980
BIOCHEMICAL SOCIETY TRANSACTIONS
of the major part of three ‘hairpin’ loops. Studies on the interaction of the protein with slightly denatured coliphage-MS2 RNA appear to support the importance of a base-paired structure at the binding site; 15-25 % denaturation by formaldehyde or by heat treatment decreases the efficiency of IF3 binding and slightly decreases the amount of RNA protected. S1 protein, which does not appear to compete with IF3 for specific sites on coliphage-MS2 RNA, but which is known to unwind double-stranded stretches in coliphage-MS2 RNA (Thomas et a/., 1978), causes a similar decrease in the protection effect of initiation factor IF3. Location of the binding site such a long way from the initiation sites appears to rule out a function of initiation factor IF3 that would include recognition of initiation signals in these regions. Preferential binding to base-paired structures and the presence of a sequence ACCUCC homologous to the 3’-terminal region of 1 6 s rRNA (Shine & Dalgarno, 1974, 1975) overlapping the binding site, suggests that initiation factor IF3 may function at the stage of mRNA-rRNA annealing, which it may stabilize by attaching to the annealed RNA stretches. M. J. F. F. thanks the Science Research Council for the award of a Research Studentship. Dahlberg, A. E. & Dahlberg, J. E. (1975) Proc. Natl. Acad. Sci. U.S.A.72,2940-2944 Fiers, W., Contreras, R., Duerinck, F., Haegeman, G . , Iserentant, D., Merregaert, J., Min Jou, W., Molemans, F., Raeymaekers, A., Van den Berghe, A,, Volckaert, G . & Ysebaert, M . (1976) Nature (London)260,500-507
Jay, G . & Kaempfer, R. (1975) J . Biol. Chem. 250,5742-5748 Johnson, B. & Szekely, M. (1977) Nature (London)267,550-552 Johnson, B. & Szekely, M. (1978) Merhods Enzymol. in the press Shine, J. & Dalgarno, L. (1974) Proc. Nutl. Acud. Sci. U.S.A.71, 1342-1346 Shine, J. & Dalgarno, L. (1975) Nature (London) 254, 34-38 Steitz, J. A., Wahba, A. J., Laughrea, M. & Moore, P. B. (1977) Nurleir Acids Res. 4, 1-15 Thomas, J. O., Kolb. A. & Szer. W. (1978)J. Mol. Biol. 123. 163-1 76 Van Duin, J., Kurland, C . G.,’Dondon, J. & Grunberg-hlanago, M. (1975) FEBS Lett. 59, 287-290
Zipori, P., Bosch, L. & Van Duin, J. (1978) Eur. J. Biochem. 92,235-241
An Explanation for Variations in the Clinical and Biochemical Symptoms of Lysosomal-Enzyme Deficiency Diseases such as GMI Gangliosidosis PETER S. J. CHEETHAM Tate and Lyle Ltd., Group Research and Development, Philip Lyle Memorial Research Laboratories, Reading University, Whiteknights, P.O. Box 68, Reading, Berkshire RG6 2BX, U.K.
GM, gangliosidosis is characterized by autosomal recessive inheritance, mental degeneration and accumulations of glycolipids in both visceral and nervous tissues. It is difficult to classify p-galactosidase-deficiency-diseasepatients into groups with the same clinical symptoms, such as age of onset and death, involvement of visceral organs, rates of degeneration, and the same biochemical features such as degree of enzyme deficiency and the amounts and types of stored metabolites. (Derry et al., 1968; Wolf et a[., 1970; Singer & Schafer, 1972; Lowden et al., 1974; Pinsky et al., 1974; Loonen et a[., 1974; Kudoh et al., 1976; Suzuki et al., 1976; Ginsburg & Long, 1977). In the present paper, explanations are based on the primary lesion, the mutation of 8-galactosidase gene(s). Heterogeneity may arise because of multiple allelic mutations, genetic compounds, and because the various mutated subunit structures may differ in activity.
