Structure of porphobilinogen synthase from Pseudomonas aeruginosa in complex with 5-fluorolevulinic acid suggests a double Schiff base mechanism

The seeds of jack fruit (Artocarpus integrifolia) contain two tetrameric lectins, jacalin and artocarpin. Jacalin was the first lectin found to exhibit the beta-prism I fold, which is characteristic of the Moraceae plant lectin family. Jacalin contains two polypeptide chains produced by a post-translational proteolysis which has been shown to be crucial for generating its specificity for galactose. Artocarpin is a single chain protein with considerable sequence similarity with jacalin. It, however, exhibits many properties different from those of jacalin. In particular, it is specific to mannose. The structures of two crystal forms, form I and form II, of the native lectin have been determined at 2.4 and 2.5 A resolution, respectively. The structure of the lectin complexed with methyl-alpha-mannose, has also been determined at 2.9 A resolution. The structure is similar to jacalin, although differences exist in details. The crystal structures and detailed modelling studies indicate that the following differences between the carbohydrate binding sites of artocarpin and jacalin are responsible for the difference in the specificities of the two lectins. Firstly, artocarpin does not contain, unlike jacalin, an N terminus generated by post-translational proteolysis. Secondly, there is no aromatic residue in the binding site of artocarpin whereas there are four in that of jacalin. A comparison with similar lectins of known structures or sequences, suggests that, in general, stacking interactions with aromatic residues are important for the binding of galactose while such interactions are usually absent in the carbohydrate binding sites of mannose-specific lectins with the beta-prism I fold.     

Buried hydrophobic side-chains essential for the folding of the parallel beta-helix domains of the P22 tailspike

The processive beta-strands and turns of a polypeptide parallel beta-helix represent one of the topologically simplest beta-sheet folds. The three subunits of the tailspike adhesin of phage P22 each contain 13 rungs of a parallel beta-helix followed by an interdigitated section of triple-stranded beta-helix. Long stacks of hydrophobic residues dominate the elongated buried core of these two beta-helix domains and extend into the core of the contiguous triple beta-prism domain. To test whether these side-chain stacks represent essential residues for driving the chain into the correct fold, each of three stacked phenylalanine residues within the buried core were substituted with less bulky amino acids. The mutant chains with alanine in place of phenylalanine were defective in intracellular folding. The chains accumulated exclusively in the aggregated inclusion body state regardless of temperature of folding. These severe folding defects indicate that the stacked phenylalanine residues are essential for correct parallel beta-helix folding. Replacement of the same phenylalanine residues with valine or leucine also impaired folding in vivo, but with less severity. Mutants were also constructed in a second buried stack that extends into the intertwined triple-stranded beta-helix and contiguous beta-prism regions of the protein. These mutants exhibited severe defects in later stages of chain folding or assembly, accumulating as misfolded but soluble multimeric species. The results indicate that the formation of the buried hydrophobic stacks is critical for the correct folding of the parallel beta-helix, triple-stranded beta-helix, and beta-prism domains in the tailspike protein.     

Structural basis for the energetics of jacalin-sugar interactions: promiscuity versus specificity

Jacalin, a tetrameric lectin, is one of the two lectins present in jackfruit (Artocarpus integrifolia) seeds. Its crystal structure revealed, for the first time, the occurrence of the beta-prism I fold in lectins. The structure led to the elucidation of the crucial role of a new N terminus generated by post-translational proteolysis for the lectin's specificity for galactose. Subsequent X-ray studies on other carbohydrate complexes showed that the extended binding site of jacalin consisted of, in addition to the primary binding site, a hydrophobic secondary site A composed of aromatic residues and a secondary site B involved mainly in water-bridges. A recent investigation involving surface plasmon resonance and the X-ray analysis of a methyl-alpha-mannose complex, had led to a suggestion of promiscuity in the lectin's sugar specificity. To explore this suggestion further, detailed isothermal titration calorimetric studies on the interaction of galactose (Gal), mannose (Man), glucose (Glc), Me-alpha-Gal, Me-alpha-Man, Me-alpha-Glc and other mono- and oligosaccharides of biological relevance and crystallographic studies on the jacalin-Me-alpha-Glc complex and a new form of the jacalin-Me-alpha-Man complex, have been carried out. The binding affinity of Me-alpha-Man is 20 times weaker than that of Me-alpha-Gal. The corresponding number is 27, when the binding affinities of Gal and Me-alpha-Gal, and those of Man and Me-alpha-Man are compared. Glucose (Glc) shows no measurable binding, while the binding affinity of Me-alpha-Glc is slightly less than that of Me-alpha-Man. The available crystal structures of jacalin-sugar complexes provide a convincing explanation for the energetics of binding in terms of interactions at the primary binding site and secondary site A. The other sugars used in calorimetric studies show no detectable binding to jacalin. These results and other available evidence suggest that jacalin is specific to O-glycans and its affinity to N-glycans is extremely weak or non-existent and therefore of limited value in processes involving biological recognition.     

