Helix Pomatia Lectin Research Articles

Further analysis of predictive value of Helix pomatia lectin binding to primary breast cancer for axillary and internal mammary lymph node metastases

We investigated the relation between Helix pomatia (HPA) staining of primary breast cancer and the presence of axillary (AX) or internal mammary (IM) metastases, and evaluated its predictive value for AX or IM metastases in comparison with the use of clinical variables. There was a significant association between the HPA staining and AX or IM metastases. When HPA staining was regarded as an indicator of AX metastases, a diagnostic accuracy of 72%, a sensitivity of 69% and a specificity of 75% were achieved. As an indicator of IM metastases, these values were 64%, 76% and 62%, respectively. In predicting the presence of AX metastases using a discriminant function with clinical AX status, location of tumour and tumour size, diagnostic accuracy, sensitivity and specificity were 79%, 69% and 87%, respectively. In predicting the presence of IM metastases using the discriminant function with clinical AX status and tumour size, these values were 74%, 71% and 75%, respectively. Therefore, it was concluded that the HPA staining may be useful, but it was equivalent with the discriminant function with clinical variables in prediction of AX or IM metastases.

DNA ploidy and helix pomatia lectin binding as predictors of regional lymph node metastases and prognostic factors in breast cancer.

This study was designed to evaluate whether DNA ploidy and/or Helix pomatia lectin (HPA) staining would be useful for predicting regional lymph node metastases and patients' prognosis in 106 patients with invasive breast cancer. The combination of DNA ploidy and HPA staining correlated better with regional lymph node metastases than DNA aneuploidy or HPA staining alone. DNA ploidy and HPA staining in combination correlated strongly with overall and disease-free survival by univariate analysis. However, the prognostic significance of DNA ploidy and HPA staining in combination was lost in multivariate analysis when regional lymph node metastases were introduced into the models. This emphasized the relationship in survival between regional lymph node metastases and the combination of DNA ploidy and HPA staining. We therefore concluded that the combination of DNA ploidy and HPA staining might provide prognostic information for breast cancer patients in whom regional lymph node dissection has not been performed.

Markers of prognosis in breast cancer--the relationship between binding of the lectin HPA and histological grade, SPF, and ploidy.

Abnormal cellular glycosylation as demonstrated by the binding of a lectin from Helix pomatia (HPA) to paraffin-embedded sections has been shown in several studies to be associated with aggressive biological behaviour and poor long-term patient prognosis in breast cancer. This study aims to address the possibility that expression of the HPA binding ligand may be of prognostic significance through an association with increased cellular proliferation (as measured by S-phase fraction and histological grade), anaplasia (reflected in histological grade), or ploidy (DNA index). In a 24 year retrospective study, paraffin-embedded sections of 366 primary breast cancers were stained for binding of HPA. All tumours were assessed for histological grade. Flow cytometry was performed on all cases for which sufficient tumour tissue was available (358/366 cases) and S-phase fraction (SPF) and ploidy calculated. Data regarding patient age at diagnosis, nodal status, and tumour size were also recorded. Life table analyses revealed survival advantage for HPA 'non stainers' in comparison to 'stainers' (p < 0.001); for patients with tumours of low grade vs. high grade (p < 0.001); and for those with tumours of low SPF vs. high SPF (p < 0.001). No survival advantage was shown for those with diploid vs. aneuploid tumours (p = 0.17). No association was apparent between HPA binding and grade, SPF, or ploidy (Chi squared values not significant). This was confirmed by multivariate analysis in which nodal status, tumour size, and SPF were independently predictive of survival. There was no confounding effect of grade, SPF, or ploidy upon the correlation between survival and HPA binding. HPA was, however, not independently predictive owing to its strong association with nodal status. The results of this study suggest that the prognostic significance of altered glycosylation, as detected by HPA binding, is unlikely to be through an association with proliferative rate, degree of anaplasia, or cellular ploidy, but may rather be through a direct association with the presence of nodal metastases.

Impact of O-glycosylation on the function of human intestinal lactase-phlorizin hydrolase. Characterization of glycoforms varying in enzyme activity and localization of O-glycoside addition.