* Abbreviations used : Upper-case letters denote individual genes, whereas corresponding letters in lower-case italic denote the proteins produced by each gene. Subscripts indicate a mutant gene or protein: rn represents the degree of polymerization of the basic subunit structure. 1979
981
583rd MEETING, CAMBRIDGE
Mutation of enzymically or conformationally important amino acids causes loss of activity and thus the severe varieties of GMI gangliosidosis; but mutation of less important amino acids should cause less severe variants. If only one mutation occurs per gene and the probability of mutation of any base in a gene containing c codons is equal, then the number of possible alleles = 9c. Deletion, frameshift or chain-termination mutations probably inactivate the enzyme and so increase the proportion of severe variants . The Hardy-Weinberg rule predicts the frequency of genetic compounds that are intermediate between and easily distinguishable from homozgous phenotypes: A’*
+Biz+BZ2+2AB1+ 2AB2 +2BIBz = 1
(1)
where B, and Bz, 2B1B2and A are the gene frequencies of the allelic mutations, genetic compound and wild type respectively. The number of possible glycosidase structures depends on the number of different polypeptides involved and the number of subunits composing the enzyme (x). When only normal (a) and mutant (al) polypeptides are formed, the number of possible subunit structures (n)= x + 1 , x - 1 being heteromeric. Thus in a heterozygote the structures aa, aInl and aal are possible for a dimer, and four, five and nine different structures for the trimeric, tetrameric and octomeric cenzymes respectively. If both subunits are equally stable, combine randomly, and are synthesized in equal quantities expansion of: (a+al)m = 1 (2) gives the relative proportion of each mutant structure. For a dimer: a2+2aal+a1z = 1
(3)
aa, aal and alal occurring in the proportions 1 :2: 1, and for a tetramer aaaa, aaaal, uaulal,aalalal and aIalulaloccur in the proportions 1 :4: 6: 4: 1 .
If the mutated enzyme retains only 10%of normal activity and provided the activities of each subunit are independent, the relative activities of aa, aa, and alaI are in the ratio 20: 11:2. If al is inactive, then aal has half the activity of aa. However, if only unmutated subunits can combine, residual activity is proportional to the amount of unmutated subunits. When heteromers are inactive or when the activity of a is decreased by combination with al, the glycosidase activity is lower and in the former case equals the proportion of enzyme without a I . If less a, than a is present, because it is less stable or synthesized more slowly, lower proportions of the structures containing al result. For the dimer the relative proportions of aa, aa, and alal are derived from the binomial equation above (2). In the heterozygote, where the concentration of a is twice that of al (z = 2), the structures occur in the proportions 1:2/z:1/z2, i.e. 4:4:1, or 9:6:1 when z = 3. Likewise for a tetramer when z = 2 , the structures aaaa, aaaa, aaala,, aalalal and alalaIaI occur in the ratio 1 :4/z :6/z2:4/z3: 1 /z4 (1 6 :32 :24 :8 : 1) Many genetic diseases including the occasional examples of dominant inheritance have a polygenetic nature (Hsia et al., 1962). As some 8-galactosidase isoenzymes are multimers (Cheetham & Dance, 1976), dominance may be explained by a requirement for two or more structural genes. Thus the greater the number of independently mutating subunits comprising (a) glycosidase(s), then the greater the number of phenotypes possible, i.e. n = 2s (4) Thus mutation of momomeric (a), dimeric (ab), trimeric (abc) and tetrameric (abcd) glycosidases composed of the polypeptides a, 6, c and d allows the possibility of two, four, eight and sixteen different types of isoenzyme respectively. Alternatively for enzymes of the structure m (ab) n = (m+l)
assuming that mutation of any subunit of the same type is equivalent.
