Hafiz Ahmed

C-type lectins and galectins mediate innate and adaptive immune functions: their roles in the complement activation pathway

In recent years, a new pathway for complement activation mediated by the mannose-binding lectin (MBL) has been described as a key mechanism for the mammalian acute phase response to infection. This complement activation pathway is initiated by a non-self recognition step: the binding of a humoral C-type lectin [mannose-binding lectin (MBL)] to microbial surfaces bearing foreign carbohydrate determinants. The recognition factor, MBL, is associated with a serine protease [MBL-associated serine protease (MASP)] which, upon MBL binding to the microbial ligand, activates the complement component C3, leading to either (a) phagocytosis of the opsonized target via the complement receptor, or (b) humoral cell killing via assembly of the membrane attack complex. Galectins (formerly known as S-type lectins) modulate activity of the complement receptor 3 (CR3), the macrophage membrane receptor for complement components C3b and iC3b, downstream products of the MBL pathway which are covalently bound to target cells. Galectins also mediate macrophage- and dendrocyte-adhesion to lymphocytes activated by signaling through another C-type lectin, the L-selectin, leading to immunoglobulin-mediated responses. Thus, the functional interplay of MBL, galectins and L-selectin in the acute phase response neutralizes the microbial challenge, and lead to further adaptive immunity. Although the observation of various components of the lectin pathway in different invertebrate species demonstrates the high conservation and ancient roots of the components of innate immunity, there has previously been no evidence supporting the possibility that the integral lectin-mediated complement activation pathway is present in invertebrates. We now have evidence for the coexistence of homologs of all the pathways key components (MBL, MASP, C3, and galectin) in the protochordate Clavelina picta, suggesting the lectin-mediated pathway of complement activation preceded the immunoglobulin pathway in evolution. Therefore, despite being new to the textbooks, experimental evidence indicates that this pathway is ancient, and has been conserved intact throughout its evolution.

Animal lectins as self/non-self recognition molecules. Biochemical and genetic approaches to understanding their biological roles and evolution

In recent years, the significant contributions from molecular research studies on animal lectins have elucidated structural aspects and provided clues not only to their evolution but also to their multiple biological functions. The experimental evidence has suggested that distinct, and probably unrelated, groups of molecules are included under the term lectin. Within the invertebrate taxa, major groups of lectins can be identified: One group would include lectins that show significant homology to membrane-integrated or soluble vertebrate C-type lectins. The second would include those beta-galactosyl-specific lectins homologous to the S-type vertebrate lectins. The third group would be constituted by lectins that show homology to vertebrate pentraxins that exhibit lectin-like properties, such as C-reactive protein and serum amyloid P. Finally, there are examples that do not exhibit similarities to any of the aforementioned categories. Moreover, the vast majority of invertebrate lectins described so far cannot yet be placed in one or another group because of the lack of information regarding their primary structure. (See Table 1.) Animal lectins do not express a recombinatorial diversity like that of antibodies, but a limited diversity in recognition capabilities would be accomplished by the occurrence of multiple lectins with distinct specificities, the presence of more than one binding site, specific for different carbohydrates in a single molecule, and by certain flexibility of the binding sites that would allow the recognition of a range of structurally related carbohydrates. In order to identify the lectins natural ligands, we have investigated the interactions between those proteins and the putative endogenous or exogenous glycosylated substances or cells that may be relevant to their biological function. Results from these studies, together with information on the biochemical properties of invertebrate and vertebrate lectins, including their structural relationships with other vertebrate recognition molecules, are discussed.

