Minimal requirements for the binding of selectin ligands to a C-type carbohydrate-recognition domain.

The C-type carbohydrate-recognition domains of E-selectin and rat serum mannose-binding protein have similar structures. Selectin/mannose-binding protein chimeras created by transfer of key sequences from E-selectin into mannose-binding protein have previously been shown to bind the selectin ligand sialyl-Lewis(X) through a Ca(2+)-dependent subsite, common to many C-type lectins, and an accessory site containing positively charged amino acid residues. Further characterization of these chimeras as well as analysis of novel constructs containing additional regions of E-selectin demonstrate that selectin-like interaction with sialyl-Lewis(X) can be faithfully reproduced even though structural evidence indicates that the mechanisms of binding to E-selectin and the chimeras are different. Selectin-like binding to the nonfucosylated sulfatide and sulfoglucuronyl glycolipids can also be reproduced with selectin/mannose-binding protein chimeras that contain the two subsites involved in sialyl-Lewis(X) binding. These results indicate that binding of structurally distinct anionic glycans to C-type carbohydrate-recognition domains can be mediated by the Ca(2+)-dependent subsite in combination with a positively charged region that forms an ionic strength-sensitive subsite.     

Evolutionary conservation of intron position in a subfamily of genes encoding carbohydrate-recognition domains

The structure of the gene encoding a chicken liver receptor, the chicken hepatic lectin, which mediates endocytosis of glycoproteins has been established. The coding sequence is divided into six exons separated by five introns. The first three exons correspond to separate functional domains of the receptor polypeptide (cytoplasmic tail, transmembrane sequence, and extracellular neck region), while the final three exons encode the Ca(2+)-dependent carbohydrate-recognition domain. These results, as well as computer-assisted multiple sequence comparisons, establish this receptor as the evolutionary homolog of the mammalian asialoglycoprotein receptors. It is interesting that the chicken receptor falls into a subfamily of proteins along with the mammalian asialoglycoprotein receptors, since the saccharide-binding specificity of the chicken receptor resembles more closely that of a different set of calcium-dependent animal lectins, which includes the mannose-binding proteins. The portions of the genes encoding the carbohydrate-recognition domains of these proteins lack introns. The results suggest that divergence of intron-containing and intron-lacking carbohydrate-recognition domains preceded shuffling events in which other functional domains were associated with the carbohydrate-recognition domains. This was followed by further divergence, generating a variety of saccharide-binding specificities.     

Identification of amino acid residues that determine pH dependence of ligand binding to the asialoglycoprotein receptor during endocytosis

The rat hepatic asialoglycoprotein receptor mediates clearance of galactose- and N-acetylgalactosamine-terminated glycoproteins by endocytosis, binding ligands through a C-type, Ca(2+)-dependent carbohydrate-recognition domain (CRD) at extracellular pH and releasing them at lower pH in endosomes. At physiological Ca(2+) concentrations, the midpoint for ligand release from the CRD of the major subunit of the receptor is pH 7.1. In contrast, the midpoint is pH 5.0 for a galactose-binding derivative of the homologous C-type CRD of serum mannose-binding protein, which would thus not efficiently release ligand at an endosomal pH of 5.4. Site-directed mutagenesis of the CRD from the major subunit of the asialoglycoprotein receptor has been used to identify residues that are essential for efficient release of ligand at endosomal pH. The effects of changes to residues His(256), Asp(266), and Arg(270) singly and in combination indicate that these residues reduce the affinity of the CRD for Ca(2+), so that ligands are released at physiological Ca(2+) concentrations. The proximity of these three residues to the ligand-binding site at Ca(2+) site 2 of the domain suggests that they form a pH-sensitive switch for Ca(2+) and ligand binding. Introduction of histidine and aspartic acid residues into the mannose-binding protein CRD at positions equivalent to His(256) and Asp(266) raises the pH for half-maximal binding of ligand to 6.1. The results, as well as sequence comparisons with other C-type CRDs, confirm the importance of these residues in conferring appropriate pH dependence in this family of domains.     

