An evolutionary view of functional diversity in family 1 glycosyltransferases

Glycosyltransferases (GTs) (EC 2.4.x.y) catalyze the transfer of sugar moieties to a wide range of acceptor molecules, such as sugars, lipids, proteins, nucleic acids, antibiotics and other small molecules, including plant secondary metabolites. These enzymes can be classified into at least 92 families, of which family 1 glycosyltransferases (GT1), often referred to as UDP glycosyltransferases (UGTs), is the largest in the plant kingdom. To understand how UGTs expanded in both number and function during evolution of land plants, we screened genome sequences from six plants (Physcomitrella patens, Selaginella moellendorffii, Populus trichocarpa, Oryza sativa, Arabidopsis thaliana and Arabidopsis lyrata) for the presence of a conserved UGT protein domain. Phylogenetic analyses of the UGT genes revealed a significant expansion of UGTs, with lineage specificity and a higher duplication rate in vascular plants after the divergence of Physcomitrella. The UGTs from the six species fell into 24 orthologous groups that contained genes derived from the common ancestor of these six species. Some orthologous groups contained multiple UGT families with known functions, suggesting that UGTs discriminate compounds as substrates in a lineage-specific manner. Orthologous groups containing only a single UGT family tend to play a crucial role in plants, suggesting that such UGT families may have not expanded because of evolutionary constraints.     

On the origin of family 1 plant glycosyltransferases

The phylogeny of highly divergent multigene families is often difficult to validate but can be substantiated by inclusion of data outside of the phylogeny, such as signature motifs, intron splice site conservation, unique substitutions of conserved residues, similar gene functions, and out groups. The Family 1 Glycosyltransferases (UGTs) comprises such a highly divergent, polyphyletic multigene family. Phylogenetic comparisons of UGTs from plants, animals, fungi, bacteria, and viruses reveal that plant UGTs represent three distinct clades. The majority of the plant sequences appears to be monophyletic and have diverged after the bifurcation of the animal/fungi/plant kingdoms. The two minor clades contain the sterol and lipid glycosyltransferases and each show more homology to non-plant sequences. The lipid glycosyltransferase clade is homologous to bacterial lipid glycosyltransferases and reflects the bacterial origin of chloroplasts. The fully sequenced Arabidopsis thaliana genome contains 120 UGTs including 8 apparent pseudogenes. The phylogeny of plant glycosyltransferases is substantiated with complete phylogenetic analysis of the A. thaliana UGT multigene family, including intron-exon organization and chromosomal localization.     

Evolutionary selective trends of insect/mosquito antimicrobial defensin peptides containing cysteine-stabilized alpha/beta motifs

Insect defensins containing cysteine-stabilized alpha/beta motifs (Cs-alpha/beta defensin) are cationic, inducible antibacterial peptides involved in humoral defence against pathogens. To examine trends in molecular evolution of these antimicrobial peptides, sequences similar to the well-characterized Cs-alpha/beta defensin peptide of Anopheles gambiae, using six cysteine residues as landmarks, were retrieved from genomic and protein databases. These sequences were derived from different orders of insects. Genes of insect Cs-alpha/beta defensin appear to constitute a multigene family in which the copy number varies between insect species. Phylogenetic analysis of these sequences revealed two main lineages, one group comprising mainly lepidopteran insects and a second, comprising Hemiptera, Coleoptera, Diptera and Hymenoptera insects. Moreover, the topology of the phylogram indicated dipteran Cs-alpha/beta defensins are diverse, suggesting diversity in immune mechanisms in this order of insects. Overall evolutionary analysis indicated marked diversification and expansion of mature defensin isoforms within the species of mosquitoes relative to non-mosquito defensins, implying the presence of finely tuned immune responses to counter pathogens. The observed higher synonymous substitution rate relative to the nonsynonymous rate in almost all the regions of Cs-alpha/beta defensin of mosquitoes suggests that these peptides are predominately under purifying selection. The maximum-likelihood models of codon substitution indicated selective pressure at different amino acid sites in mosquito mature Cs-alpha/beta defensins is differ and are undergoing adaptive evolution in comparison to non-mosquito Cs-alpha/beta defensins, for which such selection was inconspicuous; this suggests the acquisition of selective advantage of the Cs-alpha/beta defensins in the former group. Finally, this study represents the most detailed report on the evolutionary strategies of Cs-alpha/beta defensins of mosquitoes in particular and insects in general, and indicates that insect Cs-alpha/beta defensins have evolved by duplication followed by divergence, to produce a diverse set of paralogues.     

