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Circular permutation of amino acid sequences among legume lectins
100
TIBS - March 1983
within the J and LAO proteins there are segments which are much more highly conserved (>90% homology) than the overall protein sequence (70% homology). These conserved regions are believed to be involved in forming that domain of each protein molecule which is responsible for the interaction with the membrane protein P (Fig. 2) a~. Mutational alteration in one of these highly conserved regions of the J protein results in a loss of the ability of J to interact with P. The level of homology maintained between J and LAO is such that a chimeric protein resulting from a deletion fusing the amino terminal half of LAO to the carboxy terminal half of J functions perfectly normally with the membrane-bound components, though it maintains the substrate specificity of LAO 1'~. Therefore, this system, and independently each of the other systems, would have evolved as a 'package'. Thus, the evidence that a complex ancestral system would have spawned the present multiplicity of periplasmic permeases is tantalizing. However, more systems need
to be fully characterized before serious formulation of overall models for the molecular mechanism of transport and for their evolutionary history is possible. References 1 Heppel, L. A. (1971) in Structure and Function o f Biological Membranes (Rothfield, L. I., ed. ), pp. 223-247, Academic Press, New York 2 Higgins, C. F., Haag, P. D., Nikaido, K., Ardeshir, F., Garcia, G. and Ames, G. F.-L. (1982)Nature (London) 298,723-727 3 Oxender, D. L., Quay, S. C. and Anderson, J. J. (1980) in Microorganisms and Nitrogen Sources (Payne, J. W., ed.), pp. 153-169, John Wiley and Sons, New York 4 Shuman, H. A. (1982)Ann. Microbiol. (Inst. Pasteur) 133A, 153-159 5 Robbins, A. R., Guzman, R. and Rotman, B. (1976)J. Biol. Chem. 251,3112-3116 6 Clark, A. F. and Hogg, R. W. (1981 )J. Bacteriol. 147,920-924 7 Furlong, C. E. and Schellenberg, G. D. (1980) in Microorganisms and Nitrogen Sources (Payne, J. W., ed.), pp. 89-123~ John Wiley and Sons, New York 8 Robertson, D. E., Kroon, P. A. and Ho, C. (1977) Biochemistry 16, 1443-1451
Circular permutation of amino acid sequences among legume lectins John J. Hemperly and Bruce A. Cunningham Lectins, the carbohydrate-binding proteins found in a variety o f organisms, are a diverse group o f proteins. Many lectins from leguminous plants, however, are closely related to each other and are related to the jack bean lectin Concanavalin A by an unusual circular permutation of amino acid sequences. Lectins are an assortment of proteins that share the ability to bind stereospecifically and reversibly to carbohydrates, in particular to the sugar moieties of glycoproteins and glycolipids1. These proteins have been found in plants, bacteria, cellular slime molds, invertebrates and vertebrates but their normal biological functions are largely unknown. The lectin from vertebrate liver is probably the most well characterized in terms of its physiological function. This molecule plays a key role in the uptake of desialyated glycoproteins and their removal from the blood stream 2. Nearly all of the other lectins have been implicated in various types of cell--cell interactions. Bacterial lectins are presumed to provide a means by which micro-organisms can attach to epithelial cells and tissues prior to infecJohn J. Hemperly and Bruce A. Cunningham are at The Rockefeller University, 1230 York Avenue, New York, N Y 10021, U.S.A.
