Identification of a Metabolizing Enzyme in Human Kidney by Proteomic Correlation Profiling

Molecular identification of endogenous enzymes and biologically active substances from complex biological sources remains a challenging task, and although traditional biochemical purification is sometimes regarded as outdated, it remains one of the most powerful methodologies for this purpose. While biochemical purification usually requires large amounts of starting material and many separation steps, we developed an advanced method named “proteomic correlation profiling” in our previous study. In proteomic correlation profiling, we first fractionated biological material by column chromatography, and then calculated each protein''s correlation coefficient between the enzyme activity profile and protein abundance profile determined by proteomics technology toward fractions. Thereafter, we could choose possible candidates for the enzyme among proteins with a high correlation value by domain predictions using informatics tools. Ultimately, this streamlined procedure requires fewer purification steps and reduces starting materials dramatically due to low required purity compared with conventional approaches. To demonstrate the generality of this approach, we have now applied an improved workflow of proteomic correlation profiling to a drug metabolizing enzyme and successfully identified alkaline phosphatase, tissue-nonspecific isozyme (ALPL) as a phosphatase of CS-0777 phosphate (CS-0777-P), a selective sphingosine 1-phosphate receptor 1 modulator with potential benefits in the treatment of autoimmune diseases including multiple sclerosis, from human kidney extract. We identified ALPL as a candidate protein only by the 200-fold purification and only from 1 g of human kidney. The identification of ALPL as CS-0777-P phosphatase was strongly supported by a recombinant protein, and contribution of the enzyme in human kidney extract was validated by immunodepletion and a specific inhibitor. This approach can be applied to any kind of enzyme class and biologically active substance; therefore, we believe that we have provided a fast and practical option by combination of traditional biochemistry and state-of-the-art proteomic technology.Molecular identification for an enzyme reaction or biologically active substance in an organism is challenging, although molecular biological methodologies such as expression cloning (1), recombinant protein panel (2) and RNAi screening (3) have been introduced recently as alternative approaches. Conventional biochemical purification has provided a number of successes and thus still remains a powerful, though labor-intensive strategy.In the traditional protein purification, it had been necessary to purify an individual protein nearly to homogeneity at a microgram amount so that the purified protein could be analyzed by N-terminal amino acid sequencing. Protein identification by mass spectrometry subsequently revolutionized this technology by enabling identification of proteins at much lower abundances: individual proteins could then be associated with specific activities as soon as a band in SDS-PAGE could be observed, even when the purified protein was far from homogeneity (4–6). Although this streamlined the workflow by reducing the required starting materials as well as the separation steps for protein purification, a faster and more generalized approach from smaller starting material has still been desired because some proteins are physiochemically difficult for example in solubilization and stability. To solve these problems, we devised a proteomic correlation profiling methodology (7).The basic concept of proteomic correlation profiling was originally developed by Andersen et al. (8). They quantitatively profiled hundreds of proteins across several centrifugation fractions by mass spectrometry and identified centrosomal proteins by calculating the correlation of these protein expression profiles with already known centrosomal proteins. In the following study, Foster et al. applied this strategy to map more than 1400 proteins to ten subcellular locations (9). Although these studies used centrifugation as a separation method and a known marker profile as a standard for correlation, we extended this concept to use chromatography as a separation method and kinase activity as a basis for comparison; our approach successfully identified a kinase responsible for phosphorylation of peptide substrates just after one step chromatography, and was termed proteomic correlation profiling (7). Independently, Kuromitsu et al. reported identification of an active substance in the serum response element-dependent luciferase assay from interstitial cystitis urine after three-step chromatography by a similar concept (10). In theory, this general proteomic correlation profiling strategy can be adapted to any kind of separation method and activity profile but no other example has been reported thus far, therefore, actual examples