Molecular cloning and biochemical characterization of a new mouse testis soluble-zinc-metallopeptidase of the neprilysin family

2000 ◽  
Vol 347 (2) ◽  
pp. 419-429 ◽  
Author(s):  
Galia GHADDAR ◽  
Andréa Frota RUCHON ◽  
Mélanie CARPENTIER ◽  
Mieczyslaw MARCINKIEWICZ ◽  
Nabil G. SEIDAH ◽  
...  
Keyword(s):  
Amino Acid ◽  
Mammalian Cells ◽  
Biochemical Characterization ◽  
Transmembrane Domain ◽  
Amino Acid Sequences ◽  
Mouse Testis ◽  
Peptidase Activity ◽  
Converting Enzyme ◽  
C Terminus ◽  
The Family

Because of their roles in controlling the activity of several bio-active peptides, members of the neprilysin family of zinc metallopeptidases have been identified as putative targets for the design of therapeutic agents. Presently, six members have been reported, these are: neprilysin, endothelin-converting enzyme (ECE)-1 and ECE-2, the Kell blood group protein, PHEX (product of the phosphate-regulating gene with homologies to endopeptidase on the X chromosome) and X-converting enzyme (XCE). In order to identify new members of this important family of peptidases, we designed a reverse transcriptase-PCR strategy based on conserved amino acid sequences of neprilysin, ECE-1 and PHEX. We now report the cloning from mouse testis of a novel neprilysin-like peptidase that we called NL1. NL1 is a glycoprotein that, among the members of the family, shows the strongest sequence identity with neprilysin. However, in contrast with neprilysin and other members of the family which are type II integral membrane proteins, NL1 was secreted when expressed in cultured mammalian cells, likely due to cleavage by a subtilisin-like convertase at a furin-like site located 22 amino acid residues in the C-terminus of the transmembrane domain. The recombinant enzyme exhibited neprilysin-like peptidase activity and was efficiently inhibited by phosphoramidon and thiorphan, two inhibitors of neprilysin. Northern blot analysis and in situ hybridization showed that NL1 mRNA was found predominantly in testis, specifically in round and elongated spermatids. This distribution of NL1 mRNA suggests that it could be involved in sperm formation or other processes related to fertility.

scholarly journals The CFTR-derived peptides as a model of sequence-specific protein aggregation

2007 ◽  
Vol 12 (3) ◽  
Author(s):  
Daniel Bąk ◽  
Garry Cutting ◽  
Michał Milewski
Keyword(s):  
Amino Acid ◽  
Protein Aggregation ◽  
Mammalian Cells ◽  
Specific Protein ◽  
Amino Acid Sequences ◽  
Model Systems ◽  
Aggregation Pattern ◽  
C Terminus ◽  
Crucial Component ◽  
Modified Peptides

AbstractProtein aggregation is a hallmark of a growing group of pathologies known as conformational diseases. Although many native or mutated proteins are able to form aggregates, the exact amino acid sequences involved in the process of aggregation are known only in a few cases. Hence, there is a need for different model systems to expand our knowledge in this area. The so-called ag region was previously found to cause the aggregation of the C-terminal fragment of the cystic fibrosis transmembrane conductance regulator (CFTR). To investigate whether this specific amino acid sequence is able to induce protein aggregation irrespective of the amino acid context, we altered its position within the CFTR-derived C-terminal peptide and analyzed the localization of such modified peptides in transfected mammalian cells. Insertion of the ag region into a different amino acid background affected not only the overall level of intracellular protein aggregation, but also the morphology and subcellular localization of aggregates, suggesting that sequences other than the ag region can substantially influence the peptide’s behavior. Also, the introduction of a short dipeptide (His-Arg) motif, a crucial component of the ag region, into different locations within the C-terminus of CFTR lead to changes in the aggregation pattern that were less striking, although still statistically significant. Thus, our results indicate that even subtle alterations within the aggregating peptide can affect many different aspects of the aggregation process.

Yak Milk Casein as a Functional Ingredient: Preparation and Identification of Angiotensin-I-Converting Enzyme Inhibitory Peptides

2006 ◽  
Vol 74 (1) ◽  
pp. 18-25 ◽  
Author(s):  
Jingli Jiang ◽  
Shangwu Chen ◽  
Fazheng Ren ◽  
Zhang Luo ◽  
Steve S Zeng
Keyword(s):  
Amino Acid ◽  
Inhibitory Activity ◽  
Value Added ◽  
Amino Acid Sequences ◽  
Food Protein ◽  
Converting Enzyme ◽  
Angiotensin I ◽  
Yak Milk ◽  
Angiotensin I Converting Enzyme ◽  
Milk Casein

