The structure of MANF
MANF has 2 distinc and also functionally different domains, C-terminal and N-terminal, connected by an unstructured loop. There are 4 S-S bridges formed in the molecule, 3 of which in the N-terminal and 1 in the C-terminal domain. The structure suggests a dual function of the protein, since the two domains of which it consists, are rather different from one another and show structural similarity to functionally very distinct proteins. In the next chapters, we will take a closer look at the two domains.
N-terminal domain
Here is the of the protein. MANF N-terminus is a saposin-like domain (SAPLIP). SAPLIPs, although they have a rather low sequence homology, have a high structural homology and seem to all share the ability to bind lipids (Hellman et al., 2012). However, since the biological receptor and interaction partners are either completely (receptor(s)) or largely (interactions with other proteins) unknown, the precise molecular mechanisms remain elusive. However, it has been proposed, that the neuroprotective ability of MANF may reside in it's N-terminal domain. Since no hard proof is as of the moment available, the lipind-binding ability would suggest the ability of MANF to enter the cells via endocytosis and has given rise to speculations that maybe extracellular MANF does not even necessarily need an expressed receptor to elicit it's protective ability and enter the cell upon need. Of course, that would mean that using inhibitors of membrane fusion, like botulism toxin, would abolish that function, which does not seem to be the case, leaving all possibilities still open.
C-terminal domain
Here is the of the protein. It is worth mentioning that residues 138-158 are not visible in the crystal structure, probably due to being natively unstructured. This also includes a RTDL sequence, which is homologous to the KDEL sequence, an ER retention sequence. This places MANF biologically into the endoplasmic reticulum and the CKGC disulphide bridge, present also in the same region of the protein, suggests a role in ER-stress response. The same type of CKGC disulphide bridge is namely present in the reductases and disulphide isomerases. The RTDL sequence is also required for the secretion in response to ER-stress (Apostolou et al., 2008).
MANF in disease
MANF knock-out mouse has been generated and characterized, the results were published very recently, revealing a very intruiging phenotype, further confirming the beforementioned interactions and structure/function relations. Namely, the global MANF knock-out develops a severe case of diabetes due to increased ER-stress response (Lindahl et al, 2014). The roles of MANF, proposed by the crystal structure of the C-terminal domain, have thus been confirmed by the in vivo studies.
In part it makes the perfect sense - namely secretion of proteins is a very demanding procedure and the total amount of proteins that pancreas needs to produce and secrete is humongous. This means that the secretory cells have a much higher basal stress level than non-secreting cells. The more proteins are produced the higher the chance and also absolute amount of misfolded and/or unfolded proteins amassed that needs to be dealt with by the intracellular machinery. If now further damage is caused to these reparatory cascades the higher the chance for the cell to reach a point of no return and the UPR becoming too overwhelming and thus the cell goes into apoptosis. As expected thus, Lindahl et al. show the increased amount of TUNEL-positive pancreatic beta cells in MANF knock-out animals, confirming that eventually, beta cells undergo programmed cell death in response to increased ER-stress.
However, MANF was discovered as a neurotrophic factor and it's ability to protect neurons in different lesion models is remarkable and cannot simply be explained by any structural or functional features that we known of to this date. Furthermore, in Drosophila melanogaster there are no two different CDNF and MANF proteins, is just one homologue, called DmMANF. The knock-out flies of that die due to a very specific developmental defect in the dopaminergic system in the central nervous system [1]. One would thus expect to see a more brain-related phenotype also in other organisms, but that does not seem to be the case at least for mice.
That would, in my opinion, argue for the lack of knowledge regarding the unstructured regions of the MANF protein, further covered in the next chapter.
Conclusions and future perspectives
Structurally, MANF is a difficult protein to study. It has been shown, that it is secreted upon ER stress and it does contain sequences which clearly suggest a role in ER-stress (RTDL sequence in the very C-terminus, CKGC disulphide bridge in the same region) little is known about it's interaction with other proteins. The only protein which has been successfully co-immunoprecipitated (Glembotski et al., 2012) with MANF, is GRP78, an essential component of the UPR pathway. This suggest a very conserved role of MANF in the very primary regulation of ER-stress response, since the dissociation of GRP78 from PERK, IRE1 or ATF6 is required for activation of any of these different pathways. These are just a few examples of functions that are being attributed to the C-terminus of MANF.
However, exactly how is MANF able to protect neural cells from ischemic or toxin-induced damage, remains a total mystery, since while ER-stress is an essential part of the cytotoxic mechanisms in these paradigms, there is a lot more going on, most importantly the production of reactive oxygen species, unspecific necrosis due to oxygen deprivation as well as apoptosis in the penumbra region in the case of stroke. To date, also partially speculated upon in publications discussing N-terminal structure of MANF, these functions are thought to be elicited or reside in that given part of the protein. However, convincing in vitro and in vivo studies showing the neuroprotective and neurorestorative function of isolated N-terminal domain of MANF are lacking, giving rise to the speculation that there is none. There are, of course, other explanations. One possibility is that in order for the N-terminus to be biologically active, it needs to be connected to C-terminus, even if the C-terminus in the given experimental setting is not, per se, required for the rescue mechanism.
In my opinion, a far more interesting explanation would be in the so far unstructured regions of the protein. As mentioned before, the N and C termini of the MANF protein are connected to one another by a long unstructured loop and in addition to that, the very last 20 C-terminal amino acids are not visible in the crystal structure. I would propose that this is only true for the unbinded case and upon binding to any of the interaction partners the structure of MANF is changed rather principially, meaning that the natively unstructured areas form a distinct structural complex with their ligand(s), probably also being responsible for a unique, maybe even a unique set of binding regions or pockets.
All in all, the future studies of MANF biology are or should be aimed at explaining the exact intracellular mechanismis of MANF and mainly it's interaction partners. The successful co-immunoprecipitation of MANF with GRP78 is a big step forward and hopefully in the near future we shall see more publications on that matter as well as a crystal structure of MANF in complex with GRP78.