Vol. I
(5)
982
BIOCHEMICAL SOCIETY TRANSACTIONS
In heterodimeric glycosidases (ab), four structures are possible (ab, alb, albl and abl). Similarily the heterotetramer (2a2b), heterohexamer and heterooctomer may
form nine, sixteen and twenty-five possible structures. Individuals displaying very similar clinical and biochemical symptoms include double heterozygotes (AA,BB,), homozygotes for either subunit (AIAIBB or AAB,B,), the double homozygote (AIAIBIB1)or individuals homozygous for one subunit and heterozygous for the other (AAIBIB1 or AIAIBB1), the relative frequency of each genotype being calculated by multiplying the frequencies of each gene. Independent mutation of the subunits of a heteropolymeric enzyme causes variations in the genetic status, the subunit structure of the residual enzymes, and the residual activities and severity of the disease. The residual activity also depends on whether mutant subunits can combine with unmutated subunits, and on the possibility that structures such as albl may regain activity by intergenic complementation. Variation may range from complete enzyme inactivation and severe symptoms, to a very mild loss of activity in which the patient has few symptoms, such as the benign deficiency of B-galactosidase B (Cheetham et al., 1978), Non-penetrance, expressivity and environmental differences may further increase phenotypic heterogeneity and could explain the wide normal variations in glycosidase activity (Van Hoof & Hers, 1968). Variations due to point mutations are most likely, definite evidence that the other mutations occur requires precise characterization of the genetics of the disease and the enzymes involved (Galjaard et al., 1975; Koster et al., 1975). Characterization may also elucidate relationships between gene and enzyme structure, function, and control, and enable accurate genetic counselling and prenatal diagnosis. Cheetham, P. S. J. & Dance, N. E. (1976) Biochem. J. 157,189-195 Cheetham, P. S. J., Dance, N. E. & Robinson, D. (1978) Clin. Chim. Actu 83,67-74 Derry, D. M., Fawcett, J. S., Andermann, F. & Wolf, L. S. (1968) Neurology 18,340-348 Galjaard, H., Hoogeveen, A., Keijer, W., de Wit-Verbeek, H. A., Reuser, A. J. J., Ho, M. W. & Robinson, D. (1975) Nature (London) 257,60-62 Ginsburg, G. M. &Long, G. G. (1977)J. Med. Genef.14,132-134 Hsia, D. Y.-Y., Naylor, J. & Bigler, J. A. (1962) Symp. Tay-Sachs Dis. Allied Disord. 327-342 Koster, J. F., Niermeyer, M. F., Loonen, M. C. B. & Galjaard, H. (1976) Clin. Genet. 9,427-432 Kudoh, T., Orii, T., Nakao, T., Sakagami, T. (1976) Cliiz. Chim. Actu 70,277-283 Lowden, J. A., Callahan, J. W., Norman, M. G., Thain, M. & Pritchard, J. S. (1974) Arch. Neurol. 31,200-203
Loonen, M. C. B., van den Lugt, L. & Franke, C. L. (1974) Lancet ii, 785 Pinsky, L., Miller, J., Stanfield, J., Walters, G. & Wolf, L. S . (1974) Am. J . Hum. Genet. 26, 563-577
Singer, H. S. & Schafer, I. A. (1972) Am. J. Hum. Genet. 24,454-463 Suzuki, Y., Hayakawa, T., Yazaki, M. & Hiratini, Y. (1976) Eur. J. Puediut. 122, 178-187 Van Hoof, F. & Hers, H.-G. (1968) Eur. J . Biochem. 7 , 34-44 Wolf, L. S., Callahan, J., Fawcett, J. S.. Andermann, F. & Scriver, C. R. (1970) Neurology 20, 23-44
Effect of a Mononuclear-CellFactor on Prostaglandin Production by Cells Cultured from Human Gingiva, Synovium, Cartilage and Endometrium D. J. ENGLIS, S. M. DSOUZA, J. E. MEATS, M. B. McGUIRE, J. WRIGHT and R. G. G. RUSSELL Department of Human Metabolism and Clinical Biochemistry, University of Shefield Medical School, Beech Hill Road, Shefield S10 2 R X , U.K. It has been shown (Dayer et al., 1977)that normal human peripheral-blood lymphocytes and monocytes incubated in culture release a soluble factor that stimulates prostaglandin E production by adherent cells isolated from human rheumatoid synovium. The aim of the present study was to determine the specificity of this phenomenon in cells obtained from several other human tissues. 1979
