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Specific antisera against three mammalian beta-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 beta-galactoside-specific endogenous lectins therefore appears to be regulated differently in normal breast compared with mammary tumours.

The specific activities of 17 aminoacyl-tRNA synthetases in liver, lung, heart, spleen, kidney and small intestine of old female normal and leukemic (reticulum-cell sarcoma, type A) mice have been monitored. No difference appears for lung, heart and kidney; small increases with varying particular changes for liver and marked increases in spleen and small intestine of the tumor bearing mice have been found, following a similar pattern. This finding suggests a coordinated adaptation to modulation of the requirements of protein synthesis imposed by histiocytic sarcoma.

Histochemical detection of binding sites for sulfated polysaccharides believed to be important mediators within recognitive interactions was carried out by application of biotinylated probes such as heparin, native and desulfated fucoidan, dermatan sulfate, and two types of carrageenans. The probes were derivatized by mild cyanogen bromide activation and subsequent aminoalkylation to allow incorporation of biotin, inserted with an epsilon-aminocaproic acid spacer to reduce charge-related and steric impediments. Specific labeling could be detected in different cell types of human placenta, dependent on the developmental stage. Sulfated polysaccharides bound predominantly to leucocytes in full-term placenta, whereas demonstration of specific binding sites in decidua, syncytiotrophoblasts, and cytotrophoblasts was restricted primarily to heparin and, less intensely, fucoidan, although not desulfated fucoidan. Heparin binding in the placenta after 8 weeks of gestation was reduced for epithelia that, at this stage of development, revealed carrageenan binding sites. Fucoidan binding was at this developmental stage measurable only for leucocytes. These results provide definite histochemical evidence for the presence and developmental regulation of expression of receptors for sulfated polysaccharides in different cell types of human placenta.

Salt and detergent extracts of a malignant epithelial tumor, obtained by extraction of acetone powder, were fractionated on different sets of Sepharose columns covalently derivatized with lactose, asialofetuin, melibiose, mannan, fucose, and heparin. Successive elution by chelating reagent and specific sugar resulted in isolation of different Ca2+-dependent and Ca2+-independent endogenous carbohydrate-binding proteins, as analyzed by gel electrophoresis. It appears from the analysis that certain bands represent newly identified proteins capable of binding to lactose (at Mr 64,000), melibiose (at Mr 28,000), and fucose (at Mr 62,000 and 70,000). Other carbohydrate-binding proteins isolated from this human tumor have been identified in normal, especially embryonic, tissues of different nonhuman vertebrates. The carbohydrate-binding proteins are assayable as agglutinin with rabbit erythrocytes and show no detectable enzymatic activity. They can thus be defined as lectins. The presence of a complex pattern of endogenous lectins and their biochemical characteristics may contribute to an understanding of intercellular interaction during the complex process of metastatic spread and may furthermore allow a new tool for diagnosis and a lectin-based therapy.

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.

Salt and detergent extracts of xenografted human embryonic carcinoma and a human yolk sac carcinoma were fractionated on different sets of Sepharose columns covalently derivatized with lactose, asialofetuin, melibiose, mannan and fucose. Successive elution by chelating reagent and specific sugar resulted in isolation of endogenous Ca2+-dependent and Ca2+-independent carbohydrate-binding proteins, as analyzed by gel electrophoresis. The salt extracts of the embryonic carcinoma contained a Ca2+-dependent carbohydrate-binding protein at Mr66,000, that was undetectable in yolk sac tumor extracts. Also, a Ca2+-independent fucose-binding protein at Mr62,000 could be purified. In general, the pattern of carbohydrate-binding proteins showed quantitative and qualitative differences as compared to normal tissue. The carbohydrate-binding proteins were assayable as agglutinin and showed no enzymatic activity. They can thus be defined as lectins. Their presence within certain stages of differentiation and developmental regulation may contribute to improvement of the classification, diagnosis and therapy of this tumor class. These new lectins may also be functionally related to the stage-specific embryonic antigens like SSEA-1.

