Photoaffinity labeling of the adenine binding site of the lectins from lima bean, Phaseolus lunatus, and the kidney bean, Phaseolus vulgaris

8-Azidoadenine was employed as a photoaffinity probe of the adenine binding site of the seed lectin from lima beans and from Phaseolus vulgaris erythroagglutinin. This compound was shown to (a) bind competitively to the adenine binding site of these lectins and (b) exhibit enhanced binding in the presence of 1,8-anilinonaphthalenesulfonic acid in the same manner as adenine. The presence or absence of 1,8-anilinonaphthalenesulfonic acid during labeling caused no change in the peptide maps of either lectin when digested with trypsin. The peptide maps of each lectin showed one major peak of radioactivity. Sequencing of the corresponding tryptic peptide from lima bean lectin indicated the primary structure to be Val-Leu-Ile-Thr-Tyr-Asp-Ser-Ser-Thr-Lys. The sequence of the labeled peptide isolated from P. vulgaris erythroagglutinin was Thr-Thr-Thr-Trp-Asp-Phe-Val-Gly-Glu-Asn-Glu-Val-Leu-Ile-Thr-Tyr, which corresponded to residues 173-190 of the cDNA-derived sequence (Hoffman, L. M., and Donaldson, D. D. (1985) EMBO J. 4, 883-889). Residues 186-190 (italicized) are identical to the first five amino acids in the lima bean lectin peptide. The peptides are located at the COOH-terminal half of the lectin and show extensive homology with other legume lectins.     

Architecture of the sugar binding sites in carbohydrate binding proteins-a computer modeling study

Different sugars, Gal, GalNAc and Man were docked at the monosaccharide binding sites of Erythrina corallodenron (EcorL), peanut lectin (PNA), Lathyrus ochrus (LOLI), and pea lectin (PSL). To study the lectin-carbohydrate interactions, in the complexes, the hydroxymethyl group in Man and Gal favors, gg and gt conformations respectively, and is the dominant recognition determination. The monosaccharide binding site in lectins that are specific to Gal/GalNAc is wider due to the additional amino acid residues in loop D as compared to that in lectins specific to Man/Glc, and affects the hydrogen bonds of the sugar involving residues from loop D, but not its orientation in the binding site. The invariant amino acid residues Asp from loop A, and Asn and an aromatic residue (Phe or Tyr) in loop C provides the basic architecture to recognize the common features in C4 epimers. The invariant Gly in loop B together with one or two residues in the variable region of loop D/A holds the sugar tightly at both ends. Loss of any one of these hydrogen bonds leads to weak interaction. While the subtle variations in the sequence and conformation of peptide fragment that resulted due to the size and location of gaps present in amino acid sequence in the neighborhood of the sugar binding site of loop D/A seems to discriminate the binding of sugars which differ at C4 atom (galacto and gluco configurations). The variations at loop B are important in discriminating Gal and GalNAc binding. The present study thus provides a structural basis for the observed specificities of legume lectins which uses the same four invariant residues for binding. These studies also bring out the information that is important for the design/engineering of proteins with the desired carbohydrate specificity.     

The dinitrophenyl group as a selective label in high-performance liquid chromatography of peptides

1-Fluoro-2,4-dinitrobenzene can be used to selectively label histidine, tyrosine, and cysteine residues in maleylated proteins. The usefulness of the resulting chromophores for peptide mapping by high-performance liquid chromatography was demonstrated with the lectin from sainfoin (Onobrychis viciifolia). The 2,4-dinitrophenyl (Dnp) label also can be used in a hydrophobic modulation approach as the mobility of a labeled model peptide changes considerably when its Dnp group is removed by thiolysis. Application of the method for checking sequences obtained by DNA or amino acid methods was shown by experiments with Viciae lectins. The probable cleavage site that generates the pea lectin's beta-chain from the alpha-beta precursor was identified and the sequence differences between the lentil and pea lectin beta-chains were examined.     

The chemical characterization of favin, a lectin isolated from Vicia faba.

