Structural basis for the specificity of basic winged bean lectin for the Tn-antigen: a crystallographic, thermodynamic and modelling study

The crystal structure of winged bean basic agglutinin in complex with GalNAc-alpha-O-Ser (Tn-antigen) has been elucidated at 2.35 angstroms resolution in order to characterize the mode of binding of Tn-antigen with the lectin. The Gal moiety occupies the primary binding site and makes interactions similar to those found in other Gal/GalNAc specific legume lectins. The nitrogen and oxygen atoms of the acetamido group of the sugar make two hydrogen bonds with the protein atoms whereas its methyl group is stabilized by hydrophobic interactions. A water bridge formed between the terminal oxygen atoms of the serine residue of the Tn-antigen and the side chain oxygen atom of Asn128 of the lectin increase the affinity of the lectin for Tn-antigen compared to that for GalNAc. A comparison with the available structures reveals that while the interactions of the glyconic part of the antigen are conserved, the mode of stabilization of the serine residue differs and depends on the nature of the protein residues in its vicinity. The structure provides a qualitative explanation for the thermodynamic parameters of the complexation of the lectin with Tn-antigen. Modeling studies indicate the possibility of an additional hydrogen bond with the lectin when the antigen is part of a glycoprotein.     

The beta 1----2-D-xylose and alpha 1----3-L-fucose substituted N-linked oligosaccharides from Erythrina cristagalli lectin. Isolation, characterisation and comparison with other legume lectins

The carbohydrate moieties of Erythrina cristagalli lectin were released as oligosaccharides by hydrazinolysis, followed by N-acetylation and reduction with NaB3H4. Fractionation of the tritium-labelled oligosaccharide mixture by Bio-Gel P-4 column chromatography and high-voltage borate electrophoresis revealed that it is composed of five neutral oligosaccharides. Structural studies by sequential exoglycosidase digestion in combination with methylation analysis and two-dimensional 1H-NMR showed that the major component was the fucose-containing heptasaccharide Man alpha 3(Man alpha 6)(Xyl beta 2)Man beta 4GlcNAc beta 4(Fuc alpha 3)GlcNAcol. This is the first report of such a structure in plant lectins. Small amounts of the corresponding afucosyl hexasaccharide were also identified, as well as three other minor components. The structure of the heptasaccharide shows the twin characteristics of a newly established family of N-linked glycans, found to date only in plants. The characteristics are substitution of the common pentasaccharide core [Man alpha 3(Man alpha 6)Man beta 4GlcNAc beta 4GlcNAc] by a D-xylose residue linked beta 1----2 to the beta-mannosyl residue and an L-fucose residue linked alpha 1----3 to the reducing terminal N-acetylglucosamine residue. The oligosaccharide heterogeneity pattern for Erythrina cristagalli lectin was also found for the lectins from four other Erythrina species and the lectins of two other legumes, Sophora japonica and Lonchocarpus capassa.     

The high specificities of Phaseolus vulgaris erythro- and leukoagglutinating lectins for bisecting GlcNAc or beta 1-6-linked branch structures, respectively, are attributable to loop B

Despite very similar tertiary structures based upon a common framework, legume lectins exhibit an amazing variety of sugar binding specificities. While most of these lectins recognize rather discrete sugar linkages, Phaseolus vulgaris erythroagglutinating and leukoagglutinating lectins (E(4)- and L(4)-PHA) are unique in recognizing larger structures. E(4)- and L(4)-PHA are known to recognize complex type N-glycans containing bisecting GlcNAc or a beta1,6-linked branch, respectively. However, the detailed mechanisms of molecular recognition are poorly understood. In order to dissect the contributions of different portions of each lectin, we carried out region-swapping mutagenesis between E(4)- and L(4)-PHA. We prepared six chimeric lectins by exchanging different combinations of loop B and the central portion of loop C, two of four loops thought to be important for the recognition of monosaccharides (Sharma, V., and Surolia, A. (1997) J. Mol. Biol. 267, 433-445). The chimeric lectins' sugar binding activities were evaluated quantitatively by surface plasmon resonance. These comparisons indicate that the high specificities of E(4)- and L(4)-PHA toward bisecting GlcNAc and beta1,6-linked branch structures are almost solely attributable to loop B. The contribution of the central portion of loop C to the recognition of those structural motifs was found to be negligible. Instead, it modulates affinity toward LacNAc residues present at the nonreducing terminus. Moreover, some of the chimeric lectins prepared in this study showed even higher specificities/affinities than native E(4)- and L(4)-PHA toward complex sugar chains containing either a bisecting GlcNAc residue or a beta1,6-linked branch.     

