植物GT元件和GT因子的研究进展

GT元件是位于植物基因启动子区域中的串联重复DNA序列, 是启动子中富含T和A的顺式作用元件。迄今已经在不同植物中鉴定了一些GT元件并分析了其功能。GT因子是以三螺旋基序为DNA结合域, 与GT元件特异结合的一类转录因子。到目前为止, 仅在植物中发现有GT因子。GT因子与GT元件相互作用调节相关基因的转录, 进而提高植物的抗性或影响植物形态建成。文章就植物中不同的GT元件及GT因子的发现、结构及二者相互作用等方面进行了综述。     

水稻Trihelix转录因子家族全基因组分析及功能预测

Trihelix转录因子家族在植物生长发育以及响应逆境胁迫等方面发挥着重要作用,但目前基于水稻全基因组水平鉴定和分析该基因家族的研究尚未见相关报道。本文利用生物信息学方法在水稻基因组数据库中鉴定到Trihelix家族成员31个,序列聚类和功能结构域分析发现该家族均含有高度保守的、特征性的Trihelix结构域;根据亲缘关系远近和结构域特点,将其分为5个亚家族(Ⅰ~Ⅴ)。通过与拟南芥、二穗短炳草和高粱中Trihelix家族的聚类分析发现,这4个物种中Trihelix家族的分类相一致,但每个物种均含有不同亚家族的成员,表明该基因家族的分化早于物种的分化。基于MEME程序分析水稻Trihelix转录因子家族的保守基序与聚类分析结果具有较高的一致性。染色体区段复制分析表明,部分Trihelix家族成员在水稻以及水稻与其他物种之间存在种内和种间的染色体区段复制;生物芯片数据分析发现,Trihelix基因家族在水稻不同组织中、以及对6种不同植物激素的响应呈现多样化的表达谱。采用RiceFREND在线数据库分析发现,水稻Trihelix转录因子家族的20个成员与其他蛋白存在互作关系。本研究结果初步明确了水稻Trihelix转录因子家族的进化特点、染色体分布、染色体区段复制关系、组织表达、激素应答,以及该家族蛋白与其他蛋白质的互作情况,为进一步揭示Trihelix转录因子家族的分子进化规律和生物学功能奠定了基础。     

Genome wide identification of the trihelix transcription factors and overexpression of Gh_A05G2067 (GT‐2), a novel gene contributing to increased drought and salt stresses tolerance in cotton

We identified 102, 51 and 51 proteins encoded by the trihelix genes in Gossypium hirsutum, Gossypium arboreum and Gossypium raimondii, respectively. RNA sequence data and real‐time quantitative polymerase chain reaction analysis showed that Gh_A05G2067 (GT‐2) was highly upregulated under drought and salt stress conditions. Transient expression of GT‐2‐green fluorescent protein fusion protein in protoplast showed that GT‐2 was localized in the nucleus. The overexpression of GT‐2 conferred an enhanced drought tolerance to cotton, with lower malondialdehyde, hydrogen peroxide contents and higher reactive oxygen scavenging enzyme activities. Moreover, chlorophyll content, relative leaf water content (RLWC), excised leaf water loss (ELWL) and cell membrane stability (CMS) were relatively stable in the GT‐2‐overexpressed lines compared to wild‐type (WT). Similarly, stress‐responsive genes RD29A, SOS1, ABF4 and CBL1 were highly upregulated in the GT‐2‐overexpressed lines but were significantly downregulated in WT. In addition, the GT‐2‐silenced cotton plants exhibited a high level of oxidation injury, due to high levels of oxidant enzymes, in addition to negative effects on CMS, ELWL, RLWC and chlorophyll content. These results mark the foundation for future exploration of the trihelix genes in cotton, with an aim of developing more resilient, versatile and highly tolerant cotton genotypes.     

GT-2: a transcription factor with twin autonomous DNA-binding domains of closely related but different target sequence specificity.

A triplet of adjacent, highly similar GT motifs in the phyA promoter of rice functions to support maximal expression of this gene. We have obtained a recombinant clone that encodes a full-length nuclear protein, designated GT-2, which binds specifically to these target sequences. This novel protein contains acidic, basic and proline- + glutamine-rich regions, as well as two autonomous DNA-binding domains, one NH2-terminal and the other COOH-terminal, that discriminate with high resolution between the three GT motifs. A duplicated sequence of 75 amino acids, present once in each DNA-binding domain, appears likely to mediate DNA target element recognition. Each copy of this duplicated protein sequence is predicted to form three amphipathic alpha-helices separated from each other by two short loops. The absence of sequence similarity to other known proteins suggests that this predicted structural unit, which we term the trihelix motif, might be representative of a new class of DNA-binding proteins.     

