A Family of Genes of Multidomain Free Lectins from a Planarian: Structure,Expression, and the Use as Markers for Monitoring Regeneration

A family of genes of the asexual race of planarian Girardia tigrina were described that encode proteins that belong to the superfamily of C-type lectins and were demonstrated to have a unique domain organization. The genes are differentially expressed in the planarian body. The protein products of at least two genes (scarf2 and gtlec1) are expressed in specifically differentiated gland cells of the planarian and secreted into the environment through long cell necks. A comparison of the results obtained by electron microscopy and immunohistochemistry with literature data allows the assignment of these cells to the group of adhesion glands. The observation of the regeneration of the cell necks in normal and artificial two-headed planaria indicated that the dorsoventral contact at the edge of the head part of the planarian body directs and maintains the growth of the gtLec1-producing cell necks during regeneration.     

A family of genes of multidomain free lectins from a planarian: structure, expression, and use as markers for regeneration monitoring

A family of genes of the agamic race of planarian Girardia tigrina were described that encode proteins that belong to the superfamily of C-type lectins and were demonstrated to have a unique domain organization. The genes are differentially expressed in the planarian body. The protein products of at least two genes (scarf2 and gtlec1) are expressed in specifically differentiated gland cells of the planarian and secreted into the environment through long cell necks. A comparison of the results obtained by electron microscopy and immunohistochemistry with literature data allows the assignment of these cells to the group of adhesion glands. The observation of the regeneration of the cell necks in normal and artificial two-headed planaria indicated that the dorsoventral contact at the edge of the head part of the planarian body directs and maintains the growth of the gtLec1-producing cell necks during regeneration. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 6; see also http://www.maik.ru.     

Cloning of Clr, a new family of lectin-like genes localized between mouse Nkrp1a and Cd69

We report the identification of a novel family of genes, named Clr, encoding C-type lectin-like molecules, which maps in the natural killer (NK) gene complex (NKC) on mouse Chromosome 6. Genomic sequence analysis indicates the presence of at least seven members between Nkrpla and Cd69. By RT-PCR, at least three members of the family are expressed on interleukin-2-activated NK cells. Sequence analysis revealed complete open reading frames of 203-205 amino acids, with a carboxyl-terminal C-type lectin-like carbohydrate recognition domain (CRD). The CRDs of the Clr proteins exhibit a significant degree of homology with the known NKC-encoded NK-cell receptors. However, a key cysteine usually present in the CRD is missing in the Clr proteins, suggesting that their ligands and functions are distinct from other molecules encoded in the NKC.     

Characterization of a novel C-type lectin-like gene, LSECtin: demonstration of carbohydrate binding and expression in sinusoidal endothelial cells of liver and lymph node

A new C-type lectin-like gene encodes 293 amino acids and maps to chromosome 19p13.3 adjacent to the previously described C-type lectin genes, CD23, dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN), and DC-SIGN-related protein (DC-SIGNR). The four genes form a tight cluster in an insert size of 105 kb and have analogous genomic structures. The new C-type lectin-like molecule, designated liver and lymph node sinusoidal endothelial cell C-type lectin (LSECtin), is a type II integral membrane protein of approximately 40 kDa in size with a single C-type lectin-like domain at the COOH terminus, closest in homology to DC-SIGNR, DC-SIGN, and CD23. LSECtin mRNA was only expressed in liver and lymph node among 15 human tissues tested, intriguingly neither expressed on hematopoietic cell lines nor on monocyte-derived dendritic cells (DCs). Moreover, LSECtin is expressed predominantly by sinusoidal endothelial cells of human liver and lymph node and co-expressed with DC-SIGNR. LSECtin binds to mannose, GlcNAc, and fucose in a Ca(2+)-dependent manner but not to galactose. Our results indicate that LSECtin is a novel member of a family of proteins comprising CD23, DC-SIGN, and DC-SIGNR and might function in vivo as a lectin receptor.     

Are Planaria Individuals? What Regenerative Biology is Telling Us About the Nature of Multicellularity

Freshwater planaria (Platyhelminthes, Turbellaria, Tricladida) pose a challenge to current concepts of biological individuality. We review molecular and developmental evidence suggesting that mature intact planaria are not biological individuals but their totipotent stem cells (neoblasts) are individuals. Neoblasts within a single planarian body are, in particular, genetically heterogeneous, migratory, effectively immortal, and effectively autonomous. They cooperate to maintain the planarian body as an obligate environment but compete to make this environment maximally conducive to the survival of their own neoblast lineages. These results suggest that planaria have not fully completed the transition to multicellularity, but instead represent an intermediate form in which a small number of genetically-heterogeneous, reproductively-competent cells effectively “farm” their reproductively-incompetent offspring.     