tion 3. Slime mold lectins have been assumed to play a role in cell-cell adhesion during the development of multicellular colonies from discrete amoebae, although conclusive evidence to support this notion is still lacking*. Several plant lectins have been postulated to mediate the binding of symbiotic Rhizobia to legume roots (see below). Thus it is clear that the group of molecules designated as lectins includes a variety of proteins and glycoproteins with diverse structures, different origins and probably distinct biological functions. Many lectins in leguminous plants, however, appear to be closely related, despite the fact that initial studies suggested that they too would be a collection of heterogeneous molecules. Lectins were first recognized in extracts of the seeds and beans of the Leguminosae. They were initially used in
,t'~'ElsevierBiomedicalPress 1983 0376 5067/83/0000- 0000/$0100
9 Ames, G. F.-L. and Spudich, E. N. (1976)Proc. Natl Acad. Sci. U.S.A. 73, 1877-1881 10 Shuman, H. A. (1982)J. Biol. Chem. 257, 5455-5461 11 Hong, J.-H., Hunt, A. G., Masters, P. S. and Lieberman, M. A. (1979) Proc. Natl Acad. Sci. U.S.A. 76, 1213-1217 12 Landick, R., Anderson, J. J., Mayo, M. M., Gunsalus, R. P., Mavromara, P., Daniels, C. J. and Oxender, D. L. (1980)J. Supramol. Structure 14, 527-537 13 Boos, W. (1982)Ann. Microbiol. (Inst. Pasteur) 133A, 145-151 14 Strange, P. G. and Koshland, D. E., Jr (1976) Proc. Natl Acad. Sci. U.S.A. 73,762-766 15 Bavoil, P. and Nikaido, H. (1981)J. Biol. Chem. 256, 11385-11388 16 Higgins, C. F. and Ames, G. F.-L. (1981)Proc. Natl Acad. Sci. U.S.A. 78, 6038-6042 17 Mahoney, W. C., Hogg, R. W. and Hermodson, M. A. (1981)J. Biol. Chem. 256, 4350-4356 18 Rosen, P. (1971)J. Biol. Chem. 246, 3653-3662 19 Clement. J. M. and Hofnung, M. ( 1981 ) Cell 27, 507-514 20 Gilson, E., Higgins, C. F., Hofnung, M., Ames, G. F.-L. and Nikaido, H. (1982)J. Biol. Chem. 257, 9915-9918 21 Gilliland, G. L. and Quiocho, F. A. (1981) J. Mol. Biol. 146,341-362
serological blood typing because different lectins preferentially agglutinate red blood cells of different types. With the demonstration that some plant lectins could also induce mitogenesis in lymphocytes5, these molecules became widely used reagents in immunology. They have since been used extensively to detect and perturb glycoproteins on cell surfaces and as affinity reagents for purifying glycoproteins. Concanavalin A (Con A) from the jack bean is one of the most abundant legume lectins. It is also the most well characterized. The protein was f'u'st crystallized by Sumner in 1919 (Ref. 6) and its amino acid sequence and three-dimensional structure were described in the early 1970s (Refs 7-9). Its biological activities have been analysed in detail, including both its ability to stimulate mitosis in lymphocytes and its dose-dependent ability to induce or inhibit the mobility of cell surface receptors ~°. At physiological pH, Con A is a tetramer of identical polypeptide chains, each containing 237 amino acids, one Ca 2+ and one Mn 2+. The polypeptide chain of each monomer is folded into two extensive /3-pleated sheets, one at the back and one through the center of the molecule. The carbohydrate-binding site is a shallow depression at the top of the molecule near the metal atoms 1~.~. There is also a deep cavity at the back of each monomer that appears to be capable of binding hydrophobic compounds. Below pH 5 or on chemical derivatization of amino groups,
TIBS
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Fig. 1. Comparison of the amino acid sequences of Con A (Reds 7,8) (top line in each row), soybean agglutinin (SBA) ( Ref 19) (second line), and favin ( Refs 14,, 20) (bottom line). Gaps are inserted in all sequences to maximize homology. Boxes enclose identical residues in at least two of the proteins. Arrows indicate the amino termini of the a and/3 chains of ravin, and dashes indicate regions of SBA that are as yet undetermined (adapted from Ref. 19 and reproduced by permission of Academic Press, Inc.).
the Con A tetramer dissociates to dimers that retain many of the biological properties of the tetramerTM. The dimer differs from the tetramer in that it lacks the ability to inhibit mitogenesis and receptor mobility at high concentrations19. Lectins from several other Leguminosae appeared at first to be somewhat different to Con A (for a review, see Ref. 1). Carbohydrate-binding proteins from the red kidney bean (PHA), peanut (PNA), and soybean (SBA) are similar in size to the Con A tetramer and are composed of polypeptides similar in size to the Con A monomer; however, they bind specifically to galactose derivatives whereas Con A binds glucose and mannose. In addition, SBA and PHA are glycoproteins, whereas Con A contains no covalently bound carbohydrate. The lectins from lentils and peas, and subsequently the lectin favin from fava beans",ts, were shown to have sugar-binding specificities similar to those of Con A but these molecules are only half the size of the Con A tetramer. Moreover, they are made up of
two types of polypeptide chains (a and B), both smaller than the Con A monomer. Like SBA and PHA, favin is a glycoprotein. Initial studies by Foriers et al. showed that despite these differences, the aminoterminal amino acid sequences of the lentil and pea/3 chains, and of SBA, PNA, and two types of PHA are homologousTMto portions of Con A (Ref. 17). The extent of this similarity and its unusual nature were shown by the analysis of the complete amino acid sequence of favinTM, by the completion of the structure of the lentil lectinTM, and by more recent analysis of the structure ofSBA (Ref. 20). The amino acid sequences of Con A, favin and SBA are shown in Fig. 1 and are aligned according to the sequence of Con A. The similarities are extensive; moreover, the residues that contribute to the distinguishing features of the threedimensional structure of Con A are highly conserved. The unusual feature of this comparison is the manner in which favin and SBA must be aligned with Con A in
237
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Fig. 2. Schematic drawings of the alignment of ravin el and fl chains (open bars), soybean agglutinin (SBA) (stippled bar), and Con A (dark bar) showing the circular permutation that gives maximum homology among the three sequences shown in Fig 1. The dashed line transecting the ravin fl chain indicates the region where the homologous 13chain of the lentil lectin ends. The location of the coral. ently bound carbohydrate in ravin is indicated by the asterisk (adapted from Ref. 20 and reproduced by permission of Academic Press, Inc. ).
