Iron is crucial for the survival of living cells, particularly the human pathogen Mycobacterium tuberculosis (M.tb) which uses multiple strategies to acquire and store iron. M.tb synthesizes high affinity iron chelators (siderophores), these extract iron from host iron carrier proteins such as transferrin (Tf) and lactoferrin (Lf). Recent studies have revealed that M.tb may also relocate several housekeeping proteins to the cell surface for capture and internalization of host iron carrier protein transferrin. One of the identified receptors is the glycolytic enzyme Glyceraldehyde-3-phosphate dehydrogenase (GAPDH). This conserved multifunctional protein has been identified as a virulence factor in several other bacterial species. Considering the close structural and functional homology between the two major human iron carrier proteins (Tf and Lf) and the fact that Lf is abundantly present in lung fluid (unlike Tf which is present in plasma), we evaluated whether GAPDH also functions as a dual receptor for Lf. The current study demonstrates that human Lf is sequestered at the bacterial surface by GAPDH. The affinity of Lf-GAPDH (31.7 ± 1.68 nM) is higher as compared to Tf-GAPDH (160 ± 24 nM). Two GAPDH mutants were analyzed for their enzymatic activity and interaction with Lf. Lastly, the present computational studies offer the first significant insights for the 3D structure of monomers and assembled tetramer with the associated co-factor NAD+. Sequence analysis and structural modeling identified the surface exposed, evolutionarily conserved and functional residues and predicted the effect of mutagenesis on GAPDH.
Prokaryotic pathogens establish infection in mammals by capturing the proteolytic enzyme plasminogen (Plg) onto their surface to digest host extracellular matrix (ECM). One of the bacterial surface Plg receptors is the multifunctional glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). In a defensive response, the host mounts an inflammatory response, which involves infiltration of leukocytes to sites of inflammation. This requires macrophage exit from the blood and migration across basement membranes, a phenomenon dependent on proteolytic remodeling of the ECM utilizing Plg. The ability of Plg to facilitate inflammatory cell recruitment critically depends on receptors on the surface of phagocyte cells. Utilizing a combination of biochemical, cellular, knockdown, and approaches, we demonstrated that upon inflammation, macrophages recruit GAPDH onto their surface to carry out the same task of capturing Plg to digest ECM to aid rapid phagocyte migration and combat the invading pathogens. We propose that GAPDH is an ancient, evolutionarily conserved receptor that plays a key role in the Plg-dependent regulation of macrophage recruitment in the inflammatory response to microbial aggression, thus pitting prokaryotic GAPDH against mammalian GAPDH, with both involved in a conserved role of Plg activation on the surface of their respective cells, to conflicting ends.-Chauhan, A. S., Kumar, M., Chaudhary, S., Patidar, A., Dhiman, A., Sheokand, N., Malhotra, H., Raje, C. I., Raje, M. Moonlighting glycolytic protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH): an evolutionarily conserved plasminogen receptor on mammalian cells.
Lactoferrin is a crucial nutritionally important pleiotropic molecule and iron an essential trace metal for all life. The current paradigm is that living organisms have evolved specific membrane anchored receptors along with iron carrier molecules for regulated absorption, transport, storage and mobilization of these vital nutrients. We present evidence for the existence of non-canonical pathway whereby cells actively forage these vital resources from beyond their physical boundaries, by secreting the multifunctional housekeeping enzyme Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) into the extracellular milieu. This effect’s an autocrine/paracrine acquisition of target ligand into the cell. Internalization by this route is extensively favoured even by cells that express surface receptors for lactoferrin and involves urokinase plasminogen activator receptor (uPAR). We also demonstrate the operation of this phenomenon during inflammation, as an arm of the innate immune response where lactoferrin denies iron to invading microorganisms by chelating it and then itself being sequestered into surrounding host cells by GAPDH.
During physiologic stresses, like micronutrient starvation, infection, and cancer, the cytosolic moonlighting protein glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) is trafficked to the plasma membrane (PM) and extracellular milieu (ECM). Our work demonstrates that GAPDH mobilized to the PM, and the ECM does not utilize the classic endoplasmic reticulum–Golgi route of secretion; instead, it is first selectively translocated into early and late endosomes from the cytosol via microautophagy. GAPDH recruited to this common entry point is subsequently delivered into multivesicular bodies, leading to its membrane trafficking through secretion via exosomes and secretory lysosomes. We present evidence that both pathways of GAPDH membrane trafficking are up‐regulated upon iron starvation, potentially by mobilization of intracellular calcium. These pathways also play a role in clearance of misfolded intracellular polypeptide aggregates. Our findings suggest that cells build in redundancy for vital cellular pathways to maintain micronutrient homeostasis and prevent buildup of toxic intracellular misfolded protein refuse.—Chauhan, A. S., Kumar, M., Chaudhary, S., Dhiman, A., Patidar, A., Jakhar, P., Jaswal, P., Sharma, K., Sheokand, N., Malhotra, H., Raje, C. I., Raje. M. Trafficking of a multifunctional protein by endosomal microautophagy: linking two independent unconventional secretory pathways. FASEB J. 33, 5626–5640 (2019). http://www.fasebj.org
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.