scholarly journals Biochemical and Functional Characterization of GALT8, an Arabidopsis GT31 β-(1,3)-Galactosyltransferase That Influences Seedling Development

2021 ◽  
Vol 12 ◽  
Author(s):  
Joan Oñate Narciso ◽  
Wei Zeng ◽  
Kris Ford ◽  
Edwin R. Lampugnani ◽  
John Humphries ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Functional Characterization ◽  
Endosperm Development ◽  
Phenotypic Analysis ◽  
Catalytic Function ◽  
Liquid Chromatography Tandem Mass ◽  
Micropylar Endosperm ◽  
Mutant Phenotypes ◽  
Almost All

Arabinogalactan-proteins (AGPs) are members of the hydroxyproline-rich glycoprotein (HRGP) superfamily, a group of highly diverse proteoglycans that are present in the cell wall, plasma membrane as well as secretions of almost all plants, with important roles in many developmental processes. The role of GALT8 (At1g22015), a Glycosyltransferase-31 (GT31) family member of the Carbohydrate-Active Enzyme database (CAZy), was examined by biochemical characterization and phenotypic analysis of a galt8 mutant line. To characterize its catalytic function, GALT8 was heterologously expressed in tobacco leaves and its enzymatic activity tested. GALT8 was shown to be a β-(1,3)-galactosyltransferase (GalT) that catalyzes the synthesis of a β-(1,3)-galactan, similar to the in vitro activity of KNS4/UPEX1 (At1g33430), a homologous GT31 member previously shown to have this activity. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) confirmed the products were of 2-6 degree of polymerisation (DP). Previous reporter studies showed that GALT8 is expressed in the central and synergid cells, from whence the micropylar endosperm originates after the fertilization of the central cell of the ovule. Homozygous mutants have multiple seedling phenotypes including significantly shorter hypocotyls and smaller leaf area compared to wild type (WT) that are attributable to defects in female gametophyte and/or endosperm development. KNS4/UPEX1 was shown to partially complement the galt8 mutant phenotypes in genetic complementation assays suggesting a similar but not identical role compared to GALT8 in β-(1,3)-galactan biosynthesis. Taken together, these data add further evidence of the important roles GT31 β-(1,3)-GalTs play in elaborating type II AGs that decorate AGPs and pectins, thereby imparting functional consequences on plant growth and development.

scholarly journals Functional characterization of canine wild type glucocorticoid receptor and an insertional mutation in a dog

2019 ◽  
Vol 15 (1) ◽  
Author(s):  
Kosei Yamanaka ◽  
Masaru Okuda ◽  
Takuya Mizuno
Keyword(s):  
Glucocorticoid Receptor ◽  
Cushing Syndrome ◽  
Functional Characterization ◽  
Wild Type ◽  
Key Factors ◽  
Truncated Form ◽  
Glucocorticoid Sensitivity ◽  
Almost All

Abstract Background Glucocorticoids, among the most widely utilized drugs in veterinary medicine, are employed to treat a wide variety of diseases; however, their use often induces adverse events in dogs. The efficacy of glucocorticoids usually depends on dosage, although differences in sensitivity to glucocorticoids in individual animals have been reported. Glucocorticoids bind to the cytoplasmic glucocorticoid receptor (GR), which is expressed in almost all cells. These receptors are key factors in determining individual sensitivity to glucocorticoids. This study examined individual differences in glucocorticoid sensitivity in dogs, focusing on reactivity of the GR to prednisolone. Results We first molecularly cloned the GR gene from a healthy dog. We discovered a mutant GR in a dog suspected to have iatrogenic Cushing syndrome. The mutant GR had extra nucleotides between exons 6 and 7, resulting in a truncated form of GR that was 98 amino acids shorter than the wild-type dog GR. The truncated GR exhibited very low reactivity to prednisolone, irrespective of concentration. Conclusions We have identified the truncated form of canine GR in a dog with iatrogenic Cushing syndrome. This truncated form showed the very less sensitivity to glucocorticoid in vitro, unfortunately, we could not elucidate its clinical significance. However, our data is a first report about the function of canine GR, and will facilitate the analysis of canine glucocorticoid sensitivity.

