Search PubMed⌕ Search

Biomedical subjects

J Hirabayashi

Publications and source records attributed to J Hirabayashi.

At least 37 records · Page 2Linked to original sources
PubMed1997

Structure of the 32-kDa galectin gene of the nematode Caenorhabditis elegans.

Galectins are a family of soluble beta-galactoside-binding lectins distributed in both vertebrates and invertebrates and, more recently, found also in fungus. The 32-kDa galectin isolated from the nematode Caenorhabditis elegans (Hirabayashi, J., Satoh, M., and Kasai, K. (1992) J. Biol. Chem. 267, 15485-15490) was the first "tandem repeat-type" galectin, containing two homologous carbohydrate-binding sites. Here, we report the structure of the nematode 32-kDa galectin gene. Physical mapping by yeast artificial chromosome polytene filter hybridization revealed that the 32-kDa galectin gene is located on chromosome II. Analysis of the transcript (1.4 kilobases) showed the presence at its 5'-end of a 22-nucleotide trans-spliced leader sequence (SL1). The entire genomic structure spanning >5 kilobase pairs (kbp), including the 5'-noncoding region, two intervening sequences (introns 1 and 2), and the 3'-noncoding region, was completely determined by the combination of genomic polymerase chain reaction and conventional colony hybridization. Intron 1 was relatively long (2.4 kbp) and was found to be inserted after the ninth codon (TAC) from the initiation codon. This position proved to be almost homologous to the conserved first intron insertion position in the vertebrate galectin genes (i. e. genes of mammalian galectin-1, -2, and -3 and chick 14-kDa galectin). On the other hand, intron 2 was much shorter (0.6 kbp), and it was inserted into the central region of the second carbohydrate-binding site. Although such an insertion pattern has never been observed in the vertebrate galectin genes, it seems to be common in C. elegans tandem repeat-type galectin genes, as predicted by the C. elegans genome project (Coulson, A., and the C. elegans Genome Consortium (1996) Biochem. Soc. Trans. 24, 289-291). Based on extensive sequence comparison, the origin and molecular evolution of the tandem repeat-type galectins are discussed.

Amino Acid Sequence↗
PubMed1997

The two lectin domains of the tandem-repeat 32-kDa galectin of the nematode Caenorhabditis elegans have different binding properties. Studies with recombinant protein.

Some properties of recombinant proteins derived from the 32-kDa galectin isolated from the nematode Caenorhabditis elegans, which lectin is composed of two tandemly repeated homologous domains [Hirabayashi et al. (1992) J. Biol. Chem. 267, 15485], were studied in order to elucidate the function of this unique polypeptide architecture. We expressed the whole molecule (N32), the N-terminal lectin domain (Nh), and the C-terminal lectin domain (Ch) in Escherichia coli using the expression vector pET21a. All of the recombinant proteins were bound by asialofetuin-Sepharose. CD spectra of the recombinant proteins indicated all of them to be rich in beta-structure and properly refolded. Gel filtration on an HPLC column suggested that all of them existed as monomers. Neither Nh nor Ch seemed to form dimers, in contrast to vertebrate proto-type galectins. Only N32 showed hemagglutination activity towards trypsinized rabbit erythrocytes. Comparison of the affinity of N32, Nh, and Ch for asialofetuin-Sepharose by frontal affinity chromatography [Kasai et al. (1986) J. Chromatogr. 376, 33] showed that Ch has 7-fold weaker affinity than N32, and Nh proved to have still weaker affinity. Since the Asn residue in the CRD (carbohydrate recognition domain), which is conserved in all other galectins, is substituted by Ser in the case of Nh, these data suggest that the two CRDs in this tandem-repeat galectin have different sugar binding properties and that the 32-kDa galectin may serve as a heterobifunctional crosslinker.

Animals↗
PubMed1996

Purification and molecular characterization of a novel 16-kDa galectin from the nematode Caenorhabditis elegans.

