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Soluble β-galactoside binding lectins: Physicochemical and functional properties
SOLUBLE fS-GALACTOSIDE BINDING AND FUNCTIONAL PROPERTIES
LECTINS:
PHYSICOCHEMICAL
NAJMA ALl Interdisciplinary Biotechnology Unit Aligarh Muslim University Aligarh 202002, India
Introduction
Lectins were first discovered in plants but they are now known to occur in all types of organisms) They can decode the information of structural diversification of complex carbohydrates. 2'3 In general, animal lectins are classified into two major categories: (1) membrane-bound lectins, also called asialoglycoprotein receptors which show Ca 2÷dependent carbohydrate binding activity, 1'4-6 and (2) soluble [3-galactoside binding lectins which require exogenous thiol groups but not divalent metal ions for hemagglutinating activity. Soluble 13-galactoside binding lectins are widely distributed in vertebrate tissues. They are acidic, low Mr proteins having subunits of 14-16 kDa. Unlike membrane-bound lectins they do not require divalent metal ions but do need exogenous thiol groups for activity. Generally they are nonglycosylated with the exceptions of lectins from electric eel and some from mammalian liver, cDNA and protein sequence analysis have revealed that soluble lectins from various species or organisms are structurally related suggesting that these proteins are derived from a common ancestor gene and that they are members of a family of nuclear proteins like the carbohydrate binding protein-35 (CBP-35). This has a carbohydrate domain and a second domain homologous with some hrRNPs (see below). Recently, soluble lectins have been found to have an unexpected antigenic and structural relationship with myelin basic protein. The developmental regulation and tissue specific distribution of lectins suggest that they are involved in organization of extracellular matrix, embryogenesis and cellular differentiation and development. Study of these interesting proteins affords an opportunity to explore several areas of general interest to biochemistry students including carbohydrate and protein structure.
Isolation and Physicochemical Properties
Soluble 13-galactoside binding lectins have been isolated from embryonic and differentiating tissues of vertebrates including teleosts, 7'8 amphibians, 9 aves 1 and mammals. 1-10 They are acidic proteins extracted from the tissues without aid of detergent and hence are called soluble lectins) '2"11 The isoelectric points of protein are usually determined by isoelectric focusing (IEF), a method based on electrophoretic mobility as a function of pH. Soluble lectins have been purified from the tissues by affinity chromatography on lactosyl or asialofetuin Sepharose-4B column. T M In the description of the isolation of these lectins to students, advantage might be taken of the opportunity to review basic ideas about IEF and affinity chromatography as general techniques.
Molecular Properties
The molecular properties of some soluble 13-galactoside binding lectins from various tissues of different species are summarized in Table 1. Multiple, distinct forms of soluble lectins have been recognized in human and rat lung, 12A3chicken liver and muscles 14"15a n d rabbit bone-marrow. 16 Most lectins occur as 14 kDa dimers, but minor forms exist as single, 18-29 kDa polypeptide chains.12-15 The subunits of dimeric lectins are held together by noncovalent interactions. 1"hA3 Generally protein molecular weights may be determined by sedimentation equilibrium or gel filtration under native conditions and subunit molecular weights by SDS polyacrylamide gel electrophoresis under reducing conditions. However, it is well worth pointing out to students that in case of glycoproteins sedimentation equilibrium gives accurate molecular weights without any overestimation in the value due to the carbohydrate moiety. In a course of lectures, students could be given experimental data and set simple problems to determine Mr and subunit structures of lectins. Experimental data could also be given to show that glycoproteins can behave anomalously.
