Preliminary Study On The Structure And Function Of Human Augmenter Of Liver Regeneration

Human augmenter of liver regeneration ( hALR ) was cloned from human fetal liver cDNA library. It was a growth factor which can stimulate hepatocyte proliferation during liver regeneration and prevent liver damage from carbon tetrachloride and D-galactosamine. Except for the activity of protecting hepatocyte, ALR also serves as a sulfhydryl oxidiase. ALR belongs to ERV1 (essential for respiration and viability 1, ERV1)/ALR protein family. Of the family members which have the activity as growth factor and sulfhydryl oxidiase, all of them contain the conserved Cys-Xaa-Xaa-Cys (CXXC) sequence motif. They take part in introduce disulfide bonds into protein substrates, maintain the redox equilibrium and play an important role in the biogenesis of mitochondria especially in maintenance intact mitochondrial membranes, normal mitochondrial morphology and mitochondrial transcription. In order to study the relationship between the structure and function of hALR, we analyzed the spatial structure and the activity motif of hALR and designed a serial of site-directed mutagenesis. hALR mutations protein were expressed and purified. The activity of the mutations in stimulating hepatocyte proliferation, preventing hepatocyte from chemical materials, acting as sulfhydryl oxidiase and the interaction with Na⁺,K⁺-ATPase were compared. This study paved a way for the insights into the relationship between structure and function of ALR and will provied scientific basis for the mechanism study, application and exploitation of ALR.1 Site-directed mutagenesis of hALR and construction of mutated expression plasmidThe mutations at the sites of C65A, Q88C, C85S of hALR and the deletants of hALR-5 and hALR-13 were designed. To destroy the CXXC active motif of hALR, we changed the cysteine residue at position 65 in hALRp into alanine. On the contrary, in order to form another CXXC active motif of hALR at the position of 85 88, we changed glutamine residue to cysteine at position 88 in hALRp. In the third mutation, we mutated one of the two more cysteine of hALR compared with rALR at the position of 85 into serine. As to the deletants of hALR-5 and hALR-13, we respectively deleted five and thirteen amino acids in the N-terminal of hALRp. Mutant constructs of hALR were prepared with mutagenesis kit using pBV220-hALR as a template and the deletants were prepared with PCR. The plasmids were constructed successfully with gene recombinant technique after identification with restriction enzyme. After sequence analysis, all of the mutations were mutated at the expected site and the rest sequence was accorded with the wild hALR. The sequence of all the deletants were accorded with the wild hALR except the absence.2 Expression and purification of hALR mutationsWe expressed the interest proteins in E.coli DH5α. After the procedure of inclusion bodies'purification, degeneration and renaturation, the renaturing protein was purified by ion-exchange resin. hALR and its mutations were analyzed by SDS-PAGE. The purity of the mutated and deleted proteins was 93.2% (hALR-C65A), 90.7% (hALR-Q88C), 92.1% (hALR-C85S), 55.7% (hALR-5).3 Compare the bioactivity of hALR and its mutations3.1 Activity in stimulating HL-7702 hepatocyte proliferationThe cell suspension was planted in 96-well plate. The cells were treated with hALR. The concentration was ranged from 0.001μg/ml to 0.5μg/ml. The peak activity of hALR was at the concentration of 0.5μg/ml.We used MTT assay to detect the activity of hALR and its mutations at the concentration of 0.5μg/ml. pHGF and BSA were used as positive control and negative control respectively. The activity of hALR and its mutations were significantly higher than the negative control (P < 0.05). The simulation index of pHGF,hALR,hALR-Q88C,hALR-C65A,hALR-C85S and hALR-5 was 2.75,1.80,2.15,1.75,1.77 and 1.81 respectively. The stimulate activity of hALR-Q88C was higher than hALR in numerus, but there was no significant difference between them (P > 0.05). NO significant difference was observed between hALR and the other mutations (P > 0.05).3.2 Activity in preventing HepG₂ cell chronic damage from D-galactosamineHepG₂ cells were treated with the mixture of 1 mg/ml D-galactosamine and hALR protein. The concentration of hALR was ranged from 0.001μg/ml to 0.5μg/ml. The peak activity of hALR was at the concentration of 0.5μg/ml.We used MTT assay to detect the activity of hALR and its mutations at the concentration of 0.5μg/ml. pHGF and BSA were used as positive control and negative control respectively. The activity of hALR and its mutations were significantly higher than the negative control (P < 0.05). The activity of hALR-Q88C was significantly higher than hALR (P < 0.05). The results indicated that the function of hALR-Q88C protein was more effective than hALR in protecting HepG₂ cell damage from D-galactosamine. NO significant difference was