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H J Gabius

Publications and source records attributed to H J Gabius.

At least 19 recordsLinked to original sources
PubMed1992

Ligand binding characteristics of the major mistletoe lectin.

Carbohydrate binding specificity of the galactose-specific, major lectin of mistletoe extract (ML-1) was studied by an inhibition assay using monosaccharides, monosaccharide derivatives, disaccharides, and compounds containing multiple galactosyl terminals. The results indicate that 1) both alpha- and beta-galactosyl residues are recognized equally well; 2) each of the hydroxyl groups of galactose contributes to varying degrees to the binding process, the 4-OH being the most important and the 6-OH the least important hydroxyl group; 3) disaccharide sequences of Gal beta 2Gal and Gal beta 3Gal have much higher affinity than galactose, whereas affinity of all other Gal-disaccharides is only slightly better than galactose; 4) macromolecular ligands having 10 or more terminal galactosyl residues have 500-fold higher affinity than Gal; and 5) a group on ML-1 with pK alpha of 4.8 appears to be involved in the binding of ligand.

Binding Sites↗
PubMed1992

Behavior of lymphocyte subsets and expression of activation markers in response to immunotherapy with galactoside-specific lectin from mistletoe in breast cancer patients.

Cellular aspects of the immunomodulating activity of the galactoside-specific lectin from mistletoe (ML-1) were investigated in 10 cancer patients. Regular subcutaneous injections (4 weeks) of the optimal dosis of ML-1 (1 ng per kg body weight, twice a week) yielded notable increases in the apparent numbers of certain lymphocyte subsets [pan T cells; helper T cells; natural killer (NK) cells] which are generally believed to be involved in antitumor immunity. Moreover, ML-1 administration resulted in an increased level of expression of interleukin (IL)2 receptors on lymphatic cells, an indicator of cellular activation. In vitro, the exposure of human lymphocytes to ML-1 resulted in an enhanced expression of receptors for IL-2 (T cells) and HLA-DQ (B cells), which similarly substantiated the capacity of ML-1 to affect immunological parameters within the host defense system. Thorough clinical trials are now required to assess any impact of the application of the lectin on the course of the disease.

Biomarkers↗
PubMed1992

Detection and quantitation of cell-surface sugar receptor(s) of Leishmania donovani by application of neoglycoenzymes.

Promastigote culture forms of the log growth phase of Leishmania donovani stock LRC L 51 were investigated for expression of cell-surface carbohydrate-binding sites using 15 types of a chemically glycosylated enzyme termed neoglycoenzyme. Carbohydrate conjugation and coupling yield were kept constant to ensure that the type of carbohydrate moiety was the only variable feature of the applied tools. Para-aminophenyl derivatives of the following carbohydrate residues were used for the glycosylation of beta-galactosidase from Escherichia coli: beta-D-lactose, beta-D-thiogalactose, alpha-D-mannose, alpha-L-rhamnose, alpha-D-N-acetylgalactosamine, beta-D-N-acetylgalactosamine, beta-D-N-acetylglucosamine, the alpha- and beta-glucosides maltose and cellobiose, beta-D-xylose, alpha-D-mannose-6-phosphate, the alpha-galactoside melibiose, alpha-L-fucose, and beta-D-glucuronic acid as well as sialic acid. Only melibiose, fucose, and glucuronic acid showed no binding affinity for the cultured flagellates; this served as an internal control reaction to exclude any binding to the linker group. This result demonstrates that many but not all sugar types can be recognized by appropriate receptor structure(s) on the surface of the promastigote Leishmania. Transformation of the binding data for neoglycoenzymes exposing lactose, mannose, rhamnose, and N-acetylated hexose residues, which was carried out to obtain the dissociation constants and to estimate the number of binding sites at saturation, revealed KD values of around 100 mM and around 10(4) binding sites for the polyvalent ligands.

Animals↗
PubMed1992

Analysis of tumour necrosis factor alpha-specific, lactose-specific and mistletoe lectin-specific binding sites in human lung carcinomas by labelled ligands.