The first crystal structure of a Mimosoideae lectin reveals a novel quaternary arrangement of a widespread domain

The crystal structures of the apo and mannose-bound Parkia platycephala seed lectin represent the first structure of a Mimosoideae lectin and a novel circular arrangement of beta-prism domains, and highlight the adaptability of the beta-prism fold as a building block in the evolution of plant lectins. The P.platycephala lectin is a dimer both in solution and in the crystals. Mannose binding to each of the three homologous carbohydrate-recognition domains of the lectin occurs through different modes, and restrains the flexibility of surface-exposed loops and residues involved in carbohydrate recognition. The planar array of carbohydrate-binding sites on the rim of the toroid-shaped structure of the P.platycephala lectin dimer immediately suggests a mechanism to promote multivalent interactions leading to cross-linking of carbohydrate ligands as part of the host strategy against phytopredators and pathogens. The cyclic structure of the P.platycephala lectin points to the convergent evolution of a structural principle for the construction of lectins involved in host defense or in attacking other organisms.     

Unusual sugar specificity of banana lectin from Musa paradisiaca and its probable evolutionary origin. Crystallographic and modelling studies

The crystal structure of a complex of methyl-alpha-D-mannoside with banana lectin from Musa paradisiaca reveals two primary binding sites in the lectin, unlike in other lectins with beta-prism I fold which essentially consists of three Greek key motifs. It has been suggested that the fold evolved through successive gene duplication and fusion of an ancestral Greek key motif. In other lectins, all from dicots, the primary binding site exists on one of the three motifs in the three-fold symmetric molecule. Banana is a monocot, and the three motifs have not diverged enough to obliterate sequence similarity among them. Two Greek key motifs in it carry one primary binding site each. A common secondary binding site exists on the third Greek key. Modelling shows that both the primary sites can support 1-2, 1-3, and 1-6 linked mannosides with the second residue interacting in each case primarily with the secondary binding site. Modelling also readily leads to a bound branched mannopentose with the nonreducing ends of the two branches anchored at the two primary binding sites, providing a structural explanation for the lectin's specificity for branched alpha-mannans. A comparison of the dimeric banana lectin with other beta-prism I fold lectins, provides interesting insights into the variability in their quaternary structure.     

Helianthus tuberosus lectin reveals a widespread scaffold for mannose-binding lectins

BACKGROUND: Heltuba, a tuber lectin from the Jerusalem artichoke Helianthus tuberosus, belongs to the mannose-binding subgroup of the family of jacalin-related plant lectins. Heltuba is highly specific for the disaccharides Man alpha 1-3Man or Man alpha 1-2Man, two carbohydrates that are particularly abundant in the glycoconjugates exposed on the surface of viruses, bacteria and fungi, and on the epithelial cells along the gastrointestinal tract of lower animals. Heltuba is therefore a good candidate as a defense protein against plant pathogens or predators. RESULTS: The 2.0 A resolution structure of Heltuba exhibits a threefold symmetric beta-prism fold made up of three four-stranded beta sheets. The crystal structures of Heltuba in complex with Man alpha 1-3Man and Man alpha 1-2Man, solved at 2.35 A and 2.45 A resolution respectively, reveal the carbohydrate-binding site and the residues required for the specificity towards alpha 1-3 or alpha 1-2 mannose linkages. In addition, the crystal packing reveals a remarkable, donut-shaped, octahedral assembly of subunits with the mannose moieties at the periphery, suggesting possible cross-linking interactions with branched oligomannosides. CONCLUSIONS: The structure of Heltuba, which is the prototype for an extended family of mannose-binding agglutinins, shares the carbohydrate-binding site and beta-prism topology of its galactose-binding counterparts jacalin and Maclura pomifera lectin. However, the beta-prism elements recruited to form the octameric interface of Heltuba, and the strategy used to forge the mannose-binding site, are unique and markedly dissimilar to those described for jacalin. The present structure highlights a hitherto unrecognized adaptability of the beta-prism building block in the evolution of plant proteins.     