An endogenous Drosophila receptor for glycans bearing alpha 1,3-linked core fucose residues

The genome of Drosophila melanogaster encodes several proteins that are predicted to contain Ca(2+)-dependent, C-type carbohydrate-recognition domains. The CG2958 gene encodes a protein containing 359 amino acid residues. Analysis of the CG2958 sequence suggests that it consists of an N-terminal domain found in other Drosophila proteins, a middle segment that is unique, and a C-terminal C-type carbohydrate-recognition domain. Expression studies show that the full-length protein is a tetramer formed by noncovalent association of disulfide-linked dimers that are linked through cysteine residues in the N-terminal domain. The expressed protein binds to immobilized yeast invertase through the C-terminal carbohydrate-recognition domain. Competition binding studies using monosaccharides demonstrate that CG2958 interacts specifically with fucose and mannose. Fucose binds approximately 5-fold better than mannose. Blotting studies reveal that the best glycoprotein ligands are those that contain N-linked glycans bearing alpha1,3-linked fucose residues. Binding is enhanced by the additional presence of alpha1,6-linked fucose. It has previously been proposed that labeling of the Drosophila neural system by anti-horseradish peroxidase antibodies is a result of the presence of difucosylated N-linked glycans. CG2958 is a potential endogenous receptor for such neural-specific carbohydrate epitopes.     

Decoding the molecular design principles underlying Ca(2+) binding to βγ-crystallin motifs

Numerous proteins belonging to the recently expanded βγ-crystallin superfamily bind Ca(2+) at the double-clamp N/D-N/D-X(1)-X(2)-S/T-S motif. However, there have been no attempts to understand the intricacies involving Ca(2+) binding, such as the determinants of Ca(2+)-binding affinity and their contributions to gain in stability. This work is an in-depth analysis of understanding the modes and determinants of Ca(2+) binding to βγ-crystallin motifs. We have performed extensive naturally occurring substitutions from related proteins on the βγ-crystallin domains of flavollin, a low-affinity Ca(2+)-binding protein, and clostrillin, a moderate-affinity protein. We monitored the consequences of these modifications on Ca(2)(+) binding by isothermal titration calorimetry, thermal stability and conformational and crystal structure analyses. We demonstrate that Ca(2)(+) binding to the two sites of a βγ-domain is interdependent and that the presence of Arg at the fifth position disables a site. A change from Thr to Ser, or vice versa, influences Ca(2+)-binding affinity, highlighting the basis of diversity found in these domains. A subtle change in the first site has a greater influence on Ca(2)(+) binding than a similar alteration in the second site. Thus, the second site is more variable in nature. Replacing an acidic or hydrophobic residue in a binding site alters the Ca(2+)-binding properties drastically. While it appears from their binding site sequence that these domains have evolved randomly, our examination illustrates the subtlety in the design of these modules. Decoding such design schemes would aid in our understanding of the functional themes underlying differential Ca(2)(+) binding and in predicting these in emerging sequence information.     

Role of conserved and nonconserved residues in the Ca(2+)-dependent carbohydrate-recognition domain of a rat mannose-binding protein. Analysis by random cassette mutagenesis.

The carbohydrate-recognition domain of rat serum mannose-binding protein A has been subjected to random cassette mutagenesis. Mutant domains, expressed in bacteria, were initially screened for binding to invertase-coated nitrocellulose and then analyzed further for Ca2+ affinity, saccharide binding, resistance to proteolysis, and oligomerization. The results are consistent with previous evolutionary and structural studies. Six out of seven completely inactive mutants have changes in residues directly involved in ligating Ca2+. Most changes in conserved residues which form part of the hydrophobic core characteristic of Ca(2+)-dependent (C-type) animal lectins result in decreased affinity for Ca2+, even though these residues are distant from the Ca2+ sites. Changes can be made in large portions of the surface without affecting saccharide binding. The results indicate that the precise arrangement of the regular portion of the domain containing the hydrophobic core is necessary for formation of a stable Ca(2+)-ligated structure under physiological conditions. The data also suggest that the saccharide-binding site is likely to be in close proximity to the bound Ca2+.     

Crystal structure of the C-type lectin-like domain from the human hematopoietic cell receptor CD69

CD69, one of the earliest specific antigens acquired during lymphoid activation, acts as a signal-transducing receptor involved in cellular activation events, including proliferation and induction of specific genes. CD69 belongs to a family of receptors that modulate the immune response and whose genes are clustered in the natural killer (NK) gene complex. The extracellular portion of these receptors represent a subfamily of C-type lectin-like domains (CTLDs), which are divergent from true C-type lectins and are referred to as NK-cell domains (NKDs). We have determined the three-dimensional structure of human CD69 NKD in two different crystal forms. CD69 NKD adopts the canonical CTLD fold but lacks the features involved in Ca(2+) and carbohydrate binding by C-type lectins. CD69 NKD dimerizes noncovalently, both in solution and in crystalline state. The dimer interface consists of a hydrophobic, loosely packed core, surrounded by polar interactions, including an interdomain beta sheet. The intersubunit core shows certain structural plasticity that may facilitate conformational rearrangements for binding to ligands. The surface equivalent to the binding site of other members of the CTLD superfamily reveals a hydrophobic patch surrounded by conserved charged residues that probably constitutes the CD69 ligand-binding site.     