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.     

Stem cell protection mechanisms in planarians: the role of some heat shock genes

Planarians contain a large population of stem cells, named neoblasts, and use these for continuous turnover of all cell types. In addition, thanks to the amazing flexibility of these cells, planarians respond well to the effects of stressful situations, for example activating regeneration after trauma. How neoblasts respond to stress and support continuous proliferation, maintaining long-term stability, is still an open question. Heat shock proteins (HSPs) are a complex protein family with key roles in maintaining protein homeostasis, as well as in apoptosis and growth-related processes. We recently characterized some planarian homologs of hsp genes that are highly expressed in mammalian stem cells, and observed that some of them are critical for neoblast survival/maintenance. The results of these studies support the notion that some HSPs play crucial roles in the modulation of pathways regulating stem cell activity, regeneration and tissue repair. In this review we compare the evidence available for planarian hsp genes and focus on questions emerging from these results.     

Amino acid sequences and phosphorylation sites of emu and rhea eggshell C-type lectin-like proteins

Avian calcified eggshell layers contain in their organic matrix one or two C-type lectin-like proteins. Previously characterized eggshell proteins of this family are chicken ovocleidin-17 (OC-17), goose ansocalcin and ostrich struthiocalcins 1 and 2 (SCA-1, SCA-2). In this report we present the amino acid sequences of two emu (Dromaius novaehollandiae) (dromaiocalcin-1 and -2; DCA-1, DCA-2) and of two rhea (Rhea americana) (rheacalcin-1 and -2; RCA-1, RCA-2) C-type lectin-like eggshell proteins, thus doubling the data set for comparison of these major specific eggshell proteins. The ratite proteins can be divided into two groups. Group 1, comprising SCA-1, DCA-1 and RCA-1, shows by 70--77% identity of sequences, the lack of phosphorylation, and a variable number (7--9) of cysteines. Group 2, consisting of SCA-2, DCA-2 and RCA-2, shows 78--85% identical sequences, 2--3 phosphorylated serines located at almost identical sites, and contains only the common set of six conserved cysteins characteristic for this family of proteins. While goose ansocalcin fits perfectly into group 1 with a sequence identity of 63--70% to the other members, no phosphorylation, and seven cysteines, chicken OC-17 was assigned to group 2 in spite of only 42--47% sequence identity (and 37--39% to group 1) because of its two phosphorylated serines and its regular set of six cysteines. At present it remains unknown why ratites, but not goose or chicken, require two different types of C-type lectin-like proteins to construct their eggshells.     

The C-type lectin-like receptors of Dectin-1 cluster in natural killer gene complex

Natural killer gene complex (NKC) encodes a group of proteins with a single C-type lectin-like domain, (CTLD) which can be subdivided several subfamilies according to their structures and expression patterns. The receptors containing the conserved calcium binding sites in the CTLD fold belong to group II of C-type lectin superfamily and are expressed on myeloid cells and non- myeloid cells. The receptors lacking conserved calcium binding sites in the CTLD fold have evolved to bind ligands other than carbohydrates independently on calcium and thereby are named as C-type lectin-like receptors. The C-type lectin-like receptors are previously thought to be exclusively expressed on natural killer (NK) cells and enable NK cells to discriminate self, missing self or altered self. However, some C-type lectin-like receptors are identified in myeloid cells and are intensely investigated, recently. These myeloid C-type lectin-like receptors, especially Dectin-1 cluster, have a wide variety of ligands, including those of exogenous origin, and play important roles in the physiological functions and pathological processes including immune homeostasis, immune defenses, and immune surveillance. In this review, we summarize each member of the Dectin-1 cluster, including their structural profiles, expression patterns, signaling properties as well as known physiological functions.     

蛇毒C型凝集素类蛋白cDNA与基因组DNA序列分析显示特别的转录后加工(英文)

为研究蛇毒C型凝集素类蛋白的快速进化机制和结构功能关系 ,使用PCR技术扩增了若干编码C型凝集素类蛋白 β链的cDNA分子以及agkisasinβ的基因组DNA ,并将这些扩增产物进行克隆和测序 .对测序结果与试验过程中的具体条件进行了因果关系分析 ,并且进行点阵图比较和多序列比对 .结果表明 ,可能存在“转录后同源重组”等转录后的事件 ,在蛇毒C型凝集素类蛋白的多样性上起着重要的作用 .对于解释基因数目与蛋白质数目的差异这一后基因组时代的重要问题 ,具有一定的参考价值 .首次报告蛇毒C型凝集素类蛋白的基因组DNA序列 ,其中未发现有内含子     

