Purification and characterization of a β-galactoside-binding soluble lectin from rat and bovine brain

A β-galactoside-binding activity has been detected in mammalian brain extracts using a hemagglutination test and a nerve cell aggragation assay. Inhibition studies suggested the involvement of lectin-carbohydrate interactions in these processes. In an attempt to explore further the biological role of brain lectins, the β-galactoside-binding activity has been purified to apparent homogeneity from bovine and rat brain by salt extraction of the brain tissue and affinity chromatography on asialofetuin-agarose. The molecular weights determined by gel filtration, under native conditions on Ultrogel AcA-34, were 30 000 for the bovine brain lectin and 32 000 for the rat brain lectin; polyacrylamide gel electrophoresis in SDS gave molecular weights of 15 000 and 16 000, respectively, suggesting that the two brain lectins are dimers. Both lectins have an isoelectric point of 3.9. Amino acid composition data indicate that both lectins contain high proportions of glycine and acidic amino acids. The lectins are specific for β-D-galactosides and related sugars and the configuration of carbon atoms 1, 2 and 4 seems of primary importance. Moreover, the nerve cell aggregation-promoting activity of the purified lectin is 300-fold that of the crude extracts.     

Immunohistochemical localization of 14 kDa β-galactoside-binding lectin in various organs of rat

Summary Immunohistochemical localization of 14 kDa -galactoside-binding lectin in various organs of adult rat was achieved using a monospecific antibody raised against lectin purified from rat lung. The antibody-stained cells were formed into small aggregates, thin fascicles, or thick bundles in the walls of blood vessels, gastrointestinal tracts and urogenital organs. From the patterns of distribution, as well as their organization, these immunoreactive cells were regarded as smooth muscle cells. This was confirmed by a double immunofluorescence study using a mixture of anti 14 kDa lectin and anti -smooth muscle-specific actin antibodies. Strong 14 kDa lectin immunoreactivity was seen in the pericellular matrix of smooth muscle cells in intact organs as well as in detergent-treated organs from which all cellular components were extracted. From these findings, it is suggested that the 14 kDa lectin may be externalized by smooth muscle cells into their pericellular matrix and participate in the crosslinking of the complementary glycoconjugate(s) localized at that site. The macromolecular complex of glycoconjugates thus formed around smooth muscle cells may play a role in anchoring smooth muscle cells to the pericellular connective tissue thereby permitting the force of muscle contraction to be efficiently transmitted to the surrounding connective tissue proper.     

Binding of biogenic and synthetic polyamines to β-lactoglobulin

The bindings of biogenic polyamines spermine (spm), spermidine (spmd) and synthetic polyamines 3,7,11,15-tetrazaheptadecane·4HCl (BE-333) and 3,7,11,15,19-pentazahenicosane·5HCl (BE-3333) with β-lactoglobulin (β-LG) were determined in aqueous solution. FTIR, UV-vis, CD and fluorescence spectroscopic methods as well as molecular modeling were used to determine the polyamine binding sites and the effect of polyamine complexation on protein stability and secondary structure. Structural analysis showed that polyamines bind β-LG via both hydrophilic and hydrophobic contacts. Stronger polyamine-protein complexes formed with synthetic polyamines than biogenic polyamines, with overall binding constants of K_spm-β-LG = 3.2(±0.6) × 10⁴ M⁻¹, K_spmd-β-LG = 1.8(±0.5) × 10⁴ M⁻¹, K_BE-333-β-LG = 5.8(±0.3) × 10⁴ M⁻¹ and K_{BE-3333-β-LG} = 6.2(±0.05) × 10⁴ M⁻¹. Molecular modeling showed the participation of several amino acids in the polyamine complexes with the following order of polyamine-protein binding affinity: BE-3333 > BE-333 > spermine > spermidine, which correlates with their positively charged amino group content. Alteration of protein conformation was observed with a reduction of β-sheet from 57% (free protein) to 55-51%, and a major increase of turn structure from 13% (free protein) to ∼21% in the polyamine-β-LG complexes, indicating a partial protein unfolding.     