102 order to show the homology. As with the a chains o f lentil and pea lectins, the a chain o f favin is homologous to residues 70 to 119 of Con A; the/3 chain of favin is homologous to Con A beginning at residue 120. The homology in the/3 chain continues to the carboxyl terminus o f Con A and without interruption through the amino-terminal region of Con A so that the/3 chain of favin ends at a position homologous to the position where the a chain begins 2~. This circular permutation is illustrated schematically in Fig. 2. The lentil lectin is directly comparable to favin but its/3 chain is slightly shorter and lacks the region homologous to the carbohydrate-containing portion of the favin/3 chainlL (The comparable portion of Con A lacks the sequence Asn-X-Thr/Ser usually associated with asparagine-linked carbohydrate22). The pea lectin also lacks covalently bound sugar and may be directly comparable to the lentil lectin. The amino acid sequence of SBA is also permuted with regard to Con A; its amino terminus, however, is homologous to that of the favin B-chain, and the region o f SBA comparable to the favin ct chain is at the carboxyl terminus (see Fig. 2). The location of the carbohydrate in SBA has not yet been established. The two PHA glycoproteins will presumably have structures similar to that of SBA; PNA will probably be similar but may be shortened much like the lentil lectin is with respect to ravin. Several other legume iectins have amino-terminal amino acid sequences homologous to those mentioned above. In particular, the lectin from Vicia sativa is very similar to favin ~ and the D o l i c h o s b i f l o r u s lectin seems to be homologous to PHA (Ref. 24). These extensive homologies do not appear to extend to non-legume lectins such as wheat germ agglutinin 2s and the R i c i n u s c o m m u n i s lectin ~6. The comparison of favin to SBA raised the possibility that favin is synthesized as a SBA-like precursor and that the a and/3 chains are generated by proteolysis after translation (Fig. 3). To test this hypothesis, we have recently translated favin messenger RNA in vitro 27. The ravin molecule appears to be synthesized as a single polypeptide precursor of a size comparable to the sum of the a and/3 chains and is preceded by a 29-residue signal sequence. The signal sequence is followed by the sequence of the favin/3 chain; the c~ chain sequence is also included in the precursor, presumably at the carboxyl-terminal end. Such a precursor is directly comparable to a SBA-like polypeptide. Processing of the favin precursor molecule by dog pancreas membranes resulted in glycosylation, removal o f the signal sequence, and some apparently aberrant cleavage, but we were
TIBS - March 1 983
(Sda, M., ed.), Vol. 4, pp. 429-529, Academic Press, New York 2 Ashwell, G. and Mot'ell, A. G. (1974) Adv. Enzymol. Relat. Areas Mol. Biol. 41, 99-128 S ,8 CHO 30fek, l., Beachley, E. H. and Sharon, N. (1978) I a r---q I I I Trends Biochem. Sci. 3, 159-160 4 Barondes, S. H. (1981) Annu. Rev. Biochem. 50, 207-23 l B CHO I a 5 Nowell,P. D. (1960) Cancer Res. 20, 462--466 I I I I 6 Sumner, J. B. and Howell, S. F. (1936) J. Bacteriol. 32, 227-237 Fig. 3. Schematic representation o f the biosynthesis 7 Edelman, G. M., Cunningham, B. A., Reeke, o f favin. The signal peptide (S), the ct chain (cO, the G. N., Jr, Becket, J. W., Waxdal, M. J. and chain (fl), and covalently bound carbohydrate Wang, J. L. (1972)Proc. Natl Acad. Sci. U.S.A. ( CHO) are indicated. 