observed between hALR and the other mutations (P > 0.05).3.3 Activity in preventing HepG₂ cell acute damage from carbon tetrachloridehALR had the outstanding function in preventing the acute liver damage from carbon tetrachloride, but there was no relationship between the concentration and effect.We used MTT assay to detect the activity of hALR and its mutations at the concentration of 0.5μg/ml and 2μg/ml. BSA was used as negative control. The activity of all the experimental groups were significantly higher than CCl4 damage group (P < 0.05). There were no significant difference between hALR and its mutations. No relationship between the concentration and effect of all groups was observed.The results showed that introducing another CXXC motif to hALR could gently enhanced the activity in stimulating hepatocyte proliferation and significantly enhanced the ability in preventing the chronic hepatocyte damage from D-galactosamine. The mutations of C65A, C85S and deletant of hALR-5 didn't affect the activity in stimulating hepatocyte proliferation and preventing hepatocyte damage from chemical materials. 4 The sulfhydryl oxidiase activity of hALR and its mutationsAbout 1.3 2.6 units protein binding with one FAD molecule was observed. The spectrum with two absorbance maxima in the range from 300 nm to 500 nm is characteristic for absorbance of FAD molecule. As a protein, hALR didn't have absorbance maximum in this range. After combined with FAD, hALR-FAD had two absorbance maxima at the sites of 375.7 nm and 455.0 nm. The absorbance maximum in the range of 450 nm was shifted about 5nm in protein-FAD compared with FAD. But the absorbance maximum of hALR-C65A-FAD in the range of 450nm didn't shift at all.The CXXC motif and FAD are vital for the enzymatic activity of hALR as sulfhydryl oxidase. hALR had the highest enzymatic activity using dithiothreitol as substrate.Dithiothreitol was used as substrate to detect the sulfhydryl oxidiase activity of the monomer and dimer form of hALR. The dimer-hALR-FAD had the enzymatic activity as sulfhydryl oxidase. The concentration of thiol groups decreased from 12.87μmol/L to 8.09μmol/L in 6 min. Because of no change of the concentration of thiol group during the procedure, the monomer-hALR-FAD lost the enzymatic activity completely. Except hALR-C65A-FAD, the other mutations still had the enzymatic activity as sulfhydryl oxidase.The results showed that hALR depended on CXXC motif and FAD for the enzymatic activity. The protein with the CXXC motif destroyed will completely lost its sulfhydryl oxidase activity. Compared with hALR, the mutations of Q88C, C85S and hALR-5 deletant didn't change their sulfhydryl oxidase activity.5 The interaction of target protein with GST-Na⁺,K⁺-ATPase fusion proteinRecombinant expression plasmid pGEX-4T-1-Na⁺,K⁺-ATPase was constructed successfully with gene recombinant technique after identification with restriction enzyme and sequence analysis.pGEX-4T-1 and pGEX-4T-1-Na⁺,K⁺-ATPase plasmids were transformated into E.coli BL21 competent cells. GST and GST-Na⁺,K⁺-ATPase fusion protein were expressed inducing by IPTG. The interest proteins were purified by Glutathione Sepharose 4B.hALR and its mutations were incubated with the beads which binding the GST-Na⁺,K⁺-ATPase fusion protein. After washing for several times, we could detect the interest protein with SDS-PAGE. But the protein in the incubation system of target protein and GST could not detect with SDS-PAGE. The results showed that all of the mutations and deletant of hALR could interact with Na⁺,K⁺-ATPase but not with GST.The above results showed that all of the mutations and deletant of hALR could interact with Na⁺,K⁺-ATPase. The CXXC motif didn't affect the interaction of hALR with Na⁺,K⁺-ATPase. The results of the interaction of hALR and its mutations with Na⁺,K⁺-ATPase were accorded with their activity in promoting hepatocyte proliferation and preventing hepatocyte damage from chemical materials.The study successfully cloned the mutation and deletant genes and constructed their expression vector. Expressing the interest protein in E.coli DH5α, we got the mutated proteins with purity above 90%. Studying the bioactivity of hALR and its mutations, we found that the CXXC motif conduced hALR to play its activity in stimulating hepatocyte proliferation and preventing hepatocyte damage. The CXXC motif played an important role in the enzymatic activity of hALR. Destroying this motif will lead hALR completely lost the activity of sulfhydryl oxidiase. The CXXC motif didn't affect the interaction of hALR and Na⁺,K⁺-ATPase. The results of protein interaction with Na⁺,K⁺-ATPase was accorded with its activity in promoting hepatocyte proliferation and preventing hepatocyte damage from chemical materials. This study paved a way for the insights into the relationship between structure and function of ALR and will provied scientific basis for the mechanism study, application and exploitation of ALR.