CaMBP-10的cDNA克隆和表达及钙调素结合活性分析

采用RT PCR法 ,从中国大白菜中分离了编码CaMBP 1 0的cDNA克隆 .该cDNA全长 4 96bp ,编码 92个氨基酸 ,3′端含有 2 1 6bp的非编码区和poly A尾 .将此BP 1 0cDNA的成熟蛋白序列导入表达质粒pET1 5b并转化至大肠杆菌E .coliBL2 1 (DE3)condonplus RIL进行表达 .以免疫印迹和钙调素结合分析法对重组BP 1 0进行鉴定 ,证明其保持了与天然BP 1 0相同的钙调素结合活性 .氨基酸和核苷酸序列分析结果显示 ,它与植物转脂蛋白高度同源 ,特别是含有 8个保守半胱氨酸 .BP 1 0与转脂蛋白之间具极为相似的理化性质如分子量、等电点、热稳定性等 .据此认为 ,CaMBP 1 0是转脂蛋白家族的新成员 ,Ca2 + CaM信号系统可能参与植物转脂蛋白功能的调节     

Isolation and characterization of a novel calmodulin-binding protein from potato.

Tuberization in potato is controlled by hormonal and environmental signals. Ca(2+), an important intracellular messenger, and calmodulin (CaM), one of the primary Ca(2+) sensors, have been implicated in controlling diverse cellular processes in plants including tuberization. The regulation of cellular processes by CaM involves its interaction with other proteins. To understand the role of Ca(2+)/CaM in tuberization, we have screened an expression library prepared from developing tubers with biotinylated CaM. This screening resulted in isolation of a cDNA encoding a novel CaM-binding protein (potato calmodulin-binding protein (PCBP)). Ca(2+)-dependent binding of the cDNA-encoded protein to CaM is confirmed by (35)S-labeled CaM. The full-length cDNA is 5 kb long and encodes a protein of 1309 amino acids. The deduced amino acid sequence showed significant similarity with a hypothetical protein from another plant, Arabidopsis. However, no homologs of PCBP are found in nonplant systems, suggesting that it is likely to be specific to plants. Using truncated versions of the protein and a synthetic peptide in CaM binding assays we mapped the CaM-binding region to a 20-amino acid stretch (residues 1216-1237). The bacterially expressed protein containing the CaM-binding domain interacted with three CaM isoforms (CaM2, CaM4, and CaM6). PCBP is encoded by a single gene and is expressed differentially in the tissues tested. The expression of CaM, PCBP, and another CaM-binding protein is similar in different tissues and organs. The predicted protein contained seven putative nuclear localization signals and several strong PEST motifs. Fusion of the N-terminal region of the protein containing six of the seven nuclear localization signals to the reporter gene beta-glucuronidase targeted the reporter gene to the nucleus, suggesting a nuclear role for PCBP.     

A potent antimicrobial protein from onion seeds showing sequence homology to plant lipid transfer proteins.

An antimicrobial protein of about 10 kD, called Ace-AMP1, was isolated from onion (Allium cepa L.) seeds. Based on the near-complete amino acid sequence of this protein, oligonucleotides were designed for polymerase chain reaction-based cloning of the corresponding cDNA. The mature protein is homologous to plant nonspecific lipid transfer proteins (nsLTPs), but it shares only 76% of the residues that are conserved among all known plant nsLTPs and is unusually rich in arginine. Ace-AMP1 inhibits all 12 tested plant pathogenic fungi at concentrations below 10 micrograms mL-1. Its antifungal activity is either not at all or is weakly affected by the presence of different cations at concentrations approximating physiological ionic strength conditions. Ace-AMP1 is also active on two Gram-positive bacteria but is apparently not toxic for Gram-negative bacteria and cultured human cells. In contrast to nsLTPs such as those isolated from radish or maize seeds, Ace-AMP1 was unable to transfer phospholipids from liposomes to mitochondria. On the other hand, lipid transfer proteins from wheat and maize seeds showed little or no antimicrobial activity, whereas the radish lipid transfer protein displayed antifungal activity only in media with low cation concentrations. The relevance of these findings with regard to the function of nsLTPs is discussed.     

