Detection and mapping of endogenous receptors for carrier-immobilized constituents of glycoconjugates (lectins) by labelled (neo)glycoproteins and by affinity chromatography in human adult mesencephalon, pons, medulla oblongata and cerebellum

Different carrier-immobilized carbohydrate moieties were employed as tools to detect respective binding sites glycohistochemically and glycobiochemically. Besides ascertaining their presence the pattern of endogenous sugar receptors (lectins) in different regions of the human central nervous system was mapped to reveal any non-uniform expression. A strong and specific staining with biotinylated neoglycoproteins, exposing different sugar moieties as ligands, indicated the presence of sugar receptors in the nuclei, neuronal pathways and accessory structures such as ependyma cells, plexus chorioideus, intra- and extracerebral vessels and leptomeninges localized in the mesencephalon, in the pons, in the medulla oblongata and in the cerebellum. Significant differences were seen for various neuroanatomical regions like nerve cells in the basal and central regions of the nuclei pontis in the glycohistochemically detected level of expression of endogenous sugar receptors (lectins). The used approach with carbohydrate constituents of cellular glycoconjugates as ligands in search of specific receptors complemented studies on the localization of glycoconjugates with sugar-specific tools like plant lectins. Exemplary glycobiochemical investigations on the medulla oblongata and cerebellum were performed to investigate the molecular nature of sugar receptors detected glycohistochemically. Despite notable overall similarities, carbohydrate-binding proteins of differing molecular weight can be isolated from these two regions of the central nervous system, namely in the case of receptors with specificity to beta-galactoside termini, to N-acetyl-D-galactosamine and N-acetyl-D-glucosamine and to D-xylose. These combined glycohistochemical and glycobiochemical results serve as a guideline for exploring the physiological relevance of the detected regional differences.     

The effect of structural differences in the reducing terminus of sugars on the binding affinity of carbohydrates and proteins analyzed using photoaffinity labeling

Because carbohydrates and proteins bind with such low affinity, the nature of their interactions is not clear. Photoaffinity labeling with diazirin groups is useful for elucidating the roles of carbohydrates in these binding processes. However, when carbohydrate probes are synthesized according to this conventional method, the reducing terminus of the sugar is opened to provide an acyclic structure. Because greater elucidation of carbohydrate-protein interactions requires a closed-ring carbohydrate in addition to the photoreactive group, we synthesized new molecular tools. The carbohydrate ligands were synthesized in three steps (glycosylation with allyl alcohol, deprotection, and ozonolysis). Specific binding proteins for carbohydrate ligands were obtained by photoaffinity labeling. Closed ring-type carbohydrate ligands, in which the reducing sugar is closed, bound to lectins more strongly than open ring-type sugars. Carbohydrate to protein binding was observed using AFM.     

Detection of binding sites for biotinylated neoglycoproteins and heparin (endogenous lectins) during cerebellar ontogenesis in the rat

Endogenous carbohydrate-binding sites were studied during rat cerebellar development on sections of fixed tissue using synthetic tools, biotinylated neoglycoproteins, in conjunction with subsequent avidinperoxidase staining. Neoglycoproteins were constructed by chemically coupling the histochemically pivotal carbohydrate moieties to an inert carrier protein. The sugar part of the neoglycoproteins included common constituents of the carbohydrate part of cellular glycoconjugates, namely mannose, galactose, fucose, N-acetyl-glucosamine, N-acetylgalactosamine and N-acetyl-neuraminic acid to probe for the presence of respective endogenous receptors. Heparin was biotinylated after mild cyanogen bromide activation and aminoalkylation. Specific positive reactions were obtained for all neoglycoproteins and heparin. The staining pattern with the individual probes disclosed variable developmental regulation. Consequently, these results suggest that recognition processes during cerebellar development may include several types of carbohydrate determinants. In two instances, the binding of neoglycoproteins could be compared to endogenous lectin-specific antibodies. Despite a significant extent of accordance the comparison revealed notable differences. These differences were attributed primarily to fixation and the presence of physiological ligands that can mask the active endogenous carbohydrate-binding proteins. In any case, histochemical application of labeled neoglycoproteins is valuable to discern the presence, localization and developmental pattern of binding sites for the carbohydrate part of glycoconjugates, on which further biochemical and cell biological studies can consequently be based.     

