Preparation and X-ray characterization of four new crystal forms of jacalin, a lectin from Artocarpus integrifolia

Four new crystal forms of the anti-T lectin from jackfruit (Artocarpus integrifolia) have been prepared and characterized. Three of them, two monoclinic (P21, a = 59.4 A, b = 83.3 A, c = 63.5 A, beta = 107.7 degrees; C2, a = 106.1 A, b = 53.9 A, c = 128.0 A, beta = 95.0 A) and one orthorhombic (C222(1), a = 98.1 A, b = 67.3 A, c = 95.1 A) were grown with 2-methylpentan-2,4-diol (MPD) as the precipitant while the fourth, an hexagonal form (P6(1)22, a = b = 129.6 A, c = 157.9 A), was obtained in the presence of methyl-alpha-D-galactopyranoside with polyethylene glycol 4000 as the precipitant. The reported relative molecular mass (Mr) of the lectin was found to be inconsistent with the solvent content of the crystals estimated using measured densities. The Mr was redetermined using size-exclusion chromatography in the presence of methyl-alpha-D-galactopyranoside and Ferguson-plot analysis of mobilities in polyacrylamide gel electrophoresis. The redetermined Mr (66,000) is consistent with the measured crystal densities. The orthorhombic and the hexagonal forms, which have one half molecule and one molecule, respectively, in the asymmetric unit, are suitable for high-resolution X-ray analysis.     

Structural and thermodynamic studies of KM+, a d-mannose binding lectin from Artocarpus integrifolia seeds

The KM+ lectin exhibits a novel and unusual circular dichroism (CD) spectrum that could be explained by a high proline content that would be inducing deformation of the beta-structure and/or unusual turns. KM+ was shown to be a very rigid lectin, which was very stable under a broad variety of conditions (urea, guanidine, hydrolysis, pH, etc.). Only incubation for 60 min at 333-338 K and extreme basic pH were able to induce conformational changes which could be observed by CD and fluorescence measurements. Data from CD are typical for protein denaturing associated with changes in the overall secondary structure. Data from high-performance size exclusion chromatography (SEC) showed that the denatured forms produced at pH 12.0 are eluted in clusters that co-elute with the native forms. A significant contribution from the tyrosines to the fluorescence emission upon denaturation was observed above 328 K. In fact at 328 K some broadening of the emission spectrum takes place followed by the appearance of a shoulder (approx. 305 nm) at 333 K and above. The sensitivity of tryptophan fluorescence to the addition of sugar suggests a close proximity of the tryptophan residues to the sugar binding site, K(a)=(2.9+/-0.6)x10(3) M(-1). The fraction of chromophore accessible to the quencher obtained is f(a)=0.43+/-0.08, suggesting that approximately 50% of the tryptophan residues are not accessible to quenching by d-mannose. KM+ thermal denaturation was found to be irreversible and was analyzed using a two-state model (N-->D). The results obtained for the activation energy and transition temperature from the equilibrium CD studies were: activation energy, E(a)=134+/-11 kJ/mol and transition temperature, T(m)=339+/-1 K, and from the fluorescence data: E(a)=179+/-18 kJ/mol and T(m)=337+/-1 K. Kinetic studies gave the following values: E(a)=108+/-18 kJ/mol and E(a)=167+/-12 kJ/mol for CD and fluorescence data, respectively.     

Analysis of dynamics and mechanism of ligand binding to Artocarpus integrifolia agglutinin. A 13C and 19F NMR study

Binding of 13C-labeled N-acetylgalactosamine (13C-GalNAc) and N-trifluoroacetylgalactosamine (19F-GalNAc) to Artocarpus integrifolia agglutinin has been studied using 13C and 19F nuclear magnetic resonance spectroscopy, respectively. Binding of these saccharides resulted in broadening of the resonances, and no change in chemical shift was observed, suggesting that the alpha- and beta-anomers of 13C-GalNAc and 19F-GalNAc experience a magnetically equivalent environment in the lectin combining site. The alpha- and beta-anomers of 13C-GalNAc and 19F-GalNAc were found to be in slow exchange between free and protein bound states. Binding of 13C-GalNAc was studied as a function of temperature. From the temperature dependence of the line broadening, the thermodynamic and kinetic parameters were evaluated. The association rate constants obtained for the alpha-anomers of 13C-GalNAc and 19F-GalNAc (k+1 = 1.01 x 10(5) M-1.s-1 and 0.698 x 10(5) M-1.s-1, respectively) are in close agreement with those obtained for the corresponding beta-anomers (k+1 = 0.95 x 10(5) M-1.s-1 and 0.65 x 10(5) M-1.s-1, respectively), suggesting that the two anomers bind to the lectin by a similar mechanism. In addition these values are several orders of magnitude slower than those obtained for diffusion controlled processes. The dissociation rate constants obtained are 49.9, 56.9, 42, and 43 s-1, respectively, for the alpha- and beta-anomers of 13C-GalNAc and 19F-GalNAc. A two-step mechanism has been proposed for the interaction of 13C-GalNAc and 19F-GalNAc with A. integrifolia lectin in view of the slow association rates and high activation entropies. The thermodynamic parameters obtained for the association and dissociation reactions suggest that the binding process is entropically favored and that there is a small enthalpic contribution.     

