Malik HS et al. (2000) Genome Res "Poised for contagion: evolutionary origins of the infectious abilities of invertebrate retroviruses."

Henikoff S, Eickbush TH, Malik HS

[Genome Res, 2000]

Phylogenetic analyses suggest that long-terminal repeat (LTR) bearing retrotransposable elements can acquire additional open-reading frames that can enable them to mediate infection. Whereas this process is best documented in the origin of the vertebrate retroviruses and their acquisition of an envelope (env) gene, similar independent events may have occurred in insects, nematodes, and plants. The origins of env-like genes are unclear, and are often masked by the antiquity of the original acquisitions and by their rapid rate of evolution. In this report, we present evidence that in three other possible transitions of LTR retrotransposons to retroviruses, an envelope-like gene was acquired from a viral source. First, the gypsy and related LTR retrotransposable elements (the insect errantiviruses) have acquired their envelope-like gene from a class of insect baculoviruses (double-stranded DNA viruses with no RNA stage). Second, the Cer retroviruses in the Caenorhabditis elegans genome acquired their envelope gene from a Phleboviral (single ambisense-stranded RNA viruses) source. Third, the Tas retroviral envelope (Ascaris lumricoides) may have been obtained from Herpesviridae (double-stranded DNA viruses, no RNA stage). These represent the only cases in which the env gene of a retrovirus has been traced back to its original source. This has implications for the evolutionary history of retroviruses as well as for the potential ability of all LTR-retrotransposable elements to become infectious agents.

Malik HS et al. (2000) Gene "Putative telomerase catalytic subunits from Giardia lamblia and Caenorhabditis elegans."

Burke WD, Malik HS, Eickbush TH

[Gene, 2000]

Eukaryotic chromosomes end in short nucleotide repeats that are added by the enzyme telomerase. The catalytic subunit of telomerase has been shown to be most closely related in sequence to reverse transcriptases encoded by eukaryotic retrotransposable elements. This raises the question as to whether the telomerase subunit was present in the first eukaryotes or was derived during early eukaryote evolution from the replication machinery of a retrotransposable element. We present the sequence of a putative telomerase catalytic subunit from the diplomonad parasite, Giardia lamblia. The G. lamblia subunit appears to have most of the characteristics of other sequenced telomerases, except that it lacks the conserved telomerase-specific 'T' motif previously identified in other eukaryotic genes. Searching genomic databases with the G. lamblia sequence, we also identified a potential telomerase catalytic subunit from Caenorhabditis elegans. The C. elegans subunit is uncharacteristically short, and lacks several motifs found in all other telomerases. The identification of a G. lamblia telomerase similar to that of most other eukaryotes suggests that telomerase dates back to the earliest extant marker of eukaryotic evolution. The atypical C. elegans telomerase, on the other hand, raises intriguing biochemical questions concerning sub-domains of the telomerase catalytic subunit previously considered indispensable. The enzymatic machinery for telomere formation in C. elegans is likely to differ substantially from that of other eukaryotes.

Malik HS et al. (2000) Genetics "NeSL-1, an ancient lineage of site-specific non-LTR retrotransposons from Caenorhabditis elegans."

Malik HS, Eickbush TH

[Genetics, 2000]

Phylogenetic analyses of non-LTR retrotransposons suggest that all elements can be divided into 11 lineages. The 3 oldest lineages show target site specificity for unique locations in the genome and encode an endonuclease with an active site similar to certain restriction enzymes. The more "modern" non-LTR lineages possess an apurinic endonuclease-like domain and generally lack site specificity. The genome sequence of Caenorhabditis elegans reveals the presence of a non-LTR retrotransposon that resembles the older elements, in that it contains a single open reading frame with a carboxyl-terminal restriction-like endonuclease domain. Located near the N-terminal end of the ORF is a cysteine protease domain not found in any other non-LTR element. The N2 strain of C. elegans appears to contain only one full-length and several 5' truncated copies of this element. The elements specifically insert in the Spliced leader-1 genes; hence the element has been named NeSL-1 (Nematode Spliced Leader-1). Phylogenetic analysis confirms that NeSL-1 branches very early in the non-LTR lineage and that it represents a 12th lineage of non-LTR elements. The target specificity of NeSL-1 for the spliced leader exons and the similarity of its structure to that of R2 elements leads to a simple model for its expression and retrotransposition.

