Sigrun Gabius

Lectins and Cancer

The lectins, isolated from Narcissus pseudonarcissus [NPL] and Gerardia savaglia [Gerardia-lectin], are specific for terminal Man(al-3)Man units. They react very specifically with the external glycoprotein gp120 of the AIDS-virus, the human immunodeficiency virus type 1 (HIV-l). They prevent in vjtro the infection of cells with HIV. These lectins were tools for identifying antigens [mannans from Candida albicans and from Saccharomyces cerevisiae, comprising Man(al-3)Man and Man(al-2)Man linked residues] which were successfully used to raise in rabbits neutralizing antibodies against HIV infection jn vitro. The NPL was used to develop a novel enzyme-linked immunoassay [ELISA] system for quantifying HIV-gpI20 within the range of 0.6 to 20,000 ngjml.

Neoglycoenzymes: A versatile tool for lectin detection in solid-phase assays and glycohistochemistry

Carbodiimide-mediated coupling of p-aminophenyl glycosides to a naturally nonglycosylated enzyme yields a neoglycoenzyme. This compound combines inherent enzymatic activity with synthetically conferred ligand properties to lectins. Appropriate choice of the ligand allows custom-made synthesis to reliably detect various types of lectins. To exemplify practical applications of this class of compounds, glycosylated bacterial beta-galactosidase has been employed to quantitate plant lectins, immobilized on plastic surfaces as well as on nitrocellulose. Competitive inhibition by specific sugar ascertained the dependence of binding on protein--carbohydrate interactions. In view of lectins as tools, a sandwich lectin-binding assay for high mannose-type glycoprotein detection has been modified to principally facilitate wide application to other lectin-reactive sugar chains by introducing the neoglycoenzyme. In addition to lectin determination in solid-phase assays, neoglycoenzymes allow one to glycohistochemically localize endogenous lectins in tissue prints and tissue sections with a minimum number of steps. This nonradioactive, rapid, sensitive, and convenient assay concept, based on conjugation of a ligand to an enzyme with maintenance of its receptor-binding activity, may find extended application beyond lectinology in receptor analysis.

Binding of T-antigen-bearing neoglycoprotein and peanut agglutinin to cultured tumor cells and breast carcinomas.

Chemical conjugation of appropriate carbohydrate ligands to an inert labeled carrier renders probes available to screen for the presence of respective binding sites. A set with a certain plant lectin and a suitable neoglycoprotein can thus determine complementary parts of a potentially relevant glycobiological interaction system. Owing to the interest in the peanut agglutinin-reactive T-antigen, we performed chemical synthesis of the respective disaccharide structure to serve as glycohistochemical ligand and established refinements of the synthetic patway. Coupling of the derivatized monomers had to be performed in the presence of sodium sulfate for optimal results. Complete removal of the protective groups from the p-nitrophenyl derivative of the N-acetylgalactosamine moiety was achieved under mild conditions with 2,3-dichloro-5,6-dicyanobenzoquinone without affecting any other functional groups. Specific binding sites for the synthetic neoglycoprotein as well as for the plant lectin were demonstrated in cell lines of human breast carcinoma colon adenocarcinoma, and erythroleukemia. ABC reagents in conjunction with DAB as peroxidase substrate were used to visualize specific binding sites. Binding complied with the accepted criteria for specificity. Moreover, carbohydrate-specific binding sites were detected in sections of nine out of 14 cases with malignant breast lesions. The percentage of positive tumor cells with both neoglycoprotein and lectin was similar in each of the individual sections, regardless of quantitative variations between cases, lectin staining intensity often being more pronounced. The reactivity pattern in sections of primary and metastatic lesions was not significantly correlated with the lymph node status. This study emphasized that custom synthesis of saccharides and histochemical application of the resulting neoglycoprotein has a remarkable potential for complementary assessment of endogenous binding sites for carbohydrate structures, localized by external tools such as plant lectins, as a step to elucidate the importance of a putative proteincarbohydrate interaction.

PROTEIN-ZUCKER-ERKENNUNG : GRUNDLAGEN UND MEDIZINISCHE ANWENDUNG AM BEISPIEL DER TUMORLEKTINOLOGIE

Protein-carbohydrate interactions are involved in multifarious physiological processes. Together with antibodies and enzymes, lectins constitute the family of carbohydrate-binding proteins. The current research activities of tumor lectinology comprise the design of custom-made carrier-immobilized carbohydrate ligands (neoglycoconjugates), their application for the detection of specific binding sites and the evaluation of potential therapeutical approaches by blocking access or by directing drug conjugates to cell surface lectins, correlation of their expression to clinical parameters such as prognosis and their biochemical characterization. Thereby, mammalian lectins are made available to serve as tools. These experimental approaches are evaluated as regards their potential for improving cancer diagnosis and therapy.

