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Karen J. Colley - One of the best experts on this subject based on the ideXlab platform.
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Specific sequences in the signal anchor of the Beta-Galactoside alpha-2,6-sialyltransferase are not essential for Golgi localization. Membrane flanking sequences may specify Golgi retention.
The Journal of biological chemistry, 1993Co-Authors: Refka Y. Dahdal, Karen J. ColleyAbstract:The Beta-Galactoside alpha-2,6-sialyltransferase is a trans Golgi/trans Golgi network glycosyltransferase which adds sialic acid residues to Asn-linked oligosaccharides of glycoproteins. Previous results suggested that the sialyltransferase stem and signal anchor including flanking sequences may be two independent Golgi retention regions. However, other experiments demonstrated that the sequence of the signal anchor itself was not important. To investigate whether the sialyltransferase signal anchor was necessary and sufficient for Golgi retention, several mutant and chimeric proteins were expressed and localized in Cos-1 and Chinese hamster ovary cells. We found that the signal anchor and flanking sequences were able to retain the sialyltransferase catalytic domain in the Golgi. However, efficient Golgi retention was still observed when the signal anchor was altered or entirely replaced in either the presence or absence of most of the luminal stem region. Chimeric proteins consisting of the sialyltransferase cytoplasmic tail and signal anchor fused to the extracellular domains of two different cell surface proteins demonstrated poor Golgi retention. A significant increase in the Golgi retention of one of these chimeras was observed when two lysines were placed next to the signal anchor on the luminal side. Taken together these results suggest that the sialyltransferase signal anchor is not necessary or sufficient for Golgi retention, rather, appropriately spaced cytoplasmic and luminal flanking sequences are the important elements of the sialyltransferase Golgi retention region.
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The signal anchor and stem regions of the Beta-Galactoside alpha 2,6-sialyltransferase may each act to localize the enzyme to the Golgi apparatus.
The Journal of biological chemistry, 1992Co-Authors: Karen J. Colley, Eryn Ujita Lee, James C. PaulsonAbstract:Abstract The Beta-Galactoside alpha 2,6-sialyltransferase has been localized to the trans cisternae of the Golgi apparatus and the trans Golgi network where it transfers sialic acid residues to terminal positions on N-linked oligosaccharides. It is a type II transmembrane protein possessing a 9-amino acid amino-terminal cytoplasmic tail, a 17-amino acid signal anchor domain, and a 35-amino acid stem region which tethers the large luminal catalytic domain to the membrane anchor. Previous work has demonstrated that the soluble sialytransferase catalytic domain is rapidly secreted from Chinese hamster ovary cells. These results suggest that the signals for Golgi apparatus localization do not reside in the catalytic domain of the enzyme but must reside in the cytoplasmic tail, signal anchor domain, and/or stem region. To determine which amino-terminal regions are required for Golgi apparatus localization, mutant sialyltransferase proteins were constructed by in vitro oligonucleotide-directed mutagenesis, expressed in Cos-1 cells, and localized by indirect immunofluorescence microscopy. Signal cleavage-sialyltransferase mutants which consist of only the stem and catalytic domain of the enzyme are not rapidly secreted but are retained intracellularly and predominantly localized to the Golgi apparatus. However, deletion of either the stem region or the cytoplasmic tail of the membrane-bound sialyltransferase does not alter its Golgi apparatus localization. In addition, sequential replacement of the amino acids of the sialyltransferase signal anchor domain with amino acids from the signal anchor domain of a plasma membrane protein, the influenza virus neuraminidase does not alter the Golgi apparatus localization of the sialyltransferase. These observations suggest that sequences in the signal anchor region and stem region allow the Golgi apparatus localization of the membrane-bound and soluble forms of the sialytransferase, respectively, and that both regions may contain Golgi apparatus localization signals.
Ken-ichi Kasai - One of the best experts on this subject based on the ideXlab platform.