Structure
cDNA and protein sequence analysis have revealed primary structures for some soluble lectins. 17-19 Comparison of protein sequences shows that members of this class of lectin
B I O C H E M I C A L E D U C A T I O N 19(1) 1991
Table I
Molecular properties of some soluble ~-galactoside binding lectins
Source
Mr (kDa)
No of subunits
Subunit Mr (kDa)
Human lung Human placenta Rat liver Rat brain Rat lung Rabbit lung Goat lung Goat liver Bovine heart Bovine brain Bovine lung Calf heart Sheep liver Buffalo liver Pig kidney Pig spleen Chicken muscle Chicken liver Chicken intestine Electric eel
26.0 14.0 29.0 32.0 29.0 9.2 28.8 22.0 24.0 30.0 29.0 11.8 18.0 24.0 14.5 28.0 15.0 15.0 14.0 33.0
2 1 2 2 2 1 2 -2 2 1 1 --1 2 1 1 1 2
14.0 -14.5 16.0 14.5 -14.0 -13.0 15.0 -----14.0 ---16.0
Reference 37 21 17 38 12 16 16 10 39 38 12 16 10 10 40 41 1 1 1 25
from diverse tissues or organisms have extensive sequence homology (66"92%) suggesting that these proteins are derived from a common ancestor gene and have maintained through evolution part of structure of the 13-D-galactoside binding site, putatively assigned to residues (70-76) T r p - G l y - T h r - G l u - G l n - A r g - G l u . 19 They display immunological cross reactivity. 1 and the major 14 kDa lectins from various sources share identical specificity for 13-galactosides and require 2-mercapto-ethanol for activity. 11 Interestingly, these lectins possess highly-conserved cysteine residues which a r e functionally relevant: 17'2° treatment of soluble lectins with thiol blocking reagents such as NEM and pHMB causes complete inactivation of hemagglutinating activity. 1°'17'2° In addition, analysis of mRNA encoding several of these proteins shows that they lack an amino terminal signal sequence as well as internal signal sequence able to direct the protein to cell surface. 17'18 These findings suggest that the occurrence of soluble lectins extracellularly results from their release during cell rupture. 1 However, it should be noted that other proteins are able to make their way out of cells without guidance from the classical signal sequences. 22 The sequence data also indicate the absence of any domain structure of the type seen in proteins such as immunoglobulins.23 Among soluble [3-galactoside binding lectins reported thus far, the major 14 kDa rat lung lectin has been studied in detail. 21 Using the empirical procedures of Chou and Fasman, 24 Clerch and coworkers ~7 calculated the secondary structure of rat lung lectin. They predicted that the lectin contained 27% a-helix, 22% 13-sheet, 28% 13-turn and 23% random coil. Chou and Fasman's method provides a simple procedure devoid of complex computer calculations, to predict the secondary structure of proteins from their known amino acid sequence. This would be a good point at which to introduce students to these ideas and to set them some calculations. The functionally important cysteine residues mentioned above are not involved in the formation of secondary structure.17 Furthermore, biochemical data on the 14 kDa human placenta and HL-60 lectin show that despite the presence of a possible carbohydrate attachment site, the protein is not glycosylated.TM At this point, it would be worth digressing to tell students about how carbohydrate attachment sites have been identified.
BIOCHEMICAL EDUCATION 19(1) 1991
However, Levi and Teichberg 25 reported that the lectin from electric eel is a glycoprotein containing 2% neutral hexoses but lacking sialic acid residues. 25 Similarly, the three lectins from sheep, goat and buffalo livers were found to be glycoproteins with 4-6% neutral hexoses. 1° Unlike membrane-bound lectins, soluble 13-galactoside binding lectins show metal ion-independent activity. However, recent studies on hepatic soluble lectins from various mammalian liver demonstrated that the hemagglutinating activity of these lectins is stimulated in the presence of Ca 2÷ (ref 10).