Calmodulin-binding protein detection using a non-radiolabeled calmodulin fusion protein

Calmodulin-binding proteins are involved in numerous cellular signaling pathways. The biotinylated-calmodulin overlay is a nonradioactive method widely used to detect calmodulin-binding proteins in tissue and cell samples. This method has several limitations; therefore, we developed a nonradioactive calmodulin-binding protein detection overlay using an S-tag-labeled calmodulin fusion protein. An expression system was used to generate a calmodulin fusion protein with an S-tag label, a 15 amino acid sequence that binds to a 105 amino acid S-protein. The S-protein is conjugated to horseradish peroxidase for final detection with a chemiluminescent substrate. The S-tag calmodulin was compared to purified calmodulin and biotinylated calmodulin in a calmodulin-dependent phosphodiesterase assay. The results of the calmodulin-dependent phosphodiesterase assay indicate that S-tag calmodulin induces higher phosphodiesterase activity than biotinylated calmodulin and lower activity than purified calmodulin. A comparison of the biotinylated and S-tag calmodulin overlay assays indicate that S-tag calmodulin is more sensitive than biotinylated calmodulin in the detection of calcineurin, a known calmodulin-binding protein. The overlay assay results also indicate that the S-tag calmodulin and biotinylated calmodulin detect similar calmodulin-binding proteins in colon epithelial cells. In conclusion, the S-tag calmodulin overlay assay is a consistent, sensitive, and rapid nonradioactive method to detect calmodulin-binding proteins.     

A novel kinesin-like protein with a calmodulin-binding domain

Calcium regulates diverse developmental processes in plants through the action of calmodulin. A cDNA expression library from developing anthers of tobacco was screened with ³⁵S-labeled calmodulin to isolate cDNAs encoding calmodulin-binding proteins. Among several clones isolated, a kinesin-like gene (TCK1) that encodes a calmodulin-binding kinesin-like protein was obtained. The TCK1 cDNA encodes a protein with 1265 amino acid residues. Its structural features are very similar to those of known kinesin heavy chains and kinesin-like proteins from plants and animals, with one distinct exception. Unlike other known kinesin-like proteins, TCK1 contains a calmodulin-binding domain which distinguishes it from all other known kinesin genes. Escherichia coli-expressed TCK1 binds calmodulin in a Ca²⁺-dependent manner. In addition to the presence of a calmodulin-binding domain at the carboxyl terminal, it also has a leucine zipper motif in the stalk region. The amino acid sequence at the carboxyl terminal of TCK1 has striking homology with the mechanochemical motor domain of kinesins. The motor domain has ATPase activity that is stimulated by microtubules. Southern blot analysis revealed that TCK1 is coded by a single gene. Expression studies indicated that TCK1 is expressed in all of the tissues tested. Its expression is highest in the stigma and anther, especially during the early stages of anther development. Our results suggest that Ca²⁺/calmodulin may play an important role in the function of this microtubule-associated motor protein and may be involved in the regulation of microtubule-based intracellular transport.     

The primary structure of bovine cellular retinoic acid-binding protein

The complete amino acid sequence of bovine adrenal gland cellular retinoic acid-binding protein (CRABP) has been determined. The primary structure was established by analyses of cyanogen bromide fragments and peptides obtained by trypsin and Staphylococcus aureus protease digestions. The polypeptide chain of bovine CRABP comprises 136 amino acid residues. From partial sequence information, CRABP has been shown to be homologous to cellular retinol-binding protein, myelin protein P2, and the fatty acid-binding Z-protein. A comparison of the complete amino acid sequences of the members of this protein family, which also includes the rat intestinal fatty acid-binding protein, shows that CRABP is more similar to cellular retinol-binding protein and protein P2 than to the fatty acid-binding proteins. All five proteins are very similar in their NH2-terminal regions, suggesting that this part is important for a property common to the members of this protein family. This is the first report of a complete amino acid sequence of a CRABP.     

