BIS(MONOACYLGLYCERO)PHOSPHATE


STRUCTURE, OCCURRENCE AND BIOCHEMISTRY


1.  Structure and Occurrence

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. It was first termed ‘lysobisphosphatidic acid’, although it is only superficially related to phosphatidic acid per se and can in fact be considered a structural isomer of phosphatidylglycerol. It is an interesting lipid from several standpoints. For example, its stereochemical configuration 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 recently been confirmed by 1H NMR spectroscopy with chiral shift reagents.

All the initial studies from the first publication until recently suggested that positions sn-3 and 3’ in the glycerol moieties were esterified with fatty acids. On the other hand, there is an increasing school of thought to the effect that the fatty acids are esterified to position sn-2 and sn-2’ in the native molecule. Certainly, fatty acids in a lipid with the latter structure would be expected to undergo rapid acyl migration when subjected to most extraction and isolation procedures, resulting in the most thermodynamically stable form with fatty acids in the primary positions.

Formula of bis(monacylglycero)phosphate

Synthetic studies have shown how readily this can occur. Acyl migration might also be expected to take place under the acidic conditions in lysosomes (see below). Further evidence for this hypothesis comes from biosynthetic considerations, from the fact that a specific phospholipase A2 has been found that degrades bis(monoacylglycero)phosphate at the physiological pH in lysosomes, from physical chemical studies and from improved chromatographic conditions, which show three peaks for the lipid. None of this is conclusive, but together with doubts about some of the early NMR data, it suggests that a re-evaluation of the basic structure of this molecule is necessary. Those most active in the study of this lipid favour the structure illustrated below.

Probable structure of dioleoyl bis(monacylglycero)phosphate

Certainly, the 2,2’-dioleoyl form rather than the 3,3’-isomer is essential for the function of bis(monacylglycero)phosphate in cholesterol metabolism in lysosomes (see below).

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), 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); to my knowledge, no other natural lipid contains so much of this fatty acid. Lung alveolar macrophages contain mainly fatty acids of the n-6 family also. In contrast, the metabolically important lysosomal lipid contains almost 70% 22:6(n-3). Baby hamster kidney (BHK) fibroblast cells are very different in that they contain more than 80% of oleate. Such unusual compositions must confer distinctive properties in membranes and suggest quite specific functions, most of which have yet to be revealed.

Table 1. Fatty acid composition (wt% of the total) of bis(monoacylglycero)phosphate from various tissues.
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); 2, Huterer, S. and Wherrett, J. J. Lipid Res., 20, 966-973 (1979); 3, Luquain, C. et al., Biochem. J., 351, 795-804 (2000); 4, Brotherus, J. and Renkonen, O. Chem. Phys. Lipids, 13, 11-20 (1974).

Bis(monacylglycero)phosphate is usually a rather minor component of animal tissues (~1-2%), although it is easily misidentified as phosphatidic acid in many chromatographic systems. It is found at low levels in plasma, where it is associated both with the lipoprotein fractions (40%) and the lipoprotein-deficient compartment (60%).

Scottish thistleHowever, 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. Lysosomes are the digestive organelles of the cell and are rich in hydrolytic enzymes at an acidic pH (4.6 to 5). Cellular constituents, including excess nutrients, growth factors and foreign antigens are captured by receptors on the cell surface, for uptake and delivery to lysosomes. Within the cell, receptors such as the mannose-6-phosphate receptor bind and divert hydrolytic enzymes from biosynthetic pathways to the lysosomes. These molecules pass through 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. There, the hydrolases are activated and the unwanted materials are digested. It is the internal membranes of mature or ‘late’ endosomes and the lysosomes that contain the unique lipid, bis(monacylglycero)phosphate. Indeed, there appear to be inner membranes of the late endosomes that contain as much as 70% of the phospholipids as this lipid.

If the reported presence of bis(monacylglycero)phosphate in some alkalophilic strains of Bacillus species can be confirmed, this will be the only known exception to the rule that this lipid is strictly of mammalian origin and not present in prokaryotes, yeasts and higher plants.


