Glycosylation of Asn-632 and Asn-651 Is Important for Functional Expression of Endothelin-Converting Enzyme-1

Nelboeck, Peter; Fuchs, Mathias; Bur, Daniel; Löffler, Bernd–Michael

doi:10.1097/00005344-199800001-00003

Cited by 8 publications

(5 citation statements)

References 8 publications

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“…In agreement with this conclusion, deglycosylation of purified ECE-1 did not significantly alter its enzyme activity (42). Mutating two of ECE-1 glycosylation sites is, however, enough to render the enzyme inactive (43), which is expected considering the importance of glycosylation for proper protein folding in the secretory pathway (for review see (44). Our data thus suggest that molecular chaperones can efficiently compensate for the lack of glycosylation and promote proper folding of ECE-1sv in the cytosol.…”

Section: Discussionsupporting

confidence: 72%

Endothelin-converting Enzyme-1, Abundance of Isoforms a-d and Identification of a Novel Alternatively Spliced Variant Lacking a Transmembrane Domain

Meidan

Klipper

Gilboa

et al. 2005

Journal of Biological Chemistry

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Endothelin-converting enzyme-1 (ECE-1) cleaves big endothelins, as well as bradykinin and ␤-amyloid peptide. Several isoforms of ECE-1 (a-d) have been identified to date; they differ only in their NH 2 terminus but share the catalytic domain located in the COOHterminal end. Using quantitative PCR, we found ECE-1d to be the most abundant type in several endothelial cells (EC) types. In addition to full-length ECE-1 forms we have identified novel, alternatively spliced mRNAs of ECE-1 b-d. These splice variants (SVs) lack exon 3, which codes for the transmembrane region and is present in full-length forms. SVs mRNA were highly expressed in EC derived from macro and microvascular beds but much less so in other, nonendothelial cells expressing ECE-1, which suggests that the splicing mechanism is cell-specific. Analyses of ECE-1d and its SV form in stably transfected HEK-293 cells revealed that both proteins were recognized by anti COOH-terminal ECE-1 antibodies, but anti NH 2 -terminal antibodies only bound ECE-1d. The novel protein, designated ECE-1 sv, has an apparent molecular mass of 75 kDa; by using site-directed mutagenesis its start site was identified in a region common to all ECE-1 forms suggesting that ECE-1 b-d SV mRNAs are translated into the same protein. In agreement with the findings demonstrating common COOH terminus for ECE-1sv and ECE-1d, both exhibited a similar catalytic activity. However, immunofluorescence staining and differential centrifugation revealed a distinct intracellular localization for these two proteins. The presence of ECE-1sv in different cellular compartments than full-length forms of the enzyme may suggest a distinct physiological role for these proteins.

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Section: Discussionsupporting

confidence: 72%

Endothelin-converting Enzyme-1, Abundance of Isoforms a-d and Identification of a Novel Alternatively Spliced Variant Lacking a Transmembrane Domain

Meidan

Klipper

Gilboa

et al. 2005

Journal of Biological Chemistry

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“…Ten potential glycosylation signals are present in Lom ECE, suggesting that Lom ECE, like mammalian ECE, is a highly glycosylated protein with a higher molecular weight than calculated from its predicted residue masses alone (Shimada et al ., 1994). Among these glycosylation signals in Lom ECE, Asn 589 and Asn 608 are found, which are proven to be important for the functional expression of mammalian ECE‐1 (Nelboeck et al ., 1998). The putative zinc ligand Glu 624, which is located 61 residues downstream from the active site compares with a 61 AA separation in mammalian ECE (Shimada et al ., 1995a,b), in contrast to mammalian NEP which possesses a 64 residue separation (Hooper et al ., 1994).…”

Section: Discussionmentioning

confidence: 99%

“…Among these glycosylation signals in Lom ECE, Asn 589 and Asn 608 are found, which are proven to be important for the functional expression of mammalian ECE‐1 (Nelboeck et al ., 1998). The putative zinc ligand Glu 624, which is located 61 residues downstream from the active site compares with a 61 AA separation in mammalian ECE (Shimada et al ., 1995a,b), in contrast to mammalian NEP which possesses a 64 residue separation (Hooper et al ., 1994). The CEVW sequence, critical for proper enzyme maturation and enzyme activity (MacLeod et al ., 2001), conserved among all mammalian ECE sequences, is present in Lom ECE.…”

Section: Discussionmentioning

confidence: 99%

An endothelin‐converting enzyme homologue in the locust, Locusta migratoria: functional activity, molecular cloning and tissue distribution

Macours

Poels

Hens

et al. 2003

Insect Molecular Biology

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Endothelin-converting enzyme is the key enzyme in the process of endothelin production. Endothelin is a peptide that plays an important role in vasoconstriction and the development of neural crest-derived cells in vertebrates. Activity assays performed on membrane extracts from Locusta migratoria brain revealed the existence of a protease activity responsible for the formation of mature endothelin-1 from its precursor, big endothelin. Cloning experiments led to a cDNA sequence (Lom ECE) with an open reading frame of 727 amino acid residues displaying all the characteristic ECE features. A comparison of ECE activity levels among different tissues of the locust showed a high enzyme activity in the gonads and midgut. RT-PCR experiments showed a wide tissue distribution of Lom ECE mRNA, with transcription being most abundant in brain tissue.

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“…Single mutations of the glycosylated Asn632 and Asn651 of the metalloprotease endothelin-converting enzyme had no effect, while the double mutant was completely inactive [109]. A series of sequon mutants of mouse meprin-α showed that six out of ten N -glycans are critical for catalysis [110].…”

Section: Effects Of Glycosylation On Proteasesmentioning

confidence: 99%

Effects of Glycosylation on the Enzymatic Activity and Mechanisms of Proteases

Goettig

2016

IJMS

100

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Posttranslational modifications are an important feature of most proteases in higher organisms, such as the conversion of inactive zymogens into active proteases. To date, little information is available on the role of glycosylation and functional implications for secreted proteases. Besides a stabilizing effect and protection against proteolysis, several proteases show a significant influence of glycosylation on the catalytic activity. Glycans can alter the substrate recognition, the specificity and binding affinity, as well as the turnover rates. However, there is currently no known general pattern, since glycosylation can have both stimulating and inhibiting effects on activity. Thus, a comparative analysis of individual cases with sufficient enzyme kinetic and structural data is a first approach to describe mechanistic principles that govern the effects of glycosylation on the function of proteases. The understanding of glycan functions becomes highly significant in proteomic and glycomic studies, which demonstrated that cancer-associated proteases, such as kallikrein-related peptidase 3, exhibit strongly altered glycosylation patterns in pathological cases. Such findings can contribute to a variety of future biomedical applications.

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Glycosylation of Asn-632 and Asn-651 Is Important for Functional Expression of Endothelin-Converting Enzyme-1

Cited by 8 publications

References 8 publications

Endothelin-converting Enzyme-1, Abundance of Isoforms a-d and Identification of a Novel Alternatively Spliced Variant Lacking a Transmembrane Domain

Endothelin-converting Enzyme-1, Abundance of Isoforms a-d and Identification of a Novel Alternatively Spliced Variant Lacking a Transmembrane Domain

An endothelin‐converting enzyme homologue in the locust, Locusta migratoria: functional activity, molecular cloning and tissue distribution

Effects of Glycosylation on the Enzymatic Activity and Mechanisms of Proteases

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