Miller DL et al. (2011) PLoS One "HIF-1 and SKN-1 coordinate the transcriptional response to hydrogen sulfide in Caenorhabditis elegans."

Miller DL, Budde MW, Roth MB

[PLoS One, 2011]

Hydrogen sulfide (HS) has dramatic physiological effects on animals that are associated with improved survival. C. elegans grown in HS are long-lived and thermotolerant. To identify mechanisms by which adaptation to HS effects physiological functions, we have measured transcriptional responses to HS exposure. Using microarray analysis we observe rapid changes in the abundance of specific mRNAs. The number and magnitude of transcriptional changes increased with the duration of HS exposure. Functional annotation suggests that genes associated with protein homeostasis are upregulated upon prolonged exposure to HS. Previous work has shown that the hypoxia-inducible transcription factor, HIF-1, is required for survival in HS. In fact, we show that hif-1 is required for most, if not all, early transcriptional changes in HS. Moreover, our data demonstrate that SKN-1, the C. elegans homologue of NRF2, also contributes to HS-dependent changes in transcription. We show that these results are functionally important, as skn-1 is essential to survive exposure to HS. Our results suggest a model in which HIF-1 and SKN-1 coordinate a broad transcriptional response to HS that culminates in a global reorganization of protein homeostasis networks.

Attreed M et al. (2015) Methods Mol Biol "A transgenic approach to live imaging of heparan sulfate modification patterns."

Attreed M, Bulow HE

[Methods Mol Biol, 2015]

Heparan sulfate (HS) glycosaminoglycan chains contain highly modified HS domains that are separated by sections of sparse or no modification. HS domains are central to the role of HS in protein binding and mediating protein-protein interactions in the extracellular matrix. Since HS domains are not genetically encoded, they are impossible to visualize and study with conventional methods in vivo. Here we describe a transgenic approach using previously described single chain variable fragment (scFv) antibodies that bind HS in vitro and on tissue sections with different specificities. By engineering a secretion signal and a fluorescent protein to the scFvs and transgenically expressing these fluorescently tagged antibodies in Caenorhabditis elegans, we are able to directly visualize specific HS domains in live animals (Attreed et al. Nat Methods 9(5):477-479, 2012). The approach allows concomitant colabeling of multiple epitopes, the study of HS dynamics and, could lend itself to a genetic analysis of HS domain biosynthesis or to visualize other nongenetically encoded or posttranslational modifications.

Attreed, Matthew et al. (2009) International Worm Meeting "Uncovering the heparanome in C. elegans and the factors that create it."

van Kuppevelt, Toin, Attreed, Matthew, Bulow, Hannes

[International Worm Meeting, 2009]

The heparanome is composed of the entirety of heparan sulfate (HS) in C. elegans. HS are unbranched, highly modified polysaccharide chains which are attached to core proteins to form proteoglycans. We have previously shown that HS modifications display specific and instructive functions during neural development, possibly due to the interaction of ligands and receptors in the extracellular space. The HS glycosaminoglycans (GAGs) are modified by enzymes, which can add sulfates, remove acetyl groups, and epimerize the conformation of sugars in the molecule. These HS modifications are introduced non-randomly and non-uniformly to form functional domains of HS modification patterns. While we know the enzymes that introduce individual modifications, nothing is known about what regulates the enzymes to create defined HS domains. Our work sets out to first determine the temporal and spatial expression of defined HS patterns, and second, to devise a genetic approach to identify the genes that are required to establish the HS domain patterns. To this end, we have conducted immunohistochemical (IHC) studies of the heparanome using single chain variable fragment (scFv) antibodies that recognize specific HS modification patterns. We find that antibodies that recognize different HS patterns stain distinct anatomical structures, sometimes with extraordinary cell specificity, suggesting that worms harbor a complex ''sugar landscape'' in the extracellular space. We are currently in the process of defining (i) the individual modifications that are required for staining with a given antibody, and (ii) the temporal and spatial expression pattern of HS domains. To facilitate a forward genetic approach to identify genes involved in the creation of defined HS modification patterns, we have developed a novel approach that allows, for the first time, in vivo labeling of defined sugar modification patterns. This will greatly speed up the screening process and should allow the use of high throughput screening approaches to identify the genes, which create functional domains on HS.

