Protein Modifications Critical for Myonectin/Erythroferrone Secretion and Oligomer Assembly
Ashley N Stewart1, Hannah C Little1, David J Clark2
1Department of Physiology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, United States
Biochemistry
|July 1, 2020
Summary
Myonectin (CTRP15) requires specific N-linked glycosylation and cysteine modifications for proper folding, secretion, and multimerization. These protein modifications are crucial for its physiological functions.
Area of Science:
- Biochemistry
- Molecular Biology
- Endocrinology
Background:
- Myonectin/erythroferrone (CTRP15) is a secreted hormone involved in metabolism and stress erythropoiesis.
- Despite its physiological importance, its biochemical characteristics and post-translational modifications remain poorly understood.
Purpose of the Study:
- To investigate the critical protein modifications essential for myonectin secretion and multimerization.
- To elucidate the roles of N-linked glycosylation, cysteine residues, and proline hydroxylation in myonectin's biochemical properties.
Main Methods:
- Site-directed mutagenesis was used to alter potential glycosylation sites (Asn) and cysteine residues.
- Tunicamycin and glucosamine were employed to inhibit N-linked glycosylation.
- Mass spectrometry was utilized to confirm glycosylation sites.
- Mutant proteins were analyzed for secretion, folding, and oligomeric assembly.
- Proline hydroxylation was inhibited, and the collagen domain was deleted to assess their impact on secretion.
Main Results:
- Abolishing N-linked glycosylation at Asn-229 and Asn-281 blocked myonectin secretion.
- Glutamine substitution at Asn-319 led to misfolding and endoplasmic reticulum retention.
- Cysteine residues Cys-273 and Cys-278 were vital for proper folding and secretion.
- Cys-142 and Cys-194 were involved in higher-order oligomer formation (trimers, hexamers, HMW oligomers) via disulfide bonds.
- Proline hydroxylation and the collagen domain were essential for efficient protein secretion.
Conclusions:
- Multiple post-translational modifications, including N-linked glycosylation, specific cysteine disulfide bond formation, and proline hydroxylation within the collagen domain, are critical for myonectin folding, secretion, and multimeric assembly.
- These findings provide a foundation for future structure-function relationship studies of myonectin.
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