Lactase-phlorizin hydrolase (LPH) is an integral intestinal brush border membrane glycoprotein responsible for the hydrolysis of lactose, the primary carbohydrate in mammalian milk. To assess the role of N- and O-glycosylation on the function of LPH, lectin-binding experiments combined with enzymatic and chemical deglycosylation of purified LPH molecules were performed. These investigations provided evidence for the existence of two forms of brush border LPH, an N-glycosylated molecule (LPHN) and an N- and O-glycosylated molecule (LPHN/O). These two forms could be discriminated on the basis of (i) their binding capacity to Helix pomatia lectin, which has high specificity toward O-linked oligosaccharides, and (ii) their deglycosylation patterns with endo-beta-N-acetylglucosaminidase F/GF, O-glycanase, and trifluoromethanesulfonic acid. Interestingly, both forms have identical Km values (approximately 14 mM) when assayed with lactose, but hydrolyze this substrate at different rates. Thus, the N- and O-glycosylated form exhibits almost a 4-fold higher Vmax than that of the N-glycosylated enzyme (3.28 nM/min versus 0.90 nM/min) and is therefore enzymatically more active than the latter. Sequential affinity chromatography of glycopeptides derived from [3H]mannose-labeled LPHN and LPHN/O on lectin columns revealed similar patterns of N-linked glycosylation of both forms indicating that the presence of O-linked oligosaccharides did not affect or alter the processing of N-linked oligosaccharides. O-Linked glycosylation of LPH appears to occur in the Golgi apparatus, since the earliest detectable forms of LPH, the mannose-rich precursor (pro-LPH) is not O-glycosylated. In view of the fact that differentiation of intestinal crypt cells to mature epithelial cells is accompanied by significant phenotypical, morphological, and structural alterations, including changes in the levels of several Golgi glycosyl, -sialyl, galactosyl-, and N-acetylgalactosaminyltransferases, and since O-glycosylation is a Golgi event, we suggest that the generation of LPHN and LPHN/O is strongly linked to differentiation of intestinal cells. Finally, the variations in the enzymatic activity of the two forms propose a role for O-glycosylation in posttranslational regulation of LPH activity.

Mannose-induced dysmorphogenesis of metanephric kidney. Role of proteoglycans and adenosine triphosphate.

Because various fetal anomalies are seen in diabetic offspring, we examined the effects of sugars on proteoglycans (PGs): extracellular matrix (ECM) macromolecules modulating morphogenesis. 13-d-old mouse metanephric kidney explants were exposed to mannose for 7 d and labeled with [35S]sulfate, [35S]-methionine, or [3H]thymidine. Mannose exposure caused reduction in kidney size and disorganization of ureteric bud branches with inhibition of glomerulogenesis. Tissue autoradiographic and immunofluorescence studies indicated decreased expression of sulfated PGs in ECMs. Helix pomatia lectin binding to D-GalNAc residues of glomerular epithelial cells was also reduced. Biochemical studies revealed decreased synthesis of sulfated PGs. PGs were of lower molecular weight with reduced charge density and increased chondroitin/heparan sulfate ratio. Immunoprecipitation of [35S]methionine-labeled proteins confirmed the reduction of PG core peptides. Intracellular ATP levels were reduced. The addition of 0.1 mM ATP to culture media restored kidney size, the population of glomeruli, and the synthesis and characteristics of PGs to almost normal, with no detectable effect on the replication of cells as determined by [3H]thymidine incorporation. The effect of ATP could be partially blocked by the P2y-purinoreceptor, i.e., reactive blue-2. Data suggest that mannose causes energy depletion by cellular ATP consumption and thus selectively alters the synthesis of heavily glycosylated proteins with rapid turnover, such as PGs, resulting in renal dysmorphogenesis.

Quantitative changes of Ricinus communis agglutinin I and Helix pomatia lectin binding sites in the acrosome of rat spermatozoa during epididymal transit.