Txp40, a ubiquitous insecticidal toxin protein from Xenorhabdus and Photorhabdus bacteria

Xenorhabdus and Photorhabdus are gram-negative bacteria that produce a range of proteins that are toxic to insects. We recently identified a novel 42-kDa protein from Xenorhabdus nematophila that was lethal to the larvae of insects such as Galleria mellonella and Helicoverpa armigera when it was injected at doses of 30 to 40 ng/g larvae. In the present work, the toxin gene txp40 was identified in another 59 strains of Xenorhabdus and Photorhabdus, indicating that it is both highly conserved and widespread among these bacteria. Recombinant toxin protein was shown to be active against a variety of insect species by direct injection into the larvae of the lepidopteran species G. mellonella, H. armigera, and Plodia interpunctella and the dipteran species Lucilia cuprina. The protein exhibited significant cytotoxicity against two dipteran cell lines and two lepidopteran cell lines but not against a mammalian cell line. Histological data from H. armigera larvae into which the toxin was injected suggested that the primary site of action of the toxin is the midgut, although some damage to the fat body was also observed.     

Potential detoxification of gossypol by UDP-glycosyltransferases in the two Heliothine moth species Helicoverpa armigera and Heliothis virescens

The cotton bollworm Helicoverpa armigera and the tobacco budworm Heliothis virescens are closely related generalist insect herbivores and serious pest species on a number of economically important crop plants including cotton. Even though cotton is well defended by its major defensive compound gossypol, a toxic sesquiterpene dimer, larvae of both species are capable of developing on cotton plants. In spite of severe damage larvae cause on cotton plants, little is known about gossypol detoxification mechanisms in cotton-feeding insects. Here, we detected three monoglycosylated and up to five diglycosylated gossypol isomers in the feces of H. armigera and H. virescens larvae fed on gossypol-supplemented diet. Candidate UDP-glycosyltransferase (UGT) genes of H. armigera were selected by microarray studies and in silico analyses and were functionally expressed in insect cells. In enzymatic assays, we show that UGT41B3 and UGT40D1 are capable of glycosylating gossypol mainly to the diglycosylated gossypol isomer 5 that is characteristic for H. armigera and is absent in H. virescens feces. In conclusion, our results demonstrate that gossypol is partially metabolized by UGTs via glycosylation, which might be a crucial step in gossypol detoxification in generalist herbivores utilizing cotton as host plant.     

Anti-Cancer Drugs Elicit Re-Expression of UDP-Glucuronosyltransferases in Melanoma Cells

The UDP-glucuronosyltransferase (UGT) family of enzymes plays a vital role in the detoxification of carcinogens as well as clearance of anti-cancer drugs. In humans, 19 UGT family members have been identified and are expressed in a tissue specific manner throughout the body. However, the UGTs have not been previously characterized in melanocytes or melanoma. In the present study, UGT2B7, UGT2B10, and UGT2B15 were identified as being normally expressed in human melanocytes. The same three UGT family members were also expressed in the primary melanoma cell line WM115. No UGT expression was detected in another primary melanoma cell line, WM3211, or in any metastatic melanoma cell line examined. These results suggest that UGT expression is lost during melanoma progression. Treatment of WM3211 or metastatic melanoma cell lines with anti-cancer agents (including vemurafenib) induced expression of UGT2B7, UGT2B10 and UGT2B15 demonstrating that melanoma cells retain the ability to re-express these same three UGTs. The corresponding increase in glucuronidation activity in melanoma cells following anti-cancer treatment was also observed. Furthermore, knockdown of UGT2B7 in WM115 cells sensitized these cells to treatment by adriamycin and epirubicin indicating that UGT2B7 is involved in resistance to these drugs. However, knockdown of UGT2B7 had no effect on temozolomide toxicity. Taken together, these results clearly demonstrate a role for UGTs in melanoma etiology. Since the UGTs are drug metabolism enzymes, we propose that re-expression of the UGTs constitutes a previously unsuspected mechanism for intratumoral drug resistance in melanoma.     

