The complement binding-like domains of the murine homing receptor facilitate lectin activity.

The leukocyte homing receptor (HR), the endothelial leukocyte adhesion molecule, and gmp140/platelet activation-dependent granule membrane protein are members of a family of adhesion molecules, termed the lectin cell adhesion molecules (LEC-CAMS) which are unified by a multi-domain structure containing a lectin motif, an epidermal growth factor-like (egf) motif, and variable numbers of a complement binding-like (CB) motif. Previous data have indicated a predominant role for the lectin motif in cell adhesion directed by the LEC-CAMS, although the egf-like domain of the HR may also play a potential role in cell binding. While the role(s) of the CB domains in the LEC-CAMS is currently not understood, they have been hypothesized to act as rigid spacers or stalks for lectin and perhaps, egf domain presentation. In this paper, we analyze the functional characteristics of murine HR-IgG chimeras containing the lectin, lectin plus egf, and lectin plus egf plus CB domains. The Mel 14 mAb, an adhesion blocking antibody which recognizes a conformational determinant in the N-terminus of the HR lectin domain, shows a significantly decreased affinity for a HR construct which lacks the CB motifs, consistent with the possibility that the CB domains are involved with lectin domain structure. In agreement with this conjecture, HR mutants lacking the CB domains show a profound decrease in lectin-specific interaction with the carbohydrate polyphosphomannan ester, suggesting that the changes in Mel 14 affinity for the lectin domain are reflected in lectin functionality. Various assays investigating the interactions between the HR deletion mutants and the peripheral lymph node high endothelium, including cell blocking, immunohistochemical staining, and radioactively labeled ligand binding, all showed that removal of the CB domains results in a lack of HR adhesive function. These results imply that the CB domains of the HR, and, by analogy, the other members of the LEC-CAM family, may play important structural roles involving induction of lectin domain conformation and resultant functionality

Endogenous mammalian lectin localized extracellularly in lung elastic fibers.

An affinity-purified antibody preparation raised against a beta-galactoside-binding lectin from bovine lung was used to localize a similar lectin in rat lung by immunofluorescence and by electron microscopy after on-grid staining visualized with colloidal gold conjugated second antibody. The endogenous mammalian lectin was found in smooth muscle cells and squamous alveolar epithelial (type I) cells and was concentrated extracellularly in elastic fibers of pulmonary parenchyma and blood vessels. The extracellular localization of this lectin suggests that it, like others, functions by interaction with extracellular glycoconjugates

Localization of an endogenous lectin in chicken liver, intestine, and pancreas.

Extracts of adult chicken liver, pancreas, and intestine contain high levels of a lectin which appears to be identical to one previously purified from embryonic chick muscle. This lectin is virtually absent from adult muscle, but is highly concentrated in cells lining liver sinusoids, intestinal goblet cells, and the extracellular spaces surrounding pancreatic acini. These findings suggest that the lectin may play different roles in different tissues and at different times in the life of a chicken

The serlogical specificity of the lectin from Lens culinaris

Lens culinaris, the common lentil, contains a lectin which has been shown to be specific for a glycoprotein saliva antigen and a glycolipoprotein serum antigen. Both the saliva and serum precipitin reactions with the lectin are directly inhibited with saccharides, especially those related to D-mannose. Electrophoresis of the serum antigen showed that it migrates as three bands, while appearing as a single band in double diffusion precipitin patterns. Quantitative studies of the saliva antigen levels by hemagglutination inhibition titration indicated a polygenic, quantitative mode of inheritance with a minimum heritability of O. 34. Blood group ABH secretor individuals were found to have a significantly lower mean saliva antigen level than nonsecretor individuals. The lectins from Pisum sativum and Canavaliafiensiformis formed precipitin bands of identity with L.culinaris lectin against saliva. C. ensiformis and L. culinaris lectins exhibited precipitin bands of partial identity against serum; and P. sativum and L. culinaris lectins exhibited a pattern of identity against serum. In addition, precipitin patterns of partial identity with the non-H lectin from Lotus tetragonolobus has been demonstrated. Using Ulex europaeus lectin in hemagglutination inhibition experiments with saliva from blood group O secretor individuals, a minimum heritability of approximately 0.40 for H antigen levels was found. A higher frequency of nonsecretor individuals was observed in the Black population compared with the White population

Lectin based glycoprotein analysis

Many of the biopharmaceutical therapeutics entering the market and currently in clinical trails are recombinant glycoprotein molecules, the glycan moieties of which have a significant impact on efficacy and immunogenicity. The cell culture techniques required to produce these glycoproteins often result in products that are heterogeneous with respect to glycan content. This inconsistency ultimately leads to increased production costs and restricts patient accessibility to these therapeutics. To overcome these difficulties novel analytical platforms facilitating rapid in-process monitoring and product quality control are essential. Work undertaken within the Centre for Bioanalytical Sciences (CBAS) seeks to exploit the microbial world as a source of novel biorecognition elements to produce such platforms

Optimisation of the enzyme-linked lectin assay for enhanced glycoprotein and glycoconjugate analysis

Lectin’s are proteins capable of recognising and binding to specific oligosaccharide tructures found on glycoproteins and other biomoloecules. As such they have found tility for glycoanalytical applications. One common difficulty encountered in the pplication of these proteins, particularly in multi-well plate assay formats known as Enzyme Linked Lectin Assays (ELLA’s), is in finding appropriate blocking solutions to prevent non-specific binding with plate surfaces. Many commonly used blocking agents contain carbohydrates and generate significant background signals in ELLA’s, limiting the utility of the assay. In this study we examined the suitability of a range of blocking reagents, including rotein based, synthetic and commercially available carbohydrate free blocking eagents, for ELLA applications. Each blocking reagent was assessed against a panel f 19 commercially available biotinylated lectins exhibiting diverse structures and arbohydrate specificities. We identified the synthetic polymer Polyvinyl Alcohol PVA) as the best global blocking agent for performing ELLA’s. We ultimately present n ELLA methodology facilitating broad spectrum lectin analysis of glycoconjugates nd extending the utility of the ELLA

