Bis(monoacylglycero)phosphate
Bis(monoacylglycero)phosphate ('BMP') was first isolated from pig lung in 1967 and is now known to be a common if minor constituent of all animal tissues, but mainly in the lysosomal compartment of cells and in exosomes. It was first incorrectly termed ‘lysobisphosphatidic acid’, although it is only superficially related to phosphatidic acid per se and might better be considered a structural analogue of phosphatidylglycerol. A unique stereochemistry makes this lipid of special interest, but many aspects of its structure, biosynthesis and function remain uncertain. It is not found in plants and its occurrence in bacteria is problematic.
1. Structure and Occurrence
The stereochemical configuration of bis(monoacylglycero)phosphate differs from that of all other animal glycero-phospholipids in that the phosphodiester moiety is linked only to positions sn-1 and sn-1′ of glycerol, rather than to positions sn-3 and sn-3′. This has been established by many methods, including confirmation by 1H NMR spectroscopy with chiral shift reagents.
Although there has been a school of thought to the effect that the fatty acids are esterified to position sn-2 and sn-2′ in the native molecule with the latter structure could undergo rapid acyl migration when subjected to most extraction and isolation procedures, biosynthetic studies now suggest that this is unlikely. However, it canot be discounted that this could occur sppontaneously under the acidic conditions in lysosomes, and there is one report that he 2,2′-dioleoyl form rather than the more stable sn‑3,3′‑isomer is essential for the function of bis(monacylglycero)phosphate in cholesterol metabolism in lysosomes.
Bis(monacylglycero)phosphate is usually a rather minor component of animal tissues (~1-2%), but it is highly enriched in the lysosomal membranes of liver and other tissues, where it can amount to 15% or more of the phospholipids, and it is now recognized as a marker for this organelle. Cellular constituents, including excess nutrients, growth factors and foreign antigens, are captured by receptors on the cell surface, such as the mannose-6-phosphate receptor, for uptake and delivery to an intermediate heterogeneous set of organelles known as endosomes, which act as a kind of sorting station where the receptors are recycled before the hydrolases and other materials are directed to the lysosomes, the digestive organelles of the cell enriched in hydrolytic enzymes at an acidic pH (4.6 to 5). There, the hydrolases are activated, and the unwanted materials are digested. It is the internal membranes of mature or ‘late’ endosomes and lysosomes that contain bis(monacylglycero)phosphate, where it amounts to as much as 70% of the phospholipids or 11% of the phospholipids of some macrophage/microglial cell lines, reflecting the increased endo-lysosomal capacity of these cells.
Whatever the positions of the fatty acids on the glycerol molecule, their compositions can be distinctive with 18:1(n-9) and 18:2(n-6) and/or 20:4 and 22:6(n-3) being abundant, although this is highly dependent on the specific tissue, cell type or organelle (see Table 1). For example, the testis lipid contains more than 70% 22:5(n‑6), and to my knowledge, no other natural lipid contains so much of this fatty acid. Lung alveolar macrophages contain mainly C18 fatty acids, and baby hamster kidney (BHK) fibroblast cells are very different in that they contain more than 80% of oleate. In contrast, the metabolically important lipid isolated from rat liver lysosomes specifically contains almost 70% 22:6(n-3). Such unusual compositions must confer distinctive properties in membranes and suggest quite special functions, most of which have yet to be revealed. In plasma, the lipid is found at trace levels only where it is associated both with the lipoprotein fractions (40%) and the lipoprotein-deficient compartment (60%).
Table 1. Fatty acid composition (wt% of the total) of bis(monoacylglycero)phosphate from various tissues. |
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| Fatty acid | Rat liver lysosomes | Human liver | Rabbit lung macrophages | Rat uterine stromal cells | Rat testis | BHK cells |
|---|---|---|---|---|---|---|
| 16:0 | 3 | 6 | 4 | 6 | 5 | 4 |
| 18:0 | 1 | 5 | 6 | 3 | 3 | trace |
| 18:1 | 5 | 57 | 47 | 30 | 5 | 83 |
| 18:2 | 6 | 10 | 29 | 2 | 1 | 6 |
| 20:4 | 6 | 4 | 4 | |||
| 22:4 | 6 | 5 | ||||
| 22:5(n-6) | } 4 | } 2 | 3 | 70 | ||
| 22:5(n-3) | 8 | trace | ||||
| 22:6(n-3) | 69 | 9 | 1 | 36 | 5 | |
| Reference | 1 | 1 | 2 | 3 | 3 | 4 |
| 1, Wherrett, J.R. and Huterer, S. Lipids, 8, 531-533 (1973); DOI. 2, Huterer, S. and Wherrett, J. J. Lipid Res., 20, 966-973 (1979); DOI. 3, Luquain, C. et al., Biochem. J., 351, 795-804 (2000); DOI. 4, Brotherus, J. and Renkonen, O. Chem. Phys. Lipids, 13, 11-20 (1974); DOI. | ||||||
This lipid may not be uniquely of animal origin, as the plant bacterial-pathogen Agrobacterium tumefaciens can take up lyso-phosphatidylglycerol and convert it to two distinct isoforms of BMP, and it has been reported from some alkalophilic strains of Bacillus species, although it is not known whether any of these have the distinctive stereochemistry of the animal equivalent.
