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. Author manuscript; available in PMC: 2021 Dec 20.
Published in final edited form as: ACS Chem Biol. 2021 Oct 14;16(12):2757–2765. doi: 10.1021/acschembio.1c00393

Mapping sphingolipid metabolism pathways during phagosomal maturation

Neelay Mehendale 1, Roop Mallik 2,*, Siddhesh S Kamat 1,*
PMCID: PMC7612119  EMSID: EMS137024  PMID: 34647453

Abstract

Phagocytosis is an important physiological process, which, in higher organisms is a means of fighting infections and clearing cellular debris. During phagocytosis, detrimental foreign particles (e.g. pathogens, apoptotic cells) are engulfed by phagocytes (e.g. macrophages), enclosed in membrane-bound vesicles called phagosomes, and transported to the lysosome for eventual detoxification. During this well-choreographed process, the nascent phagosome (also called early phagosome, EP) undergoes a series of spatiotemporally regulated changes in its protein and lipid composition, and matures into a late phagosome (LP), that subsequently fuses with the lysosomal membrane to form the phagolysosome. While several elegant proteomics studies have identified the role of unique proteins during phagosomal maturation, corresponding lipidomics studies are sparse. Recently, we reported a comparative lipidomics analysis between EPs and LPs, and showed that ceramides are enriched on the LPs. Further, we found that this ceramide accumulation on LPs was orchestrated by ceramide synthase 2, inhibition of which, hampers phagosomal maturation. Following up on this study, here, using biochemical assays, we first show that increased ceramidase activity on EPs also significantly contributes to the accumulation of ceramides on LPs. Next, leveraging lipidomics, we show that de novo ceramide synthesis does not significantly contribute to the ceramide accumulation on LPs, while concomitant to increased ceramides, glucosylceramides are substantially elevated on LPs. We validate this interesting finding using biochemical assays, and show that LPs indeed have heightened glucosylceramide synthase activity. Taken together, our studies provide interesting insights and possible new roles of sphingolipid metabolism during phagosomal maturation.

Introduction

Phagocytosis is an evolutionarily conserved immunological process, where solid particles ≥ 500 nm are engulfed by a class of cells called phagocytes1–3. In mammals (including humans), these phagocytes, typically macrophages or immune cells from the monocyte lineage, leverage the process of phagocytosis as a means of clearing detrimental solid particles (e.g. microbial and/or viral pathogens, or apoptotic cells) and are essential innate immune cells for fighting invading infections and/or clearing damaged cells to prevent tissue (organ) and/or systemic damage2, 3. The detrimental solid particles that are engulfed and internalized by phagocytes are then enclosed tightly in membrane bound vesicles called phagosomes, and are subsequently transported to the lysosome for eventual degradation2. During phagocytosis, these phagosomes undergo a well-orchestrated sequence of events that are spatiotemporally regulated, whereby they “mature” from a nascent phagosome (also called early phagosome (EP)) to a late phagosome (LP)1, 2, 4. This LP then fuses with the lysosomal membrane to form the phagolysosome, and thus marks the end of the transport of the solid particles from the extracellular environment to the lysosome for degradation and/or detoxification1, 2. During this maturation process, both the protein and lipid composition of the phagosomes change significantly through transient interactions with various sub-cellular components and their membranes. This process is popularly known now as the “kiss and run mechanism”5, and these protein and lipid changes are thought to be critical in directing the maturing phagosome from the cellular periphery to the lysosome.

Several shotgun proteomics studies have investigated the changes in the global protein content of phagosomes during the maturation process, and these have resulted in establishing protein markers that are hallmarks of various stages of maturation6–8. For example, the EP is enriched with Rab5 (a small GTPase from the Rab family) and the early endosome antigen 1 (EEA1), that together initiate the membrane formation for the engulfment of the foreign solid particle, while the LP is enriched with the GTPase Rab7 and the lysosomal associated membrane proteins 1 (LAMP1), both of which, are essential in directing the LP to the lysosome and facilitating its fusion to form the phagolysosome7, 8. Interestingly, concomitant to the aforementioned protein changes, during maturation, the luminal pH within the phagosomes, progressively decreases from neutral (~ 7 – 7.5) to acidic (~ 4.5 – 5), and this drop in pH is thought to be mediated by the recruitment of vacuolar ATPases (vATPases) on LPs4. Contrary to the number of proteomics studies, surprisingly, lipidomics studies have until recently been limited to only a single lipid class9. For example, it has been shown that cholesterol is significantly enriched on LPs, and that physiologically, this accumulation of cholesterol is necessary to form stable lipid rafts that recruit dynein motors, which generate sufficient intracellular forces in facilitating the unidirectional motion of the ageing phagosomes (LPs) towards the lysosome10.