Animal lectins : a functional view

INTRODUCTION Introduction to Animal Lectins, G.R. Vasta and H. Ahmed MODERN APPROACHES FOR ASSESSING LECTIN FUNCTION Structural Aspects of Lectin-Ligand Interactions, M.A. Bianchet, H. Ahmed, G.R. Vasta, and L.M. Amzel Thermodynamic Approaches to the Study of Affinity of Clustered Carbohydrate Epitopes in Galectin-Glycoconjugate Interactions, T.K. Dam and C.F. Brewer Deciphering Lectin Ligands through Glycan Arrays, D.F. Smith and R.D. Cummings Chromatography and Related Approaches for Qualitative and Quantitative Analyses of Lectin Specificity, J. Hirabayashi Analysis of Whole-Genome and Other Data Resources to Characterize the Molecular, Structural, and Evolutionary Diversity of C-Lectins and Discover New Genes, A.N. Zelensky and J.E. Gready Animal Models for Assessing the Biological Roles of Lectins, H. Ahmed, G.A. Rabinovich, S.S. Jackson, M. Salatino, K. Saito, G. Bianco, S. Tasumi, S.-J. Du, and G.R. Vasta GLYCOPROTEIN FOLDING, SORTING AND SECRETION, TARGETING, DEGRADATION, AND CLEARANCE Calreticulin and Calnexin as Chaperones in Glycoprotein Folding, J.J. Caramelo and A.J. Parodi Role of L-Type Lectins in Glycoprotein Sorting and Trafficking, B. Nyfeler, M.W. Wendeler, and H.-P. Hauri P-Type Lectins and Lysosomal Enzyme Targeting, N.M. Dahms, L.J. Olson, and J.-J.P. Kim M-Type Lectins as Novel Components of Secretory Pathways, N. Hosokawa and K. Nagata Functional Aspects of the Hyaluronan and Chondroitin Sulfate Receptors, E.N. Harris and P.H. Weigel CELL ADHESION AND CELL SURFACE LATTICE FORMATION Galectin-3 and Cancer, Y. Wang, V. Balan, and A. Raz Myelin-Associated Glycoprotein (Siglec-4): A Nervous System Lectin That Regulates Axon-Myelin Stability and Axon Regeneration, R.L. Schnaar and N.R. Mehta Hyaluronan-Binding Proteoglycans, E.N. Harris and P.H. Weigel Roles of Coral Lectins in Morphological Change of Zooxanthellae, H. Kamiya, M. Jimbo, K. Koike, and R. Sakai CELL-CELL INTERACTIONS, SIGNALING, AND TRANSPORT Siglecs: Roles in Cell-Cell Interactions and Signaling, C.O. and P.R. Crocker Signaling through the Fungal b-Glucan Receptor Dectin-1, A.M. Kerrigan, K.M. Dennehy, and G.D. Brown CD22: A Regulator of B Cell Survival and Signal Transduction, S.H. Smith and T.F. Tedder Galectins and Integrins in Pre-B Cell Development, M. Espeli, L. Gauthier, S. Mancini, F. Mourcin, B. Rossi, and C. Schiff Golgi N-Glycan Processing and Galectin Functions, K.S. Lau, I.R. Nabi, M. Demetriou, and J.W. Dennis RECOGNITION AND EFFECTOR FUNCTIONS IN INNATE IMMUNITY Mannan-Binding Lectin Polymorphisms and Infectious Diseases, M. Moller-Kristensen, S. Thiel, and J.C. Jensenius Immunoregulatory Roles of Lung Surfactant Proteins A and D, N. Palaniyar, G.L. Sorensen, and U. Holmskov C-Type Lectin Receptors on Dendritic Cells, Y. van Kooyk Structural and Functional Roles of C-type Lectin Receptors on Natural Killer Cells, N. Dimasi Activation of Lepidopteran Insect Innate Immune Responses by C-type Immulectins, X.-Q. Yu and M.R. Kanost Galectins as Novel Regulators of Immune Cell Homeostasis and Inflammation, G. A. Rabinovich, M.A. Toscano, J.M. Ilarregui, and L.G. Baum Interactions of Galectins with Leukocytes, S.R. Stowell and R.D. Cummings Regulation of Immune Responses by Galectin-3, D. K. Hsu and F.-T. Liu X-Lectins: A New Family with Homology to the Xenopus laevis Oocyte Lectin XL-35, J.K. Lee and M. Pierce F-Type Lectins: A New Family of Recognition Factors, G.R. Vasta, E.W. Odom, M.A. Bianchet, L.M. Amzel, K. Saito, and H. Ahmed Biology of FREPs: Diversified Lectins with Fibrinogen-Related Domains from Freshwater Snail Biomphalaria glabrata, B.A. Stout, C.M. Adema, S.-M. Zhang, and E.S. Loker Lectins in Sand Fly-Leishmania Interactions, S. Kamhawi and J.G. Valenzuela Ficolins: The Structural Basis for Recognition Plasticity, M. Matsushita, Y. Endo, and T. Fujita Hemolytic Lectin in Marine Invertebrates, T. Hatakeyama Structure-Function Relationship in Mammalian Chitinase-Like Lectins, S. Morroll, S. Turner, and F.H. Falcone Index

Galectins in teleost fish: Zebrafish (Danio rerio) as a model species to address their biological roles in development and innate immunity

