Kenneth D. Hapner

Some phytotoxic glycopeptides from Ceratocystis ulmi, the Dutch Elm Disease pathogen

Ceratocystis ulmi, the causal agent of Dutch Elm Disease, produces phytotoxic glycopeptides in culture. A mixture of phytotoxic glycopeptides has been prepared by affinity chromatography on a concanavalin A-Sepharose column and collectively they have been termed the toxin. The polydisperse component that makes up the majority of toxin (80%) by weight has a molecular weight of about 2.7.10(5). The large molecular weight component (less than 5%) elutes at the void volume of a Bio-Gel A 50 m column. The other component (15%) appears as a trailing peak on the edge of the major component and has an approximate molecular weight of 7.10(4). The toxin is composed of 83% sugar residues, primarily rhamnose and mannose, and 7% amino acid residues. Methylation analysis coupled with mild acid hydrolysis indicates that the backbone of the polysaccharide portion of the toxin is composed of alpha -1,6-linked mannosyl residues with a 3-linked terminal rhamnosyl residue linked to C-3 of almost every mannosyl residue. The carbohydrate portion of the molecule is linked to the peptide via O-glycosidic linkages to both threonyl and seryl residues. All three components of the toxin are capable of causing wilt in stem cuttings of American elm.

Preparation and properties of haemagglutinin from haemolymph of acrididae (Grasshoppers)

Abstract The proteinaceous haemagglutinin (lectin) present in the haemolymph of Melanoplus sanguinipes (F) has been isolated and biochemically characterized. The protein was purified to homogeneity by affinity chromatography on a column of Sepharose-galactose. The purified haemagglutinin accounted for all observed haemolymphatic haemagglutination activity. Gel filtration and electrophoresis showed the haemagglutinin to be a 600–700,000 mol. wt noncovalent aggregate of 70,000 mol. wt subunits. The 70,000 subunit contained two disulfide-linked polypeptide chains of mol. wt 40,000 and 28,000, respectively. The purified haemagglutinin contained all the common amino acids and had highest amounts of acidic residues and least amounts of methionine and glucosamine. Haemagglutination activity was stable at −20°C in the presence of d -galactose but was destroyed by treatment of the haemagglutinin with trypsin, heat or EDTA. Haemagglutination inhibition studies showed that low concentrations (≤5 mM) of either d -galactosidic or d -glucosidic carbohydrates were bound by the haemagglutinin and inhibited agglutination of human asialo-erythrocytes. The purified haemagglutinin was antigenic in rabbits. Comparative experiments showed that the haemagglutinin from M. differentialis (Thomas) was identical to that from M. sanguinipes.

Haemagglutinin activity in the haemolymph of Teleogryllus commodus (Walker)

Abstract Strong haemagglutinin activity (titre 512) toward vertebrate erythrocytes is present in the haemolymph of both sexes of the adult cricket Teleogryllus commodus (Walker). Affinity chromatography on Sepharose-fetuin was used to concentrate the activity 213-fold. The haemagglutinin is a high molecular weight, water-insoluble protein which decreases in stability during purification. Purified activity is lost or diminished upon lyophilization, ultrafiltration, dialysis, heating, freezing, EDTA or trypsin treatment. In contrast, frozen crude haemolymph retains full activity for months. Insolubilized concanavalin A irreversibly absorbs the haemagglutinin activity independent of competing ligand. Human (ABO), chicken, pigeon and rat erythrocytes are strongly agglutinated, while sheep, cat and monkey red cells are weakly agglutinated. Horse and rabbit cells are not agglutinated by T. commodus haemagglutinin. Unpurified haemagglutinin activity is inhibited by low concentrations of fetuin and N-acetyl neuraminic acid. Purified activity is additionally inhibited by N- acetyl- d -glucosamine and N- acetyl- d -galactosamine . Bovine serum albumin, trehalose, sucrose, d -glucuronic acid and several other simple carbohydrates are non-inhibitory.

