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In vertebrate development the Dickkopf protein family carries out multiple functions and is represented by at least four different genes with distinct biological activities. In invertebrates such as Drosophila and Caenorhabditis, Dickkopf genes have so far not been identified. Here we describe the identification and characterization of a Dickkopf gene with a deduced amino acid sequence closely related to that of chicken Dkk-3 in the basal metazoan Hydra. HyDkk-3 appears to be the only Dickkopf gene in Hydra. The gene is expressed in the gastric region in nematocytes at a late differentiation stage. In silico searches of EST and genome databases indicated the absence of Dkk genes from the protostomes Drosophila and Caenorhabditis, whereas within the deuterostomes, a Dkk-3 gene could be identified in the genome of the urochordate Ciona intestinalis. The results indicate that at an early stage of evolution of multicellularity Dickkopf proteins have already played important roles as developmental signals. They also suggest that vertebrate Dkk-1, 2 and 4 may have originated from a common ancestor gene of Dkk-3.H. Fedders and R. Augustin contributed equally to this workEdited by D. Tautz 相似文献
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Casanova JC Kuhn J Kleesiek K Götting C 《Biochemical and biophysical research communications》2008,365(4):678-684
Human xylosyltransferase II (EC 2.4.2.26, XT-II) represents an isoform of xylosyltransferase I (XT-I). Recently, we and others provided first evidence that XT-II is capable of initiating the biosynthesis of glycosaminoglycan chains in proteoglycans. Here, a soluble form of human XT-II was expressed in the yeast Pichia pastoris and the substrate specificity for various acceptors was investigated, pointing to a modified bikunin peptide to be the optimal XT-II acceptor (KM = 1.9 μM). Furthermore, biochemical characterization of XT-II showed that this enzyme was strongly inhibited by nucleotides and glycosaminoglycans. Its temperature optimum, stability, and ion dependency were further examined, demonstrating necessity for Mg2+ or Mn2+ ions for its enzymatic activity. Our data show for the first time that XT-I and XT-II are xylosyltransferases with similar but not identical properties, pointing to their potential role in modulating the cellular proteoglycan pool. 相似文献
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Random peptide libraries can be constructed either by in vitro synthesis of random peptides, or through translation of DNA
sequences from synthetic random oligonucleotides. Here we describe an alternative way of making arbitrary peptide libraries
with high diversity that can be used in screening as random peptide libraries. Genomic DNA digested with a frequent-cutting
restriction enzyme recognizing four nucleotides will theoretically consist of small DNA pieces with average length of 256
nucleotides, and on average around 107 fragments can be generated from a genome of 3 × 109 bases. A peptide library translated from these fragments will have sufficient diversity for some protein interaction screening
experiments. Moreover, the same genome digested with a different four-cutter enzyme or ligated into different reading frames
will result in different nonoverlapping libraries. A series of such libraries could be generated with genomic DNAs from different
species. In this study, human genomic DNA was digested with four-cutter restriction enzymes DpnII and Tsp509I, respectively, and cloned into yeast expression vector pGADT7 to generate arbitrary peptide libraries. These libraries
were used in yeast two-hybrid assays to screen for binding motifs of the PDZ domain containing protein synectin. Our results
showed that in addition to various native carboxy-terminal tails, synectin could also bind to many artificial ones, some of
which contained a consensus sequence—(S/T)XC-COOH. 相似文献
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GIOVANNI GIUDICE 《Cell biology international》1996,20(1):29-32
Some general molecular mechanisms underlying development are described. Namely: those involved in the differentiation of the R7 receptor inDrosophilaembryonic retina; those involved in the determination of embryonic axes and in polar cell differentiation, inDrosophila; those involved in the determination of the AB and P cell lineage and in vulva differentiation inCaenorhabditisembryos. 相似文献
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Role of perlecan in skeletal development and diseases 总被引:4,自引:0,他引:4
