Reconocimiento sintáctico de patrones

Under normal, physiological conditions, sphingomyelin and phosphatidylcholine constitute approximately 50% of the phospholipid content in the bilayer membranes of eukaryotic cells (Niemelä et al. 2004). Although SM and PC both contain phosphocholine as a polar head group, there are significant structural differences between the two phospholipids. In the sphingolipid sphingomyelin, the glycerol diester backbone of the phosphoglyceride phosphatidylcholine is replaced by a sphingosine unit that participates in extensive intramolecular and intermolecular hydrogen bonding (Chiu et al. 2003; Niemelä et al. 2004). A second distinguishing feature concerns the fatty acid units, with sphingomyelin being significantly more saturated than phosphatidylcholine. As a result, bilayers of SM and PC have different structural and dynamic properties. This encouraged us to carry out a more extensive comparison of the two lipids. In these experiments, hydrolysis reactions consisting of 2 mM of sphingomyelin or of phosphatidylcholine, 10 mM of Ce(NH4)2(NO3)6, and 20 mM of piperazine

buffer pH 4.8 or of HEPES buffer pH 7.2 were allowed to sit at 60 ºC for 20 h (no Triton X-100). Because a therapeutic agent should also be active at normal, core body temperature, a second set of solutions was reacted at 37 ºC. The malachite green/molybdate- and Amplex® Red-based

assays were then used to detect and quantitate the average amounts of free phosphate (Figure 2.5) and free choline (Figure 2.6) released upon Ce(IV)-assisted hydrolysis.

Figure 2.5. Averaged hydrolysis yields plotted as a function pH for malachite green detection of free phosphate. A total of 2 mM of sphingomyelin (SM) or 2 mM of phosphatidylcholine (PC) was treated for 20 h at A) 60 C or at B) 37 C. Reactions were run in the presence of 10 mM of Ce(NH4)2(NO3)6 and 20 mM piperazine buffer (~ pH 4.8) or 20 mM HEPES buffer (~ pH 7.2).

The number of trials (n) appears in parenthesis. Error bars represent standard deviation. The 60

Figure 2.6. Averaged hydrolysis yields plotted as a function pH for Amplex® Red detection of free choline. A total of 2 mM of sphingomyelin (SM) or 2 mM of phosphatidylcholine (PC) was treated for 20 h at A) 60 C or at B) 37 C. Reactions were run in the presence of 10 mM of Ce(NH4)2(NO3)6 and 20 mM piperazine buffer (~ pH 4.8) or 20 mM HEPES buffer (~ pH 7.2).

The number of trials (n) appears in parenthesis. Error bars represent standard deviation. The 60

The 60 ºC and 37 ºC data shown in Figures 2.5 and 2.6 display trends consistent with our initial colorimetric experiments (Figures 2.2 and 2.3; Kassai et al. 2011). Cerium(IV) hydrolyzed pure lipid vesicles of sphingomyelin and phosphatidylcholine more efficiently at mildly acidic pH compared to near-neutral values, with the amounts of choline being significantly higher or equivalent to free phosphate. A new finding concerned the relative susceptibilities of the two phospholipids towards hydrolysis. Under all of the reaction conditions tested, phosphatidylcholine generated more choline and phosphate than sphingomyelin. Treatment of phosphatidylcholine with Ce(IV) at 60 ºC and ~ pH 4.8 accordingly released choline and inorganic phosphate in 70 ± 4% and 42 ± 5% yields (Figures 2.5A and 2.6A), values that are 1.3 and 5.5 fold higher than SM hydrolysis yields at ~ pH 4.8. When the reaction temperature was lowered from 60 ºC to 37 ºC, measurable hydrolytic activity was still observed (Figures 2.5B and 2.6B). In the phosphatidylcholine reactions, the amounts of choline and inorganic phosphate were 41 ± 3% and 17 ± 3%, yields that were 3.4 and 3.8 fold higher than SM hydrolysis at the same pH. In parallel controls in which the metal solutions were replaced with equivalent volumes of ddH2O, levels of choline and inorganic phosphate were considerably lower (Figure

2.S5 in Electronic supplementary material; Kassai et al. 2011).

A number of factors are likely to contribute to the relative susceptibilities of sphingomyelin and phosphatidylcholine towards metal-assisted hydrolysis. Published NMR and FT Raman structural data show that the polar head groups of SM and PC are approximately parallel with respect to the bilayer surface due to the gauche conformation of the choline O-C-C- N+ backbone. The head group of PC lies almost exactly along the surface. In the case of SM, however, intramolecular hydrogen bonding between the OH group of sphingosine and the phosphate ester oxygen on the ceramide side of phosphorous causes the polar head group to tilt

15 degrees towards the interior of the bilayer (Niemelä et al. 2004). The interaction between the phosphate oxygen and the sphingosine hydroxyl reduces the binding of Ca(II) to sphingomyelin relative to phosphatidylcholine (Shah and Schulman 1966). The hydrogen bond also decreases the ability of SM to hydrogen bond to water, lowering the overall hydration state of the polar region of SM (Schmidt et al. 1997; Chiu et al. 2003; Niemelä et al. 2004). Interestingly, the enzymatic activity of the acid hydrolase phospholipase A2 has been correlated to levels of

phospholipid bilayer hydration (Oliver et al. 1995). Because water is also required in metal- assisted hydrolysis reactions, the reduced levels of sphingomyelin hydration that arise from intramolecular hydrogen bonding could explain why this phospholipid less susceptible to cerium(IV) hydrolysis than phosphatidylcholine.

