scholarly journals Human carboxypeptidase E. Isolation and characterization of the cDNA, sequence conservation, expression and processing in vitro

1990 ◽  
Vol 267 (2) ◽  
pp. 517-525 ◽  
Author(s):  
E Manser ◽  
D Fernandez ◽  
L Loo ◽  
P Y Goh ◽  
C Monfries ◽  
...  
Keyword(s):  
Amino Acid ◽  
Membrane Protein ◽  
Sequence Similarity ◽  
Coding Region ◽  
Sequence Comparisons ◽  
Brain Membrane ◽  
Carboxypeptidase E ◽  
Isolation And Characterization ◽  
Microsomal Membranes

Carboxypeptidase E (CPE), which cleaves C-terminal amino acid residues and is involved in neuropeptide processing, is itself subject to intracellular processing. Human CPE cDNA was isolated and sequence comparisons were made with those of a previously isolated brain cDNA (M1622) encoding rat CPE and of other human carboxypeptidases (M and N). Human (2.5 kb) and rat (2.1 kb) CPE cDNAs approximated to the size of their respective mRNAs; additional sequences were located in putative 5′ and 3′ untranslated regions of human CPE mRNA. There is 79% sequence similarity between human and rat CPE cDNAs, with greater similarity (89%) over the coding region and short sections of the non-coding sequence. The predicted 476-amino acid-residue sequences of human and rat preproCPEs are highly conserved (96% identity), with lower degree of similarity of the N-terminal signal peptide (76%). Human CPE showed 51% and 43% sequence similarity to human CPN and CPM respectively, with discrete regions of divergence dispersed between the highly conserved mechanistically implicated regions. Antiserum generated from a fusion protein, synthesized in Escherichia coli from constructs of the human cDNA, recognized an approx. 50 kDa membrane protein and a smaller soluble protein in rat and human brain preparations, corresponding to the two forms of native CPE. Human CPE mRNA transcripts directed the synthesis in reticulocyte lysate of a 54 kDa translation product, which in the presence of dog pancreas microsomal membranes was co-translationally processed with cleavage, insertion into membranes and glycosylation. Three processed forms were generated, the largest (56 kDa) and smallest (52 kDa) being equally glycosylated. The membrane association of the processed translation products and of native brain membrane CPE, detected immunologically, was resistant to moderate alkali but not pH 11.5 extraction. These results are consistent with secondary-structure predictions that CPE is a peripheral membrane protein. The dissimilar regions of human carboxypeptidases may provide information on sequences responsible for their different cellular disposition.

Advancing RNA Virus Discovery and Biology with Whole Genome Sequencing

2021 ◽  
Author(s):  
◽  
Mariah Taylor ◽  
Keyword(s):  
Genetic Variation ◽  
Amino Acid ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Rna Virus ◽  
Animal Health ◽  
Functional Domains ◽  
Whole Genome ◽  
Coding Region

