The complete amino-acid sequence of the rieske FeS-precursor protein from spinach chloroplasts deduced from cDNA analysis

Summary SummarySeveral cDNA clones encoding the entire Rieske FeS-precursor protein of the chloroplast cytochrome b ₆ f-complex have been isolated by high density plaque immunoscreening of a phage lambda gt11 cDNA expression library, made from poly A⁺-RNA of spinach seedlings. The identity of the cDNAs has been confirmed by N-terminal amino acid sequencing of the purified protein. The nucleotide sequence indicates a protein of 247 amino acid residues including a putative transit sequence of 68 amino acids corresponding to molecular masses of 26.3 kDa (precursor) and 18.8 kDa (mature protein; 179 amino acid residues). Alignteins of the sequence with sequences from Rieske FeS-proteins of respiratory electron transport chains, two of bacterial and three of mitochondrial origin, shows little sequence homology, but remarkable similarity in secondary structure including a putative N-terminal transmembrane segment of about 25 residues and the peptides CTHLGCV and CPCHGS in the C-terminal region of the protein that are involved in the binding of the Fe₂S₂-cluster.     

Synthesis of vitamin D3 analogues with A-ring modifications to directly measure vitamin D levels in biological samples

C-3-substituted 25-hydroxyvitamin D₃ analogues were synthesized as tools to directly measure levels of vitamin D in biological samples. The strategy involves vinyloxycarbonylation of the 3β-hydroxy group and formation of a carbamate bond with a hydroxyl or amino group at the end of the alkyl chain. Biotinylated conjugates of synthesized derivatives were generated to be linked with vitamin D binding protein (DBP). The spacer group present in the alkyl chain is important in the binding of antibodies to the analogue–DBP complex. When compared to 25-hydroxyvitamin D₃-DBP, the binding of some antibodies to the analogue–DBP complex of the 25-hydroxyvitamin D₃ derivative 10 that posses an 8-aminoctyl alkyl chain is significantly reduced, but this analogue displaced [26,27-³H]-25-hydroxyvitamin D₃ from DBP. In contrast, the 8-hydroxyoctyl alkyl chain analogue 9 showed less displacement.     

The vitamin D system in iguanian lizards

The apparent plasma concentration of vitamin D binding protein (DBP) in an iguanian lizard, Pogona barbata, and the affinity of this protein for 25-hydroxyvitamin D₃ (25(OH)D₃), 25-hydroxyvitamin D₂ (25(OH)D₂), and 1,25-dihydroxyvitamin D₃ (1,25(OH)D₃) was found to resemble more closely that of the domestic hen than that of the human. The characteristics of Pogona DBP, the pattern of vitamin D metabolites derived from injected radioactive vitamin D₃ and the plasma concentrations of endogenous 25-hydroxyvitamin D (25(OH)D) in a range of iguanian lizards have been examined. The findings suggest that 25-hydroxyvitamin D (25(OH)D) is the major metabolite of vitamin D, and that it may represent the storage form of vitamin D in these species in the same way as in mammals. High concentrations of vitamin D within iguanian embryos and egg yolks suggest a role for this compound in embryogenesis in these species, and perhaps indicates that there is a mechanism for vitamin D delivery to eggs comparable to that found in the domestic chicken.     

Suppression of PTH by the vitamin D analog eldecalcitol is modulated by its high affinity for the serum vitamin D-binding protein and resistance to metabolism