In our previous study (Hirabayashi, J., Satoh, M., Ohyama, Y., and Kasai, K. (1992) J. Biochem. (Tokyo) 111, 553-555), two beta-galactoside-binding lectins (apparent subunit molecular masses, 16 and 32 kDa, respectively) were identified in the nematode Caenorhabditis elegans. The subsequent study revealed that the 32-kDa lectin is a member of the galectin family. Since the 32-kDa galectin was found to consist of two homologous domains (approximately 16 kDa), 16-kDa lectin was thought to be a degradation product of the 32-kDa galectin. To clarify this, the 16-kDa lectin was purified by an improved procedure employing extraction with a calcium-supplemented buffer. The purified 16-kDa lectin was found to exist as a dimer (approximately 30 kDa) and showed hemagglutinating activity toward trypsinized rabbit erythrocytes, which was inhibited by lactose. Almost the whole sequence of the 16-kDa polypeptide (approximately 95%, 135 amino acids) was determined after digestion with various proteases. Based on the obtained information, a full-length cDNA was cloned with the aid of RNA-polymerase chain reaction. The clone encoded 146 amino acids including initiator methionine (calculated molecular mass, 15,928 Da). Based on these results, it was concluded that the 16-kDa lectin is a novel member of the galectin family, but not a degradation product of the 32-kDa galectin as had previously thought. However, the 16-kDa galectin showed relatively low sequence similarities to both the N-terminal and the C-terminal domains of the 32-kDa galectin (28% and 27% identities, respectively) and to various vertebrate galectins (14-27%). Nonetheless, all of the critical amino acids involved in carbohydrate binding were conserved. These observations suggest that, in spite of phylogenic distance between nematodes and vertebrates, both the 16-kDa and 32-kDa nematode isolectins have conserved essentially the same function(s) as those of vertebrate galectins, probably through recognition of a key disaccharide moiety, "N-acetyllactosamine."

Amino Acid Sequence↗
PubMed1996

Applied slalom chromatography improved DNA separation by the use of columns developed for reversed-phase chromatography.

Improved resolution in slalom chromatography, a novel size-fractionation method discovered recently for relatively large DNA molecules (> 5 kpb), was obtained by using columns generally employed for reversed-phase chromatography: i.e., two types of Capcell-Pak (methyl or phenyl-derivatized 5-microns microbeads), and five types of Hypersil-3 packings (trimethylsilyl, dimethyloctyl, cyanopropyl, octadecyl or phenyl-derivatized 3-microns microbeads). The resolution of 5-15-kbp DNA was significantly improved by employing these columns, though the separation characteristics differed. When Capcell-Pak columns were used with a normal low-salt eluting solvent (10 mM sodium phosphate, pH 6.8, 1 mM EDTA), chromatograms were obtained for lambda/HindIII fragments (a mixture of 0.1, 0.5, 2.0, 2.3, 4.4, 6.6, 9.4 and 23.1-kbp fragments) similar to those obtained previously with Asahipak GS-310 5-microns size-exclusion packings. However, when up to 0.2 M NaCl was added to the solvent, the DNA was increasingly retarded, particularly the 4.4, 6.6 and 9.4-kbp fragments, resulting in improved resolution in the low to middle molecular-mass range. The effect of salt was more significant with Capcell-Pak Phe than C1, although various features characteristic of slalom chromatography were preserved with both columns; i.e., dependency on DNA size, flow-rate, and temperature. This suggests that a mixed mode of separation, that is, slalom mode and hydrophobic-interaction mode, was operating. Although all of the Hypersil-3 packings showed significant adsorption of lambda/HindIII fragments under low-salt conditions, the fragments could be eluted with satisfactory yield and resolution by adding acetonitrile (> 5%) to the solvent. Notably, these Hypersil-3 packings allowed resolution of a 4.4-kbp lambda/HindIII fragment from the flow-through fraction for the first time, possibly due to their small particle size. Thus, various packing materials developed for high-performance liquid chromatography proved to be applicable for slalom chromatography, though the eluting conditions still need to be refined. The results support the concept that slalom chromatography is based on a hydrodynamic phenomenon.

Cation Exchange Resins↗
PubMed1996

Cell calcium signalling induced by endogenous lectin carbohydrate interaction in the Jurkat T cell line.

