Identification of the amino acid residues involved in the hemolytic activity of the Cucumaria echinata lectin CEL-III

Background

CEL-III is a hemolytic lectin isolated from the sea cucumber Cucumaria echinata that shows Ca^2 +-dependent Gal/GalNAc-binding specificity. This lectin is composed of two carbohydrate-recognition domains (domains 1 and 2) and an oligomerization domain (domain 3) that facilitates CEL-III assembly in the target cell membrane to form ion-permeable pores.

Methods

Several amino acid residues in domain 3 were replaced by alanine, and hemolytic activity of the mutants was examined.

Results

K344A, K351A, K405A, K420A and K425A showed marked increases in activity. In particular, K405A had activity that was 360-fold higher than the wild-type recombinant CEL-III and 3.6-fold higher than the native protein purified from sea cucumber. Since these residues appear to play roles in the stabilization of domain 3 through ionic and hydrogen bonding interactions with other residues, the mutations of these residues presumably lead to destabilization of domain 3, which consequently induces the oligomerization of the protein through association of domain 3 in the membrane. In contrast, K338A, R378A and R408A mutants exhibited a marked decrease in hemolytic activity. Since these residues are located on the surface of domain 3 without significant interactions with other residue, they may be involved in the interaction with components of the target cell membrane.

Conclusions

Several amino acid residues, especially basic residues, are found to be involved in the hemolytic activity as well as the oligomerization ability of CEL-III.

General significance

The results provide important clues to the membrane pore-forming mechanism of CEL-III, which is also related to that of bacterial pore-forming toxins.     

草鱼胶原凝集素基因的克隆及其功能分析

从草鱼Ctenopharyngodon idella肝肾cDNA文库中克隆得到胶原凝集素基因。草鱼胶原凝集素全长cDNA为1128bp,其中5′非编码区229bp,3′非翻译区104bp,最大开放阅读框为795bp,编码264个氨基酸。系统进化分析表明草鱼胶原凝集素与斑马鱼的亲缘关系最近。根据草鱼胶原凝集素序列特征,克隆了包含糖基识别域(CRD)的cDNA,并进行原核表达、纯化获得其重组蛋白PCRD。进行PCRD与6种细菌的凝集和糖抑制实验,结果表明半乳糖、葡萄糖、甘露糖和麦芽糖4种糖都会使PCRD与嗜水气单胞菌的凝集明显下降甚至极大地干扰凝集;麦芽糖使金黄色葡萄球菌的凝集明显下降,而肽聚糖和甘露糖会使凝集受到抑制;此外,PCRD的凝集反应不依赖Ca2+。     

Crystal structure and mutational analysis of Ca2+-independent type II antifreeze protein from longsnout poacher, Brachyopsis rostratus

We recently found that longsnout poacher (Brachyosis rostratus) produces a Ca²⁺-independent type II antifreeze protein (lpAFP) and succeeded in expressing recombinant lpAFP using Phichia pastoris. Here, we report, for the first time, the X-ray crystal structure of lpAFP at 1.34 Å resolution. The lpAFP structure displayed a relatively planar surface, which encompasses two loop regions (Cys86-Lys89 and Asn91-Cys97) and a short β-strand (Trp109-Leu112) with three unstructured segments (Gly57-Ile58, Ala103-Ala104, and Pro113-His118). Electrostatic calculation of the protein surface showed that the relatively planar surface was divided roughly into a hydrophobic area (composed of the three unstructured segments lacking secondary structure) and a hydrophilic area (composed of the loops and β-strand). Site-directed mutation of Ile58 with Phe at the center of the hydrophobic area decreased activity significantly, whereas mutation of Leu112 with Phe at an intermediate area between the hydrophobic and hydrophilic areas retained complete activity. In the hydrophilic area, a peptide-swap mutant in the loops retained 60% activity despite simultaneous mutations of eight residues. We conclude that the epicenter of the ice-binding site of lpAFP is the hydrophobic region, which is centered by Ile58, in the relatively planar surface. We built an ice-binding model for lpAFP on the basis of a lattice match of ice and constrained water oxygen atoms surrounding the hydrophobic area in the lpAFP structure. The model in which lpAFP has been docked to a secondary prism (2-1-10) plane, which is different from the one determined for Ca²⁺-independent type II AFP from sea raven (11-21), appears to explain the results of the mutagenesis analysis.     