Receptor sites can be visualized by labelled ligands as an alternative to receptor-specific antibodies, as substantiated for two different receptor classes. Recombinant tumour necrosis factor alpha (TNF) was biotinylated via amino-groups and the resultant probe was applied to formalin-fixed, paraffin-embedded tissue sections of 94 primary bronchial carcinomas and to normal peripheral lung parenchyma. In addition, monoclonal antibodies specific for neuron-specific enolase (NSE) and TNF itself were used. The biotinylated beta-galactoside-specific mistletoe lectin, which exhibits dose-dependent immunomodulatory and toxic potency, and two probes that specifically detect certain types of sugar receptors were employed to illustrate further the feasibility of using ligands for receptor localisation. The tumours comprised 62 small cell lung carcinomas, 10 epidermoid carcinomas, 11 adenocarcinomas and 11 large cell anaplastic carcinomas. Expression of TNF-binding sites was found in 39 of the small cell lung carcinomas and in 13 of the non-small cell lung carcinomas. Binding capacity for the TNF-specific antibody was seen in similar proportions of small cell lung carcinomas and of non-small cell lung carcinomas. None of the normal lung parenchymas revealed significant staining. Binding capacities to mistletoe lectin were seen in all normal lung parenchymas and in nearly all cases of adenocarcinoma (10/11). A correlation between the expression of NSE and the binding capacities to TNF was detected. Endogenous lectins, specific for lactose or beta-GalNAc, were displayed in nearly one half of the small cell lung carcinoma cases (44% or 45% respectively) and in about 25% of the non-small cell lung carcinoma cases.

Acetylgalactosamine↗
PubMed1992

Changes of expression of endogenous sugar receptors by polymorphonuclear leukocytes after prolonged anaesthesia and surgery.

Anaesthesia and surgery are known to depress granulocyte function in the early postoperative period, leading to deterioration of the immune defence against infection. Carbohydrate-lectin interactions may play an important role in the activities of phagocytic cells in that they facilitate initial host defence in the event of microbial antigenic challenge. A panel of biotinylated (neo)glycoproteins (chemically glycosilated carrier proteins) was used to detect endogenous carbohydrate-binding receptors /lectins/, on peripheral blood polymorphonuclear leukocytes of patients undergoing prolonged anaesthesia for replantation surgery. Four hours after induction of anaesthesia, a progressive decline of expression of endogenous sugar receptors on granulocytes was detected using the labelled (neo)glycoproteins lactose-BSA, N-acetyl-D-glucosamine-BSA, D-mannose-BSA, sialic-acid-BSA and D-xylose-BSA. Concomitant changes in peripheral white blood cell counts and the lack of depression in the absence of general anaesthetic agents suggested the existence of a possible relationship between reduced expression of (neo)glycoprotein receptors to impaired granulocyte function and anaesthetic-induced immunodepression.

Acetylglucosamine↗
PubMed1992

Purification and properties of a fucoidan-binding protein from human placenta and its identification as immunoglobulin G.

1. A fucoidan-binding protein from human placenta was purified by affinity chromatography on immobilized fucoidan. 2. Characterization of molecular and immunological properties and peptide mapping indicated that the fucoidan-binding protein is an immunoglobulin G. 3. Cleavage with papain and transblot analysis with labelled fucoidan ascertained binding properties of the F(ab) fragments. 4. The specificity for fucoidan was furthermore substantiated by hapten inhibition of haemagglutination as well as by solid-phase assays with biotinylated fucoidan as ligand. The results emphasized the importance of structural features instead of simple ionic interactions. 5. Chemical modification with group-specific reagents to lysine, arginine, tryptophan, tyrosine and histidine resulted in substantial inactivation, their impact being markedly reduced by the presence of fucoidan in the cases of lysine, arginine and tryptophan.

Chromatography, Affinity↗
PubMed1992

Studies on carbohydrate-binding proteins using liposome-based systems--I. Preparation of neoglycoprotein-conjugated liposomes and the feasibility of their use as drug-targeting devices.

1. Five types of neoglycoprotein-coupled liposomes were prepared in order to investigate their potential utility as new types of drug-targeting devices which exploit cellular functions of carbohydrate-binding proteins. 2. These preparations were shown to be stable at 37 degrees C for 24 hr and at 7 degrees C over 4 months. 3. An inhibition assay in an in vitro system using human adenocarcinoma cells indicated the high affinity binding of neoglycoprotein-conjugated liposomes. The inhibitory potency correlated with both the type and the amount of immobilized neoglycoproteins on liposomes. 4. A tissue distribution assay in an in vivo system using Ehrlich solid tumor-bearing mice showed the feasibility of the application of [125I]neoglycoprotein-conjugated liposomes as drug-targeting devices, based on carbohydrate-protein interactions.

Carbohydrate Metabolism↗
PubMed1992

Variations in lectin localization in different parts of the bovine heart.

In order to study the distribution of endogenous sugar-binding proteins (lectins) in various areas of the adult bovine heart, we used a battery of biotinylated neoglycoproteins. These tools expose carrier-immobilized carbohydrate moieties as ligands for receptor detection. Characteristic staining patterns depending on the type of carbohydrate ligand were observed in all constituents examined. Comparison to data obtained for lectin distribution in the respective areas of the human heart indicate that the localization of certain types of endogenous sugar receptors can exhibit species-dependent variations.