Mannose-binding plant lectins: different structural scaffolds for a common sugar-recognition process

Mannose-specific lectins are widely distributed in higher plants and are believed to play a role in recognition of high-mannose type glycans of foreign micro-organisms or plant predators. Structural studies have demonstrated that the mannose-binding specificity of lectins is mediated by distinct structural scaffolds. The mannose/glucose-specific legume (e.g., Con A, pea lectin) exhibit the canonical twelve-stranded beta-sandwich structure. In contrast to legume lectins that interact with both mannose and glucose, the monocot mannose-binding lectins (e.g., the Galanthus nivalis agglutinin or GNA from bulbs) react exclusively with mannose and mannose-containing N-glycans. These lectins possess a beta-prism structure. More recently, an increasing number of mannose-specific lectins structurally related to jacalin (e.g., the lectins from the Jerusalem artichoke, banana or rice), which also exhibit a beta-prism organization, were characterized. Jacalin itself was re-defined as a polyspecific lectin which, in addition to galactose, also interacts with mannose and mannose-containing glycans. Finally the B-chain of the type II RIP of iris, which has the same beta-prism structure as all other members of the ricin-B family, interacts specifically with mannose and galactose. This structural diversity associated with the specific recognition of high-mannose type glycans highlights the importance of mannose-specific lectins as recognition molecules in higher plants.     

The crystal structure of the Calystegia sepium agglutinin reveals a novel quaternary arrangement of lectin subunits with a beta-prism fold

The high number of quaternary structures observed for lectins highlights the important role of these oligomeric assemblies during carbohydrate recognition events. Although a large diversity in the mode of association of lectin subunits is frequently observed, the oligomeric assemblies of plant lectins display small variations within a single family. The crystal structure of the mannose-binding jacalin-related lectin from Calystegia sepium (Calsepa) has been determined at 1.37-A resolution. Calsepa exhibits the same beta-prism fold as identified previously for other members of the family, but the shape and the hydrophobic character of its carbohydrate-binding site is unlike that of other members, consistent with surface plasmon resonance analysis showing a preference for methylated sugars. Calsepa reveals a novel dimeric assembly markedly dissimilar to those described earlier for Heltuba and jacalin but mimics the canonical 12-stranded beta-sandwich dimer found in legume lectins. The present structure exemplifies the adaptability of the beta-prism building block in the evolution of plant lectins and highlights the biological role of these quaternary structures for carbohydrate recognition.     

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.     

Sub-Micellar Concentration of Sodium Dodecyl Sulphate Prevents Thermal Denaturation Induced Aggregation of Plant Lectin,Jacalin

The irreversible thermal unfolding of jacalin, the lectin purified from jackfruit seeds was accompanied by aggregation, where intermolecular interactions among the subunits are favoured over intramolecular interactions. The extent of aggregation increased as a function of temperature, time and protein concentration. The anionic surfactant, sodium dodecyl sulphate (SDS) significantly suppressed the formation of aggregates as observed by turbidity measurements and Rayleigh scattering assay. Moreover, far UV-CD spectra indicate that the protein β sheet transforms into α helical structure, when denatured in the presence of 3 mM SDS. Further, jacalin when heated in the presence of SDS partially retained the hemagglutination activity when jacalin-SDS mixture was diluted to 1:8 factor since 3 mM SDS was found to lyse the red blood cells. Thus, SDS only altered the aggregation behaviour of jacalin by preventing intermolecular hydrogen bonding among the exposed residues but did not completely stabilize the native conformation.     