The C-type lectin-like domain superfamily

The superfamily of proteins containing C-type lectin-like domains (CTLDs) is a large group of extracellular Metazoan proteins with diverse functions. The CTLD structure has a characteristic double-loop ('loop-in-a-loop') stabilized by two highly conserved disulfide bridges located at the bases of the loops, as well as a set of conserved hydrophobic and polar interactions. The second loop, called the long loop region, is structurally and evolutionarily flexible, and is involved in Ca2+-dependent carbohydrate binding and interaction with other ligands. This loop is completely absent in a subset of CTLDs, which we refer to as compact CTLDs; these include the Link/PTR domain and bacterial CTLDs. CTLD-containing proteins (CTLDcps) were originally classified into seven groups based on their overall domain structure. Analyses of the superfamily representation in several completely sequenced genomes have added 10 new groups to the classification, and shown that it is applicable only to vertebrate CTLDcps; despite the abundance of CTLDcps in the invertebrate genomes studied, the domain architectures of these proteins do not match those of the vertebrate groups. Ca2+-dependent carbohydrate binding is the most common CTLD function in vertebrates, and apparently the ancestral one, as suggested by the many humoral defense CTLDcps characterized in insects and other invertebrates. However, many CTLDs have evolved to specifically recognize protein, lipid and inorganic ligands, including the vertebrate clade-specific snake venoms, and fish antifreeze and bird egg-shell proteins. Recent studies highlight the functional versatility of this protein superfamily and the CTLD scaffold, and suggest further interesting discoveries have yet to be made.     

C型凝集素

C型凝集素（C-type lectin）代表一个识别碳水化合物配体依赖于钙离子（Ca2＋）参与的糖原结合蛋白家族,含有一个或多个一级结构和二级结构同源的碳水化合物识别结构域。随着研究的深入,越来越多的C型凝集素能够识别体内的非糖类的配体,包括蛋白质和脂类等。这些C型凝集素在维持机体稳态、免疫防御以及免疫监视等重要生理病理过程中发挥着重要作用。就C型凝集素的结构、分类和在免疫系统中的功能作一介绍。     

Genomic analysis of C-type lectins

Many biological effects of complex carbohydrates are mediated by lectins that contain discrete carbohydrate-recognition domains. At least seven structurally distinct families of carbohydrate-recognition domains are found in lectins that are involved in intracellular trafficking, cell adhesion, cell-cell signalling, glycoprotein turnover and innate immunity. Genome-wide analysis of potential carbohydrate-binding domains is now possible. Two classes of intracellular lectins involved in glycoprotein trafficking are present in yeast, model invertebrates and vertebrates, and two other classes are present in vertebrates only. At the cell surface, calcium-dependent (C-type) lectins and galectins are found in model invertebrates and vertebrates, but not in yeast; immunoglobulin superfamily (I-type) lectins are only found in vertebrates. The evolutionary appearance of different classes of sugar-binding protein modules parallels a development towards more complex oligosaccharides that provide increased opportunities for specific recognition phenomena. An overall picture of the lectins present in humans can now be proposed. Based on our knowledge of the structures of several of the C-type carbohydrate-recognition domains, it is possible to suggest ligand-binding activity that may be associated with novel C-type lectin-like domains identified in a systematic screen of the human genome. Further analysis of the sequences of proteins containing these domains can be used as a basis for proposing potential biological functions.     

Characterization of sugar binding by the mannose receptor family member,Endo180

Members of the mannose receptor family, the mannose receptor, the phospholipase A(2) receptor, DEC-205, and Endo180, contain multiple C-type lectin-like domains (CTLDs) within a single polypeptide. In addition, at their N termini, all four family members contain a cysteine-rich domain similar to the R-type carbohydrate recognition domains of ricin. However, despite the common presence of multiple lectin-like domains, these four endocytic receptors have divergent ligand binding activities, and it is clear that the majority of these domains do not bind sugars. Here the functions of the lectin-like domains of the most recently discovered family member, Endo180, have been investigated. Endo180 is shown to bind in a Ca(2+)-dependent manner to mannose, fucose, and N-acetylglucosamine but not to galactose. This activity is mediated by one of the eight CTLDs, CTLD2. Competition assays indicate that the monosaccharide binding specificity of Endo180 CTLD2 is similar to that of mannose receptor CTLD4. However, additional experiments indicate that, unlike the cysteine-rich domain of the mannose receptor, the cysteine-rich domain of Endo180 does not bind sulfated sugars. Thus, although Endo180 and the mannose receptor are now both known to be mannose binding lectins, each receptor is likely to have a distinct set of glycoprotein ligands in vivo.     