Molecular evolution and expression of the CRAL_TRIO protein family in insects

CRAL_TRIO domain proteins are known to bind small lipophilic molecules such as retinal, inositol and Vitamin E and include such gene family members as PINTA, α-tocopherol transfer (ATT) proteins, retinoid binding proteins, and clavesins. In insects, very little is known about either the molecular evolution of this family of proteins or their ligand specificity. Here we characterize insect CRAL_TRIO domain proteins and present the first insect CRAL_TRIO protein phylogeny constructed by performing reciprocal BLAST searches of the reference genomes of Drosophila melanogaster, Anopheles gambiae, Apis mellifera, Tribolium castaneum, Bombyx mori, Manduca sexta and Danaus plexippus. We find several highly conserved amino acid residues in the CRAL_TRIO domain-containing genes across insects and a gene expansion resulting in more than twice as many gene family members in lepidopterans than in other surveyed insect species, but no lepidopteran homolog of the PINTA gene in Drosophila. In addition, we examined the expression pattern of CRAL_TRIO domain genes in Manduca sexta heads using RNA-Seq data. Of the 42 gene family members found in the M. sexta reference genome, we found 30 expressed in the head tissue with similar expression profiles between males and females. Our results suggest this gene family underwent a large expansion in Lepidoptera, making the lepidopteran CRAL_TRIO domain family distinct from other holometabolous insect lineages.     

Characterization of three novel SINE families with unusual features in Helicoverpa armigera

Although more than 120 families of short interspersed nuclear elements (SINEs) have been isolated from the eukaryotic genomes, little is known about SINEs in insects. Here, we characterize three novel SINEs from the cotton bollworm, Helicoverpa armigera. Two of them, HaSE1 and HaSE2, share similar 5' -structure including a tRNA-related region immediately followed by conserved central domain. The 3' -tail of HaSE1 is significantly similar to that of one LINE retrotransposon element, HaRTE1.1, in H. armigera genome. The 3' -region of HaSE2 showed high identity with one mariner-like element in H. armigera. The third family, termed HaSE3, is a 5S rRNA-derived SINE and shares both body part and 3'-tail with HaSE1, thus may represent the first example of a chimera generated by recombination between 5S rRNA and tRNA-derived SINE in insect species. Further database searches revealed the presence of these SINEs in several other related insect species, but not in the silkworm, Bombyx mori, indicating a relatively narrow distribution of these SINEs in Lepidopterans. Apart from above, we found a copy of HaSE2 in the GenBank EST entry for the cotton aphid, Aphis gossypii, suggesting the occurrence of horizontal transfer.     

Does host‐plant diversity explain species richness in insects? A test using Coccidae (Hemiptera)

1. The megadiverse herbivores and their host plants are a major component of biodiversity, and their interactions have been hypothesised to drive the diversification of both. 2. If plant diversity influences the diversity of insects, there is an expectation that insect species richness will be strongly correlated with host‐plant species richness. This should be observable at two levels (i) more diverse host‐plant groups should harbour more species of insects, and (ii) the species richness of a group of insects should correlate with the richness of the host groups it uses. However, such a correlation is also consistent with a hypothesis of random host use, in which insects encounter and use hosts in proportion to the diversity of host plants. Neither of these expectations has been widely tested. 3. These expectations were tested using data from a species‐rich group of insects – the Coccidae (Hemiptera). 4. Significant positive correlations were found between the species richness of coccid clades (genera) and the species richness of the host‐plant family or families upon which the clades occur. On a global scale, more closely related plant families have more similar communities of coccid genera but the correlation is weak. 5. Random host use could not be rejected for many coccids but randomisation tests and similarity of coccid communities on closely related plant families show that there is non‐random host use in some taxa. Overall, our results support the idea that plant diversity is a driver of species richness of herbivorous insects, probably via escape‐and‐radiate or oscillation‐type processes.     

Phylogenetic analysis of the UDP-glycosyltransferase multigene family of Arabidopsis thaliana

A class of UDP-glycosyltransferases (UGTs) defined by the presence of a C-terminal consensus sequence is found throughout the plant and animal kingdoms. Whereas mammalian enzymes use UDP-glucuronic acid, the plant enzymes typically use UDP-glucose in the transfer reactions. A diverse array of aglycones can be glucosylated by these UGTs. In plants, the aglycones include plant hormones, secondary metabolites involved in stress and defense responses, and xenobiotics such as herbicides. Glycosylation is known to regulate many properties of the aglycones such as their bioactivity, their solubility, and their transport properties within the cell and throughout the plant. As a means of providing a framework to start to understand the substrate specificities and structure-function relationships of plant UGTs, we have now applied a molecular phylogenetic analysis to the multigene family of 99 UGT sequences in Arabidopsis. We have determined the overall organization and evolutionary relationships among individual members with a surprisingly high degree of confidence. Through constructing a composite phylogenetic tree that also includes all of the additional plant UGTs with known catalytic activities, we can start to predict both the evolutionary history and substrate specificities of new sequences as they are identified. The tree already suggests that while the activities of some subgroups of the UGT family are highly conserved among different plant species, others subgroups shift substrate specificity with relative ease.     