Function of Soluble Lectins
The localization of the soluble 13-galactoside binding lectins has helped a great deal in understanding their functions. Immunohistochemical techniques have been employed to locate these lectins in the cells and tissues. One possibility consistent with the experimental data is that soluble ~-galactoside binding lectins play a role in the assembly or organization of carbohydrate structures at the cell surface, within the extracellular martrix or at the plasma membrane. Such function would be especially important in the regulation of the interaction of cells during tissue development. This area would be of especial interest to medical students and would be worth developing perhaps in the tutorial setting. Thus, the expression of the lectin, CLL-I, in chicken embryonic muscles increases 2-3-fold during embryonic development and various studies have revealed that lectins are involved in the myotube fusion at the time of myoblast formation. 1 The predominant expression of CLL-I in chicken embryonic tissue suggests that these lectins may play a role in the organization of tissues during embryogenesis. An organizational role for soluble lectin in adult tissue has been reported by Bayer et a126 who fould that in chicken intestine the localization of CLL-II and low levels of CLL-I in globlet cell together possess the capacity to bind intestinal mucin and hence may function in the organization of mucus for secretion. 1,26 In rat lung, soluble lectin has been localized in smooth muscle and squamous alveolar epithelial cells and is also concentrated extracellularly in elastic fibres of pulmonary parenchyma and blood vessels. 1 Other experimental approaches have revealed the involvement of soluble [3-galactoside binding lectins in cellular differentiation and organ formation. ~'~ In rabbit bone-marrow differentiation erythroblasts cluster around a macrophage until enucleation occurs. 3 The appearance and cellular distribution of soluble lectin correlates with the expression of complementary carbohydrate structures in many developing organs and tissues.l The best example of this coordination is the appearance of two lectins in neurons of dorsal root ganglia. Both lectins are restricted to a distinct subset of the neurons. 27'2s In addition, surface soluble [3-galactoside binding lectins are also involved in the cell-cell interactions. J Levi and Teichberg 29 demonstrated that a 13-galactoside-specific lectin in the epithelium of the thymus was responsible for holding immature thymocytes in the thymic cortex by binding galactose residues on the surface of these cells. Thymocytes are released as they mature and galactose residues become masked by sialic acid residues. However, the precise role of soluble ~-galactoside binding lectins is still uncertain.
Dual Function of Soluble Lectins
The discovery of carbohydrate binding protein (CBP-35) from 3T3 fibroblasts has given a new insight to the function of soluble lectin in the cell. This protein behaves as a galactose specific lectin. 3° Experimental evidence has indicated that CBP-35 may be component of human heteronuclear ribonucleoprotein (hr RNP) because structure analysis has shown that CBP-35 possesses two functional domains, namely (1) a C-terminal domain which is homologous with the major 14 kDa lectin from rat lung, bovine heart, chicken skin, human lung and placenta, human hepatoma clone-l, and (2) an N-terminal domain which shows sequence homology with known protein constituents of ribonuclear protein. 3° Recently an unexpected antigenic and structural relationship between basic protein and soluble [3-galactoside binding protein has been found. 31 Human myelin basic protein contains the tetrapeptide sequence Trp-Gly-Ser-Glu at positions 116-119 which reacts with antilectin monoclonal antibody. This tetrapeptide sequence is also conserved among soluble 13-galactoside-binding lectins, suggesting that it may be important functionally. The tetrapeptide sequence in myelin basic protein is a part of the main domain involved in allergic encephalomyelitis.32 Several lines of evidence have revealed that phosphorylation of the myelin protein is inhibited by adding gangliosides. 33 Myelin basic protein possesses substantial number of phosphorylation sites (15-18 sites) and hence it is used as a sub-
