Biomedical subjects

H Kaltner

Publications and source records attributed to H Kaltner.

At least 37 records · Page 2Linked to original sources

PubMed2001

Galectins are differentially expressed in supratentorial pilocytic astrocytomas, astrocytomas, anaplastic astrocytomas and glioblastomas, and significantly modulate tumor astrocyte migration.

Galectins, a family of mammalian lectins with specificity to beta-galactosides, are involved in growth-regulatory mechanisms and cell adhesion. A relationship is assumed to exist between the levels of expression of galectins and the level of malignancy in human gliomas. A comparative study of this aspect in the same series of clinical samples is required to prove this hypothesis. Using computer-assisted microscopy, we quantitatively characterized by immunohistochemistry the levels of expression of galectins-1, -3 and -8 in 116 human astrocytic tumors of grades I to IV. Extent of transcription of galectins-1, -3, and -8 genes was investigated in 8 human glioblastoma cell lines by means of RT-PCR techniques. Three of these cell lines were grafted into the brains of nude mice in order to characterize in vivo the galectins-1, -3 and -8 expression in relation to the patterns of the tumor invasion of the brain. The role of galectin-1, -3 and -8 in tumor astrocyte migration was quantitatively determined in vitro by means of computer-assisted phase-contrast videomicroscopy. The data indicate that the levels of galectin-1 and galectin-3 expression significantly change during the progression of malignancy in human astrocytic tumors, while that of galectin-8 remains unchanged. These three galectins are involved in tumor astrocyte invasion of the brain parenchyma since their levels of expression are higher in the invasive parts of xenografted glioblastomas than in their less invasive parts. Galectin-3, galectin-1, and to a lesser extent galectin-8, markedly stimulate glioblastoma cell migration in vitro. Since bands for the transcripts of human galectins-2, -4 and -9 were apparently less frequent and intense in the 8 human glioblastoma cell lines, this system provides an excellent model to assign defined roles to individual galectins and delineate overlapping and distinct functional aspects.

Adolescent↗

PubMed2001

Detection of galectin-3 in tear fluid at disease states and immunohistochemical and lectin histochemical analysis in human corneal and conjunctival epithelium.

BACKGROUND/AIM: Components of the tear fluid contribute to the biochemical defence system of the eye. To reveal whether the immune mediator and lipopolysaccharide binding galectin-3 is present in tears, tear samples were collected from eyes in healthy and pathological states. Investigation of expression of galectin-3 and galectin-3 reactive glycoligands in normal human conjunctival and corneal epithelia was also initiated as a step to understand the role of galectin-3 in ocular surface pathology. METHODS: Immunoblot analysis using either a rabbit polyclonal or a mouse monoclonal antibody against galectin-3 was employed to detect galectin-3 in tear fluid. Galectin-3 expression in tissue specimens was detected by immunocytochemistry employing A1D6 mouse monoclonal antibody, and galectin-3 reactive glycoligands were visualised by lectin histochemistry using labelled galectin-3. RESULTS: Galectin-3 was found only in tears from patients with ocular surface disorders. It was expressed in normal corneal and conjunctival epithelia but not in lacrimal glands. Inflammatory leucocytes and goblet cells found in galectin-3 containing tear fluid also expressed galectin-3. Galectin-3 binding sites were detected on the surface of conjunctival and corneal epithelial cells co-localising with desmoglein. CONCLUSIONS: This study revealed expression of galectin-3 in tear fluid obtained from patients with eye diseases. The role of this endogenous lectin (produced by inflammatory as well as epithelial cells) in antimicrobial action and inflammation modulation could be expected.

Antigens, Differentiation↗

PubMed2001

Towards defining the role of glycans as hardware in information storage and transfer: basic principles, experimental approaches and recent progress.

The term 'code' in biological information transfer appears to be tightly and hitherto exclusively connected with the genetic code based on nucleotides and translated into functional activities via proteins. However, the recent appreciation of the enormous coding capacity of oligosaccharide chains of natural glycoconjugates has spurred to give heed to a new concept: versatile glycan assembly by the genetically encoded glycosyltransferases endows cells with a probably not yet fully catalogued array of meaningful messages. Enciphered by sugar receptors such as endogenous lectins the information of code words established by a series of covalently linked monosaccharides as letters for example guides correct intra- and intercellular routing of glycoproteins, modulates cell proliferation or migration and mediates cell adhesion. Evidently, the elucidation of the structural frameworks and the recognition strategies within the operation of the sugar code poses a fascinating conundrum. The far-reaching impact of this recognition mode on the level of cells, tissues and organs has fueled vigorous investigations to probe the subtleties of protein-carbohydrate interactions. This review presents information on the necessarily concerted approach using X-ray crystallography, molecular modeling, nuclear magnetic resonance spectroscopy, thermodynamic analysis and engineered ligands and receptors. This part of the treatise is flanked by exemplarily chosen insights made possible by these techniques.