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Involvement of Galectin-1 and Galectin-3 in Proliferation and Migration of Rat Hepatic Stellate Cells in Culture
Comparative Hepatology, 2004Co-Authors: Naoto Maeda, Jun Hirabayashi, Ken-ichi Kasai, Norifumi Kawada, Shuichi Seki, Kazuo Ikeda, Hiroaki Okuyama, Katsutoshi YoshizatoAbstract:Hepatic stellate cells (HSC), liver-specific pericytes, play a pivotal role in hepatic fibrogenesis. Galectin forms a group of animal lectins characterized by their specificity for Beta-Galactosides. At present, more than 10 galectins have been identified in mammals [1]. Galectin-1 forms a homodimer of 14 kDa subunits, and galectin-3 is a monomer having molecular weights of approximately 32 kDa. Galectin-1 and galectin-3 are localized not only in intracellular space such as the cytoplasm or the nucleus but also in extracellular space such as the cell surface or the extracellular matrix. Although the biological functions of galectin-1 and galectin-3 remain speculative in individual cells, tissues, or diseases, there is evidence that they play a role in cellular proliferation, differentiation, adhesion, neoplastic transformation, apoptosis, neoplastic and extracellular matrix interaction [2-4]. These functions are thought to act by cross-linking Beta-Galactoside containing glycoconjugates, resulting in modulation of cell signaling [5,6]. Here, we detail the expression pattern of galectin-1 and galectin-3 in activated HSC and in fibrotic liver tissues. We further show that both galectins are possible mitogens for HSC activating MAP kinase pathways presumably by cross-linking extracellular Beta-Galactoside.
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evidence that caenorhabditis elegans 32 kda Beta Galactoside binding protein is homologous to vertebrate Beta Galactoside binding lectins cdna cloning and deduced amino acid sequence
Journal of Biological Chemistry, 1992Co-Authors: Jun Hirabayashi, Masahiro Satoh, Ken-ichi KasaiAbstract:Abstract We have cloned a full-length cDNA for a Beta-Galactoside-binding protein with a relative molecular mass of 32 kDa (32-kDa GBP), recently purified from a nematode, Caenorhabditis elegans (Hirabayashi, J., Satoh, M., Ohyama, Y., and Kasai, K. (1992) J. Biochem. 111, 553-555). The clone contained a single open reading frame encoding 279 amino acids, including the initiator methionine. Significant sequence homology to metal-independent Beta-Galactoside-binding lectins (25-30% identities), which had previously been found only in vertebrates, was observed. Moreover, the nematode 32-kDa GBP proved to have a unique polypeptide architecture; that is, it is composed of two tandemly repeated homologous domains, each consisting of about 140 amino acids. The internal homology was about 32%. Thus, this protein is constructed with a duplicated fundamental unit which is similar to the subunit of vertebrate 14-kDa lectins. In spite of the extreme phylogenic distance between nematodes and vertebrates (divergence greater than 6 x 10(8) years ago), both of the two repeated domains of the nematode 32-kDa GBP retained most of the amino acid residues conserved in vertebrate lectins. This means that members of the metal-independent animal lectin family are distributed much more widely than had been believed: from nematodes to vertebrates. The implication is that proteins belonging to this family have fundamental roles which are not restricted to vertebrates but are common to almost all animals.
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Purification and characterization of Beta-Galactoside-binding proteins from Caenorhabditis elegans.
Journal of biochemistry, 1992Co-Authors: Jun Hirabayashi, Motohide Satoh, Yuji Ohyama, Ken-ichi KasaiAbstract:Two carbohydrate-binding proteins (subunit molecular masses, 32 and 16 kDa, respectively) were isolated for the first time from a nematode, Caenorhabditis elegans. They were specifically extracted with lactose and adsorbed on asialofetuin-Sepharose in the absence of a metal ion. Although these two proteins were co-eluted from a gel filtration column at a position corresponding to an apparent molecular size of 30 kDa under non-denaturing conditions, they could be separated by reversed-phase chromatography. The 32 kDa protein, the main component, was further characterized. Together with its solubility, saccharide specificity and metal independence, some other structural properties, including its amino acid composition, UV spectrum, and partial amino acid sequence, strongly suggested that the 32 kDa protein is a member of a class of soluble Beta-Galactoside-binding lectins which had previously been only found in vertebrates.
