Purification and properties of a monomeric lactate dehydrogenase from yak Hypoderma sinense larva

Experimental Parasitology 134 (2013) 190–194

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Purification and properties of a monomeric lactate dehydrogenase from yak Hypoderma sinense larva Pengfei Li, Suyu Jin, Lin Huang, Haohao Liu, Zhihong Huang, Yaqiu Lin, Yucai Zheng ⇑ College of Life Science and Technology, Southwest University for Nationalities, Chengdu, Sichuan 610041, China

h i g h l i g h t s

g r a p h i c a l a b s t r a c t

" LDH from H. sinense larva consists of

A monomeric lactate dehydorgenase was purified from yak H. Sinense larva; this enzyme was thermal stable and pH insensitive, showing a very low Km for substarte lactate.

a single subunit. " LDH from H. sinense larva shows significantly lower Km for lactate than that of other animals. " LDH from H. sinense larva is a thermal stable and pH insensitive enzyme.

a r t i c l e

i n f o

Article history: Received 2 April 2012 Received in revised form 6 February 2013 Accepted 19 February 2013 Available online 6 March 2013 Keywords: Yak Lactate dehydrogenase Hypoderma sinense Larva Hypodermosis

a b s t r a c t The objective of the present study was to study the characteristics of lactate dehydrogenase (LDH) from Hypoderma sinense larva. H. sinense larvae were collected from yak (Bos grunniens) and identified by a PCR-RFLP method. Analysis of LDH activity showed that the total LDH activity in H. sinense larva was negatively correlated with the length of larva. Polyacrylamide gel electrophoresis of the extracts of H. sinense larvae revealed one band of LDH, which was then purified by affinity chromatography and gel filtration. This enzyme showed an approximately 36 kDa band on SDS-gel under both reducing and non-reducing conditions, in addition, size exclusion chromatography analysis showed that its molecular weight was smaller than bovine serum albumin (67 kDa), indicating that it contains only one subunit. Michaelis constants (Km) values assay revealed that LDH from H. sinense larva showed significantly lower Km for lactate than other animals. LDH of H. sinense larva was stable at 60 °C for 15 min, and also exhibited high catalytic efficiency in a wide range of pH. HgCl2 at the concentration of 0.1 mM significantly decreased the activity of LDH from H. sinense larva but not at the concentration of 0.01 mM. The results of the present study demonstrate that LDH from H. sinense larva is a thermal stable and pH insensitive enzyme suitable for catalyzing both forward and reverse reactions. Ó 2013 Elsevier Inc. All rights reserved.

1. Introduction Larvae of Hypoderma spp. cause myiasis to develop in warbles under the skin of domestic and wild ruminants, and thus greatly impair livestock production. In China, hypodermosis is one of the most important arthropod pest diseases of yaks (Bos grunniens) and cattle (Otranto et al., 2004, 2006). The species of Hypoderma ⇑ Corresponding author. Fax: +86 28 85522310. E-mail address: [email protected] (Y. Zheng). 0014-4894/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.exppara.2013.02.013

present in China include H. sinense, H. bovis, and H. lineatum. It was reported that H. sinense, which mainly affects yaks and cattle in China, accounted for approximately 90% in Tibetan region (Li et al., 2004). Lactate dehydrogenase (LDH, EC 1.1.1.27) constitutes a major checkpoint of anaerobic glycolysis by catalyzing the reduction of pyruvate into lactate. LDH is a tetramer composed of M and H subunit encoded by ldha and ldhb genes respectively (Li, 1989). The assembly of H and M subunits results in five isozymes of LDH (LDH1 to LDH5) in somatic tissues of nearly all vertebrates. The

P. Li et al. / Experimental Parasitology 134 (2013) 190–194

third subunit C forms LDH-C4 which is expressed only in testis of mammals (Wheat and Goldberg, 1977). LDH has recently received a great deal of attention since it may constitute a valid therapeutic target for diseases dependant on anaerobic glycolysis for energy production. Marchat et al. (1996) purified LDH isozymes from the filarial worm Molinema dessetae, and found high level of LDH activity and some structural and kinetic differences between mammalian and filarial LDH isozymes. The expression of LDH is essential for parasite such as Toxoplasma gondii (Al-Anouti et al., 2004), and therefore the study of parasite LDH is of potential significance for developing new drugs (Vander Jagt et al., 1981; Brown et al., 2004; Berwal et al., 2006). This experiment deals with the isolation and characterization of LDH from H. sinense larvae affecting yaks, in an attempt to reveal its characteristics.