During passage through the epididymis, spermatozoa undergo a number of changes which result in their acquisition of fertility and motility. Some of the changes that occur include loss of the cytoplasmic droplet and changes in sperm morphology, metabolism and properties of the nucleus and plasma membrane. Changes have also been reported in the acrosomic system of mammalian spermatozoa during their transit through the epididymis. In the present study, the quantitative changes of the glycoconjugate content in the acrosome of rat spermatozoa were examined during their passage through the epididymis using lectin-colloidal gold cytochemistry. Various regions of the epididymis (initial segment, caput, corpus and cauda epididymidis) were fixed by perfusion with 1% or 2% glutaraldehyde buffered in sodium cacodylate (0.1 M), dehydrated in ethanol and embedded without osmication in Lowicryl K4M. Lectin-colloidal gold labeling was performed on thin sections using Ricinus communis agglutinin I (RCA I) or Helix pomatia lectin (HPL) to detect D-galactose- and N-acetyl-D-galactosamine-containing glycoconjugates, respectively. The labeling density over the acrosome of the acrosomic system was evaluated as the number of gold particles per microns 2 of profile area using a Zeiss MOP-3 image analyzer. The overall mean labeling densities over the acrosome of spermatozoa for each lectin was estimated from 4 rats and over the four distinct epididymal regions. The mean labeling density of the acrosome with RCA I and HPL showed a similar pattern along the epididymis, although RCA I revealed approximately twice as many gold particles per epididymal region. In either case, there was a significant decrease in the labeling density of the acrosome of spermatozoa between the initial segment or caput epididymidis and cauda epididymidis (p less than 0.01). A similar decrease was also noted between the initial segment and corpus epididymidis (p less than 0.01). No change was found between the initial segment and caput epididymidis. Controls showed a virtual absence of labeling. These results suggest that in addition to a multitude of changes occurring to spermatozoa during epididymal transit, there are also significant quantitative changes in the glycoconjugate content within the acrosome.

Duck lymphocytes—V. Transformation responses to phorbol ester and calcium ionophore

1. Lymphocytes purified from duck blood and spleen were cultured in the presence of phorbol 12-myristate 13-acetate (PMA) and the calcium ionophore A23187. Stimulation was assessed by the incorporation of [ 3H]thymidine after 3 days' culture. 1. 2. PMA stimulated over a wide range of concentrations, with maximum stimulation at final concentrations of 5 × 10 −7−5 × 10 −8 M/litre. A23187 was effective in the range 5 × 10 −6−5 × 10 −7 M/litre and also, in some experiments using spleen lymphocytes, at 5 × 10 −11−5 × 10 −12 M litre . 2. 3. Synergism was observed between PMA and A23187, the pattern depending on the concentrations of these reagents employed. Synergism was also observed between PMA and suboptimum concentrations of phytohaemagglutinin (PHA), wheat germ agglutinin (WGA), pokeweed mitogen (PWM) and Bandeiraea simplicifolia seed extract (BSS), but not with concanavalin A (Con A), lentil lectin (LL) or Helix pomatia lectin (HP). Similarly, synergism occurred between A23187 and WGA or PWM, but not with PHA, BSS, Con A, LL or HP. 3. 4. Mitomycin C and cycloheximide inhibited the response of duck lymphocytes to PMA, A23187 and lectins. Cyclosporin A inhibited responses to lectins but not to PMA or A23187. Neither hydrocortisone nor indomethacin inhibited responses to lectins, PMA or A23187. 4. 5. These results indicate that activation of duck lymphocytes occurs by virtue of similar intracellular messenger pathways to those operating in mammalian lymphocytes.

Discrepancy between bone resorption assayed by urinary excretion of pyridinoline crosslinks and of 3H-tetracycline in the rat

