Epitope-specific antibody-induced cleavage of angiotensin-converting enzyme from the cell surface

2002 ◽  
Vol 362 (3) ◽  
pp. 585-595 ◽  
Author(s):  
Irina V. BALYASNIKOVA ◽  
Eric H. KARRAN ◽  
Ronald F. ALBRECHT ◽  
Sergei M. DANILOV
Keyword(s):  
Transmembrane Domain ◽  
Umbilical Vein ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Integral Membrane Protein ◽  
Converting Enzyme ◽  
Ovary Cells ◽  
Angiotensin I Converting Enzyme ◽  
Hydrophobic Transmembrane Domain ◽  
Umbilical Vein Endothelial Cells

Angiotensin I-converting enzyme (ACE; CD143, EC 3.4.15.1) is a type-1 integral membrane protein that can also be released into extracellular fluids (such as plasma, and seminal and cerebrospinal fluids) as a soluble enzyme following cleavage mediated by an unidentified protease(s), referred to as ACE secretase, in a process known as ‘shedding'. The effects of monoclonal antibodies (mAbs) to eight different epitopes on the N-terminal domain of ACE on shedding was investigated using Chinese hamster ovary cells (CHO cells) expressing an ACE transgene and using human umbilical vein endothelial cells. Antibody-induced shedding of ACE was strongly epitope-specific: most of the antibodies increased the shedding by 20–40%, mAbs 9B9 and 3A5 increased the shedding by 270 and 410% respectively, whereas binding of mAb 3G8 decreased ACE shedding by 36%. The ACE released following mAb treatment lacked a hydrophobic transmembrane domain anchor. The antibody-induced shedding was completely inhibited at 4°C and by zinc chelation using 1,10-phenanthroline, suggesting involvement of a metalloprotease in this process. A hydroxamate-based metalloprotease inhibitor (batimastat, BB-94) was 15 times more efficacious in inhibiting mAb-induced ACE shedding than basal (constitutive) ACE release. Treatment of CHO-ACE cells with BB-94 more effectively prevented elevation in antibody-dependent (but not basal) ACE release induced by 3,4-dichloroisocoumarin and iodoacetamide. These data suggest that different secretases might be responsible for ACE release under basal compared with antibody-induced shedding. Further experiments with more than 40 protease inhibitors suggest that calpains, furin and the proteasome may participate in this process.

Recombinant Human Soluble Thrombomodulin Delivers Bounded Thrombin to Antithrombin III: Thrombomodulin Associates with Free Thrombin and Is Recycled to Activate Protein C

1993 ◽  
Vol 70 (03) ◽  
pp. 418-422 ◽  
Author(s):  
Masaharu Aritomi ◽  
Naoko Watanabe ◽  
Rika Ohishi ◽  
Komakazu Gomi ◽  
Takao Kiyota ◽  
...  
Keyword(s):  
Protein C ◽  
Antithrombin Iii ◽  
Transmembrane Domain ◽  
Chinese Hamster Ovary Cells ◽  
Time Lag ◽  
Chinese Hamster ◽  
Ovary Cells ◽  
Soluble Thrombomodulin ◽  
Simulation Based ◽  
Recombinant Human Soluble Thrombomodulin

SummaryRecombinant human soluble thrombomodulin (rhs-TM), having no transmembrane domain or chondroitin sulfate, was expressed in Chinese hamster ovary cells. Interactions between rhs-TM, thrombin (Th), protein C (PC) and antithrombin III (ATIII) were studied. Equilibrium between rhs-TM and Th had no detectable time lag in clotting inhibition (K d = 26 nM) or PC activation (K d = 22 nM), while ATIII inhibited Th at a bimolecular rate constant = 5,200 M-1s-1 (K d <0.2 nM). A mixture of ATIII, Th and rhs-TM showed that ATIII reacted with Th slower than rhs-TM, whose presence did not affect the reaction between ATIII and Th. In a mixture of rhs-TM, ATIII and PC, the repeated addition of Th caused the repeated activation of PC; which was consistent with the Simulation based on the assumption that rhs-TM is recycled as a Th cofactor. From these results, we concluded that upon inhibition of the rhs-TM-Th complex by ATIII, rhs-TM is released to recombine with free Th and begins to activate PC, while the Th-ATIII complex does not affect rhs-TM-Th equilibrium.

scholarly journals Identification of a cysteine-rich receptor for fibroblast growth factors.

