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1.
Mol Genet Metab ; 123(4): 501-510, 2018 04.
Article in English | MEDLINE | ID: mdl-29478818

ABSTRACT

BACKGROUND: Gaucher disease is characterized by the activation of splenic and hepatic macrophages, accompanied by dramatically increased levels of angiotensin-converting enzyme (ACE). To evaluate the source of the elevated blood ACE, we performed complete ACE phenotyping using blood, spleen and liver samples from patients with Gaucher disease and controls. METHODS: ACE phenotyping included 1) immunohistochemical staining for ACE; 2) measuring ACE activity with two substrates (HHL and ZPHL); 3) calculating the ratio of the rates of substrate hydrolysis (ZPHL/HHL ratio); 4) assessing the conformational fingerprint of ACE by evaluating the pattern of binding of monoclonal antibodies to 16 different ACE epitopes. RESULTS: We show that in patients with Gaucher disease, the dramatically increased levels of ACE originate from activated splenic and/or hepatic macrophages (Gaucher cells), and that both its conformational fingerprint and kinetic characteristics (ZPHL/HHL ratio) differ from controls and from patients with sarcoid granulomas. Furthermore, normal spleen was found to produce high levels of endogenous ACE inhibitors and a novel, tightly-bound 10-30 kDa ACE effector which is deficient in Gaucher spleen. CONCLUSIONS: The conformation of ACE is tissue-specific. In Gaucher disease, ACE produced by activated splenic macrophages differs from that in hepatic macrophages, as well as from macrophages and dendritic cells in sarcoid granulomas. The observed differences are likely due to altered ACE glycosylation or sialylation in these diseased organs. The conformational differences in ACE may serve as a specific biomarker for Gaucher disease.


Subject(s)
Dendritic Cells/enzymology , Gaucher Disease/enzymology , Gaucher Disease/pathology , Granuloma/enzymology , Macrophages/enzymology , Peptidyl-Dipeptidase A/metabolism , Cells, Cultured , Humans , Liver/enzymology , Phenotype , Spleen/enzymology
2.
J Proteome Res ; 9(11): 5782-93, 2010 Nov 05.
Article in English | MEDLINE | ID: mdl-20873814

ABSTRACT

Fine epitope mapping of monoclonal antibodies (mAbs) to 16 epitopes on human angiotensin I-converting enzyme (ACE) revealed that the epitopes of all mAbs contained putative glycosylation sites. ACE glycosylation is both cell- and tissue-specific and, therefore, the local conformation of ACE produced by different cells could be also unique. The pattern of ACE binding by a set of mAbs to 16 epitopes of human ACE - "conformational fingerprint of ACE" - is the most sensitive marker of ACE conformation and could be cell- and tissue-specific. The recognition of ACEs by mAbs to ACE was estimated using an immune-capture enzymatic plate precipitation assay. Precipitation patterns of soluble recombinant ACE released from Chinese hamster ovary (CHO)-ACE cells was influenced by conditions that alter ACE glycosylation. This pattern was also strongly cell type specific. Patients with sarcoidosis exhibited conformational fingerprints of tissue ACE (lungs and lymph nodes), as well as blood ACE, which were distinct from controls. Conformational fingerprinting of ACE may detect ACE originated from the cells other than endothelial cells in the blood and when combined with elevated blood ACE levels in patients with sarcoidosis may potentially reflect extrapulmonary sarcoidosis involvement (bone marrow, spleen, liver). If proven true, this would serve as a biomarker of enormous potential clinical significance.


Subject(s)
Peptidyl-Dipeptidase A/chemistry , Sarcoidosis/enzymology , Animals , Antibodies, Monoclonal , Cell Line , Epitope Mapping/methods , Epitopes , Glycosylation , Humans , Peptidyl-Dipeptidase A/immunology , Protein Conformation , Tissue Distribution
3.
J Proteome Res ; 7(8): 3396-411, 2008 Aug.
Article in English | MEDLINE | ID: mdl-18576678

ABSTRACT

Angiotensin I-converting enzyme (ACE, CD143) has two homologous domains, each having a functional active site. Fine epitope mapping of 8 mAbs to the C-terminal domain of human ACE was carried out using plate precipitation assays, mAbs' cross-reactivity with ACE from different species, site-directed mutagenesis, and antigen- and cell-based ELISAs. Almost all epitopes contained potential glycosylation sites. Therefore, these mAbs could be used to distinguish different glycoforms of ACE expressed in different tissues or cell lines. mAbs 1B8 and 3F10 were especially sensitive to the composition of the N-glycan attached to Asn 731; mAbs 2H9 and 3F11 detected the glycosylation status of the glycan attached to Asn 685 and perhaps Asn1162; and mAb 1E10 and 4E3 recognized the glycan on Asn 666. The epitope of mAb 1E10 is located at the N-terminal end of the C domain, close to the unique 36 amino acid residues of testicular ACE (tACE). Moreover, it binds preferentially to tACE on the surface of human spermatozoa and thus may find application as an immunocontraceptive drug. mAb 4E3 was the best mAb for quantification of ACE-expressing somatic cells by flow cytometry. In contrast to the other mAbs, binding of mAb 2B11 was not markedly influenced by ACE glycosylation or by the cell culture conditions or cell types, making this mAb a suitable reference antibody. Epitope mapping of these C-domain mAbs, particularly those that compete with N-domain mAbs, enabled us to propose a model of the two-domain somatic ACE that might explain the interdomain cooperativity. Our findings demonstrated that mAbs directed to conformational epitopes on the C-terminal domain of human ACE are very useful for the detection of testicular and somatic ACE, quantification using flow cytometry and ELISA assays, and for the study of different aspects of ACE biology.


Subject(s)
Antibodies, Monoclonal/analysis , Peptidyl-Dipeptidase A/immunology , Amino Acid Sequence , Animals , CHO Cells , Cricetinae , Cricetulus , Epitope Mapping , Epitopes , Glycosylation , Humans , Macaca , Models, Molecular , Molecular Sequence Data , Peptidyl-Dipeptidase A/metabolism , Protein Structure, Tertiary , Species Specificity
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