Flow cytofluorometry and drug targeting with labelled neoglycoproteins are used as tools to probe for membrane lectins in two human adenocarcinoma cell lines. Both cell lines express activities for galactosides, glucosides and fucosides. Affinity chromatography on gels with immobilized sugar leads to purification of an alpha-galactoside-binding protein at an apparent molecular weight of 64 kDa that also binds to lactose, maltose and fucose and exhibits Ca2+-requirement for binding, a beta-galactoside-binding protein without Ca2+-requirement at an apparent molecular weight of 14 kDa, and an alpha-glucosyl-binding protein without Ca2+-requirement at an apparent molecular weight of 34 kDa from both cell lines. The description of membrane lectins, documented here for the first human tumor cell lines, is an initial step towards a lectin-based improvement of the clinical management of human colon adenocarcinoma.

Important biological functions and cellular recognition phenomena are supposedly governed by specific sugar-protein interactions. Human hematopoietic cell lines offer an excellent model for the study of the expression of endogenous receptors for the carbohydrate part of glycoconjugates with respect to cell lineage and modulation by differentiation. Initially, a panel of fluorescent (neo)glycoproteins was successfully employed to demonstrate cytologically the actual presence of such receptors on different cell lines: the B lymphoblast line, Daudi; the T cell lymphoblastic leukemia line, P12; the multipotent leukemic line, K562 and the promyelocytic line, HL060. Biochemical analyses were performed using affinity chromatography on supports with immobilized lactose and asialofetuin (simple or complex beta-galactosides), melibiose (alpha-galactoside), fucose, N-acetyl-D-galactosamine, maltose (alpha-glucoside), the mannose-rich yeast glycoprotein, mannan, glycopeptides containing sialic acid residues and heparin. Subsequently, sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis was used to detect cell lineage-dependent changes in theses parameters. Differentiation-dependent changes in the expression of receptors with specificity to galactose, N-acetylgalactosamine, maltose and heparin were similarly uncovered upon dimethyl sulfoxide-induced differentiation of HL60 cells. Differences in this type of cellular characteristic were also apparent for lymphoma cells from patients with various histological subtypes of lowgrade lymphomas. This initial description of lineage- and differentiation-dependent differences in various human hematopoietic cell lines and in cells from patients with lowgrade lymphomas suggests that advances in the knowledge of the composition of endogenous sugar receptors (lectins) may aid in understanding aspects of the biological behavior of hematopoietic cells and their related malignancies via participation of sugar-protein (lectin) interactions.

Neoglycoproteins are readily available conjugates of a histochemically inert carrier protein and histochemically crucial carbohydrate moieties which are covalently attached to the carrier protein by chemical synthesis. Biotinylation renders these conjugates detectable in formalin-fixed, paraffin-embedded tissue sections of human lung cancer by standard staining protocols, thereby localizing endogenous receptors for carbohydrate moieties. Examination of 30 cases of main types of human lung cancer revealed the presence of alpha-fucosyl-, alpha-mannosyl-, and alpha-glucosyl-specific receptors in adenocarcinomas or epidermoid carcinomas with high positivity rates. The extent of the expression of receptors for alpha- and beta-galactosides appeared to be comparatively lower. Within the standard protocol, using a concentration of the biotinylated probes of 10 micrograms/mL, this panel of probes consistently failed to detect endogenous sugar receptors in ten cases of small cell anaplastic carcinoma of the lung. Whereas none of the sections from the tumor cases bound the sulfated fucan fucoidan, the accompanying inflammatory cells, especially the granulocytes, expressed receptors for the sulfated fucan. Pronounced labeling for macrophages was observed for the alpha-galactoside-specific probe, whereas no binding to inflammatory cells and pneumocytes was detectable for the beta-galactoside-specific probe. The results indicate that expression of endogenous receptors for neoglycoproteins may be useful in discriminating between small cell and non-small cell lung carcinoma and carcinomatous cells from accompanying inflammatory cells.