Molecular dynamics simulations of glycoclusters and glycodendrimers

Protein-carbohydrate recognition plays a crucial role in a wide range of biological processes, required both for normal physiological functions and the onset of disease. Nature uses multivalency in carbohydrate-protein interactions as a strategy to overcome the low affinity found for singular binding of an individual saccharide epitope to a single carbohydrate recognition domain of a lectin. To mimic the complex multi-branched oligosaccharides found in glycoconjugates, which form the structural basis of multivalent carbohydrate-protein interactions, so-called glycoclusters and glycodendrimers have been designed to serve as high-affinity ligands of the respective receptor proteins. To allow a rational design of glycodendrimer-type molecules with regard to the receptor structures involved in carbohydrate recognition, a deeper knowledge of the dynamics of such molecules is desirable. Most glycodendrimers have to be considered highly flexible molecules with their conformational preferences most difficult to elucidate by experimental methods. Longtime molecular dynamics (MD) simulations with inclusion of explicit solvent molecules are suited to explore the conformational space accessible to glycodendrimers. Here, a detailed geometric and conformational analysis of 15 glycodendrimers and glycoclusters has been accomplished, which differ with regard to their core moieties, spacer characteristics and the type of terminal carbohydrate units. It is shown that the accessible conformational space depends strongly on the structural features of the core and spacer moieties and even on the type of terminating sugars. The obtained knowledge about possible spatial distributions of the sugar epitopes exposed on the investigated hyperbranched neoglycoconjugates is detailed for all examples and forms important information for the interpretation and prediction of affinity data, which can be deduced from biological testing of these multivalent neoglycoconjugates.     

Detection and mapping of endogenous receptors for carrier-immobilized constituents of glycoconjugates (lectins) by labelled (neo)glycoproteins and by affinity chromatography in human adult mesencephalon,pons, medulla oblongata and cerebellum

Summary Different carrier-immobilized carbohydrate moieties were employed as tools to detect respective binding sites glycohistochemically and glycobiochemically. Besides ascertaining their presence the pattern of endogenous sugar receptors (lectins) in different regions of the human central nervous system was mapped to reveal any non-uniform expression. A strong and specific staining with biotinylated neoglycoproteins, exposing different sugar moieties as ligands, indicated the presence of sugar receptors in the nuclei, neuronal pathways and accessory structures such as ependyma cells, plexus chorioideus, intra- and extracerebral vessels and leptomeninges localized in the mesencephalon, in the pons, in the medulla oblongata and in the cerebellum. Significant differences were seen for various neuroanatomical regions like nerve cells in the basal and central regions of the nuclei pontis in the glycohistochemically detected level of expression of endogenous sugar receptors (lectins). The used approach with carbohydrate constituents of cellular glycoconjugates as ligands in search of specific receptors complemented studies on the localization of glycoconjugates with sugar-specific tools like plant lectins. Exemplary glycobiochemical investigations on the medulla oblongata and cerebellum were performed to investigate the molecular nature of sugar receptors detected glycohistochemically. Despite notable overall similarities, carbohydrate-binding proteins of differing molecular weight can be isolated from these two regions of the central nervous system, namely in the case of receptors with specificity to -galactoside termini, to N-acetyl-d-galactosamine and N-acetyl-d-glucosamine and to d-xylose. These combined glycohistochemical and glycobiochemical results serve as a guideline for exploring the physiological relevance of the detected regional differences.     

Prediction of carbohydrate binding sites on protein surfaces with 3-dimensional probability density distributions of interacting atoms

Non-covalent protein-carbohydrate interactions mediate molecular targeting in many biological processes. Prediction of non-covalent carbohydrate binding sites on protein surfaces not only provides insights into the functions of the query proteins; information on key carbohydrate-binding residues could suggest site-directed mutagenesis experiments, design therapeutics targeting carbohydrate-binding proteins, and provide guidance in engineering protein-carbohydrate interactions. In this work, we show that non-covalent carbohydrate binding sites on protein surfaces can be predicted with relatively high accuracy when the query protein structures are known. The prediction capabilities were based on a novel encoding scheme of the three-dimensional probability density maps describing the distributions of 36 non-covalent interacting atom types around protein surfaces. One machine learning model was trained for each of the 30 protein atom types. The machine learning algorithms predicted tentative carbohydrate binding sites on query proteins by recognizing the characteristic interacting atom distribution patterns specific for carbohydrate binding sites from known protein structures. The prediction results for all protein atom types were integrated into surface patches as tentative carbohydrate binding sites based on normalized prediction confidence level. The prediction capabilities of the predictors were benchmarked by a 10-fold cross validation on 497 non-redundant proteins with known carbohydrate binding sites. The predictors were further tested on an independent test set with 108 proteins. The residue-based Matthews correlation coefficient (MCC) for the independent test was 0.45, with prediction precision and sensitivity (or recall) of 0.45 and 0.49 respectively. In addition, 111 unbound carbohydrate-binding protein structures for which the structures were determined in the absence of the carbohydrate ligands were predicted with the trained predictors. The overall prediction MCC was 0.49. Independent tests on anti-carbohydrate antibodies showed that the carbohydrate antigen binding sites were predicted with comparable accuracy. These results demonstrate that the predictors are among the best in carbohydrate binding site predictions to date.     