Antimicrobial properties of avian eggshell-specific C-type lectin-like proteins

C-type lectin-like proteins are major components of the calcified eggshell of multiple avian species. In this study, two representative avian C-type lectin-like proteins, ovocleidin-17 and ansocalcin, were purified from decalcified chicken and goose eggshell protein extracts and investigated for carbohydrate binding activity as well as antimicrobial activity. Purified ovocleidin-17 and ansocalcin were found to bind bacterial polysaccharides, and were bactericidal against Bacillus subtilis, Staphylococcus aureus and Pseudomona aeruginosa. Bactericidal activity was found to be enhanced in the presence of calcium but was not dependent on its presence. The results suggest that avian C-type lectin-like proteins may play an important antimicrobial role in defence of the avian embryo.     

Immune-related, lectin-like receptors are differentially expressed in the myeloid and lymphoid lineages of zebrafish

The identification of C-type lectin (Group V) natural killer (NK) cell receptors in bony fish has remained elusive. Analyses of the Fugu rubripes genome database failed to identify Group V C-type lectin domains (Zelensky and Gready, BMC Genomics 5:51, 2004) suggesting that bony fish, in general, may lack such receptors. Numerous Group II C-type lectin receptors, which are structurally similar to Group V (NK) receptors, have been characterized in bony fish. By searching the zebrafish genome database we have identified a multi-gene family of Group II immune-related, lectin-like receptors (illrs) whose members possess inhibiting and/or activating signaling motifs typical of Group V NK receptors. Illr genes are differentially expressed in the myeloid and lymphoid lineages, suggesting that they may play important roles in the immune functions of multiple hematopoietic cell lineages.     

C-type lectin-like domains.

Carbohydrate-recognition domains of C-type (Ca2+-dependent) animal lectins serve as prototypes for an important family of protein modules. Only some domains in this family bind Ca2+ or sugars. A comparison of recent structures of C-type lectin-like domains reveals diversity in the modular fold, particularly in the region associated with Ca2+ and sugar binding. Some of this diversity reflects the changes that occur during normal physiological functioning of the domains. C-type lectin-like domains associate with each other through several different surfaces to form dimers and trimers, from which ligand-binding sites project in a variety of different orientations.     

Analysis of part of the chicken<Emphasis Type="Italic"> Rfp-Y</Emphasis> region reveals two novel lectin genes,the first complete genomic sequence of a class I α-chain gene,a truncated class II β-chain gene,and a large CR1 repeat

The Rfp-Y region lies on the same microchromosome as the B-F/B-L region of the B complex, yet in contrast to the latter it is poorly characterised. To date it has been shown to contain at least two class I alpha-chain ( Y-F) genes, a class II B-chain gene and a C-type lectin-like gene. We describe the sequencing and analysis of some 20 kb of the Rfp-Y region, and identify several new genes. These include two novel C-type lectin-like genes ( Y-Lec1 and Y-Lec2) that differ strongly from the previously described C-type lectin-like gene found in the Rfp-Y region. We describe a complete genomic sequence of a class I alpha-chain ( Y-F) gene and its promoter from the Rfp-Y region. The predicted cDNA from this gene has high homology to the previously reported Y-F cDNAs. The promoter contains an altered enhancer A element. This portion of the Rfp-Y region also contains a truncated class II B-chain ( Y-LB) gene, as well as a large chicken repeat 1 (CR1) element.     

圆斑蝰蛇毒七个C-型凝集素样蛋白亚基的cDNA克隆与序列分析

按照Promega公司的mRNA提取试剂盒操作手册,从圆斑蝰蛇（Daboia russellii siamensis）的毒腺中提取mRNA;利用RT-PCR的方法进行体外扩增,获得C-型凝集素蛋白的基因,克隆到pMD18-T载体中。随机挑选14个阳性克隆进行核酸测序,获得7个编码不同蛇毒C-型凝集素样蛋白亚基的cDNA,分别命名为DRS-L1、DRS-L2、DRS-L3、DRS-L4、DRS-L5、DRS-L6和DRS-L7。由基因序列推导出的氨基酸序列表明,克隆到的7个蛇毒C-型凝集素样蛋白的亚基中均有糖识别结构域存在。BLAST分析显示,仅有DRS-L1的蛋白序列与目前已知的蛇毒C-型凝集素样蛋白的α亚基相似。序列同源性比较并结合半胱氨酸位点分析,推测DRS-L1和DRS—L2可能分别是圆斑蝰蛇毒X因子激活剂的轻链LC2和LC1。DRS-L3和DRS-L4可能是高分子量的蛇毒C-型凝集素样蛋白的β亚基,而DRS-L5和DRS-L6可能是低分子量的蛇毒C-型凝集素样蛋白的β亚基。DRS—L7可能是类似于血小板膜糖蛋白Ib结合蛋白的β亚基。     