A plant kinesin heavy chain-like protein is a calmodulin-binding protein

Calmodulin, a calcium modulated protein, regulates the activity of several proteins that control cellular functions. A cDNA encoding a unique calmodulin-binding protein, PKCBP, was isolated from a potato expression library using protein-protein interaction based screening. The cDNA encoded protein bound to biotinylated calmodulin and ³⁵S-labeled calmodulin in the presence of calcium and failed to bind in the presence of EGTA, a calcium chelator. The deduced amino acid sequence of the PKCBP has a domain of about 340 amino acids in the C-terminus that showed significant sequence similarity with the kinesin heavy chain motor domain and contained conserved ATP- and microtubule-binding sites present in the motor domain of all known kinesin heavy chains. Outside the motor domain, the PKCBP showed no sequence similarity with any of the known kinesins, but contained a globular domain in the N-terminus and a putative coiled-coil region in the middle. The calmodulin-binding region was mapped to a stretch of 64 amino acid residues in the C-terminus region of the protein. The gene is differentially expressed with the highest expression in apical buds. A homolog of PKCBP from Arabidopsis (AKCBP) showed identical structural organization indicating that kinesin heavy chains that bind to calmodulin are likely to exist in other plants. This paper presents evidence that the motor domain has microtubule stimulated ATPase activity and binds to microtubules in a nucleotide-dependent manner. The kinesin heavy chain-like calmodulin-binding protein is a new member of the kinesin superfamily as none of the known kinesin heavy chains contain a calmodulin-binding domain. The presence of a calmodulin-binding motif and a motor domain in a single polypeptide suggests regulation of kinesin heavy chain driven motor function(s) by calcium and calmodulin.     

Volkensin from Adenia volkensii Harms (kilyambiti plant), a type 2 ribosome-inactivating protein.

Volkensin, a type 2 ribosome-inactivating protein from the roots of Adenia volkensii Harms (kilyambiti plant) was characterized both at the protein and nucleotide level by direct amino acid sequencing and cloning of the gene encoding the protein. Gene sequence analysis revealed that volkensin is encoded by a 1569-bp ORF (523 amino acid residues) without introns, with an internal linker sequence of 45 bp. Differences in residues present at several sequence positions (reproduced after repeated protein sequence analyses), with respect to the gene sequence, suggest several isoforms for the volkensin A-chain. Based on the crystallographic coordinates of ricin, which shares a high sequence identity with volkensin, a molecular model of volkensin was obtained. The 3D model suggests that the amino acid residues of the active site of the ricin A-chain are conserved at identical spatial positions, including Ser203, a novel amino acid residue found to be conserved in all known ribosome-inactivating proteins. The sugar binding site 1 of the ricin B-chain is also conserved in the volkensin B-chain, whilst in binding site 2, His246 replaces Tyr248. Native volkensin contains two free cysteinyl residues out of 14 derived from the gene sequence, thus suggesting a further disulphide bridge in the B chain, in addition to the inter- and intrachain disulphide bond pattern common to other type 2 ribosome-inactivating proteins.     

Isolation, purification and characterization of a nonspecific lipid transfer protein from Cuminum cyminum

Cuminum cyminum, an aromatic plant from the family Umbelliferae, is used as a flavoring and seasoning agent in foods. This communication reports the characterization of a nonspecific lipid transfer protein nsLTP1 from its seeds. Plant nsLTPs are small basic proteins involved in transport of lipids between membranes. These proteins are known to participate in plant defense; however, the exact mechanism of their antimicrobial action against fungi or bacteria is still unclear.The cumin nsLTP1 has been purified using a combination of chromatographic procedures and further characterized using mass spectrometry, circular dichroism spectroscopy and Edman degradation. Amino acid sequence has been used to predict homology model of cumin nsLTP1 in complex with myristic acid, and lyso-myristoyl phosphatidyl choline (LMPC). Cumin nsLTP1 is a monomeric protein with a molecular weight of 9.7 kDa as estimated by SDS-PAGE and ESIMS. The protein shows an isoelectric point of 7.8 on 6% PAGE. The primary structure consists of 92 amino acids with eight conserved cysteine residues. The global fold of cumin nsLTP1 includes four α-helices stabilized by four disulfide bonds and a C-terminal tail. The role of internal hydrophobic cavity of the protein in lipid transfer is discussed.     

