Caenorhabditis elegans galectins LEC-6 and LEC-10 interact with similar glycoconjugates in the intestine

Galectins are a family of metazoan proteins that show binding to various β-galactoside-containing glycans. Because of a lack of proper tools, the interaction of galectins with their specific glycan ligands in the cells and tissues are largely unknown. We have investigated the localization of galectin ligands in Caenorhabditis elegans using a novel technology that relies on the high binding specificity between galectins and their endogenous ligands. Fluorescently labeled recombinant galectin fusions are found to bind to ligands located in diverse tissues including the intestine, pharynx, and the rectal valve. Consistent with their role as galactoside-binding proteins, the interaction with their ligands is inhibited by galactose or lactose. Two of the galectins, LEC-6 and LEC-10, recognize ligands that co-localize along the intestinal lumen. The ligands for LEC-6 and LEC-10 are absent in three glycosylation mutants bre-1, fut-8, and galt-1, which have been shown to be required to synthesize the Gal-β1,4-Fuc modifications of the core N-glycans unique to C. elegans and several other invertebrates. Both galectins pull down the same set of glycoproteins in a manner dependent on the presence of these carbohydrate modifications. Endogenous LEC-6 and LEC-10 are expressed in the intestinal cells, but they are localized to different subcellular compartments that do not appear to overlap with each other or with the location of their glycan targets. An altered subcellular distribution of these ligands is found in mutants lacking both galectins. These results suggest a model where LEC-6 and LEC-10 interact with glycoproteins through specific glycans to regulate their cellular fate.     

Crystallization and preliminary x-ray crystallographic analysis of galectin LEC-1 from Caenorhabditis elegans

Galectin LEC-1 isolated from the nematode Caenorhabditis elegans was the first galectin found in invertebrates and also the first tandem-repeat-type galectin identified, containing two homologous carbohydrate-binding sites. This galectin is localized most abundantly in the adult cuticle and possibly plays a role in the formation of epidermal layers. We succeeded in crystallizing LEC-1 composed of 279 amino acids with a calculated molecular weight of 31,809 Da under two independent sets of conditions as a result of extensive screening. The crystals grown under one set of conditions belong to the triclinic space group P1, with unit-cell parameters a = 48.44, b = 52.13, c = 64.24 A, alpha = 108.73, beta= 91.39, and gamma = 98.45 degrees and two protein molecules per unit cell. The crystals grown under the other set of conditions which included lactose belong to the monoclinic space group P2(1), with unit-cell parameters a = 52.90, b = 47.01, c = 66.16 A, and beta= 113.30 degrees and one protein molecule per asymmetric unit.     

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.     

Myosin and paramyosin of Caenorhabditis elegans: biochemical and structural properties of wild-type and mutant proteins.

Myosin and paramyosin have been purified from the nematode, Caenorhabditis elegans. The properties of the myosin in general resemble those of other myosins. The native molecule is a dimer of heavy (210,000 dalton) polypeptide chains and contains 18,000 and 16,000 dalton light chains. When rapidly precipitated from solution, it forms small, bipolar aggregates, about 150 nm long, consistent with the expected molecular structure of a rigid rod with a globular head region at one end. Its ATPase activity is stimulated by Ca2+ and EDTA. The myosin binds to F actin in a polar and ATP-sensitive manner, and the Mg2+-ATPase is activated by either F actin or nematode thin filaments. Dialysis of myosin to low ionic strength produces very long filaments. When a myosin-paramyosin mixture is dialyzed under the same condtions, co-filaments form which consist of a myosin cortex, surrounding a paramyosin core. Some properties of myosin from the mutants E675 and E190, which have functionally and structurally altered body wall muscles, are compared with those of wild-type myosin. These myosins of these results are discussed in terms of the myosin heavy chain composition.     

