Hans-Joachim Gabius

Influence of type of linkage and spacer on the interaction of β-galactoside-binding proteins with immobilized affinity ligands

Affinity chromatography provides a powerful tool for isolation of carbohydrate-binding proteins. However, the choice of the ligand and spacer has an important impact on effectiveness. The influence of several different ligands on qualitative and quantitative aspects of the purification of two beta-galactoside-specific lectins has been evaluated. Sepharose was modified by coupling four types of neoglycoproteins (galactosylated or lactosylated bovine serum albumin with increasing sugar content) and two naturally occurring asialoglycoproteins at similar densities. Carbohydrate ligands at essentially equal density were made accessible to the lectins by seven commonly used methods. The yield of mistletoe lectin was high when lactosylated neoglycoproteins were used for separation. For these resins the sugar incorporation exceeded 10 sugar groups per protein carrier molecule. The yield was similarly high with the asialoglycoproteins and with lactose; the sugar was coupled to the resin as a p-aminophenyl derivative or by means of divinyl sulfone activation. An epoxy group in linkages of galactose or lactose decreased the binding capacity. A quantitatively similar degree of protein yields was obtained for the beta-galactoside-binding protein of bovine heart, although different proteins were obtained when neoglycoproteins were used as ligand. The nature of the affinity ligand in lectin purification can increase the yield and may also influence the profile of the carbohydrate-binding proteins.

Localization of endogenous lectins in normal human breast, benign breast lesions and mammary carcinomas.

SummarySpecific antisera against three mammalian β-galactoside-specific lectins of apparent molecular weights 14.5 kDa, 18 kDa and 29 kDa have been used to localize these lectins in normal breast, and in benign and malignant mammary lesions. In normal breast tissue discrete localization of two lectins (Mrs 14.5 kDa and 18 kDa) was demonstrated in fibroblasts, smooth muscle cells, myoepithelial cells and capillary endothelium. Extracellular localization of one lectin (Mr 14.5 kDa) in collagen was apparent. The third lectin (Mr 29 kDa) labelled preferentially luminal cells and their secretory product. Two benign tumours (an analyzed fibroadenoma and a papilloma) revealed strong staining with two lectins (Mrs 18 kDa and 29 kDa). Of the 24 mammary carcinomas examined, the lectin (Mr 14.5 kDa) was expressed by only occasional tumour cells, the lectin (Mr 18 kDa) occurred in many tumour cells and the lectin (Mr 29 kDa) labelled tumour cells in nearly all cases. The expression of these β-galactoside-specific endogenous lectins therefore appears to be regulated differently in normal breast compared with mammary tumours.

An 8- to 10-fold enhancement in sensitivity for quantitation of proteins by modified application of colloidal gold.

We have modified the highly sensitive protein assay of C. M. Stoscheck (1987, Anal. Biochem. 160, 301-305), resulting in a further 8- to 10-fold enhancement of sensitivity. This assay, responding to protein quantities with a detection limit of 1 ng, involves the single step of addition of colloidal gold solution, as now commonly used in histochemistry and protein blotting, to the protein sample, followed by simple measurement of the change in absorbance at 590 nm within minutes. By increasing the concentration of the colloidal gold, by using gold sol that has been stabilized with 0.01% polyethylene glycol and adjusted to pH 3.8, and by adapting the assay to microtiter plates, this type of assay can be applied to reliably determine proteins in the complete nanogram range. This assay therefore compares favorably to other assay procedures in terms of rapidity, sensitivity, expense, and lack of interference by many laboratory reagents, although like the others it suffers from the drawback of differences in response of different proteins, which is inherent in dye-binding assays.

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.

Sugar Receptors of Different Types in Human Metastases to Lung and Liver

Endogenous sugar receptors of human tumors, supposedly involved in recognitive interactions and growth regulation, were comparatively analyzed from human metastases to lung and liver by affinity chromatography and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These profiles of sugar receptors including Ca2+-dependent and Ca2+-independent specificities to alpha- and beta-galactosides, alpha-mannosyl and alpha-fucosyl moieties from salt and detergent extracts were found to be significantly different from the profile of the corresponding normal tissue. Metastatic lesions to lung from three different types of primary tumors revealed primarily tumor-associated mannan- and galactoside-binding proteins, whereas different liver metastases showed a tendency towards preferential expression of additional beta-galactoside-binding proteins and, to a reduced extent, fucose-binding proteins. The patterns of two metastatic lesions to lung and liver from a similar primary tumor, a colon carcinoma, disclose significant differences. Each resembles the pattern of other metastases to the same target organ more than it resembles the pattern of metastatic lesions to the other target organ, derived from a similar primary tumor. Further analyses of two primary liver tumors underscore the significance of changes in such a pattern upon malignant transformation.

Age‐related changes in different steps of protein synthesis of liver and kidney of rats

Protein synthesis in cell‐free systems of rat liver and kidney decreases markedly with age. Examination of activity changes of the different steps revealed for both types of organs that reduced binding of aminoacyl‐tRNA to ribosomes and reduced peptidyl transfer might be of major importance for the decrease in overall protein synthesis whereas ageing has only little effect on translocation as well as on initiation and termination.

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 calcyclins 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.

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.

TUMORLEKTINOLOGIE-STATUS UND PERSPEKTIVEN KLINISCHER ANWENDUNG

A detailed knowledge of the mechanisms of molecular recognition is a prerequisite to rationally improved diagnostic and therapeutic procedures in diseases. In addition to sequences of amino acids, carbohydrate structures apparently store biological information that is thought to be relevant for physiologically important processes. Such ligands, namely the carbohydrate part of cellular glycoconjugates, can be recognized by specific endogenous binding proteins like lectins. If their presence can be reliably ascertained and correlated to the clinical course of the disease, e.g. in oncology, lectinology may help to define a yet undisclosed role for this class of proteins in tumor progression and spread.

(Neo)glycoproteins as tools in neuropathology: histochemical patterns of the extent of expression of endogenous carbohydrate-binding receptors, like lectins, in meningiomas.

SummaryBiotinylated (neo)glycoproteins were used to specifically detect endogenous sugar receptors such as lectins in sections of formaldehydefixed, paraffin-embedded tissue from meningiomas. The histochemical methods used consisted of the application of a carrier protein and various covalently linked sugar moieties, available mainly through chemical synthesis, in an optimized standard protocol. They proved valuable in elucidating differential binding patterns within the various meningioma subtypes.α-Fucoside-, β-galactoside-, α-mannoside-and β-xyloside-specific carbohydrate-binding receptors were detected in all the tumor subclasses examined, although the levels of expression exhibited pronounced quantitative differences. In addition, differences in the extent of histochemical staining were observed, using a labelled carrier protein, derived from N-acetylglucosamine and mannose-6-phosphate moieties, respectively. Quantitative differences in the reaction intensity were also measured in the respective subtypes. Receptors for N-acetyl-D-galactosamine were detected only in the anaplastic forms, while glucuronic acid-specific receptors were only present in the meningotheliomatous meningioma. In contrast to the other types, malignant meningiomas failed to show cytoplasmic staining with the α-glucoside-specific maltose-(BSA-biotin). Distinct differences in the pattern of expression of endogenous sugar receptors, evaluated by a standard protocol, provided further evidence for a possible additional subtype of men-ingioma, the submalignant meningioma. Our results suggest that labelled (neo)glycoproteins could be used routinely as tools for assessing the expression of endogenous sugar receptors in diagnostic neuro-oncology.