Recently, we performed a comparative mass spectrometry based lipidomics analysis between EPs and LPs, and found that along with cholesterol, certain sphingolipids also displayed differential enrichments on phagosomes across different stages of maturation11. Specifically, we found that ceramides, particularly those bearing very-long chain (VLC) lipids, were significantly elevated on LPs, with a concomitant decrease in precursor sphingosine concentrations11. Upon further biochemical characterization of this sphingolipid pathway, we found that the enzyme ceramide synthase 2 (CerS2)12 was responsible for orchestrating the enrichment of ceramides, specifically VLC lipid containing ceramides, on LPs. Of note, the pharmacological inhibition of CerS2 greatly reduced ceramide levels on LPs, and delayed the process of phagosomal maturation11. Given their complementary structure to cholesterol, ceramides are also thought to stabilize lipid rafts13, and possibly make such lipid (micro)domains rigid enough for the efficient recruitment of important protein factors (e.g. GTPases, motor proteins), necessary to promote phagosomal maturation.

Ceramides are the central lynchpin in mammalian sphingolipid metabolism that flux reversibly into various modified sphingolipid species like the sphingomyelins, ceramide phosphates, and glucosylceramides by the action of dedicated biosynthetic and degradative enzymes14–20 (Figure 1). Having shown the differential enrichment and importance of ceramides, particularly VLC lipid containing ceramides, during phagosomal maturation, and it’s possible implications and/or involvement in the stabilization of lipid rafts, we wanted to quantitatively assess the full complement of sphingolipids present on EPs and LPs. Additionally, we were also interested in mapping the possible biochemical (enzymatic) activities that might be contributing to the accumulation of different sphingolipids during various stages of phagocytosis. To address this, in this study, we show using biochemical enzymatic assays, that the ceramidase activity during phagosomal maturation also significantly contributes towards the accumulation of ceramides on LPs. Further using lipidomics measurements, we show that de novo ceramide biosynthesis contributes little to the flux of ceramides during phagocytosis, while glucosylceramides significantly increase on LPs. We validate this finding using biochemical assays, and show that heightened glucosylceramide synthase activity on LPs contributes to this sphingolipid change during phagocytosis. Our findings taken together provide new insights into sphingolipid metabolism during phagocytosis and establish new pathways for this lipid family that are likely to be important for phagosomal maturation and pathogen clearance.

Figure 1. Schematic representation of the ceramide metabolism in mammalian cells.

Figure 1

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(SM = Sphingomyelin, DHC = Dihydroceramide, Cer-1-P = Ceramide 1-Phosphate; For all the sphingolipids, R = fatty acid, for the SM class, X = choline or ethanolamine)