BIOCHEMICAL SOCIETY TRANSACTIONS
of the major part of three ‘hairpin’ loops. Studies on the interaction of the protein with slightly denatured coliphage-MS2 RNA appear to support the importance of a base-paired structure at the binding site; 15-25 % denaturation by formaldehyde or by heat treatment decreases the efficiency of IF3 binding and slightly decreases the amount of RNA protected. S1 protein, which does not appear to compete with IF3 for specific sites on coliphage-MS2 RNA, but which is known to unwind double-stranded stretches in coliphage-MS2 RNA (Thomas et a/., 1978), causes a similar decrease in the protection effect of initiation factor IF3. Location of the binding site such a long way from the initiation sites appears to rule out a function of initiation factor IF3 that would include recognition of initiation signals in these regions. Preferential binding to base-paired structures and the presence of a sequence ACCUCC homologous to the 3’-terminal region of 1 6 s rRNA (Shine & Dalgarno, 1974, 1975) overlapping the binding site, suggests that initiation factor IF3 may function at the stage of mRNA-rRNA annealing, which it may stabilize by attaching to the annealed RNA stretches. M. J. F. F. thanks the Science Research Council for the award of a Research Studentship. Dahlberg, A. E. & Dahlberg, J. E. (1975) Proc. Natl. Acad. Sci. U.S.A.72,2940-2944 Fiers, W., Contreras, R., Duerinck, F., Haegeman, G . , Iserentant, D., Merregaert, J., Min Jou, W., Molemans, F., Raeymaekers, A., Van den Berghe, A,, Volckaert, G . & Ysebaert, M . (1976) Nature (London)260,500-507
Jay, G . & Kaempfer, R. (1975) J . Biol. Chem. 250,5742-5748 Johnson, B. & Szekely, M. (1977) Nature (London)267,550-552 Johnson, B. & Szekely, M. (1978) Merhods Enzymol. in the press Shine, J. & Dalgarno, L. (1974) Proc. Nutl. Acud. Sci. U.S.A.71, 1342-1346 Shine, J. & Dalgarno, L. (1975) Nature (London) 254, 34-38 Steitz, J. A., Wahba, A. J., Laughrea, M. & Moore, P. B. (1977) Nurleir Acids Res. 4, 1-15 Thomas, J. O., Kolb. A. & Szer. W. (1978)J. Mol. Biol. 123. 163-1 76 Van Duin, J., Kurland, C . G.,’Dondon, J. & Grunberg-hlanago, M. (1975) FEBS Lett. 59, 287-290
Zipori, P., Bosch, L. & Van Duin, J. (1978) Eur. J. Biochem. 92,235-241
An Explanation for Variations in the Clinical and Biochemical Symptoms of Lysosomal-Enzyme Deficiency Diseases such as GMI Gangliosidosis PETER S. J. CHEETHAM Tate and Lyle Ltd., Group Research and Development, Philip Lyle Memorial Research Laboratories, Reading University, Whiteknights, P.O. Box 68, Reading, Berkshire RG6 2BX, U.K.
GM, gangliosidosis is characterized by autosomal recessive inheritance, mental degeneration and accumulations of glycolipids in both visceral and nervous tissues. It is difficult to classify p-galactosidase-deficiency-diseasepatients into groups with the same clinical symptoms, such as age of onset and death, involvement of visceral organs, rates of degeneration, and the same biochemical features such as degree of enzyme deficiency and the amounts and types of stored metabolites. (Derry et al., 1968; Wolf et a[., 1970; Singer & Schafer, 1972; Lowden et al., 1974; Pinsky et al., 1974; Loonen et a[., 1974; Kudoh et al., 1976; Suzuki et al., 1976; Ginsburg & Long, 1977). In the present paper, explanations are based on the primary lesion, the mutation of 8-galactosidase gene(s). Heterogeneity may arise because of multiple allelic mutations, genetic compounds, and because the various mutated subunit structures may differ in activity.
