Lactate Dehydrogenase Like Crystallin: A Potentially Protective Shield for Indian Spiny-Tailed Lizard (Uromastyx hardwickii) Lens Against Environmental Stress?

Taxon specific lens crystallins in vertebrates are either similar or identical with various metabolic enzymes. These bifunctional crystallins serve as structural protein in lens along with their catalytic role. In the present study, we have partially purified and characterized lens crystallin from Indian spiny-tailed lizard (Uromastyxhardwickii). We have found lactate dehydrogenase (LDH) activity in lens indicating presence of an enzyme crystallin with dual functions. Taxon specific lens crystallins are product of gene sharing or gene duplication phenomenon where a pre-existing enzyme is recruited as lens crystallin in addition to structural role. In lens, same gene adopts refractive role in lens without modification or loss of pre-existing function during gene sharing phenomenon. Apart from conventional role of structural protein, LDH activity containing crystallin in U. hardwickii lens is likely to have adaptive characteristics to offer protection against toxic effects of oxidative stress and ultraviolet light, hence justifying its recruitment. Taxon specific crystallins may serve as good models to understand structure–function relationship of these proteins.


Introduction:
Lenses of vertebrate eyes play vital part in maintaining transparency and refractive index [ 1]. Main component of lenses are structural proteins, called crystallins. Due to their unique properties, role in transparency, evolutionary history and distribution patterns, crystallins have been subject of interest since decades. They are mainly classified into two groups, namely ubiquitous crystallins and Enzyme or taxon specific crystallins. First group contains a, p and y crystallins which are most prevalent in ve1iebrate and invertebrate lenses while latter includes taxon specific crystallins which are identical or closely related with metabolic enzymes prevalent in scattered group of species [2]. Taxon specific crystallins were first observed in birds followed by discovery in lenses of other species [3]. These crystallins are shown to be either the product of gene sharing [4] or gene duplication phenomenon [5]. During gene sharing phenomenon, same gene adopts refractive role in lens without modification/loss of pre-existing function [4]. The 1:-crystallins of Crocodylus palustris is an example of gene sharing phenomenon containing sequence similarity with a-enolase from brain, hemi, and gonad [ 6]. In gene duplication, however, original gene is duplicated to produce two copies of gene among which one copy retains its original catalytic activity while other become structural protein in lens [5]. In avian lens, argininosuccinate lyase gene is duplicated; one copy of gene maintained its role as enzymes (o2-crystallin), while other gene has evolved as structural protein by losing little or all catalytic activity [3].
A major protein component in birds and reptiles is s crystallin which is homologous to glycolytic enzyme lactate dehydrogenase (LDH) [7,8]. Lactate dehydrogenase is responsible for converting glucose to lactate during anaerobic condition [9], subsequently, forming ATP. LDH has various isozymes which are tissues specific; such as LDH-A and LDH-B are found in muscle and heart, respectively. During evolution, the distribution of LDH isozymes has varied in different organs but function remained same [10]. Scientists suggested that distribution of LDH isozymes in different organs might be due to gene duplication or gene sharing phenomenon under selective pressures [1]. Birds and reptiles have diverged almost 200 million years ago and still share some morphological characters; however, difference in their proteins composition has been observed [11,12]. Staple et a!. [8] have reported presence of s-crystallin in many avian lenses. In case of reptiles, evidence for s-crystallin existence was found only in caiman, crocodiles and alligator which belong to order crocodylus. So far, there is only one report for presence of s-crystallin in gecko phelsuma, a member of order squamata [ 13].
In this study, Uromastix hardwickii was used as an experimental model.
Uromastix hardwickii belongs to reptilian family, order squamata, and has certain unique characteristics of amphibians, birds and mammals [14]. It is a terrestrial, hibernating, burrowing and diurnal animal commonly found in desert. Due to diversified environment Uromastix hardwickii lives in, it is a model animal to understand the gene recruitment phenomenon and biochemical adaptations. The lens crystallins of Uromastix hardwickii has not been studied so far. In the present investigation, partial purification and characterization of £-crystallin was performed using chromatographic techniques including gel filtration, RP-HPLC and affinity chromatography. Furthermore, e-Ciystallin!LDH gene expression and DNA sequencing studies were also conducted.

Materials and Methods:
Sample collection and protein extraction: The study was conducted after approval of Institutional Review Board, University of Karachi. All research procedures followed were in accordance with the standards set forth in the Guide for the Care and Use of Laboratory Animals (National Academy of Science, National Academy Press, Washington, D.C.). Fresh lenses from Uromastix hardwickii were removed and homogenized in 50 mM sodium phosphate buffer, pH 7.0 on ice in a ratio of 1110 (w/v). Homogenate was centrifuged at 15,000 x g for 20 min at 4°C. The supernatant was collected and labeled as water soluble fraction. The protein concentration was determined by Bradford protein assay kit (BioRad).