Two RNA virus families that pose a threat to human and animal health are Hantaviridae and Coronaviridae. These RNA viruses which originate in wildlife continue and will continue to cause disease, and hence, it is critical that scientific research define the mechanisms as to how these viruses spillover and adapt to new hosts to become endemic. One gap in our ability to define these mechanisms is the lack of whole genome sequences for many of these viruses. To address this specific gap, I developed a versatile amplicon-based whole-genome sequencing (WGS) approach to identify viral genomes of hantaviruses and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) within reservoir and spillover hosts. In my research studies, I used the amplicon-based WGS approach to define the genetic plasticity of viral RNA within pathogenic and nonpathogenic hantavirus species. The standing genetic variation of Andes orthohantavirus and Prospect Hill orthohantavirus was mapped out and amino acid changes occurring outside of functional domains were identified within the nucleocapsid and glycoprotein. I observed several amino acid changes in functional domains of the RNA-dependent RNA polymerase, as well as single nucleotide polymorphisms (SNPs) within the 3’ non-coding region (NCR) of the S-segment. To identify whether virus adaptation would occur within the S- and L-segments we attempted to adapt hantaviruses in vitro in a spillover host model through passaging experiments. In early passages we identified few mutations in the M-segment with the majority being identified in the S-segment 3’ NCR and the L-segment. This work suggests that hantavirus adaptation occurs in the S- and L-segments although the effect of these mutants on pathology is yet to be determined. While sequencing laboratory isolates is easily accomplished, sequencing low concentrations of virus within the reservoir is a formidable task. I further translated our amplicon-based WGS approach into a pan-oligonucleotide amplicon-based WGS approach to sequence hantavirus vRNA and mRNA from reservoir and spillover hosts in Ukraine. This approach successfully identified a novel Puumala orthohantavirus (PUUV) strain in Ukraine and using Bayesian phylogenetics we found this strain to be associated with the PUUV Latvian lineage. Early during the SARS-CoV-2 pandemic, I applied the knowledge gained in the hantavirus WGS efforts to sequencing of SARS-CoV-2 from nasopharyngeal swabs collected in April 2020. The genetic diversity of 45 SARS-CoV-2 isolates was evaluated with the methods I developed. We identified D614G, a notable mutation known for increasing transmission, in over 90% of our isolates. Two major lineages distinguish SARS-CoV-2 variants worldwide, lineages A and B. While most of our isolates were found within B lineage, we also identified one isolate within lineage A. We performed in vitro work which confirmed A lineage isolates as having poor replication in the trachea as compared to the nasal cavity. Five of these isolates presented a unique array of mutations which were assessed in the keratin 18 human angiotensin-converting enzyme 2 (K18-hACE2) mouse model for its response immunologically and pathogenically. We identified a distinction of pathogenesis between the A and B lineages with emphysema being common amongst A lineage isolates. Additionally, we discovered a small cohort of likely SNPs that defined the late induction of eosinophils during infection. In summary, this work will further define the dynamics of genetic variation and plasticity within virus populations that cause disease outbreaks and will allow a deeper understanding of the virus-host relationship.

scholarly journals Genetic Characterization of Avian Influenza A (H11N9) Virus Isolated from Mandarin Ducks in South Korea in 2018

Viruses ◽  
2020 ◽  
Vol 12 (2) ◽  
pp. 203
Author(s):  
Hien Thi Tuong ◽  
Ngoc Minh Nguyen ◽  
Haan Woo Sung ◽  
Hyun Park ◽  
Seon-Ju Yeo
Keyword(s):  
Influenza Virus ◽  
Amino Acid ◽  
Avian Influenza ◽  
South Korea ◽  
Avian Influenza Virus ◽  
Influenza A ◽  
Sequence Similarity ◽  
Basic Amino Acid ◽  
Wild Birds

In July 2018, a novel avian influenza virus (A/Mandarin duck/South Korea/KNU18-12/2018(H11N9)) was isolated from Mandarin ducks in South Korea. Phylogenetic and molecular analyses were conducted to characterize the genetic origins of the H11N9 strain. Phylogenetic analysis indicated that eight gene segments of strain H11N9 belonged to the Eurasian lineages. Analysis of nucleotide sequence similarity of both the hemagglutinin (HA) and neuraminidase (NA) genes revealed the highest homology with A/duck/Kagoshima/KU57/2014 (H11N9), showing 97.70% and 98.00% nucleotide identities, respectively. Additionally, internal genes showed homology higher than 98% compared to those of other isolates derived from duck and wild birds. Both the polymerase acidic (PA) and polymerase basic 1 (PB1) genes were close to the H5N3 strain isolated in China; whereas, other internal genes were closely related to that of avian influenza virus in Japan. A single basic amino acid at the HA cleavage site (PAIASR↓GLF), the lack of a five-amino acid deletion (residue 69–73) in the stalk region of the NA gene, and E627 in the polymerase basic 2 (PB2) gene indicated that the A/Mandarin duck/South Korea/KNU18-12/2018(H11N9) isolate was a typical low-pathogenicity avian influenza. In vitro viral replication of H11N9 showed a lower titer than H1N1 and higher than H9N2. In mice, H11N9 showed lower adaptation than H1N1. The novel A/Mandarin duck/South Korea/KNU18-12/2018(H11N9) isolate may have resulted from an unknown reassortment through the import of multiple wild birds in Japan and Korea in approximately 2016–2017, evolving to produce a different H11N9 compared to the previous H11N9 in Korea (2016). Further reassortment events of this virus occurred in PB1 and PA in China-derived strains. These results indicate that Japanese- and Chinese-derived avian influenza contributes to the genetic diversity of A/Mandarin duck/South Korea/KNU18-12/2018(H11N9) in Korea.

scholarly journals Isolation and Characterization of vicH, Encoding a New Pleiotropic Regulator in Vibrio cholerae