Glycosylated foldamers: synthesis of carbohydrate-modified beta3hSer and incorporation into beta-peptides.

Fmoc-protected beta(3)hserine (beta(3)hSer) was prepared and O-linked to suitably protected N-acetylgalactosamine (GalNAc) and N-acetylglucosamine (GlcNAc) derivatives. Glycosylation of beta(3)hSer was made by two independent routes: either by direct glycosyl linkage to the beta(3)hSer, or linkage to natural L-Ser and then utilizing the carbohydrate moiety as a protecting group in an Arndt-Eistert homologation. Both procedures gave the novel glycosylated beta(3)-amino acids Fmoc-beta(3)hSer(alpha-D-GalNAc(Ac)(3))-OH (1a), its beta-anomer (1b), and Fmoc-beta(3)hSer(beta-D-GlcNAc(Ac)(3))-OH (2), which were utilized in the solid-phase peptide synthesis of four glycosylated dipeptides (3a-d) and two heptapeptides (4a-b). The preparation of beta-amino acids bearing common post-translational modifiers represents an important step towards functionalized foldamers with broad applications in biomedical research.     

Synthesis of the glycosylated polypeptide chain of an inducible costimulator on T-cells

The glycoprotein AILIM/ICOS (Activation inducible lymphocyte immunomediately molecule/Inducible co-stimulator) on T-cells was identified in 1998 as a member of the CD28/CTLA4 family. The three-dimensional structure of the AILIM/ICOS extracellular domain has not been solved, and therefore we have examined the preparation of homogeneous glycosylated polypeptide chains of this domain having two homogeneous N-linked complex type oligosaccharides for use in folding experiments. To synthesize the glycosylated whole polypeptide chain of the AILIM/ICOS extracellular domain, the target polypeptide chain was divided into four segments, each containing a cysteine residue. Those peptide segments were synthesized by conventional SPPS, followed by thioesterification of the C-terminus. The oligosaccharide moiety, a biantennary complex type disialyloligosaccharide, was attached to the cysteine thiol in the peptide backbone using the haloacetamide method. These peptides, as well as a glycosylated peptide, were sequentially coupled by use of native chemical ligation. This process successfully afforded the desired polypeptide chain having homogeneous oligosaccharides.     

Characterization of the carbohydrate moiety of Clerodendron trichotomum lectins. Its structure and reactivity toward plant lectins

Lectins were isolated from fruits and leaves of Clerodendron trichotomum by affinity chromatography on lactamyl-Sepharose. The purified lectins (C. trichotomum agglutinin: CTA) were homogeneous on SDS/polyacrylamide gel electrophoresis, and the carbohydrate moiety was characterized by physicochemical and immunochemical methods. The asparagine-linked oligosaccharides were released by treatment with N-oligosaccharide glycopeptidase (almond, EC 3.5.1.52) of peptic glycopeptides obtained from fruit CTA, and separated by gel filtration and thin-layer chromatography. The structure of the predominant oligosaccharide was determined as Xyl beta 1----2 (Man alpha 1----6)(Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4(Fuc alpha 1----3)GlcNAc by high-performance liquid chromatography, sugar analysis and 1H-NMR spectroscopy. The reactivity of the carbohydrate moiety of CTA toward various lectins was studied. Fruit and leaf CTAs were applied to polyacrylamide gel electrophoresis, transferred to nitrocellulose sheets and detected with horseradish-peroxidase-conjugated lectins. Concanavalin A, lentil lectin, pea lectin, Vicia faba lectin and Ulex europeus agglutinin I, but not wheat germ lectin, bound to fruit CTA. The results indicate new binding properties of these plant lectins: a beta-xylosyl residue substituted at C-2 of the beta-mannosyl residue of N-linked oligosaccharide does not affect the binding with mannose-specific lectins, lentil, pea and Vicia faba lectins can bind to N-linked oligosaccharides containing an alpha-L-fucosyl residue attached to C-3 of the asparagine-linked N-acetyl-D-glucosamine residue, and Ulex europeus agglutinin I can bind to the (alpha 1----3)-linked fucose residue of the N-linked oligosaccharide.     