Cell surface glycans, such as glycocoproteins and glycolipids, encode information that modulates interactions between cells, or between cells and the extracellular matrix, by specifically regulating the binding to cell surface-associated or soluble carbohydrate-binding receptors, such as lectins. Rapid modifications of exposed carbohydrate moieties by glycosidases and glycosyltransferases, and the equally dynamic patterns of expression of their receptors during early development, suggest that both play important roles during embryogenesis. Among a variety of biological roles, galectins have been proposed to mediate developmental processes, such as embryo implantation and myogenesis. However, the high functional “redundancy” of the galectin repertoire in mammals has hindered the rigorous characterization of their specific roles by gene knockout approaches in murine models. In recent years, the use of teleost fish as alternative models for addressing developmental questions in mammals has expanded dramatically, and we propose their use for the elucidation of biological roles of galectins in embryogenesis and innate immunity. All three major galectin types, proto, chimera, and tandem-repeat, are present in teleost fish, and phylogenetic topologies confirm the expected clustering with their mammalian orthologues. As a model organism, the zebrafish (Danio rerio) may help to overcome limitations imposed by the murine models because it offers substantial advantages: external fertilization, transparent embryos that develop rapidly in vitro, a diverse toolbox of established methods to manipulate early gene expression, a growing collection of mutations that affect early embryonic development, availability of cell lines, and most importantly, an apparently less diversified galectin repertoire. Published in 2004.

Soluble beta-galactosyl-binding lectin (galectin) from toad ovary: crystallographic studies of two protein-sugar complexes.

Galectin‐1, S‐type β‐galactosyl‐binding lectins present in vertebrate and invertebrate species, are dimeric proteins that participate in cellular adhesion, activation, growth regulation, and apoptosis. Two high‐resolution crystal structures of B. arenarum galectin‐1 in complex with two related carbohydrates, LacNAc and TDG, show that the topologically equivalent hydroxyl groups in the two disaccharides exhibit identical patterns of interaction with the protein. Groups that are not equivalent between the two sugars present in the second moiety of the disaccharide, interact differently with the protein, but use the same number and quality of interactions.

Regulation of advanced glycation end product (AGE) receptors and apoptosis by AGEs in osteoblast-like cells.

Advanced glycation end products (AGEs) have been proposed as the pathological mechanisms underlying diabetic chronic complications. They may also play a role in the pathogenesis of diabetic osteopenia, although their mechanisms of action remain unclear. We investigated the protein (immunofluorescence) and gene expression (realtime RT-PCR) of two receptors for AGEs, RAGE and galectin-3, as well as their regulation by AGEs, and the apoptotic effect on osteoblast-like cells (UMR106 and MC3T3E1) in culture. AGEs up-regulated the expression of RAGE and galectin-3 in both cells lines. These effects were accompanied by an increase in the corresponding mRNA in the non-tumoral MC3T3E1 but not in the osteosarcoma UMR106 cells. Finally, we demonstrated that a 24xa0h exposure to AGEs induced apoptosis in both cell lines. Thus, AGEs-receptors may play important roles in the bone alterations described in aging and diabetic patients.

Nodavirus Infection of Sea Bass (Dicentrarchus labrax) Induces Up-Regulation of Galectin-1 Expression with Potential Anti-Inflammatory Activity,

Sea bass nervous necrosis virus is the causative agent of viral nervous necrosis, a disease responsible of high economic losses in larval and juvenile stages of cultured sea bass (Dicentrarchus labrax). To identify genes potentially involved in antiviral immune defense, gene expression profiles in response to nodavirus infection were investigated in sea bass head kidney using the suppression subtractive hybridization (SSH) technique. A total of 8.7% of the expressed sequence tags found in the SSH library showed significant similarities with immune genes, of which a prototype galectin (Sbgalectin-1), two C-type lectins (SbCLA and SbCLB) from groups II and VII, respectively, and a short pentraxin (Sbpentraxin) were selected for further characterization. Results of SSH were validated by in vivo up-regulation of expression of Sbgalectin-1, SbCLA, and SbCLB in response to nodavirus infection. To examine the potential role(s) of Sbgalectin-1 in response to nodavirus infection in further detail, the recombinant protein (rSbgalectin-1) was produced, and selected functional assays were conducted. A dose-dependent decrease of respiratory burst was observed in sea bass head kidney leukocytes after incubation with increasing concentrations of rSbgalectin-1. A decrease in IL-1β, TNF-α, and Mx expression was observed in the brain of sea bass simultaneously injected with nodavirus and rSbgalectin-1 compared with those infected with nodavirus alone. Moreover, the protein was detected in the brain from infected fish, which is the main target of the virus. These results suggest a potential anti-inflammatory, protective role of Sbgalectin-1 during viral infection.