Cell Communication↗

PubMed2001

Immunohistochemical profile of galectin-8 expression in benign and malignant tumors of epithelial, mesenchymatous and adipous origins, and of the nervous system.

This study aims to investigate whether the immunohistochemical expression of galectin-8 could be used as a diagnostic marker in tumor tissues of various histogenetic origins including specimens from epithelial (n=145), mesenchymatous (n=16), adipous (n=10) and central and peripheral nervous system (n=25) tissue, and 4 mesotheliomas. Immunohistochemical reactions were carried out with a polyclonal anti-galectin-8 antibody and histological slides from tissues derived from the files of the Laboratory of Anatomopathology of University Erasmus Hospital, Brussels. Formalin-fixed paraffin-embedded tissues of 45 normal cases as well as 41 benign and 114 malignant tumors were studied. Marked decreases in immunohistochemical galectin-8 expression were observed in colon (p=0.001), pancreas (p=0.007), liver (p=0.0008), skin (p=0.002) and larynx (p=0.02) tissue when comparing malignant tissue to normal tissue and/or benign tumors. The reverse relationship was observed for breast tissue (p=0.007). No statistically significant differences (p>0.05) were detected when comparing normal tissue and/or benign to malignant tumors in lung, bladder, kidney, prostate and stomach tissue. Significant galectin-8 expression was also measured in non-epithelial tissue including tumors of the central and peripheral nervous system as well as in skeletal muscle and mesotheliomas. Immunohistochemical monitoring of galectin-8 thus reveals an organ-type-dependent regulation of expression upon malignant transformation of various tissue types of epithelial origin. This observation will prompt further studies to delineate any relationship with prognosis.

Adipose Tissue↗

PubMed2001

Expression of galectin-3-reactive ligands in squamous cancer and normal epithelial cells as a marker of differentiation.

The definition of biological markers for oropharynx and larynx cancer is essential to predict their clinical behavior. Since cellular glycans play an important role in biological information transfer, we have employed an endogenous lectin, galectin-3, to examine in primary squamous carcinomas, lymph node metastases, and physiological squamous epithelia whether glycans recognized by this lectin are altered in relation to the state of differentiation. The expression of galectin-3 was concomitantly evaluated by immunohistochemistry using the A1D6 monoclonal antibody. In addition, other antibodies were used for the detection of cytokeratins and desmosomal proteins (desmoplakin-1 and desmoglein). The results show the expression of galectin-3-reactive ligands in moderately/highly differentiated carcinomas only in areas exhibiting a high level of keratinization. Except for one patient out of 14, metastatic cells in lymph nodes expressed no accessible binding sites for galectin-3. No galectin-3-reactivity was detected in the basal cell layer of all studied normal epithelia (which contains the proliferating cells). The suprabasal layers were positive in epidermis and epithelium of tongue and cornea and negative in epithelium of palatine tonsil. The tumor cells expressed galectin-3 with an intensity positively correlated with tumor differentiation. The position of galectin-3-reactive sites colocalized with the two tested desmosomal proteins. However, presence of these proteins was also detected in areas of tumor and suprabasal layers of tonsil epithelium where no binding reactivity for galectin-3 was found. The present study showed that expression of galectin-3-reactive glycoligands is differentiation-dependent in normal as well as malignant squamous cells. Colocalization of galectin-3-reactive sites with desmosomal proteins (desmoplakin-1 and desmoglein) suggests an association of the galectin-3 ligand(s) with the cell surface, pointing to a potential participation of galectin-3 in mediation of intercellular contacts in these tumor types.

Antigens, Differentiation↗

PubMed2000

Differences in zero-force and force-driven kinetics of ligand dissociation from beta-galactoside-specific proteins (plant and animal lectins, immunoglobulin G) monitored by plasmon resonance and dynamic single molecule force microscopy.