Unraveling the functions of glycosyltransferase family 47 in plants

The function of glycosyltransferases (GTs) from family GT47 was first identified in animal exostosins as β-glucuronyltransferase involved in the synthesis of heparan sulfate. Two recent papers report the functions of two plant members in this family as a pectin β-glucuronyltransferase and a xyloglucan β-galactosyltransferase. These findings greatly extend our understanding of the biological functions of family GT47 and also represent an important leap toward the molecular dissection of cell wall biosynthesis.     

Identification of residues involved in catalytic activity of the inverting glycosyl transferase WbbE from Salmonella enterica serovar borreze

Synthesis of the O:54 O antigen of Salmonella enterica is initiated by the nonprocessive glycosyl transferase WbbE, assigned to family 2 of the glycosyl transferase enzymes (GT2). GT2 enzymes possess a characteristic N-terminal domain, domain A. Based on structural data from the GT2 representative SpsA (S. J. Charnock and G. J. Davies, Biochemistry 38:6380-6385, 1999), this domain is responsible for nucleotide binding. It possesses two invariant Asp residues, the first forming a hydrogen bond to uracil and the second coordinating a Mn(2+) ion. Site-directed replacement of Asp41 (D41A) of WbbE, the analogue of the first Asp residue of SpsA, revealed that this is not required for activity. WbbE possesses three Asp residues near the position analogous to the second conserved residue. Whereas D95A reduced WbbE activity, activity in D93A and D96A mutants was abrogated, suggesting that either D93 or D96 may coordinate the Mn(2+) ion. Our studies also identified a C-terminal region of sequence conservation in 22 GT2 members, including WbbE. SpsA was not among these. This region is characterized by an ED(Y) motif. The Glu and Asp residues of this motif were individually replaced in WbbE. E180D in WbbE had greatly reduced activity, and an E180Q replacement completely abrogated activity; however, D181E had no effect. E180 is predicted to reside on a turn. Combined with the alignment of the motif with potential catalytic residues in the GT2 enzymes ExoM and SpsA, we speculate that E180 is the catalytic residue of WbbE. Sequence and predicted structural divergence in the catalytic region of GT2 members suggests that this is not a homogeneous family.     

Long-term evolution of the CAZY glycosyltransferase 6 (ABO) gene family from fishes to mammals--a birth-and-death evolution model

Functional glycosyltransferase 6 (GT6) family members catalyze the transfer of galactose or N-acetylgalactosamine in alpha1,3 linkage to various substrates and synthesize structures related to the A and B histo-blood group antigens, the Forssman antigen, alphaGal epitope, and iGb3 glycolipid. In rat, mouse, dog, and cow genomes, we have identified three new mammalian genes (GT6m5, GT6m6, and GT6m7) encoding putative proteins belonging to the GT6 family. Among these, GT6m6 protein does not display major alterations of the GT6 motifs involved in binding of the divalent cation and the substrate. Based on protein sequence comparison, gene structure, and synteny, GT6 homologous sequences were also identified in bird, fish, and amphibian genomes. Strikingly, the number and type of GT6 genes varied widely from species to species, even within phylogenetically related groups. In human, except ABO functional alleles, all other GT6 genes are either absent or nonfunctional. Human, mouse, and cow have only one ABO gene, whereas rat and dog have several. In the chicken, the Forssman synthase-like is the single GT6 family member. Five Forssman synthase-like genes were found in zebrafish, but are absent from three other fishes (fugu, puffer fish, and medaka). Two iGb3 synthase-like genes were found in medaka, which are absent from zebrafish. Fugu, puffer fish, and medaka have an additional GT6 gene that we termed GT6m8, which is absent from all other species analyzed here. These observations indicate that individual GT6 genes have expanded and contracted by recurrent duplications and deletions during vertebrate evolution, following a birth-and-death evolution type.     