Identification of a novel 4 kDa immunoglobulin-A-binding peptide obtained by the limited proteolysis of jacalin

Jacalin, an IgA-binding lectin from jackfruit (Artocarpus heterophyllus) seeds, was isolated by the passage of PBS extracts of seeds over an affinity matrix containing IgA-Sepharose-4B. It was further purified by HPLC. When analyzed by SDS-PAGE under both reducing and nonreducing conditions, the native jacalin was dissociated into two subunits of 12 and 15.4 kDa. Both the subunits could bind IgA. Peptide mapping performed with radioiodinated jacalin indicated that both the subunits were susceptible to proteolysis by Staphyloccous aureus V8 proteinase. One degradation product was a small peptide of 4 kDa. This small proteolytic fragment also bound IgA. The amino-termini of the two major IgA binding subunits, 12 and 15.4 kDa, were identical. The 4 kDa IgA-binding proteolytic fragment of jacalin had a different amino-terminal sequence, suggesting that the region of jacalin which binds IgA does not remain close to the amino-terminus of the peptide.     

Jacalin, an IgA-binding lectin, inhibits differentiation of human B cells by both a direct effect and by activating T-suppressor cells

Jacalin, a lectin extracted from the seeds of Artocarpus intergifolia (jackfruit), has been reported to bind specifically to IgA while inducing B-cell polyclonal immunoglobulin secretion. We confirmed that jacalin only binds to IgA and not to IgG or IgM and extended these findings by showing that it does not bind to IgE. Addition of jacalin to either unfractioned peripheral blood lymphocytes or purified B cells failed to induce immunoglobulin synthesis; indeed immunoglobulin production was diminished in the presence of jacalin. We found that jacalin directly inhibited the induction of immunoglobulin synthesis from B cells in the presence of T-cell replacing factor. Cell lines making IgG, IgM, and IgA were inhibited by jacalin. Furthermore, T cells incubated with jacalin also inhibited immunoglobulin production by stimulated B cells. Under these conditions jacalin was found to be a potent mitogen for T cells but to induce little or no activation of B cells. Jacalin appears to be a potent T-cell mitogen which can induce suppressor T cells for Ig production. It also has a direct inhibitory effect on B-cell Ig production.     

Jacalin: an IgA-binding lectin

We previously reported that seeds of Artocarpus integrifolia (jackfruit) contain a lectin, which we call jacalin, that is both a potent T cell mitogen and an apparently T cell-independent activator of human B cells for the secretion of immunoglobulins. During the above experiments we noted a massive precipitation in cell cultures stimulated with greater than or equal to 100 micrograms of lectin. In this paper, we show that the precipitate is formed after the interaction of jacalin and the serum protein added to the culture medium. More importantly, we demonstrate that IgA is probably the major serum constituent precipitated by the lectin and that no IgG or IgM can be detected in the precipitates. In secretions such as colostrum, IgA is the only protein precipitated by jacalin. On the basis of this specificity we describe a simple and reliable affinity chromatography procedure for the purification of both human serum and colostrum IgA. Jacalin is a D-Gal binding lectin and should be a useful tool for studying of serum and secretory IgA.     

Knowledge-based model of a glucosyltransferase from the oral bacterial group of mutans streptococci.

Mutans streptococci glucosyltransferases catalyze glucosyl transfer from sucrose to a glucan chain. We previously identified an aspartyl residue that participates in stabilizing the glucosyl transition state. The sequence surrounding the aspartate was found to have substantial sequence similarity with members of alpha-amylase family. Because little is known of the protein structure beyond the amino acid sequence, we used a knowledge-based interactive algorithm, MACAW, which provided significant level of homology with alpha-amylases and glucosyltransferase from Streptococcus downei gtfI (GTF). The significance of GTF similarity is underlined by GTF/alpha-amylase residues conserved in all but one alpha-amylase invariant residues. Site-directed mutagenesis of the three GTF catalytic residues are homologous with the alpha-amylase catalytic triad. The glucosyltransferases are members of the 4/7-superfamily that have a (beta/alpha)8-barrel structure and belong to family 13 of the glycohydralases.     