Eldecalcitol [1α,25‐dihydroxy‐2β‐(3‐hydroxypropyloxy)vitamin D₃], a vitamin D analog with enhanced efficacy for treatment of osteoporosis, has been found to be less potent than 1,25‐dihydroxyvitamin D₃ (calcitriol) in suppressing PTH in vivo. To define the mechanism for the latter observation, we compared the effects of eldecalcitol and calcitriol on PTH secretion by bovine parathyroid cells. While the two compounds showed similar potency when the cells were cultured in medium containing 15% newborn calf serum, eldecalcitol was 100 times more potent than calcitriol in the absence of serum. Eldecalcitol has a higher affinity for the serum vitamin D‐binding protein (DBP), and therefore binding to DBP, and possibly other serum components, appears to limit the uptake and activity of eldecalcitol in parathyroid cells, providing an explanation for the lower PTH suppressing activity in vivo (100% serum). However, the 100‐fold higher activity of eldecalcitol in the absence of serum was unexpected since the VDR affinity for eldecalcitol is eightfold lower than for calcitriol. The enhanced activity was not due to preferential uptake, but to a resistance to metabolism. While 1 nM [³H]calcitriol was completely degraded within 24 h, [³H]eldecalcitol was not metabolized, despite the induction of the vitamin D catabolic enzyme, 24‐hydroxylase (CYP24A). The resistance to metabolism is the likely explanation for the higher potency of eldecalcitol in suppressing PTH in cell culture lacking serum. Thus, the unique properties of eldecalcitol in vivo can be attributed, at least in part, to its high‐DBP affinity which increases the half‐life, but limits the uptake of eldecalcitol, and to its reduced metabolism, which prolongs the activity of this analog in target tissues. J. Cell. Biochem. 112: 1348–1352, 2011. © 2011 Wiley‐Liss, Inc.     

Rat vitamin D binding protein. Determination of the full-length primary structure from cloned cDNA

Vitamin D binding protein (DBP) is an abundant serum glycoprotein secreted by the liver which transports vitamin D sterols, binds to actin, and is found on the surface of B-lymphocytes and subpopulations of T-lymphocytes. In the current study, a cDNA to rat DBP mRNA was cloned from a bacteriophage lambda gt 11 rat liver expression library. This DBP cDNA clone was identified by immunoblotting and its identity was confirmed by immunoprecipitation of a 54-kDa protein after hybrid-assisted translation. Northern analysis and primer extension mapping of rat liver mRNA indicated that the full-length DBP mRNA contains 1700 bases. By DNA sequence analysis this 1655-base pair clone contains a single open reading frame encoding the 476-amino acid containing full-length DBP and includes its 16-amino acid signal sequence. Analysis of the sequence reveals about 40% nucleotide and 23% amino acid homology to both albumin and alpha-fetoprotein. The encoded DBP contains a characteristic placement of cysteine residues, identical to that in albumin, suggesting a similar secondary folding structure. Albumin and alpha-fetoprotein are composed of three internally homologous domains. DBP mRNA terminates 122 amino acids before the larger albumin mRNA in the third internal domain, but retains the characteristic homology among the first two domains and the truncated portion of the third domain. These data support the conclusion that DBP is a member of a multigene family which includes albumin and alpha-fetoprotein.     

Polymorphism of the vitamin D binding protein (DBP) among primates: an evolutionary analysis

The distribution of the DBP (vitamin D binding protein) polymorphism is now well characterized among human populations but for primates only limited results are known. The aim of this paper is to describe the electrophoretic polymorphism of this protein among various species. Using three different electrophoretic methods, we are able to detect an unknown polymorphism and to classify the different alleles observed. These results may be used to set an international nomenclature for further comparisons. The different electrophoretic mobilities between Old and New World Monkeys show that: 1) the Cercopithecoïdea are presenting the largest genetic heterogeneity; 2) the DBP among the Galago corresponds to the lowest isoelectric points observed among Primates; 3) during the evolution from nonhuman Primates to Man, the DBP is able to keep its affinity for vitamin D derivatives despite the occurrence of significant molecular modifications; 4) among Anthropoïdea, the electrophoretic patterns of DBP are very close to the human Gc 1 proteins. These results show that evolution at the DBP level can be considered as a continous mechanism of structural modifications. A significant transition occurs during the differentiation between Cercopithecoïdea and Anthropoïdea. It is not too speculative to consider that some electrophoretic forms detected among Gorilla, Pongo, or Pan may be identical to rare variants observed among humans.     