2. Biochemistry and Function
Biosynthesis: Although some questions remain to be answered, there is good evidence that bis(monacylglycero)phosphate is synthesised from phosphatidylglycerol via lysophosphatidylglycerol (LPG) in the endosomal system. In the first rate-limiting step, lysosomal phospholipase A2 removes the fatty acid from position sn-2 of phosphatidylglycerol, before in the second step, the lysophosphatidylglycerol produced is acylated on the sn-2′ position of the head group glycerol moiety by means of a transacylase reaction with a further molecule of lysophosphatidylglycerol to yield sn-3:sn-1′ bis(monacylglycero)phosphate. A protein designated CLN5 is the BMP synthase with, and this enzyme catalyses an energy-independent base exchange reaction between two LPG molecules with glycerophosphoglycerol as a by-product; CLN5 is deficient in Batten disease, and studies with CNL5-knockout cells show a deficiency of BMP and a substantial accumulation of lysophosphatidylglycerol (LPG) in the lysosomes.
However, the primary source of phosphatidylglycerol is unknown, possibly mitochondria via crosstalk with lysosomes, nor is it known how the novel stereospecificity of BMP arises, i.e., whether it ours spontaneously during biosynthesis or requires a separate isomerase. A further question is how the distinctive fatty acid composition is achieved. Bis(monacylglycero)phosphate with the sn-3:sn-1′ configuration has been isolated from BHK and rat uterine stromal cells, but it may be an intermediate in the biosynthetic pathway. While other biosynthetic routes may be possible, cardiolipin has been ruled out as a potential precursor.
Physical and chemical properties: The properties of bis(monacylglycero)phosphate in membranes will be highly dependent on fatty acid composition, but the function in lysosomes is of particular interest and is under active investigation. It certainly has a structural role in developing the complex intraluminal membrane system, aided by a tendency not to form a bilayer. Like cardiolipin, it is a cone-shaped molecule with a small but hydrated head group, which is negatively charged, and it encourages fusion of membranes or formation of internal vesicles (invagination) at the pH in the endosomes where it may associate with specific proteins, which carry a positive charge under the acidic conditions.
The unique stereochemistry of bis(monacylglycero)phosphate means that it is resistant to most phospholipases, and this may hinder or prevent self-digestion of the lysosomal membranes. Although the fatty acid constituents may turn over rapidly by transacylation, the glycerophosphate backbone is stable, and it is not touched by the main phospholipases that hydrolyse phosphatidylcholine and phosphatidylethanolamine. However, some phospholipases have been tentatively identified that may be involved in catabolism under acidic conditions and other local environmental factors, although the control mechanisms are not known (see below).
Various extracellular membrane-bound vesicles secreted by most living cells and are found in biological fluids that act as mediators of cell-cell communication and have roles in patho-physiological processes. Small vesicles of this kind (<200 nm), usually termed 'exosomes', are of endosomal origin with bis(monoacylglycero)phosphate as an identifying lipid marker and probably a participant in their formation. For example, exosomes shed from reticulocytes are nano-vesicles that carry a cargo of lipids and other materials into the circulation, and they can be distinguished from secreted microvesicles of a different biological origin by their content of bis(monacylglycero)phosphate. The 22:6-22:6 species is reportedly a biomarker for phagocytizing macrophages/microglia cells during cerebral ischaemia and of urinary exosomes as a consequence of drug-induced endolysosomal dysfunction.
Function in lysosomes: Bis(monoacylglycero)phosphate is negatively charged at lysosomal pH and can form a stable docking station in the endosomal membranes for luminal acid hydrolases that are positively charged at acidic pH and require a water-lipid interface for activation. BMP-enriched vesicles serve in endosomal-lysosomal trafficking and activate these enzymes. By binding in this way, it stimulates the activity of lysosomal lipid-degrading enzymes, including acid sphingomyelinase, acid ceramidase, acid phospholipase A2 and an acid lipase with the capacity to hydrolyse triacylglycerols and cholesterol esters, while by binding to the heat-shock protein Hsp70, which promotes survival of stressed cells by inhibiting lysosomal membrane permeabilization on the inner lysosomal membrane, it activates the acid sphingomyelinase for stabilization of lysosomes. Thus, bis(monoacylglycero)phosphate plays a central role in determining the fate of the lysosomal content by stimulating degradation and sorting of lipids.