Results

Early phagosomes have higher ceramidase activity

We have previously shown that the ceramide synthase activity (Figure 2A) is higher on EPs, but unexpectedly, ceramide concentrations are significantly lower on EPs compared to LPs11. This was at the time, attributed to an anticipatory function of CerS2, where ceramides, (particularly VLC containing ceramides) are biosynthesized on EPs by the enzymatic activity of CerS2, but as phagosomes mature, CerS2 is possibly shed by them, and ceramides are eventually found elevated on LPs. Following up on this finding, we also decided to investigate whether counter to the CerS2 (ceramide synthesis) activity, there exists any ceramidase (ceramide degradation) activity (Figure 2A) and if so, how does this ceramidase activity change as a function of phagosomal maturation. In mammals, there are three types of ceramidases21, 22, namely the acid ceramidase (ASAH1), the neutral ceramidase (ASAH2), and the alkaline ceramidase (ACER1-3), whose activity, as their names suggest, is pH dependent. Since previous studies on phagosomal maturation, have shown that luminal pH of EPs and LPs is neutral and acidic respectively, we decided to perform the ceramidase assays at neutral and acidic pH. For the EP, we found that the specific ceramidase activity at neutral pH (31.9 ± 2.4 pmol mg-1 min-1) was significantly more (~ 6-fold) than that observed at an acidic pH (5.2 ± 1.2 pmol mg-1 min-1), while for the LP, the specific ceramidase activities at both neutral (10.0 ± 1.0 pmol mg-1 min-1) and acidic pH (7.9 ± 1.7 pmol mg-1 min-1) were comparable (Figure 2B). Given their luminal pH values based on available literature, we found that specific ceramidase activity for EP at a neutral pH was significantly more (~ 4-fold) than that of the LP at an acidic pH (Figure 2B, 2C). On a similar note, we also measured the specific ceramide synthase activity arising from CerS2 on EPs (24.4 ± 7.7 pmol mg-1 min-1) and LPs (10.3 ± 4.0 pmol mg-1 min-1) at neutral pH, and found consistent with our previous studies11, that EP had significantly more (~ 2.5-fold) ceramide synthase activity than LPs (Figure 2C). Interestingly, we found that the relative to LPs, EPs had relatively more ceramidase activity (~ 4-fold) compared to the CerS2 mediated ceramide synthase activity (~ 2.5-fold) (Figure 2D).

Figure 2. The ceramidase activity during phagosomal maturation.

Figure 2

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(A) The biochemical reaction catalyzed by the enzymes ceramide synthase and ceramidase. (B) The specific ceramidase activity of EPs and LPs at neutral (pH 7) and acidic (pH 4.5) conditions. (C) The specific ceramide synthase activity of EPs and LPs at neutral (pH 7) conditions. (D) The relative levels of ceramide synthase (from CerS2) and ceramidase activities on EP versus LP. For (B), (C) and (D), the bar plots represent data as mean ± standard deviation from three biological replicates per experimental group. **p < 0.01, and ***p < 0.001 by Student’s t-test for the two groups compared.

In a quest to find whether ASAH1 and/or ASAH2 were differentially present on EPs and LPs, we screened several commercially antibodies for both enzymes, and unfortunately, found that none of them were selective enough to robustly report on the abundance of either protein. Hence, we decided to investigate the levels of ASAH1 and ASAH2 on EP and LP using a proteomics approach from tryptic peptide preparations previously reported by us23, 24. First, we resorted to a standard data-dependent acquisition (DDA) (also known as information dependent acquisition, IDA) method for identifying these enzymes in the proteomes obtained from EPs and LPs, but were unable to identify any unique peptides for either ASAH1 or ASAH2, possibly because of their low concentration relative to other abundant proteins in these complex proteomes. Therefore, we decided to measure the relative levels of these ceramidases on EPs and LPs by a LC-MS based targeted proteomics approach25 using publicly available peptide data information for these enzymes from the SRMAtlas26, 27 and PeptideAtlas repositories28, 29. From this targeted LC-MS based proteomics experiment, we found that consistent with its luminal pH, as expected, relative to a control protein (β-actin)11, LPs had significantly more ASAH1 (~ 3.5 fold), while EPs had increased levels of ASAH2 (~ 4-fold) (Supplementary Figure 1). Finally, we also screened for both the alkaline ceramidase activity at pH 9, and the enzyme associated with this activity i.e. ACER1-3, and were unable to detect either in our assays. Taken together, our results show that while both the EP and LP possess ceramidase activity, this enzymatic activity, particularly the neutral ceramidase activity compared to LPs, is significantly higher on EPs.

Dihydroceramide levels remain unchanged during phagosomal maturation

In mammals, ceramides are biosynthesized from dihydroceramide (DHC) precursors by the action of the enzyme DHC desaturase15, 30, 31 (Figure 1), and we wanted to determine if this de novo biosynthetic pathway (if at all) contributes to the increased ceramide levels that we observe on LPs. We had not measured this particular sphingolipid class in earlier investigations11, and using reported LC-MS based MRM methods, we quantified DHCs on EPs and LPs. From this LC-MS based lipidomics experiment, we found that we could reliably measure several DHC species, and that there was no notable change in their absolute concentrations on EPs and LPs (Figure 3).

Figure 3. DHC levels on EPs and LPs.

