Lectins Translators of the Sugar Code

The concept of a recognitive interplay between a sugar ligand and a lectin readily receives support, when the assumed ligand properties can be ascertained. As compiled in Table 1, various experimental approaches exploit the lectin's binding specificity in assays for their detection and characterization. The success in establishing these techniques and the power of affinity chromatography together with expression cloning and homology searches have spurred the transition from the early phase to categorize lectins according to their monosaccharide specificity and requirement for cations to the era to draw genealogical trees of lectin families. Having its roots in the structural definition of the folding pattern and architecture of the carbohydrate recognition domain, the classification scheme is currently agreed upon with five distinct families of animal lectins, i.e. C-type lectins, galectins, I-type lectin, P-type lectins, and pentraxins (Drickamer, 1988, 1993; Gabius, 1997a; Powell and Varki, 1995; Rini and Lobsanov, 1999). That this compilation is unlikely to be final is implied by the description of lectin sequences lacking invariant characteristics of any of the five classes (e.g. the chaperones calnexin and calreticulin mentioned in Table 2) and the detection of new folding arrangements (e.g. the five-bladed P-propeller in the invertebrate lectin tachylectin-2 (Beisel et al., 1999)).

Table 1 Methods used in the search for lectins. (Modified from Gabius, 1997a.)

Tools

Parameter

Multivalent glycans and (neo)glyco-conjugates or defined cell populations

Labelled (neo)glycoconjugates and matrix-immobilized extract fractions or purified proteins Cell populations Tissue sections

Animal (neo)glycoconjugatedrug chimera and cell populations Matrix-immobilized (neo)glycoconju-gates and cell populations Cell extracts

Homology searches with computer programs (e.g. Gene-finder or Blast), expressed sequence tags and knowledge of key structural aspects of carbohydrate recognition domains Lectin motif-reactive probe (antibody, primer sets)

Carbohydrate-dependent inhibition of lectin-mediated glycan precipitation or cell agglutination Signal intensity

Labeling intensity Staining intensity

Biodistribution of signal intensity cellular responses (cell viability etc.) Carbohydrate-inhibitable cell adhesion

Carbohydrate-elutable proteins Homology score in sequence alignment or knowledge-based modeling

Extent of cross-reactivity

In each lectin family sequence alignments and homology searches have so far been conducive to unravel the divergent pathway from an ancestral gene to the current diversity. The intrafamily genealogy of mammalian C-type lectins has elegantly been traced back in a dendrogram to common ancestors for the seven subfamilies (Drickamer, 1993). To illustrate that such domains, often a part of modular arrangements, are no rare peculiarity in animal genomes, it is telling to add that a current database lists 389 C-type lectin-like sequences in animals (Sonnhammer et al., 1998). Yeast lacks this module in its domain collection. In the nematode C. elegans, whose elaborate enzymatic system for fucosylation has already been referred to (Oriol et al., 1999), this domain is ranked on the seventh place in frequency of occurrence, excelling for example the abundance of the EGF-like motif (The C. elegans Sequencing Consortium, 1998). At present, 183 C-type lectin-like domains have been traced in 125 proteins (Drickamer and Dodd, 1999). However, it is presently unclear how many of these proteins will be actually operative in Ca2+-dependent sugar (or peptide) binding (Drickamer, 1999). Also, at least eight functional galectin genes and a tentative total of 28 candidate galectin genes among the approximately 20,000 genetic reading frames (current number predicted: 19,099) in its genome were identified in the nematode (Hirabayashi et al., 1997; Cooper and Barondes, 1999). These new insights into lectin abundance further increase the percentage of the coding genome devoted to glycan production and recognition.

Table 2 Functions of animal lectins Activity

Ligand-selective molecular chaperones in endoplasmic reticulum Intracellular routing of glycoproteins and vesicles

Intracellular transport and extracellular assembly Cell type-specific endocytosis

Recognition of foreign glycans (P1,3-glucans, LPS)

Recognition of foreign or aberrant glycosig-natures on cells (incl. endocytosis or initiation of opsonization or complement activation)

Targeting of enzymatic activity in multimodular proteins

Bridging of molecules

Effector release (H2O2, cytokines etc.)

Cell growth control and apoptosis

Cell routing

Cell-cell interactions

Cell-matrix interactions

Matrix network assembly

Example of Lectin Calnexin, calreticulin

ERGIC-53, VIP-36, P-type lectins, comitin

Non-integrin 67 kDa elastin/laminin-binding protein

Hepatic asialoglycoprotein receptor, macrophage C-type lectins, hepatic endothelial cell receptor for GalNAc-4-SO4-bearing glycoproteins CR3 (CD11b/CD18), Limulus coagulation factors C and G Collectins, C-type macrophage receptors, pentraxins (CRP, limulin), L-ficolin, tachylectins

Acrosin, Limulus coagulation factor C

Homodimeric and tandem-repeat galectins, cytokines (e.g. IL-2:IL-2R and CD3 of TCR), cerebellar soluble lectin Galectins, selectins, CD23 Galectins, C-type lectins, amphoteiin-like protein, cerebellar soluble lectin Selectins, I-type lectins, galectins Selectins and other C-type lectins, galectins,

I-type lectins Galectins, heparin- and hyaluronic acid-

binding lectins Proteoglycan core proteins (C-type CRD), galectins, non-integrin 67 kDa elastin/ laminin-binding protein

For further information, see Gabius (1997a), Gabius and Gabius (1993, 1997), Kaltner and Stierstorfer (1998), Kishore et al. (1997), Vasta et al. (1999), Zanetta (1998) for recent reviews.