Dual function of C-type lectin-like receptors in the immune system

Carbohydrate-binding C-type lectin and lectin-like receptors play an important role in the immune system. The large family can be subdivided into subtypes according to their structural similarities and functional differences. The selectins are of major importance in mediating cell adhesion and migration, and the mannose receptor subfamily is specialised in the binding and uptake of pathogens. Recent advances show that some of the type II C-type lectin-like receptors, such as DC-SIGN, can function both as an adhesion receptor and as a phagocytic pathogen-recognition receptor, similar to the Toll-like receptors. Although major differences in the cytoplasmic domains of these receptors might predict their function, recent findings show that differences in glycosylation of ligands can dramatically alter C-type lectin-like receptor usage.     

Mutational analysis of affinity and selectivity of kringle-tetranectin interaction. Grafting novel kringle affinity ontp the trtranectin lectin scaffold

C-type lectin-like domains are found in many proteins, where they mediate binding to a wide diversity of compounds, including carbohydrates, lipids, and proteins. The binding of a C-type lectin-like domain to a ligand is often influenced by calcium. Recently, we have identified a site in the C-type lectin-like domain of tetranectin, involving Lys-148, Glu-150, and Asp-165, which mediates calcium-sensitive binding to plasminogen kringle 4. Here, we investigate the effect of conservative substitutions of these and a neighboring amino acid residue. Substitution of Thr-149 in tetranectin with a tyrosine residue considerably increases the affinity for plasminogen kringle 4, and, in addition, confers affinity for plasminogen kringle 2. As shown by isothermal titration calorimetry analysis, this new interaction is stronger than the binding of wild-type tetranectin to plasminogen kringle 4. This study provides further insight into molecular determinants of importance for binding selectivity and affinity of C-type lectin kringle interactions.     

Avoidance behavior in two sympatric planaria species: effects of conspecific and heterospecific chemical alarm cues

In many aquatic animals, predator avoidance can be stimulated by chemical cues, including those released by injured prey (alarm cues). Alarm cues of both conspecific and heterospecific origin have been identified within several fish taxa, where phylogenetic conservation of the cue-response complex is common. Turbellarian flatworms (planaria) are among the simplest animals known to respond to chemical cues released by injured conspecifics. We examined how two locally sympatric planaria species respond to conspecific and heterospecific chemical cues using macerated tissue suspensions. Brown (Girardia tigrina) and black (Dugesia dorotocephala) planaria both exhibited avoidance behavior when presented conspecific cues. Despite a significant twofold difference in body size (black > brown), stimulus prepared from a single (1×) individual of either species elicited avoidance. Increasing brown planaria cue concentration by macerating two individuals (2×) produced a significant increase in conspecific avoidance. Heterospecific stimuli produced asymmetric results. Black planaria avoided the brown planaria stimulus, but only in the higher concentration (2×) trials. Brown planaria did not consistently exhibit avoidance of the black planaria stimulus and some brown subjects approached and consumed black planarian tissues. Our results expand the demonstrated occurrence of alarm cues among planaria and suggest that avoidance behavior can be mediated by multiple environmental and intrinsic factors in freshwater Turbellaria.     

The Dugesia ryukyuensis database as a molecular resource for studying switching of the reproductive system

The planarian Dugesia ryukyuensis reproduces both asexually and sexually, and can switch from one mode of reproduction to the other. We recently developed a method for experimentally switching reproduction of the planarian from the asexual to the sexual mode. We constructed a cDNA library from sexualized D. ryukyuensis and sequenced and analyzed 8,988 expressed sequence tags (ESTs). The ESTs were analyzed and grouped into 3,077 non-redundant sequences, leaving 1,929 singletons that formed the basis of unigene sets. Fifty-six percent of the cDNAs analyzed shared similarity (E-value<1E -20) with sequences deposited in NCBI. Highly redundant sequences encoded granulin and actin, which are expressed in the whole body, and other redundant sequences encoded a Vasa-like protein, which is known to be a component of germ-line cells and is expressed in the ovary, and Y-protein, which is expressed in the testis. The sexualized planarian expressed sequence tag database (http://planaria.bio.keio.ac.jp/planaria/) is an open-access, online resource providing access to sequence, classification, clustering, and annotation data. This database should constitute a powerful tool for analyzing sexualization in planarians.