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.

Affinity chromatography of salt and detergent extracts from bovine pancreas on glycosylated or glycoprotein-linked Sepharose 4B resulted in purification of different carbohydrate-binding proteins. Three species of proteins with molecular masses of 16 kDa, 35 kDa and 64 kDa exhibiting specificity for beta-galactosides, but none with preferential specificity for alpha-galactosides, were isolated from salt and detergent extracts. No Ca2+ was required for binding. Mannan-binding proteins of 37 kDa, 47 kDa and 94 kDa without Ca2+-requirement were only found in the salt extract. No other mannan-binding activity could be detected. Fucose-binding proteins of 34 kDa, 62 kDa and 70 kDa exhibiting Ca2+-requirement for binding were present in the salt extract and two proteins with 62 kDa and 70 kDa in detergent extract. The different fractions showed agglutination activity when assayed with rabbit erythrocytes. Thus they can be defined as lectins.

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 synthetase from the archaebacterium Methanosarcina barkeri activates a number of phenylalanine analogues (methionine, p-fluorophenylalanine, beta-phenylserine, beta-thien-2-ylalanine, 2-amino-4-methylhex-4-enoic acid and ochratoxin A) in the absence of tRNA, as demonstrated by Km and kcat of the ATP/PPi exchange reaction. Upon complexation with tRNA, AMP formation from the enzyme X tRNA complex in the presence of ATP, one of the above analogues or tyrosine, leucine, mimosine, N-benzyl-L- or N-benzyl-D-phenylalanine indicates activation of the analogues under conditions of aminoacylation. Natural noncognate amino acids are not transferred to tRNAPhe-C-C-A or tRNAPhe-C-C-A-(3'-NH2). This pretransfer proofreading mechanism, together with the comparatively low ratio of synthetic to successive hydrolytic steps, resembles the mechanism of liver enzymes of vertebrates. In contrast, eubacterial phenylalanyl-tRNA synthetases achieve the necessary fidelity by post-transfer proofreading, a corrective hydrolytic event after transfer to tRNAPhe. Diadenosine 5',5'''-P1,P4-tetraphosphate synthesis is shown to be a common feature for phenylalanyl-tRNA synthetases from all three lineages of descent. The immunological approach demonstrates that aminoacyl-tRNA synthetases do not belong to the group of enzymes in gene expression with high structural conservation.

The sugar part of cellular glycoconjugates and specific endogenous sugar receptors, i.e., lectins, can establish a system of biological recognition based on protein-carbohydrate interactions. An assortment of labelled (neo)glycoproteins, carrying different types of sugar moieties, is synthesized to localize respective sugar receptors. With these tools, the histochemical patterns of endogenous carbohydrate-binding receptors of the epi-, peri-, and endoneurium were analyzed in human sural and accessory nerves and in swine sciatic nerve. This approach is complementary to the application of plant lectins, focusing on endogenous carbohydrate-binding proteins (lectins). In contrast to the epi- and endoneurium, which bound certain types of carbohydrates, such endogenous sugar receptors were histochemically not detectable in the perineurial cells. Moreover, no histochemical reaction was present in the "connective tissue septa" localized in the endoneurium in which the endoneurial vessels were embedded. This common property supplies evidence that these septa are composed of perineurial cells. They may represent a barrier in addition to the capillary endothelium. Our observations suggest histogenetical differences between the cell populations of epi- and endoneurium vs. perineurium. This significant difference in the ability to bind carbohydrate residues, conjugated to a carrier protein, is contradictory to the assumption that perineurial cells and fibroblasts are functional variants of the same cell type. The histochemical patterns of endogenous carbohydrate-binding receptors found in human and swine nerves were similar but not identical, with exception of the perineurium, reflecting phylogenetic differences in the expression of sugar-binding proteins. The absence of specific sugar receptors in perineurial cells, however, seems to be a more general phenomenon.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

