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Unsaturated glycerophospholipids mediate heme crystallization: biological implications for hemozoin formation in the kissing bug Rhodnius prolixus.

Stiebler R, Majerowicz D, Knudsen J, Gondim KC, Wright DW, Egan TJ, Oliveira MF - PLoS ONE (2014)

Bottom Line: Interestingly, uPC-mediated reactions resulted in two morphologically distinct crystal populations: one less representative group of regular crystals, resembling those induced by uPE, and the other largely represented by crystals with numerous sharp edges and tapered ends.Interestingly, crystals produced by RML were homogeneous in shape and quite similar to those mediated by uPE.Thus, β-hematin formation can be rapidly and efficiently induced by unsaturated glycerophospholipids, particularly uPE and uPC, and may play a role on biological heme crystallization in R. prolixus midgut.

View Article: PubMed Central - PubMed

Affiliation: Laboratório de Bioquímica de Resposta ao Estresse, Instituto de Bioquímica Médica Leopoldo de Meis, Universidade Federal do Rio de Janeiro, RJ, Brazil ; Laboratório de Inflamação e Metabolismo, Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil ; Department of Chemistry, Vanderbilt University, Nashville, Tennessee, United States of America.

ABSTRACT
Hemozoin (Hz) is a heme crystal produced by some blood-feeding organisms, as an efficient way to detoxify heme derived from hemoglobin digestion. In the triatomine insect Rhodnius prolixus, Hz is essentially produced by midgut extracellular phospholipid membranes known as perimicrovillar membranes (PMVM). Here, we investigated the role of commercial glycerophospholipids containing serine, choline and ethanolamine as headgroups and R. prolixus midgut lipids (RML) in heme crystallization. All commercial unsaturated forms of phospholipids, as well as RML, mediated fast and efficient β-hematin formation by means of two kinetically distinct mechanisms: an early and fast component, followed by a late and slow one. The fastest reactions observed were induced by unsaturated forms of phosphatidylethanolamine (uPE) and phosphatidylcholine (uPC), with half-lives of 0.04 and 0.7 minutes, respectively. β-hematin crystal morphologies were strikingly distinct among groups, with uPE producing homogeneous regular brick-shaped crystals. Interestingly, uPC-mediated reactions resulted in two morphologically distinct crystal populations: one less representative group of regular crystals, resembling those induced by uPE, and the other largely represented by crystals with numerous sharp edges and tapered ends. Heme crystallization reactions induced by RML were efficient, with a heme to β-hematin conversion rate higher than 70%, but clearly slower (t1/2 of 9.9-17.7 minutes) than those induced by uPC and uPE. Interestingly, crystals produced by RML were homogeneous in shape and quite similar to those mediated by uPE. Thus, β-hematin formation can be rapidly and efficiently induced by unsaturated glycerophospholipids, particularly uPE and uPC, and may play a role on biological heme crystallization in R. prolixus midgut.

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Related in: MedlinePlus

Kinetics of heme crystallization promoted by different commercial and biological lipids.Heme crystallization reactions were induced in vitro mediated by uPC, uPS or uPE (100 µM), a blended phospholipid mixture of commercial uPS (14%), uPC (32%) and uPE (51%) or 10 µg/mL of total lipids isolated from PMVM of R. prolixus previously fed with plasma or blood. Data are expressed as mean ± SD, of at least three different experiments and fitted using the Avrami equation as described in the methods section. To perform the Avrami analysis, the uPC-induced kinetics were independently analyzed at early and late times, which are shown as insets.
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pone-0088976-g004: Kinetics of heme crystallization promoted by different commercial and biological lipids.Heme crystallization reactions were induced in vitro mediated by uPC, uPS or uPE (100 µM), a blended phospholipid mixture of commercial uPS (14%), uPC (32%) and uPE (51%) or 10 µg/mL of total lipids isolated from PMVM of R. prolixus previously fed with plasma or blood. Data are expressed as mean ± SD, of at least three different experiments and fitted using the Avrami equation as described in the methods section. To perform the Avrami analysis, the uPC-induced kinetics were independently analyzed at early and late times, which are shown as insets.