Hamster submaxillary glycoprotein (HSM), one of the simplest glycoproteins among mammalian salivary mucins, is composed of approximately equivalent amounts of protein, hexosamine and sialic acid. The Thr and Ser residues in the protein core account for more than half of all of the amino acid residues, while Lys, Glu, Pro and Ala are the major components of the remaining portion of amino acids. The carbohydrate side chains of this mucous glycoprotein have mainly the NeuAc-GalNAc-(sialyl-Tn) sequence (HSM), and those of the desialylated product (HSM-Tn) are almost exclusively unsubstituted GalNAc residues (Tn determinants). The binding properties of sialyl-Tn (HSM) and asialo-HSM (HSM-Tn) glycoproteins were tested by precipitin assay with Gal, GalNAc and GlcNAc specific lectins. The HSM-Tn completely precipitated Vicia villosa (WL both B4 and mixture of A and B), Maclura pomifera (MPL), and Artocarpus integrifolia (Jacalin) lectins; less than 2 μg of HSM-Tn were required for precipitating 50% of 5.0–6.3 μg lectin nitrogen added. HSM-Tn also reacted well with Helix pomatia lectin (HPL), Wistaria floribunda lectin (WFL) and Abrus precatorius agglutinin (APA) and precipitated in each case over 81% of the lectin nitrogen added. The reactivity of HSM-Tn with other lectins (Ricinus communis, RCA1; Dolichol biflorus, DBL; Viscum album, ML-I; Arachis hypogaea, PNA, and Triticum vulgaris, WGA) was weak or negligible. The activity of sialyl-Tn (HSM) was more restricted; HSM reacted well with Jacalin, moderately with MPL and VVL-B4, but was inactive or only weakly active with the other lectins used. These findings indicate that HSM and its desialylated product (HSM-Tn) are highly useful reagents for the differentiation of Tn and T/Gal specific lectins and for anti-T, Tn and Af monoclonal antibodies.

Helix Pomatia Lectin Binding Pattern of Brain Metastases Originating from Breast Cancers

The glycosylation pattern of brain metastases originating from primary breast carcinomas was investigated using Helix pomatia agglutinin (HPA), a lectin which recognises N-acetyl-galactosamine (GalNac) residues of glycoconjugates. In a previous retrospective study this lectin was shown to label only those primary breast cancers that metastasised. To explore this as a clinical marker of metastatic breast cancer behaviour it is necessary to analyse the HPA binding pattern of metastases to see if this differs from primary cancers. To test the question if brain metastases commitently retain this trait of metastatic primary tumors, we studied Helix pomatia binding pattern of brain metastases removed by surgical excision and immediately fixed and processed. Brain metastases from 16 patients with breast cancer were obtained, 13/16 metastases showed binding to the cytoplasm in the majority of cancer cells, 3/16 did not show binding to cancer cells. Normal adjacent brain showed binding to red blood cells, to capillary endothelium of several biopsies and to rare neurones; this binding did not relate to cancer cell binding. Therefore we conclude that HPA is a relatively stable marker for metastasizing breast cancer cells.

Lectin coated piezoelectric crystal biosensors

The lectins Helix pomatia and Ptilota plumosa have been covalently immobilised on the surfaces of silver electroded AT-cut quartz crystals to form sugar and erythrocyte specific sensors. Using fully immersed crystals the dissociation constant for the Helix pomatia — n-acetyl d-galactosamine complex has been found to be typically 1.2 mM. A microdelivery mode has also been investigated and using this mode the dissociation constant for Ptilota plumosa with d-galactose was found to be ∼7 mM. The lower detection limit for erythrocytes was of the order of 10 3 cells.

Agglutination of Bordetella species by lectins

Phase I cells of Bordetella pertussis but not those of B. parapertussis, B. bronchiseptica or B. avium were agglutinated by Limulus polyphemus lectin. Most strains of B. pertussis but not those of the other species were also agglutinated by Helix pomatia lectin. In precipitation reactions between lectins and purified Bordetella lipopolysaccharide (LPS) preparations a similar pattern occured. Lectin agglutination provides a rapid presumptive method for the differentiation of B. pertussis from B. parapertussis and other Bordetella species.

Early increase in lymphocyte cyclic nucleotide phosphodiesterase activity upon mitogenic activation of human peripheral blood mononuclear cells.

Cyclic nucleotide phosphodiesterase activity of human peripheral blood mononuclear cells was significantly increased following a short (30 min) incubation with the mitogenic lectin Concanavalin A. Con A stimulated phosphodiesterase activity to the same extent whatever the subcellular compartment (homogenate, cytosol or pellet). Further separation of the Con A-activated mononuclear cells into lymphocyte-enriched and monocyte-enriched populations showed that the Con A-induced increase of phosphodiesterase activity exclusively affected the lymphocyte-enriched population. In lymphocytes, cyclic GMP phosphodiesterase activity was more importantly enhanced by Con A (+275%) than cyclic AMP phosphodiesterase activity (+75%). The increase of both activities occurred as early as from 10 min of Con A incubation and proved to be maximal with Con A doses of 2.5 and 5 micrograms per 10(6) cells, lower and higher doses being less effective. Inhibition experiments with reference inhibitors suggested that, among the high affinity phosphodiesterase isoforms, the cyclic GMP-inhibited enzyme might be more selectively enhanced by Con A than the cyclic AMP-specific, Rolipram-sensitive one. The non-mitogenic lectin Helix pomatia hemagglutinin, was not able to enhance cyclic nucleotide phosphodiesterase activity of human mononuclear cells whereas anti-CD3 monoclonal antibody, although being less effective than Con A, exhibited a significant stimulatory effect. Putting together these results suggest that the early increase in phosphodiesterase activity might be a normal step involved in the mitogenic activation of human lymphocyte.

