scholarly journals Characterization of the first non-insect invertebrate functional angiotensin-converting enzyme (ACE): leech TtACE resembles the N-domain of mammalian ACE

2004 ◽  
Vol 382 (2) ◽  
pp. 565-573 ◽  
Author(s):  
Guillaume RIVIÈRE ◽  
Annie MICHAUD ◽  
Laurence DELOFFRE ◽  
Franck VANDENBULCKE ◽  
Angélique LEVOYE ◽  
...  
Keyword(s):  
Angiotensin Converting Enzyme ◽  
Active Site ◽  
Active Sites ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Inhibitory Effect ◽  
Converting Enzyme ◽  
Ovary Cells

Angiotensin-converting enzyme (ACE) is a zinc metallopeptidase that plays a major role in blood homoeostasis and reproduction in mammals. In vertebrates, both transmembrane and soluble ACE, containing one or two homologous active sites, have been characterized. So far, several ACEs from invertebrates have been cloned, but only in insects. They are soluble and display a single active site. Using biochemical procedures, an ACE-like activity was detected in our model, the leech, Theromyzon tessulatum. Annelida is the most distant phylum in which an ACE activity has been observed. To gain more insight into the leech enzyme, we have developed a PCR approach to characterize its mRNA. The approx. 2 kb cDNA has been predicted to encode a 616-amino-acid soluble enzyme containing a single active site, named TtACE (T. tessulatum ACE). Surprisingly, its primary sequence shows greater similarity to vertebrates than to invertebrates. Stable in vitro expression of TtACE in transfected Chinese-hamster ovary cells revealed that the leech enzyme is a functional metalloprotease. As in mammals, this 79 kDa glycosylated enzyme functions as a dipeptidyl carboxypeptidase capable of hydrolysing angiotensin I to angiotensin II. However, a weak chloride inhibitory effect and acetylated N-acetyl-SDKP (Ac SDAcKP) hydrolysis reveal that TtACE activity resembles that of the N-domain of mammalian ACE. In situ hybridization shows that its cellular distribution is restricted to epithelial midgut cells. Although the precise roles and endogenous substrates of TtACE remain to be identified, characterization of this ancestral peptidase will help to clarify its physiological roles in non-insect invertebrate species.

scholarly journals Shedding of somatic angiotensin-converting enzyme (ACE) is inefficient compared with testis ACE despite cleavage at identical stalk sites

2000 ◽  
Vol 347 (3) ◽  
pp. 711-718 ◽  
Author(s):  
Zenda L. WOODMAN ◽  
Sylvester Y. OPPONG ◽  
Sarah COOK ◽  
Nigel M. HOOPER ◽  
Sylva L. U. SCHWAGER ◽  
...  
Keyword(s):  
Angiotensin Converting Enzyme ◽  
Cleavage Site ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Analytical Techniques ◽  
Human Testis ◽  
Converting Enzyme ◽  
Substrate Preferences

The somatic and testis isoforms of angiotensin-converting enzyme (ACE) are both C-terminally anchored ectoproteins that are shed by an unidentified secretase. Although testis and somatic ACE both share the same stalk and membrane domains the latter was reported to be shed inefficiently compared with testis ACE, and this was ascribed to cleavage at an alternative site [Beldent, Michaud, Bonnefoy, Chauvet and Corvol (1995) J. Biol. Chem. 270, 28962-28969]. These differences constitute a useful model system of the regulation and substrate preferences of the ACE secretase, and hence we investigated this further. In transfected Chinese hamster ovary cells, human somatic ACE (hsACE) was indeed shed less efficiently than human testis ACE, and shedding of somatic ACE responded poorly to phorbol ester activation. However, using several analytical techniques, we found no evidence that the somatic ACE cleavage site differed from that characterized in testis ACE. First, anti-peptide antibodies raised to specific sequences on either side of the reported cleavage site (Arg1137/Leu1138) clearly recognized soluble porcine somatic ACE, indicating that cleavage was C-terminal to Arg1137. Second, a competitive ELISA gave superimposable curves for porcine plasma ACE, secretase-cleaved porcine somatic ACE (eACE), and trypsin-cleaved ACE, suggesting similar C-terminal sequences. Third, mass-spectral analyses of digests of released soluble hsACE or of eACE enabled precise assignments of the C-termini, in each case to Arg1203. These data indicated that soluble human and porcine somatic ACE, whether generated in vivo or in vitro, have C-termini consistent with cleavage at a single site, the Arg1203/Ser1204 bond, identical with the Arg627/Ser628 site in testis ACE. In conclusion, the inefficient release of somatic ACE is not due to cleavage at an alternative stalk site, but instead supports the hypothesis that the testis ACE ectodomain contains a motif that activates shedding, which is occluded by the additional domain found in somatic ACE.

scholarly journals Immunoglobulin fragment F(ab’)2 against RBD potently neutralizes SARS-CoV-2 in vitro

2020 ◽  
Author(s):  
Xiaoyan Pan ◽  
Pengfei Zhou ◽  
Tiejiong Fan ◽  
Yan Wu ◽  
Jing Zhang ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
High Titer ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Inhibitory Effect ◽  
Ovary Cells ◽  
Global Pandemic ◽  
Potent Inhibitory Effect