69, 2580-2584 8 Becket,J. W., Cunningham,B. A., Reeke,G. N., not able to generate polypeptides comparJr, Wang, J. L. and Edelman, G. M. (1976) in able to the favin a and/3 chains using this Concanavalin A as a Tool (Bittiger, H. and Schnebli,H. P., eds), pp. 33-54, John Wileyand system. Sons, London The comparisons o f these lectins suggest 9 Hardman, K. D. and Ainsworth, C. F. (1972) that they are closely related evolutionarily, Biochemistry I l, 4910--4919 will probably have similar threel0 Edelman,G. M. (1976)Science 192,218-226 dimensional structures, and will probably II Becket, J. W., Reeke, G. N., Jr, Cunningham, serve similar functions. From these limited B. A. and Edelman, G. M. (1976) Nature (Loncomparisons, Con A would appear to be the don) 259,406--409 unusual protein in that it is circularly per- 12 Hardman, K. D. and Ainsworth, C. F. (1976) Biochemistry 15, 1120-1128 muted with respect to the others. A number of hypotheses have been suggested to 13 Gunther,G. R., Wang, J. L., Yahara, I., Cunningham, B. A. and Edelman, G. M. (1973) Proc. account for this permutation xs.~9.we favor Natl Acad. Sci. U.S.A. 70, 1012-1016 mechanisms that implicate alteration at the 14 Wang, J. L., Becket, J. W., Reeke, G. N., Jr and level o f DNA and particularly those Edelman,G. M. (1974)J. Mol. Biol. 88,259-262 mechanisms involving gene duplication 15 Hemperly,J. J., Hopp, T. P., Becker, J. W. and followed by expression of a permuted lectin Cunningham, B. A. (1979)J. Biol. Chem. 254, 6803--6810 gene. Some valuable clues to this question will undoubtedly be obtained by examining 16 Foriers, A., Wuilmart, C., Sharon, N. and Strosberg, A. D. (1977) Biochem. Biophys. Res. the genes for these molecules using cDNA Commun. 75,980-986 and other probes. We expect that a number 17 Foriers, A., de Neve, R., Kanarek, L. and Strosof genes for at least some of these lectins berg, A. D. (1978) Proc. Nad Acad. Sci. U.S.A. will be found because we and others have 75, 1136-1139 detected closely related but non-identical 18 Canningham,B. A., Hempedy,J. J., Hopp, T. P. and Edelman,G. M. (1979)Proc. NatlAcad. Sci. polypeptides in the lectins o f some plants U.S.A. 76, 3218-3222 (for examples, see Refs 16, 28). 19 Foriers, A., Lebnm, E., Van Rapenbusch,R., de The key remaining question about these Neve, R. and Strosberg, A. D. (1981) J. Biol. lectins in L e g u m i n o s a e is their biological Chem. 256, 5550-5560 function. Some evidence has been pre- 20 Hemperly,J. J., Becker, J. W. and Cunningham, sented for a role in binding nitrogen-fixing B. A. (1982) in Proteins in Biology and Medicine (Bradshaw, R. A., Hill, R., Tang, J., bacteria to roots2L While this seems more Liang, C., Tsao, T. and Tson, C., eds), pp. plausible than a simple role as 'storage pro395-409, AcademicPress, New York teins' or in a pseudo-immune function, it is 21 Hopp, T. P., Hemperly, J. J. and Cunningham, by no means proven. In seeking a function, B. A. (1982)J. Biol. Chem. 257, 4473-4483 the temptation is to emphasize the 22 Marshall,R. D. (1972)Annu. Rev. Biochem. 41, carbohydrate-binding properties of these 673-702 molecules, but this prejudice is clearly 23 Gebauer, G., Schlitz, E. and Riidiger, H. (1981) Eur. J. Biochem. 113,319-325 based on the methods initially used to detect lectins. There is on Con A at least one addi- 24 Etzler, M. E., Talbot, C. F. and Ziaya, P. R. FEBS Lett. 82, 39-41 tional site, the hydrophobic cavity, that could be equally important in functional 25 Wright,C. S. (1977)J. Mol. Biol. I 11,439-457 26 Cawley, D. B., Hedblom, M. L. and Houston, considerations, especially since it has been L. L. (1978)Arch. Biochem. Biophys. 190, shown that Con A can bind plant auxins 3°. 744-755 With renewed interest in plant biochemistry 27 Hemperly,J. J., Mostov, K. E. and Cunningham, and the relative abundance o f lectins in B. A. (1982)J. Biol. Chem. 257, 7903-7909 important food sources, the elucidation of 28 Lolan, R., Cacan, R., Cacan, M., Debmy, H., Carter, W. G. and Sharon, N. (1975)FEBS Lett. the function o f these molecules is an obvi57, 100-103 ous and worthwhile goal. 29 Broughton,W. J. (1978)J. Appl. Bacteriol. 45, 165-194 30 Edelman,G. M. and Wang, J. L. (1978)J. Biol. References Chem. 253, 3016-3022 1 Lis, H. and Sharon, N. (1977) in The Antigens s