2000 ◽  
Vol 182 (7) ◽  
pp. 2026-2032 ◽  
Author(s):  
Christian Tendeng ◽  
Cyril Badaut ◽  
Evelyne Krin ◽  
Pierre Gounon ◽  
Saravuth Ngo ◽  
...  
Keyword(s):  
Amino Acid ◽  
Vibrio Cholerae ◽  
Genomic Library ◽  
Single Gene ◽  
Wild Type ◽  
Semisolid Medium ◽  
E Coli ◽  
Isolation And Characterization ◽  
Serovar Typhimurium

ABSTRACT During the last decade, the hns gene and its product, the H-NS protein, have been extensively studied in Escherichia coli. H-NS-like proteins seem to be widespread in gram-negative bacteria. However, unlike in E. coli and inSalmonella enterica serovar Typhimurium, little is known about their role in the physiology of those organisms. In this report, we describe the isolation of vicH, an hns-like gene in Vibrio cholerae, the etiological agent of cholera. This gene was isolated from a V. cholerae genomic library by complementation of different phenotypes associated with anhns mutation in E. coli. It encodes a 135-amino-acid protein showing approximately 50% identity with both H-NS and StpA in E. coli. Despite a low amino acid conservation in the N-terminal part, VicH is able to cross-react with anti-H-NS antibodies and to form oligomers in vitro. ThevicH gene is expressed as a single gene from two promoters in tandem and is induced by cold shock. A V. choleraewild-type strain expressing a vicHΔ92 gene lacking its 3′ end shows pleiotropic alterations with regard to mucoidy and salicin metabolism. Moreover, this strain is unable to swarm on semisolid medium. Similarly, overexpression of the vicH wild-type gene results in an alteration of swarming behavior. This suggests that VicH could be involved in the virulence process in V. cholerae, in particular by affecting flagellum biosynthesis.

Isolation and characterization of the pea cytochrome c oxidase Vb gene

Genome ◽  
2006 ◽  
Vol 49 (11) ◽  
pp. 1481-1489 ◽  
Author(s):  
Nakao Kubo ◽  
Shin-ichi Arimura ◽  
Nobuhiro Tsutsumi ◽  
Koh-ichi Kadowaki ◽  
Masashi Hirai
Keyword(s):  
Amino Acid ◽  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Fluorescent Proteins ◽  
Amino Acid Sequences ◽  
Homology Search ◽  
Coding Region ◽  
Angiosperm Evolution ◽  
Isolation And Characterization ◽  
Duplication Events

Three copies of the gene that encodes cytochrome c oxidase subunit Vb were isolated from the pea (PscoxVb-1, PscoxVb-2, and PscoxVb-3). Northern Blot and reverse transcriptase-PCR analyses suggest that all 3 genes are transcribed in the pea. Each pea coxVb gene has an N-terminal extended sequence that can encode a mitochondrial targeting signal, called a presequence. The localization of green fluorescent proteins fused with the presequence strongly suggests the targeting of pea COXVb proteins to mitochondria. Each pea coxVb gene has 5 intron sites within the coding region. These are similar to Arabidopsis and rice, although the intron lengths vary greatly. A phylogenetic analysis of coxVb suggests the occurrence of gene duplication events during angiosperm evolution. In particular, 2 duplication events might have occurred in legumes, grasses, and Solanaceae. A comparison of amino acid sequences in COXVb or its counterpart shows the conservation of several amino acids within a zinc finger motif. Interestingly, a homology search analysis showed that bacterial protein COG4391 and a mitochondrial complex I 13 kDa subunit also have similar amino acid compositions around this motif. Such similarity might reflect evolutionary relationships among the 3 proteins.

scholarly journals CONTROLLED PROTEOLYSIS OF NASCENT POLYPEPTIDES IN RAT LIVER CELL FRACTIONS

1970 ◽  
Vol 45 (1) ◽  
pp. 146-157 ◽  
Author(s):  
D. D. Sabatini ◽  
G. Blobel
Keyword(s):  
Amino Acid ◽  
Messenger Rna ◽  
Close Association ◽  
Membrane Integrity ◽  
Sucrose Density Gradient ◽  
Electron Microscopic ◽  
Microsomal Membranes ◽  
Membrane Bound ◽  
Cell Fractions