Mentions: The kinetic profiles of β-hematin formation mediated by commercial unsaturated phospholipids and R. prolixus midgut lipids were determined along 24 h of reaction (Figure 4). For this set of experiments we also included a blend of uPE (51%), uPC (32%) and uPS (14%), in an attempt to reach similar ratios of these phospholipids to those synthesized by R. prolixus midgut [52]. A brief visual inspection of the fitted curve shapes for all three commercial phospholipids, indicates that all reactions mediated fast and efficient β-hematin formation with quite distinct kinetic patterns among the groups (Figure 4). Reactions induced by uPC clearly exhibited two kinetically distinct components, both contributing substantially to crystal formation: a very fast early process, converting about 40% of heme into β-hematin and a slower late one, which converts further 48%. It is tempting to suggest that the kinetic behavior of the reaction induced by uPC may be related to the two distinct crystal morphologies exhibited by this phospholipid (Figure 2, uPC). Curiously, the brick-shaped crystals produced by uPC are quite similar to those of uPE, while the needle-like ones resemble the uPS crystals. However, this suggestion does not correlate with the very small fraction of the brick-like crystals produced in this process. Another possibility to consider is that these two kinetically distinct components of uPC were related to the way by which heme interacts with the exposed CH3 groups at the surface of uPC vesicles [46]. Interestingly, β-hematin formation induced by the phospholipid blend was revealed to be as fast as the uPE-mediated heme crystallization, suggesting that beyond representing the dominant phospholipid synthesized by R. prolixus midgut epithelia [52] uPE is able to produce crystals very similar in shape to those found biologically (Figure 2), mediating fast and efficient β-hematin formation (about 60% of heme conversion) (Figure 4). Regardless of the catalyst, we can observe that all reactions were essentially completed after 8 h (∼500 minutes) and all exhibited two kinetically distinct components: one very fast, making a major contribution to β-hematin conversion (54% to 100%), and the other a slow one. With the exception of uPC, this slow component does not greatly contribute to the overall heme conversion to β-hematin and its mechanism is uncertain. Therefore, in Table 2 we show five distinct calculated kinetic parameters of reactions induced by commercial and biological lipids, based on their pattern shown in Figure 4. This was accomplished by fitting all seven sets of kinetics data to the Avrami equation (see “data analyses” in methods section), which mathematically describes the processes of solid transition from one phase to another at constant temperature [61]. Since all data could be fitted to the Avrami equation, this indicates that β-hematin formation mediated by the different lipids involves both nucleation and growth. The Avrami constant, n, can only take on integer values between 1 and 4 and this gives an insight into the geometry of crystal growth and the nature of the nucleation process, which could be instantaneous (all nuclei are preformed at the beginning of the process), or sporadic (nuclei form throughout the process). When n = 1, which is the case for all catalysts, with exception of the late uPC component and uPS, the fits are mathematically indistinguishable from first-order processes, with exponential curves, which can be interpreted to mean that they occur through one dimensional growth at preformed nuclei. Firstly, heme conversion to β-hematin varied from 39.8% with Blend to 96.5% with the overall uPC-mediated reactions, while in lipids from R. prolixus midgut this conversion was about 74%. We speculate that the different sizes of vesicles produced by the phospholipids may explain the discrepancies of heme conversion to β-hematin. This possibility is supported by literature [31], [39] which has proposed that the β-hematin crystal nucleates at the surface of neutral lipid particles [31] or DV membranes [39], [44], [45] and grows along the lipid-water interface until the curvature of the lipid particle limits this process. Conceivably, uPC would produce the largest vesicles, and the Blend the smallest ones. Regarding the rate constants (z), the data obtained for uPS and the late component of uPC (Table 2) cannot be compared to other phospholipids because of the different value of the Avrami constant n, and hence different units. Nevertheless, it is remarkable to note the differences in reaction half-lives mediated by uPE (0.04 min.) which are orders of magnitude lower than those of uPC (0.7 min., for early, and 402 min., for the late component), uPS (225 min.), and those induced by lipids from plasma or blood fed R. prolixus biological lipids (17.7 and 9.9 min., respectively). Interestingly, the reaction half-life mediated by uPE was undistinguishable from those of reactions promoted by phospholipid blend (0.04 vs. 0.035 min., respectively, p = 0.88). Previous reports have demonstrated that phospholipids are efficient catalysts of heme crystallization [28], [48], with variable results. For instance, the study conducted by Dorn and colleagues demonstrated that PE, PS, PC, phophatidylinositol (PI) and sphingomyelin were all able to produce β-hematin, with PC being more efficient than PE in overnight reactions [48]. However, the opposite was shown in a study of Egan and colleagues, where the heme conversion to β-hematin mediated by PE was higher than PC, after only five minutes of reaction [28]. Since the exact fatty acid composition of phospholipids in both reports are unknown, a direct comparison between these data and our results cannot be made. Potentially, the kinetic behavior of uPE and uPC in mediating β-hematin formation may explain the relative quickness and extent by which heme crystallization reactions proceed in plasma and blood derived R. prolixus midgut lipids, considering that this preparation would contain uPE, uPC and uPS in different proportions [52]. Also, these data highlight the effective contribution of each phospholipid in the reaction process, in which the fast component of uPC and uPE would play a central catalytic role at early time points of β-hematin formation, whereas the slower uPC component would play a prominent role at later reaction times, increasing the conversion extent. An alternative explanation for the differences observed in the rate constant and half-life of the three phospholipids tested could be their effect in reducing the activation energy required for heme transition from the π-π dimer to reciprocal iron-carboxylate dimers of β-hematin. Conceivably, the extent of the reduction of the activation energy barrier for this transition would be provided by the chemical environment of each phospholipid headgroup, thus affecting the kinetics of reaction.