where the method can be applied to other enzyme classes are required to prove its generality.Multiple sclerosis is the most common autoimmune disorder of the central nerve system in which the fatty myelin sheaths around the axons of the brain and spinal cord are damaged, leading to demyelination and scarring (11, 12). Until recently, the standard treatments for multiple sclerosis such as interferon beta, glatiramer acetate, mitoxantrone, and natalizumab would often cause severe adverse events (13, 14), providing an opportunity for development of less dangerous treatments for this disease. However, in 2010, Food and Drug Administration approved fingolimod (Gilenya; chemical structure in Fig. 1) as the first oral medicine, and recommended this as a first-line treatment for relapsing-remitting multiple sclerosis, opening up a new therapeutic approach to the disease (15).Open in a separate windowFig. 1.The chemical structures of CS-0777, fingolimod and their phosphorylated derivatives.Sphingosine 1-phosphate receptor 1 (S1P1)¹ modulators are emerging as a new class of drugs with potential therapeutic application in multiple sclerosis (15), and fingolimod is a nonselective sphingosine 1-phosphate (S1P) receptor modulator (16–18, 21, 22). Given its structural similarity to sphingosine, fingolimod is phosphorylated in vivo by sphingosine kinase, in particular sphingosine kinase 2 (SPHK2) (19, 20), and the fingolimod-phosphate (fingolimod-P, Fig. 1) binds to and activates four G protein-coupled S1P receptors (21, 22). By this mechanism, fingolimod-P induces internalization of S1P1 on lymphocytes, blocking the ability of the receptor to support lymphocyte egress and recirculation through secondary lymphoid organs. This suppresses immune responses and is presumably the main immunomodulatory mode of action of fingolimod.CS-0777 (Fig. 1) is a novel selective S1P1 modulator (23). Although the immunomodulatory effects are supposed to be mainly mediated by S1P1, some lines of evidence suggest that the agonist activity on S1P receptor 3 (S1P3) could cause acute toxicity and cardiovascular deregulation, including bradycardia in rodents (24, 25). Thus, CS-0777 was designed to have more selectivity on S1P1 over S1P3 in contrast to fingolimod-P which has potent agonistic activity for S1P3, S1P4, and S1P5 in vitro (22). Like fingolimod, CS-0777 is also a prodrug phosphorylated in vivo, and the phosphorylated CS-0777 (CS-0777-P, Fig. 1) agonizes S1P1 with more than 300-fold selectivity relative to S1P3 whereas CS-0777-P has weaker effects on S1P5 and no activity on S1P2 (23). CS-0777 showed immunosuppressive activity in mouse and rat models of experimental autoimmune encephalitis, animal models for multiple sclerosis. In healthy volunteers, single oral doses of CS-0777 caused marked, dose-dependent decreases in numbers of circulating lymphocytes, including marked and reversible decreases in circulating T and B cells (26). Furthermore, in multiple sclerosis patients, single oral doses of CS-0777 caused dose-dependent decreases in circulating lymphocytes, with a slightly greater suppression of CD4+ versus CD8+ T cells. Therefore, CS-0777 would alter immune responses solely through activation of S1P1 without S1P3 modulation in humans, which could circumvent a bradycardia adverse effect, although the relationships associating selectivity of S1P1 to S1P3 with bradycardia in humans are not fully understood (12).Orally administrated CS-0777 is phosphorylated and rapidly reaches equilibrium with CS-0777-P as in the case of fingolimod (22), suggesting that the high kinase activity in blood is balanced by phosphatases. Therefore, identification of a phosphatase, the inactivating enzyme of an active metabolite, as well as identification of a kinase, the activating enzyme of a prodrug, are critical to fully understand the mechanism of action at the molecular level for both CS-0777 and fingolimod. Sphingosine kinase 2 (SPHK2) was identified as the major kinase of fingolimod (21, 28, 29) and lipid phosphate phosphatase 3 (LPP3) was reported to be a phosphatase for fingolimod-P dephosphorylation (30), although contribution of LPP3 in vivo has not been fully studied. In our previous work, we have identified CS-0777 kinases in human blood as fructosamine 3-kinase-related protein (FN3K-RP) and fructosamine 3-kinase (FN3K) (6), whereas the phosphatase of CS-0777-P had not been identified thus far.In this study, we have successfully identified alkaline phosphatase, tissue-nonspecific isozyme (ALPL) as the major CS-0777-P phosphatase candidate in the human kidney by proteomic correlation profiling. According to available information, this is the first report applying proteomic correlation profiling to enzyme classes other than kinases; similarly, we believe this to be first application of proteomic correlation profiling to human tissue extract, which therefore has opened up wide usage of proteomic correlation profiling for all types of enzyme identification.     