Rat lung 29 kD β-galactoside-binding lectin is secreted by bronchiolar Clara cells into airways

Summary We isolated a mixture of -galactoside-binding lectins from rat lung and raised polyclonal antibody against 14 kD lectin purified from the mixture of lectins. Immunoblotting of the mixture of lectins, which was separated with SDS-PAGE under reducing condition and transferred onto a NC paper, showed that the antibody reacted with two bands at 14 and 29 kD, indicating that these two lectins have common antigenic determinant(s). Immunohistochemically, the antibody recognized only bronchiolar Clara cells with intense immunofluorescence in their apical cytoplasmic protrusions where the secretory granules of the cells are known to be stored. Thus, to determine if the lectin(s) might be secreted into airways, we next raised antibody against airway secretions free from serum as well as surfactant proteins. By immunoblot analysis, the resulting antibody stained 29,45 and 55 kD bands, but not 14 kD band, on a NC paper transferred with the mixture of lectins. These findings suggest that at least 29 kD lung lectin is located in bronchiolar Clara cells and secreted by these cells into airways.     

Immunohistochemical localization of a β-d -galactoside-binding lectin at the human maternofetal interface

Summary 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.     

Conformational stability of LYLA1, a synthetic chimera of human lysozyme and bovine α-lactalbumin

LYLA1 is a chimeric protein mainly consisting of residues originating from human lysozyme but in which the central part (Ca²⁺-binding site and helix C) of bovine α-lactalbumin has been inserted. The equilibrium unfolding of this hybrid protein has been examined by circular dichroism and tryptophan fluorescence techniques. The reversible denaturation process induced by temperature or by addition of chemical denaturant is three-state in the case of apo-LYLA1 and two-state in the presence of Ca²⁺. The Ca²⁺-bound form of the chimera exhibits higher stability than both wild-type lysozyme and α-lactalbumin. The stability of the apo-form, however, is intermediate between that of the parent molecules. Unfolding of apo-LYLA1 involves an intermediate state that becomes populated to a different extent under various experimental conditions. Combination of circular dichroism with bis-ANS fluorescence experiments has permitted us to characterize the acid state of LYLA1 as a molten globule. Furthermore our results strongly suggest the presence of multiple denatured states depending on external conditions. Received: 24 April 1996 / Accepted: 4 September 1996     

Oligoclonal β-galactoside-binding immunoglobulins antigenically related to 14kda lectin in human serum and cerebrospinal fluid: Purification and characterization

• 1.1. An antiserum raised against a 14kDa β-galactoside specific lectin from human brain (HBL14) was used to probe blots from samples of serum and cerebrospinal fluid. The only HBL14-immunoreactive material detected was heavy and light chains of a β-galactoside-binding IgG fraction (lectin-like IgG).
• 2.2. Lectin-like IgG, as well as IgG Fab fragments, compete with HBL14 for binding either to anti-HBL14 antibody or to a lactosyl polyacrylamide-based copolymer.
• 3.3. Purification of lectin-like IgG was obtained by affinity chromatography on immobilized rabbit anti-lectin immunoglobulins. The carbohydrate-binding specificity of the purified molecules was restricted to β-Gal-containing structures and close to the HBL14 one.

     

Structural assessment of β-glucuronidase carbohydrate chains by lectin affinity chromatography

Rat liver -glucuronidase was studied by sequential lectin affinity chromatography. -Glucuronidase glycopeptides were obtained by extensive Pronase digestion followed byN-[¹⁴C]acetylation and desialylation by neuraminidase treatment. According to the distribution of the radioactivity in the various fractions obtained by chromatography on different lectins, and on the assumption that all glycopeptides were acetylated to the same specific radioactivity, a relative distribution of glycan structure types is proposed. The presence of complex biantennary and oligomannose type glycans (56.8% and 42.7%, respectively) was indicated by Concanavalin A-Sepharose chromatography.Ulex europaeus agglutinin-agarose chromatography revealed the presence of (1-3) linked fucose in some of the complex biantennary type glycans (16.6% of the total glycopeptides). Wheat germ agglutinin chromatography indicated that the minority (0.5%) were hybrid or poly (N-acetyllactosamine) type glycans. Furthermore, the absence of O-glycans, tri-, tetra- and bisected biantennary type glycans was demonstrated by analysis of Concanavalin A-Sepharose unbound fraction by chromatography on immobilized soybean agglutinin,Ricinus communis agglutinin andPhaseolus vulgaris erythroagglutinin.     