Animals↗
PubMed1992

Mammalian fetuin-binding proteins sarcolectin, aprotinin and calcyclin display differences in their apparent carbohydrate specificity.

The interferon antagonist sarcolectin, the protease inhibitor aprotinin and calcyclin whose expression is regulated by growth stimulation in quiescent fibroblasts display sialic acid-dependent binding to fetuin, visualized by solid-phase assays using biotinylated fetuin. The potential functional importance of this property prompted its comparative characterization with an array of neutral or negatively charged sugars, sulfated polysaccharides and sialoglycoproteins as inhibitors of binding of biotinylated fetuin to the immobilized proteins. The results revealed that this activity of sarcolectin and calcyclin is nearly unaffected by charge-free carbohydrates in contrast to aprotinin, calcyclin exhibits a notable affinity for Neu5Gc and together with aprotinin for phosphorylated sugars, and that sarcolectin's binding is affected to the highest extent by sulfated sugars relative to aprotinin and calcyclin.

Aprotinin↗
PubMed1992

Analysis of expression of erythropoietin-binding sites in human lung carcinoma by the biotinylated ligand.

Recombinant glycosylated erythropoietin (EPO) was biotinylated with biotin-aminocaproyl hydrazide via periodate-treated sialic acid moieties and applied to sections of 64 tumors of the lower respiratory tract, comprising 19 primary adenocarcinomas, 19 epidermoid carcinomas, 13 large cell anaplastic carcinomas, 11 small cell lung carcinomas, 11 intrapulmonary metastases, 1 mesothelioma and 1 lymphocytic interstitial pneumonia. The formalin-fixed, paraffin-embedded specimens were incubated with labelled EPO at room temperature and a concentration of 10 micrograms/ml for 60 min. The expression of the EPO-binding sites was visualized by the ABC technique. All of the analyzed large cell anaplastic carcinomas and the majority of the epidermoid carcinoma (89%), adenocarcinoma (79%), and metastases (82%) displayed binding capacities for EPO. Five out of the eleven small cell lung carcinomas, the analyzed mesothelioma and lymphocytic interstitial pneumonia revealed definite staining, too. Binding sites could, in addition, be seen in air dried, non-fixed, acetone-fixed, and ether-ethanol-fixed cytological specimens. The data indicate that the expression of binding sites with specificity for EPO can be frequently seen in human bronchial malignancies.

Binding Sites↗
PubMed1992

Detection of binding sites for biotinylated neoglycoproteins and heparin (endogenous lectins) during cerebellar ontogenesis in the rat: an ultrastructural study.

In a previous paper (Kuchler et al., Eur. J. Cell Biol. 52, 87-97 (1990)), endogenous carbohydrate-binding sites were studied at the optical level during rat cerebellar development on sections of fixed tissue using synthetic tools, biotinylated neoglycoproteins, in conjunction with subsequent avidin-peroxidase staining. It was shown that these tools were capable of revealing carbohydrate-binding sites during development of the rat cerebellum. The staining pattern with the individual probes disclosed variable developmental regulation and consequently suggested that recognition processes during cerebellar development may include several types of carbohydrate determinants. However, studies at the light microscope could not give information on potential membrane-bound localization of carbohydrate-binding sites and therefore discern the possible involvement of these molecules in cell adhesion processes. Furthermore, nuclear staining was suggested at the optical level. In order to elucidate these points, we examined the localization of mannose-, fucose- and heparin-binding sites at the electron microscopic level. Ultrastructural studies demonstrate that these tools are very efficient in detecting intracellular carbohydrate-binding sites, but failed to detect most of them expressed at the cell surface when using immunocytochemical techniques for known receptors, probably because of the interaction of these carbohydrate-binding sites with endogenous membrane-bound ligands. The significance of the nuclear staining and of part of the nucleolus found with fucose-containing neoglycoprotein and of the nuclear staining found with heparin are discussed.

Animals↗
PubMed1991

Carbohydrate-binding specificity of calcyclin and its expression in human tissues and leukemic cells.