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effect of amino acid substitution by sited directed mutagenesis on the carbohydrate recognition and stability of human 14 kda Beta Galactoside binding lectin
Journal of Biological Chemistry, 1991Co-Authors: Jun Hirabayashi, Ken-ichi KasaiAbstract:Abstract The roles of selected amino acid residues of human 14-kDa Beta-Galactoside-binding lectin were studied by site-directed mutagenesis. Ten mutant lectin proteins were produced, in each of which one of the residues regarded as possibly related to the stability of the lectin (6 cysteine residues) or one of those highly conserved in the vertebrate Beta-Galactoside-binding lectin family (Asn46, Trp68, Glu71, and Arg73), was substituted. All the mutant lectins in which one of the cysteine residues had been substituted with serine (C2S, C16S, C42S, C60S, C88S, and C130S) proved to have sugar binding ability comparable with that of the wild-type lectin. In addition, one of the mutants in which Cys2 was substituted (C2S) was found to have become considerably more stable under non-reducing conditions. It retained asialofetuin binding activity for over a week in the absence of Beta-mercaptoethanol, while the wild-type lectin lost it within a day. This suggests that oxidation of Cys2 could be a key process in the inactivation of human 14-kDa lectin. Substitution of highly conservative Trp68 to tyrosine (W68Y) slightly reduced lactose binding ability, but the mutant was still adsorbed strongly on asialofetuin-agarose. Other mutant lectins in which conservative hydrophilic amino acids were substituted (N46D, E71Q, and R73H) failed to bind to the asialofetuin agarose, with no sign of retardation. Thus, conservative hydrophilic residues proved to be more important in carbohydrate recognition than the cysteine and tryptophan residues, contrary to the widely accepted concept that these latter residues are essential.
Fu-tong Liu - One of the best experts on this subject based on the ideXlab platform.
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Galectin-3, a Beta-Galactoside-binding animal lectin, is a marker of anaplastic large-cell lymphoma.
The American journal of pathology, 1996Co-Authors: Konstantin N. Konstantinov, Bruce A. Robbins, Fu-tong LiuAbstract:Galectin-3 is a member of a newly named family of Beta-Galactoside-binding animal lectins, which has been described with a number of possible important biological functions, including the regulation of cell growth and association with tumor transformation. This protein has a wide tissue distribution but is notably not expressed by normal lymphocytes. We have previously shown that galectin-3 is markedly up-regulated in HTLV-I-infected T cells, most likely mediated by the viral transactivating protein Tax. In this study, we surveyed various lymphomas by immunohistochemistry and found the expression of galectin-3 in all of the 8 cases of Ki-1+ anaplastic large-cell lymphoma (ALCL). Immunoreactivity for galectin-3 was found in a majority of the neoplastic cells in the ALCLs studied. In contrast, only 2 of the 35 cases of other types of lymphoma, including various Hodgkin's and non-Hodgkin's lymphomas, were positive. Unlike the cases of ALCL, immunoreactivity for galectin-3 in these 3 cases was found only sporadically in a small number of neoplastic cells. Thus, galectin-3 may prove to be a useful marker for ALCL and its expression in neoplastic cells in ALCL may contribute to the biological behavior of this specific type of lymphoma.
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Human T lymphotropic virus-I infection of human T lymphocytes induces expression of the Beta-Galactoside-binding lectin, galectin-3.
The American journal of pathology, 1996Co-Authors: Daniel K. Hsu, Ichiro Kuwabara, Stephen R. Hammes, Warner C. Greene, Fu-tong LiuAbstract:Animal lectins play important roles in a variety of biological processes via their recognition of glycoconjugates. Galectin-3 is a Beta-Galactoside-binding lectin previously designated as epsilon BP (IgE-binding protein), CBP35, Mac-2, L-29, and L-34, and its expression has been associated with various physiological and pathological processes, including cell growth, tumor transformation, and metastasis. Galectin-3 is widely distributed in various tissues and cell types and is expressed in many leukocytes, with the notable exception of B and T lymphocytes. We now report that galectin-3 is abundantly expressed in a number of human T lymphotropic virus (HTLV)-I-infected human T cell lines, including F6T, HUT 102, K3T, MT-2, and SLB-I, but is not expressed in non-HTLV-I-infected T cell lines such as Jurkat, CEM, and MOLT-4. In addition, the galectin-3 level was markedly increased in human thymocytes after infection with HTLV-I as compared with uninfected thymocytes. The up-regulation of galectin-3 expression appeared to correlate well with HTLV-I gene expression, as undetectable or very low levels of galectin-3 were found in the S1T and ATL-1K cell lines, which are nonproductively infected with HTLV-I. In co-transfection experiments, the galectin-3 promoter was significantly up-regulated by expression vectors encoding the 40-kd Tax protein, a potent transactivator in HTLV-I. Analysis of various Tax mutants suggested that galectin-3 promoter induction is dependent on activation of the cyclic-AMP-responsive element binding protein/activation transcription factor family of transcription factors and, to a lesser extent, nuclear factor-kappa B/Rel induction. Transfection of human promonocytic U-937 cells with an HTLV-I Tax expression vector induced galectin-3 expression in this cell line. Functionally, galectin-3 was shown to activate interleukin-2 production in Jurkat T cells. Together, these findings raise the possibility that HTLV-I Tax production induces the transcription and subsequent synthesis and secretion of galectin-3, which in turn may further activate these T cells and contribute to the altered properties of cell growth found in adult T cell leukemia induced by HTLV-I.