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TIBS - March 1983
within the J and LAO proteins there are segments which are much more highly conserved (>90% homology) than the overall protein sequence (70% homology). These conserved regions are believed to be involved in forming that domain of each protein molecule which is responsible for the interaction with the membrane protein P (Fig. 2) a~. Mutational alteration in one of these highly conserved regions of the J protein results in a loss of the ability of J to interact with P. The level of homology maintained between J and LAO is such that a chimeric protein resulting from a deletion fusing the amino terminal half of LAO to the carboxy terminal half of J functions perfectly normally with the membrane-bound components, though it maintains the substrate specificity of LAO 1'~. Therefore, this system, and independently each of the other systems, would have evolved as a 'package'. Thus, the evidence that a complex ancestral system would have spawned the present multiplicity of periplasmic permeases is tantalizing. However, more systems need
to be fully characterized before serious formulation of overall models for the molecular mechanism of transport and for their evolutionary history is possible. References 1 Heppel, L. A. (1971) in Structure and Function o f Biological Membranes (Rothfield, L. I., ed. ), pp. 223-247, Academic Press, New York 2 Higgins, C. F., Haag, P. D., Nikaido, K., Ardeshir, F., Garcia, G. and Ames, G. F.-L. (1982)Nature (London) 298,723-727 3 Oxender, D. L., Quay, S. C. and Anderson, J. J. (1980) in Microorganisms and Nitrogen Sources (Payne, J. W., ed.), pp. 153-169, John Wiley and Sons, New York 4 Shuman, H. A. (1982)Ann. Microbiol. (Inst. Pasteur) 133A, 153-159 5 Robbins, A. R., Guzman, R. and Rotman, B. (1976)J. Biol. Chem. 251,3112-3116 6 Clark, A. F. and Hogg, R. W. (1981 )J. Bacteriol. 147,920-924 7 Furlong, C. E. and Schellenberg, G. D. (1980) in Microorganisms and Nitrogen Sources (Payne, J. W., ed.), pp. 89-123~ John Wiley and Sons, New York 8 Robertson, D. E., Kroon, P. A. and Ho, C. (1977) Biochemistry 16, 1443-1451
Circular permutation of amino acid sequences among legume lectins John J. Hemperly and Bruce A. Cunningham Lectins, the carbohydrate-binding proteins found in a variety o f organisms, are a diverse group o f proteins. Many lectins from leguminous plants, however, are closely related to each other and are related to the jack bean lectin Concanavalin A by an unusual circular permutation of amino acid sequences. Lectins are an assortment of proteins that share the ability to bind stereospecifically and reversibly to carbohydrates, in particular to the sugar moieties of glycoproteins and glycolipids1. These proteins have been found in plants, bacteria, cellular slime molds, invertebrates and vertebrates but their normal biological functions are largely unknown. The lectin from vertebrate liver is probably the most well characterized in terms of its physiological function. This molecule plays a key role in the uptake of desialyated glycoproteins and their removal from the blood stream 2. Nearly all of the other lectins have been implicated in various types of cell--cell interactions. Bacterial lectins are presumed to provide a means by which micro-organisms can attach to epithelial cells and tissues prior to infecJohn J. Hemperly and Bruce A. Cunningham are at The Rockefeller University, 1230 York Avenue, New York, N Y 10021, U.S.A.