Unsaturated glycerophospholipids mediate heme crystallization: biological implications for hemozoin formation in the kissing bug Rhodnius prolixus.

Stiebler R, Majerowicz D, Knudsen J, Gondim KC, Wright DW, Egan TJ, Oliveira MF - PLoS ONE (2014)

Kinetics of heme crystallization promoted by different commercial and biological lipids.Heme crystallization reactions were induced in vitro mediated by uPC, uPS or uPE (100 µM), a blended phospholipid mixture of commercial uPS (14%), uPC (32%) and uPE (51%) or 10 µg/mL of total lipids isolated from PMVM of R. prolixus previously fed with plasma or blood. Data are expressed as mean ± SD, of at least three different experiments and fitted using the Avrami equation as described in the methods section. To perform the Avrami analysis, the uPC-induced kinetics were independently analyzed at early and late times, which are shown as insets.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3935856&req=5

pone-0088976-g004: Kinetics of heme crystallization promoted by different commercial and biological lipids.Heme crystallization reactions were induced in vitro mediated by uPC, uPS or uPE (100 µM), a blended phospholipid mixture of commercial uPS (14%), uPC (32%) and uPE (51%) or 10 µg/mL of total lipids isolated from PMVM of R. prolixus previously fed with plasma or blood. Data are expressed as mean ± SD, of at least three different experiments and fitted using the Avrami equation as described in the methods section. To perform the Avrami analysis, the uPC-induced kinetics were independently analyzed at early and late times, which are shown as insets.
Mentions: The kinetic profiles of β-hematin formation mediated by commercial unsaturated phospholipids and R. prolixus midgut lipids were determined along 24 h of reaction (Figure 4). For this set of experiments we also included a blend of uPE (51%), uPC (32%) and uPS (14%), in an attempt to reach similar ratios of these phospholipids to those synthesized by R. prolixus midgut [52]. A brief visual inspection of the fitted curve shapes for all three commercial phospholipids, indicates that all reactions mediated fast and efficient β-hematin formation with quite distinct kinetic patterns among the groups (Figure 4). Reactions induced by uPC clearly exhibited two kinetically distinct components, both contributing substantially to crystal formation: a very fast early process, converting about 40% of heme into β-hematin and a slower late one, which converts further 48%. It is tempting to suggest that the kinetic behavior of the reaction induced by uPC may be related to the two distinct crystal morphologies exhibited by this phospholipid (Figure 2, uPC). Curiously, the brick-shaped crystals produced by uPC are quite similar to those of uPE, while the needle-like ones resemble the uPS crystals. However, this suggestion does not correlate with the very small fraction of the brick-like crystals produced in this process. Another possibility to consider is that these two kinetically distinct components of uPC were related to the way by which heme interacts with the exposed CH3 groups at the surface of uPC vesicles [46]. Interestingly, β-hematin formation induced by the phospholipid blend was revealed to be as fast as the uPE-mediated heme crystallization, suggesting that beyond representing the dominant phospholipid synthesized by R. prolixus midgut epithelia [52] uPE is able to produce crystals very similar in shape to those found biologically (Figure 2), mediating fast and efficient β-hematin formation (about 60% of heme conversion) (Figure 4). Regardless of the catalyst, we can observe that all reactions were essentially completed after 8 h (∼500 minutes) and all exhibited two kinetically distinct components: one very fast, making a major contribution to β-hematin conversion (54% to 100%), and the other a slow one. With the exception of uPC, this slow component does not greatly contribute to the overall heme conversion to β-hematin and its mechanism is uncertain. Therefore, in Table 2 we show five distinct calculated kinetic parameters of reactions induced by commercial and biological lipids, based on their pattern shown in Figure 4. This was accomplished by fitting all seven sets of kinetics data to the Avrami equation (see “data analyses” in methods section), which mathematically describes the processes of solid transition from one phase to another at constant temperature [61]. Since all data could be fitted to the Avrami equation, this indicates that β-hematin formation mediated by the different lipids involves both nucleation and growth. The Avrami constant, n, can only take on integer values between 1 and 4 and this gives an insight into the geometry of crystal growth and the nature of the nucleation process, which could be instantaneous (all nuclei are preformed at the beginning of the process), or sporadic (nuclei form throughout the process). When n = 1, which is the case for all catalysts, with exception of the late uPC component and uPS, the fits are mathematically indistinguishable from first-order processes, with exponential curves, which can be interpreted to mean that they occur through one dimensional growth at preformed nuclei. Firstly, heme conversion to β-hematin varied from 39.8% with Blend to 96.5% with the overall uPC-mediated reactions, while in lipids from R. prolixus midgut this conversion was about 74%. We speculate that the different sizes of vesicles produced by the phospholipids may explain the discrepancies of heme conversion to β-hematin. This possibility is supported by literature [31], [39] which has proposed that the β-hematin crystal nucleates at the surface of neutral lipid particles [31] or DV membranes [39], [44], [45] and grows along the lipid-water interface until the curvature of the lipid particle limits this process. Conceivably, uPC would produce the largest vesicles, and the Blend the smallest ones. Regarding the rate constants (z), the data obtained for uPS and the late component of uPC (Table 2) cannot be compared to other phospholipids because of the different value of the Avrami constant n, and hence different units. Nevertheless, it is remarkable to note the differences in reaction half-lives mediated by uPE (0.04 min.) which are orders of magnitude lower than those of uPC (0.7 min., for early, and 402 min., for the late component), uPS (225 min.), and those induced by lipids from plasma or blood fed R. prolixus biological lipids (17.7 and 9.9 min., respectively). Interestingly, the reaction half-life mediated by uPE was undistinguishable from those of reactions promoted by phospholipid blend (0.04 vs. 0.035 min., respectively, p = 0.88). Previous reports have demonstrated that phospholipids are efficient catalysts of heme crystallization [28], [48], with variable results. For instance, the study conducted by Dorn and colleagues demonstrated that PE, PS, PC, phophatidylinositol (PI) and sphingomyelin were all able to produce β-hematin, with PC being more efficient than PE in overnight reactions [48]. However, the opposite was shown in a study of Egan and colleagues, where the heme conversion to β-hematin mediated by PE was higher than PC, after only five minutes of reaction [28]. Since the exact fatty acid composition of phospholipids in both reports are unknown, a direct comparison between these data and our results cannot be made. Potentially, the kinetic behavior of uPE and uPC in mediating β-hematin formation may explain the relative quickness and extent by which heme crystallization reactions proceed in plasma and blood derived R. prolixus midgut lipids, considering that this preparation would contain uPE, uPC and uPS in different proportions [52]. Also, these data highlight the effective contribution of each phospholipid in the reaction process, in which the fast component of uPC and uPE would play a central catalytic role at early time points of β-hematin formation, whereas the slower uPC component would play a prominent role at later reaction times, increasing the conversion extent. An alternative explanation for the differences observed in the rate constant and half-life of the three phospholipids tested could be their effect in reducing the activation energy required for heme transition from the π-π dimer to reciprocal iron-carboxylate dimers of β-hematin. Conceivably, the extent of the reduction of the activation energy barrier for this transition would be provided by the chemical environment of each phospholipid headgroup, thus affecting the kinetics of reaction.