B I O C H E M I C A L EDUCATION 19(1) 1991
strate by protein kinase C. Thus, the phosphorylation of myelin basic protein regulates the phospholipid hydrolysis in the nerve cells. It has, therefore, been suggested that part of myelin basic protein which includes tetrapeptide sequence may have carbohydrate binding activity and that the interaction with carbohydrate may regulate the phosphorylation. 33 This suggests that lectins might have bifunctional properties, being involved in regulation of phosphorylation as well as binding carbohydrates. Soluble Lectins from Cancer Cells
Soluble 13-galactoside binding lectins have also been shown to occur in cancer cells) This is another area that would be very well worth mentioning to medical students. Recently two soluble lectins with molecular weights of 14.5 and 34 kDa, respectively, have been isolated from human and murine tumor cells including fibrosarcoma, melanoma and carc i n o m a . 34-36 The comparison of sequence of the 14.5 kDa-lectin with other soluble lectins such as human placental lectin, reveals extensive sequence homology. In contrast, the 34 kDa-lectin from transformed cells shows no sequence homology with known proteins. Moreover, the concentration of mRNA encoding the 34 kDa-lectin is much more abundant in transformed cells than in normal cells while the level of m R N A encoding for 14.5 kDa-lectin remains the same in both cancer and normal cells. 3 Several lines of evidence have revealed that expression of soluble lectin correlates with an increased tendency of cells to form emboli by aggregating with other tumor cells in host cells and hence enhance the metastatic potential of cancer cells. Recently, approaches are being made to use 34 kDa-lectin for diagnosis of cancer and the cell surface lectins as targets for controlled and selective delivery of drug to malignant cells)
References
1Barondes, S H (1986) in 'The Lectins' (edited by I R Liener, S Nathan and I J Goldstein), Academic Press, Orlando Florida, pp 438-462 2Drickamer, K (1988) J Biol Chem 263, 9557-9560 3Sharon, N and Lis, H (1989) Science 246, 227-234 4Ali, N and Salahuddin, A (1989) FEBS Lett 246, 163-165 SBaenziger, J A and Maynard, Y (1980) J Biol Chem 255, 4607-4613 6Herzig, M C S and Weigel, P H (1989) Biochemistry 28,600-610 7Teichberg, V I, Silman, I, Beitseh, O D and Resheff, G (1975) Proc Natl Acad Sci (USA) 72, 1383-1387 8Teichberg, V I (1978) Methods Enzymol 50, 291-302 9Bols, N C, Roberson, M M, Haywood Reid, P I, Cerra, R F and Barondes S H (1986) J Cell Bio1102, 492-499 l°Ali, N and Salahuddin, A (1989) Biochim Biophys Acta 992, 30-34 11Barondes, S H (1984) Science 223, 1259-1266 12Cerra, R F, Gitt, M A and Barondes, S H (1985) J Biol Chem 260, 10474-10477 13Sparrow, C P, Leffler, H and Barondes, S H (1987) J Biol Chem 262, 7383-7390 laDen, H and Malinzak, D A (1977) J Biol Chem 252, 5444-5448 15Bayer, E C, Zweig, S E and Barondes, S H (1980) J Biol Chem 255, 4236-4239 16Harrison, F L, Fitzgerald, J E and Catt, J W (1984) J Cell Sci 72, 147-162 17Clerch, L B, Whitney, P, Hass, M, Brew, K, Miller, T, Werner, R and Massaro, W (1988) Biochemistry 27, 692-699 18Oliver, P C, Denise, C B, Bringman, T S, Joseph, G, Michael, M C G and Nedwin, C E (1989) J Biol Chem 264, 1310-1316 19paroutaud, P, Levi, G, Teichberg, V I and Strosberg, A D (1987) Proc Natl Acad Sci (USA) 84, 6345-6348 2°Whitney, P L, Powell, J T and Sanfrod, G L (1986) Biochem J 238, 683-689 21Hirabayashi, J, Kawasaki, H, Suziki, K G C and Kasal, K (1987) J Biochem 101,987-995 22Abraham, J A, Megia, A, Whang, J L, Tumolo, A, Friedman, J, Hyerrild, K A, Gaspondarwiz, D and Fiddes, J C (1986) Science 233,545-548 23Burton, D R (1985) Mol lrnmunol 22, 606-616 24Chou, P Y and Fasman, G D (1974) Biochemistry 13, 211-222 25Levi, G and Teichberg, V I (1981) J Biol Chem 256, 12523-12527 26Bayer, E C, Zweig, S E and Barondes, S H (1980) J Biol Chem 255, 4236-4239 27Kuchier, S, Fressinaud, C, Sarlieve, L L, Vicendron, G and Zenetta, J P (1988) Dev Neurosci 10, 199-204' 28Sharon, N (1983) Adv lmmunol 34, 313-315 29Levi, G and Teichberg, V I (1983) Immttnol Lett 7, 35-39 3°Jia, S and Wang, J L (1980) J Biol Chem 263, 6009-6011 31Abbott, W M, Meller, A, Edwards, Y and Feizi, T (1989) Biochem J 259, 283-290