tion 3. Slime mold lectins have been assumed to play a role in cell-cell adhesion during the development of multicellular colonies from discrete amoebae, although conclusive evidence to support this notion is still lacking*. Several plant lectins have been postulated to mediate the binding of symbiotic Rhizobia to legume roots (see below). Thus it is clear that the group of molecules designated as lectins includes a variety of proteins and glycoproteins with diverse structures, different origins and probably distinct biological functions. Many lectins in leguminous plants, however, appear to be closely related, despite the fact that initial studies suggested that they too would be a collection of heterogeneous molecules. Lectins were first recognized in extracts of the seeds and beans of the Leguminosae. They were initially used in
,t'~'ElsevierBiomedicalPress 1983 0376 5067/83/0000- 0000/$0100
9 Ames, G. F.-L. and Spudich, E. N. (1976)Proc. Natl Acad. Sci. U.S.A. 73, 1877-1881 10 Shuman, H. A. (1982)J. Biol. Chem. 257, 5455-5461 11 Hong, J.-H., Hunt, A. G., Masters, P. S. and Lieberman, M. A. (1979) Proc. Natl Acad. Sci. U.S.A. 76, 1213-1217 12 Landick, R., Anderson, J. J., Mayo, M. M., Gunsalus, R. P., Mavromara, P., Daniels, C. J. and Oxender, D. L. (1980)J. Supramol. Structure 14, 527-537 13 Boos, W. (1982)Ann. Microbiol. (Inst. Pasteur) 133A, 145-151 14 Strange, P. G. and Koshland, D. E., Jr (1976) Proc. Natl Acad. Sci. U.S.A. 73,762-766 15 Bavoil, P. and Nikaido, H. (1981)J. Biol. Chem. 256, 11385-11388 16 Higgins, C. F. and Ames, G. F.-L. (1981)Proc. Natl Acad. Sci. U.S.A. 78, 6038-6042 17 Mahoney, W. C., Hogg, R. W. and Hermodson, M. A. (1981)J. Biol. Chem. 256, 4350-4356 18 Rosen, P. (1971)J. Biol. Chem. 246, 3653-3662 19 Clement. J. M. and Hofnung, M. ( 1981 ) Cell 27, 507-514 20 Gilson, E., Higgins, C. F., Hofnung, M., Ames, G. F.-L. and Nikaido, H. (1982)J. Biol. Chem. 257, 9915-9918 21 Gilliland, G. L. and Quiocho, F. A. (1981) J. Mol. Biol. 146,341-362
serological blood typing because different lectins preferentially agglutinate red blood cells of different types. With the demonstration that some plant lectins could also induce mitogenesis in lymphocytes5, these molecules became widely used reagents in immunology. They have since been used extensively to detect and perturb glycoproteins on cell surfaces and as affinity reagents for purifying glycoproteins. Concanavalin A (Con A) from the jack bean is one of the most abundant legume lectins. It is also the most well characterized. The protein was f'u'st crystallized by Sumner in 1919 (Ref. 6) and its amino acid sequence and three-dimensional structure were described in the early 1970s (Refs 7-9). Its biological activities have been analysed in detail, including both its ability to stimulate mitosis in lymphocytes and its dose-dependent ability to induce or inhibit the mobility of cell surface receptors ~°. At physiological pH, Con A is a tetramer of identical polypeptide chains, each containing 237 amino acids, one Ca 2+ and one Mn 2+. The polypeptide chain of each monomer is folded into two extensive /3-pleated sheets, one at the back and one through the center of the molecule. The carbohydrate-binding site is a shallow depression at the top of the molecule near the metal atoms 1~.~. There is also a deep cavity at the back of each monomer that appears to be capable of binding hydrophobic compounds. Below pH 5 or on chemical derivatization of amino groups,
TIBS
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109
Fig. 1. Comparison of the amino acid sequences of Con A (Reds 7,8) (top line in each row), soybean agglutinin (SBA) ( Ref 19) (second line), and favin ( Refs 14,, 20) (bottom line). Gaps are inserted in all sequences to maximize homology. Boxes enclose identical residues in at least two of the proteins. Arrows indicate the amino termini of the a and/3 chains of ravin, and dashes indicate regions of SBA that are as yet undetermined (adapted from Ref. 19 and reproduced by permission of Academic Press, Inc.).
the Con A tetramer dissociates to dimers that retain many of the biological properties of the tetramerTM. The dimer differs from the tetramer in that it lacks the ability to inhibit mitogenesis and receptor mobility at high concentrations19. Lectins from several other Leguminosae appeared at first to be somewhat different to Con A (for a review, see Ref. 1). Carbohydrate-binding proteins from the red kidney bean (PHA), peanut (PNA), and soybean (SBA) are similar in size to the Con A tetramer and are composed of polypeptides similar in size to the Con A monomer; however, they bind specifically to galactose derivatives whereas Con A binds glucose and mannose. In addition, SBA and PHA are glycoproteins, whereas Con A contains no covalently bound carbohydrate. The lectins from lentils and peas, and subsequently the lectin favin from fava beans",ts, were shown to have sugar-binding specificities similar to those of Con A but these molecules are only half the size of the Con A tetramer. Moreover, they are made up of
two types of polypeptide chains (a and B), both smaller than the Con A monomer. Like SBA and PHA, favin is a glycoprotein. Initial studies by Foriers et al. showed that despite these differences, the aminoterminal amino acid sequences of the lentil and pea/3 chains, and of SBA, PNA, and two types of PHA are homologousTMto portions of Con A (Ref. 17). The extent of this similarity and its unusual nature were shown by the analysis of the complete amino acid sequence of favinTM, by the completion of the structure of the lentil lectinTM, and by more recent analysis of the structure ofSBA (Ref. 20). The amino acid sequences of Con A, favin and SBA are shown in Fig. 1 and are aligned according to the sequence of Con A. The similarities are extensive; moreover, the residues that contribute to the distinguishing features of the threedimensional structure of Con A are highly conserved. The unusual feature of this comparison is the manner in which favin and SBA must be aligned with Con A in
237
] Con A
'70
]20
Fig. 2. Schematic drawings of the alignment of ravin el and fl chains (open bars), soybean agglutinin (SBA) (stippled bar), and Con A (dark bar) showing the circular permutation that gives maximum homology among the three sequences shown in Fig 1. The dashed line transecting the ravin fl chain indicates the region where the homologous 13chain of the lentil lectin ends. The location of the coral. ently bound carbohydrate in ravin is indicated by the asterisk (adapted from Ref. 20 and reproduced by permission of Academic Press, Inc. ).