The effects of the beta-galactoside-binding lectin from human placenta (HPL14) on intracellular calcium concentration ([Ca2+]i) were examined in the human Jurkat T cell line. The lectin induces a concentration dependent increase in [Ca2+]i. This calcium signalling effect is clearly mediated through complementary cell surface galactoglycoconjugates because it can be blocked by beta-galactosides. The observed Ca2+ - response involves both the release of calcium from intracellular stores and a calcium influx from the extracellular space. It is sustained in the presence of 1 mM extracellular calcium whereas it becomes transient when the influx of extracellular calcium was blocked by calcium chelation to EGTA. Voltage-sensitive calcium channel blockers like verapamil and prenylamine were without effect on the action of HPL14. Protection of the sugar binding activity of HPL14 in the absence of a thiol-reducing reagent by carboxamidomethylation (CM-HPL14) or by substitution Cys2 with serine (C2S) results in lectin proteins with considerably decreased calcium signalling efficiency. The recombinant lectin (Rec H) and the mutant protein obtained by substitution of highly conservative Trp68 with tyrosine (W68Y) induce lower levels of [Ca2+]i compared to wild type lectin.

Binding Sites↗
PubMed1996

An immunohistochemical study of the 32-kDa galectin (beta-galactoside-binding lectin) in the nematode Caenorhabditis elegans.

The localization of the 32-kDa galectin (beta-galactoside-binding lectin) of the nematode Caenorhabditis elegans, which is the first lectin to be found in a nematode, was examined immunohistochemically using an anti-lectin antiserum. The lectin was found to be localized most abundantly in the adult cuticle and also in the terminal bulb of the pharynx. However, it was difficult to locate the galectin in larval animals, though immunochemical experiments suggested its presence. These results suggest that one of the fundamental roles of the galectin may be as a component of the durable outer barrier, as in the case of the morphogenesis of chick embryonic skin.

Animals↗
PubMed1996

On the origin of elementary hexoses.

A possible scenario on the origin of elementary hexoses (fructose, glucose, mannose, and galactose) is proposed: (1) Formol reaction, an autocatalytic polymerization reaction, occurred on the primitive earth under weakly alkaline conditions to generate various small molecular compounds. (2) Among them, glyceraldehyde and dihydroxyacetone undergo aldol condensation to produce stable ketohexoses, fructose and sorbose. (3) Subsequently, Lobry de Bruyn rearrangement converts fructose into stable aldohexoses, glucose, and mannose. (4) Thus, prebiotically synthesized ¿first triplet¿ hexoses would have been available for utilization by ancestral primitive microorganisms. (5) After the development of biochemical pathways, various saccharides, including galactose, were biosynthesized from glucose and mannose as ¿bricolage products¿; the utilization of galactose as a key recognition molecule, based on its distinctive axial 4-OH and its outermost location in glycoconjugates, owing to its late arrival, may have evolved concomitantly with the evolution of multicellular organisms.

Animals↗
PubMed1996

Galectins: a family of animal lectins that decipher glycocodes.

Galectins, animal lectins exhibiting specificity for galactosides, are now known to be widely distributed from lower invertebrates, such as sponges and nematodes, to higher vertebrates. The origin of the family can be traced back to the Precambrian era. They are classified into proto-, chimera-, and tandem-repeat types on the basis of protein architecture. The molecular functions of these types should be different because they can cross-link pairs of biomolecules of different combinations. Their biological significance, however, is not yet fully understood because they are involved in too many phenomena, such as differentiation, morphogenesis, metastasis, etc., and too many problems remain unsolved, such as those regarding their controversial cellular localization, mechanism of externalization, etc. Nevertheless, such difficulties seem to indicate their importance as household equipment and their common roles throughout the animal kingdom. They are likely to be responsible for recognizing the N-acetyllactosamine (LacNAc) structure, which is included in various glycoconjugates and considered to be an important glycocode, and then carry out appropriate tasks under given circumstances. Recently, crystallographic studies revealed that galectins and legume lectins such as concanavalin A have a common topology in spite of the absence of sequence homology. This suggests a possible relationship between animal and plant lectins, and the existence of a lectin super family. Studies on the galectin family are becoming increasingly important for glycobiology.

Amino Sugars↗
PubMed1995

Changes in expression of two endogenous beta-galactoside-binding isolectins in the dermis of chick embryonic skin during development in ovo and in vitro.