We have determined the subunit structure of the glucose- and mannose-binding lectin favin, from Vicia faba. The molecule is composed of two nonidentical polypeptide chains held together by noncovalent interactions. We have determined the complete amino acid sequence of the smaller alpha chain (Mr = 5,571) and shown that it is homologous to the alpha chain of the lectins from lentil and pea and to residues 72 to 120 of concanavalin A (Con A). The larger beta chain (Mr = 20,000) contains carbohydrate and is homologous to the beta chain of lentil, pea, soybean, peanut, and red kidney bean lectins and is homologous to a portion of the Con A molecule beginning at residue 122. Favin also contains a minor component, beta' (Mr = 18,700), that closely resembles the beta chain but lacks carbohydrate and may, on the basis of apparent molecular weight, lack some part of the COOH-terminal region of the polypeptide chain. Although favin is similar to Con A, it, like the lentil and pea lectins, appears to lack residues corresponding to positions 1 to 71 of Con A. Because these residues contribute significantly to the carbohydrate binding site of Con A, the lack of this region in the otherwise homologous lectin favin suggests that the carbohydrate binding site of favin differs from that of Con A or that the region represented by residues 1 to 71 of Con A is located in a different portion (i.e. in the beta chain) of the favin molecule.     

Enzymic and immunochemical properties of lysozyme. XI. Conformation and immunochemistry of the two-disulfide peptide and the tryptophan and lysine residues in its antigenic reactivity.

The previously described peptide 62-68 (Cys 64-Cys 80) 74-96 (Cys 76-Cys 94) (Atassi, M.Z., Suliman, A.M. and Habeeb, A.F.S.A. (1975) Biochim. Biophys. Acta 405, 452-463), which accounted for about one-third of the total antigenic reactivity of native lysozyme, was isolated here with lysine 97 attached to it. The peptide was subjected to specific modification reactions in order to determine some of the residues which formed part of its antigenic reactive site. ORD measurements showed that the peptide was greatly unfolded in solution relative to its expected mode of folding within the intact lysozyme molecule. Modification of the two tryptophan residues in the peptide by reaction with 2,3-dioxo-5-indolinesulfonic acid provided a derivative which possessed similar conformational parameters to those of the unmodified peptide. However, the derivative retained only about half the immunochemical reactivity of the peptide. Succinylation of the amino groups afforded a derivative whose conformational parameters were identical to those of the unmodified peptide but in which half of the immunochemical reactivity was lost. Modification of the two tryptophan residues followed by succinylation of the amino groups resulted in almost complete loss of the antigenic reactivity, and the loss was not due to conformational differences. The antigenic reactivity of the peptide was also destroyed on removal of tryptophans 62 and 63, of sequence 84-93 from the loop 74-79 and of sequence 74-75 by chymotryptic digestion. From these and previous results it was concluded that the antigenic reactive site in this part of the lysozyme molecule incorporates one or both of tryptophans 62 and 63 as well as one or both lysines 96 and 97. The two disulfides 64-80 and 76-94 bring these two parts of the lysozyme molecule into a single reactive site. The intactness of the disulfides is essential for maintenance and reactivity of the site.     

The primary structure of the Cytisus scoparius seed lectin and a carbohydrate-binding peptide.

The complete amino acid sequence of 2-acetamido-2-deoxy-D-galactose-binding Cytisus scoparius seed lectin II (CSII) was determined using a protein sequencer. After digestion of CSII with endoproteinase Lys-C or Asp-N, the resulting peptides were purified by reversed-phase high performance liquid chromatography (HPLC) and then subjected to sequence analysis. Comparison of the complete amino acid sequence of CSII with the sequences of other leguminous seed lectins revealed regions of extensive homology. The amino acid residues of concanavalin A (Con A) involved in the metal binding site are highly conserved among those of CSII. A carbohydrate-binding peptide of CSII was obtained from the endoproteinase Asp-N digest of CSII by affinity chromatography on a column of GalNAc-Gel. This peptide was retained on the GalNAc-Gel column and was presumed to have affinity for the column. The amino acid sequence of the retarded peptide was determined using a protein sequencer. The retarded peptide was found to correspond to the putative metal-binding region of Con A. These results strongly suggest that this peptide represents the carbohydrate-binding and metal ion-binding sites of CSII.     