Tn-syndrome

The idiopathic Tn-syndrome, formerly called 'permanent mixed-field polyagglutinability', is a rare hematological disorder characterized by the expression of the Tn-antigen on all blood cell lineages. The immunodominant epitope of the Tn-antigen is terminal alpha-N-acetylgalactosamine, O-glycosidically linked to protein. Normally this residue is 3'-substituted by 5-galactose thereby forming the core 1 structure known as the Thomsen-Friedenreich (TF) antigen (Galbeta1 ==> 3GalNAcalpha1 ==> Thr/Ser). The cause of the exposure of the Tn-antigen appears to be due to the silencing of the gene expression of beta1,3galactosyltransferase, since treatment of deficient Tn(+) lymphocyte T clones with 5'azacytidine or Na butyrate leads to reexpression of enzyme activity and the sialylated TF-antigen. The Tn-syndrome is acquired and permanent and affects both sexes at any age. Its origin is unknown. Pluripotent stem cells are affected since all lineages are involved but each one to a variable extent. Therefore, normal cells co-exist with Tn-transformed cells. Clinically, patients suffering from the Tn-syndrome appear healthy. Laboratory findings usually reveal moderate thrombocyto- and leukopenia and some signs of hemolytic anemia not warranting any treatment.     

Structure Predictions of Two Bauhinia variegata Lectins Reveal Patterns of C-Terminal Properties in Single Chain Legume Lectins

Bauhinia variegata lectins (BVL-I and BVL-II) are single chain lectins isolated from the plant Bauhinia variegata. Single chain lectins undergo post-translational processing on its N-terminal and C-terminal regions, which determines their physiological targeting, carbohydrate binding activity and pattern of quaternary association. These two lectins are isoforms, BVL-I being highly glycosylated, and thus far, it has not been possible to determine their structures. The present study used prediction and validation algorithms to elucidate the likely structures of BVL-I and -II. The program Bhageerath-H was chosen from among three different structure prediction programs due to its better overall reliability. In order to predict the C-terminal region cleavage sites, other lectins known to have this modification were analysed and three rules were created: (1) the first amino acid of the excised peptide is small or hydrophobic; (2) the cleavage occurs after an acid, polar, or hydrophobic residue, but not after a basic one; and (3) the cleavage spot is located 5-8 residues after a conserved Leu amino acid. These rules predicted that BVL-I and –II would have fifteen C-terminal residues cleaved, and this was confirmed experimentally by Edman degradation sequencing of BVL-I. Furthermore, the C-terminal analyses predicted that only BVL-II underwent α-helical folding in this region, similar to that seen in SBA and DBL. Conversely, BVL-I and -II contained four conserved regions of a GS-I association, providing evidence of a previously undescribed X4+unusual oligomerisation between the truncated BVL-I and the intact BVL-II. This is the first report on the structural analysis of lectins from Bauhinia spp. and therefore is important for the characterisation C-terminal cleavage and patterns of quaternary association of single chain lectins.     

Two MHC surface amino acid differences distinguish foreign peptide recognition from autoantigen specificity

KRN T cells can recognize two self MHC alleles with differing biological consequences. They respond to the foreign peptide RN(42--56) bound to I-A(k) or alternatively initiate autoimmune arthritis by interacting with a self Ag, GPI(282--294), on I-A(g7). Five surface amino acid differences between the two MHC molecules collectively alter which peptide side chains are recognized by the KRN TCR. In this study, it is shown that mutation of only two of these residues, alpha 65 and beta 78, in I-A(k) to their I-A(g7) counterparts is sufficient to allow recognition of the TCR contacts from GPI(282--294). To provide a detailed mechanism for the specificity change, the distinct contributions of each of these two mutations to the global effect on peptide specificity were analyzed. The alpha65 mutation is shown to broaden the spectrum of amino acids permissible at P8 of the peptide. In contrast, the beta 78 mutation alone blocks KRN TCR interaction with I-A(k) and requires the simultaneous presence of the alpha 65 mutation to preserve recognition. In the presence of the alpha 65 mutation, the beta 78 residue broadens peptide recognition at P3 and prevents recognition of the P8 L in RN(42--56), thus producing the observed specificity shift. These results localize the functionally relevant differences between the surfaces of two self-restricted MHC molecules to two residues that have counterbalanced positive and negative contributions to interaction with a single TCR. They highlight how subtle structural distinctions attributable to single amino acids can stand at the interface between foreign Ag responsiveness and pathogenic autoreactivity.     