Figure 3

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The bar plots represent data as mean ± standard deviation from three biological replicates per experimental group. No statistically significant changes were observed between EP and LP for this lipid class.

Next, we attempted to determine the levels of the enzyme DHC desaturase on EPs and LPs, and initially screened a few commercially available antibodies to detect this enzyme. Unfortunately, like ASAH1/2, the commercially available antibodies were not specific to DHC desaturase, and hence, could not be used for this purpose. Like the ceramidases, we also looked for the presence of unique peptides for DHC desaturase on EPs and LPs, using DDA/IDA and/or targeted proteomics approaches, but we were unable to detect any such unique peptides for this enzyme on phagosomes, suggesting that on phagosomes, this enzyme (and therefore its activity) are likely absent or present at concentrations below detection limits of our experiments. Nonetheless, taken together, these results suggest that phagosomes possibly obtain DHC from various cellular membranes that they make contacts with during the maturation process, and the absence of DHC desaturase (and its enzymatic activity) imply that the de novo biosynthesis of ceramides is not a major pathway that contributes to increased ceramide concentration on LPs.

Late phagosomes have higher levels of glucosylceramides

As described earlier, during the metabolism of sphingolipids, ceramides flux into sphingomyelins, ceramide 1-phosphate and glucosylceramides by the action of dedicated enzymes14–20 (Figure 1). Previously, we have measured levels of sphingomyelins and ceramide 1-phosphates on EP and LP preparations, finding no significant differences in the concentrations of sphingomyelins or ceramide 1-phosphates between EPs and LPs11. However, we had not measured the levels of glucosylceramides during phagosomal maturation, and given the increased ceramides on ageing phagosomes, we were interested in seeing if glucosylceramides also change during phagosomal maturation. We first decided to generate a MS/MS fragmentation map for the commercially available unnatural C17:0 glucosylceramide (Figure 4), towards developing a LC-MS based high-resolution multiple reaction monitoring (MRM-HR) method for quantitatively measuring glucosylceramide levels on EPs and LPs. From this high-resolution LC-MS based MS/MS fragmentation study, we found that at a collision energy of 70 V in the positive ionization mode, C17:0 glucosylceramide ([M+H+]: m/z = 714.5878) formed 4 major fragments (Figure 4). Based on the mass to charge (m/z) ratio of these MS/MS fragments, we were successfully able to assign their chemical structures based on the chemical structure of the parent C17:0 glucosylceramide (Figure 4). Of note, from this MS/MS fragmentation map, we found the characteristic “Fragment O” ([M+H+]: m/z = 264.2685)32 (Figure 4), that is a signature for all ceramide species, and used the fragmentation of the parent glucosylceramide m/z to this “Fragment O” as the MRM-HR transition (Supplementary Table 1) for the absolute quantification of glucosylceramides on EPs and LPs. Additionally, we also used another MS/MS fragment, the dehydrated parent mass fragment (parent mass – 18 Da) as an additional confirmatory fragment for checking the validity of absolute quantification of glucosylceramides on EPs and LPs (Supplementary Table 1).

Figure 4. MS/MS fragmentation study for C17:0 glucosylceramide.

Figure 4

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The 4 major fragments generated from the C17:0 glucosylceramide standard from the LC-MS based MS/MS study are marked with different asterisks and their chemical structures are described below the displayed MS/MS spectrum. This MS/MS experiment was repeated thrice with the starting compound to ensure the fragments were reproducibly obtained each time for the given MS parameters. Complete details of the MS parameters can be found in Table S1.

Leveraging established protocols11, 24, 33, we extracted lipids from EP and LP preparations, and measured the glucosylceramide concentrations on them using the above described LC-MS based MRM-HR method. We found from this quantitative LC-MS analysis, that like ceramides, the glucosylceramides were significantly enriched on LPs (~ 5-fold) relative to EPs (Figure 5). We also found that while the magnitude of the fold-change (~ 5-fold) for the enrichment of the ceramides and glucosylceramides on LPs was almost similar (Figure 5), for a particular ceramide species (e.g. C24:0 ceramide), the concentration of the corresponding glucosylceramide (e.g. C24:0 glucosylceramide) was ~ 5-fold higher on both EP and LP. This suggests that glucosylceramide might indeed be the stable end sphingolipid product during phagosomal maturation. Interestingly, we also observed that, concomitant to VLC chain containing ceramides, VLC containing glucosylceramides were found to increase most profoundly (Figure 5), further suggesting that these are likely biosynthesized from ceramide precursors on LPs. We also attempted to look for glucosylsphingosine, but were unable to reliably detect this sphingolipid in any of our lipidomics experiments.