The
endosomal membranes are a continuation of the lysosomal membranes, and they have the same function in sorting and recycling material back to
the plasma membrane and endoplasmic reticulum, and in particular, they are an important element of
cholesterol homeostasis.
Thus, low-density lipoproteins (LDL) internalized in the liver reach the late endosomes where the constituent cholesterol esters are
hydrolysed by an acidic cholesterol ester hydrolase.
The characteristic network of bis(monacylglycero)phosphate-rich membranes contained within multivesicular late endosomes regulates cholesterol
transport by acting as a collection and re-distribution point for the free cholesterol generated in this way via intralumenal vesicles
membranes before being redistributed to the endosomal/lysosomal limiting membrane and then to the rest of the cell.
The process is under the control of Alix/AlP1, a cytosolic protein that interacts specifically with this lipid and is involved in sorting
into the multivesicular endosomes.
In macrophages such as those in foam cells, bis(monacylglycero)phosphate is likewise involved in the regulation of intracellular cholesterol
traffic where it is reported to have a protective effect by inhibiting the production of pro-apoptotic oxysterols, while in alveolar
macrophages, it has a dynamic role in the provision of arachidonate for eicosanoid production.
In animal models, it has been demonstrated that different tissues have characteristic bis(monacylglycero)phosphate profiles that adapt to the nutritional and metabolic state, especially in hepatocytes, brown adipocytes and pancreatic cells, suggesting that this lipid has a role in how these adapt to nutrient availability and ambient temperatures.
Disease: In consequence of its role in lysosomes, it has become evident that bis(monacylglycero)phosphate is involved in the pathology of lysosomal storage diseases such as Niemann-Pick C disease, where cholesterol accumulation is a distinctive feature. Similarly, high levels of bis(monacylglycero)phosphate enriched in docosahexaenoic acid are found in the retinal pigment epithelium in Stargardt disease, which is characterized by juvenile onset retinal degeneration, and they are presumed to be a consequence of late endosomal/lysosomal dysfunction. It also accumulates as a secondary storage material in the brain of a broad range of mammals with gangliosidoses. In these circumstances, its concentration can increase substantially, probably as a secondary event, and its composition may change to favour molecular species that contain less of the polyunsaturated components. Reduced levels of bis(monacylglycero)phosphate lower the activity of the lysosomal enzyme GCase to cause an accumulation of glucosylsphingosine, a factor in frontotemporal dementia. Dysregulation of bis(monacylglycero)phosphate metabolism and thence of cholesterol homeostasis may be relevant to atherosclerosis.
Bis(monacylglycero)phosphate is an antigen recognized by autoimmune sera from patients with a rare and poorly understood disease known as antiphospholipid syndrome, so it is obviously a factor in the pathological basis of this illness. In general in such diseases, bis(monoacylglycerol)phosphate levels are elevated in the circulation and its fatty acid composition changes, so that this can be used as a diagnostic biomarker that enables a clear distinction between lipid overload and drug-induced lysosomal storage diseases. An elevated concentration of di-docosahexaenoyl bis(monoacylglycerol)phosphate in urine be a biomarker of drug-induced phospholipidosis, and it may be a marker for metastatic cancers of macrophage origin. It has been suggested that high levels of this molecular species in breast cancer function to scavenge reactive oxygen species in lysosomes to protect them from oxidant-induced lysosomal membrane permeabilization.
Non-enveloped viruses of the family Reoviridae, which include mammalian pathogens, enter cells without the aid of a limiting membrane and thus cannot fuse with host cell membranes. The bluetongue virus was the first to be shown to use bis(monoacylglycero)phosphate in late endosomes and endolysosomes for membrane penetration and entry into host cells, and it has since been established that this mechanism may influence the infectivity of many other viruses, including COVID-19, influenza and Lassa virus, by enabling them to hijack the endosomal machinery leading to fusion of viral and endosomal membranes and release of viral RNA into the cytosol.
Catabolism: Although bis(monoacylglycero)phosphate is resistant to hydrolysis by many of the common phospholipases because of its unique stereochemistry. it is now known to be hydrolysed with high specificity in liver by a hydrolase designated α/β-hydrolase domain-containing 6 or ABHD6, once thought to be mainly a monoacylglycerol lipase capable of degrading the endocannabinoid 2‑arachidonoylglycerol. An enzyme designated ABHD12 may function in a similar manner to ABHD6 in brain, while some hydrolysis has been observed in vitro at least by the lysosomal acid sphingomyelinase.
3. Related Lipids
'Semilysobisphosphatidic
acid', i.e., with three moles of fatty acid per mole of lipid, is occasionally found in tissues.
It is sometimes found in the Golgi membranes, where the relative amount varies in different regions,
but can attain as much as 15% of the total phospholipids in those compartments that are most active biologically.