Further characterization and immunochemical studies on the carbohydrate specificity of jackfruit (Artocarpus integrifolia) lectin

The lectin from jackfruit (Artocarpus integrifolia) seeds has been purified by Rivanol (6,9-diamino-2-ethoxyacridine lactate) treatment. The specific activity, molecular weights of parent lectin and its subunit, its glycoprotein nature, and hemagglutination-inhibition assays suggest that this preparation is identical to that obtained by affinity chromatography on melibiose-agarose adsorbent (Ahmed, H., and Chatterjee, B. P. (1986) in Lectins, Biology, Biochemistry, Clinical Biochemistry (B?g-Hansen, T. C., and van Driessche, E., eds) Vol. 5, pp. 125-133, Walter de Gruyter, New York). The lectin strongly agglutinates human and several animal erythrocytes. The lectin contains five isolectins of pI values 7.1, 6.85, 5.5, 5.3, and 5.1. It is thermally stable and loses its activity above 75 degrees C. The hemagglutinating activity remains unchanged in the presence of bivalent cations viz., Ca2+, Mg2+, Mn2+, etc. It is a metalloprotein. The lectin retains its activity by dialysis with acetic acid followed by EDTA. It agglutinates Ehrlich ascites cells. Equilibrium dialysis of lectin with melibiose and quenching of fluorescence of 4-methylumbelliferyl-alpha-D-galactopyranoside by the lectin show that homotetrameric jackfruit lectin has two sugar-binding sites. The lectin precipitates well several galactomannans and glycoproteins having terminal D-Gal-alpha-(1----6)- or D-Gal-beta-(1----3)-D-GalNAc residues. It hardly or does not precipitate polysaccharides having terminal D-Gal-alpha-(1----3) residues. Quantitative precipitin-inhibition studies using various haptens suggest that the -OCH2- group at C-1 and -OH groups at C-4 and partially at C-6 in the alpha-glycoside of D-galactose configuration are important for lectin-sugar interaction.     

Crystallization and preliminary crystallographic data of a neutrophil migration-inducing lectin (KM+) extracted from the seed of Artocarpus integrifolia

The tetrameric KM+ lectin from the seeds of Artocarpus integrifolia has, when compared to other plant lectins, the singular property of directly inducing neutrophil migration into the peritoneal cavity or into the air pouch of rats. This protein crystals have been grown and they belong to the orthorhombic system with space group C222₁. The unit cell parameters are a = 54.4 Å, b = 127.9 Å and c = 99.8 Å. A native diffraction dataset to 2.8 Å was collected and an analysis of the self-rotation function has shown the presence of only one independent non-crystallographic 2-fold axis orthogonal to the crystal b-axis, compatible with a dimer in the asymmetric unit. Proteins 27:157–159 © 1997 Wiley-Liss, Inc.     

cDNA cloning and functional expression of KM+, the mannose-binding lectin from Artocarpus integrifolia seeds