A C-type lectin of Caenorhabditis elegans: its sugar-binding property revealed by glycoconjugate microarray analysis

C-type lectins are a family of proteins with an affinity to carbohydrates in the presence of Ca²⁺. In the genome of Caenorhabditis elegans, almost 300 genes encoding proteins containing C-type lectin-like domains (CTLDs) have been assigned. However, none of their products has ever been shown to have carbohydrate-binding activity. In the present study, we selected 6 potential C-type lectin genes and prepared corresponding recombinant proteins. One of them encoded by clec-79 was found to have sugar-binding activity by using a newly developed glycoconjugate microarray based on evanescent-field excited fluorescence. CLEC-79 exhibited affinity to sugars containing galactose at the non-reducing terminal, especially to the Galβ1-3GalNAc structure, in the presence of Ca²⁺. Combined with structural information of the glycans of C. elegans, these results suggest that CLEC-79 preferentially binds to O-glycans in vivo.     

Calcium-binding EGF-like modules in coagulation proteinases: function of the calcium ion in module interactions

Epidermal growth factor (EGF)-like modules are involved in protein-protein interactions and are found in numerous extracellular proteins and membrane proteins. Among these proteins are enzymes involved in blood coagulation, fibrinolysis and the complement system as well as matrix proteins and cell surface receptors such as the EGF precursor, the low density lipoprotein receptor and the developmentally important receptor, Notch. The coagulation enzymes, factors VII, IX and X and protein C, all have two EGF-like modules, whereas the cofactor of activated protein C, protein S, has four EGF-like modules in tandem. Certain of the cell surface receptors have numerous EGF modules in tandem. A subset of EGF modules bind one Ca(2+). The Ca(2+)-binding sequence motif is coupled to a sequence motif that brings about beta-hydroxylation of a particular Asp/Asn residue. Ca(2+)-binding to an EGF module is important to orient neighboring modules relative to each other in a manner that is required for biological activity. The Ca(2+) affinity of an EGF module is often influenced by its N-terminal neighbor, be it another EGF module or a module of another type. This can result in an increase in Ca(2+) affinity of several orders of magnitude. Point mutations in EGF modules that involve amino acids which are Ca(2+) ligands result in the biosynthesis of biologically inactive proteins. Such mutations have been identified, for instance, in factor IX, causing hemophilia B, in fibrillin, causing Marfan syndrome, and in the low density lipoprotein receptor, causing hypercholesterolemia. In this review the emphasis will be on the coagulation factors.     

EGF-like module pair 3-4 in vitamin K-dependent protein S: modulation of calcium affinity of module 4 by module 3, and interaction with factor X.

Calcium-binding epidermal growth factor (EGF)-like modules are found in numerous extracellular and membrane proteins involved in such diverse processes as blood coagulation, lipoprotein metabolism, determination of cell fate, and cell adhesion. Vitamin K-dependent protein S, a cofactor of the anticoagulant enzyme activated protein C, has four EGF-like modules in tandem with the three C-terminal modules each harbouring a Ca(2+)-binding consensus sequence. Recombinant fragments containing EGF modules 1-4 and 2-4 have two Ca(2+)-binding sites with dissociation constants ranging from 10(-8) to 10(-5) M. Module-module interactions that greatly influence the Ca(2+) affinity of individual modules have been identified. As a step towards an analysis of the structural basis of the high Ca(2+) affinity, we expressed the Ca(2+)-binding EGF pair 3-4 from human protein S. Correct folding was shown by (1)H NMR spectroscopy. Calcium-binding properties of the C-terminal module were determined by titration with chromophoric chelators; binding to the low-affinity N-terminal site was monitored by (1)H-(15)N NMR spectroscopy. At physiological pH and ionic strength, the dissociation constants for Ca(2+) binding were 1.0x10(-6) M and 4. 8x10(-3) M for modules 4 and 3, respectively, i.e. the calcium affinity of the C-terminal site was about 5000-fold higher than that of the N-terminal site. Moreover, the Ca(2+) affinity of EGF 4, in the pair 3-4, was about 9000-fold higher than that of synthetic EGF 4. The EGF modules in protein S are known to mediate the interaction with factor Xa. We have now found modules 3-4 to be involved in this interaction. However, the individual modules 3 and 4 manifested no measurable activity.     