The pattern of sugar inhibition of rosette formation, a model for intercellular interaction between cultured cells and glutaraldehyde-fixed, trypsinated rabbit erythrocytes, served to infer the presence of carbohydrate-binding proteins. This profile from cell extracts for the two murine macrophage-like cell lines, P388D1 and J774A.1, was comparatively analyzed by affinity chromatography on supports with immobilized carbohydrates (lactose, L-fucose, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, and maltose) or with the immobilized mannose-rich yeast glycoprotein mannan or fetuin-derived glycopeptides containing sialic acid residues. After elution with specific sugar in the absence of Ca2+ ions, the proteins were separated by sodium dodecyl sulfate - polyacrylamide slab gel electrophoresis. The composition of carbohydrate-binding proteins of the two lines clearly exhibited quantitative and qualitative differences. Moreover, the pattern of P388D1 cells was also demonstrated to change significantly in response to alterations in the conditions of the physiological environment. These alterations were imposed by in vitro growth, by subsequent in vivo growth in nude mice, and by re-adaptation of cells to culture after in vivo passage. Collectively, our observations and other physiological and biochemical reports on macrophage lectins indicate that the presence of sugar receptors with different specificities may be an indicator of macrophage differentiation, being reversibly modulated to a considerable extent by external factors, e.g., microenvironment. Extensive but selective alterations in this respect could play an important role in the control of recognition and effector mechanisms within diverse functions of macrophage subpopulations.

Protein-carbohydrate recognition has been found to play an important role in phagocytosis. Labelled (neo)glycoproteins were employed to comparatively analyze the histochemical pattern and ultrastructural localization of endogenous carbohydrate-binding proteins (lectins) of mononuclear macrophages and multinucleate giant cells involved in the granulomatous foreign body reaction. Sugar receptors having an affinity to simple alpha- and beta-galactoside-structures, to alpha-mannose residues, to N-acetylglucosamine, to N-acetylgalactosamine and to glucuronic acid, respectively, were detected in both cell types. However, alpha-fucoside- and beta-xyloside-specific receptors were present only in the mononuclear macrophages. Pronounced differences were seen with labelled, suitably modified glycoproteins, exposing different complex sugar parts with common beta-galactoside-termini. Among the population of multinucleate giant cells, a positive histochemical reaction was observed with mannose-6-phosphate-, galactose-6-phosphate- and glucuronic acid-(BSA-biotin), respectively, only in giant cells in which fusing mononuclear cells were recognizable. This transient expression indicates changes within the profile of endogenous sugar receptors in the stages from fusion to establishment of giant cells. Aside from the diffuse intracytoplasmic distribution of carbohydrate-binding proteins, a prominent accumulation of various types of glycosylated ferritin, used as a marker for electron microscopic evaluation, was ultrastructurally found in membranous subcellular structures and vesicles. This study is a basis for further investigation of the potential involvement of various sugar receptors in the process of macrophage fusion, resulting in multinucleate giant cells of foreign body type, and the process of phagocytosis.

Phenylalanyl-tRNA synthetase from beans (Phaseolus vulgaris) was purified 2 800-fold to homogeneity with a 16% overall yield by salting-out chromatography, salting-out affinity chromatography, gel filtration and chromatography on DEAE-cellulose and hydroxylapatite. This combination minimizes potentially harmful effects of proteinases and products of the secondary metabolism of a green plant during the early steps. The molecular mass is 260 000 Da with a subunit structure of alpha 2 beta 2 (alpha = 59 000, beta = 70 000 Da). Enzymatic activity was optimal with 20mM Mg2+ and 10mM KCl at pH 6.5 and pH 8.5, depending on the buffer substance. Kinetic measurements at low temperature and steady-state kinetics indicate that the esterification of tRNA or a step preceding it, but not the activation, are rate-determining at pH 7.65. The cognate tRNAPhe is exclusively aminoacylated at the 2'-OH group. tRNAs from Escherichia coli and bean chloroplasts are not aminoacylated. No immunological relationship of the plant enzyme to other phenylalanyl-tRNA synthetases was revealed by immuno-diffusion and immunotitration with polyclonal antibodies raised against the enzymes from E. coli, yeast and hen liver. ATP analogs revealed a unique pattern of substrate properties with indication of conservation of ATP binding in the form of an ATP-Mg2+ complex in the anti-conformation with a coordination of the cation to the nitrogen in position 7 of the purine moiety.

