Hans-J. Gabius

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

SummaryMolecular 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 physicologically valid conclusions in this field for mammalian tissues. Consequently, experiments were prompted to employ the abundant β-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 β-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. Overall, the introduction of biotinylated mammalian lectins as well as the lectin-binding glycoproteins will aid to critically evaluate the physiological significance of the glycobiological interplay between endogenous lectins and distinct carbohydrate parts of cellular glycoconjugates.

Endogenous sugar-binding proteins in human breast tissue and benign and malignant breast lesions

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‐acetyl‐glucosamine 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.

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.

Targeting of neoglycoprotein-drug conjugates to cultured human embryonal carcinoma cells

SummaryFluorescent neoglycoproteins were used to screen for the presence and sugar specificities of cell surface lectins in two human embryonal carcinoma cell lines. Efficient labeling correlated with extent of lectin-mediated uptake of neoglycoproteins, as measured by inhibition of DNA synthesis by drug-neoglycoprotein conjugates. These conjugates contain covalently linked carbohydrate moieties on the carriers to render them accessible to the membrane lectins, most effectively galactosides and α-glucosides. They furthermore contain chemically linked cytotoxic drugs (etoposide, cis-diamminedichloroplatinum II and methotrexate) which are intracellularly released after lysosomal breakdown of the carrier, as indicated by the effect of leupeptin. Sugars can confer a greater than 10-fold increase in cytotoxic capacity to the nonglycosylated carrier-drug conjugate, nearly reaching the level of toxicity of the freely diffusible drug. Two different neoglycoproteins, reacting with independently targeted membrane lectins, were shown to be useful in a model for combination chemotherapy. These results therefore suggest potential usefulness of custom-made glycosylated carriers in the targeting of therapeutic agents to human embryonal carcinoma cells.

Comparative Histochemical and Biochemical Analysis of Endogenous Receptors for Glycoproteins in Human and Pig Peripheral Nerve

Abstract: Endogenous sugar‐binding proteins were localized in sections of human and pig peripheral nerves by the application of two types of labelled ligands: neoglycopro‐teins (chemically glycosylated carrier proteins that had proven to be histochemically inert) and desialylated, naturally occurring glycoproteins. These proteins allowed evaluation of the presence and distribution of endogenous receptors for carbohydrates, commonly present in cellular glycoconjugates. (Neo)glycoprotein binding was similar, but not identical, for the two types of mammalian peripheral nerves. The pig nerve differed from the human nerve in more pronounced staining when using different types of β‐galactoside‐terminated (neo)glycoproteins and charge‐carrying neoglycoproteins, such as bovine serum albumin, bearing galactose‐6‐phosphate residues, glucuronic acid residues, and sialic acid residues. Comparative biochemical analysis of certain classes of sugar receptors by affinity chromatography and gel electrophoresis revealed the presence of sugar receptors that can contribute to the histochemical staining in a pattern with certain significant differences among rather similar expression for the two species. The assessment of sugar receptor distribution by application of (neo)glycoprotein binding among morphologically defined regions in nerves may hold promise in detecting developmental regulation and changes during nerve degeneration and subsequent regeneration after trauma or pathological states. Correlation of these results to changes in the structure and abundance of glycoconjugates, which are the potential physiological ligands of endogenous sugar receptors commonly detected by plant lectins, may help to infer functional relationships.

Are there Characteristic Alterations of Lung Tissue Associated with Crohn's Disease?

Two male patients aged 12 and 31 years suffered from Crohns disease for more than six years and were treated with Cortison for more than four years. Surgical excision of parts of the terminal ileum was performed in both patients. They suffered from pulmonary symptoms as dyspnoea, shortness of breath and ventilation disturbances two years after operation. Wedge biopsies of the lungs revealed the following histomorphological findings: 1. Granulomatous interstitial lymphocyte infiltrates 2. Acute alveolitis with severe dysplasia of pneumocytes 3. Moderate interstitial fibrosis. Immunohistology performed in one case showed predominantly lambda chains expressed by lymphocytes associated with IgA and IgM. IgG was missing, furthermore kappa chains could not be detected. Macrophages contained endogenous lectins (sugar receptors) for fucose, maltose, and N-acetyl-D-glucosamine (glcNAc). No receptors specific for mannose, lactose, and heparin could be found. Pneumocytes did not bind the neoglycoproteins but were found to express HLA-DR receptors detectable by the monoclonal antibody LN 3 in dysplastic pneumocytes only. The histomorphological and immunohistochemical findings suggest that the analyzed alterations of lung tissue are related to the underlying disease of enteritis regionalis.

Lineage- and differentiation-dependent alterations in the expression of receptors for glycoconjugates (lectins) in different human hematopoietic cell lines and low grade lymphomas

SummaryImportant 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, P 12; the multipotent leukemic line, K 562 and the promyelocytic line, HL 060. Biochemical analyses were performed using affinity chromatography on supports with immobilized lactose and asialofetuin (simple or complex β-galactosides), melibiose (α-galactoside), fucose, N-acetyl-D-galactosamine, maltose (α-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 these 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 HL 60 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.

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.

Endogenous tumor lectins: A new class of tumor markers and targets for therapy?

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.

Localization of Endogenous β-Galactoside-Specific Lectins by Neoglycoproteins, Lectin-Binding Tissue Glycoproteins and Antibodies and of Accessible Lectin-Specific Ligands by Mammalian Lectin in Human Breast Carcinomas

Protein-carbohydrate interactions constitute a system of molecular interaction with relevance to pathologic conditions. Carrier-immobilized carbohydrate structures enable the histochemical investigation of the protein part of this recognitive system. However, thorough systematic studies are inevitably required for standardized application of this relatively novel class of markers. Consequently, serial sections of 21 cases of malignant breast lesion were comparatively analyzed with three different types of probe, specific for beta-galactoside-binding lectins. In addition to the chemically lactosylated neoglycoprotein, human lectin-binding glycoproteins, purified by affinity chromatography on resins with an immobilized beta-galactoside-specific lectin, and a lectin-specific antibody were employed to answer the question whether differences occur in their capacity for lectin localization. The patterns of staining were qualitatively similar, the lectin-binding glycoproteins yielding the most intense reaction. Having assured the reliable applicability of the neoglycoprotein, structural alterations of the subterminal carbohydrate residue on the labelled carrier addressed the issue, whether selectivity of binding can be inferred histochemically, allowing rational synthetic tailoring. An N-acetylglucosamine residue in beta-1,3-linkage proved to be a less favorable extension than this type of sugar in beta-1,4-linkage or an N-acetylgalactosamine moiety in beta-1,3-linkage. Binding was clearly reduced in cells of normal breast tissue with this probe. In order to gain evidence on the expression of potential carbohydrate ligands for the glyco- and immunohistochemically localized binding activity, a labelled mammalian beta-galactoside-specific lectin was similarly used as histochemical tool. It effectively bound to accessible sites in the sections. The binding pattern was different to that of plant lectins with specificity to beta-galactosides. This result underscores that caution is necessary in the functional interpretation of results of studies with plant, not mammalian lectins.(ABSTRACT TRUNCATED AT 250 WORDS)