Yak milk casein derived from Qula, a traditional Tibetan acid curd cheese, was hydrolyzed by six commercially available proteases (Trypsin, Pepsin, Alcalase, Flavourzyme, Papain and Neutrase). These hydrolysates were assayed for their inhibitory activity of Angiotensin-I-converting enzyme (ACE). The hydrolysates obtained by Neutrase from Bacillus amyloliquefaciens showed the highest ACE inhibitory activity. The IC50 value of Neutrase-hydrolysate was 0·38 mg/ml. The hydrolysate obtained by Neutrase was further separated by consecutive ultra-filtration with 10 kDa and then with 6 kDa molecular weight cut-offs into different permeated parts and fractionated by gel filtration chromatography with a Sephadex G-25 column. The active fraction was subjected to RP-HPLC, in which five peaks were purified and identified. Amino acid sequence analysis confirmed that the peptides and origins were as follows: YQKFPQY (αs2-CN; f89–95), LPQNIPPL (β-CN; f70–77), SKVLPVPQK (β-CN; f168–176), LPYPYY (κ-CN; f56–61) and FLPYPYY (κ-CN; f55–61). Their amino acid sequences matched well with those of known bioactive peptides from bovine casein. The results indicated that yak milk casein could be a resource to generate antihypertensive peptides and be used as multifunctional active ingredients for many value-added functional foods as well as a traditional food protein.

Complete Genome Sequence of a Novel Ourmia-like Mycovirus Infecting the Phytopathogenic Fungus Botryosphaeria Dothidea

2021 ◽  
Author(s):  
Liying Sun ◽  
Ziqian Lian ◽  
Subha Das ◽  
Jingxian Luo ◽  
Ida Bagus Andika
Keyword(s):  
Amino Acid ◽  
Genome Sequence ◽  
Amino Acid Sequences ◽  
Shanxi Province ◽  
Phytopathogenic Fungus ◽  
Botryosphaeria Dothidea ◽  
Reading Frame ◽  
Ring Rot ◽  
Ssrna Virus ◽  
The Family

Abstract In this study, we describe the full-length genome sequence of a novel ourmia-like mycovirus, tentatively designated Botryosphaeria dothidea ourmia-like virus 1 (BdOLV1), isolated from the phytopathogenic fungus, Botryosphaeria dothidea strain P8, associated with apple ring rot in Shanxi province, China. The complete BdOLV1 genome is comprised of 2797 nucleotides, a positive-sense (+) single-stranded RNA (ssRNA) with a single open reading frame (ORF). The ORF putatively encodes a 642-amino acid polypeptide with conserved RNA-dependent RNA polymerase (RdRp) motifs, related to viruses of the family Botourmiaviridae. Phylogenetic analysis based on the RdRp amino acid sequences showed that BdOLV1 is grouped with oomycete-infecting unclassified viruses closely related to the genus Botoulivirus in Botourmiaviridae. This is the first report of a novel (+)ssRNA virus in B. dothidea related to the genus Botoulivirus in the family Botourmiaviridae.

scholarly journals Taxonomic status of Bhanja and Kismayo viruses (family Bunyaviridae)

2012 ◽  
Vol 17 (4) ◽  
pp. 4-8
Author(s):  
A. S Klimentov ◽  
A. P Gmyl ◽  
A. M Butenko ◽  
L. V Gmyl ◽  
O. V Isaeva ◽  
...  
Keyword(s):  
Phylogenetic Analysis ◽  
Amino Acid ◽  
Nucleotide Sequence ◽  
Taxonomic Status ◽  
Amino Acid Sequences ◽  
The Family ◽  
L Segment

The nucleotide sequence of M= (1398 nucleotides and L= (6186 nucleotides) segments of the genome of Bhanja virus and L-segment (1297 nucleotides) of Kismayo virus has been partially determined. Phylogenetic analysis of deduced amino acid sequences showed that these viruses are novel members of the Flebovirus (Phlebovirus) genus in the family Bunyaviridae

scholarly journals The Extracellular Transport Signal of the Vibrio cholerae Endochitinase (ChiA) Is a Structural Motif Located between Amino Acids 75 and 555

2002 ◽  
Vol 184 (8) ◽  
pp. 2225-2234 ◽  
Author(s):  
Jason P. Folster ◽  
Terry D. Connell
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Vibrio Cholerae ◽  
Structural Information ◽  
Amino Acid Sequences ◽  
Structural Motif ◽  
Terminal Branch ◽  
C Terminus ◽  
N Terminus ◽  
Extracellular Transport