菜花烙铁头蛇毒C-型凝集素基因的克隆与序列分析

从菜花烙铁头蛇（Trimeresurus jerdonii)的毒腺中提取mRNA,采用RT-PCR技术进行体外扩增,将扩增产物克隆到PMD18-T载体中,最后筛选出一个编码凝集素的基因,命名为TJL。由TJL基因序列推导的氨基酸序列中包含分别由23和135个氨基酸残基组成的信号肽和成熟肽。氨基酸序列比较分析表明,TJL含有半乳糖结合位点和钙离子结合位点,与蝰科蛇毒凝集素TSL、PAL、APL和RSL的同源性较高（87.4%-90.4%）,与眼镜蛇科蛇毒凝集素BML的同源性较低（61.5%)。     

Molecular characterization of limulin, a sialic acid binding lectin from the hemolymph of the horseshoe crab, Limulus polyphemus.

The sialic acid binding lectin, limulin, was isolated by gel filtration and ion-exchange chromatography from the hemolymph of Limulus polyphemus. When the purified protein was characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of beta-mercaptoethanol, two major protein bands were observed. These two bands, subsequently found to contain carbohydrate as well, corresponded to molecular weights of 25 000 and 27 000. Amino acid sequence analyses were performed on both the intact protein and isolated cyanogen bromide fragments. The following primary structural features were noted in the amino-terminal region of limulin: (1) the absence of histidine and alanine from the NH2-terminal 50 residues; (2) the presence of five of the total eight prolines of the molecule between positions 13 and 30; and (3) a possible carbohydrate attachment site consisting of only the amino acids proline and serine between residues 13 and 19. The resultsof cyanogen bromide cleavage studies confirmed the presence of 2 methionine residues per subunit, at positions 25 and 58 respectively. No sequence heterogeneity was observed in this study. While it is quite possible that limulin plays some role in the defense mechanisms of the horseshoe crab, there is no obvious sequence homology between this invertebrate lectin and vertebrate immunoglobulins.     

Chemical modification studies on Abrus agglutinin. Involvement of tryptophan residues in sugar binding.

The galactose-binding lectin from the seeds of the jequirity plant (Abrus precatorius) was subjected to various chemical modifications in order to detect the amino acid residues involved in its binding activity. Modification of lysine, tyrosine, arginine, histidine, glutamic acid and aspartic acid residues did not affect the carbohydrate-binding activity of the agglutinin. However, modification of tryptophan residues carried out in native and denaturing conditions with N-bromosuccinimide and 2-hydroxy-5-nitrobenzyl bromide led to a complete loss of its carbohydrate-binding activity. Under denaturing conditions 30 tryptophan residues/molecule were modified by both reagents, whereas only 16 and 18 residues/molecule were available for modification by N-bromosuccinimide and 2-hydroxy-5-nitrobenzyl bromide respectively under native conditions. The relative loss in haemagglutinating activity after the modification of tryptophan residues indicates that two residues/molecule are required for the carbohydrate-binding activity of the agglutinin. A partial protection was observed in the presence of saturating concentrations of lactose (0.15 M). The decrease in fluorescence intensity of Abrus agglutinin on modification of tryptophan residues is linear in the absence of lactose and shows a biphasic pattern in the presence of lactose, indicating that tryptophan residues go from a similar to a different molecular environment on saccharide binding. The secondary structure of the protein remains practically unchanged upon modification of tryptophan residues, as indicated by c.d. and immunodiffusion studies, confirming that the loss in activity is due to modification only.     

Primary structure, physicochemical properties, and chemical modification of NAD(+)-dependent D-lactate dehydrogenase. Evidence for the presence of Arg-235, His-303, Tyr-101, and Trp-19 at or near the active site.