The Primary Structure and Carbohydrate Specificity of a β-Galactosyl-binding Lectin from Toad (Bufo arenarum Hensel) Ovary Reveal Closer Similarities to the Mammalian Galectin-1 than to the Galectin from the Clawed Frog Xenopus laevis

The detailed characterization of a galectin from the toad (Bufo arenarum Hensel) ovary in its primary structure, carbohydrate specificity, and overall biochemical properties has provided novel information pertaining to structural and evolutionary aspects of the galectin family. The lectin consists of identical single-chain polypeptide subunits composed of 134 amino acids (calculated mass, 14,797 daltons), and its N-terminal residue, alanine, is N-acetylated. When compared to the sequences of known galectins, the B. arenarum galectin exhibited the highest identity (48% for the whole molecule and 77% for the carbohydrate recognition domain (CRD)) with the bovine spleen galectin-1, but surprisingly less identity (38% for the whole molecule and 47% for the CRD) with a galectin from Xenopus laevis skin (Marschal, P., Herrmann, J., Leffler, H., Barondes, S. H., and Cooper, D. N. W. (1992) J. Biol. Chem. 267, 12942-12949). Unlike the X. laevis galectin, the binding activity of the B. arenarum galectin for N-acetyllactosamine, the human blood group A tetrasaccharide and Galβ1,3GalNAc relative to lactose, was in agreement with that observed for the galectin-1 subgroup and those galectins having “conserved” (type I) CRDs (Ahmed, H., and Vasta, G. R. (1994) Glycobiology 4, 545-549). Moreover, the toad galectin shares three of the six cysteine residues that are conserved in all mammalian galectins-1, but not in the galectins from X. laevis, fish, and invertebrates described so far. Based on the homologies of the B. arenarum galectin with the bovine spleen galectin-1 and X. laevis skin galectin, it should be concluded that within the galectin family the correlation between conservation of primary structure and phylogenetic distances among the source species may not be a direct one as proposed elsewhere (Hirabayashi, J., and Kasai, K. (1993) Glycobiology 3, 297-304). Furthermore, galectins with conserved (type I) CRDs, represented by the B. arenarum ovary galectin, and those with “variable” (type II) CRDs, represented by the X. laevis 16-kDa galectin, clearly constitute distinct subgroups in the extant amphibian taxa and may have diverged early in the evolution of chordate lineages.

Superoxide dismutases from the oyster parasite Perkinsus marinus: purification, biochemical characterization, and development of a plate microassay for activity

We have isolated and biochemically characterized superoxide dismutase (SOD) activity in cell extracts of clonally cultured Perkinsus marinus, a facultative intracellular parasite of the Eastern oyster, Crassostrea virginica. In order to assess the SOD activity throughout the purification, we developed and optimized a 96-well-plate microassay based on the inhibition of pyrogallol oxidation. The assay was also adapted to identify SOD activity type (Cu/Zn-, Mn-, or FeSOD), even in mixtures of more than one type of SOD. All SOD activity detected in the cell extracts was of the FeSOD type. Most of the SOD activity in P. marinus trophozoites resides in a major component of subunit molecular weight 24 kDa. The protein was purified by affinity chromatography on an anti-SOD antibody-Sepharose column. Amino-terminal peptide sequence of the affinity-purified protein corresponds to the predicted product of the PmSOD1 gene and indicates that amino-terminal processing has taken place. The results are discussed in the context of processing of mitochondrially targeted SODs.

Biological Roles of Lectins in Innate Immunity: Molecular and Structural Basis for Diversity in Self/Non-Self Recognition

Lectins and other pattern recognition proteins are critical components of innate immune mechanisms in invertebrates and vertebrates. Unlike immunoglobulins, TCRs, and VLRs, which generate diversity in recognition by genetic recombination, lectins like most innate immune receptors are “hard-wired” in the germline. Therefore, one of the outstanding questions is how the innate immune system is able to cope with the great diversity of potential microbial infectious challenges. Although the concept of pattern recognition proposes that only a handful of microbial conserved surface molecules need to be recognized for successful innate immune defense, the highly diversified microbial communities to which all organisms are exposed to and the dynamic changes in surface expression components suggests that a substantial diversity in non-self recognition mechanisms may be required for immune protection. The detailed analysis of the structural basis of lectin ligand binding and the diversity and complexity of the lectin repertoires in taxa that lack adaptive immunity, such as invertebrates, strongly suggests that this is the case. Further, recent studies have extended these observations to ectothermic vertebrates. In this review we focus on