It would not be at all surprising if this lipid were found to have a distinctive role in the Golgi complex,
but this is a matter of speculation.
The fully acylated lipid, bis(diacylglycero)phosphate or 'bisphosphatidic acid', has been found in lysosomes from cultured hamster fibroblasts (BHK21 cells). In addition, it has been detected in bacteria, where it presumably has a different stereochemistry because of the mechanism of its synthesis from phosphatidylglycerol.
Although Archaeal glycerolipids have the phosphate moiety linked to position sn-1 of the glycerol moiety, the biosynthetic mechanism and functions of these lipids are entirely different from those of bis(monoacylglycerol)phosphate.
4. Analysis
Bis(monoacylglycero)phosphate is easily misidentified as phosphatidic acid in many chromatographic systems. Modern mass spectrometric methods involving electrospray ionization now appear to be well suited to analysis, but especially when used in conjunction with liquid chromatography to ensure separation from phosphatidylglycerol with which it is isobaric.
Recommended Reading
- Akgoc, Z., Sena-Esteves, M., Martin, D.R., Han, X., d'Azzo, A. and Seyfried, T.N. Bis(monoacylglycero)phosphate: a secondary storage lipid in the gangliosidoses. J. Lipid Res., 56, 1006-1013 (2015); DOI.
- Christie, W.W. and Han, X. Lipid Analysis - Isolation, Separation, Identification and Lipidomic Analysis (4th edition), 446 pages (Oily Press, Woodhead Publishing and now Elsevier) (2010) - see Science Direct.
- Czolkoss, S., Borgert, P., Poppenga, T., Holzl, G., Aktas, M. and Narberhaus, F. Synthesis of the unusual lipid bis(monoacylglycero)phosphate in environmental bacteria. Environm. Microbiol., 23, 6993-7008 (2021); DOI.
- Goursot, A. Mineva, T., Bissig, C., Gruenberg, J. and Salahub, D.R. Structure, dynamics, and energetics of lysobisphosphatidic acid (LBPA) isomers. J. Phys. Chem. B, 114, 15712-15720 (2010); DOI.
- Grabner, G.F, and 12 others. Metabolic regulation of the lysosomal cofactor bis(monoacylglycero)phosphate in mice. J. Lipid Res., 61, 995-1003 (2020); DOI.
- Gruenberg, J. Life in the lumen: The multivesicular endosome. Traffic., 21, 76-93 (2020); DOI.
- Hullin-Matsuda, F., Colosetti, P., Rabia, M., Luquain-Costaz, C. and Delton, I. Exosomal lipids from membrane organization to biomarkers: Focus on an endolysosomal-specific lipid. Biochimie, 203, 77-92 (2022); DOI.
- Luquain, C., Dolmazon, R., Enderlin, J.M., Laugier, G., Lagarde, M. and Pageaux, J.F. Bis(monoacylglycerol) phosphate in rat uterine stromal cells: structural characterization and specific esterification of docosahexaenoic acid. Biochem. J., 351, 795-804 (2000); DOI.
- Medoh, U.N., Hims, A., Chen, J.Y., Ghoochani, A., Nyame, K., Dong, W.T. and Abu-Remaileh, M. The Batten disease gene product CLN5 is the lysosomal bis(monoacylglycero)phosphate synthase. Science, 381, 1182-1188 (2023); DOI.
- Pribasnig, M.A. and 18 others. α/β Hydrolase domain-containing 6 (ABHD6) degrades the late endosomal/lysosomal lipid bis(monoacylglycero)phosphate. J. Biol. Chem., 290, 29869-29881 (2015); DOI.
- Schulze, H. and Sandhoff, K. Sphingolipids and lysosomal pathologies. Biochim. Biophys. Acta, Lipids, 1841, 799-810 (2014); DOI.
- Showalter, M.R., Berg, A.L., Nagourney, A., Heil, H., Carraway, K.L. and Fiehn, O. The emerging and diverse roles of bis(monoacylglycero) phosphate lipids in cellular physiology and disease. Int. J. Mol. Sci., 21, 8067 (2020); DOI.
- Tan, H.H., Makino, A., Sudesh, K., Greimel, P. and Kobayashi, T. Spectroscopic evidence for the unusual stereochemical configuration of an endosome-specific lipid. Angew. Chemie Int. Ed., 51, 533-535 (2012); DOI.
- Wang, X.Y., Schmitt, M.V., Xu, L.N., Jiao, Y.P., Guo, L.J., Lienau, P., Reichel, A. and Liu, X.H. Quantitative molecular tissue atlas of bis(monoacylglycero)phosphate and phosphatidylglycerol membrane lipids in rodent organs generated by methylation assisted high resolution mass spectrometry. Anal. Chim. Acta, 1084, 60-70 (2019); DOI.
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© Author: William W. Christie |  |
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