赤子爱胜蚓蛋白磷酸酶2A类似激活剂样蛋白cDNA和氨基酸序列分析

目的分析赤子爱胜蚓蛋白磷酸酶2A(protein phosphatase 2A,PP2A)类似激活剂样蛋白的cDNA和氨基酸序列。方法从赤子爱胜蚓cDNA文库中随机测序得到目标cDNA序列,应用DNAMAN、NCBI ORF finder、BLAST、Conserved Domains、GOR、SWISS-MODEL、PDB等基因和蛋白质分析软件进行该目标基因测序及氨基酸序列分析。结果赤子爱胜蚓PP2A类似激活剂样蛋白cDNA序列长547 bp,编码87个氨基酸残基;与该蛋白基因序列相似度较高的多是不同物种中PP2A类似激活剂蛋白基因序列,其中与Crassostrea virginica serine/threonine-protein phosphatase 2A activator-like mRNA核苷酸相似度为67%;与该蛋白氨基酸序列相似度较高的多是不同物种中的PP2A类似激活剂蛋白氨基酸序列,其中与serine/threonine-protein phosphatase 2A activator(Crassostrea gigas)氨基酸相似度为70%;目标序列中存在一段PTPA超家族保守结构域,蛋白质二级结构以α螺旋以及无规则卷曲为主,三级预测结果与二级结构一致。结论本实验分析的赤子爱胜蚓PP2A类似激活剂样蛋白与牡蛎PP2A类似激活剂蛋白及存在于所有真核生物中且高度保守的PTPA同源度较高,可能具有特异性激活肿瘤抑制因子PP2A的间接抑制肿瘤作用。     

Does a backwardly read protein sequence have a unique native state?

Amino acid sequences of native proteins are generally not palindromic.Nevertheless, the protein molecule obtained as a result of readingthe sequence backwards, i.e. a retro-protein, obviously hasthe same amino acid composition and the same hydrophobicityprofile as the native sequence. The important questions whicharise in the context of retro-proteins are: does a retro-proteinfold to a well defined native-like structure as natural proteinsdo and, if the answer is positive, does a retro-protein foldto a structure similar to the native conformation of the originalprotein? In this work, the fold of retro-protein A, originatedfrom the retro-sequence of the B domain of Staphylococcal proteinA, was studied. As a result of lattice model simulations, itis conjectured that the retro-protein A also forms a three-helixbundle structure in solution. It is also predicted that thetopology of the retro-protein A three-helix bundle is that ofthe native protein A, rather than that corresponding to themirror image of native protein A. Secondary structure elementsin the retro-protein do not exactly match their counterpartsin the original protein structure; however, the amino acid sidechain contact pattern of the hydrophobic core is partly conserved.     

Molecular Cloning and Expression Analysis of P-Selectin from Zebrafish (Danio rerio)

The glycoprotein P-selectin belongs to the selectin family of cell adhesion molecules. In this study, we cloned the full-length cDNA of P-selectin from zebrafish (Danio rerio) by the method of rapid amplification of cDNA ends polymerase chain reaction (RACE-PCR). Zebrafish P-selectin cDNA is 2,800 bp and encodes a putative 868 amino acid protein with a theoretical molecular weight of 122.36 kDa and isoelectric point of 6.27. A signal peptide of 25 amino acids is predicted at the N-terminus of the putative protein. All structural domains involved in P-selectin function are conserved in the putative protein. The amino acid sequence of zebrafish P-selectin is 37% to 39% identical to that of mammalian P-selectins. Real-time quantitative PCR and whole-mount in situ hybridization analysis revealed that P-selectin was expressed in early embryonic development, the expression increased from 0.2 hpf (1-cell stage) to 72 hpf, and the expression significantly upregulated within 30 minutes of ADP induction. The results indicate that the structure of P-selectin protein is highly conserved among species and zebrafish P-selectin plays an important role in early embryonic development and probably has similar biological function to mammalian P-selectins.     