The primary structure of coagulation factor IX/factor X-binding protein isolated from the venom of Trimeresurus flavoviridis. Homology with asialoglycoprotein receptors, proteoglycan core protein, tetranectin, and lymphocyte Fc epsilon receptor for immunoglobulin E

An anticoagulant protein, factor IX/factor X-binding protein (IX/X-bp), isolated from the venom of Trimeresurus flavoviridis, binds with factor IX and factor X in the presence of Ca2+ with a 1 to 1 stoichiometry (Atoda, H., and Morita, T. (1989) J. Biochem. (Tokyo) 106, 808-813). Analysis of S-pyridylethylated IX/X-bp by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a 16.0-kDa band (designated the A chain) and a 15.5-kDa band (designated the B chain). These two chains were separated by reversed-phase high performance liquid chromatography, and their complete amino acid sequences were determined by sequencing of the peptides obtained after digestion with lysyl endopeptidase, chymotrypsin, and V8 protease from Staphylococcus aureus and after chemical cleavage with cyanogen bromide. The A chain had an amino-terminal sequence of Asp-Cys-Leu-Ser-Gly- and consisted of 129 residues with Mr 14,830. The B chain has an amino-terminal sequence of Asp-Cys-Pro-Ser-Asp- and consists of 123 residues of Mr 14,440. There was 47% identity between the A and the B chain. The sequence of IX/X-bp showed 25-37% identity with that of the C-type carbohydrate recognition domain-like structure of acorn barnacle lectin, human and rat asialoglycoprotein receptors, the human lymphocyte Fc epsilon receptor for immunoglobulin E, proteoglycan core protein, pancreatic stone protein, and tetranectin. The sequences of the first 18 amino acid residues of both the A and B chains were also, to a certain extent, homologous to the partial amino acid sequence of the b subunit of factor XIII, a member of the beta 2-glycoprotein I-like family. In this region, some similarity with the amino-terminal amino acid sequence of botrocetin was also observed.     

The X-ray structure of a mutant eye lens beta B2-crystallin with truncated sequence extensions.

beta-Crystallins are oligomeric eye lens proteins that are related to monomeric gamma-crystallins by domain swapping: like gamma-crystallins, they are comprised of two similar domains but they differ in having long sequence extensions. beta B2, a major component of beta-crystallin oligomers, self-associates to a homodimer in solution. In two crystal structures of native beta B2, the protein is a 222-symmetric tetramer of eight domains. It has previously been shown that a mutant of rat beta B2-crystallin, in which the bulk of the N- and C-terminal sequence extensions has been deleted, assembles into dimers and tetramers. Here we present the 3.0 A resolution X-ray structure of the tetramer, beta B2 delta NC1. The mutant tetramer has a very similar set of domain interactions to the native structure. However, the structures differ in the relative orientation of the two sets of four domains. The paired N- and C-terminal domain interface, which is at the heart of the dimer structure, is very similar to the native structure. However, the truncation of the C-terminal extension removes an important tryptophan residue, which prevents the extension from acting as a (non-covalent) linker, as it does in native beta B2. There is a knock-on structural effect that removes a contact between extension and covalent linker, and this appears to cause a small twist in the linker that is amplified into a 20 degrees rotation between sets of paired domains.     

Crystal-state conformation of Calpha,alpha-dialkylated peptides containing chiral beta-homo-residues.

Secondary structure formation and stability are essential features in the knowledge of complex folding topology of biomolecules. To better understand the relationships between preferred conformations and functional properties of beta-homo-amino acids, the synthesis and conformational characterization by X-ray diffraction analysis of peptides containing conformationally constrained Calpha,alpha-dialkylated amino acid residues, such as alpha-aminoisobutyric acid or 1-aminocyclohexane-1-carboxylic acid and a single beta-homoamino acid, differently displaced along the peptide sequence have been carried out. The peptides investigated are: Boc-betaHLeu-(Ac6c)2-OMe, Boc-Ac6c-betaHLeu-(Ac6c)2-OMe and Boc-betaHVal-(Aib)5-OtBu, together with the C-protected beta-homo-residue HCl.H-betaHVal-OMe. The results indicate that the insertion of a betaH-residue at position 1 or 2 of peptides containing strong helix-inducing, bulky Calpha,alpha-disubstituted amino acid residues does not induce any specific conformational preferences. In the crystal state, most of the NH groups of beta-homo residues of tri- and tetrapeptides are not involved in intramolecular hydrogen bonds, thus failing to achieve helical structures similar to those of peptides exclusively constituted of Calpha,alpha-disubstituted amino acid residues. However, by repeating the structural motifs observed in the molecules investigated, a beta-pleated sheet secondary structure, and a new helical structure, named (14/15)-helix, were generated, corresponding to calculated minimum-energy conformations. Our findings, as well as literature data, strongly indicate that conformations of betaH-residues, with the micro torsion angle equal to -60 degrees, are very unlikely.     

