Bovine Liver

6.1 Summary

The oxidosqualene-lanosterol cyclase (OSC) from bovine liver has been successfully purified to homogeneity by using ultracentrifugation, Q-Sepharose, hydroxyapatite, and HiTrap heparin chromatographies. The detergent requirement for its activity in the microsomal fraction was also determined. The purified OSC cyclase displayed a single band with the molecular weight of ~70 and ~140 kDa on the silver-stained SDS-PAGE and the Coomassie-stained Native-PAGE, respectively.

Peptide mapping coupled with tandem mass spectrometric determination identified three peptide fragments, ILGVGPDDPDLVR, LSAEEGPLVQSLR, and NPDGGFATYETK, which are highly homologous to human, rat, and mouse OSCs.

After successful purification and tandem mass characterization of bovine liver OSC, the encoded gene was determined via PCR based strategy. The deduced amino acid sequence showed >80% identity to the other three mammalian lanosterol cyclases.

Genetic complementation of bovine liver OSC gene into a yeast erg7 knockout strain, CBY57, demonstrated its functional expression and enzymatic activity. The inhibitor binding site of bovine liver OSC was probed with a potent Ro48-8071 inhibitor and determined via LC/MS/MS. However, due to the low peptide coverage, the precise inhibitor binding site still could not be identified at this time. A monoclonal antibody

was also produced and exhibited its activity against the purified OSC via the rational design of an immunogenic oligopeptide antigen based on the homology modeling structure of cyclase.

6.2 Introduction

As previously described in the Chapter 1, the purification of oxidosqualene cyclase has always met with difficulties in obtaining the functionally active and soluble enzyme. These difficulties were mainly caused by the detergent- or salt-dependence of cyclase activity. Moreover, due to the instability of the solubilized enzyme, the acquisition of a large amount of purified cyclase protein for the crystal structure determination remains a challenge.² Even though the purification of oxidosqualene-lanosterol cyclase has been hampered, some OSCs have been purified to homogeneity. After successful purification of OSCs, the amino acid sequences could be obtained via different methodologies. Recently, numerous cyclase genes were successfully cloned and sequenced, made it is possible to deduce the primary amino acid sequence analysis of OSC. Comparison of amino acids sequence among different eukaryotic oxidosqualene cyclases and prokaryotic squalene cyclases revealed the moderate overall sequence identity and several highly conserved motifs.

These results suggested that the cyclase enzymes might maintain the general structure from their common ancestral protein, but alter the critical functional residues in the specific position for developing their diverse products. Thus, from the concept of diverse evolution, understanding the primary amino acid sequence conservation in OSCs is a prerequisite for the future investigation.

On the other hand, photoaffinity labeling is a technique to study the interaction between two different molecules. It has been used broadly in different biological

fields during the past several years. After irradiation reaction occurred, the covalent bond was formed between two molecules. This covalent labeling technology has become a popular tool to investigate the protein-ligand, protein-peptide, or protein-protein interactions in the proteomics studies recently. One of the features in this technique is that the covalent linkage between photoreactive ligand and its binding protein remains even under the denaturing conditions. These covalent binding information could be elucidated by different levels of analysis.¹⁶² In the protein level, SDS-PAGE or Native-PAGE is used to identify the intact proteins, which have been covalent labeled. After enzymatic or chemical degradation, the labeled fragments could be isolated and identified via different liquid chromatographies.¹⁶² The LC/MS/MS instrument provides a very powerful tool to determine the exact photolabeled amino acid.¹⁶³

In contrast, although the X-ray crystallography and NMR spectroscopic methods are the most powerful tools to study the structure of the ligand binding, the photoaffinity labeling is thought to be independent from above two approaches and provides another evidence for the ligand binding mode.^164-166 The conformation alteration of the binding ligand might happen during crystallization process from the soluble phase to solid phase; or the binding mode of ligand is absolutely different in these two phases. Consequently, the photoaffinity labeling could fit the requirement complementarily for the structural elucidation of protein-ligand binding events.

Various criteria for photoaffinity probes design were discussed recently.¹⁶⁷ First, it should be stable under the ambient light; second, a photogenerated long-life excitation state should be formed for the covalent linkage reaction; third, a single unambiguous covalent adduct should be generated once; fourth, the covalent linkage products should be avoided for the solvent trapping or releasing. Additionally, the

protein. There are three kinds of photoreactive compounds, including aryl azides, aryl diazirine, and benzophenone derivatives extensively used in chemical biology. Their chemical structure and photoinduced mechanism are shown in Scheme 6.1. Among these photoaffinity probes, benzophenone (BP) derivatives are most stable, and the carbonyl oxygen of BPs react preferentially with the neighboring carbon atom within the distance of 3.1Å even in the presence of solvent. This highly efficient covalent modification and the site-specific labeling property of BPs, let it be applied in many biochemical systems.^{167, 168} The mechanism of the covalent bond formation between carbon atom and BPs contain three steps: the triple state excitation, the proton abstraction, and the radical recombination (Scheme 6.2).¹⁶⁸