Dynamics of galectin-3 in the nucleus and cytoplasm

This review summarizes selected studies on galectin-3 (Gal3) as an example of the dynamic behavior of a carbohydrate-binding protein in the cytoplasm and nucleus of cells. Within the 15-member galectin family of proteins, Gal3 (M_r ∼ 30,000) is the sole representative of the chimera subclass in which a proline- and glycine-rich NH₂-terminal domain is fused onto a COOH-terminal carbohydrate recognition domain responsible for binding galactose-containing glycoconjugates. The protein shuttles between the cytoplasm and nucleus on the basis of targeting signals that are recognized by importin(s) for nuclear localization and exportin-1 (CRM1) for nuclear export. Depending on the cell type, specific experimental conditions in vitro, or tissue location, Gal3 has been reported to be exclusively cytoplasmic, predominantly nuclear, or distributed between the two compartments. The nuclear versus cytoplasmic distribution of the protein must reflect, then, some balance between nuclear import and export, as well as mechanisms of cytoplasmic anchorage or binding to a nuclear component. Indeed, a number of ligands have been reported for Gal3 in the cytoplasm and in the nucleus. Most of the ligands appear to bind Gal3, however, through protein–protein interactions rather than through protein–carbohydrate recognition. In the cytoplasm, for example, Gal3 interacts with the apoptosis repressor Bcl-2 and this interaction may be involved in Gal3's anti-apoptotic activity. In the nucleus, Gal3 is a required pre-mRNA splicing factor; the protein is incorporated into spliceosomes via its association with the U1 small nuclear ribonucleoprotein (snRNP) complex. Although the majority of these interactions occur via the carbohydrate recognition domain of Gal3 and saccharide ligands such as lactose can perturb some of these interactions, the significance of the protein's carbohydrate-binding activity, per se, remains a challenge for future investigations.     

Glycans as endocytosis signals: the cases of the asialoglycoprotein and hyaluronan/chondroitin sulfate receptors

Animal cells internalize specific extracellular macromolecules (ligands) by using specialized cell surface receptors that operate through a complex and highly regulated process known as receptor-mediated endocytosis, which involves the binding, internalization, and transfer of ligands through a series of distinct intracellular compartments. For the uptake of a variety of carbohydrate-containing macromolecules, such as glycoproteins, animal cells use specialized membrane-bound lectins as endocytic receptors that recognize different sugar residues or carbohydrate structures present on various ligands. The hepatic asialoglycoprotein receptor, which recognizes glycoconjugates containing terminal galactose or N-acetylgalactosamine residues, was the first membrane lectin discovered and has been a classical system for studying receptor-mediated endocytosis. Studies of how the asialoglycoprotein receptor functions have led to the discovery of two functionally distinct, parallel pathways of clathrin-mediated endocytosis (called the State 1 and State 2 pathways), which may also be utilized by all the other endocytic recycling receptor systems. Another endocytic membrane lectin, the hyaluronan/chondroitin sulfate receptor, which has recently been purified and cloned, is responsible for the turnover in mammals of these glycosaminoglycans, which are important components of extracellular matrices. We discuss the characteristics and physiological importance of these two proteins as examples of how lectins can function as endocytic receptors.     

