Role of two sequence motifs of mesencephalic astrocyte-derived neurotrophic factor in its survival-promoting activity

Abstract

Mesencephalic astrocyte-derived neurotrophic factor (MANF) is a prosurvival protein that protects the cells when applied intracellularly in vitro or extracellularly in vivo. Its protective mechanisms are poorly known. Here we studied the role of two short sequence motifs within the carboxy-(C) terminal domain of MANF in its neuroprotective activity: the CKGC sequence (a CXXC motif) that could be involved in redox reactions, and the C-terminal RTDL sequence, an endoplasmic reticulum (ER) retention signal. We mutated these motifs and analyzed the antiapoptotic effect and intracellular localization of these mutants of MANF when overexpressed in cultured sympathetic or sensory neurons. As an in vivo model for studying the effect of these mutants after their extracellular application, we used the rat model of cerebral ischemia. Even though we found no evidence for oxidoreductase activity of MANF, the mutation of CXXC motif completely abolished its protective effect, showing that this motif is crucial for both MANF's intracellular and extracellular activity. The RTDL motif was not needed for the neuroprotective activity of MANF after its extracellular application in the stroke model in vivo. However, in vitro the deletion of RTDL motif inactivated MANF in the sympathetic neurons where the mutant protein localized to Golgi, but not in the sensory neurons where the mutant localized to the ER, showing that intracellular MANF protects these peripheral neurons in vitro only when localized to the ER.

Mesencephalic astrocyte-derived neurotrophic factor (MANF), a new player in endoplasmic reticulum diseases: structure, biology, and therapeutic roles

“…MANF contains eight conserved cysteines that form four intramolecular disulfide bonds including two in CXXC sequences 28 . Although the CXXC motif can be found in thiol-disulfide oxidoreductase 26, 30 , MANF is not involved in redox reactions. The CKGC disulfide bridge in the MANF C-terminus is crucial for MANF’s neuroprotective activity 30 .…”

“…Although the CXXC motif can be found in thiol-disulfide oxidoreductase 26, 30 , MANF is not involved in redox reactions. The CKGC disulfide bridge in the MANF C-terminus is crucial for MANF’s neuroprotective activity 30 . In addition, the four amino acids, RTDL at the extreme C terminus of MANF (Fig 2) can bind to the KDEL receptors (KDELRs), which is localized primarily in the cis -Golgi, to facilitate the cycling of MANF from the Golgi to the ER 30, 31 .…”

“…The CKGC disulfide bridge in the MANF C-terminus is crucial for MANF’s neuroprotective activity 30 . In addition, the four amino acids, RTDL at the extreme C terminus of MANF (Fig 2) can bind to the KDEL receptors (KDELRs), which is localized primarily in the cis -Golgi, to facilitate the cycling of MANF from the Golgi to the ER 30, 31 . In vitro deletion of RTDL in SCG neurons almost completely abrogates the intracellular pro-survival effect of MANF by causing it to localize to the Golgi complex 30 .…”

“…In addition, the four amino acids, RTDL at the extreme C terminus of MANF (Fig 2) can bind to the KDEL receptors (KDELRs), which is localized primarily in the cis -Golgi, to facilitate the cycling of MANF from the Golgi to the ER 30, 31 . In vitro deletion of RTDL in SCG neurons almost completely abrogates the intracellular pro-survival effect of MANF by causing it to localize to the Golgi complex 30 . Interestingly, the interaction between RTDL and KDELRs can also be found at the plasma membrane of SH-SY5Y cells, which is required for MANF binding at cell surface 31 .…”

Mesencephalic astrocyte‐derived neurotrophic factor affords neuroprotection to early brain injury induced by subarachnoid hemorrhage via activating Akt‐dependent prosurvival pathway and defending blood‐brain barrier integrity

“…It was, therefore, speculated that the cell protection of MANF might be produced by relieving ER stress and modulating UPR (2,13). In recent years, researchers have reported that increased MANF is able to reduce neuronal apoptosis (9,(14)(15)(16). However, the underlying mechanisms that enable cell protection have not been elucidated.…”