The Hoffmann-La Roche group has developed a series of BP-containing orally active cholesterol-lowering compounds targeting at OSC. Among them, Ro48-8071 is the most potent OSC (SHC) inhibitor.¹⁶⁹ The tritium-labeled Ro48-8071 was then synthesized and specifically labeled with bacterial SHC or rat liver OSC in either purified or crude extract protein. Competitive displacement experiment also showed that Ro48-8071 might bind in the same location with other two mechanism-based cyclase inhibitors or one nonterpenoid inhibitor.^{169, 170} Moreover, the photoaffinity experiment suggested that Ro48-8071 binds at the junction between the central active cavity and substrate entry channel, and then hinders the enzymatic substrate uptaking ability.¹⁷¹ On the other hands, co-crystallized structure containing Ro48-8071 in SHC or OSC were also established recently.^{89, 105} Interestingly, the binding site of Ro48-8071 in these two structures are very different from the previous photoaffinity labeling experiments. The structural conformations were also contradicted with the well-known noncompetitive inhibition mechanism of Ro48-8071. Due to the difference between these experiment results and the overall difference in the solid or liquid phase system, some more extensive experiments are need to clarify these

divergences.

Scheme 6.1 Three major photoreactive groups.

Scheme 6.2 Formation of covalent adducts from photoexcited benzophenone group.

Therefore, the above described importance or puzzlements, involved in the studies of oxidosqualene-lanosterol synthase, provoked us to purify the oxidosqualene-lanosterol synthase from bovine liver and characterize its catalytic activity in the native environment. The peptide mapping coupled with tandem mass spectrometric determination could provide a tool for identifying the N-terminal amino acid or internal amino acid sequence. The information obtained from the peptide sequencing and mass characterization will be used to clone the OSC gene from the

N₃ Light N

bovine liver cDNA library. After cloning and sequencing of the OSC gene, the cyclase activity is confirmed by using the genetic complementation in a yeast erg7 knockout strain. The sequence comparison will also be performed among the mammalian OSC genes. Moreover, the respective monoclonal antibody will also be developed via the rational designed oligopeptide antigene. In the last part of this chapter, the photoaffinity labeling experiment coupled with the mass spectrometry will be used to further explore the Ro48-8071 binding mode.

6.3 Results and Discussion

6.3.1 Purification of bovine liver OSC

To date, no more than five oxidosqualene cyclases, mainly cycloartenol synthase and β-amyrin synthase from higher plants, have been purified to homogeneity (Table 6.1). In order to obtain the homologous lanosterol cyclase from the mammalian source, we try to purify the oxidosqualene-lanosterol cyclase from bovine liver. Using knowledge from purification of oxidosqualene-lanosterol cyclase in different species, microsomes from bovine liver homogenates were first treated with various concentrations of Triton X-100. The specific activity of the starting microsomal suspension was apparent in the buffer containing 0.5% (w/v) Triton X-100. Therefore, the concentration of detergent was maintained at this level for subsequent purification steps.

Following recovery of OSC from the microsomal fraction, the crude enzyme solution was subjected to chromatographic separation using Q-Sepharose Fast Flow, hydroxyapatite, and HiTrap heparin columns. In each chromatographic separation, the cyclase enzymatic activity was determined by a standard procedure, and the cyclase-containing fractions were collected for the next column chromatography. The

detailed purification procedure and the cyclase activity assay were described in the experimental section.

In brief, the enzyme adsorbed on the Q-Sepharose Fast Flow column was eluted with IEB buffer containing 20, 50, 70, and 100 mM potassium chloride, and the cyclase activity appeared in fractions containing 70 mM salt. Next, the cyclase containing fractions were applied for hydroxyapatite chromatography fractionation.

Interestingly, the cyclase was not retained on hydroxyapatite resin, and exhibited the activity in the flow-through fraction. However, the hydroxyapatite, a mineral material with the formula Ca5(PO4)3(OH), effectively removed other impurities that occupied abundantly in previous Q-Sepharose fractions. Finally, oxidosqualene-lanosterol cyclase was substantially purified by HiTrap heparin chromatography via elution with HB buffer containing 50 mM potassium chloride. The hydroxyapatite and heparin steps significantly enhanced the fold of purification, yielding 728-fold enrichment in specific activity.

Electrophoresis of the fractions after the different purification steps revealed a single protein band with a molecular weight of about 70 kDa, as visualized by silver stained sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). A single band of about 140 kDa also was revealed on the non-denaturing polyacrylamide gel electrophoresis (PAGE).

The purified homogeneous enzyme was stable for up to 6 months at -80°C in the presence of Triton X-100, DTT, and phosphate buffer. Interestingly, the HiTrap heparin chromatography, which was applied to purify oxidosqualene cyclase for the first time, provided an effective method for purifying multi-milligram quantities of apparently homogeneous mammalian oxidosqualene cyclases (Figure 6.1 and Table 6.2).

Table 6.1 Purification and properties of 2,3-oxidosqualene cyclase from vertebrates, higher plants, and yeast.⁶⁴

Table 6.2 The purification of oxidosqualene-lanosterol cyclase from bovine liver

Fraction

S.cerevisiae

26 160 4400

High Plant (R. japonica)

Cycloartenol synthase

54 139 16

β-amyrin synthase

28 541 41

(Pea)

Cycloartenol synthase

55 235 167

β-amyrin synthase

35 1072 28

a: nmol/h/mg ; b: mkat/kg; c: from homogenate; d: pmol/min/mg;

e: mg protein necessary for 20% conversion ratio; f: units/mg; g: pkat/mg