Identification of a Drosophila gene encoding a calmodulin-binding protein with homology to the trp phototransduction gene.

We have isolated a number of Drosophila cDNAs on the basis of their encoding calmodulin-binding proteins. A full-length cDNA clone corresponding to one of these genes has been cloned and sequenced. Conservation of amino acid sequence and tissue-specific expression are observed between this gene and the transient receptor potential (trp) gene. We propose the name transient receptor potential-like (trpl) to describe this newly isolated gene. The trpl protein contains two possible calmodulin-binding sites, six transmembrane regions, and a sequence homologous to an ankyrin-like repeat. Structurally, the trpl and trp proteins resemble cation channel proteins, particularly the brain isoform of the voltage-sensitive Ca2+ channel. The identification of a protein similar to the trp gene product, yet also able to bind Ca2+/calmodulin, allows for a reinterpretation of the phenotype of the trp mutations and suggests that both genes may encode light-sensitive ion channels.     

Complementary DNA sequence of rabbit CAP18--a unique lipopolysaccharide binding protein

CAP18 is a novel 18 kDa cationic protein [pI approximately 10] originally purified from rabbit granulocytes using as an assay the agglutination of lipopolysaccharide (LPS) coated erythrocytes. cDNA clones encoding CAP18 were isolated from a rabbit bone marrow cDNA library using a PCR generated oligonucleotide probe derived from the N-terminal amino acid sequence. The deduced amino acid sequence reveals a putative signal sequence of 29 amino acids and a mature protein of 142 amino acid residues. The predicted size of the encoded protein is 16.6 kDa with a pI of 10. There are no N-linked glycosylation sites. The CAP18 sequence bears no homology with other known LPS-binding proteins including human bacterial permeability increasing protein (BPI)(1) and rabbit LPS binding protein (LBP)(2).     

Amino acid sequence of P-57, a neurospecific calmodulin-binding protein

The amino acid sequence was determined for bovine brain P-57, a neurospecific, membrane-associated, calmodulin-binding protein. It consists of a single 239-residue polypeptide chain blocked at its amino terminus and containing an unusually hydrophilic amino acid composition. Seventy percent of the molecule is composed of Glu/Gln, Ala, Lys, Asp/Asn, and Pro; there is only one aromatic residue. A lack of favorable cleavage sites required that a particularly wide variety of digests and subdigests be performed to obtain appropriate sets of overlapping peptides. This protein is clearly homologous with the cDNA-derived sequence of mouse brain P-57, although the bovine protein is 12 amino acid residues longer; the homology is less obvious in the middle sections of the two sequences. Bovine brain P-57 lacks homology with any other protein in an updated sequence database. A segment reported to interact with calmodulin (Arg-Gly-His-Ile-Thr-Arg-Lys-Lys-Leu) is placed at residues 43-51 within the only extended segment of P-57 that carries the net positive charge that would favor that interaction. There is no hydrophobic segment characteristic of many proteins that interact with membranes.     

Phosphorylation-regulated calmodulin binding to a prominent cellular substrate for protein kinase C