Binding of biotinylated fetuin in a solid-phase assay served as activity assay for purification of calcyclin, the product of a cell growth-related cDNA with homologies to Ca(2+)-binding proteins. Asialofetuin failed to bind to calcyclin, emphasizing the importance of sialic acids. Binding of fetuin was most effectively reduced by N-glycolylneuraminic acid within a panel of mostly negatively charged sugars. Bovine submaxillary mucin and the ganglioside GM1, but not asialo-GM1, proved more effective than neoglycoproteins, carrying negatively charged carbohydrate moieties. Extension of N-acetyl-neuraminic acid to its lactosyl derivative increased its inhibitory potency. Among charge-free carbohydrate residues, only N-acetylglucosamine, lactose, and mannose, but not fucose, melibiose, or N-acetylgalactosamine affected fetuin binding, substantiating the inherent selectivity. Chemical modification with group-specific reagents revealed that lysine and arginine residues appear to be involved in ligand binding that is optimal in the presence of Ca2+, but not Zn2+ and stable up to 1 m NaCl. Biotinylation of calcyclin by modification of carboxyl groups facilitated performance of solid-phase assays with calcyclin in solution, yielding similar results with (neo)glycoproteins in relation to assays with immobilized calcyclin, thereby excluding an impact of binding to nitrocellulose on calcyclin's specificity. Subcellular fractionation disclosed the presence of fetuin-binding activity in all fractions, the specific activity decreasing from the nuclear to the particulate cytoplasmic fraction and the cytoplasmic supernatant. Affinity-purified antibodies were employed to detect high levels of calcyclin expression in acute lymphoblastic, myelogenous, and monocytic leukemia cell lines, but not in myeloma or lymphoblastoid cells. In comparison, most cells were nearly devoid of an O-acetylsialic acid-specific protein that is more abundant in various tissue types than calcyclin.

Acetylglucosamine↗
PubMed1991

Heparin-binding lectin from human placenta: further characterization of ligand binding and structural properties and its relationship to histones and heparin-binding growth factors.

We have previously demonstrated that the heparin-binding lectin of human placenta dissociates into up to four distinct polypeptides with molecular weights of 14,400, 15,000, 16,200, and 16,700 (Kohnke-Godt, B., & Gabius, H.-J. (1989) Biochemistry 28, 6531-6538). Stable complexes to ligands can shift the molecular weight appearance of the lectin to higher values. They can be dissociated in the additional presence of 9 M urea or by enzymatic degradation of heparin in model studies. The binding of heparin is rather stable over a range of salt concentrations from 1 to 3 M NaCl. Chemical modification with group-specific reagents to arginine, lysine, histidine, tyrosine, and tryptophan results in substantial inactivation of binding activity. Further amino-terminal sequence analyses point to a high-scoring relationship in this region to histone sequences, namely, histone H2B, but to no published sequences for any heparin-binding growth factor. Calculation of relatedness on the basis of differences in amino acid composition corroborates the conclusion of molecular distinction between the lectin, histones H2A and H2B, and the fibroblast growth factor as well as angiogenin. Histones only weakly agglutinate type II erythrocytes in contrast to the lectin. The immobilized lectin exhibits two classes of binding sites with KD values of 3 and 110 nM in contrast to one estimated KD value of 250 nM with a commercially available histone fraction. Both fractions retain binding activity to biotinylated heparin in transblots and are immunologically cross-reactive to antibodies, raised against the lectin as antigen. Subcellular fractionation clearly demonstrates that heparin-inhibitable hemagglutination activity and immunologically cross-reactive protein bands, characteristic for the lectin, but not unequivocally distinguishable from certain histone fractions in blots, are not confined to the nuclear fraction in the human placenta.

Amino Acid Sequence↗
PubMed1991

Lectin localization in human nerve by biochemically defined lectin-binding glycoproteins, neoglycoprotein and lectin-specific antibody.

Molecular recognition can be mediated by protein (lectin)-carbohydrate interaction, explaining the interest in this topic. Plant lectins and, more recently, chemically glycosylated neoglycoproteins principally allow to map the occurrence of components of this putative recognition system. Labelled endogenous lectins and the lectin-binding ligands can add to the panel of glycohistochemical tools. They may be helpful to derive physiologically valid conclusions in this field for mammalian tissues. Consequently, experiments were prompted to employ the abundant beta-galactoside-specific lectin of human nerves in affinity chromatography and in histochemistry to purify and to localize its specific glycoprotein ligands. In comparison to the beta-galactoside-specific plant lectins from Ricinus communis and Erythrina cristagalli, notable similarities were especially detectable in the respective profiles of the mammalian and the Erythrina lectin. They appear to account for rather indistinguishable staining patterns in fixed tissue sections. Inhibitory controls within affinity chromatography, within solid-phase assays for each fraction of lectin-binding glycoproteins and within histochemistry as well as the demonstration of crossreactivity of the three fractions of lectin-binding glycoproteins with the biotinylated Erythrina lectin in blotting ascertained the specificity of the lectin-glycoprotein interaction. In addition to monitoring the accessible cellular ligand part by the endogenous lectin as probe, the comparison of immunohistochemical and glycohistochemical detection of the lectin in serial sections proved these methods for receptor analysis to be rather equally effective. The observation that the biotinylated lectin-binding glycoproteins are also appropriate ligands in glycohistochemical analysis warrants emphasis.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Correlations between the binding of neoglycoproteins, bovine serum albumin (BSA) and lectins in 10 to 13-day-old mouse embryos.