Sequence of a cDNA Encoding Turtle High Mobility Group 1 Protein

In order to understand sequence information about turtle HMG1 gene, a cDNA encoding HMG1 protein of the Chinese soft-shell turtle (Pelodiscus sinensis) was amplified by RT-PCR from kidney total RNA, and was cloned, sequenced and analyzed. The results revealed that the open reading frame (ORF) of turtle HMG1 cDNA is 606 bp long. The ORF codifies 202 amino acid residues, from which two DNA-binding domains and one polyacidic region are derived. The DNA-binding domains share higher amino acid identity with homologous sequences of chicken (96.5%) and mammals (74%) than homologous sequence of rainbow trout (67%). The polyacidic region shows 84.6% amino acid homology with the equivalent region of chicken HMG1 cDNA. Turtle HMG1 protein contains 3 Cys residues located at completely conserved positions. Conservation in sequence and structure suggests that the functions of turtle HMG1 cDNA may be highly conserved during evolution. To our knowledge, this is the first report of HMG1 cDNA sequence in any reptilian.From Genetika, Vol. 41, No. 7, 2005, pp. 925–930.Original English Text Copyright © 2005 by Jifang Zheng, Bi Hu, Duansheng Wu.The text was submitted by the authors in English.     

Corticosteroid binding globulin, testosterone-estradiol binding globulin, and androgen binding protein belong to protein families distinct from steroid receptors

The cDNA nucleotide sequences and the deduced amino acid sequences of human corticosteroid binding globulin (hCBG), human testosterone-estradiol binding globulin (hTeBG), and rat androgen binding protein (rABP) were determined. Studies of the steroid binding sites suggest they are toward the carboxy-terminus in hTeBG and rABP and more central in hCBG. hCBG has remarkable sequence homology with members of a superfamily whose functions have diverged; these include thyroxine-binding protein, serine protease inhibitors, egg white proteins, and angiotensinogen. hTeBG and rABP have a 68% amino acid sequence identity. Hybridization studies suggest that hTeBG is probably even more closely related, if not identical, to hABP. The carboxy-terminal sequences of hTeBG and rABP are also similar to that of protein S, a vitamin-K-dependent clotting factor. There were no nucleotide or amino acid sequence homologies between hCBG, hTeBG, or rABP and other steroid binding proteins such as steroid receptors, albumin, alpha-fetoprotein, and vitamin D binding protein. We conclude that the "extracellular steroid binding proteins" and steroid receptors do not appear to have descended from a common ancestor.     

Cross-talk among structural domains of human DBP upon binding 25-hydroxyvitamin D

Serum vitamin D-binding protein (DBP) is structurally very similar to serum albumin (ALB); both have three distinct structural domains and high cysteine-content. Yet, functionally they are very different. DBP possesses high affinity for vitamin D metabolites and G-actin, but ALB does not. It has been suggested that there may be cross-talk among the domains so that binding of one ligand may influence the binding of others. In this study we have employed 2-p-toluidinyl-6-sulfonate (TNS), a reporter molecule that fluoresces upon binding to hydrophobic pockets of DBP. We observed that recombinant domain III possesses strong binding for TNS, which is not influenced by 25-hydroxyvitamin D₃ (25-OH-D₃), yet TNS fluorescence of the whole protein is quenched by 25-OH-D₃. These results provide a direct evidence of cross-talk among the structural domains of DBP.     

Phenotype frequencies of vitamin D binding protein (Gc) and of posttransferrin-2 (Ptf-2) in Belgian cattle breeds*

The vitamin D binding protein (Gc) and posttransferrin-2 (Ptf-2) phenotypes have been determined in a number of Belgian cattle breeds. A very slow migrating variant of the Gc protein — Gc C — has been found in White and Red East Flemish breed. This variant was absent from the other breeds studied. This slow variant was identified as a vitamin D binding protein by autoradiography. The Gc C protein was shown to be controlled by a codominant autosomal allele Gc C at the Gclocus. The Gc C protein is probably identical with a fraction previously described in buffalo and an Italian cattle breed. The allele frequencies for the Gc and Pft-2 systems are reported for several Belgian breeds of cattle.     