Molecular cloning and immune responsive expression of a novel C-type lectin gene from bay scallop Argopecten irradians

C-type lectins are Ca(2+)-dependent carbohydrate-recognition proteins that play crucial roles in innate immunity. The cDNA of C-type lectin (AiCTL1) in the bay scallop Argopecten irradians was cloned by expressed sequence tag (EST) and RACE techniques. The full-length cDNA of AiCTL1 was 660 bp, consisting of a 5'-terminal untranslated region (UTR) of 30 bp and a 3' UTR of 132 bp with a polyadenylation signal sequence AATAAA and a poly(A) tail. The AiCTL1 cDNA encoded a polypeptide of 166 amino acids with a putative signal peptide of 20 amino acid residues and a mature protein of 146 amino acids. The deduced amino acid sequence of AiCTL1 was highly similar to those of the C-type lectins from other animals and contained a typical carbohydrate-recognition domain (CRD) of 121 residues, which has four conserved disulfide-bonded cysteine residues that define the CRD and two additional cysteine residues at the amino terminus. AiCTL1 mRNA was dominantly expressed in the hemocytes of the bay scallop. The temporal expression of AiCTL1 mRNA in hemocytes was increased by 5.7- and 4.9-fold at 6h after injury and 8h after injection of bacteria, respectively. The structural features, high similarity and expression pattern of AiCTL1 indicate that the gene may be involved in injury healing and the immune response in A. irradians.     

Characterization of a novel C. elegans RGS protein with a C2 domain: evidence for direct association between C2 domain and Galphaq subunit

RGS (regulator of G protein signaling) proteins are GTPase-activating proteins (GAPs) for heterotrimeric G protein alpha subunits and negatively regulate G protein-mediated signal transduction. In this study, we determined the cDNA sequence of a novel Caenorhabditis elegans (C. elegans) RGS protein. The predicted protein, termed C2-RGS, consists of 782 amino acids, and contains a C2 domain and an RGS domain. C2 domains are typically known to be Ca(2+) and phospholipid binding sites, found in many proteins involved in membrane traffic or signal transduction, and most of their biological roles are not identified. To study the function of C2-RGS protein, a series of six truncated versions of C2-RGS were constructed. When the full-length protein of C2-RGS was expressed transiently in AT1a-293T cells, ET-1-induced Ca(2+) responses were strongly suppressed. When each of the mutants with either RGS domain or C2 domain was expressed, the Ca(2+) responses were suppressed moderately. Furthermore, we found that C2 domain of PLC-beta1 also had a similar moderate inhibitory effect. RGS domain of C2-RGS bound to mammalian and C. elegans Galphai/o and Galphaq subunits only in the presence of GDP/AlF(4)(-), and had GAP activity to Galphai3. On the other hand, C2 domains of C2-RGS and PLC-beta1 also bound strongly to Galphaq subunit, in the presence of GDP, GDP/AlF(4)(-), and GTPgammaS, suggesting the stable persistent association between these C2 domains and Galphaq subunit at any stage during GTPase cycle. These results indicate that both the RGS domain and the C2 domain are responsible for the inhibitory effect of the full-length C2-RGS protein on Galphaq-mediated signaling, and suggest that C2 domains of C2-RGS and PLC-beta1 may act as a scaffold module to organize Galphaq and the respective whole protein molecule in a stable signaling complex, both in the absence and presence of stimulus.     

Ca(2+)/Calmodulin-binding proteins from the C. elegans proteome

Calmodulin (CaM) is the primary Ca(2+)-sensor that regulates a wide variety of cellular processes in eukaryotes. Although many Ca(2+)/CaM-binding proteins have been identified, very few such proteins could be found from the genome-wide protein-protein interaction maps of Caenorhabditis elegans constructed by yeast two-hybrid screening. Using a genotype-phenotype conjugation method called mRNA-display, we performed a selection for Ca(2+)/CaM-binding proteins from a proteome library of C. elegans. The method allowed the identification of 9 known and 47 previously uncharacterized Ca(2+)-dependent CaM-binding proteins from the adult worm proteome. The Ca(2+)/CaM-binding properties of these proteins were characterized and their binding motifs were identified. The availability of such information could facilitate our understanding of the signaling pathways mediated by Ca(2+)/CaM in C. elegans. Due to its simplicity and efficiency, the method could be readily applied to examine the Ca(2+)-dependent binding partners of numerous other Ca(2+)-binding proteins, which may play important roles in many signaling pathways in C. elegans.