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tion system. Flow rate was set at 1 ml/min. Bound LDH was then eluted with NAD+-sodium pyruvate adduct (Zheng et al., 2008). The eluate was condensed to 3.75 ml by freeze drying and further separated by gel filtration (Superdex 75, 1.5 cm  15 cm) and eluted with 0.02 mol/L phosphate buffer (pH 7.0), and the fraction containing LDH was assayed by SDS–PAGE and gel filtration (Superdex 75, 1 cm  20 cm) to estimate the molecular weight of LDH (Laemmli, 1970). Protein concentration was determined using a protein–dye binding method (Bradford, 1976) with bovine serum albumin as standard. The fidelity of the purified LDH was confirmed by its catalytic ability and the inhibition of its activity by oxalate (substrate analogue). In addition, substrate sodium D-lactate (Sigma–Aldrich, Switzerland) and cofactor NADPH (Roche, France) were used respectively to test the specificity of the purified LDH.

2. Materials and methods 2.5. Enzyme property assay 2.1. Collection and species identification of Hypoderma larvae In the winter season (around November), about 40 subdermal Hypoderma larvae of second- or third-stages were collected from several yaks (Bos grunniens) at slaughter house in Sichuan province (China). The larvae were collected mainly at the inner skin surface of yaks, and their developmental stages were checked under a stereomicroscope based on their morphological features (mainly pattern of spinulation) as describe by Zumpt (1965) for Hypoderma spp. Larvae with spinulation on body surface from the first to eleventh segments were regarded as the second and third larval stages. After being washed in phosphate saline buffer, the weight and length of larva were recorded and the larvae were stored at 80 °C. Meanwhile, five longissimus muscle samples of yaks were collected for the comparison of LDH isozyme profiles with those of Hypoderma larvae. Genomic DNA was isolated from whole Hypoderma larvae extracts using Chelex-100 protocol (Amills et al., 1997), and a PCR-RFLP method was used for the species identification of Hypoderma larva (Otranto et al., 2003). 2.2. Enzyme extraction Each H. sinense larva was homogenized separately with electric homogenizer in 10-fold volume of 20 mmol/L Tris–HCl (pH 7.5) at 4 °C, and then centrifuged at 10000 g for 20 min at 4 °C (Jurie et al., 2006). The supernatant was subjected to electrophoresis of LDH isozymes, total LDH activity assay and genomic DNA extraction aforementioned. 2.3. Total LDH activity and isozyme assay LDH activity was measured spectrophotometrically by recording the change of A340 nm of reaction mixture (Marchat et al., 1996). One unit of activity is defined as the amount of enzyme required to oxidize/reduce 1 lmol of coenzyme min 1 at 25 °C. LDH isozymes from the above extracts were separated with native polyacrylamide electrophoresis (PAGE) using 7% separating gel at 4 °C. After electrophoresis, the gel was activity stained according to the method of Dietz and Lubrano (1967).

The Michaelis constants (Km) of purified LDH from H. sinense larvae were measured from Lineweaver–Burk plots (Marchat et al., 1996). The Km values of LDH for substrates (NADH, pyruvate, NAD+ and lactate) were determined, respectively. The activity of LDH was analyzed at varied temperature and pH, as well as different concentration of HgCl2 using forward reaction system (the reaction media contained 0.2 mM NADH, 5 mM sodium pyruvate). The thermo stability of LDH was assayed by incubating the purified LDH for 15 min at varied temperature. In addition, the inhibition of the LDH activity by substrate analogue oxalate was also evaluated. All measurements were carried out with three repeats. 3. Results 3.1. Identification of larva species A 700 bp PCR product for the most variable region of the mitochondrial cytochrome oxidase I (COI) gene was obtained from the larvae of Hypoderma, this product could be digested into two fragments (420 bp and 270 bp) by BfaI restriction enzyme (Supplementary material S1). Direct sequencing of the PCR product showed that it shared 99.56% homology with that of Hypoderma sinense (GenBank accession number: AY350769), suggesting that the larvae examined belongs to Hypoderma sinense. 3.2. Total LDH activities in Hypoderma sinense larva The lengths of the experimental H. sinense larvae ranged from 0.5 cm to 2.1 cm. The total LDH activity (for the reduction of pyruvate) in H. sinense larvae were negatively correlated with the lengths of larvae (Supplementary material S2). PAGE analysis re-

2.4. Purification of LDH from H. sinense larva Extract mixtures of H. sinense larvae were used as the crude extract and incubated at 60 °C for 15 min, and then centrifuged at 20000 g for 15 min at 4 °C. The supernatant (11 ml) was then loaded onto a HiTrap™ Blue HP affinity column (GE lifescience; bed volume 5 ml) and washed free of unbound proteins with 0.02 mol/L phosphate buffer (pH 7.0) using ÄKTA prime purifica-

Fig. 1. Native PAGE profile of LDH in larva of H. sinense. LDH isozymes in the extracts of H. sinense larvae were separated by native PAGE and enzyme activity stained. Y, yak muscle extract; lanes 1–8, extracts of H. sinense larvae.