Sequence of a cDNA encoding turtle high mobility group 1 protein

In order to understand sequence information about turtle HMG1 gene, a cDNA encoding HMG1 protein of the Chinese soft-shell turtle (Pelodiscus sinensis) was amplified by RT-PCR from kidney total RNA, and was cloned, sequenced and analyzed. The results revealed that the open reading frame (ORF) of turtle HMG1 cDNA is 606 bp long. The ORF codifies 202 amino acid residues, from which two DNA-binding domains and one polyacidic region are derived. The DNA-binding domains share higher amino acid identity with homologues sequences of chicken (96.5%) and mammalian (74%) than homologues sequence of rainbow trout (67%). The polyacidic region shows 84.6% amino acid homology with the equivalent region of chicken HMG1 cDNA. Turtle HMG1 protein contains 3 Cys residues located at completely conserved positions. Conservation in sequence and structure suggests that the functions of turtle HMG1 cDNA may be highly conserved during evolution. To our knowledge, this is the first report of HMG1 cDNA sequence in any reptilian.     

The extrinsic 33 kDa polypeptide of the oxygen-evolving complex of photosystem II is a putative calcium-binding protein and is encoded by a multi-gene family in pea

The extrinsic 33 kDa polypeptide of the water-oxidizing complex has been extracted from pea photosystem II particles by washing with alkaline-Tris and purified by ion-exchange chromatography. The N-terminal amino acid sequence has been determined, and specific antisera have been raised in rabbits and used to screen a pea leaf cDNA library in gt11. Determination of the nucleotide sequence of positive clones revealed an essentially full-length cDNA for the 33 kDa polypeptide, the deduced amino acid sequence showing it to code for a mature protein of 248 amino acids with an N-terminal transit peptide of 81 amino acids. The protein showed a high degree of conservation with previously reported sequences for the 33 kDa protein from other species and the sequence contained a putative Ca²⁺-binding site with homology to mammalian intestinal calcium-binding proteins. Northern analysis of total pea RNA indicated a message of approximately 1.4 kb, in good agreement with the size of the cDNA obtained at 1.3 kbp. Southern blots of genomic DNA probed with the labelled cDNA give rise to several bands suggesting that the 33 kDa polypeptide is coded by a multi-gene family.Abbreviations ATZ - anilinothiazolinone - DITC - p-phenylenediisothiocyanate - PTH - phenylthiohydantoin - TFA - trifluoroacetic acid - Tris - tris (hydroxymethyl) aminomethane - bis-Tris - bis (2-hydroxyethyl) imino-tris (hydroxymethyl)-methane - p.f.u. - plaque-forming units     

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     

Chick kidney ferredoxin: Complementary DNA cloning and vitamin D effects on mRNA levels

Vertebrate ferredoxin is non-heme iron-sulfur protein found in steroideogenic tissues that serves as an electron shuttle in mitochondrial mixed function oxidase systems such as the 25-hydroxyvitamin D₃-1α-hydroxylase. A 2530-bp chick kidney ferredoxin cDNA was cloned, and the association between ferredoxin mRNA levels and the regulation of 1α-hydroxylase activity by vitamin D status was examined. The cDNA sequence indicates that the chick kidney mitochondrial mixed function oxidases use the same ferredoxin as do those in the chick testis and that the chick ferredoxin shares greater than 92% amino acid homology with mammalian ferredoxins. Southern blot analysis of genomic DNA indicates that there is a single copy of the ferredoxin gene present in the chick genome. Three species of mRNA, 1.8, 3.5 and 5.5 kb, were identified by Northern analysis. Slot blot analysis of poly A⁺ RNA from kidneys of vitamin D-deficient or -replete chicks indicates a 40% induction of ferredoxin message levels in the vitamin D-deficient chick kidney. This suggests that gene regulation of ferredoxin may be part of the mechanism of regulation for 25-hydroxyvitamin D₃-1α-hydroxylase activity in the chick kidney.     