Diurnal patterns of growth and transient reserves of sink and source tissues are affected by cold nights in barley

Barley is described to mostly use sucrose for night carbon requirements. To understand how the transient carbon is accumulated and utilized in response to cold, barley plants were grown in a combination of cold days and/or nights. Both daytime and night cold reduced growth. Sucrose was the main carbohydrate supplying growth at night, representing 50–60% of the carbon consumed. Under warm days and nights, starch was the second contributor with 26% and malate the third with 15%. Under cold nights, the contribution of starch was severely reduced, due to an inhibition of its synthesis, including under warm days, and malate was the second contributor to C requirements with 24–28% of the total amount of carbon consumed. We propose that malate plays a critical role as an alternative carbon source to sucrose and starch in barley. Hexoses, malate, and sucrose mobilization and starch accumulation were affected in barley elf3 clock mutants, suggesting a clock regulation of their metabolism, without affecting growth and photosynthesis however. Altogether, our data suggest that the mobilization of sucrose and malate and/or barley growth machinery are sensitive to cold.     

Ranging behavior of Mahale chimpanzees: a 16 year study

We have analyzed the ranging patterns of the Mimikire group (M group) of chimpanzees in the Mahale Mountains National Park, Tanzania. During 16 years, the chimpanzees moved over a total area of 25.2 or 27.4 km², as estimated by the grid-cell or minimum convex polygon (MCP) methods, respectively. Annually, the M group used an average of 18.4 km², or approximately 70 %, of the total home-range area. The chimpanzees had used 80 % of their total home range after 5 years and 95 % after 11 years. M group chimpanzees were observed more than half of the time in areas that composed only 15 % of their total home range. Thus, they typically moved over limited areas, visiting other parts of their range only occasionally. On average, the chimpanzees used 7.6 km² (in MCP) per month. Mean monthly range size was smallest at the end of the rainy season and largest at the end of the dry season, but there was much variability from year to year. The chimpanzees used many of the same areas every year when Saba comorensis fruits were abundant between August and January. In contrast, the chimpanzees used several different areas of their range in June. Here range overlap between years was relatively small. Over the 16 years of the study we found that the M group reduced their use of the northern part of their range and increased their frequency of visits to the eastern mountainous side of their home range. Changes in home-range size correlated positively with the number of adult females but not with the number of adult males. This finding does not support a prediction of the male-defended territory model proposed for some East African chimpanzee unit-groups.     

Binding of a pleurotolysin ortholog from Pleurotus eryngii to sphingomyelin and cholesterol-rich membrane domains

A mixture of sphingomyelin (SM) and cholesterol (Chol) exhibits a characteristic lipid raft domain of the cell membranes that provides a platform to which various signal molecules as well as virus and bacterial proteins are recruited. Several proteins capable of specifically binding either SM or Chol have been reported. However, proteins that selectively bind to SM/Chol mixtures are less well characterized. In our screening for proteins specifically binding to SM/Chol liposomes, we identified a novel ortholog of Pleurotus ostreatus, pleurotolysin (Ply)A, from the extract of edible mushroom Pleurotus eryngii, named PlyA2. Enhanced green fluorescent protein (EGFP)-conjugated PlyA2 bound to SM/Chol but not to phosphatidylcholine/Chol liposomes. Cell surface labeling of PlyA2-EGFP was abolished after sphingomyelinase as well as methyl-β-cyclodextrin treatment, removing SM and Chol, respectively, indicating that PlyA2-EGFP specifically binds cell surface SM/Chol rafts. Tryptophan to alanine point mutation of PlyA2 revealed the importance of C-terminal tryptophan residues for SM/Chol binding. Our results indicate that PlyA2-EGFP is a novel protein probe to label SM/Chol lipid domains both in cell and model membranes.     