We have evaluated the possibility that a major, abundant cellular substrate for protein kinase C might be a calmodulin-binding protein. We have recently labeled this protein, which migrates on sodium dodecyl sulfate-gel electrophoresis with an apparent Mr of 60,000 from chicken and 80,000-87,000 from bovine cells and tissues, the myristoylated alanine-rich C kinase substrate (MARCKS). The MARCKS proteins from both species could be cross-linked to 125I-calmodulin in a Ca2+-dependent manner. Phosphorylation of either protein by protein kinase C prevented 125I-calmodulin binding and cross-linking, suggesting that the calmodulin-binding domain might be located at or near the sites of protein kinase C phosphorylation. Both bovine and chicken MARCKS proteins contain an identical 25-amino acid domain that contains all 4 of the serine residues phosphorylated by protein kinase C in vitro. In addition, this domain is similar in sequence and structure to previously described calmodulin-binding domains. A synthetic peptide corresponding to this domain inhibited calmodulin binding to the MARCKS protein and also could be cross-linked to 125I-calmodulin in a calcium-dependent manner. In addition, protein kinase C-dependent phosphorylation of the synthetic peptide inhibited its binding and cross-linking to 125I-calmodulin. The peptide bound to fluorescently labeled 5-dimethylaminonaphthalene-1-sulfonyl-calmodulin with a dissociation constant of 2.8 nM, and inhibited the calmodulin-dependent activation of cyclic nucleotide phosphodiesterase with an IC50 of 4.8 nM. Thus, the peptide mimics the calmodulin-binding properties of the MARCKS protein and probably represents its calmodulin-binding domain. Phosphorylation of these abundant, high affinity calmodulin-binding proteins by protein kinase C in intact cells could cause displacement of bound calmodulin, perhaps leading to activation of Ca2+-calmodulin-dependent processes.     

A study on the selective binding of apoprotein B- and E-containing human plasma lipoproteins to immobilized rat serum phosphorylcholine-binding protein

Rat serum phosphorylcholine-binding protein (PCBP), a member of the pentraxin family of proteins, was previously shown to bind multilamellar liposomes prepared with egg phosphatidylcholine and lysophosphatidylcholine. The results suggested that the phosphorylcholine groups on the surface of liposomes play an important role in the binding process (Nagpurkar, A., Saxena, U., and Mookerjea, S. (1983) J. Biol Chem. 258, 10518-10523). A study on the binding of human plasma lipoproteins to PCBP immobilized on Sepharose has now been initiated. Very low density lipoproteins were partially bound to a Sepharose-PCBP column, and the bound fraction contained higher concentrations of apoprotein B and E. All the low density lipoproteins applied were bound to the column. In the case of high density lipoproteins, only a small fraction was retained on the column (based on protein analysis), and that bound fraction contained all the apoprotein E and Lp(a) lipoprotein. The binding of very low, low, and high density lipoproteins to Sepharose-PCBP was Ca2+-dependent, and the bound lipoproteins were quantitatively eluted by a phosphorylcholine gradient. Apoprotein B and E were also bound when whole human plasma was applied to Sepharose-PCBP. The effect of selective modification of lysine residues by acetoacetylation and of arginine residues by cyclohexanedione on the binding of low density lipoproteins to Sepharose-PCBP was examined. Modification of arginyl residues resulted in marked reduction of binding, whereas modification of lysine had no effect. Removal of sialic acid from PCBP also had no effect on the binding of low density lipoproteins to immobilized-desialylated PCBP column. The preferential binding of apoprotein B- and E-containing lipoproteins to Sepharose-PCBP indicates a possible physiological role of PCBP and other similar circulating phosphorylcholine-binding proteins of the pentraxin family in lipoprotein metabolism.     

Characterization of a novel plant PP2C-like protein Ser/Thr phosphatase as a calmodulin-binding protein

Protein phosphatases regulated by calmodulin (CaM) mediate the action of intracellular Ca2+ and modulate functions of various target proteins by dephosphorylation. In plants, however, the role of Ca2+ in the regulation of protein dephosphorylation is not well understood due to a lack of information on characteristics of CaM-regulated protein phosphatases. Screening of a cDNA library of the moss Physcomitrella patens by using 35S-labeled calmodulin as a ligand resulted in identification of a gene, PCaMPP, that encodes a protein serine/threonine phosphatase with 373 amino acids. PCaMPP had a catalytic domain with sequence similarity to type 2C protein phosphatases (PP2Cs) with six conserved metal-associating amino acid residues and also had an extra C-terminal domain. Recombinant GST fusion proteins of PCaMPP exhibited Mn2+-dependent phosphatase activity, and the activity was inhibited by pyrophosphate and 1 mm Ca2+ but not by okadaic acid, orthovanadate, or beta-glycerophosphate. Furthermore, the PCaMPP activity was increased 1.7-fold by addition of CaM at nanomolar concentrations. CaM binding assays using deletion proteins and a synthetic peptide revealed that the CaM-binding region resides within the basic amphiphilic amino acid region 324-346 in the C-terminal domain. The CaM-binding region had sequence similarity to amino acids in one of three alpha-helices in the C-terminal domain of human PP2Calpha, suggesting a novel role of the C-terminal domains for the phosphatase activity. These results provide the first evidence showing possible regulation of PP2C-related phosphatases by Ca2+/CaM in plants. Genes similar to PCaMPP were found in genomes of various higher plant species, suggesting that PCaMPP-type protein phosphatases are conserved in land plants.     