Hydrolysis of GM1-ganglioside by human liver β-galactosidase isoenzymes

1. GM(1)-ganglioside, specifically tritiated in the terminal galactose, was hydrolysed by two forms of ;acid' methylumbelliferyl beta-galactosidase isolated on gel filtration. 2. Identification of GM(1)-ganglioside beta-galactosidase activity with the ;acid' methyl-umbelliferyl beta-galactosidases was based on the following: coincident elution profiles on gel filtration; simultaneous inactivation by heat and other treatments; stabilization of both activities by chloride ions; mutual inhibition of hydrolysis by the two substrates. 3. The two isoenzymes (I) and (II) showed general requirements for a mixture of anionic and nonionic detergents in the hydrolysis of the natural substrate. 4. Isoenzyme (I) differed from (II) in molecular size, pH-activity profile, relative resistance to dilution and in sensitivity to various inhibitors. 5. The most significant difference between the isoenzymes is in substrate saturation kinetics: (I) was hyperbolic whereas (II) was sigmoid. The apparent Michaelis constants were 28mum for (I) and 77mum for (II). Isoenzyme (I) was insensitive to GM(2)-ganglioside whereas (II) was inhibited, consistent with the hypothesis that GM(1)-ganglioside (and its analogue) acts as modifier in isoenzyme (II) but not in (I). 6. Isoenzyme (I) was membrane-bound whereas (II) was soluble; the former probably represents isoenzyme (II) bound to membrane components, thereby becoming activated. 7. Membranes may serve a dual role in enzyme catalysis involving lipids: as a medium where both enzyme and substrate are effectively concentrated, and as actual activator of enzymes through binding of the latter to specific membrane components.     

Reversible heme-dependent regulation of human cystathionine β-synthase by a flavoprotein oxidoreductase

Human CBS is a PLP-dependent enzyme that clears homocysteine, gates the flow of sulfur into glutathione, and contributes to the biogenesis of H(2)S. The presence of a heme cofactor in CBS is enigmatic, and its conversion from the ferric- to ferrous-CO state inhibits enzyme activity. The low heme redox potential (-350 mV) has raised questions about the feasibility of the ferrous-CO state forming under physiological conditions. Herein, we provide the first evidence of reversible inhibition of CBS by CO in the presence of a human flavoprotein and NADPH. These data provide a mechanism for cross talk between two gas-signaling systems, CO and H(2)S, via heme-mediated allosteric regulation of CBS.     

Binding and aggregation of human μ-calpain by terbium ion

Human μ-calpain is activated maximally by 100–200 μM Ca²⁺. Both the 80 kDa and 29 kDa subunits of μ-calpain have a EF-hand type calcium-binding domain. It is known that trivalent terbium ion (Tb³⁺) mimics Ca²⁺ in many biological systems. We found that Tb³⁺ alone transiently activated calpain. However, in the presence of Ca²⁺, Tb³⁺ inhibited μ-calpain with an IC₅₀ of about 100 μM. As high as 10 mM Ca²⁺ did not significantly shift the IC₅₀ of Tb³⁺. Preincubating μ-calpain by Ca²⁺ (before Tb³⁺ and substrate were added) did not diminish the inhibition by Tb³⁺. On the other hand, pretreating μ-calpain with Tb³⁺ produced that Tb³⁺ has a slow dissociation rate for the calcium-binding sites when compared to Ca²⁺. Electrophoretic analysis revealed that terbium ion transiently activated μ-calpain followed by the aggregation of the proteinase.     

Crystal structure of a β-prism II lectin from Remusatia vivipara

The crystal structure of a β-prism II (BP2) fold lectin from Remusatia vivipara, a plant of traditional medicinal value, has been determined at a resolution of 2.4??. This lectin (RVL, Remusatia vivipara lectin) is a dimer with each protomer having two distinct BP2 domains without a linker between them. It belongs to the "monocot mannose-binding" lectin family, which consists of proteins of high sequence and structural similarity. Though the overall tertiary structure is similar to that of lectins from snowdrop bulbs and garlic, crucial differences in the mannose-binding regions and oligomerization were observed. Unlike most of the other structurally known proteins in this family, only one of the three carbohydrate recognition sites (CRSs) per BP2 domain is found to be conserved. RVL does not recognize simple mannose moieties. RVL binds to only N-linked complex glycans like those present on the gp120 envelope glycoprotein of HIV and mannosylated blood proteins like fetuin, but not to simple mannose moieties. The molecular basis for these features and their possible functional implications to understand the different levels of carbohydrate affinities in this structural family have been investigated through structure analysis, modeling and binding studies. Apart from being the first structure of a lectin to be reported from the Araceae/Arum family, this protein also displays a novel mode of oligomerization among BP2 lectins.     