Attreed, Matthew et al. (2011) International Worm Meeting "Direct visualization and functional modulation suggest a role for specific heparan sulfate motifs in cholinergic synaptic transmission in C. elegans."

Attreed, Matthew, Buelow, Hannes

[International Worm Meeting, 2011]

Heparan sulfates (HS) are unbranched, variably modified polysaccharide chains in the extracellular space that are invariably attached to proteins. The modification process of the sugars results in HS chains harboring different motifs of specifically arranged modifications. We and others have shown that individual HS modifications are crucial for various aspects of neural development in metazoans. Yet, little is known about the dynamics and function of defined HS motifs in the nervous system. In order to address this question experimentally, we have developed a tool that allows for live imaging of HS sugar motifs. By transgenically expressing secreted single chain variable fragment (scFv) antibody::GFP fusions, which recognize different HS motifs, we can visualize for the first time HS sugars with distinct modification patterns in living animals. We detect a highly refined distribution of HS motifs in both neuronal and non-neuronal tissues that often exhibit dynamic changes during development. Specifically, we find different HS motifs associated with the nervous system from late embryonic stages into adulthood. Intriguingly, our data suggest the existence of neuron-specific HS motifs. Genetic experiments and colocalization studies show that some HS motifs are associated with synaptic markers in the nerve cords. The possibility of functional neutralization of the HS motifs recognized by different scFv antibodies prompted us to study the nervous system of the transgenic animals in more detail. We find that the gross neuroanatomy in transgenic animals expressing different scFv antibody constructs is indistinguishable from wild type animals. However, based on aldicarb and levamisole assays, we detect defects in cholinergic synaptic transmission in animals expressing certain antibodies, possibly as a result of functional neutralization by the antibody fusions. These findings suggest that specific HS motifs serve a role in cholinergic transmission. In conclusion, we have developed a novel tool for the visualization and modulation of HS sugar motifs in vivo. Our experiments indicate that HS motifs are expressed dynamically and, possibly, in a neuron-specific manner. Moreover, distinct HS motifs may serve a role in modulating synaptic transmission.

Attreed M et al. (2010) Neuronal Development, Synaptic Function and Behavior, Madison, WI "Visualizing Heparan Sulfate Domains in Living C. elegans"

Attreed M, Buelow H

[Neuronal Development, Synaptic Function and Behavior, Madison, WI, 2010]

Heparan sulfate (HS) are unbranched, highly modified polysaccharide chains of the extracellular space. These chains are attached to proteins forming proteoglycans. HS modifications occur non-randomly and non-uniformly thereby creating specific, functional domains believed to mediate protein-protein interactions. We have previously shown that these HS modifications can serve specific and instructive functions during neural development. However, the dynamics of these sugar molecules in vivo are not known. While in vivo imaging of genetically encoded molecules is routine since the introduction of green fluorescent protein, in vivo visualization of molecules with defined covalent modifications and/or non-genetically encoded molecules in the extracellular space is at best difficult or not possible at all. Using single chain variable fragment (scFv) antibodies, which recognize domains of modified HS, we have developed a tool that allows for the imaging of HS sugar domains in vivo. We find specific HS motifs associated with distinct anatomical structures throughout development of C. elegans. From late embryonic stages, HS is associated with the nervous system and pharyngeal muscle as well as select basement membranes. Additionally, the same HS motif is also found associated with body wall muscle. Using genetic analyses we demonstrate that the recognized HS structures associated with the nerve ring are (1) attached to the syndecan core protein and (2) contain both HS-3O-sulfated and HS-6O-sulfated sugar residues. In conclusion, we have developed a novel tool for the visualization of HS sugar domains in vivo. This tool should allow for investigation of the dynamics of HS domain formation and remodeling. Our approach is in principle applicable to visualize any type of molecule (genetically encoded or not) in the extracellular space.