ABSTRACT ChiA, an 88-kDa endochitinase encoded by the chiA gene of the gram-negative enteropathogen Vibrio cholerae, is secreted via the eps-encoded main terminal branch of the general secretory pathway (GSP), a mechanism which also transports cholera toxin. To localize the extracellular transport signal of ChiA that initiates transport of the protein through the GSP, a chimera comprised of ChiA fused at the N terminus with the maltose-binding protein (MalE) of Escherichia coli and fused at the C terminus with a 13-amino-acid epitope tag (E-tag) was expressed in strain 569B(chiA::Kanr), a chiA-deficient but secretion-competent mutant of V. cholerae. Fractionation studies revealed that blockage of the natural N terminus and C terminus of ChiA did not prevent secretion of the MalE-ChiA-E-tag chimera. To locate the amino acid sequences which encoded the transport signal, a series of truncations of ChiA were engineered. Secretion of the mutant polypeptides was curtailed only when ChiA was deleted from the N terminus beyond amino acid position 75 or from the C terminus beyond amino acid 555. A mutant ChiA comprised of only those amino acids was secreted by wild-type V. cholerae but not by an epsD mutant, establishing that amino acids 75 to 555 independently harbored sufficient structural information to promote secretion by the GSP of V. cholerae. Cys77 and Cys537, two cysteines located just within the termini of ChiA(75-555), were not required for secretion, indicating that those residues were not essential for maintaining the functional activity of the ChiA extracellular transport signal.

A head-activator binding protein is present in hydra in a soluble and a membrane-anchored form

Development ◽  
1999 ◽  
Vol 126 (18) ◽  
pp. 4077-4086 ◽  
Author(s):  
W. Hampe ◽  
J. Urny ◽  
I. Franke ◽  
S.A. Hoffmeister-Ullerich ◽  
D. Herrmann ◽  
...  
Keyword(s):  
Stem Cells ◽  
Amino Acid ◽  
Transmembrane Domain ◽  
Binding Protein ◽  
Amino Acid Sequences ◽  
Secreted Protein ◽  
Specific Antibodies ◽  
Head Activator ◽  
Carboxy Terminal

The neuropeptide head activator plays an important role for proliferation and determination of stem cells in hydra. By affinity chromatography a 200 kDa head-activator binding protein, HAB, was isolated from the multiheaded mutant of Chlorohydra viridissima. Partial amino acid sequences were used to clone the HAB cDNA which coded for a receptor with a unique alignment of extracellular modules, a transmembrane domain, and a short carboxy-terminal cytoplasmic tail. A mammalian HAB homologue with identical alignment of these modules is expressed early in brain development. Specific antibodies revealed the presence of HAB in hydra as a transmembrane receptor, but also as secreted protein, both capable of binding head activator. Secretion of HAB during regeneration and expression in regions of high determination potential hint at a role for HAB in regulating the concentration and range of action of head activator.

Physiological role and regulation of the Na+/H+ exchanger

2006 ◽  
Vol 84 (11) ◽  
pp. 1081-1095 ◽  
Author(s):  
Mackenzie E. Malo ◽  
Larry Fliegel
Keyword(s):  
Plasma Membrane ◽  
Amino Acid ◽  
Mammalian Cells ◽  
Cell Movement ◽  
Physiological Role ◽  
Membrane Domain ◽  
Post Translational Modifications ◽  
Homologous Protein ◽  
The Family ◽  
Extracellular Sodium

In mammalian eukaryotic cells, the Na+/H+ exchanger is a family of membrane proteins that regulates ions fluxes across membranes. Plasma membrane isoforms of this protein extrude 1 intracellular proton in exchange for 1 extracellular sodium. The family of Na+/H+ exchangers (NHEs) consists of 9 known isoforms, NHE1–NHE9. The NHE1 isoform was the first discovered, is the best characterized, and exists on the plasma membrane of all mammalian cells. It contains an N-terminal 500 amino acid membrane domain that transports ions, plus a 315 amino acid C-terminal, the intracellular regulatory domain. The Na+/H+ exchanger is regulated by both post-translational modifications including protein kinase-mediated phosphorylation, plus by a number of regulatory-binding proteins including phosphatidylinositol-4,5-bisphosphate, calcineurin homologous protein, ezrin, radixin and moesin, calmodulin, carbonic anhydrase II, and tescalcin. The Na+/H+ exchanger is involved in a variety of complex physiological and pathological events that include regulation of intracellular pH, cell movement, heart disease, and cancer. This review summarizes recent advances in the understanding of the physiological role and regulation of this protein.

scholarly journals Biochemical Properties of a Putative Signal Peptide Peptidase from the Hyperthermophilic Archaeon Thermococcus kodakaraensis KOD1