Diversity investigation by application of DNA barcoding: A case study of lepidopteran insects in Xinjiang wild fruit forests,China

To investigate the species diversity of lepidopteran insects in Xinjiang wild fruit forests, establish insect community monitoring systems, and determine the local species pool, we test the applicability of DNA barcoding based on cytochrome c oxidase subunit I (COI) gene for accurate and rapid identification of insect species. From 2017 to 2019, a total of 212 samples with ambiguous morphological identification were selected for DNA barcoding analysis. Five sequence‐based methods for species delimitation (ABGD, BINs, GMYC, jMOTU, and bPTP) were conducted for comparison to traditional morphology‐based identification. In total, 2,422 samples were recorded, representing 143 species of 110 genera in 17 families in Lepidoptera. The diversity analysis showed that the richness indices for Noctuidae was the highest (54 species), and for Pterophoridae, Cossidae, Limacodidae, Lasiocampidae, Pieridae, and Lycaenidae were the lowest (all with 1 species). The Shannon–Wiener species diversity index (H′) and Pielou''s evenness (J′) of lepidopteran insects first increased and then decreased across these 3 years, while the Simpson diversity index showed a trend of subtracted then added. For molecular‐based identification, 67 lepidopteran species within 61 genera in 14 families were identified through DNA barcoding. Neighbor‐joining (NJ) analysis showed that conspecific individuals were clustered together and formed monophyletic groups with a high support value, except for Lacanobia contigua (Denis & Schiffermüller, 1775) (Noctuidae: Hadeninae). Sixty‐seven morphospecies were classified into various numbers of MOTUs based on ABGD, BINs, GMYC, jMOTU, and bPTP (70, 96, 2, 71, and 71, respectively). In Xinjiang wild fruit forests, the family with the largest number of species is Noctuidae, followed by Geometridae, Crambidae, and the remaining families. The highest Shannon diversity index is observed for the family Noctuidae. Our results indicate that the distance‐based methods (ABGD and jMOTU) and character‐based method (bPTP) outperform GMYC. BINs is inclined to overestimate species diversity compared to other methods.     

Triterpenoid-biosynthetic UDP-glycosyltransferases from plants

Triterpenoid saponins are naturally occurring structurally diverse glycosides of triterpenes that are widely distributed among plant species. Great interest has been expressed by pharmaceutical and agriculture industries for the glycosylation of triterpenes. Such modifications alter their taste and bio-absorbability, affect their intra?/extracellular transport and storage in plants, and induce novel biological activities in the human body. Uridine diphosphate (UDP)-glycosyltransferases (UGTs) catalyze glycosylation using UDP sugar donors. These enzymes belong to a multigene family and recognize diverse natural products, including triterpenes, as the acceptor molecules. For this review, we collected and analyzed all of the UGT sequences found in Arabidopsis thaliana as well as 31 other species of triterpene-producing plants. To identify potential UGTs with novel functions in triterpene glycosylation, we screened and classified those candidates based on similarity with UGTs from Panax ginseng, Glycine max, Medicago truncatula, Saponaria vaccaria, and Barbarea vulgaris that are known to function in glycosylate triterpenes. We highlight recent findings on UGT inducibility by methyl jasmonate, tissue-specific expression, and subcellular localization, while also describing their catalytic activity in terms of regioselectivity for potential key UGTs dedicated to triterpene glycosylation in plants. Discovering these new UGTs expands our capacity to manipulate the biological and physicochemical properties of such valuable molecules.     

Expression of UDP-glucuronosyltransferases (UGTs) 2B7 and 1A6 in the human brain and identification of 5-hydroxytryptamine as a substrate

The extrahepatic expression of UDP-glucuronosyltransferases (UGTs) is important in the detoxification of a number of endogenous and exogenous compounds, including 5-hydroxytryptamine and morphine. Studies were designed to investigate the extrahepatic expression of human UGTs using RT-PCR techniques and to determine the UGTs involved in the glucuronidation of 5-hydroxytryptamine. Human UGT2B7 expression was found in the human liver, kidney, pancreas, and brain, while UGT1A6 expression is found in the liver, kidney, and brain. This is the first observation of UGTs present in the human central nervous system. Using glucuronidation assays, a significant amount of 5-hydroxytryptamine glucuronide was found to be catalyzed by UGT1A6. These studies suggest that UGT2B7 may play an important role in the overall contribution of morphine analgesia by serving to generate the potent morphine-6-O-glucuronide in situ. UGT1A6 could play an important role in the glucuronidation of 5-hydroxytryptamine in vivo, therefore terminating the actions of the neurotransmitter.     