家蝇C-型凝集素的克隆与功能分析

【目的】从家蝇Musca domestica中克隆一种C-型凝集素(C-type lectin)基因,并分析其在家蝇免疫过程中的功能。【方法】根据家蝇转录组信息,克隆了一条C型凝集素基因,将其命名为Mdlectin-C1。构建Mdlectin-C1的原核表达载体,在大肠杆菌Escherichia coli中表达并纯化r Mdlectin-C1蛋白,制备多克隆抗体。利用实时定量PCR(quantitative real-time PCR,qRT-PCR)及Western blot技术对家蝇不同发育阶段和细菌感染前后Mdlectin-C1的表达水平进行检测。运用RNAi技术敲低家蝇1龄幼虫Mdlectin-C1基因表达,然后进行细菌刺激并观察幼虫存活率变化。【结果】Mdlectin-C1 c DNA包含一个546 bp完整开放阅读框,编码181个氨基酸残基,所推导多肽N端包含由21个氨基酸残基组成的信号肽,成熟肽中含有一个保守的碳水化合物识别结构域(carbohydrate-recognition domain,CRD)。qRT-PCR及Western blot结果显示,家蝇从1龄幼虫到蛹的发育过程中,Mdlectin-C1表达量逐步上升,蛹期达到最大值。在革兰氏阴性菌大肠杆菌E.coli和革兰氏阳性菌金黄色葡萄球菌Staphylococcus aureus刺激后,家蝇2龄幼虫Mdlectin-C1均上调表达,并分别在36和3 h时达到最高值。利用RNAi技术成功敲低Mdlectin-C1表达。对Mdlectin-C1基因敲低的1龄幼虫进行细菌刺激,敲低实验组幼虫存活率显著低于正常对照组。【结论】Mdlectin-C1可能参与家蝇抗菌免疫应答,并在此过程中具有重要作用。     

Structure of the fungal beta-glucan-binding immune receptor dectin-1: implications for function

The murine molecule dectin-1 (known as the beta-glucan receptor in humans) is an immune cell surface receptor implicated in the immunological defense against fungal pathogens. Sequence analysis has indicated that the dectin-1 extracellular domain is a C-type lectin-like domain, and functional studies have established that it binds fungal beta-glucans. We report several dectin-1 crystal structures, including a high-resolution structure and a 2.8 angstroms resolution structure in which a short soaked natural beta-glucan is trapped in the crystal lattice. In vitro characterization of dectin-1 in the presence of its natural ligand indicates higher-order complex formation between dectin-1 and beta-glucans. These combined structural and biophysical data considerably extend the current knowledge of dectin-1 structure and function, and suggest potential mechanisms of defense against fungal pathogens.     

High affinity sugar ligands of C-type lectin receptor langerin

Background

Langerin, a C-type lectin receptor (CLR) expressed in a subset of dendritic cells (DCs), binds to glycan ligands for pathogen capture and clearance. Previous studies revealed that langerin has an unusual binding affinity toward 6-sulfated galactose (Gal), a structure primarily found in keratan sulfate (KS). However, details and biological outcomes of this interaction have not been characterized. Based on a recent discovery that the disaccharide L4, a KS component that contains 6-sulfo-Gal, exhibits anti-inflammatory activity in mouse lung, we hypothesized that L4-related compounds are useful tools for characterizing the langerin-ligand interactions and their therapeutic application.

亚洲玉米螟C型凝集素CTL6的克隆和功能分析

【目的】鉴定出一种新的亚洲玉米螟Ostrinia furnacalis (Guenée) C型凝集素(C-type lectin),并对其功能进行初步研究。【方法】通过生物信息学分析,从亚洲玉米螟转录组中筛选得到一个可能的C型凝集素基因,命名为CTL6。利用RT-PCR技术分析该基因在亚洲玉米螟不同龄期、不同组织及不同病原物诱导下的表达模式。借助原核及杆状病毒真核表达系统产生重组CTL6蛋白,并利用细菌凝集实验对其功能进行初步研究。【结果】CTL6基因cDNA全长序列为1 034 nt,其中完整开放阅读框为945 nt。推导的CTL6多肽序列包括314个氨基酸残基,N端含有由22个氨基酸残基组成的信号肽。CTL6成熟肽中含有两个串联的糖识别结构域,与烟草天蛾Manduca sexta的IML-2 (Immulectin-2)同源性最高。RT-PCR结果显示,CTL6在亚洲玉米螟5龄幼虫期转录水平最高,卵期其次,不同组织中则是在脂肪体中转录水平最高,呈诱导性表达。纯化的CTL6重组蛋白对大肠杆菌Escherichia coli具有一定的凝集作用。【结论】鉴定到的亚洲玉米螟CTL6是一种典型的C型凝集素,重组CTL6蛋白可能参与了亚洲玉米螟对病原菌的凝集作用。     