Bottom Line: Interestingly, uPC-mediated reactions resulted in two morphologically distinct crystal populations: one less representative group of regular crystals, resembling those induced by uPE, and the other largely represented by crystals with numerous sharp edges and tapered ends.Interestingly, crystals produced by RML were homogeneous in shape and quite similar to those mediated by uPE.Thus, β-hematin formation can be rapidly and efficiently induced by unsaturated glycerophospholipids, particularly uPE and uPC, and may play a role on biological heme crystallization in R. prolixus midgut.

View Article: PubMed Central - PubMed

Affiliation: Laboratório de Bioquímica de Resposta ao Estresse, Instituto de Bioquímica Médica Leopoldo de Meis, Universidade Federal do Rio de Janeiro, RJ, Brazil ; Laboratório de Inflamação e Metabolismo, Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil ; Department of Chemistry, Vanderbilt University, Nashville, Tennessee, United States of America.

ABSTRACT
Hemozoin (Hz) is a heme crystal produced by some blood-feeding organisms, as an efficient way to detoxify heme derived from hemoglobin digestion. In the triatomine insect Rhodnius prolixus, Hz is essentially produced by midgut extracellular phospholipid membranes known as perimicrovillar membranes (PMVM). Here, we investigated the role of commercial glycerophospholipids containing serine, choline and ethanolamine as headgroups and R. prolixus midgut lipids (RML) in heme crystallization. All commercial unsaturated forms of phospholipids, as well as RML, mediated fast and efficient β-hematin formation by means of two kinetically distinct mechanisms: an early and fast component, followed by a late and slow one. The fastest reactions observed were induced by unsaturated forms of phosphatidylethanolamine (uPE) and phosphatidylcholine (uPC), with half-lives of 0.04 and 0.7 minutes, respectively. β-hematin crystal morphologies were strikingly distinct among groups, with uPE producing homogeneous regular brick-shaped crystals. Interestingly, uPC-mediated reactions resulted in two morphologically distinct crystal populations: one less representative group of regular crystals, resembling those induced by uPE, and the other largely represented by crystals with numerous sharp edges and tapered ends. Heme crystallization reactions induced by RML were efficient, with a heme to β-hematin conversion rate higher than 70%, but clearly slower (t1/2 of 9.9-17.7 minutes) than those induced by uPC and uPE. Interestingly, crystals produced by RML were homogeneous in shape and quite similar to those mediated by uPE. Thus, β-hematin formation can be rapidly and efficiently induced by unsaturated glycerophospholipids, particularly uPE and uPC, and may play a role on biological heme crystallization in R. prolixus midgut.

Show MeSH
Related in: MedlinePlus