Equaling the strides being taken in the structural research on lectins, elucidation of their in vivo significance has steadily moved forward in the last decade. Summarized in Table 2, our present status of knowledge bears witness to the versatility to ply glycan recognition for a variety of purposes. In addition to mediating a physical contact between molecules and cells their initial recognition can trigger postbinding signaling with impact, for example, on growth regulation (Villalobo and Gabius, 1998). With focus on the homodimeric galectin-1 its mediation of downregulation of cell growth of responsive human neuroblastoma cells and of T-cell apoptosis to alleviate collagen-induced arthritis depicts representative examples with potential clinical relevance (Kopitz et al., 1998; Rabinovich et al., 1999).

Albeit necessarily centered in basic science, such cases illustrate the conceivable future potential to turn an endogenous lectin into a pharmaceutical.

Having already moved closer to applied science, the participation of lectins and glycoconjugates in cell adhesion has prompted attempts to rationally interfere with the molecular rendezvous, conceptually visualized as anti-adhesion therapy in Fig. 4. This approach mimics the natural strategy for success achieved with the complex cocktail of milk glycoconjugates.

They are protective by blocking docking of pathogens such as enteropathogenic and hemorrhagic Escherichia coli, Campylobacter jejuni, or rotavirus (Newburg, 1999). Although realization of this approach can prove tedious, because the pattern of recognition pairs is often not restricted to very few lectins (Helicobacter pylori with at least ten different carbohydrate-binding activities compared to the single type of influenza siali-dase whose inhibition will noticeably affect virus propagation (Karlsson, 1999; Lingwood, 1998; von Itzstein and Thomson, 1997) ), the custom-made design of tools, drawn as symbols in the strategy-outlining Fig. 4, justifies efforts to first localize binding partners and then to interfere with their activity aimed at therapy.

Notably, the first method can be used independently, e.g. in diagnostic procedures to characterize cell features. The visualization of carbohydrate-specific activities is commonly performed with carrier-immobilized sugar structures. Covalent attachment of a suitable derivative furnishes the versatility to produce neoglycoconjugates tailored to the experimental requirements (Bovin and Gabius, 1995; Lee Y.C. and Lee, 1994). Compared to a single carbohydrate unit the affinity of the multivalent ligand "is often beyond that expected from the increase in sugar concentration due to the presence of multiple residues on the protein (or polymeric backbone). Such an affinity enhancement is termed the glycoside cluster effect" (Lee R.T. and Lee, 1994). The geometrical increase in affinity with a numerical increase in valence for

Fig. 4 Interference in lectin-mediated cell contact formation or recognition processes in general with target-specific blocking reagents, i.e. antibodies, sugar receptors, and oligosaccharides or mimetics thereof. (Kindly provided by Priv.-Doz. Dr. H. Kaltner, Munich. Details on the current status of anti-adhesion therapy are given by Cornejo et al., 1997; Gabius, 1997b; Gabius and Gabius, 1997; Karlsson, 1998; Simon, 1996; Zopf and Roth, 1996.)

Fig. 4 Interference in lectin-mediated cell contact formation or recognition processes in general with target-specific blocking reagents, i.e. antibodies, sugar receptors, and oligosaccharides or mimetics thereof. (Kindly provided by Priv.-Doz. Dr. H. Kaltner, Munich. Details on the current status of anti-adhesion therapy are given by Cornejo et al., 1997; Gabius, 1997b; Gabius and Gabius, 1997; Karlsson, 1998; Simon, 1996; Zopf and Roth, 1996.)

mono-, bi-, and trivalent Gal-terminated oligosaccharides and mammalian asialoglycoprotein receptor, a C-type lectin, has been attributed to the topological complementarity between multiple ligand and receptor sites (Lee and Lee, 1997). Membrane solubilization by detergent treatment will in this case disrupt the essential spatial arrangement. An important caveat for approaches to detect the cluster effect concerns the use of agglutination assays. In contrast to affinity measurements in direct binding assays, the ongoing aggregation of multivalent receptors and ligands in solution can lead to erroneous conclusions. Indeed, under these circumstances isothermal titration calorimetry failed to record enhancements of Gibbs' free energy of binding but measured an endothermic, entropically favored process, its extent correlating with the inhibitory potency (IC50-values) of tetra- and hexavalent ligands (Dimick et al., 1999).

Adding a label to the neoglycoconjugates renders them serviceable for detection of ligand-specific sites in cells and tissues, as listed in Table 1 with special practical emphasis being currently placed in tumor diagnosis (Gabius et al. 1995; Danguy et al., 1998; Gabius et al., 1998; Kayser and Gabius, 1999). In view of common lectin histo-chemistry with plant agglutinins, this method has been designated as "reverse lectin histochemistry" (Gabius et al., 1993). Following the description of a relevant clinical correlation, e.g. binding of histo-blood group A- and H-trisaccharides to lung cancer cells and survival of patients (Kayser et al., 1994), further work will aim to define the tissue target and to refine the ligand for optimal selectivity and specificity (Mammen et al., 1998) en route to running assays to unveil, if possible, therapeutic benefit in lec-tin-directed anti-adhesion therapy (see references given in legend for Fig. 4) and drug targeting (Gabius, 1989, 1997b). To attain this objective, it is indispensable to comprehend the how and why of protein-carbohydrate recognition. Thus, it is inostructive to proceed with a brief outline of these principles relevant for drug design.

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