In the present work we compared the appearance of carbohydrate binding sites for mannose, maltose, sialic acid and N-acetyl-glucosamine in the 11 to 13-day-old mouse embryo with the appearance of BSA and lectin binding sites. The carbohydrate-binding sites were localized with FITC-coupled neoglycoproteins, synthesized by chemical glycosylation of bovine serum albumin (BSA). These localizations were compared with binding of the FITC-labelled unglycosylated BSA. Furthermore the localizations of neoglycoprotein and BSA binding sites were correlated with binding of the FITC-labelled lectins WGA, RCA I and Con A. Initial appearance of neoglycoprotein binding sites occurred in the lens capsule of the 13 day old mouse embryo. Binding sites for the unglycosylated BSA appeared earlier, i.e. already in the 12-day-old embryo, in the basement membranes of the choroid plexus and the lung bud and lectin binding sites were seen in these structures in the 11-day-old embryo. The staining of the basement membrane and the lens capsule for BSA binding sites in the 12-and 13-day-old embryos correspond to WGA binding to these membranes. From these results we concluded that 1) specific carbohydrates which are probably involved in embryonic development appear much earlier in the embryo than the endogenous lectins which are able to react with these carbohydrates and 2) BSA is a protein which like WGA probably binds N-acetylglucosamine or sialic acid moieties.

Animals↗
PubMed1991

Identification of endogenous sugar-binding proteins in the accessory sex glands of NMRI mice. A histochemical and biochemical study.

In the present study we report on the histotopographical distribution of carbohydrate-binding proteins in the prostate and seminal vesicle of sexually mature NMRI mice using a panel of fluorescein-isothiocyanate labelled neoglycoproteins and asialoglycoproteins. Additionally, biochemical analysis using affinity chromatography and SDS-gel electrophoresis was performed to purify and characterize the respective proteins from the tissue. Our histochemical results clearly demonstrate the presence of endogenous receptors for the carbohydrate part of glycoconjugates in both glands. In the prostate a distinct staining was seen after incubation with melibiose-BSA-FTC, glucuronic acid-BSA-FTC and asialofetuin-FTC (only in the ventral prostate). In the epithelium of the seminal vesicle a weak staining occurred after incubation with asialofetuin-FTC and maltose-FTC. In the stroma of both accessory sex glands a distinct binding of several (neo)glycoproteins specific for beta-galactoside-binding proteins was observed which could be attributed to a beta-galactoside-binding lectin. Indeed biochemical analysis ascertained presence of such a histochemically detectable activity. We assume that the carbohydrate-binding proteins of the stroma, which were obviously linked to the elastic fibers, could play a role in the organisation of the extracellular matrix in the interstitium of the glands.

Animals↗
PubMed1991

Glycopeptide-albumin derivative: it preparation and histochemical ligand properties.

Carrier-immobilized mono- or disaccharides and other carbohydrate structures, derived by custom-made chemical synthesis, have already proven to be valuable ligands for localizing carbohydrate-binding proteins in tissue sections. Defined purified glycopeptides, as components of neoglycoproteins, offer the possibility of increasing their structural complexity and, thereby, their receptor selectivity. To test the feasibility of this approach, the glycopeptide man6-glcNAc2-asparagine derived from ovalbumin was purified after pronase digestion. It was coupled to bovine serum albumin as carrier protein with the homobifunctional linking agent bis-(sulphosuccinimidyl)suberate to yield the diglycosylated concanavalin A-reactive product. Following biotinylation, it was used to detect mannose-specific binding sites in fixed cells of seven human leukemia or lymphoma lines and in fixed, paraffin-embedded sections of human breast cancer. In comparison to chemically mannosylated bovine serum albumin with ten sites of glycosylation or to ovalbumin, this derivative produced a similar pattern of reaction with a quantitatively lower extent of staining in most cases. Remarkably, the presence of potential endogenous ligands for the detected receptor sites was ascertained using the plant lectin concanavalin A. Thus, the conjugation of a purified, deliberately selected glycopeptide to a suitable carrier produces a histochemical tool for detecting glycopeptide-specific binding sites.

Asparagine↗