Comparative analysis of carbohydrate binding properties of Sambucus nigra lectins and ribosome-inactivating proteins

In the past three decades a lot of research has been done on the extended family of carbohydrate-binding proteins from Sambucus nigra, including several so-called type 2 RIPs as well as hololectins. Although all these proteins have been studied for their carbohydrate-binding properties using hapten inhibition assays, detailed carbohydrate specificity studies have only been performed for a few Sambucus proteins. In particular SNA-I, has been studied extensively. Because of its unique binding characteristics this lectin was developed as an important tool in glycoconjugate research to detect sialic acid containing glycoconjugates. At present much less information is available with respect to the detailed carbohydrate binding specificity of other S. nigra lectins and RIPs, and as a consequence their applications remain limited. In this paper we report a comparative analysis of several lectins from S. nigra using the glycan microarray technology. Ultimately a better understanding of the ligands for each lectin can contribute to new/more applications for these lectins in glycoconjugate research. Furthermore, the data from glycan microarray analyses combined with the previously obtained sequence information can help to explain how evolution within a single lectin family eventually yielded a set of carbohydrate-binding proteins with a very broad specificity range.     

Ligand identification of carbohydrate-binding proteins employing a biotinylated glycan binding assay and tandem mass spectrometry

Characterization of protein-carbohydrate interactions at the molecular level is important for understanding many glycan-mediated processes. Here we present a method for the identification of glycan ligands of carbohydrate-binding proteins. The glycans released from natural sources are labeled with biotinamidocaproyl hydrazide (BACH) and subsequently fractionated by high-performance liquid chromatography. Glycan fractions are screened for binding to carbohydrate-binding proteins (CBPs) using a microtitration plate binding assay; CBPs are immobilized, BACH-glycan fractions are added, and bound BACH-glycans are detected using alkaline phosphatase-conjugated streptavidin. The glycan structures in binding fractions are studied by (tandem) mass spectrometry, exoglycosidase treatment, and rechromatography, thereby revealing the glycan motifs recognized by the CBPs. Subsequent surface plasmon resonance experiments using a reverse setup with immobilization of the BACH-glycan ligands on streptavidin-coated surfaces provide more information on glycan-CBP interactions via association and dissociation curves. The presented method is easy and fast, and the required instrumentation is available in many laboratories. The assay is very sensitive given that both the mass spectrometric analysis and the microtitration plate binding assay can be performed on femtomole amounts of BACH-glycans. This approach should be generally applicable to study and structurally identify carbohydrate ligands of anti-glycan antibodies and lectins.     

Carbohydrate microarrays - a new set of technologies at the frontiers of glycomics

Carbohydrate microarray technologies are new developments at the frontiers of glycomics. Results of 'proof of concept' experiments with carbohydrate-binding proteins of the immune system - antibodies, selectins, a cytokine and a chemokine - and several plant lectins indicate that microarrays of carbohydrates (glycoconjugates, oligosaccharides and monosaccharides) will greatly facilitate not only surveys of proteins for carbohydrate-binding activities but also elucidation of their ligands. It is predicted that both naturally occurring and synthetic carbohydrates will be required for the fabrication of microarrays that are sufficiently comprehensive and representative of entire glycomes. New leads to biological pathways that involve carbohydrate-protein interactions and new therapeutic targets are among biomedically important outcomes anticipated from applications of carbohydrate microarrays.     

The development of new molecular tools containing a chemically synthesized carbohydrate ligand for the elucidation of carbohydrate roles via photoaffinity labeling: Carbohydrate–protein interactions are affected by the structures of the glycosidic bonds and the reducing-end sugar

Photoaffinity labeling technology is a highly efficient method for cloning carbohydrate-binding proteins. When the carbohydrate probes are synthesized according to conventional methods, however, the reducing terminus of the sugar is opened to provide an acyclic structure. Our continued efforts to solve this problem led to the development of new molecular tools with an oligosaccharide structure that contains a phenyldiazirine group for the elucidation of carbohydrate–protein interactions. We investigated whether carbohydrate–lectin interactions are affected by differences in the glycosidic formation and synthesized three types of molecular tools containing Galp–GlcpNAc disaccharide ligands and a photoreactive group (1, 2, 3). Photoaffinity labeling validated the recognition of the new ligand by different glycosidic bonds. Photoaffinity labeling also demonstrated that both the reducing end sugar and non-reducing end sugar recognized the Erythrina cristagalli agglutinin.     

Lectin binding in human skeletal muscle: a comparison of 15 different lectins

Summary Fifteen lectin-horseradish peroxidase conjugates have been used in a comprehensive histochemical study of human skeletal muscle. The staining patterns of many lectins were found to be coincident with the known distributions of types I, III, IV and V collagen, fibronectin and laminin. One lectin,Bandeiraea simplicifolia (BSA I), selectively stained capillaries in a blood group-specific manner, the significance of which is unknown. The results show that although lectins are useful cytochemical probes for identifying tissue glycoconjugates, lectin binding is not solely determined by monosaccharide specificity as lectins which interact with the same sugars may have completely different staining patterns. Factors such as accessibility, glycan conformation and oligosaccharide sequence also affect lectin binding in tissues. For these reasons, we conclude that a comprehensive histochemical investigation of tissue glycoconjugates should employ a large number of lectins, preferably with overlapping sugar specificities.     