102 order to show the homology. As with the a chains o f lentil and pea lectins, the a chain o f favin is homologous to residues 70 to 119 of Con A; the/3 chain of favin is homologous to Con A beginning at residue 120. The homology in the/3 chain continues to the carboxyl terminus o f Con A and without interruption through the amino-terminal region of Con A so that the/3 chain of favin ends at a position homologous to the position where the a chain begins 2~. This circular permutation is illustrated schematically in Fig. 2. The lentil lectin is directly comparable to favin but its/3 chain is slightly shorter and lacks the region homologous to the carbohydrate-containing portion of the favin/3 chainlL (The comparable portion of Con A lacks the sequence Asn-X-Thr/Ser usually associated with asparagine-linked carbohydrate22). The pea lectin also lacks covalently bound sugar and may be directly comparable to the lentil lectin. The amino acid sequence of SBA is also permuted with regard to Con A; its amino terminus, however, is homologous to that of the favin B-chain, and the region o f SBA comparable to the favin ct chain is at the carboxyl terminus (see Fig. 2). The location of the carbohydrate in SBA has not yet been established. The two PHA glycoproteins will presumably have structures similar to that of SBA; PNA will probably be similar but may be shortened much like the lentil lectin is with respect to ravin. Several other legume iectins have amino-terminal amino acid sequences homologous to those mentioned above. In particular, the lectin from Vicia sativa is very similar to favin ~ and the D o l i c h o s b i f l o r u s lectin seems to be homologous to PHA (Ref. 24). These extensive homologies do not appear to extend to non-legume lectins such as wheat germ agglutinin 2s and the R i c i n u s c o m m u n i s lectin ~6. The comparison of favin to SBA raised the possibility that favin is synthesized as a SBA-like precursor and that the a and/3 chains are generated by proteolysis after translation (Fig. 3). To test this hypothesis, we have recently translated favin messenger RNA in vitro 27. The ravin molecule appears to be synthesized as a single polypeptide precursor of a size comparable to the sum of the a and/3 chains and is preceded by a 29-residue signal sequence. The signal sequence is followed by the sequence of the favin/3 chain; the c~ chain sequence is also included in the precursor, presumably at the carboxyl-terminal end. Such a precursor is directly comparable to a SBA-like polypeptide. Processing of the favin precursor molecule by dog pancreas membranes resulted in glycosylation, removal o f the signal sequence, and some apparently aberrant cleavage, but we were
TIBS - March 1 983
(Sda, M., ed.), Vol. 4, pp. 429-529, Academic Press, New York 2 Ashwell, G. and Mot'ell, A. G. (1974) Adv. Enzymol. Relat. Areas Mol. Biol. 41, 99-128 S ,8 CHO 30fek, l., Beachley, E. H. and Sharon, N. (1978) I a r---q I I I Trends Biochem. Sci. 3, 159-160 4 Barondes, S. H. (1981) Annu. Rev. Biochem. 50, 207-23 l B CHO I a 5 Nowell,P. D. (1960) Cancer Res. 20, 462--466 I I I I 6 Sumner, J. B. and Howell, S. F. (1936) J. Bacteriol. 32, 227-237 Fig. 3. Schematic representation o f the biosynthesis 7 Edelman, G. M., Cunningham, B. A., Reeke, o f favin. The signal peptide (S), the ct chain (cO, the G. N., Jr, Becket, J. W., Waxdal, M. J. and chain (fl), and covalently bound carbohydrate Wang, J. L. (1972)Proc. Natl Acad. Sci. U.S.A. ( CHO) are indicated. 