scholarly journals Cell Biological and Functional Characterization of the Vaccinia Virus F10 Kinase: Implications for the Mechanism of Virion Morphogenesis

2005 ◽  
Vol 79 (4) ◽  
pp. 2171-2190 ◽  
Author(s):  
Almira Punjabi ◽  
Paula Traktman
Keyword(s):  
Enzymatic Activity ◽  
Vaccinia Virus ◽  
Catalytic Domain ◽  
De Novo ◽  
Functional Characterization ◽  
Site Formation ◽  
Phenotypic Analysis ◽  
Virion Morphogenesis

ABSTRACT The vaccinia virus F10 protein is one of two virally encoded protein kinases. A phenotypic analysis of infections involving a tetracycline-inducible recombinant (vΔiF10) indicated that F10 is involved in the early stages of virion morphogenesis, as previously reported for the mutants ts28 and ts15. The proteins encoded by ts28 and ts15 have primary defects in enzymatic activity and thermostability, respectively. Using a transient complementation assay, we demonstrated that the enzymatic activity of F10 is essential for its biological function and that both its enzymatic and biological functions depend upon N-terminal sequences that precede the catalytic domain. An execution point analysis indicated that in addition to its role at the onset of morphogenesis, F10 is also required at later stages, when membrane crescents surround virosomal contents and develop into immature virions. The F10 protein is phosphorylated in vivo, appears to be tightly associated with intracellular membranes, and can bind to specific phosphoinositides in vitro. When F10 is repressed or impaired, the phosphorylation of several cellular and viral proteins appears to increase in intensity, suggesting that F10 may normally intersect with cellular signaling cascades via the activation of a phosphatase or the inhibition of another kinase. These cascades may drive the F10-induced remodeling of membranes that accompanies virion biogenesis. Upon the release of ts28-infected cultures from a 40°C-induced block, a synchronous resumption of morphogenesis that culminates in the production of infectious virus can be observed. The pharmacological agents H89 and cerulenin, which are inhibitors of endoplasmic reticulum exit site formation and de novo lipid synthesis, respectively, block this recovery.

scholarly journals Indole Biodegradation in Acinetobacter sp. Strain O153: Genetic and Biochemical Characterization

2017 ◽  
Vol 83 (19) ◽  
Author(s):  
Mikas Sadauskas ◽  
Justas Vaitekūnas ◽  
Renata Gasparavičiūtė ◽  
Rolandas Meškys
Keyword(s):  
Biochemical Characterization ◽  
Functional Characterization ◽  
Future Research ◽  
Signal Molecule ◽  
Major Drawback ◽  
Content Type ◽  
Enzymatic Function ◽  
Wide Range ◽  
Microbial Biodegradation

ABSTRACT Indole is a molecule of considerable biochemical significance, acting as both an interspecies signal molecule and a building block of biological elements. Bacterial indole degradation has been demonstrated for a number of cases; however, very little is known about genes and proteins involved in this process. This study reports the cloning and initial functional characterization of genes (iif and ant cluster) responsible for indole biodegradation in Acinetobacter sp. strain O153. The catabolic cascade was reconstituted in vitro with recombinant proteins, and each protein was assigned an enzymatic function. Degradation starts with oxidation, mediated by the IifC and IifD flavin-dependent two-component oxygenase system. Formation of indigo is prevented by IifB, and the final product, anthranilic acid, is formed by IifA, an enzyme which is both structurally and functionally comparable to cofactor-independent oxygenases. Moreover, the iif cluster was identified in the genomes of a wide range of bacteria, suggesting the potential of widespread Iif-mediated indole degradation. This work provides novel insights into the genetic background of microbial indole biodegradation. IMPORTANCE The key finding of this research is identification of the genes responsible for microbial biodegradation of indole, a toxic N-heterocyclic compound. A large amount of indole is present in urban wastewater and sewage sludge, creating a demand for an efficient and eco-friendly means to eliminate this pollutant. A common strategy of oxidizing indole to indigo has the major drawback of producing insoluble material. Genes and proteins of Acinetobacter sp. strain O153 (DSM 103907) reported here pave the way for effective and indigo-free indole removal. In addition, this work suggests possible novel means of indole-mediated bacterial interactions and provides the basis for future research on indole metabolism.