Protein-carbohydrate interactions are involved in diverse regulatory processes. To help understand the mechanics and kinetics of dissociation of receptor-ligand complexes, we have analyzed the separation of lactose and the N-glycan chains of asialofetuin (ASF) from three lectins and an immunoglobulin G fraction by surface plasmon resonance at zero force and by atomic force microscopy with variations of the external force. While the (AB)2 agglutinins from Ricinus communis (RCA) and Viscum album (VAA) show structural homology, the homodimeric galectin-1 from bovine heart (BHL) has no similarity to the two plant lectins except for sharing this monosaccharide specificity. The beta-galactoside-binding immunoglobulin G (IgG) fraction from human serum provides a further model system with distinct binding-site architecture. The k(off) constants for the two plant agglutinins were independent of the nature of the ligand at 1.1-1.3 x 10(-3) s(-1), whereas the geometry of ligand and binding site presentation affected this parameter for BHL (0.5 x 10(-3) s(-1) for lactose and 1 x 10(-3) s(-1) for ASF) and IgG (1.3 x 10(-3) s(-1) for lactose and 0.55 x 10(-3) s(-1) for ASF). When assessing comparatively the rupture forces at a loading rate of 3 nN/s with lactose as ligand, 34 +/- 6 pN (BHL), 36 +/- 4 pN (IgG), 47 +/- 7 pN (VAA), and 58 +/- 9 pN (RCA) were measured. For the same loading rate the rupture forces for the receptor-ASF interactions were found to be 37 +/- 3 pN (BHL), 43 +/- 5 pN (VAA), 45 +/- 6 pN (IgG), and 65 +/- 9 pN (RCA). The variation of the pulling velocity revealed in all cases a linear dependence between the rupture force and the natural logarithm of the loading rate. Performing probability density and Monte Carlo calculations, the potential barrier widths, which determine the inverse dynamic dependence with the rate of force elevation, increased from 4 A (RCA) and 7 A (VAA and IgG) to 10 A (BHL) for the receptor-lactose interactions. Presenting ASF as ligand potential widths of 4 A for RCA and IgG and 6 A for VAA and BHL were obtained. Since the dissociation kinetics at zero force apparently cannot predict the behavior in force-driven experiments, these results reveal new insights into biological functions. The dissociation kinetics under force helps to explain the difference in the toxic potency of VAA and RCA and points to a function of the galectin in cis-crosslinking and in transient trans-bridging.

Animals↗

PubMed2000

A new combined computational and NMR-spectroscopical strategy for the identification of additional conformational constraints of the bound ligand in an aprotic solvent.

This study documents the feasibility of switching to an aprotic medium in sugar receptor research. The solvent change offers additional insights into mechanistic details of receptor--carbohydrate ligand interactions. If a receptor retained binding capacity in an aprotic medium, solvent-exchangeable protons of the ligand would not undergo transfer and could act as additional sensors, thus improving the level of reliability in conformational analysis. To probe this possibility, we first focused on hevein, the smallest lectin found in nature. The NMR-spectroscopic measurements verified complexation, albeit with progressively reduced affinity by more than 1.5 orders of magnitude, in mixtures of up to 50% dimethyl sulfoxide (DMSO). Since hevein lacks the compact beta-strand arrangement of other sugar receptors, such a structural motif may confer enhanced resistance to solvent exchange. Two settings of solid-phase activity assays proved this assumption for three types of alpha- and/or beta-galactoside-binding proteins, that is, a human immunoglobulin G (IgG) subfraction, the mistletoe lectin, and a member of the galectin family of animal lectins. Computer-assisted calculations and NMR experiments also revealed no conspicuous impact of the solvent on the conformational properties of the tested ligands. To define all possible nuclear Overhauser effect (NOE) contacts in a certain conformation and to predict involvement of exchangeable protons, we established a new screening protocol applicable during a given molecular dynamics (MD) trajectory and calculated population densities of distinct contacts. Experimentally, transferred NOE (tr-NOE) experiments with IgG molecules and the disaccharide Gal'alpha1-3Galbeta1-R in DMSO as solvent disclosed that such an additional crosspeak, that is, Gal'OH2--GalOH4, was even detectable for the bound ligand under conditions in which spin diffusion effects are suppressed. Further measurements with the plant lectin and galectins confirmed line broadening of ligand signals and gave access to characteristic crosspeaks in the aprotic solvent and its mixtures with water. Our combined biochemical, computational, and NMR-spectroscopical strategy is expected to contribute notably to the precise elucidation of the geometry of ligands bound to compactly folded sugar receptors and of the role of water molecules in protein--ligand (carbohydrate) recognition, with relevance to areas beyond the glycosciences.