KM+, a mannose-binding lectin present in the seeds of Artocarpus integrifolia, has interesting biological properties and potential pharmaceutical use [A. Panunto-Castelo, M.A. Souza, M.C. Roque-Barreira, J.S. Silva, KM(+), a lectin from Artocarpus integrifolia, induces IL-12 p40 production by macrophages and switches from type 2 to type 1 cell-mediated immunity against Leishmania major antigens, resulting in BALB/c mice resistance to infection, Glycobiology 11 (2001) 1035-1042. ; L.L.P. daSilva, A. Panunto-Castelo, M.H.S. Goldman, M.C. Roque-Barreira, R.S. Oliveira, M.D. Baruffi, J.B. Molfetta-Machado, Composition for preventing or treating appearance of epithelia wounds such as skin and corneal wounds or for immunomodulating, comprises lectin, Patent number WO20041008.]. Here, we have isolated clones encoding the full-length KM+ primary sequence from a cDNA library, through matrix PCR-based screening methodology. Analysis of KM+ nucleotide and deduced amino acid sequences provided strong evidence that it neither enters the secretory pathway nor undergoes post-translational modifications, which is in sharp contrast with jacalin, the more abundant lectin from A. integrifolia seeds. Current investigations into the KM+ properties are often impaired by the difficulty in obtaining sufficient quantities of jacalin-free KM+ through direct seed extraction. To obtain active recombinant protein (rKM+) in larger amounts, we tested three different expression systems. Expression vectors were constructed to produce: (a) rKM+ in E. coli in its native form, (b) rKM+ with GST as an N-terminal tag and (c) native rKM+ in Saccharomyces cerevisiae. The presence of the GST-tag significantly improved the overall rKM+ yield; however, most of the obtained rGST-KM+ was insoluble. Production of rKM+ in the yeast host yielded the highest quantities of soluble lectin that retained the typical high-mannose oligosaccharide-binding properties of the natural protein. The possible biotechnological applications of recombinant KM+ are discussed.     

Production of lectin from jack fruit (Artocarpus integrifolia) seeds and its interaction with carbohydrates and glycoproteins

A method is developed to obtain lectin from jack fruit (Artocarpus integrifolia) seeds using an affinity chromatography on a sorbent prepared from the egg white. The minimum agglutination concentration of human erythrocytes is 80 ng/ml, the molecular weight of the preparation is about 39 kDa, it contains 1.8% of neutral hexoses and 3.1% of hexosamines. PAAG electrophoresis in the alkali system has revealed several molecular forms of lectin isolated by preparative electrophoresis, their properties are investigated. SDS-PAAG electrophoresis has revealed several types of polypeptide chains among which two chains (12 and 14 kDa) are predominant. Lectin possesses affinity to galactosides (not to free galactose) and N-acetylgalactosamine and interacts with O-glycans with high affinity. The preparation has mitogenic activity in optimal concentration 50 micrograms/ml.     

The gene for stinging nettle lectin (Urtica dioica agglutinin) encodes both a lectin and a chitinase.

Chitin-binding proteins are present in a wide range of plant species, including both monocots and dicots, even though these plants contain no chitin. To investigate the relationship between in vitro antifungal and insecticidal activities of chitin-binding proteins and their unknown endogenous functions, the stinging nettle lectin (Urtica dioica agglutinin, UDA) cDNA was cloned using a synthetic gene as the probe. The nettle lectin cDNA clone contained an open reading frame encoding 374 amino acids. Analysis of the deduced amino acid sequence revealed a 21-amino acid putative signal sequence and the 86 amino acids encoding the two chitin-binding domains of nettle lectin. These domains were fused to a 19-amino acid "spacer" domain and a 244-amino acid carboxyl extension with partial identity to a chitinase catalytic domain. The authenticity of the cDNA clone was confirmed by deduced amino acid sequence identity with sequence data obtained from tryptic digests, RNA gel blot, and polymerase chain reaction analyses. RNA gel blot analysis also showed the nettle lectin message was present primarily in rhizomes and inflorescence (with immature seeds) but not in leaves or stems. Chitinase enzymatic activity was found when the chitinase-like domain alone or the chitinase-like domain with the chitin-binding domains were expressed in Escherichia coli. This is the first example of a chitin-binding protein with both a duplication of the 43-amino acid chitin-binding domain and a fusion of the chitin-binding domains to a structurally unrelated domain, the chitinase domain.     

A spectroscopic and thermodynamic study of porphyrin/DNA supramolecular assemblies.