scholarly journals Characterization of murine amniotic fluid B cells in normal pregnancy and in preterm birth

Reproduction ◽  
2019 ◽  
Vol 158 (4) ◽  
pp. 369-376
Author(s):  
Imke Bommer ◽  
Lorena Juriol ◽  
Damián Muzzio ◽  
Natalin Valeff ◽  
Jens Ehrhardt ◽  
...  
Keyword(s):  
Preterm Birth ◽  
B Cells ◽  
Amniotic Fluid ◽  
Functional Characterization ◽  
Normal Pregnancy ◽  
Immune Defense ◽  
Limited Information ◽  
Phenotypic Analysis

The amniotic fluid provides mechanical protection and immune defense against pathogens to the fetus. Indeed, components of the innate and adaptive immunity, including B cells, have been described in the amniotic fluid. However, limited information concerning phenotype and functionality of amniotic fluid B cells is available. Hence, we aimed to perform a full phenotypical and functional characterization of amniotic fluid B cells in normal pregnancy and in a mouse model of preterm birth. Phenotypic analysis depicted the presence of two populations of amniotic fluid B cells: an immature population, resembling B1 progenitor cells and a more mature population. Further isolation and in vitro co-culture with a bone marrow stroma cell line demonstrated the capacity of the immature B cells to mature. This was further supported by spontaneous production of IgM, a feature of the B1 B cell sub-population. An additional in vitro stimulation with lipopolysaccharide induced the activation of amniotic fluid B cells as well as the production of pro and anti-inflammatory cytokines. Furthermore, amniotic fluid B cells were expanded in the acute phase of LPS-induced preterm birth. Overall our data add new insight not only on the phenotype and developmental stage of the amniotic fluid B1 B cells but especially on their functionality. This provides important information for a better understanding of their role within the amniotic fluid as immunological protective barrier, especially with regard to intraamniotic infection and preterm birth.

Biochemical and functional characterization of the human tissue kallikrein 9

2017 ◽  
Vol 474 (14) ◽  
pp. 2417-2433 ◽  
Author(s):  
Panagiota S. Filippou ◽  
Sofia Farkona ◽  
Davor Brinc ◽  
Yijing Yu ◽  
Ioannis Prassas ◽  
...  
Keyword(s):  
Human Tissue ◽  
Biochemical Characterization ◽  
Functional Characterization ◽  
Squamous Carcinoma ◽  
Biochemical Properties ◽  
Tissue Kallikrein ◽  
Related Family ◽  
Squamous Carcinoma Cells

Human tissue kallikrein 9 (KLK9) is a member of the kallikrein-related family of proteases. Despite its known expression profile, much less is known about the functional roles of this protease and its implications in normal physiology and disease. We present here the first data on the biochemical characterization of KLK9, investigate parameters that affect its enzymatic activity (such as inhibitors) and provide preliminary insights into its putative substrates. We show that mature KLK9 is a glycosylated chymotrypsin-like enzyme with strong preference for tyrosine over phenylalanine at the P1 cleavage position. The enzyme activity is enhanced by Mg2+ and Ca2+, but is reversibly attenuated by Zn2+. KLK9 is inhibited in vitro by many naturally occurring or synthetic protease inhibitors. Using a combination of degradomic and substrate specificity assays, we identified candidate KLK9 substrates in two different epithelial cell lines [the non-tumorigenic human keratinocyte cells (HaCaT) and the tumorigenic tongue squamous carcinoma cells (SCC9)]. Two potential KLK9 substrates [KLK10 and midkine (MDK)] were subjected to further validation. Taken together, our data delineate some functional and biochemical properties of KLK9 for future elucidation of the role of this enzyme in health and disease.