Molecular cloning of the 18-kDa growth-associated protein of developing brain

An 18-kDa protein (designated herein as the heparin-binding growth-associated molecule, HB-GAM), the expression of which in rat brain correlates to the rapid postnatal developmental phase, was previously isolated and suggested to have a role in the maturation and growth of brain (Rauvala, H. (1989) EMBO J. 8, 2933-2941). A protein with a similar molecular mass, similar heparin-binding properties, and the same N-terminal sequence was more recently also isolated as a mitogen for NIH 3T3 cells (Milner, P. G., Li, Y.-T., Hoffman, R. M., Kodner, C. M., Siegel, N. R., and Deuel, T. F. (1989) Biochem. Biophys. Res. Commun. 165, 1096-1103). This study reports the cloning and sequencing of the cDNA that encodes HB-GAM. The sequence that precedes the structure of the mature molecule has the characteristics of a signal sequence found in secretory proteins. The sequence of HB-GAM is a novel structure that contains 136 amino acid residues. The sequence is very rich in cationic amino acids (24% of the residues); lysine cluster sequences are found in the N-terminal and C-terminal ends of the structure. Cysteine is also abundant in the sequence (7% of the residues). The only homologous sequence found in computer searches is the retinoic acid-induced differentiation factor. The mRNA of HB-GAM detected by the cloned cDNA shows the same kind of developmental regulation as the protein; the mRNA is strongly expressed during the early postnatal growth phase of rat brain as compared with embryonic or adult tissue.     

The classical arabinogalactan protein gene family of arabidopsis

Arabinogalactan proteins (AGPs) are extracellular proteoglycans implicated in plant growth and development. We searched for classical AGPs in Arabidopsis by identifying expressed sequence tags based on the conserved domain structure of the predicted protein backbone. To confirm that these genes encoded bona fide AGPs, we purified native AGPs and then deglycosylated and deblocked them for N-terminal protein sequencing. In total, we identified 15 genes encoding the protein backbones of classical AGPs, including genes for AG peptides-AGPs with very short backbones (10 to 13 amino acid residues). Seven of the AGPs were verified as AGPs by protein sequencing. A gene encoding a putative cell adhesion molecule with AGP-like domains was also identified. This work provides a firm foundation for beginning functional analysis by using a genetic approach.     

65-kilodalton protein phosphorylated by interleukin 2 stimulation bears two putative actin-binding sites and two calcium-binding sites

We have previously characterized a 65-kilodalton protein (p65) as an interleukin 2 stimulated phosphoprotein in human T cells and showed that three endopeptide sequences of p65 are present in the sequence of l-plastin [Zu et al. (1990) Biochemistry 29, 1055-1062]. In this paper, we present the complete primary structure of p65 based on the cDNA isolated from a human T lymphocyte (KUT-2) cDNA library. Analysis of p65 sequences and the amino acid composition of cleaved p65 N-terminal peptide indicated that the deduced p65 amino acid sequence exactly coincides with that of l-plastin over the C-terminal 580 residues [Lin et al. (1988) Mol. Cell. Biol. 8, 4659-4668] and has a 57-residue extension at the N-terminus to l-plastin. Computer-assisted structural analysis revealed that p65 is a multidomain molecule involving at least three intriguing functional domains: two putative calcium-binding sites along the N-terminal 80 amino acid residues; a putative calmodulin-binding site following the calcium-binding region; and two tandem repeats of putative actin-binding domains in its middle and C-terminal parts, each containing approximately 240 amino acid residues. These results suggest that p65 belongs to actin-binding proteins.