Rough microsomes were incubated in an in vitro amino acid-incorporating system for labeling the nascent polypeptide chains on the membrane-bound ribosomes. Sucrose density gradient analysis showed that ribosomes did not detach from the membranes during incorporation in vitro. Trypsin and chymotrypsin treatment of microsomes at 0° led to the detachment of ribosomes from the membranes; furthermore, trypsin produced the dissociation of released, messenger RNA-free ribosomes into subunits. Electron microscopic observations indicated that the membranes remained as closed vesicles. In contrast to the situation with free polysomes, nascent chains contained in rough microsomes were extensively protected from proteolytic attach. By separating the microsomal membranes from the released subunits after proteolysis, it was found that nascent chains are split into two size classes of fragments when the ribosomes are detached. These were shown by column chromatography on Sephadex G-50 to be: (a) small (39 amino acid residues) ribosome-associated fragments and (b) a mixture of larger membrane-associated fragments excluded from the column. The small fragments correspond to the carboxy-terminal segments which are protected by the large subunits of free polysomes. The larger fragments associated with the microsomal membranes depend for their protection on membrane integrity. These fragments are completely digested if the microsomes are subjected to proteolysis in the presence of detergents. These results indicate that when the nascent polypeptides growing in the large subunits of membrane-bound ribosomes emerge from the ribosomes they enter directly into a close association with the microsomal membrane.

scholarly journals Extracellular-matrix degradation at acid pH. Avian osteoclast acid collagenase isolation and characterization

1993 ◽  
Vol 290 (3) ◽  
pp. 873-884 ◽  
Author(s):  
H C Blair ◽  
S L Teitelbaum ◽  
L E Grosso ◽  
D L Lacey ◽  
H L Tan ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Substrate Specificity ◽  
Cathepsin B ◽  
Type I Collagen ◽  
Sequence Similarity ◽  
Bone Matrix ◽  
Collagenolytic Activity ◽  
Type I ◽  
Isolation And Characterization

Osteoclasts degrade bone matrix, which is mainly type I collagen and hydroxyapatite, in an acidic extracellular compartment. Thus we reasoned that osteoclasts must produce an acid collagenase. We purified this enzyme, a 31 kDa protein, from avian osteoclast lysates (in 100 mM acetate/1 mM CHAPS/1 mM dithiothreitol, pH 4.4), fractionated by (NH2)2SO4 precipitation, gelatin-affinity, cation exchange, and gel filtration. Fraction activity was measured using diazotized collagen or 3H-labelled cross-linked collagen (decalcified and trypsin-treated metabolically L-[4,5-3H]proline-labelled bone) as substrates. Iodoacetate, leupeptin, antipain, pepstatin and mercurials inhibited collagenolysis by the isolated proteinase; mercurial derivatives could not be re-activated by dithiothreitol. Collagen degradation was maximal at pH 4.4; purified proteinase reproduced the collagenolytic activity of cell lysates. The N-terminal amino acid sequence from the isolated protein and its CNBr degradation fragments showed sequence similarity to mammalian cathepsin Bs, and near-identity with avian liver cathepsin B. Peptide substrate specificity of the osteoclastic enzyme resembled those of mammalian cathepsin B and its avian liver counterpart, but degradation of low-molecular-mass substrates by the osteoclastic enzyme was slower, reflecting generally lower kcat. values. Further, kcat/Km varied less between arginine-containing substrates than for previously reported cathepsin Bs, indicating different substrate specificity of the osteoclast enzyme. Polyclonal antibody raised to a 25 kDa fragment of the enzyme recognized a single 31 kDa band in SDS/PAGE of osteoclast lysates blotted to poly(vinylidene difluoride), adsorbed collagenolytic activity of osteoclast lysates, and stained avian osteoclasts in tissue sections. Degenerate sense- and antisense-oligonucleotide primers, predicted from segments of primary amino acid sequence, amplified a 486 bp DNA fragment; this was cloned and sequenced. Of 162 amino acids encoded, 77% are identical with those of human cathepsin B; hybridization identified a 2.4 kb RNA in osteoclast lysates. We conclude that the major avian osteoclast collagenolytic enzyme is a cathepsin B, whose activity varies from other enzymes of its class.

scholarly journals Isolation and characterization of pig enamelins

1987 ◽  
Vol 243 (2) ◽  
pp. 385-390 ◽  
Author(s):  
H Limeback
Keyword(s):  
Amino Acid ◽  
Gel Electrophoresis ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Binding Studies ◽  
Isolation And Characterization ◽  
Sequential Extraction Procedures ◽  
Enamel Proteins