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Expression and function of galectin-3, a Beta-Galactoside-binding lectin, in human monocytes and macrophages.
The American journal of pathology, 1995Co-Authors: Fu-tong Liu, Riaz I. Zuberi, Daniel K. Hsu, Ichiro Kuwabara, E. Y. Chi, W. R. HendersonAbstract:A family of Beta-Galactoside-binding animal lectins has recently been designated as galectins. One member of this family, galectin-3, has been known as epsilon BP for its IgE-binding activity and as Mac-2, a macrophage surface antigen, CBP35, CBP30, L-29, and L-34. Although much information has accumulated on the expression of this lectin in murine macrophages and human monocytic cell lines, little is known about the expression and function of this protein in normal human monocytes/macrophages. We now report that galectin-3 is expressed in normal human peripheral blood monocytes and its level increases dramatically as human monocytes differentiate into macrophages upon culturing in vitro. Immunoblot analysis showed that there was a 5-fold increase in the level of galectin-3 after 1 day of culture and greater than a 12-fold increase after 5 days. Immunocytochemical analysis confirmed this progressive increase of galectin-3 expression in cultured monocytes. Immunogold cytochemistry/electron microscopy analysis revealed that galectin-3 was expressed on the surface of human monocytes and that the level of cell surface galectin-3 increased progressively as these cells differentiated into macrophages. The level of galectin-3 in human monocytes/macrophages was modulated by stimuli such as lipopolysaccharide and interferon-gamma, and galectin-3 was secreted when monocytes were stimulated by calcium ionophore A23187 Soluble galectin-3 caused superoxide release from human monocytes; this activity was dependent on the lectin property of galectin-3, as it was inhibitable by lactose. Thus, galectin-3 may modulate the function of this cell type in an autocrine or paracrine fashion through binding to cell surface glycoconjugates.
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Human keratinocytes release the endogenous Beta-Galactoside-binding soluble lectin immunoglobulin E (IgE-binding protein) which binds to Langerhans cells where it modulates their binding capacity for IgE glycoforms.
The Journal of experimental medicine, 1993Co-Authors: Andreas Wollenberg, Fu-tong Liu, Daniel Hanau, Thomas BieberAbstract:A better understanding of the pathophysiological role of Langerhans cells (LC) in atopic diseases is dictated by the characterization of the structures involved in immunoglobulin (IgE)-binding on their cell surface. We previously reported that human LC express the high affinity receptor for IgE (Fc epsilon RI), as well as the low affinity receptor for IgE (Fc epsilon RII/CD23). In the present study, we document the presence of a third IgE-binding structure on human LC, the IgE-binding protein (epsilon BP), an endogenous soluble Beta-Galactoside binding lectin. Immunohistochemical studies performed on normal human skin revealed an anti-epsilon BP reactivity in the cytoplasm of keratinocytes and in that of acinous cells of eccrine sweat glands. epsilon BP was also found on the cell surface of LC, as shown by anti-epsilon BP/anti-CD1a double labeling and flow cytometric analysis. Anti-epsilon BP binding to the surface of LC was completely abolished by preincubation with lactose and restored by addition of recombinant human epsilon BP, indicating that epsilon BP binds to LC surface by virtue of its lectin property. Immunoblot analysis of anti-epsilon BP-reactive material in keratinocytes and purified LC disclosed a protein with an apparent molecular weight of 33,000 consistent with epsilon BP. Interestingly, mRNA transcripts for epsilon BP were detected only in keratinocytes but not in purified LC isolated from normal skin. epsilon BP was found to be released in culture supernatants of keratinocytes. Incubation of LC with these supernatants resulted in epsilon BP-binding to LC surface via protein-carbohydrate interaction. Most importantly, we could show that binding of human myeloma IgE to LC was inhibited by epsilon BP. In contrast, neuraminidase-treated human myeloma IgE binds to LC only in the presence of epsilon BP. In situ binding studies revealed that keratinocytes, although containing epsilon BP intracytoplasmatically, failed to exhibit any IgE-binding properties. Collectively, our results suggest that human keratinocytes produce the Beta-Galactoside-binding lectin epsilon BP, which subsequently binds to the surface of LC where it is functional in modulating their binding capacity for IgE glycoforms.