B I O C H E M I C A L E D U C A T I O N 19(1) 1991
3ZEylar, E H, Estall, F C and Brostoff, S (1971) J Biol Chem 246, 3418-3424 33Kreutter, D, Kim, J Y H, Goldenring, J R, Rasmussin, H, Ukomada, C, Delorenzo, R J and Yu, R K (1987) J Biol Chem 262, 1633-1637 34Raz, A and Lotan, R (1980) Cancer Metastasis Res 6, 433-438 35Lotan, R and Raz, A (1988) J Cell Biochem 37, 107 36Lotan, R, Lotan, D and Carraleno, D M (1989) Cancer Lett 48, 115-122 37Sparrow, C P, Leffier, H and Barondes, S H (1987) J Biol Chem 262, 7383-7390 3SCaron, M, Joubert, R and Bladier, D (1987) Biochim Biophys Acta 925,290-296 39Southan, C, Aitken, A, Robert, C A, Abboat, N W and Feizi, T (1987) FEBS Lett 214, 301-304 4°Masstsumoto, I, Kitagaki, H, Iida, N, Saita, Y and Seno, Y (1986) Carbohydrate Res 151,262-270 41Allen, H J, Cytlwinski, M, Palmberg, R and Dicioccoio, R A (1987) Arch Biochem Biophys 252, 523-533
Announcement
Science and T e c h n o l o g y Education: Responsible C h a n g e for the 21st C e n t u r y
The sixth International Symposium on World Trends in Science and Technology Education, will take place in August 12-22, 1991, in the Wyndham Palm Springs Hotel and Conference Center, Palm Springs, California, USA. IOSTE (International Organization for Science and Technology Education) was established to advance the cause of education in science and technology as a vital part of the general education of the peoples of all countries and to provide scholarly exchange and discussion. The theme of the forthcoming symposium is: Responsible Change for the 21st Century. Exchanges and discussions will be held in the sub-themes (or contexts) of: - - science education issues - - environmental issues - - technology issues - - socio-cultural issues.
Call for presenters and papers Submit proposals as soon as possible to: IOSTE 6th Symposium Organizer and Chairman Dr Herbert K Brunkhorst Institute for Science Education California State University, San Bernardino 5500 University Parkway San Bernardino, CA 92407-2397, USA Fax: 714-880-5988
B I O C H E M I C A L E D U C A T I O N 19(1) 1 9 9 1
LECTINS:
PHYSICOCHEMICAL
NAJMA ALl Interdisciplinary Biotechnology Unit Aligarh Muslim University Aligarh 202002, India
Introduction
Lectins were first discovered in plants but they are now known to occur in all types of organisms) They can decode the information of structural diversification of complex carbohydrates. 2'3 In general, animal lectins are classified into two major categories: (1) membrane-bound lectins, also called asialoglycoprotein receptors which show Ca 2÷dependent carbohydrate binding activity, 1'4-6 and (2) soluble [3-galactoside binding lectins which require exogenous thiol groups but not divalent metal ions for hemagglutinating activity. Soluble 13-galactoside binding lectins are widely distributed in vertebrate tissues. They are acidic, low Mr proteins having subunits of 14-16 kDa. Unlike membrane-bound lectins they do not require divalent metal ions but do need exogenous thiol groups for activity. Generally they are nonglycosylated with the exceptions of lectins from electric eel and some from mammalian liver, cDNA and protein sequence analysis have revealed that soluble lectins from various species or organisms are structurally related suggesting that these proteins are derived from a common ancestor gene and that they are members of a family of nuclear proteins like the carbohydrate binding protein-35 (CBP-35). This has a carbohydrate domain and a second domain homologous with some hrRNPs (see below). Recently, soluble lectins have been found to have an unexpected antigenic and structural relationship with myelin basic protein. The developmental regulation and tissue specific distribution of lectins suggest that they are involved in organization of extracellular matrix, embryogenesis and cellular differentiation and development. Study of these interesting proteins affords an opportunity to explore several areas of general interest to biochemistry students including carbohydrate and protein structure.