Assemblies of trans-bis(N-methylpyridinium-4-yl)diphenylporphine ions on the surface of calf thymus DNA have been studied using several spectroscopic techniques: absorbance, circular dichroism, and resonance light scattering. The aggregation equilibrium can be treated as a two-state system-monomer and assembly-each bound to the nucleic acid template. The aggregate absorption spectrum in the Soret region is resolved into two bands of Lorentzian line shape, while the DNA-bound monomer spectrum in this region is composed of two Gaussian bands. The Beer-Lambert law is obeyed by both porphyrin forms. The assembly is also characterized by an extremely large, bisignate induced circular dichroism (CD) profile and by enhanced resonance light scattering (RLS). Both the CD and RLS intensities depend linearly on aggregate concentration. The RLS result is consistent with a model for the aggregates as being either of a characteristic size or of a fixed distribution of sizes, independent of total porphyrin concentration or ionic strength. Above threshold values of concentration and ionic strength, the mass action expression for the equilibrium has a particularly simple form: K' = cac-1; where cac is defined as the "critical assembly concentration."offe dependence of the cac upon temperature and ionic strength (NaCl) has been investigated at a fixed DNA concentration. The value of the cac scales as the inverse square of the sodium chloride concentration and, from temperature dependence studies, the aggregation process is shown to be exothermic.     

Preliminary crystallographic study of the alpha-D-galactose-specific lectin from jack fruit (Artocarpus integra) seeds

Crystals of the alpha-D-galactose-specific lectin from Jack fruit (Artocarpus integra) have been obtained from polyethylene glycol 400 solutions. The crystals are orthorhombic, space group P2(1)2(1)2 with a = 77.09 A, b = 123.3 A and c = 78.73 A (1 A = 0.1 nm) and have one 39,500 Mr tetramer per asymmetric unit. The crystals diffract to at least 2.8 A on precession photographs.     

A spectroscopic and thermodynamic study of Taxol nucleic acid complexes

The interactions of natural and synthetic polynucleotide double strands with the antitumor agent paclitaxel and the oncological product "Taxol for Injection Concentrate" (abbreviated as taxol) were examined in diluted aqueous solutions by thermal denaturation profiles (Tm), CD spectra and UV-absorption measurements. Furthermore, DNA-paclitaxel and -taxol complexes in condensed nucleic acid solutions were studied by differential scanning calorimetry. As polynucleotides alternating and homologous poly[d(AT)] and poly[d(GC)] and calf thymus DNA were used. The results point to stabilizing interactions of paclitaxel to AT nucleotides, whereas in the presence of GC base pairings no interaction took place. Thereby the interaction to homologous (dA).(dT)-tracts seems to be preferred.     

KM+, a mannose-binding lectin from Artocarpus integrifolia: amino acid sequence, predicted tertiary structure, carbohydrate recognition, and analysis of the beta-prism fold

The complete amino acid sequence of the lectin KM+ from Artocarpus integrifolia (jackfruit), which contains 149 residues/mol, is reported and compared to those of other members of the Moraceae family, particularly that of jacalin, also from jackfruit, with which it shares 52% sequence identity. KM+ presents an acetyl-blocked N-terminus and is not posttranslationally modified by proteolytic cleavage as is the case for jacalin. Rather, it possesses a short, glycine-rich linker that unites the regions homologous to the alpha- and beta-chains of jacalin. The results of homology modeling implicate the linker sequence in sterically impeding rotation of the side chain of Asp141 within the binding site pocket. As a consequence, the aspartic acid is locked into a conformation adequate only for the recognition of equatorial hydroxyl groups on the C4 epimeric center (alpha-D-mannose, alpha-D-glucose, and their derivatives). In contrast, the internal cleavage of the jacalin chain permits free rotation of the homologous aspartic acid, rendering it capable of accepting hydrogen bonds from both possible hydroxyl configurations on C4. We suggest that, together with direct recognition of epimeric hydroxyls and the steric exclusion of disfavored ligands, conformational restriction of the lectin should be considered to be a new mechanism by which selectivity may be built into carbohydrate binding sites. Jacalin and KM+ adopt the beta-prism fold already observed in two unrelated protein families. Despite presenting little or no sequence similarity, an analysis of the beta-prism reveals a canonical feature repeatedly present in all such structures, which is based on six largely hydrophobic residues within a beta-hairpin containing two classic-type beta-bulges. We suggest the term beta-prism motif to describe this feature.     

A circular-dichroism study of epidermolytic toxins A and B from Staphylococcus aureus.

The far-u.v. circular-dichroism spectra of the two epidermolytic toxins was analysed into fractional contributions of 0.09 helix and 0.46 beta-sheet to each toxin structure. Trifluoroethanol perturbation caused an initial increase in dichroic absorption at 205 nm and then a change characterized as a beta-sheet-to-alpha-helix transition. The intense near-u.v. spectra suggested that the toxins have unusually rigid, though different, aromatic-side-chain arrangements.     