The expression of endogenous lectins was compared in mouse Lewis lung carcinomas growing in the kidney or subcutaneously. A panel of carbohydrates coupled to a biotinylated carrier molecule (bovine serum albumin), biotinylated desialylated glycoproteins and sulfated polysaccharides were used in histochemical assays to detect the presence and distribution of carbohydrate receptors. Heterogeneous staining patterns were observed with most carbohydrates in sections of tumor tissue from both anatomic sites, and staining intensities also varied within each section. At least 2 populations of cells were identified at each site, of which one had receptors for all carbohydrates, while the other had no receptors for melibiose, sialic acid, or alpha-glucosides (maltose and glucose). There were quantitative differences in expression of endogenous lectins by tumors growing s.c. or in the kidney; 3LL cells growing in the kidney bound 6 out of 10 carbohydrates to a greater extent than 3LL cells in s.c. tumors. Conversely, 3LL cells in s.c. tumors bound heparin and asialofetuin to greater extents than cancer cells in kidney tumors. Biochemical analyses of detergent extracts of tissues subjected to affinity chromatography and subsequent SDS-PAGE revealed quantitative and also qualitative differences in lectins between tumors growing in the 2 anatomic sites.

Endogenous lectins of normal tissues can play a functional role in recognition processes and cell adhesion. These functions are areas of particular relevance to tumor growth and metastasis. Our initial results on endogenous lectins of different tumors lead to the working hypothesis that the pattern of endogenous lectins is qualitatively and quantitatively different between different types of tumors and between tumors and normal, nonmalignant tissues. The endogenous lectins may thus prove to be potentially important in establishing a new concept for a rational lectin-based type of diagnosis and therapy of various tumors.

A tumor model system of clones of myeloproliferative sarcoma virus (MPV)-transformed rat fibroblasts (NRK) with different growth properties and metastatic potential was studied. The relationship between metastatic behavior and composition of carbohydrate-binding proteins (lectins) was analyzed by affinity chromatography. The metastatic variant differs qualitatively from its parental clone in the presence of galactoside-binding proteins at apparent molecular weight of 42 kDa. The alpha-glucosyl-binding proteins at apparent molecular weights of 67 kDa and 53 kDa and a galactoside-binding protein of apparent molecular weight of 34 kDa, however, are not detectable in the metastatic variant in comparison to its parental clone. In this respect the parental clone shows closer resemblance to the clone 5-8#1 with different growth properties and low metastatic potential than to its own metastatic variant. Furthermore, only the parental clone has a melibiose- and a mannan-binding protein of an apparent molecular weight of 64 kDa and 14 kDa, respectively. Rosette formation as model system for intercellular interaction reveals differences in the inhibition pattern with sugar between the two clones 5-8#1 and 5-20#20, whereas the metastatic variant 5-20#20 (s) exhibits drastically reduced capability to form rosettes. Initial experiments demonstrate the feasibility of drug targeting to transformed fibroblasts via carbohydrate-binding proteins.

