The Molecular Mechanism Of Serum ALT Elevation In Mice Treated With Carbon Tetrachloride And Human ALT Isoenzymes: Molecular Cloning, Protein Expression And Purification And Related Study

IntroductionAlanine aminotransferase(ALT)[EC 2.6.1.2.,formerly glutamate-pyruvate transaminase],is a pyridoxal enzyme catalyzing reversible transamination between alanine and 2-oxoglutarate to form pyruvate and glutamate.Through producing those four important intermediary metabolites,ALT plays an important role in gluconeogenesis and amino acid metabolism,particularly during fasting and exercise where glucose is made from pyruvate converted from alanine through de-transamination by ALT.ALT has been used as a marker for liver injury in people.Serum ALT activity is significantly elevated in a variety of liver conditions including occupational and environmental pollutants,such as carbon tetrachloride,viral infection,cirrhosis, non-alcohol steatohepatitis(NASH)and drug toxicity.However,the increase of serum ALT activity is also observed in conditions other than liver damage,such as muscle disease,celiac disease and in apparently healthy people.Conversely,serum ALT is not increased in some patients with histopathologically confirmed liver diseases,e.g.,in cirrhosis,NASH,hepatitis C infection.Thus,interpretation of serum ALT data can sometimes be challenging in clinical diagnostics and in preclinical drug toxicity evaluation.The reason ALT has historically served as a major marker for liver damage is attributed to its abundant expression in liver and low levels present in other tissues in both rats and people,The "leakage" of the enzyme is thought to occur during damage of the liver resulting in the serum ALT elevation.These ALT elevations have been measured by the catalytic activity of the enzyme.However,whether liver damage indeed results in the increase of ALT protein in serum has not been evaluated.The genes of ALT isoenzymes,ALT1 and ALT2,have been cloned in the mouse and human and found that the two isoenzymes are expressed differently in tissues and probably in the subcellular compartments.We have speculated that both ALT isoenzymes are present in the serum and contribute to the total ALT activity,and thus the measurement of ALT isoenzyme may provide new valuable information in understanding of molecular basis of ALT elevation.To this end,we,at first,measured ALT isoenzymes expression in multiple tissues at the protein level in rats.Moreover,we examined their sex-differences.And we treated C57B16 mice with carbon tetrachloride,a classic liver toxicant to observe the changes of their levels in the serum.Secondly,we expressed,purified the recombinant hALT1 and hALT2 in insect cells,and did preliminarily characterization of the two isoenzymes,which will not only facilitate the understanding of their biological significance,but also provide a valuable ALT reference for clinical chemistry.Finally, we develop specific and selective human ALT1 and ALT2 polyclonal antibodies.These antibodies allowed us to analyze the two proteins in human serum of normal volunteers and hepatic damage patients.Material and Methods1,Animal samples10 Snap-frozen tissues of Sprague-Dawley rats were minced and then homogenized by Dounce homogenizer in lysis buffer.Lysate supernatants were collected after centrifugation at 3,000×g for 10 min.Four C57B16 mice were injected CCl4 mixed with corn oil intraperitoneally at 2ml per kilogram of body weight.Blood was collected before and 24 hours after injection for measuring ALT activity and for ALT proteins.2,ALT activity assay ALT activity was determined by using the ALT kit according to the manufacturer's instruction.10μl of serum or tissue lysate was incubated with 200μl mixture of reagent A and B.Absorbance at 340 nm was recorded for 5 min at every 30 sec interval after the addition of protein fraction or serum.The slope of absorbance decrease is proportional to ALT activity.Final ALT activities were corrected by protein concentration of cell lysates.One unit of ALT activity was defined as the amount of enzyme which catalyzes the formation of 1μmol/L of NAD per minute under conditions of the assay at 25℃.3,Western analysisFor Western analysis,reduced cell lysates were separated by electrophoresis on 10%polyacrylamide gels.Following electrophoresis,proteins were transferred onto PVDF membranes and bound proteins were probed with primary ALT antibody, developed with electrochemiluminescence(ECL).Concentrations of ALT in each sample were obtained by measuring the density of Western signals by FluorChem using recombinant rat ALT as standards.For human serum,1.5μl samples were loaded,and other protocol is the