Haemagglutinin activity in Acrididae (grasshopper) haemolymph

Abstract The haemolymph of Acrididae causes haemagglutination of human and animal erythrocytes. Thirteen of seventeen species tested had detectable activity and gave agglutination titres in the range 2–64, Melanoplus bivittatus, and M. sanguinipes showed greatest activity. Haemagglutinin activity is continuously present in male and female insects from 4th instar and throughout adulthood. Females contain slightly more activity than do males. M. sanguinipes haemolymph agglutinates rabbit, calf, human (all ABO types) guinea pig, mouse, chicken, cat, pig and sheep erythrocytes. Rabbit red cells are agglutinated most strongly and sheep and chicken cells least. M. sanguinipes haemolymph also agglutinates the protozoan Nosema locustae, a natural grasshopper pathogen. Preabsorption of haemolymph with different erythrocyte types selectively removes haemagglutinin activity suggesting the presence of multiple or heteroagglutinins. M. sanguinipes haemagglutinin is inhibited by glycoproteins, simple carbohydrates and carbohydrate derivatives. The inhibitory pattern is complex and among the sugars tested only mannose and derivatives of mannose are exclusively non-inhibitory. Haemolymph haemagglutinin activity is destroyed by heat and EDTA. It is totally precipitated by dialysis against water and may be partially recovered in phosphate or Tris buffer. Activity is stable in frozen haemolymph.

Transfer of toxin susceptibility to plant protoplasts via the Helminthosporoside binding protein of sugarcane

Abstract The eyespot disease of sugarcane is caused by Helminthosporium sacchari . Helminthosporoside, a host-specific toxin produced by H. sacchari , is essential for the pathogenicity of this fungus. The presence of the helminthosporoside-binding protein in sugarcane likewise appears to be essential for susceptibility to the toxin. The results of this report show that leaf cell protoplasts of tobacco and toxin resistant sugarcane effectively adsorbed the toxin-binding protein derived from membranes of susceptible sugarcane. These protoplasts then became susceptible to the helminthosporoside. They also functioned to takeup raffinose, a trisaccharide structurally related to the toxin. Tobacco protoplasts were treated with [ 14 C] - binding protein, ruptured, and fractionated on a sucrose density gradient column. A peak of radioactivity was associated with the enriched plasma membrane fraction. The results support the hypothesis that the binding protein is the primary recognition site governing susceptibility of sugarcane to helminthosporoside.

Grasshopper haemagglutinin: immunochemical localization in haemocytes and investigation of opsonic properties

The haemagglutinin (lectin) present in the haemolymph of the grasshoppers Melanoplus differentialis and M. sanguinipes has been localized to the haemocytes, and the in vitro opsonic capacity of purified grasshopper haemagglutinin has been investigated. Immunocytochemical labelling of fixed haemocyte monolayers with monoclonal antibody revealed that agglutinin is present in approx. 25% of the granular cells. Plasmatocytes (phagocytes) do not contain agglutinin. Potential haemocytic membrane receptors for haemagglutinin were indicated by the binding of fluorescein-conjugated peanut agglutinin to granular haemocytes. However, comparable binding of purified grasshopper haemagglutinin could not be demonstrated by immunochemical labelling. Haemocytes, in vitro, interact with asialo human erythrocytes, Bacillus thuringiensis bacteria (vegetative cells) and spores of Nosema locustae. No increase in binding or phagocytic (opsonic) activity was observed when these three foreign particle types were incubated in either grasshopper serum or purified haemagglutinin prior to overlayering the haemocyte monolayers, or when monolayers were exposed to purified agglutinin prior to overlayering with foreign partices. The results indicate that although grasshopper haemolymphatic haemagglutinin is present in a subpopulation of grasshopper haemocytes, it does not contribute to the in vitro association of haemocytes with these foreign particles.