Exchangeability of the flagellin (FliC) and the cap protein (FliD) among different species in flagellar assembly

Flagellar filament self‐assembles from the component protein, flagellin or FliC, with the aid of the capping protein, HAP2 or FliD. Depending on the helical parameters of filaments, flagella from various species are divided into three groups, family I, II, and III. Each family coincides with the traditional classification of flagella, peritrichous flagella, polar flagella, and lateral flagella, respectively. To elucidate the physico‐chemical properties of flagellin to separate families, we chose family I flagella and family II flagella and examined how well the exchangeability of a combination of FliC and/or FliD from different families is kept in filament formation. FliC or FliD of Salmonella enterica serovar Typhimurium (Salty; family I) were exchanged with those of Escherichia coli (Escco; family I) or Pseudomonas aeruginosa (Pseae; family II). In a Salty fliC deletion mutant, Escco FliC formed short filaments, but Pseae FliC did not form filaments. In a Salty fliD deletion mutant, both Escco FliD and Pseae FliD allowed Salty FliC to polymerize into short filaments. In conclusion, FliC can be exchanged among the same family but not between different families, while FliD serves as the cap protein even in different families, confirming that FliC is essential for determining families, but FliD plays a subsidiary role in filament formation. © 2012 Wiley Periodicals, Inc.     

Identification of the redox partners of ERdj5/JPDI,a PDI family member,from an animal tissue

ERdj5 (also known as JPDI) is a member of PDI family conserved in higher eukaryotes. This protein possesses an N-terminal J domain and C-terminal four thioredoxin domains each having a redox active site motif. Despite the insights obtained at the cellular level on ERdj5, the role of this protein in vivo is still unclear. Here, we present a simple method to purify and identify the disulfide-linked complexes of this protein efficiently from a mouse tissue. By combining acid quenching and thiol-alkylation, we identified a number of potential redox partners of ERdj5 from the mouse epididymis. Further, we show that ERdj5 indeed interacted with two of the identified proteins via formation of intermolecular disulfide bond. Thus, this approach enabled us to detect and identify redox partners of a PDI family member from an animal tissue.     

Interaction Involving Disulfide Bridges between Subunits of Soybean Seed Globulin and between Subunits of Soybean and Sesame Seed Globulins

Native subunit proteins of glycinin, the acidic and the basic subunits designated as AS₁₊₂, AS₂₊₃, AS₄, AS₅, and AS₆ and BS, respectively, were isolated by DEAE-Sephadex A-50 column chromatography in the presence of 6 m urea and 0.2 m 2-mercaptoethanol.

Reconstitution of intermediary subunits involving a disulfide bridge from native acidic and basic subunits was investigated. Formation of the intermediary subunit was observed in combinations between BS and each acidic subunit except AS₆. The yields of the reconstituted intermediary subunits differed from one another.

Further, formation of the intermediary complexes was observed when native acidic and basic subunits of soybean glycinin and sesame 13 S globulin, respectively (or reverse combinations), were mixed under reductively denatured condition and subjected to the reconstitution procedure. Considerring the overall evidence, we may conclude that the complexes are probably a hybrid intermediary subunit.     

Aromatic Amino Acid Residues in Japanese-radish Peroxidase a and Apoenzyme†

The nature of aromatic amino acid residues in Japanese-radish peroxidase a and the apoprotein was investigated by means of spectrophotometry and fluorospectrophotometry. The tyrosine residues in the holoenzyme were masked in the alkali-titration, giving an abnormally high value of 12.6, while they were exposed in the apoenzyme, exhibiting a value of 10.8. The difference spectra in the ultraviolet region between the holo-and apo-enzyme showed characteristic bands of tryptophan and phenylalanine as well as tyrosine. The perturbation of the aromatic amino acid residues by 50% ethyleneglycol was observed in the apoenzyme but not in the holoenzyme. The fluorophotometric experiments also revealed that the aromatic amino acid residues were in different environments in the holo- and apoenzyme. The difference between the conformation of peroxidase and that of the apoprotein was discussed.