The hamster transferrin receptor contains Ser/Thr-linked oligosaccharides: use of a lectin-resistant CHO cell line to identify glycoproteins containing these linkages

We recently reported that the transferrin receptor (TfR) contains O-linked GalNAc residues [1]. To investigate whether this modification is shared by transferrin receptors in other mammals, we investigated the glycosylation of TfR in hamster cells. To facilitate our analysis the lectin-resistant Chinese hamster ovary (CHO) cell line Lec8 was used. These cells are unable to galactosylate glycoproteins, resulting in truncation of the Ser/Thr-linked oligosaccharides to a single residue of terminal α-linked GalNAc. This structure is bound with high affinity by the lectin Helix pomatia agglutinin (HPA). The TfR was affinity purified from Lec8 cells metabolically radiolabeled with [3H]glucosamine and the receptor was found to bind tightly to HPA-Sepharose. Treatment of the purified TfR with mild alkalne/borohydride released [3H]GalNAcitol, demonstrating the presence of O-linked GalNAc. We also found that many other unidentified [3H]glucosamine-labeled glycoproteins from Lec8 cells were bound by HPA-Sepharose. The bound and unbound glycoproteins were separated by SDS/PAGE and individual species were selected for treatment with mild base/borohydride. Treatment of glycoproteins bound by HPA, but not those unbound, resulted in the release of [3H]GalNAcitol. These studies demonstrate both that the hamster TfR contains O-linked oligosaccharides and that this approach may have general utility for identifying the presence of these oligosaccharides in other glycoproteins.

Presence of terminal N-acetylgalactosamine residues in subregions of the endoplasmic reticulum is influenced by cell differentiation in culture.

Using Helix pomatia lectin as a specific probe for terminal, nonreducing N-acetylgalactosamine residues, glycoprotein precursors bearing newly initiated O-linked oligosaccharides have been localized in the lumen of the endoplasmic reticulum and cis-Golgi cisternae. This pattern contrasts with the detection of the terminal disaccharide galactose beta-1,3-N-acetylgalactosamine by Arachis hypogaea lectin in middle and trans-Golgi compartments, which are considered elongation sites for O-glycosylation. Distribution of H. pomatia ligands in the endoplasmic reticulum is confined to specialized regions or subcompartments in both human colonic adenocarcinoma cells and cultured chicken chondrocytes. Since in cartilage, chondrocytes contain H. pomatia-binding sites exclusively concentrated in cis-Golgi cisternae, primary cultures of this cell type have been used to study those conditions that promote initiation of O-glycosylation in the endoplasmic reticulum. A correlation has been found between the age of the culture and the extent of reactivity of the endoplasmic reticulum with either H. pomatia lectin or antibody against the sequence GalNAc alpha-serine/threonine (Tn antigen). Cells showing an extensive reaction are not hindered in their secretory activity and still maintain the chondrocyte phenotype. Taken together the results suggest that the intracellular distribution of the glycosylation enzymes is not only cell type-specific as previously shown (Roth, J., Taatjes, D. J., Weinstein, J., Paulson, J. C., Greenwell, P., and Watkins, W. M. (1986) J. Biol. Chem. 261, 14307-14312) but it might also vary depending on the stage of cell differentiation.