Binding pathway determines norepinephrine selectivity for the human β1AR over β2AR

Beta adrenergic receptors (βARs) mediate physiologic responses to the catecholamines epinephrine and norepinephrine released by the sympathetic nervous system.W...     

Cloning and characterization of a novel member of human β-1, 4-galactosyltransferase gene family

By using the EST strategy for identifying novel members belonging to homologous gene families, a novel full-length cDNA encoding a protein significantly homologous to UDP-Gal: N-acetylglucosamine β-1, 4-galactosyltransferase (GAlT) was isolated from a human testis cDNA library. A nucleotide sequence of 2 173 bp long was determined to contain an open reading frame of 1032 nucleotides (344 amino acids). In view of the homology to members of the galactosyltransferase gene family and especially the closest relationship to Gallus gallus GalT type I (CK I), the predicted product of the novel cDNA was designated as human β-1, 4-galactosyltransferase homolog I (HumGT-H1). Its mRNA is present in different degrees in 16 tissues examined. Southern analysis of human genomic DNA revealed its locus on chromosome 3.     

Hydrolysis of natural and synthetic substrates by α-l-fucosidase, β-d-glucuronidase and β-n-acetylhexosaminidase purified from molluscs

• 1.1. Several mollusc glycosidases have been studied for their activities towards natural substrates. α-l-Fucosidases from Chamelea gallina, Tapes rhomboideus and Mytilus edulis hydrolyze oligosaccharides (di, tri and pentasaccharides) with α1 → 2, α1 → 3 and α1 → 4 bonds, fucose-containing glycopeptides from bovine thyroglobulin and the porcine submandibular mucin (devoid of sialic acid); α-l-fucosidase from Littorina littorea hydrolyzes fucose-containing glycopeptides from bovine thyroglobulin.
• 2.2. β-d-Glucuronidase from L. littorea hydrolyzes hyaluronic acid, chondroitin 4-sulfate and heparin with a very low activity; however, it is much more active on oligosaccharides (from the above-mentioned macromolecules) containing non-reducing terminal glucuronyl residues.
• 3.3. β-N-Acetylhexosaminidase from Helicella ericetorum acts mainly with an endo-hydrolase activity on β1 → 4N-acetylhexosamine linkages of ovalbumin, ovomucoid, chitin, hyaluronic acid and chondroitin
• 4.4-sulfate; it has also a secondary exo-hydrolase activity on these substrates.

     

Purification of β-glucuronidase and structural assessment of the carbohydrate chains by lectin affinity immunoelectrophoresis

The purification of rat liver -glucuronidase from a lysosomal fraction by methods including affinity chromatography, chromatofocusing and preparative PAGE steps is described. Molecular weights of 300 000 and 150 000 were estimated by two dimensional gradient PAGE/immunoelectrophoresis of the lysosomal extract. Isoelectrofocusing in agarose gel followed by immunoelectrophoresis in the second dimension revealed the presence of at least five maxima in the range pH 4.3–7.4.The structural assessment of the carbohydrate chains of lysosomal and microsomal -glucuronidase was performed by lectin affinity immunoelectrophoresis. Reaction with Concanavalin A indicated the presence of bi-antennary complex, oligomannosidic and hybrid type structures, whereas the absence of tri- and tetra-antennary complex type structures was deduced from the lack of interaction with phytohemagglutinin-L. The reaction withLens culinaris agglutinin, Pisum sativum agglutinin andLotus tetragonolobus lectin revealed that part of the glycans contained a fucose (1-6)-linked to theN-acetylglucosamine attached to asparagine. The presence of terminal (1-4)-galactose residues was detected withRicinus communis agglutinin I.     