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Table 1 Purification steps for LDH of H. sinense larva. Purification step

Volume (ml)

Total Protein (mg)

Total activity (U)

Specific activity (U/mg protein)

Purification (fold)

Crude extract Heat treatment Affinity chromatography Gel filtration

11 11 3.75 7.5

44.8 32.5 0.15 0.06

30.84 30.69 15.03 11.70

0.69 0.94 100 195

1 1.4 145 283

Fig. 2. SDS–PAGE of LDH from H. sinense larva. The separating gel contained 12.5% acrylamide and run under reducing condition. Lane 1, crude extract; lane 2, heattreated crude extract; lane 3, Blue HP affinity chromatography eluate; lane 4, Superdex 75 fraction 1; lane 5, Superdex 75 fraction 2; lane 6, Superdex 75 fraction 2 (non-reducing condition, b-mercaptoethnol is absent in the sample buffer); M, protein standards (kDa). The gel was stained with Coomassie brilliant blue R250.

vealed a major band of LDH in the extracts of H. sinense larvae (Fig. 1), and this band migrated much slower than yak LDH1 on the gel. Several minor bands with the same electrophoretic mobilities as yak LDH1 and LDH2 respectively were also observed in most samples (Fig. 1, lanes 1, 2, 4, 5, 7 and 8). In addition, we discovered LDH1 and LDH2 minor bands with different mobilities in some larva extract (Supplementary material S3, lane 3) 3.3. Purification of LDH from H. sinense larvae LDH of H. sinense larva was purified by approximately 280 fold using a three purification steps, resulting in a purified larva LDH with relative activity of 195 U/mg protein (Table 1). Blue HP affinity chromatography removed most of the proteins in the crude extract (Fig. 2). SDS–PAGE showed that the purified LDH of H. sinense larva has a molecular weight of approximately 36 kDa as calculated from a plot of the log mol.wt of protein standards versus relative mobility (Fig. 2). Size exclusion chromatography (Superdex 75) analysis showed that larva LDH had longer elution time than bovine serum albumin (67 kDa), confirming that it is not dimer or tetramer (Fig. 3). 3.4. Properties of LDH from H. sinense larvae The purified H. sinense larva LDH was specific for L(+)-lactate and did not react with D( )-lactate. In addition, it could not utilize NADPH as cofactor. The purified larva LDH could catalyze the conversion of pyruvate and L-lactate, which was inhibited by substrate analogue oxalate (Table 2), confirming the identity of purified LDH. LDH of H. sinense larvae was thermo stable, and incubation of the purified enzyme at 65 °C for 15 min resulted in only about 15% loss of LDH activity (Fig. 4a). Additionally, LDH of H. sinense larvae exhibited high activity in a wide range of pH (Fig. 4b). HgCl2

Fig. 3. Elution profile of Blue HP affinity chromatography eluate on Superdex 75 column. Two protein standards (bovine serum albumin, BSA, 67 kDa; lysozyme, 14.7 kDa) were used to estimate the molecular weight of LDH. The second peak of LDH protein curve was formed by the NAD+-sodium pyruvate adduct in affinity chromatography elution buffer. The three curves indicate that larval LDH protein and LDH activity peaks are recovered at the same retention time and longer than BSA peak.

Table 2 Inhibition of the LDH activity of H. sinense larva by substrate analogue oxalate. Reaction direction

Pyruvate reduction Lactate oxidation

Oxalate concentration (mmol/L) 0.5

1

5

42.7 84.6

28.0 55.3

6.7 23.1

LDH activity in the table is expressed as residual activity (%).