Interaction of aromatic imino glycoconjugates with jacalin: experimental and computational docking studies

Altering the lectin properties by chemically modified glycoconjugates can have profound effect on their biological applications. In the present case, jacalin has been chosen to study the binding aspects toward glycoconjugates modified by connecting aromatic moieties through imine conjugation at their C-1- or C-2-positions. Out of 10 glycoconjugates, the galactosyl-naphthyl imine (1c) was found to be most effective toward agglutination inhibition (260 times better than galactose), quenching fluorescence intensity, and exhibiting greater binding (K_a, 1.3 × 10⁴ M⁻¹) with jacalin. The specific binding of galactose conjugates and the nonspecific binding of other conjugates have been demonstrated based on ITC. Changes in the secondary structures have been addressed by far- and near-UV CD spectroscopy. The present studies demonstrated that galactose-based conjugates bind at carbohydrate recognition domain (CRD) mainly through polar interactions in addition to exhibiting some nonpolar/hydrophobic interactions, whereas the conjugates other than galactose primarily interact through hydrophobic interactions. Binding of galactosyl conjugates at CRD has been further demonstrated by rigid docking.     

Crystallographic and in silico analysis of the sialoside-binding characteristics of the Siglec sialoadhesin

The Siglec family of receptors mediates cell-surface interactions through recognition of sialylated glycoconjugates. Previously reported structures of the N-terminal domain of the Siglec sialoadhesin (SnD1) in complex with various sialic acid analogs revealed the structural template for sialic acid binding. To characterize further the carbohydrate-binding properties, we have determined the crystal structures of SnD1 in the absence of ligand, and in complex with 2-benzyl-Neu5NPro and 2-benzyl-Neu5NAc. These structures reveal that SnD1 undergoes very few structural changes on ligand binding and detail how two novel classes of sialic acid analogs bind, one of which unexpectedly can induce Siglec dimerization. In conjunction with in silico analysis, this set of structures informs us about the design of putative ligands with enhanced binding affinities and specificities to different Siglecs, and provides data with which to test the effectiveness of different computational drug design protocols.     

Binding sub-site dissection of a carbohydrate-binding module reveals the contribution of entropy to oligosaccharide recognition at "non-primary" binding subsites

The optimal ligands for many carbohydrate-binding proteins are often oligosaccharides comprising two, three, or more monosaccharide units. The binding affinity for these sugars is increased incrementally by contributions from binding subsites on the protein that accommodate the individual monosaccharide residues of the oligosaccharide. Here, we use CsCBM6-1, a xylan-specific type B carbohydrate-binding module (CBM) from Clostridium stercorarium falling into amino acid sequence family CBM6, as a model system to investigate the structural and thermodynamic contributions of binding subsites in this protein to carbohydrate recognition. The three-dimensional structures of uncomplexed CsCBM6-1 (at 1.8 A resolution) and bound to the oligosaccharides xylobiose, xylotriose, and xylotetraose (at 1.70 A, 1.89 A, and 1.69 A resolution, respectively) revealed the sequential occupation of four subsites within the binding site in the order of subsites 2, 3, 4 then 1. Overall, binding to all of the xylooligosaccharides tested was enthalpically favourable and entropically unfavourable, like most protein-carbohydrate interactions, with the primary subsites 2 and 3 providing the bulk of the free energy and enthalpy of binding. In contrast, the contributions to the changes in entropy of the non-primary subsites 1 and 4 to xylotriose and xylotetraose binding, respectively, were positive. This observation is remarkable, in that it shows that the 10-20-fold improvement in association constants for oligosaccharides longer than a disaccharide is facilitated by favourable entropic contributions from the non-primary binding subsites.     