Mutations in the FAD-binding fold of alcohol oxidase from Hansenula polymorpha

Alcohol oxidase of methylotrophic yeast is an FAD-containingenzyme. When in its active form, the enzyme is an octamer andlocated in the peroxisomes. To study the importance of FAD-bindingon the activity, octamerization and intracellular localizationof the enzyme, alcohol oxidase of Hansenula polymorpha was mutatedin its presumed nucleotide-binding domain, which is formed bythe N-terminal sequence. Whereas mutations of a glutamic acidresidue (E42) reduced thestability of the octamer, it hardlyaffected enzyme activity and expression. However, replacementsof three conserved glycines (G13, G15 and G18) and a conservedglutamic acid (E39) within the fold had severe effects. The utations not only resulted in loss of enzyme activity but inreduced protein levels as well, probably due to decreased stabilityof the mutant alcohol oxidase. However, octamerization of theprotein still occurred. The existence of inactive octamericproteins provides information about the formation pathway ofthis octameric flavoprotein.     

Alteration of product specificity of Aeropyrum pernix farnesylgeranyl diphosphate synthase (Fgs) by directed evolution

Directed evolution of the C25 farnesylgeranyl diphosphate synthase of Aeropyrum pernix (Fgs) was carried out by error-prone PCR with an in vivo color complementation screen utilizing carotenoid biosynthetic pathway enzymes. Screening yielded 12 evolved clones with C20 geranylgeranyl diphosphate synthase activity which were isolated and characterized in order to understand better the chain elongation mechanism of this enzyme. Analysis of these mutants revealed three different mechanisms of product chain length specificity. Two mutants (A64T and A64V) have a single mutation at the 8th amino acid upstream of a conserved first aspartate-rich motif (FARM), which is involved in the mechanism for chain elongation reaction of all prenyl diphosphate synthases. One mutant (A135T) carries a single mutation at the 7th amino acid upstream of another conserved region (141GQ142), which was recently found to be another important region controlling chain elongation of a type III C20 geranylgeranyl diphosphate synthase and Escherichia coli C15 farnesyl diphosphate synthase. Finally, one mutant carrying four mutations (V84I, H88R, I177 M and M191V) is of interest. Molecular modeling, site-directed mutagenesis and in vitro assays of this mutant suggest that product chain-length distribution can be also controlled by a structural change provoked by a cooperative interaction of amino acids.     

Fold recognition and molecular modeling of a lectin-like domain in UDP- GalNac:polypeptide N-acetylgalactosaminyltransferases

By use of threading methods, the C-terminal region of uridine diphospho- N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferases (ppGalNAc-transferases) was predicted to have the same fold as the lectin-domain of the plant cytotoxins ricin and abrin-a, for which crystal structure are available. The sequence identities are very low. Nevertheless, the amino acids involved in the hydrophobic core essential for the structure stability and the cysteine residues are conserved. In addition, the amino-acids involved in carbohydrate binding are conserved in ppGalNAc-transferases. The extra C-terminal domain of these enzymes is therefore a putative glycan-binding domain. A model of the lectin-like domain of human ppGalNAc-transferase T1 was built using knowledge based methods. Geometry optimization of the complex with galactose allowed prediction that this domain could bind this monosaccharide. However, the interaction seems to be rather weak, and at the moment there is no evidence that ppGalNAc-transferases displays a lectin activity in vivo.      

Mutational Analysis of the C–C Bond Cleaving Enzyme Phloretin Hydrolase from Eubacterium ramulus