* Abbreviations used : Upper-case letters denote individual genes, whereas corresponding letters in lower-case italic denote the proteins produced by each gene. Subscripts indicate a mutant gene or protein: rn represents the degree of polymerization of the basic subunit structure. 1979
981
583rd MEETING, CAMBRIDGE
Mutation of enzymically or conformationally important amino acids causes loss of activity and thus the severe varieties of GMI gangliosidosis; but mutation of less important amino acids should cause less severe variants. If only one mutation occurs per gene and the probability of mutation of any base in a gene containing c codons is equal, then the number of possible alleles = 9c. Deletion, frameshift or chain-termination mutations probably inactivate the enzyme and so increase the proportion of severe variants . The Hardy-Weinberg rule predicts the frequency of genetic compounds that are intermediate between and easily distinguishable from homozgous phenotypes: A’*
+Biz+BZ2+2AB1+ 2AB2 +2BIBz = 1
(1)
where B, and Bz, 2B1B2and A are the gene frequencies of the allelic mutations, genetic compound and wild type respectively. The number of possible glycosidase structures depends on the number of different polypeptides involved and the number of subunits composing the enzyme (x). When only normal (a) and mutant (al) polypeptides are formed, the number of possible subunit structures (n)= x + 1 , x - 1 being heteromeric. Thus in a heterozygote the structures aa, aInl and aal are possible for a dimer, and four, five and nine different structures for the trimeric, tetrameric and octomeric cenzymes respectively. If both subunits are equally stable, combine randomly, and are synthesized in equal quantities expansion of: (a+al)m = 1 (2) gives the relative proportion of each mutant structure. For a dimer: a2+2aal+a1z = 1
(3)
aa, aal and alal occurring in the proportions 1 :2: 1, and for a tetramer aaaa, aaaal, uaulal,aalalal and aIalulaloccur in the proportions 1 :4: 6: 4: 1 .
If the mutated enzyme retains only 10%of normal activity and provided the activities of each subunit are independent, the relative activities of aa, aa, and alaI are in the ratio 20: 11:2. If al is inactive, then aal has half the activity of aa. However, if only unmutated subunits can combine, residual activity is proportional to the amount of unmutated subunits. When heteromers are inactive or when the activity of a is decreased by combination with al, the glycosidase activity is lower and in the former case equals the proportion of enzyme without a I . If less a, than a is present, because it is less stable or synthesized more slowly, lower proportions of the structures containing al result. For the dimer the relative proportions of aa, aa, and alal are derived from the binomial equation above (2). In the heterozygote, where the concentration of a is twice that of al (z = 2), the structures occur in the proportions 1:2/z:1/z2, i.e. 4:4:1, or 9:6:1 when z = 3. Likewise for a tetramer when z = 2 , the structures aaaa, aaaa, aaala,, aalalal and alalaIaI occur in the ratio 1 :4/z :6/z2:4/z3: 1 /z4 (1 6 :32 :24 :8 : 1) Many genetic diseases including the occasional examples of dominant inheritance have a polygenetic nature (Hsia et al., 1962). As some 8-galactosidase isoenzymes are multimers (Cheetham & Dance, 1976), dominance may be explained by a requirement for two or more structural genes. Thus the greater the number of independently mutating subunits comprising (a) glycosidase(s), then the greater the number of phenotypes possible, i.e. n = 2s (4) Thus mutation of momomeric (a), dimeric (ab), trimeric (abc) and tetrameric (abcd) glycosidases composed of the polypeptides a, 6, c and d allows the possibility of two, four, eight and sixteen different types of isoenzyme respectively. Alternatively for enzymes of the structure m (ab) n = (m+l)
assuming that mutation of any subunit of the same type is equivalent.