Detection of inflammation- and neoplasia-associated alterations in human large intestine using plant/invertebrate lectins, galectin-1 and neoglycoproteins

Commonly, plant and invertebrate lectins are accepted glycohistochemical tools for the analysis of normal and altered structures of glycans in histology and pathology. Mammalian lectins and neoglycoproteins are recent additions to this panel for the detection of lectin-reactive carbohydrate epitopes and glycoligand-binding sites. The binding profiles of these three types of probes were comparatively analyzed in normal, inflamed and neoplastic large intestine. In normal colonic mucosa the intracellular distribution of glycoconjugates and carbohydrate ligand-binding sites in enterocytes reveals a differential binding of lectins with different specificity and of neoglycoproteins to the Golgi apparatus, the rough and smooth endoplasmic reticulum and the apical cell surface. The accessible glycoligand-binding sites and the lectin-reactive carbohydrate epitopes detected by galectin-1 show the same pattern of intracellular location excluding the apical cell surface. Lectin-reactive carbohydrate epitopes detected by plant lectins of identical monosaccharide specificity as the endogenous lectin {[}Ricinus communis agglutinin-I (RCA-I), Viscum album agglutinin (VAA)], however, clearly differ with respect to their intracellular distribution. Maturation-associated differences and heterogeneity in glycohistochemical properties of epithelial cells and non-epithelial cells (macrophages, dendritic cells, lymphocytes) are found. Dissimilarities in the fine structural Ligand recognition of lectins with nominal specificity to the same monosaccharide have been demonstrated for the galactoside-specific lectins RCA-I, VAA and galectin-1 as well as the N-acetylgalactosamine (GalNAc)-specific lectins Dolichos biflorus agglutinin (DBA), soybean agglutinin (SBA) and Helix pomatia agglutinin in normal mucosa and in acute appendicitis. Acute inflammation of the intestinal mucosa found in acute phlegmonous appendicitis is associated with selective changes of glycosylation of mucin in goblet cells mainly of lower and middle crypt segments resulting in an increase of DBA- and SEA-binding sites in the goblet cell population. Appendicitis causes no detectable alteration of neoglycoprotein binding. In contrast, tumorigenesis of colonic adenoma is characterized by increases in lectin-reactive galactose (Gal; Gal-beta 1,3-GalNAc), fucose and N-acetylglucosamine moieties and by enhanced presentation of respective carbohydrate ligand-binding capacity. This work reveals that endogenous lectins and neoglycoproteins are valuable glycohistochemical tools supplementing the well-known analytic capacities of plant lectins in the fields of gastrointestinal anatomy and gastroenteropathology

FGB1 and WSC3 are in planta-induced beta-glucan-binding fungal lectins with different functions

In the root endophyte Serendipita indica, several lectin-like members of the expanded multigene family of WSC proteins are transcriptionally induced in planta and are potentially involved in beta-glucan remodeling at the fungal cell wall. Using biochemical and cytological approaches we show that one of these lectins, SiWSC3 with three WSC domains, is an integral fungal cell wall component that binds to long-chain beta 1-3-glucan but has no affinity for shorter beta 1-3- or beta 1-6-linked glucose oligomers. Comparative analysis with the previously identified beta-glucan-binding lectin SiFGB1 demonstrated that whereas SiWSC3 does not require beta 1-6-linked glucose for efficient binding to branched beta 1-3-glucan, SiFGB1 does. In contrast to SiFGB1, the multivalent SiWSC3 lectin can efficiently agglutinate fungal cells and is additionally induced during fungus-fungus confrontation, suggesting different functions for these two beta-glucan-binding lectins. Our results highlight the importance of the beta-glucan cell wall component in plant-fungus interactions and the potential of beta-glucan-binding lectins as specific detection tools for fungi in vivo

Mutational analysis of the carbohydrate binding activity of the tobacco lectin

At present the three-dimensional structure of the tobacco lectin, further referred to as Nictaba, and its carbohydrate-binding site are unresolved. In this paper, we propose a three-dimensional model for the Nictaba domain based on the homology between Nictaba and the carbohydrate-binding module 22 of Clostridium thermocellum Xyn10B. The suggested model nicely fits with results from circular dichroism experiments, indicating that Nictaba consists mainly of beta-sheet. In addition, the previously identified nuclear localization signal is located at the top of the protein as a part of a protruding loop. Judging from this model and sequence alignments with closely related proteins, conserved glutamic acid and tryptophan residues in the Nictaba sequence were selected for mutational analysis. The mutant DNA sequences as well as the original Nictaba sequence have been expressed in Pichia pastoris and the recombinant proteins were purified from the culture medium. Subsequently, the recombinant proteins were characterized and their carbohydrate binding properties analyzed with glycan array technology. It was shown that mutation of glutamic acid residues in the C-terminal half of the protein did not alter the carbohydrate-binding activity of the lectin. In contrast, mutation of tryptophan residues in the N-terminal half of the Nictaba domain resulted in a complete loss of carbohydrate binding activity. These results suggest that tryptophan residues play an important role in the carbohydrate binding site of Nictaba