南美藜种皮凝集素的初步研究

南美藜种皮中存在着凝集素。最适ｐＨ７—９,热稳定性较差。南美藜种皮凝集素（ＱＳＬ）的血凝活性可以被Ｄ－半乳糖,Ｌ－鼠李糖抑制,其中前者抑制能力强。ＱＳＬ硫酸铵分级沉淀血凝活性６０—８０组分最高。     

A simple cell labeling technique by means of lectins linked to fluorochromes for the detection of cells on tissue sections

Summary— We designed a protocol for cell labeling with the lectin wheat germ agglutinin (WGA) linked to the fluorochrome tetramethyl-rhodamine isothiocyanate (TRITC) for effective detection of the B16F10 melanoma and Lewis lung carcinoma (LLc) cells on pulmonary histological sections from C57BL6; mice. We have also determined a suitable concentration of WGA-TRITC (10 μg/ml), which leads to a very intense and homogeneous labeling of the cells, as it avoids cell clumping due to the presence of the lectin WGA. In order to determine to what extent the method affects these tumor cells, we have studied some important aspects related to their metastatic behavior, taking into account three parameters: a) viability and rate of proliferation of the cells cultured in vitro; b) percentage of animals C57BL6 mice) bearing metastasis 15 days after intravenous inoculation with 10⁵ B16F10 or LLc cells; and c) pattern of distribution of tumor foci in lung. There were no significant differences in these three parameters between the WGA-TRITC labeled-cells compared to the cultures of non-labeled cells in either of the cell lines (B16F10, LLc). Thus, we conclude that B16F10 and LLc tumor cells can be labeled following the protocol set-up in our study, as it allows these cells to be neatly identified on tissue sections and it causes no important physiological changes in the cells, with regard to metastatic behavior. These points make this technique very suitable for the detection of B16F10 and LLc cells on histological sections in studying their behavior during the first stages of the metastatic process.     

Cassettes for seed-specific expression tested in transformed embryogenic cultures of soybean

Soybean (Glycine max [L.] Merrill) lectin is a seed protein that accumulates in protein bodies of cotyledons during seed development. We have constructed two expression cassettes containing the 5′ and 3′ regions of the soybean lectin gene connected by aNot I restriction site. One vector also contains the 32 amino acid signal sequence. Using polymerase chain reaction (PCR), the coding region of the β-glucuronidase (uidA) gene was inserted into theNot I site of each vector. We tested the function of the expression cassettes in transformed embryogenic cultures of soybean. Development-specific GUS expression was observed in developing somatic embryos transformed with the chimeric lectin promoter-GUS constructs as determined by histochemical assays. Our data indicate that these cassettes could be used to drive expression of foreign genes to modify embryo-specific traits of soybean as protein quality or quantity in the seed.     

Comparative modeling of the three-dimensional structure of type II antifreeze protein.

Type II antifreeze proteins (AFP), which inhibit the growth of seed ice crystals in the blood of certain fishes (sea raven, herring, and smelt), are the largest known fish AFPs and the only class for which detailed structural information is not yet available. However, a sequence homology has been recognized between these proteins and the carbohydrate recognition domain of C-type lectins. The structure of this domain from rat mannose-binding protein (MBP-A) has been solved by X-ray crystallography (Weis WI, Drickamer K, Hendrickson WA, 1992, Nature 360:127-134) and provided the coordinates for constructing the three-dimensional model of the 129-amino acid Type II AFP from sea raven, to which it shows 19% sequence identity. Multiple sequence alignments between Type II AFPs, pancreatic stone protein, MBP-A, and as many as 50 carbohydrate-recognition domain sequences from various lectins were performed to determine reliably aligned sequence regions. Successive molecular dynamics and energy minimization calculations were used to relax bond lengths and angles and to identify flexible regions. The derived structure contains two alpha-helices, two beta-sheets, and a high proportion of amino acids in loops and turns. The model is in good agreement with preliminary NMR spectroscopic analyses. It explains the observed differences in calcium binding between sea raven Type II AFP and MBP-A. Furthermore, the model proposes the formation of five disulfide bridges between Cys 7 and Cys 18, Cys 35 and Cys 125, Cys 69 and Cys 100, Cys 89 and Cys 111, and Cys 101 and Cys 117.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phaseolus vulgaris phytohemagglutinin contains high-mannose and modified oligosaccharide chains