The NAD(+)-dependent D-lactate dehydrogenase was purified to apparent homogeneity from Lactobacillus bulgaricus and its complete amino acid sequence determined. Two gaps in the polypeptide chain (10 residues) were filled by the deduced amino acid sequence of the polymerase chain reaction amplified D-lactate dehydrogenase gene sequence. The enzyme is a dimer of identical subunits (specific activity 2800 +/- 100 units/min at 25 degrees C). Each subunit contains 332 amino acid residues; the calculated subunit M(r) being 36,831. Isoelectric focusing showed at least four protein bands between pH 4.0 and 4.7; the subunit M(r) of each subform is 36,000. The pH dependence of the kinetic parameters, Km, Vm, and kcat/Km, suggested an enzymic residue with a pKa value of about 7 to be involved in substrate binding as well as in the catalytic mechanism. Treatment of the enzyme with group-specific reagents 2,3-butanedione, diethylpyrocarbonate, tetranitromethane, or N-bromosuccinimide resulted in complete loss of enzyme activity. In each case, inactivation followed pseudo first-order kinetics. Inclusion of pyruvate and/or NADH reduced the inactivation rates manyfold, indicating the presence of arginine, histidine, tyrosine, and tryptophan residues at or near the active site. Spectral properties of chemically modified enzymes and analysis of kinetics of inactivation showed that the loss of enzyme activity was due to modification of a single arginine, histidine, tryptophan, or tyrosine residue. Peptide mapping in conjunction with peptide purification and amino acid sequence determination showed that Arg-235, His-303, Tyr-101, and Trp-19 were the sites of chemical modification. Arg-235 and His-303 are involved in the binding of 2-oxo acid substrate whereas other residues are involved in binding of the cofactor.     

Mutational analysis of the carbohydrate binding activity of the tobacco lectin

At present the three-dimensional structure of the tobacco lectin, further referred to as Nictaba, and its carbohydrate-binding site are unresolved. In this paper, we propose a three-dimensional model for the Nictaba domain based on the homology between Nictaba and the carbohydrate-binding module 22 of Clostridium thermocellum Xyn10B. The suggested model nicely fits with results from circular dichroism experiments, indicating that Nictaba consists mainly of β-sheet. In addition, the previously identified nuclear localization signal is located at the top of the protein as a part of a protruding loop. Judging from this model and sequence alignments with closely related proteins, conserved glutamic acid and tryptophan residues in the Nictaba sequence were selected for mutational analysis. The mutant DNA sequences as well as the original Nictaba sequence have been expressed in Pichia pastoris and the recombinant proteins were purified from the culture medium. Subsequently, the recombinant proteins were characterized and their carbohydrate binding properties analyzed with glycan array technology. It was shown that mutation of glutamic acid residues in the C-terminal half of the protein did not alter the carbohydrate-binding activity of the lectin. In contrast, mutation of tryptophan residues in the N-terminal half of the Nictaba domain resulted in a complete loss of carbohydrate binding activity. These results suggest that tryptophan residues play an important role in the carbohydrate binding site of Nictaba.     

豌豆外源凝集素基因的克隆及序列分析

从豌豆幼叶分离基因组ＤＮＡ,设计特异引物,用聚合酶链式反应方法扩增出豌豆外源凝集素基因并克隆到Ｅ．ｃｏｌｉ质粒ｐＢｌｕｅｓｃｒｉｐｔＳＫ（＋）的ＥｃｏＲＶ位点。进一步亚克隆至ｐＵＣ１９。序列分析表明,克隆到的片段大小为８３２ｂｐ,包含了豌豆外源凝集素基因完整的编码序列。该基因无内含子,同报道的已知序列相比,其核苷酸序列及推测的氨基酸序列的同源率分别为９９．６％和９８．９％。     

Conformation and inhibitory properties of peptides based on the tissue kallikrein-aprotinin complex.

A series of peptides encompassing the primary binding segment (residues 12-19) of aprotinin has been synthesized and tested for their ability to inhibit porcine pancreatic kallikrein. A minimum sequence of five amino acids spanning residues 12-16 of aprotinin is necessary for inhibition of porcine pancreatic kallikrein. An octapeptide homologous with the binding segment of aprotinin has a Ki-value of 1.2 x 10(-4) M. The solution structure of the octapeptide was studied by one- and two-dimensional NMR methods for comparison with the known structure of the segment of aprotinin that contacts tissue kallikrein. NMR experiments suggest that the peptide is either a random coil or that it samples several conformations on the NMR time scale. Analysis of the molecular dynamics trajectory of the octapeptide also suggests that the peptide is highly flexible. Thus, inhibition by the octapeptide occurs because of its homology with residues 12-19 of aprotinin. Moreover, the absence of a stable solution conformation similar to that of the binding segment of aprotinin is consistent with the 150,000-fold increase in Ki of the octapeptide compared to intact aprotinin.     

Analysis of the amino acid sequence of the Pseudomonas aeruginosa galactophilic PA-I lectin.