Antimicrobial Cationic Peptides↗

PubMed2000

An enzyme-linked lectin assay for alpha1,3-galactosyltransferase.

UDP-Gal:Galbeta1-4GlcNAc alpha1,3-galactosyltransferase (alpha3GalT) is responsible for the synthesis of carbohydrate xenoantigen Galalpha1-3Galbeta1-4GlcNAc. In this work a convenient and sensitive assay system for quantification of alpha3GalT activity by enzyme-linked lectin assay (ELLA) with colorimetric detection is described. Microtiter plate wells whose surface had been coated with the polyacrylamide conjugate of the disaccharide Galbeta1-4GlcNAc (acceptor) are incubated with alpha3GalT in the presence of "cold" UDP-Gal as glycosyl donor. Formation of product by enzymatic extension of the glycan chain is detected by the biotinylated plant lectin Viscum album agglutinin. The standard curve for correct quantification of alpha3GalT activity is completed after running standard assays with no (background) or known quantities of enzyme activity. Product formation detected in this manner is proportional to enzyme activity and the concentrations of the acceptor and the glycosyl-donor UDP-Gal. In accordance with the known specificity of alpha3GalT, no enzymatic conversion of Le(x) into GalalphaLe(x) was observed using this assay. Human alphaGal antibodies were isolated using a disaccharide-exposing affinity adsorbent and their specificity was studied. Relative to the application of these natural immunoglobulins as product-detecting tool, the ELLA proved to be more sensitive.

Animals↗

PubMed2000

Glycohistochemical characteristics of nasal polyps from patients with and without cystic fibrosis.

OBJECTIVE: To investigate whether cystic fibrosis (CF)-related nasal polyps exhibit significantly distinct glycohistochemical characteristics when compared with single vs massive nasal polyps obtained from patients without CF. DESIGN: Glycohistochemical characteristics were identified by means of 8 histochemical probes, including 5 plant lectins (peanut, gorse seed, wheat germ, Maackia amurensis, and Sambucus nigra agglutinins), 2 animal lectins (14- and 16-kd galectins), and 1 neoglycoprotein (exposing the Thomsen-Friedenreich antigen). The binding of the 8 glycohistochemical markers was determined by means of computer-assisted microscopy. For each probe, 3 quantitative parameters were computed: the labeling index, which describes the percentage of tissue area specifically stained by a given marker; the mean optical density, which reflects the staining intensity; and the concentrational heterogeneity, which characterizes the level of heterogeneity of the staining intensity. SUBJECTS: A series of 61 nasal mucosa specimens was analyzed, including 6 normal cases, 23 single and 18 massive polyposis cases without CF, and 14 nasal polyps associated with CF. RESULTS: Normal and polyposal nasal mucosa differed in terms of the amounts and linkage types of sialic acids (revealed by the wheat germ, M amurensis, and S nigra agglutinins) rather than the characteristics of galactoside expression (monitored with the peanut agglutinin and 2 animal galectins). In contrast, nasal polyps markedly differed between patients with and without CF with respect to galactoside expression (revealed by the peanut agglutinin and the 14-kd galectin) and the display of binding site(s) for the neoglycoprotein. CONCLUSION: Normal and polyposal nasal mucosa differ essentially in sialic acid presentation, while nasal polyps from patients with CF have a higher level of various lectin-reactive galactoside residues than nasal polyps from those without CF.

Cystic Fibrosis↗

PubMed2000

Benign metastasizing leiomyoma of the uterus: documentation of clinical, immunohistochemical and lectin-histochemical data of ten cases.