scholarly journals Structural and biochemical characterization of human orphan DHRS10 reveals a novel cytosolic enzyme with steroid dehydrogenase activity

2007 ◽  
Vol 402 (3) ◽  
pp. 419-427 ◽  
Author(s):  
Petra Lukacik ◽  
Brigitte Keller ◽  
Gabor Bunkoczi ◽  
Kathryn Kavanagh ◽  
Wen Hwa Lee ◽  
...  
Keyword(s):  
Dehydrogenase Activity ◽  
Active Site ◽  
Biochemical Characterization ◽  
Functional Characterization ◽  
Significant Proportion ◽  
Tissue Expression ◽  
Intact Cells ◽  
Variable Loops ◽  
The Central Nervous System

To this day, a significant proportion of the human genome remains devoid of functional characterization. In this study, we present evidence that the previously functionally uncharacterized product of the human DHRS10 gene is endowed with 17β-HSD (17β-hydroxysteroid dehydrogenase) activity. 17β-HSD enzymes are primarily involved in the metabolism of steroids at the C-17 position and also of other substrates such as fatty acids, prostaglandins and xenobiotics. In vitro, DHRS10 converts NAD+ into NADH in the presence of oestradiol, testosterone and 5-androstene-3β,17β-diol. Furthermore, the product of oestradiol oxidation, oestrone, was identified in intact cells transfected with a construct plasmid encoding the DHRS10 protein. In situ fluorescence hybridization studies have revealed the cytoplasmic localization of DHRS10. Along with tissue expression data, this suggests a role for DHRS10 in the local inactivation of steroids in the central nervous system and placenta. The crystal structure of the DHRS10 apoenzyme exhibits secondary structure of the SDR (short-chain dehydrogenase/reductase) family: a Rossmann-fold with variable loops surrounding the active site. It also reveals a broad and deep active site cleft into which NAD+ and oestradiol can be docked in a catalytically competent orientation.

scholarly journals Functional Characterization of the Initiation Enzyme of S-Layer Glycoprotein Glycan Biosynthesis in Geobacillus stearothermophilus NRS 2004/3a

2007 ◽  
Vol 189 (7) ◽  
pp. 2590-2598 ◽  
Author(s):  
Kerstin Steiner ◽  
René Novotny ◽  
Kinnari Patel ◽  
Evgenij Vinogradov ◽  
Chris Whitfield ◽  
...  
Keyword(s):  
Salmonella Enterica ◽  
Biochemical Characterization ◽  
Functional Characterization ◽  
Geobacillus Stearothermophilus ◽  
Polysaccharide Biosynthesis ◽  
Glycan Chain ◽  
Galactose Residue ◽  
Serovar Typhimurium

ABSTRACTThe glycan chain of the S-layer glycoprotein ofGeobacillus stearothermophilusNRS 2004/3a is composed of repeating units [→2)-α-l-Rhap-(1→3)-β-l-Rhap-(1→2)-α-l-Rhap-(1→], with a 2-O-methyl modification of the terminal trisaccharide at the nonreducing end of the glycan chain, a core saccharide composed of two or three α-l-rhamnose residues, and a β-d-galactose residue as a linker to the S-layer protein. In this study, we report the biochemical characterization of WsaP of the S-layer glycosylation gene cluster as a UDP-Gal:phosphoryl-polyprenol Gal-1-phosphate transferase that primes the S-layer glycoprotein glycan biosynthesis ofGeobacillus stearothermophilusNRS 2004/3a. Our results demonstrate that the enzyme transfers in vitro a galactose-1-phosphate from UDP-galactose to endogenous phosphoryl-polyprenol and that the C-terminal half of WsaP carries the galactosyltransferase function, as already observed for the UDP-Gal:phosphoryl-polyprenol Gal-1-phosphate transferase WbaP fromSalmonella enterica. To confirm the function of the enzyme, we show that WsaP is capable of reconstituting polysaccharide biosynthesis in WbaP-deficient strains ofEscherichia coliandSalmonella entericaserovar Typhimurium.