2.  Biosynthesis and Function

There is good evidence that bis(monacylglycero)phosphate is synthesised from phosphatidylglycerol, primarily in the endosomal system. Although the later steps have still to be demonstrated experimentally, the scheme outlined below is believed to be the primary route. In the first step, a phospholipase A2 removes the fatty acid from position sn-2 of phosphatidylglycerol. In the second, the lysophosphatidylglycerol is acylated on the sn-2’ position of the head group glycerol moiety to yield sn-3:sn-1’ bis(monacylglycero)phosphate, by means of a transacylase reaction with lysophosphatidylglycerol as both the acyl donor and acyl acceptor. The third step leading to the stereospecific conversion of the precursor molecule to the unusual sn-1:sn-1' S-configuration has still to be adequately described but must involve removal of the fatty acid from position sn-1 of the primary glycerol unit and a rearrangement of the phosphoryl ester from the sn-3 to the sn-1 position. Finally position sn-2 of the primary glycerol unit is esterified, probably by a transacylation reaction with another phospholipid as donor (thence the distinctive fatty acid compositions).

Biosynthesis scheme for bis(monacylglycero)phosphate

The intracellular site of this synthesis has still to be confirmed. 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. Other biosynthetic routes may be possible, but cardiolipin has been ruled out as a potential precursor.

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. It is a cone-shaped molecule, like cardiolipin, 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. It also may associate with specific proteins, which carry a positive charge under the acidic conditions in lysosomes.

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. The fatty acid constituents may turn over rapidly by transacylation, but the glycerophosphate backbone is stable. For example, it is not touched by the main phospholipases that hydrolyse phosphatidylcholine and phosphatidylethanolamine. However, several phospholipases have been identified that may be involved in catabolism under acidic conditions and other local environmental factors, although the control mechanisms are not known.

Scottish thistleThe endosomal membranes are a continuation of the lysosomal membranes, and their function as discussed above is to sort and recycle material back to the plasma membrane and endoplasmic reticulum. 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 is an important element of cholesterol homeostasis in that it regulates cholesterol transport by acting as a collection and re-distribution point for the free cholesterol generated in this way. For example, when lysosomal membranes are incubated with antibodies to bis(monacylglycero)phosphate, substantial amounts of cholesterol accumulate. The process is under the control of Alix/AlP1, which is a cytosolic protein that interacts specifically with this lipid and is involved in sorting into multivesicular endosomes.

Bis(monacylglycero)phosphate is known to greatly stimulate the enzymes involved in the degradation of glycosylceramides, such as the sphingolipid activator proteins like the saposins. In this instance, it may simply function to provide a suitable environment for the interaction of the glycosphingolipid hydrolases and their activator. In addition, it has a dynamic role in the provision of arachidonate for eicosanoid production in alveolar macrophages.

In consequence, it has become evident that bis(monacylglycero)phosphate is involved in the pathology of lysosomal storage diseases such as Niemann-Pick C disease (cholesterol accumulation) and certain drug-induced lipidoses. In these circumstances, its composition tends to change to favour molecular species that contain less of the polyunsaturated components. Dysregulation of bis(monacylglycero)phosphate metabolism and thence of cholesterol homeostasis may also 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.


3.  Related Lipids

Formula of 'semilysobisphosphatidic acid''Semilysobisphosphatidic acid', i.e. with three moles of fatty acid per mole of lipid, is occasionally found in tissues also. In particular, it is concentrated 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 at the moment 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).

Archaeal glycerolipids also have the phosphate moiety linked to position sn-1 of the glycerol moiety, but the biosynthetic mechanism (and function) of these lipids is entirely different from that of bis(monoacylglycerol)phosphate.

Modern mass spectrometric methods involving electrospray ionization appear to be well suited to the analysis of of bis(monoacylglycero)phosphate, but especially when used in conjunction with liquid chromatography as it is isobaric with phosphatidylglycerol.


Recommended Reading


W.W. Christie

James Hutton Institute (and Mylnefield Lipid Analysis), Invergowrie, Dundee (DD2 5DA), Scotland.

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