Peptides derived from human galectin-3 N-terminal tail interact with its carbohydrate recognition domain in a phosphorylation-dependent manner

Galectin-3 (Gal-3) is a multi-functional effector protein that functions in the cytoplasm and the nucleus, as well as extracellularly following non-classical secretion. Structurally, Gal-3 is unique among galectins with its carbohydrate recognition domain (CRD) attached to a rather long N-terminal tail composed mostly of collagen-like repeats (nine in the human protein) and terminating in a short non-collagenous terminal peptide sequence unique in this lectin family and not yet fully explored. Although several Ser and Tyr sites within the N-terminal tail can be phosphorylated, the physiological significance of this post-translational modification remains unclear. Here, we used a series of synthetic (phospho)peptides derived from the tail to assess phosphorylation-mediated interactions with ¹⁵N-labeled Gal-3 CRD. HSQC-derived chemical shift perturbations revealed selective interactions at the backface of the CRD that were attenuated by phosphorylation of Tyr 107 and Tyr 118, while phosphorylation of Ser 6 and Ser 12 was essential. Controls with sequence scrambling underscored inherent specificity. Our studies shed light on how phosphorylation of the N-terminal tail may impact on Gal-3 function and prompt further studies using phosphorylated full-length protein.     

The Structure of Physarum polycephalum hemagglutinin I suggests a minimal carbohydrate recognition domain of legume lectin fold

Physarum polycephalum hemagglutinin I (HA1) is a 104-residue protein that is secreted to extracellular space. The crystal structure of HA1 has a β-sandwich fold found among lectin structures, such as legume lectins and galectins. Interestingly, the β-sandwich of HA1 lacks a jelly roll motif and is essentially composed of two simple up-and-down β-sheets. This up-and-down β-sheet motif is well conserved in other legume lectin-like proteins derived from animals, plants, bacteria, and viruses. It is more noteworthy that the up-and-down β-sheet motif includes many residues that make contact with the target carbohydrates. Our NMR data demonstrate that HA1 lacking a jelly roll motif also binds to its target glycopeptide. Taken together, these data show that the up-and-down β-sheet motif provides a fundamental scaffold for the binding of legume lectin-like proteins to the target carbohydrates, and the structure of HA1 suggests a minimal carbohydrate recognition domain.     

Proteomic analysis of ribosomes: translational control of mRNA populations by glycogen synthase GYS1

Ribosomes exist as a heterogenous pool of macromolecular complexes composed of ribosomal RNA molecules, ribosomal proteins, and numerous associated “nonribosomal” proteins. To identify nonribosomal proteins that may modulate ribosome activity, we examined the composition of translationally active and inactive ribosomes using a proteomic multidimensional protein identification technology. Notably, the phosphorylated isoform of glycogen synthase, glycogen synthase 1 (GYS1), was preferentially associated with elongating ribosomes. Depletion of GYS1 affected the translation of a subset of cellular mRNAs, some of which encode proteins that modulate protein biosynthesis. These findings argue that GYS1 abundance, by virtue of its ribosomal association, provides a feedback loop between the energy state of the cells and the translation machinery.     

Development of highly stable galectins: truncation of the linker peptide confers protease-resistance on tandem-repeat type galectins

Galectin-9 and galectin-8, members of beta-galactoside-binding animal lectin family, are promising agents for the treatment of immune-related and neoplastic diseases. The proteins consist of two carbohydrate recognition domains joined by a linker peptide, which is highly susceptible to proteolysis. To increase protease resistance, we prepared mutant proteins by serial truncation of the linker peptide. As a result, mutant forms lacking the entire linker peptide were found to be highly stable against proteolysis and retained their biological activities. These mutant proteins might be useful tools for analyzing the biological functions and evaluating the therapeutic potential of galectin-9 and galectin-8.     