The in vivo distribution and dynamics of DNA topoisomerase II in Drosophila embryonic nuclei and chromosomes

1993 ◽  
Vol 51 ◽  
pp. 74-75
Author(s):  
Jason R. Swedlow ◽  
Neil Osheroff ◽  
Tim Karr ◽  
John W. Sedat ◽  
David A. Agard
Keyword(s):  
Topoisomerase Ii ◽  
Biochemical Characterization ◽  
Developmental Cycle ◽  
Dna Topoisomerase Ii ◽  
Chromosomal Distribution ◽  
Dna Topoisomerase ◽  
Ideal System ◽  
Dnase Treatment

DNA topoisomerase II is an ATP-dependent double-stranded DNA strand-passing enzyme that is necessary for full condensation of chromosomes and for complete segregation of sister chromatids at mitosis in vivo and in vitro. Biochemical characterization of chromosomes or nuclei after extraction with high-salt or detergents and DNAse treatment showed that topoisomerase II was a major component of this remnant, termed the chromosome scaffold. The scaffold has been hypothesized to be the structural backbone of the chromosome, so the localization of topoisomerase II to die scaffold suggested that the enzyme might play a structural role in the chromosome. However, topoisomerase II has not been studied in nuclei or chromosomes in vivo. We have monitored the chromosomal distribution of topoisomerase II in vivo during mitosis in the Drosophila embryo. This embryo forms a multi-nucleated syncytial blastoderm early in its developmental cycle. During this time, the embryonic nuclei synchronously progress through 13 mitotic cycles, so this is an ideal system to follow nuclear and chromosomal dynamics.

Insights into the biochemical and functional characterization of sortase E transpeptidase of Corynebacterium glutamicum

2019 ◽  
Vol 476 (24) ◽  
pp. 3835-3847 ◽  
Author(s):  
Aliyath Susmitha ◽  
Kesavan Madhavan Nampoothiri ◽  
Harsha Bajaj
Keyword(s):  
Protein Engineering ◽  
Cell Attachment ◽  
Functional Characterization ◽  
Protein Structures ◽  
Structural Similarity ◽  
Surface Proteins ◽  
Sortase A ◽  
Peptide Cleavage ◽  
New Findings

Most Gram-positive bacteria contain a membrane-bound transpeptidase known as sortase which covalently incorporates the surface proteins on to the cell wall. The sortase-displayed protein structures are involved in cell attachment, nutrient uptake and aerial hyphae formation. Among the six classes of sortase (A–F), sortase A of S. aureus is the well-characterized housekeeping enzyme considered as an ideal drug target and a valuable biochemical reagent for protein engineering. Similar to SrtA, class E sortase in GC rich bacteria plays a housekeeping role which is not studied extensively. However, C. glutamicum ATCC 13032, an industrially important organism known for amino acid production, carries a single putative sortase (NCgl2838) gene but neither in vitro peptide cleavage activity nor biochemical characterizations have been investigated. Here, we identified that the gene is having a sortase activity and analyzed its structural similarity with Cd-SrtF. The purified enzyme showed a greater affinity toward LAXTG substrate with a calculated KM of 12 ± 1 µM, one of the highest affinities reported for this class of enzyme. Moreover, site-directed mutation studies were carried to ascertain the structure functional relationship of Cg-SrtE and all these are new findings which will enable us to perceive exciting protein engineering applications with this class of enzyme from a non-pathogenic microbe.

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