Phloretin hydrolase from Eubacterium ramulus (Phy) catalyzes the hydrolysis of the dihydrochalcone phloretin to phloroglucinol and phloretic acid, performing a formal retro- Friedel–Crafts acylation reaction on its substrate. Its closest sequence homolog, of 25 % amino acid sequence identity, is diacetyl phloroglucinol hydrolase (Phlg) from Pseudomonas fluorescens, which catalyses a similar, hydrolytic, de-acylation of its substrate. The structure of Phlg has been determined and a catalytic mechanism proposed (J Biol Chem 285:4603–4611, 2010). In order to compare the catalytic characteristics of Phy with Phlg, the gene encoding Phy was expressed and the enzyme purified and crystallised. An X-ray fluorescence scan identified zinc within the crystals. A homology model of Phy, based on the structure of Phlg (PDB code 3HWP), informed the construction of a point mutant library of the enzyme, targeting residues shared with Phlg that are thought to be involved in zinc binding and the recognition of acyl and phenol functionality on the aromatic ring of the substrates. Mutation of His123, His251, Glu154 and Glu255 (conserved zinc binding residues) resulted in variants that were either poorly expressed, or of much reduced activity; Mutation of Tyr115 and His203, thought to bind the phenol groups in the 1-and 3-positions of the phloroglucinol ring respectively, resulted in variants of 15-fold reduced activity and an inactive variant. These results are suggestive of conservation of some aspects of mechanism and substrate recognition between Phy and Phlg, and of the catalytic characteristics of Zn-dependent C–C hydrolases of this type in general.     

Annexin-Phospholipid Interactions. Functional Implications

Annexins constitute an evolutionary conserved multigene protein superfamily characterized by their ability to interact with biological membranes in a calcium dependent manner. They are expressed by all living organisms with the exception of certain unicellular organisms. The vertebrate annexin core is composed of four (eight in annexin A6) homologous domains of around 70 amino acids, with the overall shape of a slightly bent ring surrounding a central hydrophilic pore. Calcium- and phospholipid-binding sites are located on the convex side while the N-terminus links domains I and IV on the concave side. The N-terminus region shows great variability in length and amino acid sequence and it greatly influences protein stability and specific functions of annexins. These proteins interact mainly with acidic phospholipids, such as phosphatidylserine, but differences are found regarding their affinity for lipids and calcium requirements for the interaction. Annexins are involved in a wide range of intra- and extracellular biological processes in vitro, most of them directly related with the conserved ability to bind to phospholipid bilayers: membrane trafficking, membrane-cytoskeleton anchorage, ion channel activity and regulation, as well as antiinflammatory and anticoagulant activities. However, the in vivo physiological functions of annexins are just beginning to be established.     

Regulation of Amylose Content by Single Mutations at an Active Site in the Wx-B1 Gene in a Tetraploid Wheat Mutant

The granule-bound starch synthase I (GBSSI) encoded by the waxy gene is responsible for amylose synthesis in the endosperm of wheat grains. In the present study, a novel Wx-B1 null mutant line, M3-415, was identified from an ethyl methanesulfonate-mutagenized population of Chinese tetraploid wheat landrace Jianyangailanmai (LM47). The gene sequence indicated that the mutated Wx-B1 encoded a complete protein; this protein was incompatible with the protein profile obtained using sodium dodecyl sulfate–polyacrylamide gel electrophoresis, which showed the lack of Wx-B1 protein in the mutant line. The prediction of the protein structure showed an amino acid substitution (G470D) at the edge of the ADPG binding pocket, which might affect the binding of Wx-B1 to starch granules. Site-directed mutagenesis was further performed to artificially change the amino acid at the sequence position 469 from alanine (A) to threonine (T) (A469T) downstream of the mutated site in M3-415. Our results indicated that a single amino acid mutation in Wx-B1 reduces its activity by impairing its starch-binding capacity. The present study is the first to report the novel mechanism underlying Wx-1 deletion in wheat; moreover, it provided new insights into the inactivation of the waxy gene and revealed that fine regulation of wheat amylose content is possible by modifying the GBSSI activity.     