Agglutinin mediated opsonization of fungal blastospores in Melanoplus differentialis (Insecta)

Abstract Agglutinin from hemolymph of the grasshopper Melanoplus differentialis is prepared by affinity absorption to d -galactose-Sepharose followed by elution with EDTA into buffer containing CaCl 2 . The agglutinin enhances the association of fungal blastospores from Beauvaria bassiana with hemocyte monolayers four- to sixfold. Blastospores from Nomuraea rileyi are not opsonized. The opsonic stimulation occurs when either blastospores or the monolayer is treated with agglutinin prior to incubation. The opsonic activity is greatly reduced (approx. 76%) by α- d -methyl galactoside, palatinose and EDTA, all inhibitors of agglutinin carbohydrate binding, and less so (approx. 39%) by agglutinin-specific polyclonal antibody. Trehalose shows no inhibitory effect. In vivo clearance experiments with live insects show that injected B. bassiana blastospores treated with agglutinin are removed from the hemolymph 2.2-fold faster than those not treated. It is concluded that grasshopper agglutinin is an opsonin toward B. bassiana blastospores and acts as a molecular bridge between the fungal cell and the hemocyte. Grasshopper agglutinin appears to have a role in the immuno recognition of this fungus by cells functioning in defense against invading pathogens.

Haemagglutinin activity in the haemolymph of individual Acrididae (grasshopper) specimens

Abstract Twenty-four individual grasshopper specimens representing four Melanoplus spp. contained similar broad-spectrum haemolymphatic haemagglutinin. The agglutinin activity showed highest titre toward human ABO and rabbit cells among nine types of erythrocytes tested. Titre values differed between individual insects but agglutination specificity toward different erythrocytes was similar. Agglutination of type-O red cells by individual grasshopper haemolymph was inhibited by 34 of 41 tested carbohydrates, carbohydrate derivatives, alcohols and chelating agents. Individual insects showed similar patterns of haemagglutination inhibition. Non-inhibitory compounds were mannose and mannose derivatives (excepting N -acetylneuraminate), several glucose derivatives, amino sugars and ethanol. The observations indicated that haemolymph from an individual grasshopper contained complex heteroagglutinin activity similar to that found in haemolymph pooled from several insects. Determination of minimal effective inhibitor concentrations confirmed the presence of heteroagglutinin activity primarily directed toward galactose and glucose and related α-linked glycosidic derivatives.

Isolation and properties of a lectin from sainfoin (Onobrychis vichfolia, Scop.)

A glycoprotein capable of binding simple carbohydrates and causing hemagglutination has been isolated from seeds of the legume plant sainfoin (Onobrychis viciifolia, Scop. var Eski). The phytolectin was prepared by affinity chromatography of pH 7.0 sodium phosphate extracts on columns of Sepharose-4B containing covalently attached D-mannose. Molecular weight determinations showed the lectin to be a dimer consisting of 26 000 dalton, non-covalently associated monomers. Amino acid analyses indicated high amounts of aspartate, glutamate, threonine and serine which accounted for 41% of all amino acids. One residue of cysteine was present and methionine was totally absent. The lectin contained 2.6% (w/w) neutral carbohydrate and two residues of N-acetylglucosamine/monomer. Carbohydrate-binding specificity was directed toward D-mannose and D-glucose and their alpha-glycosidic derivatives. The purified protein agglutinated cat erythrocytes at 5 micrograms/ml. Antiserum to seed lectin showed a single common immunoprecipitation line in Ouchterlony double diffusion against both the seed and root antigen. Lectin isolated from sainfoin seedling roots showed molecular weight, amino acid and carbohydrate values similar to that of the seed lectin.

Site of synthesis of the haemolymph agglutinin of Melanoplus differentialis (Acrididae: Orthoptera)

Abstract Several species of Melanoplus grasshoppers contain a haemolymph agglutinin exhibiting both d -galactosidic and d -glucosidic binding specificities. With the exception of the flesh fly Sarcophaga peregrina , the site(s) of synthesis of insect agglutinins is uncertain. Primary cultures of M. differentialis fat body, haemocytes, ovary and testes were established. An ELISA assay demonstrated that the fat body, ovary and testes tissues released significant amounts of agglutinin into the culture medium over a 4-day period. Immunoprecipitated agglutinin from the culture medium was radiolabelled when the cultures were metabolically labelled with [ 35 S] methionine. Haemocyte culture medium and cell lysates of the 4 tissues did not contain detectable amounts of radiolabelled agglutinin. Attempts to alter the kinetics of agglutinin release from fat body cultures through addition of microbial cell wall components (lipopolysaccharide, peptidoglycan, zymosan, or laminarin) were unsuccessful.