Glycosylation status of vitamin D binding protein in cancer patients

On the basis of the results of activity studies, previous reports have suggested that vitamin D binding protein (DBP) is significantly or even completely deglycosylated in cancer patients, eliminating the molecular precursor of the immunologically important Gc macrophage activating factor (GcMAF), a glycosidase‐derived product of DBP. The purpose of this investigation was to directly determine the relative degree of O‐linked trisaccharide glycosylation of serum‐derived DBP in human breast, colorectal, pancreatic, and prostate cancer patients. Results obtained by electrospray ionization‐based mass spectrometric immunoassay showed that there was no significant depletion of DBP trisaccharide glycosylation in the 56 cancer patients examined relative to healthy controls. These results suggest that alternative hypotheses regarding the molecular and/or structural origins of GcMAF must be considered to explain the relative inability of cancer patient serum to activate macrophages.     

Adhesive protein cDNA sequence of the musselMytilus coruscus and its evolutionary implications

cDNA encoding the adhesive protein of the musselMytilus coruscus (Mgfpl) was isolated. The coding region encoded 848 amino acids (a.a.) comprising the 20-a.a. signal peptide, the 21-a.a. nonrepetitive linker, and the 805-a.a. repetitive domain. Although the first 204 nucleotides and the 3′-untranslated region of Mgfpl cDNA were homologous to corresponding parts ofM. galloprovincialis adhesive protein (Mgfpl) cDNA, the other parts diverged. The representative repeat motif of the repetitive domain, YKPK(1/P)(S/T)YPP(T/S), was similar but slightly different from the repeat motif of Mgfpl. The codon usage patterns for the same amino acids were different in different positions of the decapeptide motif. Almost identical nucleotide sequences encoding the two to 13 repeats appeared several times in the repetitive region, which suggests that the adhesive protein genes of mussels have evolved through the duplication of these repeat units. The nucleotide sequence data reported in this paper will appear in the GSDB, DDBJ, EMBL, and NCBI nucleotide sequence databases with the accession number D63777 Correspondence to: K. Inoue     

Comparison of partial sequence of the cap binding protein (eIF4E) isolated from Agaricus bisporus and its pathogen Verticillium fungicola

The 3 regions of the gene encoding the cap binding protein eIF4E were successfully isolated from Agaricus bisporus and Verticillium fungicola using a degenerate primer within the eIF4E gene and an anchored oligo d(T) primer. The deduced amino acid sequences contained 173 residues for A. bisporus and 171 residues V. fungicola. Analysis of these sequences shows that despite conserved regions of homology, centering around tryptophan residues, A. bisporus and V. fungicola are very diverse at the amino acid and DNA level. Percentage homology between the two fungi is low at the nucleotide, 35%, and amino acid level, 29%. The highest degree of similarity between the A. bisporus sequence and other published sequences is with the Homo sapiens eIF4E sequence (32%). V. fungicola exhibited highest homology with the eIF4E sequence from Caenorhabditis elegans (34%). Southern analysis of genomic DNA indicated a single copy of the gene within the A. bisporus genome.This revised version was published online in October 2005 with corrections to the Cover Date.     

Partial genomic organization of ribosomal protein S7 gene from malaria vector Anopheles stephensi

In this study, we describe the partial genomic organization of ribosomal protein S7 gene isolated from the mosquito Anopheles stephensi. Initially a 558 bp partial cDNA sequence was amplified as precursor mRNA sequence containing 223 bp long intron. 5＇ and 3＇ end sequences were recovered using end specific rapid amplification of cDNA ends （RACE） polymerase chain reaction. The full-length cDNA sequence was 914 nucleotide long with an open reading frame capable of encoding 192 amino acid long protein with calculated molecular mass of 22174 Da and a pI point of 9.94. Protein homology search revealed 〉75% identity to other insect＇s S7 ribosomal proteins. Analysis of sequence alignment revealed several highly conserved domains, one of which is related to nuclear localization signal （NLS） region of human rpS7. Interestingly, intron nucleotide sequence comparison with A. gambiae showed a lesser degree of conservation as compared to coding and untranslated regions. Like this, early studies on the genomic organization and cDNA/ Expressed sequence tag analysis （EST） could help in genome annotation ofA. stephensi, and would be likely to be sequenced in the future.