at the concentration of 0.1 mM significantly decreased the activity of LDH but not at the concentration of 0.01 mM (Fig. 4c). Substrate analogue oxalate decrease LDH activity of both pyruvate reduction and lactate oxidation significantly at a concentration of 0.5 mmol/L (Table 2). Km values were measured for substrates of both forward and reverse reaction (Supplementary material S4). LDH from H. sinense larvae showed significantly lower Km value for lactate than other animals (Table 3). 4. Discussion Molecular identification of Hypoderma species is a useful tool in research (Weigl et al., 2010). A simple and cheap method was developed in this experiment to extract genomic DNA from Hypoderma larvae, and used as template for the species identification of Hypoderma larvae by PCR-RFLP (Otranto et al., 2003). This method is applicable for large scale identification of Hypoderma larva species. In this experiment, we revealed a negative correlation between the total LDH activities and the lengths of H. sinense larvae (Supplementary material S2). LDH is a key enzyme in anaerobic glycolysis, therefore we propose that larva with smaller size is more dependant on glycolysis to supply metabolic energy. The LDH isozyme

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Fig. 4. Influence of temperature (a), pH (b) and HgCl2 (c) on LDH activity of H. sinense larva. LDH activity assay was carried out using the reduction reaction of pyruvate, and was expressed as residual activity.

Table 3 Km values (mM) determined for pyruvate, NADH, lactate and NAD+. LDH origin

H. sinense larva M. dessetaea D. melanogaster larvab Bovine LDH-H4c Lizard LDH-M4d a b c d

Pyruvate reduction

Lactate oxidation

pH

Km Pyruvate

Km NADH

pH

Km lactate

Km NAD+

7.0 6.8 6.8

0.155 0.34 0.154

0.043 0.25 0.027

8.8 8.8 8.8

0.26 2.5 29.4

0.31 0.18 1.33

6 6

0.05 0.02

0.014 0.04

9 9

7.4 8.1

0.11 0.04

Marchat et al. (1996). Onoufriou and Alahiotis (1982). Winer and Schwert (1958). Al-Jassabi (2002).

profile of H. sinense larvae (Fig. 1) is obviously different from mammalian LDH, which is contributed by five electrophoretically distinguishable isozymes (LDH1 through LDH5) as a result of the combination of two types of subunits (Holbrook et al., 1975). The minor bands at the position exactly corresponding to yak LDH1 and LDH2 observed in most samples (Fig. 1) are most likely originated from yak tissue fluid intake by the larvae, since the minor

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bands in the extracts of some H. sinense larvae migrated faster on the gel (Supplementary material S3), exactly corresponding to the unique F variant of yak LDH1 (Kuang et al., 2010). The reason for the absence of the minor yak LDH1/LDH2 bands in lanes 3 and 6 of Fig. 1 should be related to the intake amount of yak blood or tissue fluid by the H. sinense larvae in lanes 3 and 6. The LDH of H. sinense larva was purified by affinity and gel filtration chromatography, and confirmed by its catalytic function and the specific inhibition of substrate analogue oxalate. The purified LDH of H. sinense larvae showed only one band with molecular weight of approximately 36 kDa on SDS-gel (Fig. 2), meanwhile, size exclusion chromatography analysis showed that larva LDH exhibited longer elution time than bovine serum albumin (67 kDa) (Fig. 3), indicating that it contained only one subunit with a conserved molecular weight similar to H or M subunits of LDH from mammals (Holbrook et al., 1975; Al-Jassabi, 2002). The estimated molecular weight of H. sinense larvae LDH (36 kDa) is similar to that of D. melanogaster larva (Onoufriou and Alahiotis, 1982) and consistent with the theoretical molecular weight (35.5 kDa) of D. melanogaster LDH (GenBank: U68038.1). However, D. melanogaster LDH was reported to be tetrameric and composed of four identical subunits (Onoufriou and Alahiotis, 1982). While most LDHs from mammals are tetramers (Holbrook et al., 1975), LDH from M. dessetae seems to be dimmer of two subunits with molecular weight of 58 kDa (Marchat et al., 1996). Bacterial LDH also exhibits tetramer (Auerbach et al., 1998). Thus the tetramer structure is the most common form of LDH. Another interesting property of LDH from H. sinense larvae is that it showed a much lower Km value for lactate than other animals (Marchat et al., 1996; Onoufriou and Alahiotis, 1982; Winer and Schwert, 1958; Al-Jassabi, 2002), and the Km values for lactate and pyruvate did not differ considerably (Table 3), while in other animals, the Km value for pyruvate is significantly lower that the Km value for lactate (Marchat et al., 1996; Onoufriou and Alahiotis, 1982; Winer and Schwert, 1958; Al-Jassabi, 2002), and pyruvate reduction is the favored reaction. This means that LDH from H. sinense larvae has similar affinity for lactate and pyruvate, and can effectively catalyze the formation of pyruvate from lactate at a relatively low concentration, making it possible for the larvae to utilize lactate under fully aerobic condition. Therefore, we suppose that the lower Km value for lactate and the similar Km values for lactate and pyruvate of LDH from H. sinense larvae are of special significance for the utilization of lactate in the aerobic environment it inhabits. In fact, the utilization of lactate as fuel has also been reported in animal tissues (Brooks, 1986, O’Brien et al., 2007). This experiment showed that H. sinense larva LDH was thermo stable; it also maintained high activity in a wide pH range and was more resistant to HgCl2 inhibition than M. dessetae (Marchat et al., 1996). These properties suggest that H. sinense larvae LDH has a stable three-dimensional structure, perhaps related to its characteristic of single subunit composition. Human testis-specific LDH-C4 was also reported to be heat-stable relative to the somatic LDH isozymes (LeVan and Goldberg, 1991), and LDH5 (M4) of lizard was stable even at 70 °C (Al-Jassabi, 2002). The physiological significance of the thermo and pH stability of H. sinense larva LDH is not clear. LDH-A inhibition is a potential strategy for the treatment of diseases with extensive anaerobic glycolysis (Xie et al., 2009). The rational routes to parasite drug discovery require the identification of targets that have distinct properties between host and parasite. Further investigation of inhibition kinetic of LDH from H. sinense larva will be helpful for the development of specific inhibitors. Such research has been carried out in other parasites (Dando et al., 2001; Yang et al., 2006). Our present experiment demonstrated that the specific substrate analogue oxalate exerted strong inhibition effect on H. sinense larva LDH activity at a low concentra-