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Epsilon BP, a Beta-Galactoside-binding animal lectin, recognizes IgE receptor (Fc epsilon RI) and activates mast cells.
Biochemistry, 1993Co-Authors: Luciano G. Frigeri, Riaz I. Zuberi, Fu-tong LiuAbstract:IgE-binding protein (epsilon BP) was originally identified in rat basophilic leukemia (RBL) cells by virtue of its affinity for IgE. epsilon BP is now known to be a Beta-Galactoside-binding lectin containing an S-type carbohydrate recognition domain. It is identical to a macrophage surface antigen, Mac-2, and lectins designated as CBP35, L-34, and RL-29, for which various functions have been suggested. Studies from other groups as well as ours have indicated that epsilon BP is secreted by cells such as macrophages and is present in extracellular fluids. We demonstrated previously that binding sites for epsilon BP are present on the surface of RBL cells. In this report, we show that epsilon BP binds to a small number of glycoprotein species on the surface of RBL cells. Significantly, one of these glycoproteins is the high-affinity IgE receptor (Fc epsilon RI). Preliminary studies showed that epsilon BP causes mediator release from RBL cells, possibly through cross-linking of Fc epsilon RI. The results suggest a function of epsilon BP as an activator of mast cells.
Jun Hirabayashi - One of the best experts on this subject based on the ideXlab platform.
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Involvement of Galectin-1 and Galectin-3 in Proliferation and Migration of Rat Hepatic Stellate Cells in Culture
Comparative Hepatology, 2004Co-Authors: Naoto Maeda, Jun Hirabayashi, Ken-ichi Kasai, Norifumi Kawada, Shuichi Seki, Kazuo Ikeda, Hiroaki Okuyama, Katsutoshi YoshizatoAbstract:Hepatic stellate cells (HSC), liver-specific pericytes, play a pivotal role in hepatic fibrogenesis. Galectin forms a group of animal lectins characterized by their specificity for Beta-Galactosides. At present, more than 10 galectins have been identified in mammals [1]. Galectin-1 forms a homodimer of 14 kDa subunits, and galectin-3 is a monomer having molecular weights of approximately 32 kDa. Galectin-1 and galectin-3 are localized not only in intracellular space such as the cytoplasm or the nucleus but also in extracellular space such as the cell surface or the extracellular matrix. Although the biological functions of galectin-1 and galectin-3 remain speculative in individual cells, tissues, or diseases, there is evidence that they play a role in cellular proliferation, differentiation, adhesion, neoplastic transformation, apoptosis, neoplastic and extracellular matrix interaction [2-4]. These functions are thought to act by cross-linking Beta-Galactoside containing glycoconjugates, resulting in modulation of cell signaling [5,6]. Here, we detail the expression pattern of galectin-1 and galectin-3 in activated HSC and in fibrotic liver tissues. We further show that both galectins are possible mitogens for HSC activating MAP kinase pathways presumably by cross-linking extracellular Beta-Galactoside.