The adsorption of bacteria to immobilized lectins

The agglutination of a selection of bacteria by some lectins was examined. The lectin from Codium fragile agglutinated seven strains of Salmonella typhimurium. The lectin from Helix pomatia agglutinated eight of 12 strains of Listeria monocytogenes and a further two strains gave a weak agglutination reaction. Helix pomatia lectin conjugated to magnetic microspheres enabled the adsorption of L. monocytogenes from suspension with subsequent elution by the competing ligand N-acetyl galactosamine. Affinity chromatography of a suspension of L. monocytogenes through a column of H. pomatia lectin immobilized on agarose, also adsorbed cells and enabled subsequent elution with N-acetyl galactosamine. The column technique enabled the more rapid adsorption of bacteria perhaps because of improved interactions between bacteria and immobilized lectin.

Prediction of lymph node involvement in breast cancer by detection of altered glycosylation in the primary tumour

Axillary lymph node metastases at the time of diagnosis of breast cancer is the most accurate predictor of long-term prognosis. However, in patients treated by conservative surgery lymph node status often remains unknown. We have investigated the relation between changes in glycosylation of primary breast cancer cells, as judged by lectin binding, and the presence of axillary lymph node metastases. In a 24-year retrospective study, paraffin-embedded sections of 373 primary breast cancers were stained for the binding of Helix pomatialectin (HPA). There was a strong association between HPA binding and presence of lymph node metastases, but no association with tumour size, histological grade, S-phase fraction, or patient age at diagnosis. This relation was confirmed by multiple regression analysis (in both survival and relapse free survival models) in which the prognostic significance of HPA binding was lost once nodal status had been introduced into the models. Life tables calculated for lymph-node positive versus lymph-node negative and HPA staining versus non-staining patients were almost identical over 15 years of follow-up. We propose that H PA recognises a glycoprotein that is associated with metastasis (to axillary lymph nodes and elsewhere) and poor prognosis in breast cancer. HPA binding to paraffin sections of primary tumour could aid difficult treatment decisions by providing an additional assessment of staging and likely long-term patient prognosis.

Coding and classification of d-galactose, N-acetyl- d-galactosamine, and β- d-Gal p-[1→3(4)]-β- d-Glc pNAc, specificities of applied lectins

Grouping of lectin-binding properties, based on determinant structure rather than monosaccharide-inhibition pattern, should facilitate the selection of lectins as structural probes for glycans, as well as for the interpretation of the distribution and the properties of the carbohydrate chains on the cell surface. Based on the binding specificities studied with glycan by precipitin-inhibition, competitive-binding, and hemagglutinin-inhibition assays, twenty d-galactose-or N-acetyl- d-galactosamine-(or both)-specific lectins have been divided into six classes according to their specificity for a disaccharide unit, as all or part of the determinants, and the α- d-Gal pNAc-(1→3)-Ser(Thr) unit of the glycopeptide chain. A scheme of classification is shown as follows: (a) F-specific lectins [α- dGal pNAc-(1→3)- d-GalNAc, Forssman specific disaccharide]: Dolichos biflorus (DBL), Helix pomatia (HPL), hog peanut (ABL, Amphicarpaea bracteata), and Wistaria floribunda (WFL) lectins. (b) A-specific lectins [α- d-Gal pNAc-(1→3)- d-Gal blood group A-specific disaccharide]: Griffonia (Bondeiraea) simplicifolia-A 4 (GSI-A 4), lima bean (LBL), soy bean(SBL), Vicia villosa (VVL), Wistaria floribunda (WFL), Dolichos biflorus (DBL), and Helix pomatia (HPL) lectins. (c) Tn-specific lectins [α- d-Gal pNAc-(1→3)-Ser(Thr) of the protein core]: Vicia villosa B 4 (VVL-B 4), Salvia sclarea (SSL), Machura pomifera (MPL), Bauhinia purpurea alba (BPL), HPL, and WFL, lectins. (d) T-specific lectins [β- d-Gal p-(1→3)- d-GalNAc, the mucin-type sugar sequences on human erythrocyte membrane and T antigen, or the terminal, nonreducing disaccharide end-groups of the gangliosides]: Peanut (PNA), Bauhinia purpurea alba (BPL), Machura pomifera (MPL), Sophora japonica (SJL), Artocarpus integrifolia (Jacalin, AIL), and Artocarpus lakoocha (Artocarpin) lectins. (e) Type I and II specific lectins [β- d-Gal p-(1→3 or 4)- d-GlCNAc, the disaccharide residues at the nonreducing end of the carbohydrate chains derived from either N- or O-glycans]: Ricinus communis agglutinin (RCAl), Datura stramonium (TAL, Thorn apple), Erythrina cristagalli (ECL, Coral tree), and Geodia cydonium (GCL), lectins. (f) B-specific lectin [α- d-Gal p-(1→3)-β- d-Gal p, human blood group B-specific disaccharide]: Griffonia (Bandeiraea) simplicifolia B 4 (GSI-B 4) lectin. Many other GalNAc- or Gal-(or both)-specific lectins that can be used as tools are also described.