AbstractCOVID-19 caused by the emerging human coronavirus, SARS-CoV-2, has become a global pandemic, leading a serious threat to human health. So far, there is none vaccines or specific antiviral drugs approved for that. Therapeutic antibodies for SARS-CoV-2, was obtained from hyper immune equine plasma in this study. Herein, SARS-CoV-2 RBD with gram level were obtained through Chinese hamster ovary cells high-density fermentation. The binding of RBD to SARS-CoV-2 receptor, human ACE2, was verified and the efficacy of RBD in vivo was tested on mice and then on horses. As a result, RBD triggered high-titer neutralizing antibodies in vivo, and immunoglobulin fragment F(ab’)2 was prepared from horse antisera through removing Fc. Neutralization test demonstrated that RBD-specific F(ab’)2 inhibited SARS-CoV-2 with EC50 at 0.07 μg/ml, showing a potent inhibitory effect on SARS-CoV-2. These results highlights as RBD-specific F(ab’)2 as therapeutic candidate for SARS-CoV-2.

scholarly journals Functional characterization of the human 1-acylglycerol-3-phosphate-O-acyltransferase isoform 10/glycerol-3-phosphate acyltransferase isoform 3

2009 ◽  
Vol 42 (6) ◽  
pp. 469-478 ◽  
Author(s):  
Suja Sukumaran ◽  
Robert I Barnes ◽  
Abhimanyu Garg ◽  
Anil K Agarwal
Keyword(s):  
Enzymatic Activity ◽  
De Novo ◽  
Chinese Hamster Ovary Cells ◽  
Functional Characterization ◽  
Chinese Hamster ◽  
Protein Product ◽  
Ovary Cells ◽  
293 Cells

Synthesis of phospholipids can occur de novo or via remodeling of the existing phospholipids. Synthesis of triglycerides, a form of energy storage in cells, is an end product of these pathways. Several 1-acylglycerol-3-phosphate-O-acyltransferases (AGPATs) acylate lysophosphatidic acid (LPA) at the sn-2 (carbon 2) position to produce phosphatidic acid (PA). These enzymes are involved in phospholipids and triglyceride synthesis through an evolutionary conserved process involving serial acylations of glycerol-3-phosphate. We cloned a cDNA predicted to be an AGPAT isoform (AGPAT10). This cDNA has been recently identified as glycerol-3-phosphate-O-acyltransferase isoform 3 (GPAT3). When this AGPAT10/GPAT3 cDNA was expressed in Chinese Hamster ovary cells, the protein product localizes to the endoplasmic reticulum. In vitro enzymatic activity using lysates of human embryonic kidney-293 cells infected with recombinant AGPAT10/GPAT3 adenovirus show that the protein has a robust AGPAT activity with an apparent Vmax of 2 nmol/min per mg protein, but lacks GPAT enzymatic activity. This AGPAT has similar substrate specificities for LPA and acyl-CoA as shown for another known isoform, AGPAT2. We further show that when overexpressed in human Huh-7 cells depleted of endogenous AGPAT activity by sh-RNA-AGPAT2-lentivirus, the protein again demonstrates AGPAT activity. These observations strongly suggest that the cDNA previously identified as GPAT3 has AGPAT activity and thus we prefer to identify this clone as AGPAT10 as well.

Activity and stability of centrosomes in Chinese hamster ovary cells in nucleation of microtubules in vitro

1984 ◽  
Vol 66 (1) ◽  
pp. 277-295
Author(s):  
R. Kuriyama
Keyword(s):  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Temperature Sensitive ◽  
Ovary Cells ◽  
Protease Digestion ◽  
The Difference

Mitotic centrosomes were prepared from Chinese hamster ovary cells and their capacity to nucleate microtubules in vitro was demonstrated by incubation with exogenous brain microtubule protein. The number of microtubules polymerized onto centrosomes was directly counted by electron microscopy of whole-mount preparations. This simple and accurate quantitative assay has permitted characterization of the microtubule nucleating activity of centrosomes in vitro. The number of microtubules polymerized onto centrosomes varied according to the structure of the centrosome. The activity was roughly proportional to the centriole number. The number and length of microtubules nucleated by centrosomes depended both on the concentration of tubulin and the incubation time with tubulin. Under saturating conditions, an average of 200–250 microtubules were initiated by single centrosomes. Centrosomal activity is unstable (t 1/2 = 8 h) and could easily be irreversibly disrupted by a medium of high ionic strength. The activity is stabilized by the addition of glycerol. Centrosomes can be stored at −80 degrees C. The optimum pH for microtubule nucleation is 6.8. Activity is sensitive to protease digestion, but neither DNase or RNase affected the nucleating activity of centrosomes. The activity is temperature-sensitive, but addition of phenylmethylsulphonyl fluoride (PMSF) induces thermal stability. At an optimal concentration of 0.5 mg/ml, this drug increased the half-life of the activity (t 1/2 = 95 h) and made it resistant to salt extraction. Protease inhibitors other than PMSF or dansyl fluoride did not have any stabilizing effect on the activity. The difference between the centrosomal structure of polymerized microtubules in vivo and in vitro is discussed.

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