Phytohemagglutinin, the major lectin in the seeds of the common bean Phaseolus vulgaris L., was isolated by affinity chromatography from cotyledons of nearly mature seeds and from developing cotyledons labeled with [³H]glucosamine, [³H]mannose or [³H]fucose. The protein was subjected to exhaustive proteolysis and the carbohydrate composition of the resulting glycopeptides examined. Two classes of oligosaccharide side-chains were found. The sidechains of the first class are of the high-mannose type, containing two residues of N-acetylglucosamine and 8 or 9 mannose residues. The sidechains of the second class are of the modified type containing N-acetylglucosamine, mannose, fucose, xylose in molar ratios of 2:3.8:0.6:0.5. Two-dimensional gel electrophoresis shows that phytohemagglutinin can be fractionated into seven different glycosylated polypeptides, and that each one contains at least one modified oligosaccharide chain. The results indicate that most glycosylated polypeptides probably contain one chain of each class. The carbohydrate composition of the two types of chains is similar to that found in other plant glycoproteins, but this is the first report of a plant glycoprotein with both highmannose and modified oligosaccharides on the same polypeptide chain.Abbreviations endo H endo--N-acetylglucosaminidase H - GlcN glucosamine - GlcNAc N-acetylglucosamine - Man mannose - PHA phytohemagglutinin This work was done while A.V. was on leave from the Istituto Biosintesi Vegetali, C.N.R., via Bassini 15, I-20133 Milano, Italy     

Endogenous lectins in chickens and slime molds: Transfer from intracellular to extracellular sites

Endogenous lectins in both cellular slime molds and chicken tissues have been localized primarily intracellularly, in contrast with the predominantly extracellular localization of the glycoproteins, glycolipids, and glycosaminoglycans with which they might interact. Here we present evidence that lectins in both of these organisms may be externalized and become associated with the cell surface and/or extracellular materials. In chicken intestine, chicken-lactose-lectin-II is shown to be localized in the secretory granules of the goblet cells, along with mucin, and to be secreted onto the intestinal surface. In embryonic muscle, chicken-lactose-lectin-I is shown to be externalized with differentiation, ultimately becoming localized on the surface of myotubes and in the extracellular spaces. In a cellular slime mold, Dictyostelium purpureum, externalization of lectin is elicited by either polyvalent glycoproteins that bind the small amount of endogenous cell surface lectin, or by slime mold or plant lectins that bind unoccupied complementary cell surface oligosaccharides. These results suggest that externalization of endogenous lectin may be a response to specific external signals. We conclude that lectins are frequently held in intracellular reserves awaiting release for specific external functions.     

Synthesis of a polymide containing a glucose unit in the main chain

A new type polyamide containing a glucose unit in the main chain has been synthesized by the polymerization of C₁, C₃, C₄ blocked C₆-carboxymethylglucosamine, prepared from chitin. The deblocking procedure gave the water-soluble polyamide, of MW 1.5 × 10⁴, which can be regarded as a model for the recognition site of lectin.     

Lectin Binding to Radopholus citrophilus and R. similis Proteins

Lectin-binding glycoproteins in seven populations of two burrowing nematode sibling species were probed with five different biotinylated lectins on Western blots, and differences were correlated with nematode ability to parasitize citrus and to overcome citrus rootstock resistance. Banding patterns of molecular weight standards were fit best by an exponential decay function, and a predictive equation was used to estimate molecular weights (r² = 0.999). A band (131 kDa) that labeled with the lectin Concanavalin A (Con A) occurred in extracts from cuticles and egg shells of populations of Radopholus citrophilus that parasitize citrus. Wheat germ agglutin labeled a band (58 kDa) in aqueous homogenates of populations that reproduce in roots of citrus rootstock normally resistant to burrowing nematodes. The two sibling species R. citrophilus and R. similis were distinguished by a high molecular weight Con A-labeled band (608 kDa) from cuticle and egg shells. Probing blots with the lectin Limulus polyphemus agglutinin indicated that each population contained a band (12-16 kDa) specifically inhibited by the addition of 25 mM neuraminic acid, suggesting that glycoproteins with sialic acid moieties are present in burrowing nematodes.     

Ion exchange and affinity chromatography in the scaleup of the purification of alpha-galactosidase from soybean seeds

Soybeans (Glycine max) contain an alpha-galactosidase that makes up a small fraction of the total protein of the seed. The properties of this enzyme are of interest because of its potential to convert the galactooligosaccharides, stachyose and raffinose, in soybean meal to sugars digestible in the human gastro intestinal tract and thereby increase potential uses of this vegetable protein source in human and animal foods. Study of this enzyme required the isolation of milligram quantities of electrophoretically pure protein from ground soybeans and therefore, scaleup of laboratory procedures by a factor of 300 times. Large scale acid precipitation, ammonium sulfate precipitation, and centrifugal recovery of the precipitated protein allowed alpha-galactosidase to be isolated from 45.5 kg soybean meal containing 17.1 kg protein, to obtain an enzyme extract with a specific activity of 90 to 100. A novel combination of strong anion exchange and cation exchange chromatography followed by Concanavalin-A affinity chromatography with a methyl alpha-D mannoside gradient gave alpha-galactosidase with an average specific activity of 56,000. Ion exchange chromatography preceding Concanavalin-A affinity chromatography allowed elimination of a relatively costly melibiose affinity chromatography step (which followed the Concanavalin-A column In the laboratory procedure) thereby making scaleup practical.