69, 2580-2584 8 Becket,J. W., Cunningham,B. A., Reeke,G. N., not able to generate polypeptides comparJr, Wang, J. L. and Edelman, G. M. (1976) in able to the favin a and/3 chains using this Concanavalin A as a Tool (Bittiger, H. and Schnebli,H. P., eds), pp. 33-54, John Wileyand system. Sons, London The comparisons o f these lectins suggest 9 Hardman, K. D. and Ainsworth, C. F. (1972) that they are closely related evolutionarily, Biochemistry I l, 4910--4919 will probably have similar threel0 Edelman,G. M. (1976)Science 192,218-226 dimensional structures, and will probably II Becket, J. W., Reeke, G. N., Jr, Cunningham, serve similar functions. From these limited B. A. and Edelman, G. M. (1976) Nature (Loncomparisons, Con A would appear to be the don) 259,406--409 unusual protein in that it is circularly per- 12 Hardman, K. D. and Ainsworth, C. F. (1976) Biochemistry 15, 1120-1128 muted with respect to the others. A number of hypotheses have been suggested to 13 Gunther,G. R., Wang, J. L., Yahara, I., Cunningham, B. A. and Edelman, G. M. (1973) Proc. account for this permutation xs.~9.we favor Natl Acad. Sci. U.S.A. 70, 1012-1016 mechanisms that implicate alteration at the 14 Wang, J. L., Becket, J. W., Reeke, G. N., Jr and level o f DNA and particularly those Edelman,G. M. (1974)J. Mol. Biol. 88,259-262 mechanisms involving gene duplication 15 Hemperly,J. J., Hopp, T. P., Becker, J. W. and followed by expression of a permuted lectin Cunningham, B. A. (1979)J. Biol. Chem. 254, 6803--6810 gene. Some valuable clues to this question will undoubtedly be obtained by examining 16 Foriers, A., Wuilmart, C., Sharon, N. and Strosberg, A. D. (1977) Biochem. Biophys. Res. the genes for these molecules using cDNA Commun. 75,980-986 and other probes. We expect that a number 17 Foriers, A., de Neve, R., Kanarek, L. and Strosof genes for at least some of these lectins berg, A. D. (1978) Proc. Nad Acad. Sci. U.S.A. will be found because we and others have 75, 1136-1139 detected closely related but non-identical 18 Canningham,B. A., Hempedy,J. J., Hopp, T. P. and Edelman,G. M. (1979)Proc. NatlAcad. Sci. polypeptides in the lectins o f some plants U.S.A. 76, 3218-3222 (for examples, see Refs 16, 28). 19 Foriers, A., Lebnm, E., Van Rapenbusch,R., de The key remaining question about these Neve, R. and Strosberg, A. D. (1981) J. Biol. lectins in L e g u m i n o s a e is their biological Chem. 256, 5550-5560 function. Some evidence has been pre- 20 Hemperly,J. J., Becker, J. W. and Cunningham, sented for a role in binding nitrogen-fixing B. A. (1982) in Proteins in Biology and Medicine (Bradshaw, R. A., Hill, R., Tang, J., bacteria to roots2L While this seems more Liang, C., Tsao, T. and Tson, C., eds), pp. plausible than a simple role as 'storage pro395-409, AcademicPress, New York teins' or in a pseudo-immune function, it is 21 Hopp, T. P., Hemperly, J. J. and Cunningham, by no means proven. In seeking a function, B. A. (1982)J. Biol. Chem. 257, 4473-4483 the temptation is to emphasize the 22 Marshall,R. D. (1972)Annu. Rev. Biochem. 41, carbohydrate-binding properties of these 673-702 molecules, but this prejudice is clearly 23 Gebauer, G., Schlitz, E. and Riidiger, H. (1981) Eur. J. Biochem. 113,319-325 based on the methods initially used to detect lectins. There is on Con A at least one addi- 24 Etzler, M. E., Talbot, C. F. and Ziaya, P. R. FEBS Lett. 82, 39-41 tional site, the hydrophobic cavity, that could be equally important in functional 25 Wright,C. S. (1977)J. Mol. Biol. I 11,439-457 26 Cawley, D. B., Hedblom, M. L. and Houston, considerations, especially since it has been L. L. (1978)Arch. Biochem. Biophys. 190, shown that Con A can bind plant auxins 3°. 744-755 With renewed interest in plant biochemistry 27 Hemperly,J. J., Mostov, K. E. and Cunningham, and the relative abundance o f lectins in B. A. (1982)J. Biol. Chem. 257, 7903-7909 important food sources, the elucidation of 28 Lolan, R., Cacan, R., Cacan, M., Debmy, H., Carter, W. G. and Sharon, N. (1975)FEBS Lett. the function o f these molecules is an obvi57, 100-103 ous and worthwhile goal. 29 Broughton,W. J. (1978)J. Appl. Bacteriol. 45, 165-194 30 Edelman,G. M. and Wang, J. L. (1978)J. Biol. References Chem. 253, 3016-3022 1 Lis, H. and Sharon, N. (1977) in The Antigens s
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