The influence of chemical glycosylation on clearance of plant lectins, employed as radiopharmaceuticals, was assessed. Controlled chemical modification of the antibody to concanavalia ensiformis agglutinin did not affect its immunologic activity, but led to rapid clearance from circulation. In tumor-bearing mice, notable increases of selected tumor/non tumor ratios even after only 1 h of presence of the galactosylated antibody to the lectin in circulation were observed. Similarly, galactosylated antibody to pisum sativum agglutinin caused significant increases of tumor/non tumor ratios.

Aminoacyl-tRNA synthetases are indispensable components of protein synthesis in all three lines of evolutionary descent, eubacteria, archaebacteria and eukaryotes. Furthermore they are also present in the translational apparatus of the semi-autonomous organelles, mitochondria and chloroplasts, of the eukaryotic cell. Therefore aminoacyl-tRNA synthetases are appropriate objects for comparative molecular biology in order to obtain a comprehensive picture of the evolution of the translational process. The analysis of the phenylalanyl-tRNA synthetase in a large variety of organisms and organelles in this respect is the most advanced. In addition to comparison of quaternary structure, analysis includes functional aspects of accuracy mechanisms (proofreading) and comparison of structural features by means of substrate analogs. Evolutionary relationships are furthermore elucidated using the immunological approach and heterologous aminoacylation.

The presence and location of specific endogenous lectins in the anterior epithelia of the porcine eye were determined by histochemical staining using biotinylated glycosylated carrier molecules. Endogenous lectins with specificities for galactosides, alpha-mannosides and beta-xylosides were present in the conjunctival epithelium, but the corneal anterior epithelium appeared essentially free of histochemically demonstrable endogenous lectins. The corneal posterior epithelium contained a rich and complex set of endogenous lectins. The transitional area between conjunctival and corneal anterior epithelia at the limbus cornea contained, in striking contrast to either of the epithelia bordering it, cells expressing endogenous lectins in considerable wealth and variety.

Clinically applicable markers for tumor progression may be uncovered by selective analysis of biochemical parameters, supposedly participating in this complex process. Owing to the importance of specific protein-carbohydrate interactions in diverse biological processes, the pattern of the receptor part in this glycobiochemical recognition system, the sugar receptors (lectins), conceivably reflects biological properties of tumor cells in glycobiochemical terms. Therefore, we established and characterized xenografts from surgically removed specimens of a human primary colon adenocarcinoma and its metastatic lesions to liver of the same patient and of a histomorphologically similar primary colon adenocarcinoma of another patient in nude mice. Xenotransplantation and subsequent glycobiochemical analysis of material from early passages with a standardized procedure had been preferred to cell culture in monolayer on account of maintenance of a higher degree of organized histotypic assembly. Despite histomorphological similarities, the sugar receptor profile revealed significant differences in tumor-tumor and tumor-metastasis comparison, especially for alpha- and beta-galactoside-binding proteins. Tumor-metastasis differences were substantiated by a second successfully xenotransplanted pair of specimens. Comprehensive expansion of these initial data may eventually lead to desirable functional correlations with the different biological properties of histomorphologically similar primary colon adenocarcinomas and of the metastatic phenotype and to a rational development of therapeutic modalities to restrict tumor growth and spread.

Immunologically important mediators have been shown to exhibit ability to specifically bind distinct carbohydrates. This type of protein-carbohydrate interaction is one mechanism how to explain involvement of glycochemical interactions in regulatory processes. Interference of certain saccharides with murine multipotential colony-stimulating factor (multi-CSF)-dependent colony formation from progenitor cells in semisolid agar raised evidence for similar potential involvement of protein-carbohydrate interactions. Affinity depletion of conditioned WEHI-3B-medium on resins, bearing saccharides that have been elucidated to be effective inhibitors (mannose and lactose), resulted in preparations with significantly reduced capability to sustain development and proliferation. Sequence comparison of multi-CSF to carbohydrate-binding proteins (lectins) with this specificity failed to uncover extended homologies in diagonal plots. But detailed sequence alignments revealed confined, high-scoring stretches of homology between various lectins and two types of CSF. These results prove the importance of protein-carbohydrate interactions in stem cell proliferation.