Antigenic and calcium binding properties of a Peptide containing the essential cysteine in lima bean lectin

Polyclonal antisera were raised against a peptide containing the cysteine residue required for carbohydrate binding activity in the lima bean lectin. The antisera were tested for cross-reactivity with (a) synthetic peptide analogs to the essential cysteine containing peptide, (b) proteolytic digests of related lectins, (c) native lectins. The antisera were specifically inhibited from binding to a peptide conjugate by free synthetic peptides. The degree of inhibition by lectin digests correlated approximately along evolutionary relationships and the degree of sequence conservation. One antiserum was found to cross-react with certain lectins in the native state. In a second set of experiments, the calcium binding properties of the synthetic peptides were investigated using metal ion-chelate chromatography and UV-difference spectroscopy. The nonapeptide and undecapeptide bound to a Ca²⁺ iminodiacetic acid agarose column and were eluted with EDTA. Ultraviolet difference spectral titrations with Ca²⁺ performed on the synthetic undecapeptide and a related favin derived peptide resulted in dissociation constants of approximately 6 × 10³ per molar.     

The C-type lectin MGL expressed by dendritic cells detects glycan changes on MUC1 in colon carcinoma

The epithelial mucin MUC1 is a high molecular weight membrane glycoprotein frequently overexpressed and aberrantly glycosylated in adenocarcinoma. Mucins normally contain high amounts of O-linked carbohydrate structures that may influence immune reactions to this antigen. During malignant transformation, certain glyco-epitopes of MUC1, such as Tn-antigen, TF-antigen and their sialylated forms become exposed. The role of these glycan structures in tumor biology is unknown, but their presence is known to correlate with poor prognosis in several adenocarcinomas. We analyzed the potency of MUC1 containing Tn-antigens (MUC1-Tn) to target C-type lectins that function as carbohydrate recognition and uptake molecules on dendritic cells (DC). We identified the macrophage galactose type C-type lectin (MGL), expressed by both DC and macrophages, as the receptor for recognition and binding of MUC1-Tn. To validate the occurrence of MGL–MUC1 interactions in situ, we studied the binding of MGL to MUC1 in primary colon carcinoma tissue. Isolation of MUC1 out of colon carcinoma tissue showed strong binding activity to MGL. Interestingly, MGL binding to MUC1 was highly correlated to binding by the lectin Helix pomatia agglutinin (HPA), which is associated with poor prognosis in colorectal cancer. The detection of MGL positive cells in situ at the tumor site together with the modified glycosylation status of MUC1 to target MGL on DC suggests that MGL positive antigen presenting cells may play a role in tumor progression.     

The influence of flanking sequence on the O-glycosylation of threonine in vitro.

To investigate the influence of flanking amino acid sequence on the O-glycosylation of a single threonine residue in vitro, we have examined a series of 52 related peptides. The substrates were based upon a sequence from human von Willebrand factor which is known to be glycosylated in vivo (-6PHMAQVTVGPGL+5). Each residue of the parent peptide was substituted, in turn, with isoleucine, alanine, proline, glutamic acid, or arginine. Peptides were glycosylated using a UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase purified 15,000-fold from bovine colostrum by chromatography on DEAE-Sephacel, SP-Sephadex, Sephacryl S-300, Affi-Gel Blue, and 5-mercuri-UDP-GalNAc thiopropyl-Sepharose. Single amino acid changes in the sequences flanking the threonine could profoundly alter the glycosylation of the substrate peptides. Substitution of any amino acid tested at positions +3, -3, and -2 markedly decreased O-glycosylation, as did the presence of a charged residue at position -1. The substitution of amino acids at the other positions of the peptide substrate had little effect on the incorporation of GalNAc. Statistical analysis of sequences flanking known glycosylated threonine and serine residues suggests that they should be glycosylated with equal efficiency in the same sequence context (O'Connell et al., 1991). However, the bovine colostrum transferase failed to glycosylate a peptide derived from human erythropoietin which contains a serine that is glycosylated in vivo (-5PPDAASAAPLR+5). When a threonine was substituted for the serine in this peptide (-5PPDAATAAPLR+5), the substrate proved to be an excellent acceptor of GalNAc. These observations indicate that although flanking amino acid sequence is important for the O-glycosylation of specific hydroxyamino acids, discrete threonine- and serine-specific transferases may exist.     