Based on the NH2-terminal 30-amino acid sequence of Pseudomonas aeruginosa galactophilic PA-I lectin, two degenerate primer oligonucleotides were synthesized and used in polymerase chain reaction with the bacterial chromosomal DNA as a template. A predominant DNA fragment of the appropriate size was radiolabeled and used as a probe for screening a P. aeruginosa genomic lambda gt11 library. One positive clone carrying an insert of about 630 base pairs encompassing the entire PA-I lectin gene was isolated and found to contain a 369-base pair open reading frame between an initiation codon (19 base pairs downstream from the insertion site, subsequent to a Shine-Dalgarno sequence) and two consecutive stop codons, followed by an oligo (seven) A sequence, in a partial dyad symmetry. The deduced amino acid sequence shows excellent agreement with the quantitative amino acid analysis and a perfect match with the NH2-terminal amino acid sequence of the purified lectin. It reveals that the PA-I lectin subunit contains 121 amino acids (M(r) 12,754; pI 4.94) with a predominant central hydrophilic core between two hydrophobic domains. Secondary structure algorithms predict that it is rich in beta sheets and contains several highly antigenic epitopes, but no signal peptide. In the carboxyl region a potential glycosylation site (Asn-Asn-Ser) was identified. Comparative analyses of this lectin sequence with those of lectins from other sources, reported in the protein and gene data banks, did not reveal any extensive homology.     

Adsorption mechanism at the molecular level between polymers and uremic octapeptide by the 2D 1H NMR Technique

To remove uremic octapeptide from the blood stream of uremic patients, various modified polyacylamide cross-linked absorbents were prepared. Adsorption experiments showed these absorbents have significant differences in adsorption capacity to the target peptide. In this paper, two-dimension proton nuclear magnetic resonance (2D 1H NMR) spectroscopy was used to investigate the interaction mechanism between the peptide and the adsorbents. Because of the insolubility of the absorbent, some soluble linear polymers with the same functional groups as the absorbents were employed as the model adsorbents in 2D 1H NMR. The preferred binding site for the peptide and polymers was identified to be at the C-terminal carboxyl group of the octapeptide via chemical shift perturbation effects. In this study, we found that hydrogen bonding, electrostatic, and hydrophobic interactions all play a role in the interaction force but had different contributions. Especially, the great chemical shift changes of the aromatic amino acid residues (Trp) during the interaction between butyl-modified polyacrylamide and octapeptide suggested the hydrophobic interaction, incorporated with the electrostatic force, played an important role in the binding reaction in aqueous solutions. This information not only rationally explained the results of the adsorption experiments, but also identified the effective binding site and mechanism, and shall provide a structural basis for designing better affinity-type adsorbents for the target peptide.     

Molecular dynamics simulations of pea (Pisum sativum) lectin structure with octyl glucoside detergents: the ligand interactions and dynamics

The mitogenic pea (Pisum sativum) lectin is a legume protein of non-immunoglobulin nature capable of specific recognition of glucose derivatives without altering its structure. Molecular dynamics simulations were performed in a realistic environment to investigate the structure and interaction properties of pea lectin with various concentrations of n-octyl-beta-d-glucopyranoside (OG) detergent monomers distributed inside explicit solvent cell. In addition, the diffusion coefficients of the ligands (OG, Ca2+, Mn2+, and Cl-) and the water molecules were also reported. The structural flexibility of the lectin was conserved in all simulations. The self-assembly of OG monomers into a small micelle at the hydrophobic site of the lectin was noticed in the simulation with 20 OG monomers. The interaction energy analysis concludes that the lectin was appropriately termed an adaptive structure. One or rarely two binding sites were observed at an instant in each simulation that were electrostatically favoured for the OG to interact with the surface amino acid residues. Enhanced binding of OG to the pea lectin was quantified in the system containing only Ca2+ divalent ions. Interestingly, no binding was observed in the simulation without divalent ions. Furthermore, the lectin-ligand complex was stabilized by multiple hydrogen bonds and at least one water bridge. Finally, the work was also in accordance with the published work elsewhere that the simulations performed with different initial conditions and using higher nonbonded cutoffs for the van der Waals and electrostatic interactions provide more accurate information and clues than the single large simulation of the biomolecular system of interest.