Sustained mitogenic effect on K562 human chronic myelogenous leukemia cells by dietary lectin,jacalin

Dietary lectins have been shown to affect the proliferation of human cancer cell lines. The anti-proliferative effects of lectins from varied sources have been extensively studied and in some cases, the underlying mechanism has been explored. Except for peanut agglutinin (PNA), the mitogenic effects of no other lectins have been studied in detail. In the present study, we have shown that jacalin, lectin purified from jackfruit (Artocarpus integrifolia) seeds act as a mitogen for K562, the Bcr-Abl expressing erythroleukemia cell line (K562) and the effect was found to be dose dependent. K562 cells remained in the proliferative state for a longer period even after the withdrawal of jacalin stimulation, thus jacalin was found to induce sustained mitogenic effect on K562 cells. Further, conditioned media from K562 cells treated with jacalin were observed to have the similar mitogenic effect even in the presence of galactose. Importantly, galactose which is a known ligand for jacalin will interact with functionally active jacalin present in the conditioned media and neutralise its effect. In addition, jacalin treatment also resulted in increased mRNA expression levels of pro-inflammatory cytokines including IL-1β, IL-6 and IFN-γ. Our results indicate that jacalin induces secretion of soluble molecules, which maybe responsible for this observed increased proliferation of K562 cells.     

Prediction of secondary structure by evolutionary comparison: application to the alpha subunit of tryptophan synthase

The amino acid sequences of the a subunits of tryptophan synthase from ten different microorganisms were aligned by standard procedures. The alpha helices, beta strands and turns of each sequence were predicted separately by two standard prediction algorithms and averaged at homologous sequence positions. Additional evidence for conserved secondary structure was derived from profiles of average hydropathy and chain flexibility values, leading to a joint prediction. There is good agreement between (1) predicted beta strands, maximal hydropathy and minimal flexibility, and (2) predicted loops, great chain flexibility, and protein segments that accept insertions of various lengths in individual sequences. The a subunit is predicted to have eight repeated beta-loop-alpha-loop motifs with an extra N-terminal alpha helix and an intercalated segment of highly conserved residues. This pattern suggests that the territory structure of the a subunit is an eightfold alpha/beta barrel. The distribution of conserved amino acid residues and published data on limited proteolysis, chemical modification, and mutagenesis are consistent with the alpha/beta barrel structure. Both the active site of the a subunit and the combining site for the beta 2 subunit are at the end of the barrel formed by the carboxyl-termini of the beta strands.     

木菠萝凝集素结合部位糖的特异性

用定量免疫沉淀法和定量免疫沉淀抑制法研究了从木菠萝(Arthrocarpus integrifolia)种子提取的凝集素(jacalin)结合部位糖的特异性。Jacalin最强烈地沉淀含有DGalβ1→3DGalNAc结构的无活性抗冻糖蛋白,不同程度非特异地沉淀各种血型物质。研究发现凝集素结合部位对DGalβ→3DGalNAc有最高特异性。最强抑制剂是DGalβ1→3DGalNAcal→φNO_2,其抑制活性分别比DGalNAc和DGal高380倍和1000倍。对于各种甲基化或对硝基酚化的糖苷以及寡糖,除methylaDGalNAc_f外,仅α-构型表现出抑制活性,所有β-构型的糖苷均无抑制活性,jacalin结合部位对糖的结合是构型依赖性的。