Nematode chromosomal proteins--III. Some structural properties of the histones of Caenorhabditis elegans

The histones of Caenorhabditis elegans (Nematoda) have been identified by correlating criteria of electrophoresis and amino acid composition with the five main histones from calf thymus. C. elegans H1(1) consists of at least two subtypes with approximate molecular weights of 20,000 and 18,500 daltons as resolved by SDS polyacrylamide gel electrophoresis. They are some 10% smaller than the two subtypes of calf histone H1. The differences are also corrobated by the amino acid composition of the nematode and calf H1 complements. Nematode H2A resembles calf H2A in chromatographic and electrophoretic properties and in the amino acid composition, although it lacks histidine, which seems to be replaced by lysine. Like calf H2A, it is dimorphic as shown by Triton/acid/urea polyacrylamide gel electrophoresis. The H2B complement from C. elegans consists of two proteins with a molecular weight of approximately 12,500. They can be separated by ion-exchange chromatography, but they are very analogous to each other and to calf H2B in amino acid composition. Each form is also resolved into two more subtypes by Triton/acid/urea polyacrylamide gel electrophoresis. Nematode H3 resembles calf thymus H3 in its electrophoretic behaviour; three subfractions can be distinguished in Triton/acid/urea gels. C. elegans H4 is very similar to calf H4 in its chromatographic, electrophoretic and solubility properties, but differs significantly in composition. The meaning of this difference is discussed with regard to the generally observed stringent conservation of H4 sequences between distantly related species.     

Caenorhabditis elegans N-glycans containing a Gal-Fuc disaccharide unit linked to the innermost GlcNAc residue are recognized by C. elegans galectin LEC-6

We report a detailed structural analysis of the N-glycans of Caenorhabditis elegans recognized by C. elegans galectin LEC-6. Glycoproteins of C. elegans captured by an immobilized LEC-6 affinity adsorbent were isolated. The N-glycans of these glycoproteins were then liberated by hydrazinolysis and labeled with the fluorophore 2-aminopyridine (PA). The derived pyridylaminated (PA)-sugars were further fractionated by rechromatography on immobilized LEC-6 adsorbent and by reversed-phase high-performance liquid chromatography (HPLC). The structures of the PA-sugars thus obtained were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS/MS) in conjunction with glycosidase digestion. We confirmed that all PA-sugars having affinity for LEC-6 contain a Gal-Fuc disaccharide unit, and that this unit is bound to the innermost GlcNAc residue of the N-glycan chain. The dissociation constants of LEC-6 for these glycans were measured by frontal affinity chromatography. LEC-6 exhibited higher affinity for oligosaccharides having a Gal-Fuc unit linked to position 6 of the innermost GlcNAc residue than for those having Galbeta1-4GlcNAc units. Affinity for the former disappeared, however, following treatment with beta-galactosidase. If the glycan contained a Hex-Fuc disaccharide linked to the penultimate GlcNAc residue, the affinity would be diminished. We propose, therefore, that the galectins of C. elegans utilize the Gal-Fuc disaccharide unit for recognition instead of the Gal-GlcNAc unit that is common in vertebrates.     

Neural network features distinguish chemosensory stimuli in Caenorhabditis elegans

Nervous systems extract and process information from the environment to alter animal behavior and physiology. Despite progress in understanding how different stimuli are represented by changes in neuronal activity, less is known about how they affect broader neural network properties. We developed a framework for using graph-theoretic features of neural network activity to predict ecologically relevant stimulus properties, in particular stimulus identity. We used the transparent nematode, Caenorhabditis elegans, with its small nervous system to define neural network features associated with various chemosensory stimuli. We first immobilized animals using a microfluidic device and exposed their noses to chemical stimuli while monitoring changes in neural activity of more than 50 neurons in the head region. We found that graph-theoretic features, which capture patterns of interactions between neurons, are modulated by stimulus identity. Further, we show that a simple machine learning classifier trained using graph-theoretic features alone, or in combination with neural activity features, can accurately predict salt stimulus. Moreover, by focusing on putative causal interactions between neurons, the graph-theoretic features were almost twice as predictive as the neural activity features. These results reveal that stimulus identity modulates the broad, network-level organization of the nervous system, and that graph theory can be used to characterize these changes.     

