Reinhild Engelhardt

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.

Pattern of endogenous lectins of a human sarcoma (Ewing's sarcoma) reveals differences to human normal tissues and tumors of epithelial and germ cell origin

A human sarcoma, Ewings sarcoma, contains activities of endogenous lectins. Fractionation of salt and detergent extracts by affinity chromatography on columns with immobilized sugars or glycoproteins results in the pattern of endogenous lectins for alpha- and beta-galactosides, alpha-mannosyl- and alpha-fucosyl-moieties. Whereas some lectins are known from normal, non-malignant human tissues or from a human epithelial tumor or a human germ cell tumor, a Ca2+-independent alpha- and beta-galactoside-binding protein at apparent molecular weight of 58 kilodaltons has so far not been characterized from any human source. The patterns for the tumors and human normal tissues reveal various differences in comparison between each other. These differences, documented for the first human sarcoma, human tumors of different histogenetic lineage and normal tissues are a first step to a lectin-based diagnosis and therapy of certain human cancer types.

Preparation of neoglycoprotein-enzyme conjugate using a heterobifunctional reagent and its use in solid-phase assays and histochemistry

A conjugate of a neoglycoprotein (chemically lactosylated bovine serum albumin) and an enzyme (horseradish peroxidase) has been prepared in solution using a heterobifunctional reagent, N-succinimidyl-3-(2-pyridyldithio)propionate, and has been purified by gel filtration on an Ultrogel AcA-44 column. To preclude any carbohydrate-dependent binding to the sugar residues on the glycoprotein peroxidase, the enzyme has to be treated with sodium periodate and sodium cyanoborohydride prior to coupling, which results in oxidative cleavage of the carbohydrates and reduction of the aldehydes thus formed to primary alcohols. Lactosylated bovine serum albumin-peroxidase conjugate has been employed to detect plastic-bound Ricinus communis agglutinin with dependence of the concentration of the lectin and with dependence of the presence of specific inhibitors. Enzyme-labeled conjugates with unmodified bovine serum albumin are completely ineffective in this assay. Localization of beta-galactoside-specific sugar receptors in connective tissue is used to demonstrate the feasibility of application of such neoglycoprotein-enzyme conjugates in histochemistry with a minimum number of steps.

Carbohydrate-binding proteins of tumor lines with different growth properties

SummaryThree clones of myeloproliferative virus (MPV)-transformed rat fibroblasts (NRK) with different growth properties and morphology were transplanted to athymic nude mice. Presence of carbohydrate-binding proteins was inferred by fluorescence microscopy using fluorescent, glycosylated markers. Salt and detergent extracts of tumors from this model system were fractionated under identical conditions on different sets of Sepharose columns, to which lactose, asialofetuin, melibiose, mannan and fucose had been covalently linked. Successive elution by chelating reagent and specific sugar resulted in isolation of the different Ca2+-dependent and Ca2+-independent endogenous carbohydrate-binding proteins that were assayable as agglutinins. In comparison, the different tumors displayed a pattern with qualitative and quantitative alterations. Since protein-carbohydrate interaction mediated by carbohydrate-binding proteins (lectins) is of importance for cognitive processes, it is remarkable that the pattern of membrane glycoproteins, isolated by affinity chromatography on resins with immobilized plant lectins, had also been found to reveal certain individual properties for receptors specific for peanut agglutinin (PNA) and Ulex europaeus agglutinin (UEA). These demonstrated differences within the system of protein-carbohydrate interaction suggest that endogenous lectins and their ligands have potential significance as markers defining a certain phenotype within this tumor model system.

Expression of endogenous lectins in human small-cell carcinoma and undifferentiated carcinoma of the lung

Endogenous carbohydrate-binding proteins (lectins) were detected in specimens of tumor tissue (undifferentiated carcinoma and xenografted small-cell carcinoma) from human lung. Fractionation of salt and detergent extracts on different sets of Sepharose columns covalently derivatized with lactose, asialofetuin, melibiose, mannan, and fucose, successive elution with a chelating agent and a specific sugar, and analysis of the eluates by gel electrophoresis, resulted in the characterization of the profiles of endogenous carbohydrate-binding proteins. All preparations were devoid of enzymatic activity. Comparison between the patterns of the two types of lung carcinoma showed significant qualitative differences, e.g. the presence of fucose-binding proteins of apparent molecular weights 60,000 and 80,000 in the undifferentiated carcinoma, and the presence of beta-galactoside-binding proteins of apparent molecular weights 18,000 and 22,000 in the small-cell lung carcinoma. These proteins were not detectable in normal lung tissue. Such differences, documented for the first time for human lung tumors, are of potential importance as a step towards a lectin-based refinement of lung-cancer diagnosis and therapy.

Phenylalanyl-tRNA synthetases from yeast cytoplasm and mitochondria. The presence of a carbohydrate moiety in the mitochondrial enzyme and immunological evidence for structural relationship.

Homogeneous yeast cytoplasmic and mitochondrial phenylalanyl-tRNA synthetases (L-phenylalanine:tRNAPhe ligase (AMP-forming), EC 6.1.1.20) are analysed for structural differences. Only the large subunit of the mitochondrial enzyme is a glycoprotein with nearly 3% carbohydrate by weight. The carbohydrates present are: glucose, N-acetylglucosamine, mannose, galactose and N-acetylneuraminic acid. Removal of the sugar moieties yields an activity increase, but no significant change of sensitivity to proteolytic degradation. Antibodies to both homogeneous enzymes demonstrate a structural similarity for both types of subunit using the highly sensitive immunoblotting technique.