A second explanation takes into account the effects of membrane fluidity on hydrolysis yields. The fatty acid chains of the sphingomyelin and phosphatidylcholine preparations used in this study have an average of 0.2 and 1.28 double bonds per molecule, respectively. The higher fatty acid saturation state of SM coupled with extensive intramolecular and intermolecular hydrogen bonding afforded by the NH and OH groups of sphingosine result in significant differences in the dynamic properties of SM and PC bilayers (Niemelä et al. 2004). For example, sphingomyelin membranes have reduced fluidity, with suppressed lateral and rotational diffusion rates (Niemelä et al. 2004) and average gel-to-fluid transition temperatures (~ 37 ºC; Bar et al. 1997) that are significantly higher than phosphatidylcholine. Ruiz-Argüello et al. (2002) have demonstrated that the rate of acid sphingomyelinase hydrolysis can be enhanced by using phosphoglycerides to increase the fluidity of SM bilayers. The authors proposed that higher rates of sphingomyelin diffusion increased the probability of an interaction between the enzyme and substrate. Similarly, in our Ce(IV) reactions with SM (Figures 2.2 and 2.3) and PC (Kassai et al.

2011), hydrolysis yields were substantially increased upon the addition of sub-micellar concentrations of Triton X-100, a non-ionic surfactant that increases the fluidity of PC bilayers (Goñi et al. 1986). Taken together, the above information suggests that the differences in membrane dynamics exhibited by sphingomyelin and phosphatidylcholine have an influence on Ce(IV) hydrolysis levels. When using Ln(III) cations to cleave unilamellar vesicles of a p- nitrophenol-activated anionic lipid, Moss and co-workers observed that cleavage yields could be increased by the high transverse diffusion (flip-flop) rates occurring above the Tm of the lipid

vesicles (Scrimin et al. 1998). In contrast to anionic and other charged lipids that promote flip- flop by repelling one another electrostatically, the translocation of PC, SM, and other neutral, zwitterionic phospholipids across the bilayer is extremely slow, even at temperatures above the Tm (Moss 1994; Contreras et al. 2010). Furthermore, hydrolysis of phosphate ester bonds of SM

and PC would yield ceramide and diacylglycerol, respectively (bonds A and B; Figure 2.1). Ceramide increases the flip-flop rates of other lipids in the bilayer, but diacylglycerol has no effect (Contreras et al. 2010). While it is possible that accelerated lateral diffusion makes a significant contribution to the relatively high levels of phosphatidylcholine hydrolysis produced by Ce(IV), it is less likely that transverse diffusion of PC from the inner to the outer membrane leaflet plays an major role.

Figure 2.7. Ratio of averaged hydrolysis yields at ~ pH 4.8 to averaged hydrolysis yields at ~ pH 7.2 for 2 mM of sphingomyelin (SM) or 2 mM of phosphatidylcholine (PC) treated with 10 mM of Ce(NH4)2(NO3)6. The averaged hydrolysis yields used to calculate the ratios and the number

of trials (n) are in Figs. 5 and 6. Error bars represent standard deviation.

In Figures 2.5 and 2.6, we have established that cleavage of phosphatidylcholine proceeds in higher yield than sphingomyelin. In order to be effective in the treatment of lysosomal storage disease, a small-molecule, hydrolytic agent should display optimal levels of activity at lysosomal pH (~ 4.8) accompanied by low amounts of cleavage under near-neutral conditions. This prompted us to compare the differential levels of phospholipid hydrolysis produced by Ce(NH4)2(NO3)6 at the two pH values. In Figure 2.7 are hydrolysis ratios calculated

using the data from Figures 2.5 and 2.6. Thus, for sphingomyelin and phosphatidylcholine reactions at 60 °C and 37 °C, yields of inorganic phosphate and choline hydrolysis at ~ pH 4.8 have been divided by corresponding yields at ~ pH 7.2 (Figure 2.7). A high ratio is desirable, because it indicates that phospholipid cleavage at ~ pH 7.2 is suppressed with respect to cleavage

at ~ pH 4.8. All of the ratios in Figure 2.7 are above 1, indicating that cerium(IV) hydrolyzes lipid vesicles of sphingomyelin and phosphatidylcholine more efficiently at lysosomal pH. Interestingly, under any given set of reaction conditions, the averaged sphingomyelin ratios of ~ pH 4.8 hydrolysis to ~ pH 7.2 hydrolysis are usually greater (Figure 2.7). The causes underlying the latter phenomenon have yet to be determined and continue as a subject of research in our laboratory. Factors such as phospholipid bilayer hydration, cation binding, and gel-to-fluid transition temperature are sensitive to changes in pH (Hauser and Phillips 1979; Chemin et al. 2008) and, in theory, can play a role. However, sphingomyelin and phosphatidylcholine are predominately zwitterionic over a wide pH range (~ pH 3 to pH 13), with minimal protonation of the phosphate ester oxygen. (In PC monolayers, only 2.6% of the phospholipid molecules are protonated at pH 2.5 (Moncelli et al. 1994).) As a result, binding of Ln(III) cations to PC is independent of pH between pH 3.0 and pH 10.0 (Hauser and Phillips 1979), the hydration of SM is unaffected from pH 3.0 to pH 7.0 (Chemin et al. 2008), and the gel-to-fluid transition temperature of PC is constant from ~ pH 4.5 to pH 7.0 (Furuike et al. 1999). It is therefore conceivable that many of the physical parameters pertinent to SM and PC bilayers remain relatively constant over the ~ 4.8 to ~ 7.2 pH range employed in our study.