The solution conformation of poly(L-lysine). A Raman and infrared spectroscopic study.

The Raman and infrared spectra of poly(L -lysine) and poly(DL -lysine) in solution are reported and the effects of various salts are investigated. The results demonstrate that α-helix formation in solution is induced by specific salts and the spectral data support the hypothesis of regions of local order for poly(L -lysine) in aqueous solutions of low ionic strength.     

Biochemical and structural analysis of Helix pomatia agglutinin. A hexameric lectin with a novel fold

Helix pomatia agglutinin (HPA) is a N-acetylgalactosamine (GalNAc) binding lectin found in the albumen gland of the roman snail. As a constituent of perivitelline fluid, HPA protects fertilized eggs from bacteria and is part of the innate immunity system of the snail. The peptide sequence deduced from gene cloning demonstrates that HPA belongs to a family of carbohydrate-binding proteins recently identified in several invertebrates. This domain is also present in discoidin from the slime mold Dictyostelium discoideum. Investigation of the lectin specificity was performed with the use of glycan arrays, demonstrating that several GalNAc-containing oligosaccharides are bound and rationalizing the use of this lectin as a cancer marker. Titration microcalorimetry performed on the interaction between HPA and GalNAc indicates an affinity in the 10(-4) M range with an enthalpy-driven binding mechanism. The crystal structure of HPA demonstrates the occurrence of a new beta-sandwich lectin fold. The hexameric quaternary state was never observed previously for a lectin. The high resolution structure complex of HPA with GalNAc characterizes a new carbohydrate binding site and rationalizes the observed preference for alphaGalNAc-containing oligosaccharides.     

Glycoconjugates of human sperm surface. A study with fluorescent lectin conjugates and lens culinaris agglutinin affinity chromatography

Distribution of glycocompounds in human spermatozoa was studied by using fluorescent lectin-conjugates. Con A bound predominantly to acrosomal and posterior head regions whereas RCA I bound to the acrosomal region of intact spermatozoa, stained in suspension. Other lectins used (LCA, WGA, SBA, PNA) stained the the entire sperm surface. In airdried sperm smears binding of both Con A and RCA I were identical with the staining pattern obtained with living cells whereas LCA, WGA, SBA and PNA now bound heavily into acrosomal region. As a similar staining pattern was obtained with permeabilized sperm cells, this staining is apparently due to binding to intracellular structures. The efficiency of Lens culinaris agglutinin affinity chromatography in purification of human sperm glycoproteins was tested after their external radiolabelling with the neuraminidase/galactose oxidase/sodium borohydride method. 22% of applicated radioactivity could be eluted from the column with the specific inhibitory saccharide, and most of the radiolabelled surface glycoproteins of the whole sperm lysate, were also present in the LCA affinity column eluate. LCA affinity chromatography seems thus be an effective method to enrich membrane glycoproteins of human spermatozoa.     

A morphological study of the Petunia integrifolia complex (Solanaceae)

BACKGROUND AND AIMS: Petunia inflata has been treated taxonomically in various ways: it has been described as an independent species, treated as a synonym of P. integrifolia, and also regarded as a subspecies of P. integrifolia. The present study was designed to resolve the ambiguity involving the P. integrifolia complex (P. integrifolia plus P. inflata). METHODS: Tentative identification (either integrifolia group or inflata group) was carried out in the field based on the observation of live specimens at the restricted type localities. The accuracy of the tentative identification was later tested with principal component and cluster analyses of data obtained by measuring 21 morphological characters on cultivated live specimens sourced from 113 natural populations of the P. integrifolia complex in Argentina, Brazil, Paraguay and Uruguay. KEY RESULTS: There was a clear, statistically significant gap between the morphological measurements of the two groups, ensuring the accuracy of identification carried out in the field except for a probable hybrid swarm. Previously, the condition of the pedicel in the fruiting state was considered an important character distinguishing between these two groups; however, the condition of the pedicel was rather variable in the integrifolia group. The two groups were found to have geographically distinct distributions: the integrifolia group occurred in southern regions, whereas the inflata group occurred in northern regions. CONCLUSIONS: Based on the available evidence, it is suggested that the two groups are allopatric species, P. integrifolia and P. inflata, in agreement with the opinion of Fries (1911).