In order to elucidate the roles of metal-independent animal lectins, we systematically investigated changes in expression of 2 kinds of beta-galactoside-binding isolectins (MW 14 and 16 kDa) in the dermis of chick embryonic tarsometatarsal skin during the course of development. These lectins were immunohistochemically located at different stages of development both in ovo and in vitro by light and electron microscopy. Light-microscopic observation showed that while positive staining for the 14-kDa lectin was weak at days 8 and 10 it became intense after day 13. In contrast, staining for the 16-kDa lectin was intense at days 8, 10, and 13, but it became weak after day 17 when keratinization of the epidermis was completed. Immuno-electron-microscopic observation revealed that both the 14 and 16-kDa lectins were located on the basement membrane, in the extracellular matrix, and in both the cytoplasm and the nucleus of dermal fibroblasts. Distribution of the 2 isolectins was also examined in cultured skin explants in vitro. The results were almost the same as those obtained in ovo when the skin explant was keratinized in the presence of hydrocortisone. However, in the skin explant where keratinization was prevented and mucous metaplasia was induced by the addition of vitamin A, the distribution of the 14-kDa lectin in the epidermis was significantly affected. These results indicate that (1) the expression of the 2 isolectins is differently regulated in both the dermis and epidermis, (2) the 16-kDa lectin is involved in the early stage of the formation of the dermis and the basement membrane and is replaced by the 14-kDa lectin as keratinization of the epidermis occurs, and (3) the expression of the 2 isolectins in the dermis is not significantly affected by the induction of mucous metaplasia, in contrast to their drastic changes in the epidermis.

Animals↗
PubMed1995

Expression of the endogenous 14-kDa beta-galactoside-binding lectin galectin in normal human skin.

The localization of an endogenous 14-kDa beta-galactoside-binding lectin (galectin) and its pattern of gene expression were examined in normal human skin by light- and electron microscopy. Under the light microscope, immunostaining of 14-kDa galectin was observed in the cell membrane of cells in the basal and spinous layers of the epidermis. Galectin was also found in the Langerhans cells, as shown by double labeling using anti-14-kDa galectin and anti-CD1a antibodies. In the dermis, immunostaining for the 14-kDa galectin was positive in the extracellular matrix and fibroblasts. At the electron-microscopic level of resolution, galectin was located primarily along the plasma membrane of keratinocytes, and in both the cytoplasm and nucleus of Langerhans cells in the epidermis, whereas in the dermis it was detected in the extracellular matrix and in both the nucleus and cytoplasm of fibroblasts. The gene expression of 14-kDa galectin was visualized by the HRP-staining method following in situ hybridization techniques. The expression was detected in the cytoplasm of cells in the basal and spinous layers of the epidermis; whereas, in the dermis, it was detected in the cytoplasm of fibroblasts. Moreover, SDS-polyacrylamide gel electrophoresis and lectin-blot analysis revealed that this galectin bound to glycoproteins of approximately 17, 62, and 72 kDa in the epidermis and to those of 29, 54, and 220 kDa in the dermis. The present study indicates that 1) normal human skin produces the beta-galactoside-binding 14-kDa galectin, and 2) this galectin is located in both the epidermis, particularly in the keratinocytes and Langerhans cells, and in the dermis. These results suggest that galectin is important for cell-cell contact and/or adhesion in the epidermis and for cell-extracellular matrix interaction in the dermis.

Base Sequence↗
PubMed1994

Immunohistochemical localization of a beta-D-galactoside-binding lectin at the human maternofetal interface.

The 14 kD S-type lectin from human placenta may have a role in regulating the maternal immune response to fetal antigens. In this study, an immunoperoxidase technique was used to determine the distribution of the lectin at the human maternofetal interface. Tissue obtained during the first trimester of pregnancy and at term was used. The lectin was not detectable in either the villous syncytiotrophoblast or the underlying cytotrophoblast in first-trimester tissue, although some cells of the cytotrophoblast columns were reactive. It was also not detectable in villous or extravillous trophoblast populations at term. In contrast, strong reactivity was found in decidual stromal cells throughout gestation, and endometrial stromal cells were also positive. The lectin is, therefore, not a component of the immunosuppressive factors associated with syncytiotrophoblast membranes, but may have a role in either the decidual control of trophoblast migration or some functions unrelated to pregnancy, or both.

Chorionic Villi↗
PubMed1994

Further evidence by site-directed mutagenesis that conserved hydrophilic residues form a carbohydrate-binding site of human galectin-1.