Substrate specificity of the protease that processes human interleukin-1 beta

The substrate specificity of the protease which generates mature human interleukin-1 beta (IL-1 beta) from pro-interleukin-1 beta was investigated using synthetic peptide substrates and recombinant pro-IL-1 beta. The requirement of an L-aspartate in the P-1 position was confirmed together with the need for a small hydrophobic residue in the P-1' position (Gly or Ala). It was shown that the enzyme can tolerate conservative substitutions in the P-2 and P-2' positions. We found little difference in the enzyme's ability to cleave denatured and native pro-IL-1 beta, indicating that tertiary structure recognition is not involved in binding. The enzyme did, however, require a peptide of more than six amino acids for cleavage to occur. These results conclusively demonstrate the unusual specificity of this protease.     

Purification and characterization of a Neu5Ac alpha 2-->6Gal beta 1-->4GlcNAc and HSO3(-)-->6Gal beta 1-->GlcNAc specific lectin in tuberous roots of Trichosanthes japonica.

Two lectins were purified from tuberous roots of Trichosanthes japonica. The major lectin, which was named TJA-II, interacted with Fuc alpha 1-->2Gal beta/GalNAc beta 1-->groups, and the other one, which passed through a porcine stomach mucin-Sepharose 4B column, was purified by sequential chromatography on a human alpha 1-antitrypsin-Sepharose 4B column and named TJA-I. The molecular mass of TJA-I was determined to be 70 kDa by sodium dodecyl sulfate gel electrophoresis. TJA-I is a heterodimer of 38-kDa (36-kDa) and 32-kDa (30-kDa) subunits with disulfide linkage(s), and the difference between 38 and 36 kDa, and between 32 and 30 kDa, is due to secondary degradation of the carboxyl-terminal side. It was determined by equilibrium dialysis that TJA-I has four equal binding sites per molecule, and the association constant toward tritium-labeled Neu5Ac alpha 2-->6Gal beta 1-->4GlcNAc beta 1-->3Gal beta 1-->4GlcOT is Ka = 8.0 x 10(5) M-1. The precise carbohydrate binding specificity was studied using hemagglutinating inhibition assay and immobilized TJA-I. A series of oligosaccharides possessing a Neu5Ac alpha 2-->6Gal beta 1-->4GlcNAc or HSO3(-)-->6Gal beta 1-->4GlcNAc group showed tremendously stronger binding ability than oligosaccharides with a Gal beta 1-->4GlcNAc group, indicating that TJA-I basically recognizes an N-acetyllactosamine residue and that the binding strength increases on substitution of the beta-galactosyl residue at the C-6 position with a sialic acid or sulfate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparative study of the post-translational processing of the mannose-binding lectins in the bulbs of garlic (Allium sativum L.) and ramsons (Allium ursinum L.)

The biosynthesis and processing of the homodimeric and heterodimeric lectins from the bulbs of garlic (Allium sativum) and ramsons (wild garlic;Allium ursinum) were studied using pulse and pulse-chase labelling experiments on developing bulbs. By combining the results of thein vivo biosynthesis studies and the cDNA cloning of the respective lectins, the sequence of events leading from the primary translation products into the mature lectin polypeptides could be reconstructed. From this it is demonstrated that garlic and ramsons use different schemes of post-translational modifications in order to synthesize apparently similar lectins from totally different precursors. Both the homomeric garlic lectin (ASAII) and its homologue in ramsons (AUAII) are synthesized on the endoplasmic reticulum (ER) as nonglycosylated 13.5 kDa precursors, which, after their transport out of the ER are converted into the mature 12.0 kDa lectin polypeptides by the cleavage of a C-terminal peptide. The heterodimeric garlic lectin ASAI is synthesized on the ER as a single glycosylated precursor of 38 kDa, which after its transport out of the ER undergoes a complex processing which gives rise to two mature lectin subunits of 11.5 and 12.5 kDa. In contrast, both subunits of the heterodimeric ramsons lectin AUAI are synthesized separately on the ER as glycosylated precursors, which after their transport out of the ER are deglycosylated and further processed into the mature lectin polypeptides by the cleavage of a C-terminal peptide.     