Differential oligosaccharide recognition by evolutionarily-related beta-1,4 and beta-1,3 glucan-binding modules

Enzymes active on complex carbohydrate polymers frequently have modular structures in which a catalytic domain is appended to one or more carbohydrate-binding modules (CBMs). Although CBMs have been classified into a number of families based upon sequence, many closely related CBMs are specific for different polysaccharides. In order to provide a structural rationale for the recognition of different polysaccharides by CBMs displaying a conserved fold, we have studied the thermodynamics of binding and three-dimensional structures of the related family 4 CBMs from Cellulomonas fimi Cel9B and Thermotoga maritima Lam16A in complex with their ligands, beta-1,4 and beta-1,3 linked gluco-oligosaccharides, respectively. These two CBMs use a structurally conserved constellation of aromatic and polar amino acid side-chains that interact with sugars in two of the five binding subsites. Differences in the length and conformation of loops in non-conserved regions create binding-site topographies that complement the known solution conformations of their respective ligands. Thermodynamics interpreted in the light of structural information highlights the differential role of water in the interaction of these CBMs with their respective oligosaccharide ligands.     

Neoglycoconjugates Based on Dendrimer Poly(aminoamides)

Neoglycoconjugates containing 4, 8, 32, and 64 terminal residues of B-disaccharide (B_DI) or N-acetylneuraminic acid (Neu5Ac) attached to poly(aminoamide)-type dendrimers (PAMAMs) were synthesized. The ability of B_DI conjugates to bind natural xenoantibodies (anti-B_DI antibodies) and the ability of Neu5Ac conjugates to inhibit the hemagglutinin-mediated adhesion of influenza virus were studied. The biological activity of PAMAM conjugates turned out to be higher than that of free carbohydrate ligands, but less than that of multivalent glycoconjugates based on other types of synthetic polymeric carriers. A conformational analysis of PAMAM matrices and resulting conjugates was performed to determine the statistical distances between carbohydrate ligands. The computations revealed the tendency of the PAMAM chains toward compaction and formation of dense globules. The process results in a decrease in the distances between the carbohydrate ligands in the conjugates and, hence, could affect the ability of glycoconjugates to efficiently bind the polyvalent carbohydrate-recognizing proteins.     

Fishing for lectins from diverse sequence libraries by yeast surface display - an exploratory study

The establishment of a robust technology platform for the expression cloning of carbohydrate-binding proteins remains a key challenge in glycomics. Here we explore the utility of using yeast surface display (YSD) technology in the interaction-based lectin cloning from complete cDNA libraries. This should pave the way for more detailed studies of protein-carbohydrate interactions. To evaluate the performance of this system, lectins representing three different subfamilies (galectins, siglecs, and C-type lectins) were successfully displayed on the surface of Saccharomyces cerevisiae and Pichia pastoris as a-agglutinin and/or alpha-agglutinin fusions. The predicted carbohydrate-binding activity could be detected for three out of five lectins tested (galectin-1, galectin-3, and siaoadhesin). For galectin-4 and E-selectin, no specific carbohydrate-binding activity could be detected. We also demonstrate that proteins with carbohydrate affinity can be specifically isolated from complex metazoan cDNA libraries through multiple rounds of FACS sorting, employing multivalent, fluorescent-labeled polyacrylamide-based glycoconjugates.     

Regional differences in the distribution of endogenous receptors for carbohydrate constituents of cellular glycoconjugates, especially lectins, in cortex, hippocampus, basal ganglia and thalamus of adult human brain

Ten different types of labelled neoglycoproteins, exposing glycohistochemically pivotal carbohydrate moieties that mostly are constituents of naturally occurring glycoconjugates with an aromatic spacer, were synthesized. The panel was applied to fixed, paraffin-embedded sections of different cortical regions and white matter, of hippocampal gyrus, basal ganglia, thalamus nuclei and adjacent areas of adult human brain to comprehensively map the presence of respective binding sites in these parts. Compliance with accepted criteria for specificity of binding was routinely ascertained. Overall, not a uniform binding pattern, but a distinct distribution with regional differences on the level of specific cytoplasmic and nuclear staining in nerve cells was determined, fiber structures being generally labelled with medium or strong intensity. For example, among the neurons localized in the five cortical laminae the binding of N-acetyl-D-galactosamine varied from strong to undetectable. Biochemical analysis, employing carbohydrate residues as affinity ligands in chromatography, proved that the neuroanatomically different regions exhibited a pattern of receptors with notable similarities. These results on endogenous binding sites for glycoconjugates, especially lectins, are complementary to assessment of localization of cellular glycoconjugates by plant lectins and carbohydrate-specific monoclonal antibodies. They are thus a further obligatory step to substantiate the physiological roles of recognitive protein-carbohydrate interactions in the central nervous system.