Protein purification:
Gel filtration chromatography was performed for the separation of lens proteins. 100 mg protein was loaded on Sephacryl S-300 gel filtration column (90x2.5cm) and eluted at room temperature with O.OlM phosphate buffer saline (pH 7.4).
Fractions were collected at the flow rate of 12-15 mllhr. Both crude water soluble lens proteins and peak-2 from gel filtration chromatography were fractionated by RP-HPLC (Perkin Elmer USA). !50 fll sample was injected to RP CIS (25x46mm) column equilibrated with 0.1 % Trifluoroacetic acid (solvent A). Proteins were eluted using a gradient of solvent A and solvent B (Acetonitrile containing 0.1% Trifluoroacetic acid) attaining 70 % B in 40 minutes. Elution was monitored at 280 mn. Affinity chromatography was also employed to separate proteins from gel filtration chromatography peak-2 and crude lens protein extract. Fresh lenses (5) were homogenized in 20 mM Tris-HCl buffer, pH 8.0 (equilibration buffer) and centrifuged at 15,000 x g for 20 min at 4 °C. Approximately 7.2 mg protein from water soluble fraction in equilibration buffer was applied on gel affinity column (10 x 1.5 em) packed with Affi-gel Blue (BioRad). After elution of unbound fractions in equilibration buffer, bound proteins were eluted with same buffer containing 1M NaCI. Same procedure was used for affinity purification of catalytically active gel filtration fraction (peak-2) after an overnight dialysis in equilibration buffer at 4 °C.

Measurement of enzymatic activity:
Lactate dehydrogenase activity was determined in water soluble fraction, individual peaks of gel filtration chromatography and affinity chromatography purified fractions usmg Randox Ld pyruvate lactate assay kit (Cat no. LD 40 I). The activity was determined by monitoring the decrease in absorbance at 340 nm for 3 min [ 15].

N-terminal sequencing:
Partially purified protein fraction of affinity clu·omatography was subjected to SDS-PAGE and electroblotted onto a PVDF membrane. The bands were excised and analyzed for N-terminal amino acid sequencing using a Procise automated protein sequencer (Applied Biosystems, Inc).

nLC-MS/MS analysis:
Bands from SDS-PAGE gel of partially purified affinity chromatography fraction water, 0.1% Formic acid). Peptide separation was achieved with multi-step gradient from 3.2% to 80% solution B at the flow rate of 300 nL/min over 70 min. Mass spectra of nLC-MS/MS data were analyzed for protein identification using Mascot search engine against NCBim (www.ncbi.nlm.nih.gov/RefSegD.

Gene expression analysis:
Total RNA was extracted from Uromastix hardwickii lenses by using SV total RNA isolation system kit (Promega, USA) according to manufacturer's protocol.
Total RNA was reverse transcribed into eDNA by using reverse transcription kit (Invitrogen, UK). PCR amplification of total lens eDNA was performed by using

7 Agarose gel electrophoresis:
PCR product was visualized by using agarose gel electrophoresis. Agarose (2%) gel was prepared in TBE buffer (45mM Iris, 45mM Boric acid, 1mM EDTA) pH 8.5. Agarose was heated in TBE buffer until solution became clear. O.SfLg/ml ethidium bromide was added in warm agmose solution. After agarose gel solidification, PCR products (12ft!) were loaded in wells. TBE buffer was used as running buffer at 80 rnA for 40 min. DNA ladder (100 bp) was used as marker.
The DNA bands were visualized under UV light using gel documentation system (Bio-Rad).

Results:
Total protein content determined by Bradford protein assay from water soluble fraction was found to be 1.5 mg/lens. Elution profile of crude lens homogenate from Uromastix hardwickii on Sephacryl S-300 column is presented in Fig. I.
Total lens homogenate was fractionated into five peaks which were all examined for LDH activity. Only peak-2 was found to be catalytically active (37.14 U/L).
SDS-PAGE of peak-2 revealed more than one protein bands is shown in Fig. 2.
Elution profile of total lens homogenate and rechromatography of peak-2 from Sephacryl S-300 by reverse phase HPLC is depicted in Fig.3. Peak-2 rechromatography yielded a peak which was subjected to nLC-MS/MS analysis which revealed a match with ys crystallin from Iguana iguana (Ac. No: AAV54036) with 7% coverage. Affi-gel purified fraction had an activity of 284.76 U/L LDH (Fig. 4). SDS-P AGE analysis of affi-gel fraction revealed two bands of 22 kDa and 14 kDa which showed 22% and 20% band density, respectively (Fig. 5).
N-termina1 sequencing analysis of 22 kDa protein band resulted in sequence of 9 residues. BLASTp analysis of observed sequence identified 22 kDa band to be truncated ~A2-crystallin (Fig. 6) Gene expression from lens and liver using o-crystallin/LDH primers is shown in Fig. 7. LDH expression was found to be higher in liver as compared to the lens.