Defining the Specificity of Anti‐A/B IgM/G Antibodies with Different Antigens and Lectins1

The specificity of anti-histo blood group A/B antibodies is defined by the antigen used for their production and selection (in the case of mouse monoclonal antibodies, mAbs) or by the antigens present on various intestinal bacteria (for polyclonal human IgM and/or IgG, phAbs). Absorption experiments with red blood cells and free antigen have been used earlier to define specificity; here we use A/B substances of human, animal and synthetic origin as well as known reactivities of mAbs or lectins as tools to further characterize epitope properties of the A/B antigens. The signal response patterns obtained with identical phAbs anti-A/B IgM/G on three different antigens are superposable. In contrast, commercially available mAb anti-A/B IgM, when tested on the same antigens, revealed different response curves. The chemical specificity of lectins for distinct monosaccharides in terminal position was exploited to delineate the specificity of mouse ascites anti-A IgG1 and of phAbs anti-A/B IgM/G. Helix pomatia lectin inhibited the access of MB9 to porcine A almost completely whereas binding capacities of human anti-A IgM/G were inhibited by 50 and 62%, respectively. In similar experiments Bandeiraea simplicifolia lectin was seen to inhibit access of anti-B IgM/G to horse B by 34 and 58%, respectively. No inhibition was seen with lectin from Ulex europaeus or concanavalin A on plates coated with the three different A or B substances. Thin-layer chromatography (TLC) of antigens revealed one spot for the synthetic trisaccharides, whereas the human and animal blood group substances showed 2 and 3 spots, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Wie dick ist die Glykokalyx des menschlichen Erythrocyten?

The aim of the experiment was to determine the thickness of the glycocalyx of human erythrocytes of the blood group A. For that purpose, the distance between the electron dense contrasted lipid layer of the plasmalemma and gold sol particles loaded with Helix pomatia lectin was determined. The mean thickness of the glycocalyx under this conditions was 5.9 nm.

Binding of Platelet Agglutinating Protein p 37 from the Plasma of a Patient with Thrombotic Thrombocytopenic Purpura to Human Platelets

We have previously reported the purification of a 37 kDa platelet agglutinating protein (PAP p37) from the plasma of a patient with thrombotic thrombocytopenic purpura (TTP) that was shown to be present in a subset of TTP patients. To gain further insight into the interaction between PAP p37 and platelets, we have studied the properties of PAP p37 binding to platelets. Washed human platelets from two normal donors and two TTP patients after recovery were used for the experiments. The PAP p37 binding to platelets was specific, concentration dependent and saturable. Scatchard analysis demonstrated about 20,564-27,090 PAP p37 binding sites per platelet. Stimulation of platelets with thrombin or ADP did not have any significant effect on its binding. Thiol- and serine-specific protease inhibitors did not inhibit PAP p37 binding to the platelets. Sugars such as glucose, fructose, mannose, and sialic acid, at 40 mM, inhibited its binding to platelets by 44%, 73%, 79%, and 91% respectively, but galactose and amino sugars did not have any significant effect. At 250 micrograms/ml, Concanavalin-A inhibited 42% of binding, but other lectins, such as phytohemagglutinin-P, potato lectin and helix pomatia lectin (snail), did not. Pre-incubation of 125I-PAP p37 with the adult human IgG, decreased its binding to the platelets. The monoclonal antibodies to GP Ib (6D1) and GP IIb-IIIa complex (10E5) did not inhibit the binding of 125I-PAP p37 to platelets. Fibrinogen and von Willebrand factor did not affect the binding either. These results suggest that PAP p37 binds to platelets on the sites other than GP Ib or GP IIb-IIIa complex.