Isolation and Physicochemical Properties
Soluble 13-galactoside binding lectins have been isolated from embryonic and differentiating tissues of vertebrates including teleosts, 7'8 amphibians, 9 aves 1 and mammals. 1-10 They are acidic proteins extracted from the tissues without aid of detergent and hence are called soluble lectins) '2"11 The isoelectric points of protein are usually determined by isoelectric focusing (IEF), a method based on electrophoretic mobility as a function of pH. Soluble lectins have been purified from the tissues by affinity chromatography on lactosyl or asialofetuin Sepharose-4B column. T M In the description of the isolation of these lectins to students, advantage might be taken of the opportunity to review basic ideas about IEF and affinity chromatography as general techniques.
Molecular Properties
The molecular properties of some soluble 13-galactoside binding lectins from various tissues of different species are summarized in Table 1. Multiple, distinct forms of soluble lectins have been recognized in human and rat lung, 12A3chicken liver and muscles 14"15a n d rabbit bone-marrow. 16 Most lectins occur as 14 kDa dimers, but minor forms exist as single, 18-29 kDa polypeptide chains.12-15 The subunits of dimeric lectins are held together by noncovalent interactions. 1"hA3 Generally protein molecular weights may be determined by sedimentation equilibrium or gel filtration under native conditions and subunit molecular weights by SDS polyacrylamide gel electrophoresis under reducing conditions. However, it is well worth pointing out to students that in case of glycoproteins sedimentation equilibrium gives accurate molecular weights without any overestimation in the value due to the carbohydrate moiety. In a course of lectures, students could be given experimental data and set simple problems to determine Mr and subunit structures of lectins. Experimental data could also be given to show that glycoproteins can behave anomalously.
Structure
cDNA and protein sequence analysis have revealed primary structures for some soluble lectins. 17-19 Comparison of protein sequences shows that members of this class of lectin
B I O C H E M I C A L E D U C A T I O N 19(1) 1991
Table I
Molecular properties of some soluble ~-galactoside binding lectins
Source
Mr (kDa)
No of subunits
Subunit Mr (kDa)
Human lung Human placenta Rat liver Rat brain Rat lung Rabbit lung Goat lung Goat liver Bovine heart Bovine brain Bovine lung Calf heart Sheep liver Buffalo liver Pig kidney Pig spleen Chicken muscle Chicken liver Chicken intestine Electric eel
26.0 14.0 29.0 32.0 29.0 9.2 28.8 22.0 24.0 30.0 29.0 11.8 18.0 24.0 14.5 28.0 15.0 15.0 14.0 33.0
2 1 2 2 2 1 2 -2 2 1 1 --1 2 1 1 1 2
14.0 -14.5 16.0 14.5 -14.0 -13.0 15.0 -----14.0 ---16.0
Reference 37 21 17 38 12 16 16 10 39 38 12 16 10 10 40 41 1 1 1 25
from diverse tissues or organisms have extensive sequence homology (66"92%) suggesting that these proteins are derived from a common ancestor gene and have maintained through evolution part of structure of the 13-D-galactoside binding site, putatively assigned to residues (70-76) T r p - G l y - T h r - G l u - G l n - A r g - G l u . 19 They display immunological cross reactivity. 1 and the major 14 kDa lectins from various sources share identical specificity for 13-galactosides and require 2-mercapto-ethanol for activity. 11 Interestingly, these lectins possess highly-conserved cysteine residues which a r e functionally relevant: 17'2° treatment of soluble lectins with thiol blocking reagents such as NEM and pHMB causes complete inactivation of hemagglutinating activity. 1°'17'2° In addition, analysis of mRNA encoding several of these proteins shows that they lack an amino terminal signal sequence as well as internal signal sequence able to direct the protein to cell surface. 17'18 These findings suggest that the occurrence of soluble lectins extracellularly results from their release during cell rupture. 1 However, it should be noted that other proteins are able to make their way out of cells without guidance from the classical signal sequences. 22 The sequence data also indicate the absence of any domain structure of the type seen in proteins such as immunoglobulins.23 Among soluble [3-galactoside binding lectins reported thus far, the major 14 kDa rat lung lectin has been studied in detail. 21 Using the empirical procedures of Chou and Fasman, 24 Clerch and coworkers ~7 calculated the secondary structure of rat lung lectin. They predicted that the lectin contained 27% a-helix, 22% 13-sheet, 28% 13-turn and 23% random coil. Chou and Fasman's method provides a simple procedure devoid of complex computer calculations, to predict the secondary structure of proteins from their known amino acid sequence. This would be a good point at which to introduce students to these ideas and to set them some calculations. The functionally important cysteine residues mentioned above are not involved in the formation of secondary structure.17 Furthermore, biochemical data on the 14 kDa human placenta and HL-60 lectin show that despite the presence of a possible carbohydrate attachment site, the protein is not glycosylated.TM At this point, it would be worth digressing to tell students about how carbohydrate attachment sites have been identified.