T-RMSD: A Fine-grained, Structure-based Classification Method and its Application to the Functional Characterization of TNF Receptors

This study addresses the relation between structural and functional similarity in proteins. We introduce a novel method named tree based on root mean square deviation (T-RMSD), which uses distance RMSD (dRMSD) variations to build fine-grained structure-based classifications of proteins. The main improvement of the T-RMSD over similar methods, such as Dali, is its capacity to produce the equivalent of a bootstrap value for each cluster node. We validated our approach on two domain families studied extensively for their role in many biological and pathological pathways: the small GTPase RAS superfamily and the cysteine-rich domains (CRDs) associated with the tumor necrosis factor receptors (TNFRs) family. Our analysis showed that T-RMSD is able to automatically recover and refine existing classifications. In the case of the small GTPase ARF subfamily, T-RMSD can distinguish GTP- from GDP-bound states, while in the case of CRDs it can identify two new subgroups associated with well defined functional features (ligand binding and formation of ligand pre-assembly complex). We show how hidden Markov models (HMMs) can be built on these new groups and propose a methodology to use these models simultaneously in order to do fine-grained functional genomic annotation without known 3D structures. T-RMSD, an open source freeware incorporated in the T-Coffee package, is available online.     

A cold-induced thioredoxin h of rice, OsTrx23, negatively regulates kinase activities of OsMPK3 and OsMPK6 in vitro

Cytosolic thioredoxins are small conserved proteins that are involved in cellular redox regulation. Here, we report that a major and cold-induced thioredoxin h of rice, OsTrx23, has an inhibitory activity on stress-activated mitogen-activated protein kinases (MAPKs), OsMPK3 and OsMPK6 in vitro. This inhibition effects were redox-dependent and did not involve stable physical interaction. The data suggested a novel mechanism for redox regulation of MAPKs in plants.

Structured summary

MINT-7234362: MPK3 (uniprotkb:Q10N20) phosphorylates (MI:0217) MBP (uniprotkb:P02687) by protein kinase assay (MI:0424)MINT-7234435: MPK6 (uniprotkb:Q336X9) phosphorylates (MI:0217) MBP (uniprotkb:P02687) by protein kinase assay (MI:0424)     

A novel dimer interface and conformational changes revealed by an X-ray structure of B. subtilis SecA

The SecA ATPase moves polypeptides post-translationally across the plasma membrane of eubacteria, but the mechanism of transport is still unclear. We describe the crystal structure of a novel dimeric form of Bacillus subtilis SecA. Dimerization of SecA occurs at the prominent groove formed by the nucleotide binding domain 2 (nbd2) and the preprotein cross-linking (ppx) domain. The dimer interface is very large, burying approximately 5400 A(2) of solvent accessible surface per monomer. Single cysteine disulfide cross-linking shows the presence of this novel SecA dimer in solution. In addition, other dimers also exist in solution, arguing that they all are in equilibrium with monomeric SecA and supporting the idea that the monomer may be the functional species. Dimerization of SecA causes an alpha-helix of one subunit to convert to a short beta-strand that participates in beta-sheet formation with strands in the other subunit. This conversion of secondary structure elements occurs close to the connection between the nbd1 and ppx domains, a potential site of interaction with translocation substrate. Comparing the different X-ray structures of B. subtilis SecA suggests that small changes in the nucleotide binding domains could be amplified via helix 1 of the helical scaffold domain (hsd) to generate larger movements of the domains involved in polypeptide binding.     

Surfactant protein A modulates the lipopolysaccharide-induced inflammatory response related to preterm birth

Surfactant protein A (SP-A) functions in homeostasis of lung surfactant and in innate immunity. SP-A is secreted by the fetal lung into amniotic fluid. Additionally it has been detected in gestational tissues. We propose that SP-A influences intrauterine inflammation that is commonly associated with preterm birth, the main underlying cause of neonatal mortality and morbidity. We used our previously established mouse model of LPS-induced preterm birth of live-born pups to investigate the role of SP-A in preterm birth. Mice overexpressing rat SP-A (rSP-A) under the control of human SP-C promoter were used. Cytokine concentrations in maternal and fetal serum and in amniotic fluid and mRNA levels of several inflammatory mediators in lungs and in intrauterine tissues were quantified using Cytometric Bead Array and RNase Protection Assay, respectively. Higher levels of SP-A mRNA were observed in fetal lungs and intrauterine tissues of rSP-A mice compared with wild-type. Using Western blot we detected excess of SP-A protein in fetal lung and in amniotic fluid of rSP-A animals. Despite some differences in the basal levels of TNF-α and IL-10 between rSP-A and wild-type animals, there were no differences in the duration of pregnancy. However, the levels of TNF-α, IL-10 and some other inflammatory mediators in intrauterine tissues and in amniotic fluid differed significantly between the mouse lines after maternal LPS given at 17 dpc. We conclude that SP-A modulates the levels of intrauterine inflammatory mediators involved in preterm birth and may contribute to inflammatory processes related to spontaneous preterm labor.