Unacylated ghrelin binds heparan-sulfate proteoglycans which modulate its function

in Journal of Molecular Endocrinology
Authors:
Patric J D Delhanty Laboratory of Metabolism and Reproduction, Department of Internal Medicine, Erasmus MC, University Medical Center Rotterdam, Rotterdam, The Netherlands

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Martin Huisman Laboratory of Metabolism and Reproduction, Department of Internal Medicine, Erasmus MC, University Medical Center Rotterdam, Rotterdam, The Netherlands

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Karina Prins Laboratory of Metabolism and Reproduction, Department of Internal Medicine, Erasmus MC, University Medical Center Rotterdam, Rotterdam, The Netherlands

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Cobie Steenbergen Laboratory of Metabolism and Reproduction, Department of Internal Medicine, Erasmus MC, University Medical Center Rotterdam, Rotterdam, The Netherlands

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Rosinda Mies Laboratory of Metabolism and Reproduction, Department of Internal Medicine, Erasmus MC, University Medical Center Rotterdam, Rotterdam, The Netherlands

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Sebastian J C M M Neggers Laboratory of Metabolism and Reproduction, Department of Internal Medicine, Erasmus MC, University Medical Center Rotterdam, Rotterdam, The Netherlands

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A J van der Lely Laboratory of Metabolism and Reproduction, Department of Internal Medicine, Erasmus MC, University Medical Center Rotterdam, Rotterdam, The Netherlands

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Jenny A Visser Laboratory of Metabolism and Reproduction, Department of Internal Medicine, Erasmus MC, University Medical Center Rotterdam, Rotterdam, The Netherlands

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Correspondence should be addressed to P J D Delhanty: p.delhanty@erasmusmc.nl
DOI:
https://doi.org/10.1530/JME-20-0286
Volume/Issue:
Volume 66: Issue 2
Article Type:
Research Article
Page Range:
83–96
Online Publication Date:
Feb 2021
Copyright:
© 2021 Society for Endocrinology 2021
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Acylated ghrelin (AG) is a gut-derived peptide with growth hormone secretagogue (GHS), orexigenic and other physiological activities mediated by GHS receptor-1a (GHSR). Ghrelin occurs in unacylated form (UAG) with activities opposing AG, although its mechanism of action is unknown. UAG does not antagonize AG at GHSR, and has biological effects on cells that lack this receptor. Because UAG binds to cells, it has been hypothesized that UAG acts via a cell-surface receptor, although this has not been confirmed. This study aimed to identify cell surface proteins to which UAG binds that could modulate or mediate its biological effects. The MCF7 cell-line was used as a model because UAG induces ERK signaling in these cells in the absence of GHSR. Using ligand–receptor capture and LC-MS/MS we identified specific heparan-sulfate proteoglycans (HSPGs) to which UAG interacts on cell surfaces. In line with this, UAG, as well as AG, bind with high affinity to heparin, and heparin and heparinase treatment suppress, whereas HSPG overexpression increases, UAG binding to MCF7 cell surfaces. Moreover, heparin suppresses the ERK response to UAG. However, conversion of the lysines in UAG to alanine, which prevents its binding to heparin and cell surface HSPGs, does not prevent its activation of ERK. Our data show that the interaction of UAG with HSPGs modulates its biological activity in cells. More broadly, the interaction of UAG and AG with HSPGs could be important for the specificity and potency of their biological action in vivo.

Supplementary Materials

    • Supplemental Figure 1. UAG 6-13 (AZP502) does not bind to cells or heparin. A. FACS plots of MCF7 cells incubated with AZP502 (C-terminal biotin, AZP502-bio; N- and C-terminal biotins, bio-AZP502-bio) at different concentrations . (black, unstained; blue, APC stained; range, 200 nM AZP502; red, 2 μM AZP502; green, 20 μM AZP502). B. FPLC of biotinylated UAG and AZP502 on a heparin-sepharose column. Dot-blots of 2 ul samples from 1 ml fractions. Biotinylated peptides were detected using streptavidin-IrDye800CW fluorescent conjugates and imaged using an Odyssey CLx infra-red scanner. Fractions 1-4 are the flow through, followed by a linear NaCl gradient (0-2 M) from fractions 5-10. AZP502 was eluted in the flow through.

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Online ISSN:
1479-6813
Print ISSN:
0952-5041
Copyright:
© 2021 Society for Endocrinology 2021
Page(s):
14
Article Type:
Research Article
Received Date:
21 Oct 2020
Accepted Date:
26 Nov 2020
Print Publication Date:
01 Feb 2021
Online Publication Date:
Feb 2021
Keywords:
extracellular-signal-regulated kinase (ERK); heparin-binding protein; heparan-sulfate proteoglycan (HSPG); heparin; cell-surface protein; peptide hormone; ghrelin; unacylated; ligand-receptor capture

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