Three 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.

Discoidin I, a developmentally regulated lectin in Dictyostelium discoideum, has been implicated in cell-substratum adhesion and ordered cell migration during aggregation. This depends on the cell binding site of discoidin I, which is distinct from its carbohydrate binding site. We have isolated a receptor for the cell binding site by affinity chromatography. The receptor binds immobilized discoidin I in the presence of 0.3 M galactose and can be eluted with gly-arg-gly-asp-his-asp, a synthetic peptide the sequence of which is found in discoidin I, and which blocks cell migration into aggregates. The receptor is a developmentally regulated cell-surface glycoprotein of apparent Mr approximately 67,000. Univalent antibodies specific for this glycoprotein block aggregation.

Chloroplastic phenylalanyl-tRNA synthetase from bean leaves is purified under optimal protective conditions over 4,900-fold. Its apparent molecular weight is 78,000, as determined by gel filtration, with a dimeric subunit structure of alpha beta (alpha = 33,000 and beta = 42,000), as determined by sodium dodecyl sulfate gel electrophoresis. This indicates a drastic size reduction of 40% for each subunit compared to the corresponding cytoplasmic enzyme and a unique quaternary structure. Heterologous aminoacylation and substrate properties of ATP analogs indicate substantial differences in the topographies of the substrate binding domains of these two heterotopic intracellular plant enzymes. No common antigenic determinants with the bean cytoplasmic enzyme were detected by polyclonal antibodies against the chloroplastic enzyme. The same negative result applies to the immunological comparison with the partially purified enzymes from the cyanobacterium Anacystis nidulans and the photosynthetic green sulfur bacterium Chlorobium limicola that both have a molecular weight of 260,000.

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.

Biotinylated (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. alpha-Fucoside-, beta-galactoside-, alpha-mannoside- and beta-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 analplastic 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 alpha-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 meningioma, 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.

Biotinylation of chemically glycosylated bovine serum albumin, yielding a panel of neoglycoproteins, and of desialylated, naturally occurring glycoproteins allowed to systematically evaluate presence and distribution of various types of endogenous sugar receptors in the sections of human glioblastomas and gangliocytomas by a routine histochemical procedure. Pronounced cytoplasmic staining with markers, carrying constituents of natural glycoconjugates, e.g. for beta-galactoside-specific receptors, contrasted with the different intensities, noticed for alpha- and beta-glucoside-specific receptors. Significant qualitative differences between the two tumor types were detected with N-acetyl-D-galactosamine- and sialic acid-carrying probes. Nuclear staining with only a part of the applied panel underscored the specificity of the protein-carbohydrate interaction. Fine structural features of the synthetic neoglycoproteins, e.g. the mode of coupling of the carbohydrate moiety to the protein, were found to exert a significant influence on their suitability as histochemical markers. On the basis of the histochemical results, exemplary biochemical analysis of certain classes of endogenous sugar receptors by affinity chromatography and subsequent gel electrophoresis, namely of beta-galactoside-, alpha-fucoside-, alpha-mannoside- and alpha-glucoside-specific proteins, revealed presence and characteristics of respective sugar receptors that can contribute to the histochemical staining. Similar extent of histochemical staining with the respective probes notwithstanding, the different tumor types exhibited qualitative differences in the expression of individual endogenous sugar receptors. The combined histochemical and biochemical analysis is supposed to be of conspicuous value for biological and clinical investigations on endogenous sugar receptors.