Recognition of the N-terminal modules of thrombospondin-1 and thrombospondin-2 by alpha6beta1 integrin

In addition to its recognition by alpha3beta1 and alpha4beta1 integrins, the N-terminal pentraxin module of thrombospondin-1 is a ligand for alpha6beta1 integrin. alpha6beta1 integrin mediates adhesion of human microvascular endothelial and HT-1080 fibrosarcoma cells to immobilized thrombospondin-1 and recombinant N-terminal regions of thrombospondin-1 and thrombospondin-2. alpha6beta1 also mediates chemotaxis of microvascular cells to thrombospondin-1 and thrombospondin-2. Using synthetic peptides, LALERKDHSG was identified as an alpha6beta1-binding sequence in thrombospondin-1. This peptide inhibited alpha6beta1-dependent cell adhesion to thrombospondin-1, thrombospondin-2, and the E8 fragment of murine laminin-1. The Glu residue in this peptide was required for activity, and the corresponding residue (Glu90) in the N-terminal module of thrombospondin-1 was required for its recognition by alpha6beta1, but not by alpha4beta1. alpha6beta1 was also expressed in human umbilical vein endothelial cells; but in these cells, only certain agonists could activate the integrin to recognize thrombospondins. Selective activation of alpha6beta1 integrin in microvascular endothelial cells by the anti-beta1 antibody TS2/16 therefore accounts for their adhesion responses to thrombospondins and explains the distinct functions of alpha4beta1 and alpha6beta1 integrins as thrombospondin receptors in microvascular and large vessel endothelial cells.     

The molecular basis of class II MHC allelic control of T cell responses.

To identify the molecular basis for the effects of MHC molecule polymorphism on T cell responses, we have combined functional T cell response testing with measurements of peptide binding to the class II MHC molecules on transfected cells. Our studies identify a small subset of spatially localized polymorphic residues of the E alpha E beta dimer (strand residue beta 29, and helix residues beta 72 and beta 75) regulating cytochrome c peptide presentation by two distinct mechanisms. The first effect is on quantitative control of net peptide binding. The replacement of the valine found at position beta 29 in E beta k with the glutamic acid found in E beta b results in a selective loss of pigeon cytochrome peptide but not moth cytochrome peptide binding to the resultant mutant E alpha E beta k molecule. Reciprocally, the replacement of glutamic acid at beta 29 in E beta b with valine results in a gain of pigeon peptide binding. These changes in binding parallel changes in T cell responses in vitro to these peptide-E alpha E beta combinations and mirror the in vivo immune response gene phenotypes of mice expressing E alpha E beta k and E alpha E beta b. E alpha E beta s molecules, which have a beta 29 glutamic acid, are nevertheless able to bind and present pigeon cytochrome peptides, and this is due to changes in helix residues beta 72 and beta 75 that compensate for the negative effect of the beta 29 glutamic acid. The second activity is a critical change in the conformation of the peptide bound to the same extent by distinct MHC molecules, as revealed by changes in T cell responses to moth cytochrome peptides presented by two E alpha E beta molecules differing only at position beta 29. Both of these effects can be ascribed to a single polymorphic residue modeled to be inaccessible to TCR contact (beta 29), providing a striking demonstration of how MHC molecule polymorphism can modify T cell-dependent immune responses without direct physical participation in the receptor recognition event.     