Neural development features: spatio-temporal development of the Caenorhabditis elegans neuronal network

The nematode Caenorhabditis elegans, with information on neural connectivity, three-dimensional position and cell linage, provides a unique system for understanding the development of neural networks. Although C. elegans has been widely studied in the past, we present the first statistical study from a developmental perspective, with findings that raise interesting suggestions on the establishment of long-distance connections and network hubs. Here, we analyze the neuro-development for temporal and spatial features, using birth times of neurons and their three-dimensional positions. Comparisons of growth in C. elegans with random spatial network growth highlight two findings relevant to neural network development. First, most neurons which are linked by long-distance connections are born around the same time and early on, suggesting the possibility of early contact or interaction between connected neurons during development. Second, early-born neurons are more highly connected (tendency to form hubs) than later-born neurons. This indicates that the longer time frame available to them might underlie high connectivity. Both outcomes are not observed for random connection formation. The study finds that around one-third of electrically coupled long-range connections are late forming, raising the question of what mechanisms are involved in ensuring their accuracy, particularly in light of the extremely invariant connectivity observed in C. elegans. In conclusion, the sequence of neural network development highlights the possibility of early contact or interaction in securing long-distance and high-degree connectivity.     

Structural properties of the Caenorhabditis elegans neuronal network

Despite recent interest in reconstructing neuronal networks, complete wiring diagrams on the level of individual synapses remain scarce and the insights into function they can provide remain unclear. Even for Caenorhabditis elegans, whose neuronal network is relatively small and stereotypical from animal to animal, published wiring diagrams are neither accurate nor complete and self-consistent. Using materials from White et al. and new electron micrographs we assemble whole, self-consistent gap junction and chemical synapse networks of hermaphrodite C. elegans. We propose a method to visualize the wiring diagram, which reflects network signal flow. We calculate statistical and topological properties of the network, such as degree distributions, synaptic multiplicities, and small-world properties, that help in understanding network signal propagation. We identify neurons that may play central roles in information processing, and network motifs that could serve as functional modules of the network. We explore propagation of neuronal activity in response to sensory or artificial stimulation using linear systems theory and find several activity patterns that could serve as substrates of previously described behaviors. Finally, we analyze the interaction between the gap junction and the chemical synapse networks. Since several statistical properties of the C. elegans network, such as multiplicity and motif distributions are similar to those found in mammalian neocortex, they likely point to general principles of neuronal networks. The wiring diagram reported here can help in understanding the mechanistic basis of behavior by generating predictions about future experiments involving genetic perturbations, laser ablations, or monitoring propagation of neuronal activity in response to stimulation.     

UbiA, the major polyubiquitin locus in Caenorhabditis elegans, has unusual structural features and is constitutively expressed.

Ubiquitin is a multifunctional 76-amino-acid protein which plays critical roles in many aspects of cellular metabolism. In Caenorhabditis elegans, the major source of ubiquitin RNA is the polyubiquitin locus, UbiA. UbiA is transcribed as a polycistronic mRNA which contains 11 tandem repeats of ubiquitin sequence and possesses a 2-amino-acid carboxy-terminal extension on the final repeat. The UbiA locus possesses several unusual features not seen in the ubiquitin genes of other organisms studied to date. Mature UbiA mRNA acquires a 22-nucleotide leader sequence via a trans-splicing reaction involving a 100-nucleotide splice leader RNA derived from a different chromosome. UbiA is also unique among known polyubiquitin genes in containing four cis-spliced introns within its coding sequence. Thus, UbiA is one of a small class of genes found in higher eucaryotes whose heterogeneous nuclear RNA undergoes both cis and trans splicing. The putative promoter region of UbiA contains a number of potential regulatory elements: (i) a cytosine-rich block, (ii) two sequences resembling the heat shock regulatory element, and (iii) a palindromic sequence with homology to the DNA-binding site of the mammalian steroid hormone receptor. The expression of the UbiA gene has been studied under various heat shock conditions and has been monitored during larval moulting and throughout the major stages of development. These studies indicate that the expression of the UbiA gene is not inducible by acute or chronic heat shock and does not appear to be under nutritional or developmental regulation in C. elegans.     