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evidence that caenorhabditis elegans 32 kda Beta Galactoside binding protein is homologous to vertebrate Beta Galactoside binding lectins cdna cloning and deduced amino acid sequence
Journal of Biological Chemistry, 1992Co-Authors: Jun Hirabayashi, Masahiro Satoh, Ken-ichi KasaiAbstract:Abstract We have cloned a full-length cDNA for a Beta-Galactoside-binding protein with a relative molecular mass of 32 kDa (32-kDa GBP), recently purified from a nematode, Caenorhabditis elegans (Hirabayashi, J., Satoh, M., Ohyama, Y., and Kasai, K. (1992) J. Biochem. 111, 553-555). The clone contained a single open reading frame encoding 279 amino acids, including the initiator methionine. Significant sequence homology to metal-independent Beta-Galactoside-binding lectins (25-30% identities), which had previously been found only in vertebrates, was observed. Moreover, the nematode 32-kDa GBP proved to have a unique polypeptide architecture; that is, it is composed of two tandemly repeated homologous domains, each consisting of about 140 amino acids. The internal homology was about 32%. Thus, this protein is constructed with a duplicated fundamental unit which is similar to the subunit of vertebrate 14-kDa lectins. In spite of the extreme phylogenic distance between nematodes and vertebrates (divergence greater than 6 x 10(8) years ago), both of the two repeated domains of the nematode 32-kDa GBP retained most of the amino acid residues conserved in vertebrate lectins. This means that members of the metal-independent animal lectin family are distributed much more widely than had been believed: from nematodes to vertebrates. The implication is that proteins belonging to this family have fundamental roles which are not restricted to vertebrates but are common to almost all animals.
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Purification and characterization of Beta-Galactoside-binding proteins from Caenorhabditis elegans.
Journal of biochemistry, 1992Co-Authors: Jun Hirabayashi, Motohide Satoh, Yuji Ohyama, Ken-ichi KasaiAbstract:Two carbohydrate-binding proteins (subunit molecular masses, 32 and 16 kDa, respectively) were isolated for the first time from a nematode, Caenorhabditis elegans. They were specifically extracted with lactose and adsorbed on asialofetuin-Sepharose in the absence of a metal ion. Although these two proteins were co-eluted from a gel filtration column at a position corresponding to an apparent molecular size of 30 kDa under non-denaturing conditions, they could be separated by reversed-phase chromatography. The 32 kDa protein, the main component, was further characterized. Together with its solubility, saccharide specificity and metal independence, some other structural properties, including its amino acid composition, UV spectrum, and partial amino acid sequence, strongly suggested that the 32 kDa protein is a member of a class of soluble Beta-Galactoside-binding lectins which had previously been only found in vertebrates.
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effect of amino acid substitution by sited directed mutagenesis on the carbohydrate recognition and stability of human 14 kda Beta Galactoside binding lectin
Journal of Biological Chemistry, 1991Co-Authors: Jun Hirabayashi, Ken-ichi KasaiAbstract:Abstract The roles of selected amino acid residues of human 14-kDa Beta-Galactoside-binding lectin were studied by site-directed mutagenesis. Ten mutant lectin proteins were produced, in each of which one of the residues regarded as possibly related to the stability of the lectin (6 cysteine residues) or one of those highly conserved in the vertebrate Beta-Galactoside-binding lectin family (Asn46, Trp68, Glu71, and Arg73), was substituted. All the mutant lectins in which one of the cysteine residues had been substituted with serine (C2S, C16S, C42S, C60S, C88S, and C130S) proved to have sugar binding ability comparable with that of the wild-type lectin. In addition, one of the mutants in which Cys2 was substituted (C2S) was found to have become considerably more stable under non-reducing conditions. It retained asialofetuin binding activity for over a week in the absence of Beta-mercaptoethanol, while the wild-type lectin lost it within a day. This suggests that oxidation of Cys2 could be a key process in the inactivation of human 14-kDa lectin. Substitution of highly conservative Trp68 to tyrosine (W68Y) slightly reduced lactose binding ability, but the mutant was still adsorbed strongly on asialofetuin-agarose. Other mutant lectins in which conservative hydrophilic amino acids were substituted (N46D, E71Q, and R73H) failed to bind to the asialofetuin agarose, with no sign of retardation. Thus, conservative hydrophilic residues proved to be more important in carbohydrate recognition than the cysteine and tryptophan residues, contrary to the widely accepted concept that these latter residues are essential.
Ten Feizi - One of the best experts on this subject based on the ideXlab platform.
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Subunit molecular mass assignment of 14,654 Da to the soluble Beta-Galactoside-binding lectin from bovine heart muscle and demonstration of intramolecular disulfide bonding associated with oxidative inactivation.