BIOCHEMICAL EDUCATION 19(1) 1991
However, Levi and Teichberg 25 reported that the lectin from electric eel is a glycoprotein containing 2% neutral hexoses but lacking sialic acid residues. 25 Similarly, the three lectins from sheep, goat and buffalo livers were found to be glycoproteins with 4-6% neutral hexoses. 1° Unlike membrane-bound lectins, soluble 13-galactoside binding lectins show metal ion-independent activity. However, recent studies on hepatic soluble lectins from various mammalian liver demonstrated that the hemagglutinating activity of these lectins is stimulated in the presence of Ca 2÷ (ref 10).
Function of Soluble Lectins
The localization of the soluble 13-galactoside binding lectins has helped a great deal in understanding their functions. Immunohistochemical techniques have been employed to locate these lectins in the cells and tissues. One possibility consistent with the experimental data is that soluble ~-galactoside binding lectins play a role in the assembly or organization of carbohydrate structures at the cell surface, within the extracellular martrix or at the plasma membrane. Such function would be especially important in the regulation of the interaction of cells during tissue development. This area would be of especial interest to medical students and would be worth developing perhaps in the tutorial setting. Thus, the expression of the lectin, CLL-I, in chicken embryonic muscles increases 2-3-fold during embryonic development and various studies have revealed that lectins are involved in the myotube fusion at the time of myoblast formation. 1 The predominant expression of CLL-I in chicken embryonic tissue suggests that these lectins may play a role in the organization of tissues during embryogenesis. An organizational role for soluble lectin in adult tissue has been reported by Bayer et a126 who fould that in chicken intestine the localization of CLL-II and low levels of CLL-I in globlet cell together possess the capacity to bind intestinal mucin and hence may function in the organization of mucus for secretion. 1,26 In rat lung, soluble lectin has been localized in smooth muscle and squamous alveolar epithelial cells and is also concentrated extracellularly in elastic fibres of pulmonary parenchyma and blood vessels. 1 Other experimental approaches have revealed the involvement of soluble [3-galactoside binding lectins in cellular differentiation and organ formation. ~'~ In rabbit bone-marrow differentiation erythroblasts cluster around a macrophage until enucleation occurs. 3 The appearance and cellular distribution of soluble lectin correlates with the expression of complementary carbohydrate structures in many developing organs and tissues.l The best example of this coordination is the appearance of two lectins in neurons of dorsal root ganglia. Both lectins are restricted to a distinct subset of the neurons. 27'2s In addition, surface soluble [3-galactoside binding lectins are also involved in the cell-cell interactions. J Levi and Teichberg 29 demonstrated that a 13-galactoside-specific lectin in the epithelium of the thymus was responsible for holding immature thymocytes in the thymic cortex by binding galactose residues on the surface of these cells. Thymocytes are released as they mature and galactose residues become masked by sialic acid residues. However, the precise role of soluble ~-galactoside binding lectins is still uncertain.