Dissection of lin-11 enhancer regions in Caenorhabditis elegans and other nematodes

The Caenorhabditis elegans LIM homeobox gene lin-11 plays crucial roles in the morphogenesis of the reproductive system and differentiation of several neurons. The expression of lin-11 in different tissues is regulated by enhancer regions located upstream as well as within lin-11 introns. These regions are functionally separable suggesting that multiple regulatory inputs operate to control the spatiotemporal pattern of lin-11 expression. To further dissect apart the nature of lin-11 regulation we focused on three Caenorhabditis species C. briggsae, C. remanei, and C. brenneri that are substantially diverged from C. elegans but share almost identical vulval morphology. We show that, in these species, the 5′ region of lin-11 possesses conserved sequences to activate lin-11 expression in the reproductive system. Analysis of the in vivo role of these sequences in C. elegans has led to the identification of three functionally distinct enhancers for the vulva, VC neurons, and uterine π lineage cells. We found that the π enhancer is regulated by FOS homolog FOS-1 and LIN-12/Notch pathway effectors, LAG-1 (Su(H)/CBF1 family) and EGL-43 (EVI1 family). These results indicate that multiple factors cooperate to regulate π-specific expression of lin-11 and together with other findings suggest that the mechanism of lin-11 regulation by LIN-12/Notch signaling is evolutionarily conserved in Caenorhabditis species. Our work demonstrates that 4-way comparison is a powerful tool to study conserved mechanisms of gene regulation in C. elegans and other nematodes.     

Sugar binding properties of the two lectin domains of the tandem repeat-type galectin LEC-1 (N32) of Caenorhabditis elegans. Detailed analysis by an improved frontal affinity chromatography method

The 32-kDa galectin (LEC-1 or N32) of the nematode Caenorhabditis elegans is the first example of a tandem repeat-type galectin and is composed of two domains, each of which is homologous to typical vertebrate 14-kDa-type galectins. To elucidate the biological meaning of this unique structure containing two probable sugar binding sites in one molecule, we analyzed in detail the sugar binding properties of the two domains by using a newly improved frontal affinity chromatography system. The whole molecule (LEC-1), the N-terminal lectin domain (Nh), and the C-terminal lectin domain (Ch) were expressed in Escherichia coli, purified, and immobilized on HiTrap gel agarose columns, and the extent of retardation of various sugars by the columns was measured. To raise the sensitivity of the system, we used 35 different fluorescence-labeled oligosaccharides (pyridylaminated (PA) sugars). All immobilized proteins showed affinity for N-acetyllactosamine-containing N-linked complex-type sugar chains, and the binding was stronger for more branched sugars. Ch showed 2-5-fold stronger binding toward all complex-type sugars compared with Nh. Both Nh and Ch preferred Galbeta1-3GlcNAc to Galbeta1-4GlcNAc. Because the Fucalpha1-2Galbeta1-3GlcNAc (H antigen) structure was found to interact with all immobilized protein columns significantly, the K(d) value of pentasaccharide Fucalpha1-2Galbeta1-3GlcNAcbeta1-3Galbeta1-4Glc-PA for each column was determined by analyzing the concentration dependence. Obtained values for immobilized LEC-1, Nh, and Ch were 6.0 x 10(-5), 1.3 x 10(-4), and 6.5 x 10(-5) m, respectively. The most significant difference between Nh and Ch was in their affinity for GalNAcalpha1-3(Fucalpha1-2)Galbeta1-3GlcNAcbeta1-3Galbeta1-4Glc-PA, which contains the blood group A antigen; the K(d) value for immobilized Nh was 4.8 x 10(-5) m, and that for Ch was 8.1 x 10(-4) m. The present results clearly indicate that the two sugar binding sites of LEC-1 have different sugar binding properties.     