Perlecan, a large heparan sulfate proteoglycan (HSPG), is present in the basement membrane and other extracellular matrices. Its protein core is 400 kDa in size and consists of five distinct structural domains. A number of in vitro studies suggest multiple functions of perlecan in cell growth and differentiation and tissue organization. Recent studies with gene knockout mice and human diseases revealed critical in vivo roles of perlecan in cartilage development and neuromuscular junction activity. Published in 2003. 相似文献
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Medina-Rivero E Balderas-Hernández VE Ordoñez-Acevedo LG Paz-Maldonado LM Barba-De la Rosa AP De León-Rodríguez A 《Biotechnology letters》2007,29(9):1369-1374
Production of periplasmic human interferon-γ (hINF-γ) and human interleukin-2 (hIL-2) by the Tat translocation pathway in
Escherichia coli BL21-SI was evaluated. The expression was obtained using the pEMR vector which contains the Tat-dependent modified penicillin
acylase signal peptide (mSPpac) driven by the T7 promoter. The mSPpac-hINF-γ was processed and the protein was transported to periplasm. Up to 30.1% of hINF-γ was found in the periplasmic soluble
fraction, whereas only 15% of the mSPpac-hIL-2 was processed, but hIL-2 was not found in the periplasmic soluble fraction. 相似文献
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Structural characterization of genes corresponding to cotton fiber mRNA, E6: reduced E6 protein in transgenic plants by antisense gene 总被引:13,自引:0,他引:13
Maliyakal E. John 《Plant molecular biology》1996,30(2):297-306
Two genes, each corresponding to fiber mRNA E6, were isolated from cotton cultivars Coker 312 (Gossypium hirsutum L.) and Sea Island (G. barbadense L.). E6 is one of the predominant fiber-specific mRNAs present during early fiber development. The distinguishing feature of the nucleotide-derived E6 protein is the presence of a motif where a dimer, Ser-Gly, is repeated several times. Two of the Sea Island genes contained a pentameric motif, Ser-Gly, while one of the Coker genes had one and the other had four motifs each. cDNA clones containing one or five Ser-Gly motifs were also identified. Thus, it appears that the strict conservation of this motif may not be critical to E6 protein function. Sequence characterizations of the genes and cDNAs showed that multiple members of the E6 family are transcribed in fiber and may result in proteins 238 to 246 amino acids long. The 3 ends of the genes and cDNAs showed considerable heterology among them. Transgenic plants containing antisense genes were generated to decipher E6 function. Transgenic cotton with reduced E6 protein levels in the range of 60 to 98% were identified. However, no discernible phenotypic changes in fiber development or properties were apparent. This result leads to the conclusion that E6 is not critical to the normal development or structural integrity of cotton fibers. 相似文献
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Jonathan D. Dorigatti Kevin M. Thyne Brett C. Ginsburg Adam B. Salmon 《Biochemistry and Biophysics Reports》2021
Activation of AMP activated protein kinase (AMPK) signaling has been demonstrated to extend lifespan and improve healthspan across multiple species. This suggests pharmaceutical approaches to increase AMPK hold the potential to modify the aging process and promote healthy aging. Beta-guanidinopropionic acid (GPA) is a naturally occurring metabolite structurally similar to creatine. GPA is capable of activating AMPK signaling in mammalian models via competitive inhibition of cytosolic creatine kinase. A previous report suggested that dietary GPA supplementation increased lifespan in Drosophila through its effect on AMPK signaling and regulation of autophagy. However, studies in Caenorhabditis have found no beneficial effect of this compound on worm lifespan and that GPA may actually diminish lifespan in at least one Caenorhabditis species. To confirm previous reports of increased longevity in Drosophila, we tested a wide range of GPA concentrations on lifespan and healthspan in both male and female W1118 flies. We report here that GPA does not extend lifespan in Drosophila as previously reported. Moreover, high doses of GPA are detrimental to Drosophila lifespan and stress resistance in male flies. These results suggest the lack of a robust effect of GPA on Drosophila lifespan and highlight the importance of replication studies within the field of aging. 相似文献
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Human and porcine recombinant deoxyribonucleases I (DNases I) were expressed in COS-7 cells, and purified by a single-step
procedure. Since affinities for concanavalin A (Con A) and wheatgerm agglutinin (WGA) were strong in these recombinant DNases
I, purification using Con A–WGA mixture-agarose column was performed. By this method, the enzymes in culture medium could