The clinical histories of 10 women suffering from benign metastasizing leiomyoma (BML) after hysterectomy and information on lung lesions detected in these women are presented, together with corresponding data for 2 women with metastasizing leiomyosarcoma of the uterus for comparison: gross appearance, survival, and light microscopical, immunohistochemical and lectin-histochemical findings are reported. All patients with BML had undergone hysterectomy for uterus leiomyomatosus without any detection of sarcomatous lesions in the uterus wall. After a median period of 14.9 years intrapulmonary masses were detected by imaging techniques. On average, six nodules with a mean diameter of 1.8 cm were seen. Resection of the lesions was performed in all cases. The immunohistochemical and lectin-histochemical examination of the tumors included analysis of the proliferation-associated protein Ki-67, the p53 protein, estrogen and progesterone receptor, sarcolectin as an indicator of the presence of lymphokine macrophage migration inhibitory factor, antibodies and the labeled protein to assess galectin (galactoside-binding animal lectin)-dependent parameters, analysis of tumor vascularization (CD-34), and expression of bcl-2, vimentin, smooth muscle actin, desmin, and keratin. The lesions were characterized by low proliferation activity of 2.9% (measured with Ki-67), frequent hormone receptor expression (8 of the 10 cases presented hormone-specific receptors), low to moderate vascularization compared with metastases from the two uterine sarcomas, remarkable p53 overexpression and frequent expression of the lymphokine, the galectins and accessible binding sites. The median survival of the BML patients was 94 months after excision of the intrapulmonary lesions, and the maximum survival of the two sarcoma patients was 22 months. The results recorded in this patient sample with the methodology applied suggest that benign metastasizing leiomyomas are a slow-growing variant of leiomyosarcoma of the uterus, which becomes clinically apparent at a young age and progresses with low velocity.

Adult↗

PubMed2000

Association of concentration of asbestos and asbestos-like fibers with the patient's survival and the binding capacity of lung parenchyma to galectin-1 and natural alpha-galactoside- and alpha-mannoside-binding immunoglobulin G subfractions from human serum.

Our aim in this study was to search for lung parenchyma alterations associated with asbestos and/or asbestos-like fiber concentration. This was done by means of immuno- or glycohistochemistry. The hot-ashing technique determined the asbestos and asbestos-like fiber concentrations in the lung tissues of 100 patients of whom 52 were treated for primary lung and 25 for secondary lung tumors; fiber concentration was also measured for 23 patients whose disease was benign. The results were correlated to smoking habits, survival of the patients, and expression of binding capacities for endogenous lectins, natural carbohydrate-binding and lectin-specific antibodies. The cohort with proven asbestos exposure revealed a mean fiber concentration 114 f/g compared to 95 f/g in the non-exposed group. An increased asbestos fiber concentration was correlated to galectin-1-binding and the presence of epitopes for natural immunoglobulin G subfractions with selectivity to alpha-galactosides and alpha-mannosides. The survival of patients with primary and secondary lung tumors was negatively associated with the fiber concentration. The data indicate that increased presence of asbestos is correlated with an alteration of defined glycohistochemical features of alveolar lining cells.

Adult↗

PubMed2000

Temporal and spatial regulation of expression of two galectins during kidney development of the chicken.

Organogenesis and the establishment of the mature phenotype require an interplay between diverse recognition systems. Concerning protein-carbohydrate interactions, galectins are known to be involved in several extra- and intracellular functions. Due to the occurrence of two avian galectins in liver (chicken galectin-16 CG-16) and intestine (chicken galectin-14; CG-14) with different developmental regulation. the questions addressed are to what extent and where these galectins are present during chicken kidney development. Using Western blot analysis, the presence of both activities in tissue extracts was ascertained. A solid-phase assay showed peak levels at day 12 followed by a decline. A histochemical analysis was carried out in combination with routine staining. Epithelial cells of the mesonephric proximal tubules were immunoreactive in the cytoplasm for CG-14 from day 5 of incubation onwards. Additionally, epithelial cells of the metanephric collecting ducts were stained. For CG-16 a rather similar pattern of staining was seen, additional positivity in early glomerular podocytes being notable. At the electron microscopical level, a diffuse staining for CG-14 was seen in the cytoplasm, whereas immunoreactivity for CG-16 was observed mainly in mitochondria. These results demonstrate quantitative differences in the developmental regulation of the two avian galectins with obvious similarities in the cell-type pattern but with a disparate intracellular localisation profile.

Animals↗

PubMed2000

Lectin-mediated drug targeting: selection of valency, sugar type (Gal/Lac), and spacer length for cluster glycosides as parameters to distinguish ligand binding to C-type asialoglycoprotein receptors and galectins.