The Journal of biological chemistry, 1992Co-Authors: B M Tracey, Ten Feizi, W M Abbott, Robert A. Carruthers, B N Green, Alexander M. LawsonAbstract:Abstract The soluble dimeric Beta-Galactoside-binding lectin (subunit molecular mass, approximately 14 kDa) of bovine heart muscle, in common with the 14-kDa lectins of several other animal species, displays carbohydrate-binding activity when it is in the reduced state, but the purified lectin loses this activity upon oxidation. In the present study, the presence of any post-translational modification and the mechanism of the oxidative inactivation have been investigated by analyses of the reduced and oxidized forms of the purified bovine lectin by electrospray ionization-mass spectrometry (ESI-MS) and by liquid secondary ion mass spectrometry (LSIMS) of tryptic and peptic peptides. By ESI-MS, the molecular mass of the reduced lectin is determined to be 14,654.6 +/- 0.9 Da, and that of the oxidized lectin is 14,649.3 +/- 1.1 Da. These masses correspond to the amino acid sequence of the protein with the cysteines having free sulfhydryl groups in the reduced state and forming disulfide bonds in the oxidized state. There is no evidence of post-translational modification in either lectin form except for monoacetylation already predicted for alanine at the blocked N-terminal end. Pronounced differences in charge distribution in the electrospray ionization mass spectra of the reduced and oxidized lectin, reflecting a change in the number of accessible protonation sites in the oxidized protein, are consistent with the protein being held in an altered conformation by covalent bonding. The results of LSIMS analyses of tryptic and peptic peptides in conjunction with Edman sequencing indicate that disulfide bonding occurs predominantly between Cys2 and Cys130, Cys16 and Cys88, and Cys42 and Cys60. There is no evidence of oxidation of Trp68. These results, taken together with observations that almost the complete polypeptide chain is necessary for the functional integrity of the carbohydrate recognition domain (Abbott, W. M., and Feizi, T. (1991) J. Biol. Chem. 266, 5552-5557) point to intramolecular disulfide bonding with a change in protein folding and conformation as the mechanism of oxidative inactivation of the purified bovine lectin.
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Soluble 14-kDa Beta-Galactoside-specific bovine lectin. Evidence from mutagenesis and proteolysis that almost the complete polypeptide chain is necessary for integrity of the carbohydrate recognition domain.
The Journal of biological chemistry, 1991Co-Authors: W M Abbott, Ten FeiziAbstract:The soluble Beta-Galactoside-specific bovine lectin of subunit 14 kDa has been expressed in vitro by transcription and then translation in a rabbit reticulocyte lysate. The protein thus expressed shows the predicted binding to lactose coupled to Sepharose. Several mutants of the 134 amino acid protein have been expressed and insight gained into (a) the polypeptide length required to form the carbohydrate recognition domain and (b) the functional importance of some of the highly conserved amino acids. The following amino acids have been deleted: 1-9, 1-23, 88-122, 88-134, 107-134, or 124-134. In addition, a frame-shift mutant has been made in which the 23 amino acids at the C-terminal end were completely changed. Among these seven mutants only mutant 1-9 shows carbohydrate binding but with congruent to 30% of the activity of the wild-type protein (as assessed by the percentage of the protein bound to lactose-Sepharose). On the other hand, carbohydrate binding is relatively well preserved (75-90%) in mutant proteins where the C-terminal octapeptide sequence of the bovine lectin has been changed to sequences that resemble those in the chick 14-kDa lectin. When the single tryptophan at position 68 is changed by point mutagenesis to phenylalanine or to a leucine residue, a weak binding activity (congruent to 20%) is retained only with the former. When either of the cysteines 2 or 60 is changed to serine, binding activity is reduced to congruent to 60%, and when both are changed, to congruent to 20% of that for the wild-type protein. The susceptibility of the lectin to oxidative inactivation is unaffected when these 2 cysteines and cysteine 130 are changed to serine individually or in tandem (cysteines 2 and 60). In a second approach we show that the natural protein isolated from bovine heart is protected from proteolysis by trypsin and V8-protease in the presence of saccharide ligand. Although further work is required to identify residues which come into contact with the carbohydrate ligand, these results indicate that almost the complete polypeptide chain is necessary for the integrity of the carbohydrate recognition domain.