2005 ◽  
Vol 187 (20) ◽  
pp. 7072-7080 ◽  
Author(s):  
Rie Matsumi ◽  
Haruyuki Atomi ◽  
Tadayuki Imanaka
Keyword(s):  
Substrate Specificity ◽  
Signal Peptide ◽  
Biochemical Characterization ◽  
Transmembrane Domain ◽  
Biochemical Properties ◽  
Peptidase Activity ◽  
Amino Acid Residues ◽  
Hyperthermophilic Archaeon ◽  
Signal Peptide Peptidase ◽  
Putative Signal Peptide

ABSTRACT We have performed the first biochemical characterization of a putative archaeal signal peptide peptidase (SppATk) from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. SppATk, comprised of 334 residues, was much smaller than its counterpart from Escherichia coli (618 residues) and harbored a single predicted transmembrane domain near its N terminus. A truncated mutant protein without the N-terminal 54 amino acid residues (ΔN54SppATk) was found to be stable against autoproteolysis and was examined further. ΔN54SppATk exhibited peptidase activity towards fluorogenic peptide substrates and was found to be highly thermostable. Moreover, the enzyme displayed a remarkable stability and preference for alkaline pH, with optimal activity detected at pH 10. ΔN54SppATk displayed a Km of 240 ± 18 μM and a V max of 27.8 ± 0.7 μmol min−1 mg−1 towards Ala-Ala-Phe-4-methyl-coumaryl-7-amide at 80°C and pH 10. The substrate specificity of the enzyme was examined in detail with a FRETS peptide library. By analyzing the cleavage products with liquid chromatography-mass spectrometry, ΔN54SppATk was found to efficiently cleave peptides with a relatively small side chain at the P-1 position and a hydrophobic or aromatic residue at the P-3 position. The positively charged Arg residue was preferred at the P-4 position, while substrates with negatively charged residues at the P-2, P-3, or P-4 position were not cleaved. When predicted signal sequences from the T. kodakaraensis genome sequence were examined, we found that the substrate specificity of ΔN54SppATk was in good agreement with its presumed role as a signal peptide peptidase in this archaeon.

scholarly journals Amino acid sequences around the cystine residues in horse growth hormone

1968 ◽  
Vol 109 (1) ◽  
pp. 19-24 ◽  
Author(s):  
Louise Oliver ◽  
Anne Stockell Hartree
Keyword(s):  
Amino Acids ◽  
Growth Hormone ◽  
Amino Acid ◽  
Growth Hormones ◽  
Amino Acid Sequences ◽  
The Other ◽  
Published Data ◽  
C Terminus

The cystine-containing peptides of horse growth hormone were isolated and their amino acid sequences determined. Four unique half-cystine residues occur in two peptides, one containing 11 and the other, at the C-terminus of the protein, 15 amino acids. These sequences are compared with published data on growth hormones from other species.

Targeting motifs and functional parameters governing the assembly of connexins into gap junctions

2000 ◽  
Vol 349 (1) ◽  
pp. 281-287 ◽  
Author(s):  
Patricia E. M. MARTIN ◽  
James STEGGLES ◽  
Claire WILSON ◽  
Shoeb AHMAD ◽  
W. Howard EVANS
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Gap Junctions ◽  
Gap Junction ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Lucifer Yellow ◽  
Amino Acid Sequences ◽  
Amino Acid Residues ◽  
Green Fluorescent

To study the assembly of gap junctions, connexin-green-fluorescent-protein (Cx-GFP) chimeras were expressed in COS-7 and HeLa cells. Cx26- and Cx32-GFP were targeted to gap junctions where they formed functional channels that transferred Lucifer Yellow. A series of Cx32-GFP chimeras, truncated from the C-terminal cytoplasmic tail, were studied to identify amino acid sequences governing targeting from intracellular assembly sites to the gap junction. Extensive truncation of Cx32 resulted in failure to integrate into membranes. Truncation of Cx32 to residue 207, corresponding to removal of most of the 78 amino acids on the cytoplasmic C-terminal tail, led to arrest in the endoplasmic reticulum and incomplete oligomerization. However, truncation to amino acid 219 did not impair Cx oligomerization and connexon hemichannels were targeted to the plasma membrane. It was concluded that a crucial gap-junction targeting sequence resides between amino acid residues 207 and 219 on the cytoplasmic C-terminal tail of Cx32. Studies of a Cx32E208K mutation identified this as one of the key amino acids dictating targeting to the gap junction, although oligomerization of this site-specific mutation into hexameric hemichannels was relatively unimpaired. The studies show that expression of these Cx-GFP constructs in mammalian cells allowed an analysis of amino acid residues involved in gap-junction assembly.

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