A novel cysteine-rich domain of Sep15 mediates the interaction with UDP-glucose:glycoprotein glucosyltransferase

Selenium is an essential trace element with potent cancer prevention activity in mammals. The 15-kDa selenoprotein (Sep15) has been implicated in the chemopreventive effect of dietary selenium. Although the precise function of Sep15 remains elusive, Sep15 co-purifies with UDP-glucose:glycoprotein glucosyltransferase (GT), an essential regulator of quality control mechanisms within the endoplasmic reticulum. Recent studies identified two GT and two Sep15 homologues in mammals. We characterize interactions between these protein families in this report. Sep15 and GT form a tight 1:1 complex, and these interactions are conserved between mammals and fruit flies. In mammalian cells, Sep15 co-immunoprecipitates with both GT isozymes. In contrast, a Sep15 homologue, designated selenoprotein M (SelM), does not form a complex with GT. Sequence analysis of members of the Sep15 family identified a novel N-terminal cysteine-rich domain in Sep15 that is absent in SelM. This domain contains six conserved cysteine residues that form two CxxC motifs that do not coordinate metal ions. If this domain is deleted or the cysteines are mutated, Sep15 no longer forms a complex with GT. Conversely, if the cysteine-rich domain of Sep15 is fused to the N-terminus of SelM, the resulting chimera is capable of binding GT. These data indicate that the cysteine-rich domain of Sep15 exclusively mediates protein-protein interactions with GT.     

The plant glycosyltransferase clone collection for functional genomics

The glycosyltransferases (GTs) are an important and functionally diverse family of enzymes involved in glycan and glycoside biosynthesis. Plants have evolved large families of GTs which undertake the array of glycosylation reactions that occur during plant development and growth. Based on the Carbohydrate‐Active enZymes (CAZy) database, the genome of the reference plant Arabidopsis thaliana codes for over 450 GTs, while the rice genome (Oryza sativa) contains over 600 members. Collectively, GTs from these reference plants can be classified into over 40 distinct GT families. Although these enzymes are involved in many important plant specific processes such as cell‐wall and secondary metabolite biosynthesis, few have been functionally characterized. We have sought to develop a plant GTs clone resource that will enable functional genomic approaches to be undertaken by the plant research community. In total, 403 (88%) of CAZy defined Arabidopsis GTs have been cloned, while 96 (15%) of the GTs coded by rice have been cloned. The collection resulted in the update of a number of Arabidopsis GT gene models. The clones represent full‐length coding sequences without termination codons and are Gateway^® compatible. To demonstrate the utility of this JBEI GT Collection, a set of efficient particle bombardment plasmids (pBullet) was also constructed with markers for the endomembrane. The utility of the pBullet collection was demonstrated by localizing all members of the Arabidopsis GT14 family to the Golgi apparatus or the endoplasmic reticulum (ER). Updates to these resources are available at the JBEI GT Collection website http://www.addgene.org/ .     

A mutant Shiga-like toxin IIe bound to its receptor Gb(3): structure of a group II Shiga-like toxin with altered binding specificity

BACKGROUND: Shiga-like toxins (SLTs) are produced by the pathogenic strains of Escherichia coli that cause hemorrhagic colitis and hemolytic uremic syndrome. These diseases in humans are generally associated with group II family members (SLT-II and SLT-IIc), whereas SLT-IIe (pig edema toxin) is central to edema disease of swine. The pentameric B-subunit component of the majority of family members binds to the cell-surface glycolipid globotriaosyl ceramide (Gb(3)), but globotetraosyl ceramide (Gb(4)) is the preferred receptor for SLT-IIe. A double-mutant of the SLT-IIe B subunit that reverses two sequence differences from SLT-II (GT3; Gln65-->Glu, Lys67-->Gln, SLT-I numbering) has been shown to bind more strongly to Gb(3) than to Gb(4). RESULTS: To understand the molecular basis of receptor binding and specificity, we have determined the structure of the GT3 mutant B pentamer, both in complex with a Gb(3) analogue (2.0 A resolution; R = 0.155, R(free) = 0.194) and in its native form (2.35 A resolution; R = 0.187, R(free) = 0.232). CONCLUSIONS: These are the first structures of a member of the medically important group II Shiga-like toxins to be reported. The structures confirm the previous observation of multiple binding sites on each SLT monomer, although binding site 3 is not occupied in the GT3 structure. Analysis of the binding properties of mutants suggests that site 3 is a secondary Gb(4)-binding site. The two mutated residues are located appropriately to interact with the extra betaGalNAc residue on Gb(4). Differences in the binding sites provide a molecular basis for understanding the tissue specificities and pathogenic mechanisms of members of the SLT family.