Caenorhabditis elegans num-1 negatively regulates endocytic recycling

Much of the material taken into cells by endocytosis is rapidly returned to the plasma membrane by the endocytic recycling pathway. Although recycling is vital for the correct localization of cell membrane receptors and lipids, the molecular mechanisms that regulate recycling are only partially understood. Here we show that in Caenorhabditis elegans endocytic recycling is inhibited by NUM-1A, the nematode Numb homolog. NUM-1AGFP fusion protein is localized to the baso-lateral surfaces of many polarized epithelial cells, including the hypodermis and the intestine. We show that increased NUM-1A levels cause morphological defects in these cells similar to those caused by loss-of-function mutations in rme-1, a positive regulator of recycling in both C. elegans and mammals. We describe the isolation of worms lacking num-1A activity and show that, consistent with a model in which NUM-1A negatively regulates recycling in the intestine, loss of num-1A function bypasses the requirement for RME-1. Genetic epistasis analysis with rab-10, which is required at an early part of the recycling pathway, suggests that loss of num-1A function does not affect the uptake of material by endocytosis but rather inhibits baso-lateral recycling downstream of rab-10.     

Mos1-mediated insertional mutagenesis in Caenorhabditis elegans

We describe a protocol for mutating genes in the nematode Caenorhabditis elegans using the Mos1 transposon of Drosophila mauritiana. Mutated genes containing a Mos1 insertion are molecularly tagged by this heterologous transposable element. Mos1 insertions can therefore be identified in as little as 3 weeks using only basic molecular biology techniques. Mutagenic efficiency of Mos1 is tenfold lower than classical chemical mutagens. However, the ease and speed with which mutagenic insertions can be mapped compares favorably with the vast amount of work involved in classical genetic mapping. Therefore, Mos1 could be the tool of choice when screening procedures are efficient. In addition, Mos1 mutagenesis can greatly simplify the mapping of mutations that exhibit low penetrance, subtle or synthetic phenotypes. The recent development of targeted engineering of C. elegans loci carrying Mos1 insertions further increases the attractiveness of Mos1-mediated mutagenesis.     

Ligand-binding properties of annexin from Caenorhabditis elegans (annexin XVI, Nex-1)

Annexins are structurally related proteins that bind phospholipids in a calcium-dependent manner. Recently, we showed that annexins IV, V, and VI also bind glycosaminoglycans in a calcium-dependent manner. Annexins are widely distributed from lower to higher eukaryotes, and the nematode Caenorhabditis elegans has been found to contain Nex-1, an annexin homologue. Here, we characterize the ligand-binding properties of Nex-1 using recombinant Nex-1. Nex-1 binds to liposomes containing phosphatidylserine. The apparent K(d) was calculated by Biacore to be 4.4 nM. Compared to mammalian annexins, the Nex-1 phospholipid-binding specificities were similar whereas the K(d) values were one order of magnitude larger. The Nex-1 glycosaminoglycan-binding specificities were investigated by affinity chromatography and solid-phase assays. Nex-1 binds to heparin, heparan sulfate, and chondroitin sulfate but not to chondroitin and chemically N- or O-desulfated heparin. Besides phospholipids, heparan sulfate and/or chondroitin (sulfate), probably on perlecan, could be endogenous ligands of Nex-1.     

Species differentiation in Caenorhabditis briggsae and Caenorhabditis elegans

Identification of five laboratory strains (1-5) of putative Caenorhabditis briggsae was undertaken. Examination of the male bursal ray arrangement, mating tests with males of Caenorhabditis elegans, malate dehydrogenase zymograms, and SDS polyacrylamide electrophoresis demonstrated that strain 4 was C. briggsae and the others were C. elegans.     

KCNQ-like potassium channels in Caenorhabditis elegans. Conserved properties and modulation

The human KCNQ gene family encodes potassium channels linked to several genetic syndromes including neonatal epilepsy, cardiac arrhythmia, and progressive deafness. KCNQ channels form M-type potassium channels, which are critical regulators of neuronal excitability that mediate autonomic responses, pain, and higher brain function. Fundamental mechanisms of the normal and abnormal cellular roles for these channels may be gained from their study in simple model organisms. Here we report that a multigene family of KCNQ-like channels is present in the nematode, Caenorhabditis elegans. We show that many aspects of the functional properties, tissue expression pattern, and modulation of these C. elegans channels are conserved, including suppression by the M1 muscarinic receptor. We also describe a conserved mechanism of modulation by diacylglycerol for a subset of C. elegans and vertebrate KCNQ/KQT channels, which is dependent upon the carboxyl-terminal domains of channel subunits and activated protein kinase C.