Figure 5. Glucosylceramide concentrations on EPs and LPs.

Figure 5

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The bar plots represent data as mean ± standard deviation from three biological replicates per experimental group. ***p < 0.001 by Student’s t-test for the two groups compared.

Late phagosomes have higher glucosylceramide synthase activity

Intrigued by the enrichment of glucosylceramides on LPs during phagocytosis (Figure 5), we next wanted to assess if phagosomes also possess the enzymatic activities to make glucosylceramides (via glucosylceramide synthase)34–36 and/or break glucosylceramides (via glucosylceramidase)34, 37, 38 and if so, how do these enzyme activities change and/or enrich as a function of phagosomal maturation (Figure 6A). Like the ceramidase assays, we decided to perform the glucosylceramide synthase and the glucosylceramidase activity assays at pH 4.5 and 7 on both EP and LP preparations. From these substrate assays, we found that relative to the glucosylceramide synthase activity, the glucosylceramidase activity was negligible (~ 40-fold lower) for both EPs and LPs (Figure 6B). Next, corroborating the increased glucosylceramide levels on LPs, we found that LP preparations (specific rate: 2.0 ± 0.3 nmol mg-1 min-1 at pH 4.5, and 2.2 ± 0.2 nmol mg-1 min-1 at pH 7) had significantly more (~ 4-fold) glucosylceramide synthase activity compared to EPs (specific rate: 0.5 ± 0.1 nmol mg-1 min-1 at pH 4.5, and 0.5 ± 0.1 nmol mg-1 min-1 at pH 7), and that pH did not have much effect on this activity (Figure 6B).

Figure 6. LPs have greater glucosylceramide synthase activity.

Figure 6

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(A) The biochemical reaction catalyzed by the enzymes glucosylceramide synthase and glucosylceramidase towards making and breaking glucosylceramides respectively. (B) The specific glucosylceramidase (from GBA2 and GBA1) and glucosylceramide synthase (GCS) activity of EPs and LPs at neutral (pH 7) and acidic (pH 4.5) conditions. (C) Levels of unique peptides from the glucosylceramide synthase (3 unique peptides) on EP versus LP as determined by targeted proteomics relative to the unique peptides of a control protein (Actin, 2 unique peptides). The grey bars represent data from EP preparations, while the red bars represent data from LP preparations. Complete details of the peptides from the glucosylceramide synthase (i.e. GCS_1.1, GCS_2.1, GCS_3.1), and the control protein Actin (ACT_1.3 and ACT_2.2) can be found in Supplementary Table 2. For (B), (C) and (D), the bar plots represent data as mean ± standard deviation from three biological replicates per experimental group. *p < 0.05, **p < 0.01, and ***p < 0.001 by Student’s t-test for the two groups compared.

In mammals, the enzyme glucosylceramide synthase (GCS) is solely responsible for the biosynthesis of glucosylceramides35, 36, while two glucosylceramidases, i.e. the non-lysosomal glucosylceramidase (GBA2)34, 39 and lysosomal glucosylceramidase (GBA1)40–43 are involved in the degradation of glucosylceramides. Given the distinct activities observed, we wanted to determine the relative levels of these enzymes during phagosomal maturation. Like we did for the ceramidases, here, we also screened several commercially available antibodies, and found again, that none of them were selective enough to robustly report on the abundance of either enzyme from phagosomal preparations. Further, also ASAH1/2, we were unable to detect any unique peptides for GCS, GBA1 or GBA2 from LC-MS based DDA/IDA proteomics approaches, and hence, resorted to the targeted proteomics approach mentioned earlier. From this LC-MS targeted proteomics experiment, we found that consistent with the increased glucosylceramide synthase activity compared to EPs (Figure 6B), the protein levels of GCS were significantly higher on LPs (~ 4-fold), when normalized to the control protein β-actin11 (Figure 6C), while the protein levels of GBA2 and GBA1 did not change much between EP and LP preparations (Supplementary Figure 2), consistent with the enzyme activity profile observed by us (Figure 6B). Taken together, the results from our biochemical assays, and targeted proteomics experiments suggest that compared to EPs, LPs have greater amounts of the glucosylceramide synthase GCS, that makes glucosylceramides from ceramides using UDP-glucose as the glucose donor, and this activity eventually results in the heightened levels of glucosylceramides on LPs.