Tarja Kinnunen (2008) European Worm Meeting "Functions of heparan sulphate proteoglycans (HSPGs) in C. elegans neuronal development"

Tarja Kinnunen

[European Worm Meeting, 2008]

Heparan sulphate proteoglycans (HSPGs) are ubiquitous glycoproteins of the. cell surface and of the extracellular matrix that contain a protein core. substituted with heparan sulphate (HS) polysaccharides. HS chains encode. highly specific sugar sequences with variant sulphation patterns that. confer their biological functions as protein regulators. HS/HSPGs play. essential roles in controlling cell differentiation, tissue morphogenesis. and homeostasis. Furthermore, HS/HSPGs control amyloid formation thus. contributing to neurodegenerative diseases such as Alzheimer''s. The tissue. and developmental stage specificity of HS structures is in many cases well. documented. However, how this specificity is regulated remains vastly. unresolved.. I have analysed the role of HS/HSPGs for neuron migration and axon guidance. using C. elegans the hermaphrodite specific neurons, HSNs, as model.. Multiple HSPGs are required in parallel for the development of functional. HSNs, and defects in HSPG core proteins show additive effects for HSN. development. These HSPG dependent pathways utilize specific HS. modifications, suggesting that there is specificity in terms of both the. core protein and the HS structures required. Finally, double and triple. mutant analysis of HS modification enzymes reveals that there is a delicate. balance in the regulation of the HS structures, perturbations of which lead. to dramatic consequences in development.. Do not add objects such as pictures, boxes, headers, footers, footnotes,. etc.

Mirina, Alexandra A. et al. (2009) International Worm Meeting "A genetic approach towards structure function analyses of Heparan Sulfate."

Mirina, Alexandra A., Koh, Matthew, Townley, Robert A., Bhattacharya, Raja, Buelow, Hannes E.

[International Worm Meeting, 2009]

Heparan sulfates (HS) are polysaccharides of the cell surface and extracellular matrix that are attached to proteins to form HS proteoglycans (HSPG). The relatively simple heparan polymers undergo complex modifications involving de-acetylation, epimerization and sulfations to generate structural motifs that mediate the diverse functions of HSPGs. There is both biochemical and genetic evidence that these HS motifs have instructive functions in modulating ligand/receptor interactions during cell-cell communication. However, structure function analyses of HS domains in vivo has to date been limited due to the absence of an experimental system to characterize HS sugars with sufficient resolution in a genetically tractable system. We have developed an approach to directly correlate HS structure with function in vivo. To this end we have engineered transgenic animals that express an affinity tagged HS proteoglycan under a cell specific promoter. We have affinity purified the HS proteoglycan and, subsequently analyzed the sugars from this defined proteoglycan and tissue. In a complementary approach, we have begun to phenotypically characterize the strains misexpressing tagged HS proteoglycans. We observe that hypodermal expression of a tagged HSPG (syndecan-1) in strains lacking 2-O (hst-2) or 6-O (hst-6) sulfation leads to a severe body morphology defect not unlike the dumpy (dpy) phenotype. Using point mutant alleles of the transgene, we show that the synthetic dpy phenotype is dependent on the putative sugar attachment sites in the HS proteoglycan indicating that the synthetic phenotype is dependent on HS sugars. To gain further insight into the genetic pathways that govern the HS sugar dependent phenotype we have conducted a genetic screen for loci that produce a synthetic dpy phenotype in the syndecan-1 misexpressing strain. To date we have screened 14,000 haploid genomes and isolated 116 mutants. We expect the synthetic dpy mutants to define loci that control the synthesis of specific HS motifs or their effectors. This integrated biochemical and genetic approach will allow us to determine the relationship between HS structure and function in vivo.