Backbone assignment of the apo-form of the human C-terminal domain of UDP-glucuronosyltransferase 1A (UGT1A)

A major component of phase II drug metabolism is the covalent addition of glucuronic acid to metabolites and xenobiotics. This activity is carried out by UDP-glucuronosyltransferases (UGT) which bind the UDP-glucuronic acid donor and catalyze the covalent addition of glucuronic acid sugar moieties onto a wide variety of substrates. UGTs play important roles in drug detoxification and were recently shown to act in an inducible form of multi-drug resistance in cancer patients. Despite their biological importance, structural understanding of these enzymes is limited. The C-terminal domain is identical for all UGT1A family members and required for binding to UDP-glucuronic acid as well as involved in contacts with substrates. Here, we report the backbone assignments for the C-terminal domain of UGT1A. These assignments are a critical tool for the development of a deeper biochemical understanding of substrate specificity and enzymatic activity.     

Genetic Diversity in Cymbopogon Species using PCR-Based Functional Markers

Genetic diversity among 25 accessions (involving 8 species, 2 interspecific hybrids and one hybrid mutant) of medicinally important genus Cymbopogon was assessed using 17 PCR-based functional markers, that were designed from members of three different multigene families. We developed 16 primer pairs from two multigene families, 8 primer pairs each from cytochrome P450 and UDP-glucosyltransferase (UGT); one primer pair was derived from 5S rRNA gene family. A total of 119 fragments were visualized, of which 108 (91%) were polymorphic. The level of diversity among different taxa/accessions observed during the present study was, however, low relative to the diversity level obtained due to RAPD markers in two earlier studies. The pattern of genetic diversity neither matched with the known taxonomic classification, nor did it always match with the distribution of chemical constituents of the essential oils available in these accessions. Thus, present investigation though revealed poor correlation between the molecular and chemical diversity, these gene-based markers may prove useful in the development of perfect markers for association mapping of genes involved in controlling agronomically important traits.     

Preferential expression of biotransformation enzymes in the olfactory organs of Drosophila melanogaster, the antennae

Biotransformation enzymes have been found in the olfactory epithelium of vertebrates. We now show that in Drosophila melanogaster, a UDP-glycosyltransferase (UGT), as well as a short chain dehydrogenase/reductase and a cytochrome P450 are expressed specifically or preferentially in the olfactory organs, the antennae. The evolutionarily conserved expression of biotransformation enzymes in olfactory organs suggests that they play an important role in olfaction. In addition, we describe five Drosophila UGTs belonging to two families. All five UGTs contain a putative transmembrane domain at their C terminus as is the case for vertebrate UGTs where it is required for enzymatic activity. The primary sequence of the C terminus, including part of the transmembrane domain, differs between the two families but is highly conserved not only within each Drosophila family, but also between the members of one of the Drosophila families and vertebrate UGTs. The partial overlap of the conserved primary sequence with the transmembrane domain suggests that this part of the protein is involved in specific interactions occurring at the membrane surface. The presence of different C termini in the two Drosophila families suggests that they interact with different targets, one of which is conserved between Drosophila and vertebrates.     

东北地区早春蜜源植物传粉昆虫多样性研究

为了揭示蜜源植物与传粉昆虫的关系,科学的保护和利用传粉昆虫资源,本研究选取东北地区早春常见蜜源植物柳树Salix、延胡索Corydalis和银莲花Anemone为研究对象,对其传粉昆虫种类和访花行为进行调查,分析不同蜜源植物的传粉昆虫种类、优势类群、多样性和访花行为。结果表明：东北地区早春蜜源植物传粉昆虫共采集726头,隶属于5目14科53种,其中膜翅目4科35种,双翅目5科13种,鳞翅目3科3种,同翅目1科 1种,鞘翅目1科1种。柳树的优势传粉昆虫为蜂类,延胡索的优势传粉昆虫为熊蜂类,银莲花的优势传粉昆虫为蝇类。延胡索的传粉昆虫多样性、均匀度和丰富度均最高。蜜蜂对柳树单位时间内访花次数和单花停留时间最高,为94.33次和18.90 s。熊蜂对延胡索单位时间内访花次数和单花停留时间最高,为76.42次和15.37 s,熊蜂在采集延胡索花时有明显的盗蜜行为,该行为有利于其它传粉昆虫生存。东北地区保护生物多样性和传粉昆虫资源,应结合蜜源植物保护,可在早春时期补种柳树、延胡索和银莲花,并合理搭配,为传粉昆虫提供良好食物源和栖息地。