Binding of carbohydrate moieties was detected in tissue sections of human breast by employing two types of labeled ligands: neoglycoproteins (chemically glycosylated, histochemically inert carrier protein) and desialylated naturally occurring glycoproteins. Paraffin-embedded, formalin-fixed sections from 40 benign and malignant breast lesions were examined for the presence and distribution of endogenous sugar receptors, employing a panel of 13 biotinylated neoglycoproteins, representing carbohydrates commonly found in naturally occurring glycoconjugates, and four biotinylated glycoproteins. Benign and malignant breast lesions revealed staining with mannosylated carrier neoglycoprotein in comparison to normal breast. A mixed pattern of staining localization and intensity was seen for different types of malignancy with this neoglycoprotein. Similarly, receptors for lactose and N-acetylglucosamine could only be detected within the cytoplasm for certain types of malignancy. Their nuclear localization, however, could also be seen in normal breast specimens. The extent of staining with different glycoproteins, containing different types of galactoside-terminal sugar chains, also appeared to differ between various types of breast cancer. The detection of endogenous sugar receptors by neoglycoproteins is proposed to contribute to an understanding of malignancy-associated alterations in the structure of their potential physiological ligands, the glycoconjugates. Changes in the structure and abundance of such glycoconjugates have commonly been detected with the use of plant lectins in histopathologic studies.

Fractionation of detergent extracts of transplanted tumors of human teratocarcinoma cells by affinity chromatography yields one predominant protein with apparent molecular weight of 14,000 and further, for less abundant protein with apparent molecular weight of 35,000 from lactose-sepharose and one protein with apparent molecular weight of 68,000 from mannan-sepharose. No further carbohydrate-binding protein can be isolated on columns derivatized with asialofetuin, melibiose and L-fucose, to which the extract is applied successively. Both proteins agglutinate trypsinized, glutaraldehyde-fixed rabbit erythrocytes in the absence of Ca2+ and can thus be defined as endogenous human teratocarcinoma lectins. Inhibition of heterotypic and homotypic aggregation of human teratocarcinoma cells by D-mannose, D-galactose and glycoproteins rich in one of these sugars is consistent with a functional role of these Ca2+-independent lectins in cell aggregation. Visualization of these activities by fluorescent mannosylated and lactosylated markers on the cell surface further supports the cell surface localization of these detergent extractable lectins. The mannan-specific lectin, in particular, has so far not been detected in any mammalian tissue or tumor and is of potential value for a lectin-based diagnosis and therapy of embryonal carcinomas.

Protein-carbohydrate interactions are supposed to play a pivotal role in mediation of recognitive interactions, relevant to cellular interactions and transport. The brain microvasculature is the site of numerous cell-cell and cell-matrix recognitive interactions. Assuming carbohydrate-protein interactions play important physiological roles here, then specific carbohydrate-binding proteins should be prominent components of this microvasculature, in addition to the wealth of endogenous glycoconjugates reported by other authors. Human brain and brain tumor microvessels were analyzed histochemically for expression of endogenous sugar-binding proteins using a panel of biotin-conjugated, chemically glycosylated probes with specificities for alpha-/beta-D-galactosides and -D-glucosides, alpha-L-fucosides, alpha-D-mannosides, and beta-D-xylosides, and for expression of endogenous glycoconjugates using a panel of biotin-conjugated plant lectins with specificities for fucoside, galactoside, mannoside and glucoside moieties. Wax-embedded aldehyde- or Bouin-fixed tissues or acetone-fixed frozen sections were examined. Blood-brain barrier function was checked by ascertaining immunohistochemically the extravasation of serum albumin in these tissues. Microvessels in normal human brain tissues (with intact blood-brain barrier) contained abundant endogenous sugar-binding proteins with specificities for beta-galactosides, alpha-mannosides and beta-xylosides, and lesser amounts of proteins binding alpha-galactosides, alpha-fucosides and glucosides. Endogenous glycoconjugates bearing beta-galactoside, alpha-D-mannoside/alpha-D-glucoside or alpha-L-fucoside moieties were also abundant. In brain tumors (with defective blood brain barrier) the microvessels contained altered patterns of endogenous sugar-binding proteins, particularly noticeable in glioblastomas, in which there were notable alterations in galactoside-binding proteins in the microvessels.