Binding of N-Acetylglucosamine (GlcNAc) β1-6-branched Oligosaccharide Acceptors to β4-Galactosyltransferase I Reveals a New Ligand Binding Mode

N-Acetyllactosamine is the most prevalent disaccharide moiety in the glycans on the surface of mammalian cells and often found as repeat units in the linear and branched polylactosamines, known as i- and I-antigen, respectively. The β1-4-galactosyltransferase-I (β4Gal-T1) enzyme is responsible for the synthesis of the N-acetyllactosamine moiety. To understand its oligosaccharide acceptor specificity, we have previously investigated the binding of tri- and pentasaccharides of N-glycan with a GlcNAc at their nonreducing end and found that the extended sugar moiety in these acceptor substrates binds to the crevice present at the acceptor substrate binding site of the β4Gal-T1 molecule. Here we report seven crystal structures of β4Gal-T1 in complex with an oligosaccharide acceptor with a nonreducing end GlcNAc that has a β1-6-glycosidic link and that are analogous to either N-glycan or i/I-antigen. In the crystal structure of the complex of β4Gal-T1 with I-antigen analog pentasaccharide, the β1-6-branched GlcNAc moiety is bound to the sugar acceptor binding site of the β4Gal-T1 molecule in a way similar to the crystal structures described previously; however, the extended linear tetrasaccharide moiety does not interact with the previously found extended sugar binding site on the β4Gal-T1 molecule. Instead, it interacts with the different hydrophobic surface of the protein molecule formed by the residues Tyr-276, Trp-310, and Phe-356. Results from the present and previous studies suggest that β4Gal-T1 molecule has two different oligosaccharide binding regions for the binding of the extended oligosaccharide moiety of the acceptor substrate.     

Tumor cytotoxicity of human monocyte membrane-bound interleukin-1α induced by synergistic actions of interferon-γ and synthetic acyltripeptide,FK-565

Summary Human blood monocytes were isolated by counter-flow centrifugal elutriation from healthy donors and these noncytotoxic monocytes were rendered tumoricidal to allogeneic melanoma (A375) cells by activation with a synthetic acyltripeptide (FK-565), as assessed by measuring release of [¹²⁵I]iododeoxyuridine in 72 h. When monocytes were treated with FK-565 for 16 h, and then fixed with paraformaldehyde, they showed cytotoxicity to A375 melanoma cells. The fixed-monocyte-mediated cytotoxicity to A375 cells was induced by the synergistic actions of FK-565 and recombinant interferon- (rIFN-), but not other cytokines [rIFN-A, rIFN-, tumor necrosis factor (TNF), interleukin (IL)-2, -3 and -6]. For synergistic activation of monocytes with induction of a membrane-associated antitumor monokine, the monocytes had to be incubated first with rIFN- and then with FK-565. FK-565 also acted synergistically with rIFN- to stimulate monocytes to produce membrane-associated IL-1 activity, which induced C3H/HeJ thymocyte blastogenesis in response to phytohemagglutinin P. The tumoricidal and thymocytestimulating activities of the fixed monocytes were almost completely inhibited by a specific anti-(IL-1) antiserum, but not by a specific anti-(IL-1) antiserum or monoclonal anti-TNF antibody. These results suggest that membrane-associated IL-1 of human blood monocytes can be induced by two activation signals (rIFN- then FK-565) at their suboptimal concentrations.Abbreviations IL interleukin - IFN interferon - TNF tumor necrosis factor     

Characterization of a panel of mouse single-chain antibodies against human recombinant interferon β1b

A panel of single-chain Fv-antibodies (ScFv’s) against recombinant human interferon beta 1b (rhIFN-β1b) has been obtained from immune and naïve combinatorial cDNA libraries of the mouse variable immunoglobulin genes. ScFv’s were expressed in Escherichia coli cells. For producers isolated from the immune library a difference in production yield of ScFv’s in periplasm and incubation medium as well as their expression and storage stability have been demonstrated. After sequencing of target DNA the multiple alignment and structural analysis of ScFv’s sequences with different primary structures were carried out and significant difference in both complementarity-determining (CDR) and framework (FR) regions of theirs variable domains has been shown. For the ScFv’s isolated from the immune library, specificity of their binding with native and denatured rhIFN-β1b in ELISA and Western-blotting as well as their high storage stability have been shown. The affinity constants for each representatives of the ScFv’s panel were in the range from 1.96 × 10^?8 to 1.69 × 10^?9 M.