quickly be isolated to apparent homogeneity in approx. 10 min. From 1 ml of culture medium, about 20–30 μg of purified DNase I with a specific activity ranging from 22000 to 41000 units/mg were obtained. The purified DNases I were
subjected to enzymatic deglycosylation by either peptide N-glycosidase F (PNGase F) or endoglycosidase H (Endo H). The recombinant enzyme was cleaved by PNGase F, but not by Endo H,
indicating that the recombinant enzymes are modified by N-linked complex-type carbohydrate moieties. In the human recombinant DNase I, activity was decreased by PNGase F-treatment,
while that of the porcine DNase I remained unaffected. The thermal stability of the human enzyme was extremely susceptible
to heat following PNGase F-treatment, as was the porcine enzyme to a lesser extent. This study suggests that N-linked complex-type carbohydrate moieties may contribute to the enzymatic activity and/or thermal stability of recombinant
DNases I. 相似文献
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A novel peptide sequence in perlecan domain IV supports cell adhesion, spreading and FAK activation. 总被引:1,自引:0,他引:1
Mary C Farach-Carson Anissa J Brown Megan Lynam Jeffrey B Safran Daniel D Carson 《Matrix biology》2008,27(2):150-160
Perlecan/HSPG2 is a large, multi-domain, multifunctional heparan sulfate proteoglycan with a wide tissue distribution. With the exception of its unique domain I, each of perlecan's other four domains shares sequence similarity to other protein families including low density lipoprotein (LDL) receptor, laminin alpha chain, neural cell adhesion molecule (NCAM), immunoglobulin (Ig) superfamily members, and epidermal growth factor (EGF). Previous studies demonstrated that glycosaminoglycan-bearing perlecan domain I supports early chondrogenesis and growth factor delivery. Other sites in the core protein interact with other matrix molecules and support cell adhesion, although the peptide sequences involved remain unidentified. To identify novel functional motifs within perlecan, we used a bioinformatics approach to predict regions likely to be on the exterior of the folded protein. Unique hydrophilic sequences of about 18 amino acids were selected for testing in cell adhesion assays. A novel peptide sequence (TWSKVGGHLRPGIVQSG) from an immunoglobulin (Ig) repeat in domain IV supported rapid cell adhesion, spreading and focal adhesion kinase (FAK) activation when compared to other peptides, a randomly scrambled sequence of the domain IV peptide or a negative control protein. MG-63 human osteosarcoma cells, epithelial cells and multipotent C(3)H10T1/2 cells, but not bone marrow cells, rapidly, i.e., within 30 min, formed focal adhesions and assembled an actin cytoskeleton on domain IV peptide. Cell lines differentially adhered to the domain IV peptide, suggesting adhesion is receptor specific. Adhesion was divalent cation independent and heparin sensitive, a finding that may explain some previously poorly understood observations obtained with intact perlecan. Collectively, these studies demonstrate the feasibility of using bioinformatics-based strategies to identify novel functional motifs in matrix proteins such as perlecan. 相似文献
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North SJ Koles K Hembd C Morris HR Dell A Panin VM Haslam SM 《Glycoconjugate journal》2006,23(5-6):345-354
With the complete genome sequence of Drosophila melanogaster defined a systematic approach towards understanding the function of glycosylation has become possible. Structural assignment
of the entire Drosophila glycome during specific developmental stages could provide information that would shed further light on the specific roles
of different glycans during development and pinpoint the activity of certain glycosyltransferases and other glycan biosynthetic
genes that otherwise might be missed through genetic analyses. In this paper the major glycoprotein N- and O-glycans of Drosophila embryos are described as part of our initial undertaking to characterize the glycome of Drosophila melanogaster. The N-glycans are dominated by high mannose and paucimannose structures. Minor amounts of mono-, bi- and tri-antennary complex
glycans were observed with GlcNAc and Galβ1–4GlcNAc non-reducing end termini. O-glycans were restricted to the mucin-type
core 1 Galβ1-3GalNAc sequence. 相似文献
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Chris Spahr Justin J. Kim Sihong Deng Paul Kodama Zhen Xia Jay Tang Richard Zhang Sophia Siu Noi Nuanmanee Bram Estes Jennitte Stevens Mingyue Zhou Hsieng S. Lu 《Protein science : a publication of the Protein Society》2013,22(12):1739-1753