Dual Function of Soluble Lectins
The discovery of carbohydrate binding protein (CBP-35) from 3T3 fibroblasts has given a new insight to the function of soluble lectin in the cell. This protein behaves as a galactose specific lectin. 3° Experimental evidence has indicated that CBP-35 may be component of human heteronuclear ribonucleoprotein (hr RNP) because structure analysis has shown that CBP-35 possesses two functional domains, namely (1) a C-terminal domain which is homologous with the major 14 kDa lectin from rat lung, bovine heart, chicken skin, human lung and placenta, human hepatoma clone-l, and (2) an N-terminal domain which shows sequence homology with known protein constituents of ribonuclear protein. 3° Recently an unexpected antigenic and structural relationship between basic protein and soluble [3-galactoside binding protein has been found. 31 Human myelin basic protein contains the tetrapeptide sequence Trp-Gly-Ser-Glu at positions 116-119 which reacts with antilectin monoclonal antibody. This tetrapeptide sequence is also conserved among soluble 13-galactoside-binding lectins, suggesting that it may be important functionally. The tetrapeptide sequence in myelin basic protein is a part of the main domain involved in allergic encephalomyelitis.32 Several lines of evidence have revealed that phosphorylation of the myelin protein is inhibited by adding gangliosides. 33 Myelin basic protein possesses substantial number of phosphorylation sites (15-18 sites) and hence it is used as a sub-
B I O C H E M I C A L EDUCATION 19(1) 1991
strate by protein kinase C. Thus, the phosphorylation of myelin basic protein regulates the phospholipid hydrolysis in the nerve cells. It has, therefore, been suggested that part of myelin basic protein which includes tetrapeptide sequence may have carbohydrate binding activity and that the interaction with carbohydrate may regulate the phosphorylation. 33 This suggests that lectins might have bifunctional properties, being involved in regulation of phosphorylation as well as binding carbohydrates. Soluble Lectins from Cancer Cells
Soluble 13-galactoside binding lectins have also been shown to occur in cancer cells) This is another area that would be very well worth mentioning to medical students. Recently two soluble lectins with molecular weights of 14.5 and 34 kDa, respectively, have been isolated from human and murine tumor cells including fibrosarcoma, melanoma and carc i n o m a . 34-36 The comparison of sequence of the 14.5 kDa-lectin with other soluble lectins such as human placental lectin, reveals extensive sequence homology. In contrast, the 34 kDa-lectin from transformed cells shows no sequence homology with known proteins. Moreover, the concentration of mRNA encoding the 34 kDa-lectin is much more abundant in transformed cells than in normal cells while the level of m R N A encoding for 14.5 kDa-lectin remains the same in both cancer and normal cells. 3 Several lines of evidence have revealed that expression of soluble lectin correlates with an increased tendency of cells to form emboli by aggregating with other tumor cells in host cells and hence enhance the metastatic potential of cancer cells. Recently, approaches are being made to use 34 kDa-lectin for diagnosis of cancer and the cell surface lectins as targets for controlled and selective delivery of drug to malignant cells)
References
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Announcement
Science and T e c h n o l o g y Education: Responsible C h a n g e for the 21st C e n t u r y
The sixth International Symposium on World Trends in Science and Technology Education, will take place in August 12-22, 1991, in the Wyndham Palm Springs Hotel and Conference Center, Palm Springs, California, USA. IOSTE (International Organization for Science and Technology Education) was established to advance the cause of education in science and technology as a vital part of the general education of the peoples of all countries and to provide scholarly exchange and discussion. The theme of the forthcoming symposium is: Responsible Change for the 21st Century. Exchanges and discussions will be held in the sub-themes (or contexts) of: - - science education issues - - environmental issues - - technology issues - - socio-cultural issues.
Call for presenters and papers Submit proposals as soon as possible to: IOSTE 6th Symposium Organizer and Chairman Dr Herbert K Brunkhorst Institute for Science Education California State University, San Bernardino 5500 University Parkway San Bernardino, CA 92407-2397, USA Fax: 714-880-5988
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