PURPOSE: Common oligosaccharides of cellular glycoconjugates are ligands for more than one type of endogenous lectin. Overlapping specificities to beta-galactosides of C-type lectins and galectins can reduce target selectivity of carbohydrate-ligand-dependent drug targeting. The purpose of this study is to explore distinct features of ligand presentation and structure for design of cluster glycosides to distinguish between asialoglycoprotein-specific (C-type) lectins and galectins. METHODS: Extent of binding of labeled sugar receptors to two types of matrix-immobilized (neo)glycoproteins and to cells was evaluated in the absence and presence of competitive inhibitors. This panel comprised synthetic mono-, bi-, and trivalent glycosides with two spacer lengths and galactose or lactose as ligand part. RESULTS: In contrast to C-type lectins of hepatocytes and macrophages, bi- and trivalent glycosides do not yield a notable glycoside cluster effect for galectins-1 and -3. Also, these Ca2+-independent galactoside-binding proteins prefer to home in on lactose-bearing glycosides relative to galactose as ligand, while spacer length requirements were rather similar. CONCLUSIONS: Trivalent cluster glycosides with Gal/GalNAc as ligand markedly distinguish between C-type lectins and galectins. Undesired side reactivities to galectins for C-type lectin drug delivery will thus be minimal.

Asialoglycoproteins↗

PubMed2000

Characterization of ligands for galectins, natural galactoside-binding immunoglobulin G subfractions and sarcolectin and also of the expression of calcyclin in thyroid lesions.

The purpose of this study was to characterize ligands for galectins, natural galactoside-binding immunoglobulin G subfractions and sarcolectin and also the expression of calcyclin in various benign and malignant thyroid lesions. The extent of the binding of eight glycochemical probes was quantitatively assessed using computer-assisted microscopy on 76 thyroid lesions including 10 not-otherwise-specified multinodular goiters (S_MNG), 11 multinodular goiters with adenomatous hyperplasia (AH_MNG), 8 normomacrovesicular (NM_ADE) and 12 microvesicular (MIC_ADE) adenomas, and 9 papillary (P_CAR), 10 follicular variants of papillary (FvarP_CAR), 7 follicular (F_CAR) and 9 anaplastic (A_CAR) carcinomas. The 8 histochemical probes included 5 animal lectins (including galectins and sarcolectin), 1 polyclonal antibody (raised against calcyclin) and 2 immunoglobulin G subfractions from human serum with selectivity to alpha- and beta-galactosyl residues. The results show that multinodular goiters with adenomatous hyperplasia exhibited histochemical characteristics intermediate to those of normal multinodular goiters and microvesicular adenomas. Normomacrovesicular adenomas behaved very distinctly from microvesicular ones. Microvesicular adenomas were more closely related to differentiated thyroid carcinomas than any other type of benign thyroid lesions of epithelial origin. Papillary and follicular carcinomas seemed to represent the two extremes of the same biological entity with the follicular variant of the papillary carcinoma serving as a biological link between these two extremes. Anaplastic carcinomas behaved in a significantly different manner when compared to the differentiated forms of thyroid carcinomas. The results suggest that the patterns of expression of the glycoconjugates investigated in the present study may constitute useful tools for characterizing lesions in the human thyroid.

Adenocarcinoma↗

PubMed2000

Human epidermal Langerhans cells are selectively recognized by galectin-3 but not by galectin-1.

Langerhans cells are dendritic antigen-presenting cells residing predominantly in the epidermis. Since endogenous galactoside-binding lectins with the jelly-roll motif (galectins) are known to trigger cellular responses, including mediator release, we investigated by lectin histochemistry the cells' capacity to bind two common members of this family, i.e. galectin-1 and -3. Actually, surrounding keratinocytes express a high level of galectin-3, and these cells can be considered as donors of this lectin to Langerhans cells. Employing biotinylated galectin-1 and -3, and concomitantly an antibody against CD1a as a second marker, to visualize the position of Langerhans cells in the human epidermis, the expression of galectin-3-reactive glycoligands in contrast to the lack of binding of galectin-1 was observed. Although the functional consequences of this selectivity are unclear, these results reveal an example for differential cellular reactivity towards two related endogenous lectins.

Adult↗

PubMed2000

Endogenous lectins (galectins-1 and -3) as probes to detect differentiation-dependent alterations in human squamous cell carcinomas of the oropharynx and larynx.