1992 ◽  
Vol 12 (12) ◽  
pp. 5600-5609 ◽  
Author(s):  
L W Burrus ◽  
M E Zuber ◽  
B A Lueddecke ◽  
B B Olwin
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Integral Membrane Protein ◽  
Fibroblast Growth ◽  
Fgf Receptor ◽  
Ovary Cells ◽  
Multiple Receptors ◽  
High Degree

The fibroblast growth factor (FGF) family consists of seven members whose activities are thought to be mediated by multiple receptors. Here we describe the cDNA cloning, expression, and characterization of a cysteine-rich FGF receptor (CFR) that is distinct from previously identified FGF receptors. The deduced amino acid sequence for CFR suggests that it is an integral membrane protein containing a large extracellular domain comprising 16 cysteine-rich repeated units and an intracellular domain of 13 amino acids. No reported sequences exhibit significant homologies to either the repeated extracellular motif or to the entire CFR amino acid sequence. Several CFR transcripts are present in embryonic chick tissue, suggesting that CFR undergoes alternate mRNA splicing or that related genes are present. Chinese hamster ovary cells transfected with the CFR cDNA express a 150-kDa polypeptide that binds FGF-1, FGF-2, and FGF-4 but does not bind several non-FGF family members. The high degree of evolutionary conservation among vertebrate CFRs and its ability to bind three different FGFs with high affinity suggest that this unique receptor plays an important role in FGF biology.

Regulation of intestinal Cl−/HCO3− exchanger SLC26A3 by intracellular pH

2009 ◽  
Vol 296 (6) ◽  
pp. C1279-C1290 ◽  
Author(s):  
Hisayoshi Hayashi ◽  
Kazuhito Suruga ◽  
Yukari Yamashita
Keyword(s):  
Intracellular Ph ◽  
Transmembrane Domain ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Functional Coupling ◽  
Intestinal Epithelial ◽  
Transport Activity ◽  
Ovary Cells ◽  
Nacl Absorption ◽  
Reverse Mode

SLC26A3, a Cl−/HCO3− exchanger, is highly expressed in intestinal epithelial cells, and its mutations cause congenital chloride diarrhea. This suggests that SLC26A3 plays a key role in NaCl absorption in the intestine. Electroneutral NaCl absorption in the intestine is mediated by functional coupling of the Na+/H+ exchanger and Cl−/HCO3− exchanger. It is proposed that the coupling of these exchangers may occur as a result of indirect linkage by changes of intracellular pH (pHi). We therefore investigated whether SLC26A3 is regulated by pHi. We generated a hemagglutinin epitope-tagged human SLC26A3 construct and expressed it in Chinese hamster ovary cells. Transport activities were measured with a fluorescent chloride-sensitive dye dihydro-6-methoxy- N-ethylquinolinium iodide (diH-MEQ). pHi was clamped at a range of values from 6.0 to 7.4. We monitored the transport activity of SLC26A3 by reverse mode of Cl−/HCO3− and Cl−/NO3− exchange. None of these exchange modes induced membrane potential changes. At constant external pH 7.4, Cl−/HCO3− exchange was steeply inhibited with pHi decrease between 7.3 and 6.8 as opposed to thermodynamic prediction. In contrast, however, Cl−/NO3− exchange was essentially insensitive to pHi within physiological ranges. We also characterized the pHi dependency of COOH-terminal truncation mutants. Removal of the entire COOH-terminal resulted in decrease of the transport activity but did not noticeably affect pHi sensitivity. These results suggest that Cl−/HCO3− exchange mode of human SLC26A3 is controlled by a pH-sensitive intracellular modifier site, which is likely in the transmembrane domain. These observations raise the possibility that SLC26A3 activity may be regulated via Na+/H+ exchanger 3 (NHE3) through the alteration of pHi under physiological conditions.

Identification of a cysteine-rich receptor for fibroblast growth factors

1992 ◽  
Vol 12 (12) ◽  
pp. 5600-5609
Author(s):  
L W Burrus ◽  
M E Zuber ◽  
B A Lueddecke ◽  
B B Olwin
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Integral Membrane Protein ◽  
Fibroblast Growth ◽  
Fgf Receptor ◽  
Ovary Cells ◽  
Multiple Receptors ◽  
High Degree

The fibroblast growth factor (FGF) family consists of seven members whose activities are thought to be mediated by multiple receptors. Here we describe the cDNA cloning, expression, and characterization of a cysteine-rich FGF receptor (CFR) that is distinct from previously identified FGF receptors. The deduced amino acid sequence for CFR suggests that it is an integral membrane protein containing a large extracellular domain comprising 16 cysteine-rich repeated units and an intracellular domain of 13 amino acids. No reported sequences exhibit significant homologies to either the repeated extracellular motif or to the entire CFR amino acid sequence. Several CFR transcripts are present in embryonic chick tissue, suggesting that CFR undergoes alternate mRNA splicing or that related genes are present. Chinese hamster ovary cells transfected with the CFR cDNA express a 150-kDa polypeptide that binds FGF-1, FGF-2, and FGF-4 but does not bind several non-FGF family members. The high degree of evolutionary conservation among vertebrate CFRs and its ability to bind three different FGFs with high affinity suggest that this unique receptor plays an important role in FGF biology.

Sign in / Sign up

Export Citation Format

Share Document