Discussion

Sphingolipids are an essential class of lipids that regulate several important biological functions in all organisms14. In mammals, including humans, ceramides form the central node of sphingolipid metabolism, and metabolically fluxes into various other sphingolipids (e.g. sphingomyelins, ceramide phosphates, glucosylceramides), by the action of dedicated enzymes14–20 (Figure 1). Given its conical shape and ability to complementarily partition with cholesterol, ceramides have many important physiological functions in mammals, including humans. It facilitates the tighter packing membrane bilayers, stabilizes negative membrane curvatures and in doing so, imparts rigidity to other plastic cellular membrane bilayers13, 44–47. Together with cholesterol, ceramides and its sphingolipid derivatives are form ordered lipid microdomains on membranes, called lipid rafts13, 44–47. The formation of these lipid rafts have important consequences in phagocytosis, as these rigid membrane structures on LPs are where the dynein motor proteins cluster, and collectively produce enough unidirectional force, to transport the maturing phagosome on the underlying cellular microtubule network towards the lysosome for eventual degradation10. Given the role of lipids in phagocytosis, we recently performed a comparative lipidomics analysis on EPs and LPs, and found that as phagosomes mature, concomitant to their increased cholesterol content, the concentration of ceramides, particularly VLC lipid containing ceramides, also significantly increases11. Upon further characterization, we found that this accumulation of ceramides on LPs was orchestrated by the enzyme CerS2, and the pharmacological inhibition of CerS2 by fumonisin (a general ceramide synthase inhibitor), resulted in hampered (and/or delayed) phagosomal maturation11. Interestingly, and paradoxically, we found from this study that even though the ceramides were enriched on LPs, the protein levels and enzymatic activity of CerS2 was more on EPs, and we attributed this to an anticipatory mechanism of CerS2, where by ceramides are found on LPs during the maturation process.

Following up on our previous lipidomics study, we first wanted to understand the biochemical basis for the accumulation of ceramides on LPs, and determine if there exists a physiological balance between ceramide synthase (CerS2) and ceramidase activities that eventually leads to the differential ceramide content as phagosomes mature. Using enzyme activity assays, we show that EPs have significantly more ceramidase activity compared to LPs (~ 4-fold) (Figure 2), and that the relative ceramidase activity on EPs is greater than the biosynthetic ceramidase synthase (CerS2) activity (Figure 2). Together, our data implies that even though biosynthesis of ceramides is greater on EPs, so is its degradation, and hence, an interplay between the ceramide synthase (CerS2) and ceramidase (ASAH1/2) activities eventually result in increased ceramides on LPs. Though not fully commensurate with the magnitude of the changes seen in the activity assays, possibly because of effects like post-translational modifications or spatiotemporal localization of ASAH2, or the need for co-factors and/or protein partners for optimal enzymatic activity, we strongly think that the recruitment and/or enrichment neutral ceramidase ASAH2 is possibly the key enzyme responsible for the degradation of ceramides on EPs (Supplementary Figure 1).