Enamel proteins were extracted from pig developing enamel by sequential extraction procedures. Two proteins identified as enamelins by slab-gel electrophoresis (Mr 67,000 and 63,000) were separated from amelogenins by gel sieving and ion-exchange chromatography. Their enamelin characteristic was confirmed by hydroxyapatite-binding studies and amino acid analysis. Degradation of extracted enamel proteins was also studied in vitro. The larger of the two enamelins appeared to be resistant to degradation by endogenous enamel proteinases. Hydroxyapatite showed strong binding with the enamelins, but did not prevent the degradation of the Mr-63,000 enamelin. These results indicate that at least one high-Mr enamelin in pig developing enamel is a source of enamelin breakdown products.

scholarly journals Amino acid permeases require COPII components and the ER resident membrane protein Shr3p for packaging into transport vesicles in vitro.

1996 ◽  
Vol 135 (3) ◽  
pp. 585-595 ◽  
Author(s):  
M J Kuehn ◽  
R Schekman ◽  
P O Ljungdahl
Keyword(s):  
Plasma Membrane ◽  
Amino Acid ◽  
Membrane Protein ◽  
Integral Membrane Protein ◽  
Plasma Membrane Atpase ◽  
Amino Acid Permease ◽  
Transport Vesicles ◽  
Amino Acid Permeases ◽  
Histidine Permease

In S. cerevisiae lacking SHR3, amino acid permeases specifically accumulate in membranes of the endoplasmic reticulum (ER) and fail to be transported to the plasma membrane. We examined the requirements of transport of the permeases from the ER to the Golgi in vitro. Addition of soluble COPII components (Sec23/24p, Sec13/31p, and Sar1p) to yeast membrane preparations generated vesicles containing the general amino acid permease. Gap1p, and the histidine permease, Hip1p. Shr3p was required for the packaging of Gap1p and Hip1p but was not itself incorporated into transport vesicles. In contrast, the packaging of the plasma membrane ATPase, Pma1p, and the soluble yeast pheromone precursor, glycosylated pro alpha factor, was independent of Shr3p. In addition, we show that integral membrane and soluble cargo colocalize in transport vesicles, indicating that different types of cargo are not segregated at an early step in secretion. Our data suggest that specific ancillary proteins in the ER membrane recruit subsets of integral membrane protein cargo into COPII transport vesicles.

scholarly journals Expression and developmental regulation of two unique mRNAs specific to brain membrane-bound polyribosomes

1987 ◽  
Vol 244 (2) ◽  
pp. 359-366 ◽  
Author(s):  
C Hall ◽  
C M Lowndes ◽  
T K Leung ◽  
D N Cooper ◽  
A M Goate ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Developmental Regulation ◽  
Post Partum ◽  
Single Copy ◽  
Initial Increase ◽  
Neuronal Cultures ◽  
Brain Membrane ◽  
Isolation And Characterization ◽  
Membrane Bound

Translation in vitro of membrane-bound polyribosomal mRNAs from rat brain has shown several to be developmentally regulated [Hall & Lim (1981) Biochem. J. 196, 327-336]. Here we describe the isolation and characterization of cDNAs corresponding to two such brain mRNAs. One cDNA (M444) hybrid-selected a 0.95 kb mRNA directing the synthesis in vitro of a 21 kDa pI-6.3 polypeptide, which was processed in vitro by microsomal membranes. A second cDNA (M1622) hybridized to a 2.2 kb mRNA directing the synthesis of a 55 kDa pI-5.8 polypeptide. Both mRNAs were specific to membrane-bound polyribosomes. Restriction maps of the corresponding genomic DNA sequences are consistent with both being single copy. The two mRNAs were present in astrocytic and neuronal cultures, but not in liver or spleen or in neuroblastoma or glioma cells. The two mRNAs were differently regulated during brain development. In the developing forebrain there was a gradual and sustained increase in M444 mRNA during the first 3 weeks post partum, whereas M1622 mRNA appeared earlier and showed no further increase after day 10. In the cerebellum the developmental increase in M444 mRNA was biphasic. After a small initial increase there was a decrease in this mRNA at day 10, coincident with high amounts of M1622 mRNA. This was followed by a second, larger, increase in M444 mRNA, when amounts of M1622 mRNA were constant. The contrasting changes in these two mRNAs in the developing cerebellum are of particular interest, since they occur during an intensive period of cell proliferation, migration and altering neural connectivity. As these mRNAs are specific to differentiated neural tissue, they represent useful molecular markers for studying brain differentiation.

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