Maintenance of the hydrophobic face of the diphtheria toxin amphipathic transmembrane helix 1 is essential for the efficient delivery of the catalytic domain to the cytosol of target cells

Abstract The transmembrane (T) domain of diphtheria toxin (DT) comprisesnine -helices and has been shown to play an essential role inthe efficent delivery of the catalytic (C) domain ofDT acrossthe eukaryotic cell membrane and into the cytosol. We have demonstratedrecently thatthe first three amphipathic helixes of the T domain,although not necessary for either channel formation or receptorbinding, are required for the efficient transmembrane deliveryof the Cdomain.In the present study,we have performed a detailedstructure-function analysis of T domainhelix 1 (TH1) of theDT-related fusion protein DAB₃₈₉lL-2. We performed exchangeandsite-directed mutagenesis of TH1 and the resulting mutantfusion toxins were analyzed by gel electrophoresis and testedfor their efficiencies in the delivery of the C domain to thecell cytosol. We demonstrate that the overall charge distributionand hydrophobicity of amino acids in the amphipathic helix TH1,rather than a specific amino acid sequence, are critical forthe function of this helix. The insertion of a charged residuein the hydrophobic face of TH1 abolishes cytotoxic activity,whereas replacement of a hydrophobic residue by a charged aminoacid in the hydrophilic face of the helix has little, if any,effect on cytotoxic activity. In addition,we have identifiedSer220 by site-directed mutagenesis as a residue that appearsto be criticalfor correct folding of the fusion toxin. Mutationsin this position result in fusion proteins that are extremelysensitive to proteolytic attack.     

SMoS: a database of structural motifs of protein superfamilies

The Structural Motifs of Superfamilies (SMoS) database providesinformation about the structural motifs of aligned protein domainsuperfamilies. Such motifs among structurally aligned multiplemembers of protein superfamilies are recognized by the conservationof amino acid preference and solvent inaccessibility and areexamined for the conservation of other features like secondarystructural content, hydrogen bonding, non-polar interactionand residue packing. These motifs, along with their sequenceand spatial orientation, represent the conserved core structureof each superfamily and also provide the minimal requirementof sequence and structural information to retain each superfamilyfold. Received April 25, 2003; revised September 9, 2003; accepted September 24, 2003.     

Functional diversity of the phosphoglucomutase superfamily: structural implications

Three-dimensional structural models of three members of thephosphoglucomutase (PGM) superfamily, parafusin, phosphoglucomutase-relatedprotein and sarcoplasmic reticulum phosphoglucomutase, wereconstructed by homology modeling based on the known crystalstructure of rabbit muscle phosphoglucomutase. Parafusin, phosphoglucomutase-relatedprotein and sarcoplasmic reticulum phosphoglucomutase each have50% or more identity with rabbit muscle phosphoglucomutase atthe amino acid level and all are reported to exhibit no or minorphosphoglucomutase activity. There are four major insertionsand two deletions in the parafusin sequence relative to PGM,all of which are located in surface-exposed loops connectingsecondary structural elements. The remaining amino acid substitutionsare distributed throughout the sequence and are not predictedto alter the polypeptide fold. Parafusin contains a putativeprotein kinase C site located on a surface loop in domain IIthat is not present in the homologs. Although the general domainstructure and the active site of rabbit muscle phosphoglucomutaseare preserved in the model of phosphoglucomutase-related protein,a major structural difference is likely to occur in domain 1due to the absence of 55 amino acid residues in PGM-RP. Thisdeletion predicts the loss of three -helices and one ß-strandfrom an anti-parallel ß-sheet in this domain as comparedwith the rabbit muscle phosphoglucomutase.     

The molecular mechanism of the termination of insect diapause, part 1: A timer protein, TIME-EA4, in the diapause eggs of the silkworm Bombyx mori is a metallo-glycoprotein

TIME-EA4 is an ATPase that measures time intervals, functioning as a diapause duration clock in diapause eggs of the silkworm, Bombyx mori. Characterization of the primary and higher structures of the TIME-EA4 would be desirable to clarify the mechanism by which the protein measures the time intervals. In our current studies, the whole sequence of TIME-EA4 has been established as that of a metallo-glycoprotein by combinational means involving peptide sequence analysis, nano-HPLC-ESI-Q-TOF-MS and MS/MS, and cDNA dictation. The amino acid sequence of TIME-EA4 showed 46-55 % homology with the reported proteins of the Cu,Zn-SOD (superoxide dismutase) family; in particular, the SOD active site (core domain) includes metal-binding amino acid ligands and a disulfide bond, and these structures are completely identical in Bombyx SOD, bovine SOD, and TIME-EA4 proteins. We found, however, that TIME-EA4 contains one more copper ion than other SODs, as was proven under neutral nondenaturing conditions. ESI mass spectrometry revealed that the timer function was not in the SOD core domain. In addition, TIME-EA4 has an attached sugar chain, which is indispensable to its functioning as a timer protein.     