We have previously shown that during phagosomal maturation, concentrations of sphingomyelins and ceramide 1-phosphates don’t change11, but we had not investigated other sphingolipids such as DHC or glucosylceramides during this process. We found from lipidomics experiments reported in this study that the levels of DHC do not change during phagosomal maturation (Figure 3), and given the lack of DHC desaturase (and in turn its activity) of phagosomes, it appears that de novo ceramide synthesis route contributes little (if any) to the accumulation of ceramides of LPs. We could not find a reliable LC-MS method for quantitatively assessing glucosylceramides, and hence developed one for our studies (Figure 4). Performing quantitative lipidomics using our LC-MS MRM-HR method, we found that concomitant to ceramides, glucosylceramides increased profoundly on LPs (Figure 5). Interestingly, we found that VLC lipid containing glucosylceramides, exactly similar to their ceramide counterparts, most notably increased and were most abundant in terms of absolute concentrations on LPs (Figure 5), leading us to speculate that these glucosylceramides are possibly biosynthesized on LPs from precursor ceramides by the activity of GCS35, 36. Indeed, using biochemical assays, and targeted proteomics approaches, we found that LPs had significantly more GCS activity and concentration of this enzyme compared to EPs, and that both EPs and LPs had negligible counter glucosylceramidase activities and the significantly lower amounts of the corresponding enzymes, GBA2 and GBA1 (Figure 6, Supplementary Figure 2). Even though the functions of glucosylceramides are not well understood in cell biology, like ceramides, they form lipid rafts by partitioning with cholesterol and serve as stable platforms for localization of protein and even enrichment of receptors. Recent reports have suggested that glucosylceramides are also essential for the localization and functioning of vATPases48, and publicly available proteomics datasets show that presence of vATPases on LPs7, 8. Therefore, we think that the accumulation of glucosylceramides on LPs possibly results in the formation of rigid lipid rafts, which facilitate the recruitment and localization of vATPases, that are eventually responsible for the hyperacidification of the phagosomes during the maturation process (Figure 7).

Figure 7. Model for changes in sphingolipid content during phagosomal maturation.

Figure 7

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A summary of the sphingolipid changes during the maturation of phagosomes by the interplay of various enzymes that regulate their metabolism. As per the model, the formation of ordered lipid rafts (or lipid microdomains) because of accumulation of ceramide and glucosylceramides along with cholesterol on LPs, causes the recruitment of vATPases, that acidify maturing phagosomes.

Projecting ahead, the accumulation of glucosylceramides on ageing phagosomes opens up several new questions and research avenues. For instance, it would be interesting to figure out, whether glucosylceramides (and perhaps even ceramides) are indeed needed for the formation of stable lipid rafts (lipid microdomains) on LPs, and effects of their depletion on the recruitment of important proteins that promote phagocytosis (e.g. dynein mediated unidirectional motion of LPs to lysosomes10, or acidification by vATPases48). However, to the best of our knowledge, there are no specific inhibitors described in literature or available commercially for GCS (or the ceramidases ASAH1/2). Hence, to probe specific effects of glucosylceramides during phagocytosis (and other cellular processes), it would be beneficial to develop specific cell active GCS inhibitors and/or pharmacological tools (e.g. multifunctional glucosylceramide chemical probes) that besides lipidomics, might also enable complementary chemoproteomics and cellular imaging studies. We also found from our studies that several commercially available antibodies for enzymes involved in the sphingolipid metabolism are not very specific and amenable to cellular studies. Therefore, along with pharmacological tools, the development of specific and high-quality antibodies for these enzymes involved in sphingolipid metabolism, particularly those discussed in this study, would greatly facilitate cell biology studies, and expand our understanding of the spatiotemporal regulation of sphingolipids and their synergy with cholesterol during phagocytosis.

Methods

Enzyme activity assays

All biochemical assays were performed at pH 4.5 and pH 7, using sodium acetate buffer and phosphate buffered saline (PBS) respectively, in a final volume of 100 μL at 37 °C with constant shaking, using 25 μg of EP or LP proteome, and quenched at the desired time point by adding 500 μL of LC-MS grade methanol. Heat denatured proteomes of EPs/LPs and substrate-only reactions (only substrate, no proteome added) were used as controls for all biochemical enzymatic assays to account for the non-enzymatic rate of reactions. The ceramidase assay49 was done using C18:1 ceramide (substrate, 20 μM) for 180 mins. The concentration of sphingosine released from C18:1 ceramide was obtained by measuring the area under the curve for sphingosine relative to an unnatural sphingosine (C17:1 sphingosine, 100 pmol) used as an internal standard in the assay, by a LC-MS MRM-HR method reported by us11. The glucosylceramidase assay50 was done using C17:0 glucosylceramide (substrate, 20 μM) for 120 mins. The concentration of C17:0 glucosylceramide consumed in this assay was obtained by measuring the area under the curve for C17:0 glucosylceramide in the enzymatic assay relative to the controls (heat denatured proteomes and substrate only reaction) used in the assay, by the LC-MS MRM-HR method described in this study (see Supplementary Table 1). The glucosylceramide synthase assay51 was done 7 using C18:1 ceramide (100 μM) and UDP-glucose (250 μM) as substrates for 120 mins. The concentration C18:1 glucosylceramide (product) formed in this assay was obtained by measuring the area under the curve of C18:1 glucosylceramide relative to an unnatural C17:0 glucosylceramide (100 pmol) internal standard used in the assay, by the LC-MS MRM-HR method described in this study (see Supplementary Table 1). The specific enzymatic activity was obtained by normalizing the concentration of substrate consumed or product released in the assay to the assay time and the amount of proteome used.