Identification of a cell cycle-dependent gene product as a sialic acid-binding protein

A Ca2+-dependent sialic acid-binding protein was purified on fetuin-Sepharose from various types of human tissue. The molecular mass was determined to be 10,315 Da by laser desorption mass spectrometry. Partial sequence analysis after cyanogen bromide cleavage that yielded one N-terminus accessible for Edman degradation revealed an identity to an internal stretch following the only methionine residue within a putative amino acid sequence (Mr 10,048), deduced from the cDNA of a cell cycle-specific gene. The reported biochemical identification is a prerequisite to infer the biological role of the so far undetected gene product. Initial glycohistochemical studies with sialic acid-(BSA-biotin) raised evidence for nuclear localization of sialic acid-binding sites that might reflect, at least in part, detection of this protein.

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.

Carrier-immobilized derivatized lysoganglioside GM1 is a ligand for specific binding sites in various human tumor cell types and peripheral blood lymphocytes and monocytes

Biotinylation of ganglioside-protein conjugates, derived from selective N-acylation of the sphingoid amino group of deacylated ganglioside GM1 or a ganglioside mixture, yielded probes to detect specific binding sites in fixed specimens. GM1-containing neoligandoprotein significantly bound to tumor cells in sections of 15 out of 16 cases of human lung cancer, while the probe, derived from the mixture, was ineffective under these conditions. The same graduation of staining was under identical conditions observed with these two probes on fixed human tumor cells and on peripheral blood lymphocytes and monocytes. Attempts of biochemical isolation of proteins, responsible for this binding capacity, from tumor cell extracts in the presence of the abundant endogenous ligands led to protein bands with apparent molecular weights of 44,000, 68,000 and 72,000 with yields of 0.1-0.24 micrograms/mg protein, after the detergent extracts had been passed over a resin, exposing gangliosides of the markedly less efficient mixture, to exclude binding by non-specific ionic or hydrophobic interactions.

[4] Detection and quantification of carbohydrate-binding sites on cell surfaces and in tissue sections by neoglycoproteins

Publisher Summary This chapter discusses a procedure defining the presence of carbohydrate-binding sites that are neither occupied by endogenous high-affinity ligands, nor impaired by processing of the specimen. Availability of sensitive substrates and easy access are the key factors in choosing the enzyme for conjugation. Selection of the coupling agent depends on the presence of suitable functional groups on the enzyme and the neoglycoprotein. When the chosen enzyme itself or the carrier for the carbohydrate ligands are glycoproteins, the glycol part must be removed either by extensive enzymatic treatment with glycosidases or by chemical oxidation with periodate. Neoglycoprotein-enzyme conjugates and neoglycoenzymes are used to detect and quantify cell surface sites with corresponding carbohydrate specificity in a two-step procedure, whereas biotinylated neoglycoproteins require a three-step method as well as a commercial reagent. Owing to the availability of commercial avidin–biotin complexes (ABC) kit reagents, which contain complexes of avidin and biotinylated enzymes with the capacity to bind to biotin of the applied probe that has specifically bound in the section, glycocytological staining of cells by biotinylated markers can be amplified relative to application of neoglycoenzymes. These kits provide a high level of sensitivity to visualize cellular binding sites.

Neoglycoprotein binding to colorectal tumour cells: Comparison between primary and secondary lesions

Biotinylated neoglycoproteins are useful to determine the expression of sugar receptors (lectins) histochemically in routinely processed tissue sections. Assessment of the presence of distinct receptor classes with specificity toβ-galactosides and toα- orβ-N-acetylgalactosamine, selected on the basis of their potential relevance for recognition processes within the metastatic cascade in murine model systems, was performed for a common human tumour type, colorectal cancer. The four different types of neoglycoproteins, derived from covalent attachment of commercially available derivatives ofβ-N-acetylgalactosamine, differed only quantitatively in their capacity to detect specific binding on cultured cells and tissue sections, thus posing no major restriction on the choice of synthetic process for histochemical efficiency of the product. Glycocytological application revealed specific probe binding and a regulation of level of receptor expression for a human colon carcinoma cell line primarily forN-acetylgalactosamine-specific receptors upon retinoic acid-induced differentiation. Monitoring of sections of the 12 cases of primary and secondary colorectal lesions invariably disclosed the presence of the respective receptors, the extent of cell labelling in primary tumours and metastases being similar. Establishment of metastases, even in different target organs, is apparently not followed by a major phenotypic variation in this feature.

Glycopeptide-albumin derivative: Its preparation and histochemical ligand properties

SummaryCarrier-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.