Vol. I
(5)
982
BIOCHEMICAL SOCIETY TRANSACTIONS
In heterodimeric glycosidases (ab), four structures are possible (ab, alb, albl and abl). Similarily the heterotetramer (2a2b), heterohexamer and heterooctomer may
form nine, sixteen and twenty-five possible structures. Individuals displaying very similar clinical and biochemical symptoms include double heterozygotes (AA,BB,), homozygotes for either subunit (AIAIBB or AAB,B,), the double homozygote (AIAIBIB1)or individuals homozygous for one subunit and heterozygous for the other (AAIBIB1 or AIAIBB1), the relative frequency of each genotype being calculated by multiplying the frequencies of each gene. Independent mutation of the subunits of a heteropolymeric enzyme causes variations in the genetic status, the subunit structure of the residual enzymes, and the residual activities and severity of the disease. The residual activity also depends on whether mutant subunits can combine with unmutated subunits, and on the possibility that structures such as albl may regain activity by intergenic complementation. Variation may range from complete enzyme inactivation and severe symptoms, to a very mild loss of activity in which the patient has few symptoms, such as the benign deficiency of B-galactosidase B (Cheetham et al., 1978), Non-penetrance, expressivity and environmental differences may further increase phenotypic heterogeneity and could explain the wide normal variations in glycosidase activity (Van Hoof & Hers, 1968). Variations due to point mutations are most likely, definite evidence that the other mutations occur requires precise characterization of the genetics of the disease and the enzymes involved (Galjaard et al., 1975; Koster et al., 1975). Characterization may also elucidate relationships between gene and enzyme structure, function, and control, and enable accurate genetic counselling and prenatal diagnosis. Cheetham, P. S. J. & Dance, N. E. (1976) Biochem. J. 157,189-195 Cheetham, P. S. J., Dance, N. E. & Robinson, D. (1978) Clin. Chim. Actu 83,67-74 Derry, D. M., Fawcett, J. S., Andermann, F. & Wolf, L. S. (1968) Neurology 18,340-348 Galjaard, H., Hoogeveen, A., Keijer, W., de Wit-Verbeek, H. A., Reuser, A. J. J., Ho, M. W. & Robinson, D. (1975) Nature (London) 257,60-62 Ginsburg, G. M. &Long, G. G. (1977)J. Med. Genef.14,132-134 Hsia, D. Y.-Y., Naylor, J. & Bigler, J. A. (1962) Symp. Tay-Sachs Dis. Allied Disord. 327-342 Koster, J. F., Niermeyer, M. F., Loonen, M. C. B. & Galjaard, H. (1976) Clin. Genet. 9,427-432 Kudoh, T., Orii, T., Nakao, T., Sakagami, T. (1976) Cliiz. Chim. Actu 70,277-283 Lowden, J. A., Callahan, J. W., Norman, M. G., Thain, M. & Pritchard, J. S. (1974) Arch. Neurol. 31,200-203
Loonen, M. C. B., van den Lugt, L. & Franke, C. L. (1974) Lancet ii, 785 Pinsky, L., Miller, J., Stanfield, J., Walters, G. & Wolf, L. S . (1974) Am. J . Hum. Genet. 26, 563-577
Singer, H. S. & Schafer, I. A. (1972) Am. J. Hum. Genet. 24,454-463 Suzuki, Y., Hayakawa, T., Yazaki, M. & Hiratini, Y. (1976) Eur. J. Puediut. 122, 178-187 Van Hoof, F. & Hers, H.-G. (1968) Eur. J . Biochem. 7 , 34-44 Wolf, L. S., Callahan, J., Fawcett, J. S.. Andermann, F. & Scriver, C. R. (1970) Neurology 20, 23-44
Effect of a Mononuclear-CellFactor on Prostaglandin Production by Cells Cultured from Human Gingiva, Synovium, Cartilage and Endometrium D. J. ENGLIS, S. M. DSOUZA, J. E. MEATS, M. B. McGUIRE, J. WRIGHT and R. G. G. RUSSELL Department of Human Metabolism and Clinical Biochemistry, University of Shefield Medical School, Beech Hill Road, Shefield S10 2 R X , U.K. It has been shown (Dayer et al., 1977)that normal human peripheral-blood lymphocytes and monocytes incubated in culture release a soluble factor that stimulates prostaglandin E production by adherent cells isolated from human rheumatoid synovium. The aim of the present study was to determine the specificity of this phenomenon in cells obtained from several other human tissues. 1979