Recombinant human lecithin‐cholesterol acyltransferase Fc fusion (huLCAT‐Fc) is a chimeric protein produced by fusing human Fc to the C‐terminus of the human enzyme via a linker sequence. The huLCAT‐Fc homodimer contains five N‐linked glycosylation sites per monomer. The heterogeneity and site‐specific distribution of the various glycans were examined using enzymatic digestion and LC‐MS/MS, followed by automatic processing. Almost all of the N‐linked glycans in human LCAT are fucosylated and sialylated. The predominant LCAT N‐linked glycoforms are biantennary glycans, followed by triantennary sugars, whereas the level of tetraantennary glycans is much lower. Glycans at the Fc N‐linked site exclusively contain typical asialobiantennary structures. HuLCAT‐Fc was also confirmed to have mucin‐type glycans attached at T407 and S409. When LCAT‐Fc fusions were constructed using a G‐S‐G‐G‐G‐G linker, an unexpected +632 Da xylose‐based glycosaminoglycan (GAG) tetrasaccharide core of Xyl‐Gal‐Gal‐GlcA was attached to S418. Several minor intermediate species including Xyl, Xyl‐Gal, Xyl‐Gal‐Gal, and a phosphorylated GAG core were also present. The mucin‐type O‐linked glycans can be effectively released by sialidase and O‐glycanase; however, the GAG could only be removed and localized using chemical alkaline β‐elimination and targeted LC‐MS/MS. E416 (the C‐terminus of LCAT) combined with the linker sequence is likely serving as a substrate for peptide O‐xylosyltransferase. HuLCAT‐Fc shares some homology with the proposed consensus site near the linker sequence, in particular, the residues underlined PPP E416GS418G G G GDK. GAG incorporation can be eliminated through engineering by shifting the linker Ser residue downstream in the linker sequence. 相似文献
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In this work we describe the production of site-specific biotinylated human myeloid differentiation factor 88 (MyD88). A vector
containing a coding sequence for a peptide derived from the carboxyl terminus of the Klebsiella pneumoniae oxalacetate decarboxylase α subunit was used to allow expression and biotinylation of MyD88 in Drosophila melanogaster Schneider 2 cell cytoplasm. As estimated by a comparison of Schneider 2 lysate with standard protein, the maximum expression
level was 1.3 μg 107 cells−1. About 4 mg of biotinylated protein was purified by affinity chromatography on monomeric avidin from a I-L culture. Exogenous
biotin added to the culture medium increased the biotinylation efficiency of the expressed protein. Biotinylated MyD88 produced
in Drosophila cells was able to precipitate recombinant MyD88 expressed in human embryonic kidney cells. The stable expression of MyD88
in Drosophila Schneider 2 cells offers a convenient and attractive method for large-scale production, which may be required to clarify
the role of MyD88 in the inflammatory response. Moreover, site-specific biotinylation of MyD88 provides a useful tag for interaction
assays where high sensitivity is required. 相似文献
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Tim Smilda Anne H. Kamminga Peter Reinders Wia Baron Johan E.T. van Hylckama Vlieg Jaap J. Beintema 《Journal of molecular evolution》2001,52(5):457-466
Enzymic and structural studies on Drosophila alcohol dehydrogenases and other short-chain dehydrogenases/reductases (SDRs) are presented. Like alcohol dehydrogenases
from other Drosophila species, the enzyme from D. simulans is more active on secondary than on primary alcohols, although ethanol is its only known physiological substrate. Several
secondary alcohols were used to determine the kinetic parameters kcat and Km. The results of these experiments indicate that the substrate-binding region of the enzyme allows optimal binding of a short
ethyl side-chain in a small binding pocket, and of a propyl or butyl side-chain in large binding pocket, with stereospecificity
for R(−) alcohols. At a high concentration of R(−) alcohols substrate activation occurs. The kcat and Km values determined under these conditions are about two-fold, and two orders of magnitude, respectively, higher than those
at low substrate concentrations.
Sequence alignment of several SDRs of known, and unknown three-dimensional structures, indicate the presence of several conserved
residues in addition to those involved in the catalyzed reactions. Structural roles of these conserved residues could be derived
from observations made on superpositioned structures of several SDRs with known structures. Several residues are conserved
in tetrameric SDRs, but not in dimeric ones. Two halohydrin-halide-lyases show significant homology with SDRs in the catalytic
domains of these enzymes, but they do not have the structural features required for binding NAD+. Probably these lyases descend from an SDR, which has lost the capability to bind NAD+, but the enzyme reaction mechanisms may still be similar.
Received: 23 May 2000 / Accepted: 4 January 2001 相似文献