletal systems of microtubules and microfilaments which are thought to interact with the inner surface of the membrane affecting cell shape, motility and influencing the mobility of the membrane proteins3; erythrocytes have a cytoskeletal matrix at the inner membrane surface'; and eubacteria have external components, in particular peptidoglycan, which maintain cell shape and rigidity 5. Organisms of the kingdom Archaebacteriae have neither a cytoskeletal system nor peptidoglycan 1.2. At least one m e m b e r of this group, Halobacterium salinarium, has a cell surface glycoprotein which appears to play a structural role in stabilizing the membrane and maintaining cell morphology. The similarities in structure and biosynthesis between this glycoprotein and those of eukaryotic cell surfaces have interesting evolutionary implications. Matthew F. Mescher is at the Department o f Pathology, Harvard Medical School, Boston, Massachusetts
02115, U.S.A.
The cell surface glycoprotein of H. salinarium About 52% of the neutral hexose present in the H. salinarium cell envelope is in the form of glycolipid. The remaining hexose and all of the amino sugar is attached to a high molecular weight glycoprotein which accounts for about half of the total envelope protein and is exposed at the cell surface 8.7. The carbohydrate units consist of di- and trisaccharides attached via O-glycosidic linkages between galactose and threonine, and a relatively large heterosaccharide attached via an N-glycosidic linkage to asparagine 7 (Fig. 1). N- and O-glycosidic linkages to amino and hydroxyl groups are the major means of attachment of carbohydrate to eukaryotic glycoproteins. The H. salinarium glycoprotein also resembles those of eukaryotes with respect to the number, size and composition of the carbohydrate units 7. Attachment of the N-linked carbohydrate, and possibly the O-linked units, of the H. salinarium glycoprotein occurs via isoprenol lipid-linked intermediates 8.~(Fig. 2). Similar pathways involving isoprenol lipid-linked intermediates were first demonstrated in peptidoglycan and lipopolysaccharide synthesis in eubacteria. They are now known to be involved in the glycosylation of eukaryotic glycoproteins 1° as well. The H. salinarium glycoprotein forms a rigid matrix at the cell surface and is responsible for maintenance of cell shape 6. These organisms are rod-shaped under normal growth conditions, despite the
absence of the peptidoglycan layer necessary for the maintenance of shape in most other bacteria. Lactoperoxidase-catalyzed iodination of surface proteins and treatment of intact cells with proteolytic enzymes show that the glycoprotein is the major cell surface component. It is visible in thin-section electron micrographs as a densely staining layer approximately 15 nm thick lying external to the plasma membrane. Removal of the glycoprotein from the surface of intact cells with proteolytic enzymes results in the rod-shaped cells becoming spherical. The spherical ceils remain viable, will grow and will regain the normal rod-shape on removal of the protease and the addition of fresh growth medium. When the glycoprotein content of the cells was examined during treatment with protease and subsequent regrowth, it was found that the glycoprotein disappeared concomitantly with the conversion from rods to spherical cells and reappeared at the same time as the cells returned to rod shape. Further evidence for the structural role of the glycoprotein was provided by experiments using bacitracin, an antibiotic which blocks the lipidintermediate pathway for glycosylation 200,000
5~ooo GOI ic
GOl GIc',XU
2-14
Fig. 1. Structureof the Halobacteriumsalinariumcell surface glycoprotein=. XUA is an unidentified hexuronic acid and XNAc an unidentified amino sugar. ~ Elsevier/North-Holland
Biomedical Press 1981