Targeted proteomics

200 μg of EP/LP proteome was denatured, reductively alkylated and digested to yield tryptic peptides, and subjected to LC-MS analysis as reported earlier23, 24. Since we could not detect any unique peptides in DDA/IDA proteomics methods, we decided to develop a targeted proteomics platform for (semi)quantitatively assessing the candidate enzymes involved in sphingolipid metabolism. First, by in silico peptide predictions using Skyline52 and other available data repositories (e.g. NCBI)25, we generated a library of possible tryptic peptides for our enzymes of interest. In general, the full tryptic peptides without any missed cleavages, ranging from 8 – 25 amino acids were shortlisted, and post-translational and/or artificial static modifications were excluded. Next, these theoretical predictions were cross checked in the SRMAtlas26, 27 and PeptideAtlas28, 29 repositories, to determine which of these peptides have previously been identified, have reliable spectral data and have validated MRM data available. Based on these two criteria, a final list of all the possible peptides for the candidate enzymes involved in sphingolipid metabolism was surveyed, and the unique peptides for the enzymes of interest, that were identified by us in in this study in all the three biological replicates for both EP/LP (describing their precursor parent ion mass and the product ion targeted) can be found in Supplementary Table 2. For quantifying the relative abundance of a particular enzyme involved in sphingolipid metabolism on the EP or LP, at least two detected unique peptides for that respective enzyme were selected for this analysis, and the area under the curve for such unique peptides was normalized to the area under the curve for unique peptides for the control protein actin (see Supplementary Table 2 for complete details). The LC-MS system, columns, and MS parameters were identical to those reported by us previously23, 24. A typical LC-MS MRM run was 60 min with a flow rate of 300 nL min–1, with the chromatographic solvents (solvent A = Water + 0.1% formic acid; solvent B = acetonitrile) and following run sequence post injection: 5% solvent B for 2 min, 10% solvent B for 2 min, linear gradient of solvent B from 10–30% over 46 min, 90% solvent B for 5 min, and reequilibration with 5% solvent B for 5 min.

Supplementary Material

The Supporting Information is available free of charge via the Internet.

Supplementary Figures as referenced in the manuscript showing the targeted proteomics data for the enzymes involved in sphingolipid metabolism, a supplementary table listing the MRM parameters for the different sphingolipid species assessed in this study, and additional experimental methods for preparation of phagosomes and lipidomics samples (PDF).

Complete targeted proteomics data along with quantification (XLSX).

Figures and Table
Table S2

Acknowledgements

Members of the S.S.K. and R.M. labs are thanked for providing critical comments and discussion on this study. S. Ghosh is thanked for help with preliminary experiments. S. Shekh and S. Singh (both from IISER Pune) are thanked for technical assistance.

Funding

This work has been supported by the DBT/Wellcome Trust India Alliance Fellowships (grant number IA/I/15/2/502058 to S.S.K., and IA/S/11/2500255 to R.M.) and the Department of Science and Technology Fund for Improvement of S&T Infrastructure (DST-FIST) (grant number SR/FST/LSII-043/2016) to the IISER Pune Biology Department for building a biological mass spectrometry facility. N.M. is supported by a graduate student fellowship from the Department of Biotechnology, Govt. of India.

Footnotes

Author Contributions

N.M. performed all the studies. All authors conceived the project and analyzed the data. S.S.K. and R.M. conceived, supervised the project and acquired funding. N.M. and S.S.K wrote the manuscript with inputs from R.M.

Notes

The authors declare no competing financial interests.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figures and Table
EMS137024-supplement-Figures_and_Table.pdf (353.4KB, pdf)
Table S2
EMS137024-supplement-Table_S2.xlsx (34.2KB, xlsx)

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