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tion (<0.5 mM, Table 2). Similar compounds might be potential drugs for the treatment of myiasis in yaks and cattle. In conclusion, our study made a contribution to the basic study of LDH from H. sinense larvae in an attempt to demonstrate characteristics of this enzyme. LDH from H. sinense larva is composed of single subunit and showed a much lower Km value for lactate. The high thermo stability and insensitivity to pH and Hg2+ inhibition indicate that this enzyme has a more stable three-dimensional structure than LDH from other animals. Acknowledgments This work was supported by the Application Basic Research of Sichuan Province (Grant No. 2009JY0018) and Animal Science Discipline Program of Southwest University for Nationalities (Grant No. 2011XWD-S0905). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.exppara.2013. 02.013. References Amills, M., Francino, O., Jansa, M., Sanchez, A., 1997. Isolation of genomic DNA from milk sample by using Chelex resin. The Journal of Dairy Research 64, 231–238. Al-Anouti, F., Tomavo, S., Parmley, S., Ananvoranich, S., 2004. The expression of lactate dehydrogenase is important for the cell cycle of Toxoplasma gondii. The Journal of Biological Chemistry 279, 52300–52311. Al-Jassabi, S., 2002. Purification and kinetic properties of skeletal muscle lactate dehydrogenase from the lizard Agama stellio stellio. Biochemistry (Mosc) 67, 786–789. Auerbach, G., Ostendorp, R., Prade, L., Korndörfer, I., Dams, T., Huber, R., Jaenicke, R., 1998. Lactate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima: the crystal structure at 2.1 Å resolution reveals strategies for intrinsic protein stabilization. Structure 6, 769–781. Berwal, R., Gopalan, N., Chandel, K., Prakash, S., Sekhar, K., 2006. Amplification of LDH gene from indian strains of Plasmodium vivax. Journal of Vector Borne Diseases 43, 109–114. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72, 248–254. Brooks, G.A., 1986. Lactate production under fully aerobic conditions: the lactate shuttle during rest and exercise. Federation Proceedings 45, 2924–2929. Brown, W.M., Yowell, C.A., Hoard, A., Vander Jagt, T.A., Hunsaker, L.A., Deck, L.M., Royer, R.E., Piper, R.C., Dame, J.B., Makler, M.T., Vander Jagt, D.L., 2004. Comparative structural analysis and kinetic properties of lactate dehydrogenases from the four species of human malarial parasites. Biochemistry 43, 6219–6229. Dando, C., Schroeder, E.R., Hunsaker, L.A., Deck, L.M., Royer, R.E., Zhou, X., Parmley, S.F., Vander Jagt, D.L., 2001. The kinetic properties and sensitivities to inhibitors of lactate dehydrogenases (LDH1 and LDH2) from Toxoplasma gondii: comparisons with pLDH from Plasmodium falciparum. Molecular and Biochemical Parasitology 118, 23–32. Dietz, A.A., Lubrano, T., 1967. Separation and quantitation of lactic dehydrogenase isoenzymes by disc electrophoresis. Analytical Biochemistry 20, 246–257.

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