To identify critical amino acid residues for carbohydrate binding of galectins (soluble beta-galactoside-binding lectins found in the animal kingdom). site-directed mutagenesis was performed on human galectin-1. On the basis of the previous results (Hirabayashi and Kasai (1992) J Biol Chem 266:23648-53), more systematic mutagenesis experiments were performed in order to confirm the concept that conserved hydrophilic residues play a central role. When a homologous substitution was made for highly conserved His44, Arg48 or Asn61, the resultant mutant (H44Q, R48H or N61D, respectively) almost completely lacked carbohydrate-binding ability, as found previously for Asn46, Glu71 and Arg73 mutants. This suggests these six hydrophilic residues are essential. On the other hand, when less conserved Lys63, Arg111 or Asp125 were substituted, the resultant mutant (K63H, R111H or D125E, respectively) retained almost the same affinities to asialofetuin and lactose as the wild-type galectin. Therefore, none of these residues is directly involved in the binding. These results, together with the previous observation that the above six essential residues are all encoded in the largest exon of the gene and are located close to each other in the central, most hydrophilic region of the protein, suggest that the residues form a carbohydrate-binding site of galectin.

Amino Acid Sequence↗
PubMed1993

Secretion of endogenous 16-kDa beta-galactoside-binding lectin from vitamin A-pretreated chick embryonic cultured skin.

Localization of endogenous beta-galactoside-binding 16-kDa lectin and its gene expression pattern were investigated during differentiation of chick embryonic skin in vivo and in vitro and were compared with those of a 14-kDa lectin. By light microscopy, immunostaining of the 16-kDa lectin was weak in the undifferentiated epidermis, while it became intense in the keratinized epidermis, particularly in the intermediate cells. Essentially the same staining pattern was observed both in vivo and in vitro. The gene expression pattern was consistent with these immunohistochemical observations. These results were similar to those of the 14-kDa lectin [1]. On the other hand, in the vitamin A-pretreated cultured skin, mucous metaplasia of the epidermis was induced and marked changes in localization of these two isolectins were observed. The 16-kDa lectin expression was increased in the epidermis, especially in the superficial cells, and the gene expression was detected in all layers of the epidermis. However, 14-kDa lectin expression was markedly reduced in the metaplastic epidermis. In the keratinized epidermis, detailed localization of these lectins under the electron microscope was almost the same. Both of them were located primarily along the plasma membrane, in the intercellular space, and in the desmosomes. In the mucous metaplastic epidermis, however, the localization of the 16-kDa lectin was completely different from that of the 14-kDa lectin; e.g., the former was detected in the mucous granules and on the microvilli of the superficial cells, while the latter was scarcely observed in the epidermis. The present results revealed that (1) the 16-kDa lectin as well as 14-kDa lectin were expressed during the epidermal differentiation and (2) much of the 16-kDa lectin was produced and secreted from the vitamin A-pretreated cultured epidermis, while 14-kDa lectin disappeared. In the view of these facts, it is suggested that (1) both 14- and 16-kDa lectins may play an important role in differentiation of the skin and (2) the gene expression of 14- and 16-kDa lectins is regulated independently.

Animals↗
PubMed1993

The family of metazoan metal-independent beta-galactoside-binding lectins: structure, function and molecular evolution.

Animal metal-independent beta-galactoside-binding lectins were initially found in vertebrates, but they have recently been isolated from much lower invertebrates, such as nematode and sponge, as well. Further, an eosinophilic lysophospholipase associated with various inflammatory reactions was very recently found to be a new member of this protein family. It appears that beta-galactoside-binding lectins and some non-lectin proteins form a superfamily whose members are widely distributed from vertebrates to invertebrates. From the viewpoints of protein architecture, the superfamily members can be subdivided into three types; i.e. 'proto type' (the relatively well-studied 14 kDa lectins), 'chimera type' (29-35 kDa lectins also known as epsilon BP/CBP35/Mac2/laminin-binding protein) and 'tandem-repeat type' (a newly found nematode 32 kDa lectin). Comparison of their amino acid sequences and mutagenesis studies have suggested the functional importance of some conservative hydrophilic residues (His44, Asn46, Arg48, Glu71 and Arg73 of human 14 kDa lectin). Several non-charged residues (Gly14, Phe45, Pro47, Phe49, Val59, Trp68, Pro78 and Phe79) are also well conserved, and are probably important to maintain the structural framework of these proteins. A consideration of molecular evolution suggests that lectins belonging to this family probably existed in the Precambrian era. Ubiquitous occurrence of these homologous lectins with shared sugar specificity suggests that they are involved in 'essential minimum' functions of multicellular animals, possibly in cooperation with their partner glycoconjugates.

Amino Acid Sequence↗
PubMed1992

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.

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.

Amino Acid Sequence↗