Investigations on the thermostability and function of truncated Thermus aquaticus DNA polymerase fragments

The thermostable DNA polymerase from Thermus aquaticus (Taq polymerase) has been truncated to molecular regions essential for polymerase activity. Two truncated forms of the full-length 832 amino acid Taq polymerase have been constructed according to sequence alignments and the known domain structure of the homologous Escherichia coli DNA polymerase I (E.coli pol I): variant delta288 (lacking the N-terminal 288 amino acid portion) and variant delta413 (lacking the N-terminal 413 amino acid portion). Both protein fragments were stable and showed polymerase activity, albeit specific activity and thermostability of the variant delta413 were significantly decreased compared with the full length Taq polymerase. In order to increase the thermostability of the variant delta413, a three-dimensional model of the polymerase domain of Taq polymerase was built by homology with a model of the Klenow fragment of the E.coli pol I based on the available Calpha coordinates. Consequently two variants were designed and constructed using site-directed mutagenesis. The strategies used were deletion of 10 flexible amino acids and replacement of two hydrophobic amino acids on the surface by more hydrophilic ones. Compared with the initial protein fragment, both variant enzymes showed an increase in polymerase activity and thermostability. After the completion of this work, X-ray coordinates of the Taq polymerase became available from the protein structure data bank. A comparison between the homology model and the experimental three-dimensional structure proved the quality of the model.      

Mutations affecting the activity of urokinase-type plasminogen activator

Mutagenesis throughout the single-chain urokinase-type plasminogenactivator (scu-PA) cDNA molecule, followed by expression ofthe mutant genes and secretion of the resulting mutant proteinsfrom yeast, has been used to determine the amino acid residuesimportant for activity of scu-PA molecules. Twelve out of 13colonies secreting variant scu-PA molecules with decreased abilityto form a zone of fibrinolysis had mutant genes with a singlecodon alteration in the serine protease encoding domain (B-chain).Many of these changes are of highly conserved residues in theserine proteases and are consequently of considerable interest.A model three-dimensional structure of the protease domain ofurokinase was used to explain the basis for the effects of thesedown mutations. The model showed that the strongest down mutationsresult from either interference of the mutated side chain withsubstrate binding at the active site or the introduction ofbulky or charged groups at structurally sensitive internal positionsin the molecule. Attempts to find second site revertants offive down mutants, altered either at the plasmin activationsite or near the serine at the active site, only resulted insame-site revertants, with the original or closely related aminoacids restored.     

Comparative modeling of the three CP modules of the {beta}-chain of C4BP and evaluation of potential sites of interaction with protein S

A computer model of the ß-chain of C4b-binding protein(C4BP) was constructed, using the backbone fold of the NMR structuresof the sixteenth CP module of factor H (H16) and of a pair ofmodules consisting of the fifteenth and sixteenth CPs of factorH (H15-16). The characteristic hydrophobic core responsiblefor dictating the three-dimensional structure of the CP familyis conserved in the amino acid sequence of C4BP ßl, ß2and ß3. The distribution of the electrostatic potentialshows that the model is mainly covered by a negative contour.Interestingly, a positive area is observed in the C-terminalregion of the first CP module, enclosing peptide 31-45, knownto be a binding site for protein S. This observation suggeststhat electrostatic interactions can be of importance for theinteraction of C4BP to protein S. A solvent-accessible hydrophobicpatch, located nearby and involving the peptide 31-45, was alsofound in the model, further confirming that this area is involvedin the interaction with protein S. The contribution of ß-chainresidues 31-45 to the affinity for protein S was studied furtherby means of synthetic mutant peptides. The results suggest thatboth electrostatic and hydrophobic interactions are importantfor the binding to protein S.