Expression of glycan determinants for in situ binding is the prerequisite for a productive protein (lectin)-carbohydrate recognition. Labeled tissue lectins as tools are preferable to plant lectins to assess this parameter, because plant and animal lectins with identical saccharide specification can well differ in their profiles of oligosaccharide binding pattern. Due to their relevance in growth control and matrix adhesion the family of galectins (galactoside-binding metal ion independent animal lectins) is receiving increasing utilization in human biology. Employing biotinylated galectin-1 and galectin-3 we studied the expression of binding sites for these galectins in normal human squamous epithelium and human carcinomas from the oropharyngeal region and larynx in relation to the expression of LP-34+ cytokeratins by the procedure of double labeling. Tissue sites accessible for galectin-1 were located in all layers of normal epithelium and in tumor cells. In contrast, galectin-3 binding was suprabasal in the normal epithelium and in tumor cells exhibiting signs of keratinization. These results reveal differences in the localization of accessible sites for the two galectins. Relating to cell development galectin-3 appeared to display affinity to areas with increased extent of differentiation.

Antigens, Differentiation↗

PubMed2000

Gene expression of galectin-9/ecalectin, a potent eosinophil chemoattractant, and/or the insertional isoform in human colorectal carcinoma cell lines and detection of frame-shift mutations for protein sequence truncations in the second functional lectin domain.

The family of Ca2+-independent galactoside-binding lectins with the beta-strand topology of the jelly-roll, referred to as galectins, is known to mediate and modulate a variety of cellular activities. Their functional versatility explains the current interest in monitoring their expression in cancer research, so far primarily focused on galectin-1 and -3. Tandem-repeat-type galectin-9 and its (most probably) allelic variant ecalectin, a potent eosinophil chemoattractant, are known to be human leukocyte products. We show by RT-PCR with primers specific for both that their mRNA is expressed in 17 of 21 human colorectal cancer lines. As also indicated by restriction analysis, in addition to the expected transcript of 571 bp an otherwise identical isoform coding for a 32-amino acid extension of the link peptide was detected. Positive cell lines differentially expressed either one (7 lines) or both transcripts (10 lines). Sequence analysis of RT-PCR products, performed in four cases, allowed to assign the standard transcript to ecalectin in the case of SW480 cells and detected two point mutations in the insert of the link peptide-coding sequence in WiDr and Colo205. Furthermore, this analysis identified the insertion of a single nucleotide into the coding sequence generating a frame-shift mutation, an event which has so far not been reported for any galectin. This alteration encountered in both transcripts of the WiDr line and the isoform transcript of Colo205 cells will most likely truncate the protein part within the second (C-terminal) carbohydrate recognition domain. Our results thus reveal the presence of mRNA for a galectin-9-isoform or a potent eosinophil chemoattractant (ecalectin) or a truncated version thereof with preserved N-terminal carbohydrate recognition domain in established human colon cancer cell lines.

Adenocarcinoma↗

PubMed1999

The 2.15 A crystal structure of CG-16, the developmentally regulated homodimeric chicken galectin.

Differential developmental regulation of expression, fine-specificity differences in ligand recognition and disparate capacity for homodimerization are characteristics of the two currently known proto-type chicken galectins. The X-ray crystal structure of the first avian galectin, the homodimeric agglutinin from chicken liver (CG-16), has been solved in the absence of ligand in two crystal forms. Although the arrangement of lectin dimers in the two crystals is different, the structure of the monomers and their association into the extended beta-sandwich that characterises the dimer are virtually identical. The fold establishes a beta-sandwich motif composed of a five-stranded and a six-stranded beta-sheet evocative of proto-type mammalian galectins. The carbohydrate-binding site is occupied by six water molecules that take the place of the sugar in the complex. They help to stabilise in the absence of the ligand the spatial arrangement of the amino acid side-chains involved in sugar recognition. Docking of N-acetyllactosamine into the binding site reveals that three of these water molecules, which are in direct contact with the protein, occupy positions equivalent to the key sugar hydroxyl groups, namely the hydroxyls at positions 4 and 6 of the galactose unit and at position 3 of the N-acetylglucosamine unit. Crystallographic data are fully consistent with the binding features in solution previously derived from chemical mapping with deoxy, fluoro and O-methyl derivatives and laser photo-CIDNP (chemically induced dynamic nuclear polarisation) studies. The possible molecular basis for the monomeric character of the chicken intestinal galectin as well as potential mechanisms of oxidative inactivation by disulphide bridging are evaluated on the basis of the given structural information concerning the CG-16 dimer interface and the cysteine residues, respectively.

Amino Acid Sequence↗