Structural mechanism governing the quaternary organization of monocot mannose-binding lectin revealed by the novel monomeric structure of an orchid lectin

Two isoforms of an antifungal protein, gastrodianin, were isolated from two subspecies of the orchid Gastrodia elata, belonging to the protein superfamily of monocot mannose-specific lectins. In the context that all available structures in this superfamily are oligomers so far, the crystal structures of the orchid lectins, both at 2.0 A, revealed a novel monomeric structure. It resulted from the rearrangement of the C-terminal peptide inclusive of the 12th beta-strand, which changes from the "C-terminal exchange" into a "C-terminal self-assembly" mode. Thus, the overall tertiary scaffold is stabilized with an intramolecular beta-sheet instead of the hybrid observed on subunit/subunit interface in all known homologous dimeric or tetrameric lectins. In contrast to the constrained extended conformation with a cis peptide bond between residues 98 and 99 commonly occurring in oligomers, a beta-hairpin forms from position 97 to 101 with a normal trans peptide bond at the corresponding site in gastrodianin, which determines the topology of the C-terminal peptide and thereby its unique fold pattern. Sequence and structure comparison shows that residue replacement and insertion at the position where the beta-hairpin occurs in association with cis-trans inter-conversion of the specific peptide bond (97-98) are possibly responsible for such a radical structure switch between monomers and oligomers. Moreover, this seems to be a common melody controlling the quaternary states among bulb lectins through studies on sequence alignment. The observations revealed a structural mechanism by which the quaternary organization of monocot mannose binding lectins could be governed. The mutation experiment performed on maltose-binding protein-gastrodianin fusion protein followed by a few biochemical detections provides direct evidence to support this conclusion. Potential carbohydrate recognition sites and biological implications of the orchid lectin based on its monomeric state are also discussed in this paper.     

O-GalNAc incorporation into a cluster acceptor site of three consecutive threonines. Distinct specificity of GalNAc-transferase isoforms.

O-Glycosylation of three consecutive Thr residues in a fluorescein-conjugated peptide PTTTPLK - which mimics a portion of mucin 2 - by four isozymes of UDP-N-acetylgalactosaminyltransferases (pp-GalNAc-T1, T2, T3, or T4) was investigated. Partially glycosylated versions of this peptide, PT*TTPLK, PTTT*PLK, PT*TT*PLK, PTT*T*PLK, PT* degrees TTPLK, and PTTT* degrees PLK (*, N-acetylgalactosamine; degrees, galactose), were also tested. The products were separated by RP-HPLC and characterized by MALDI-TOF MS and peptide sequencing. The first and the third Thr residues act as the peptide's initial glycosylation sites for pp-GalNAc-T4, which were different from the sites for pp-GalNAc-T1 and T2 (the first Thr residue) or T3 (the third Thr residue) shown in our previous report. All pp-GalNAc-T isozymes tested exhibited distinct specificities toward glycopeptides. The most notable findings were: (a) prior incorporation of an N-acetylgalactosamine residue at the third Thr greatly enhanced N-acetylgalactosamine incorporation into the other Thr residues when pp-GalNAc-T2, T3, or T4 were used; (b) the enhancing effect of the N-acetylgalactosamine residue on the third Thr was completely abrogated by galactosylation of this N-acetylgalactosamine; (c) prior incorporation of an N-acetylgalactosamine at the first Thr did not have any enhancing effect; (d) pp-GalNAc-T2 was unique as it transferred N-acetylgalactosamine into the second Thr residue only when N-acetylgalactosamine was attached to the third one.     

The insecticidal activity of recombinant garlic lectins towards aphids

The heterodimeric and homodimeric garlic lectins ASAI and ASAII were produced as recombinant proteins in the yeast Pichia pastoris. The proteins were purified as functional dimeric lectins, but underwent post-translational proteolysis. Recombinant ASAII was a single homogenous polypeptide which had undergone C-terminal processing similar to that occurring in planta. The recombinant ASAI was glycosylated and subject to variable and heterogenous proteolysis. Both lectins showed insecticidal effects when fed to pea aphids (Acyrthosiphon pisum) in artificial diet, ASAII being more toxic than ASAI at the same concentration. Acute toxicity (mortality at 3d exposure) was observed over the concentration range 0.125-2.0mgml(-1). The recombinant lectins caused mortality in both symbiotic and antibiotic-treated aphids, showing that toxicity is not dependent on the presence of the bacterial symbiont (Buchnera aphidicola), or on interaction with symbiont proteins, such as the previously identified lectin "receptor" symbionin. A pull-down assay coupled with peptide mass fingerprinting identified two abundant membrane-associated aphid gut proteins, alanyl aminopeptidase N and sucrase, as "receptors" for lectin binding.