same above.4,hALT1 and h ALT2 Vector constructionThe full-length cDNA of human ALT1 and human ALT2 cDNA without the mitochondrial signal sequence,were constructed to pBAC-2cp transfer vector with encodes 6×histidine tag(His-tag),enterokinase cleavage site,respectively.Human ALT1 is referred to as pBAC-2cp-hALT1,and human ALT2 is referred to as pBAC-2cp-hALT25,hALT1 and hALT2 recombinant virus productionpBAC-2cp-hALT1 and pBAC-2cp-hALT2 together with Baculovirus linearize DNA co-transfected Sf9 insect cells,respectively.Produced recombinant virus was amplified till generation 4 and viral titration was measured.6,Protein expression and purificationThe recombinant hALT1 and hALT2 enzymes were expressed and purified from Sf9 insect cells as His-tag fusion proteins using nickel affinity chromatography, followed by the removal of His tag for functional analysis by enterokinase cleavage.7,Effect of temperature,storage conditions and pH on ALT activityThe aliquots were stored at -80℃,-20℃,4℃,25℃and 37℃for the indicated periods of time in 20mM Tris-HCl buffer and assayed for enzymatic activity, respectively.The aliquots of purified recombinant hALT1 and hALT2 proteins were added to various concentrations of glycerol(0～30%)in 20 mM Tris-HCl buffer,pH7.5 at -20℃for 24 and 48 hours respectively.The activities of recombinant hALT1 and hALT2 were determined in the pH range from 5.0 to 9.0(125 mM Tris-HCl)in the presence of 575 mM alanine and 16.3 mM 2-ketoglutarate.Assays were conducted in triplicate for each point and an average activity was taken.8,Antibody purificationPolyclonal antisera against hALT1 or hALT2 were raised through standard protocol(Abbomax,CA)by using the purified recombinant proteins,respectively.The following alternate antigen-based depletion and absorption protocols were used to remove cross-reactivities of ALT antiserum against each other isoenzyme.9,Serum ALT activity and protein expression in normal and liver injury subjectsSerum was obtained from 4 health volunteers and 4 liver damaged patients from medical center of Maryland University,respectively.Serum ALT activity and ALT isoenzyme proteins were determined.10,StatisticsData were expressed as mean+/-S.E.Statistical analyses to compare the difference of ALT activity,protein concentrations between groups were performed with Student's t test.A p value less than 0.05 was considered as significance.Results1,ALT1 and ALT2 protein expressed in rats multiple tissues Both ALT1 and ALT2 are highly expressed in the liver and muscle in the male and female rats.Striking difference in ALT isoenzyme expression is observed in the intestine where ALT1 is highly expressed in the small intestine versus ALT2 where there is minimal expression in this tissue.2,Sex-dependent difference in liver and muscle of ratsALT2 expression in male rats is about 4 fold and 2-fold higher in the liver and muscle,respectively,than female rats.Likewise,hepatic ALT activity is 30%higher in males than females.No sex-dependent difference was seen in the liver for ALT1,but it appears 20%higher in muscle in females.3,Serum ALT protein in mice pre- and post-treated with CCl4Serum ALT1 and ALT2 protein levels were elevated in mice treated with CCl4. ALT activity was dramatically increased by 66-fold.ALT1 protein was significantly elevated by 4.1-fold and ALT2 by 2.9-fold based on densitometric analysis of the Western blot.4,hALT1 and hALT2 cDNA cloning,protein expression and purificationHALT1 and hALT2 cDNA were cloned to pBAC-2cp plasmid,respectively,and together with Baculovirus DNA co-transfected to Sf9 insect cells successfully.Then we purified the His-tagged hALT1 and hALT2 fusion protein into homogeneity in one step. His-tag free recombinant proteins were obtained by enterokinase cleavage and subsequently separated by Ni-NTA agarose.5,Effect of temperature on ALT activityThe effect of temperature on ALT activity of purified recombinant human ALT proteins was studied for 20 days.In 20mM Tris-HCl(pH 7.5)buffer at 37℃,ALT1 activity decreased rapidly and lost 50%of the activity in one day and all the activity by day 6.At 4℃,25℃and -80℃,ALT1 activity was relative stable with 80%-90%of the initial activity remaining through day 10,when ALT1 began to drop to 60%by day 20 at 25℃,whereas the activity remained stable at 4℃and -80℃through day 20. Interestingly at -20℃,ALT1 lost about 43%of the activity in first 2 days,but remained relative stable thereafter till day 20.In comparison,ALT2 activity decreased quickly at all the tested temperature except -80℃and the lower the storage temperature,the better the activity was preserved.Nevertheless,the ALT2 activity lost about 72%,56%,53% and 44%at 37℃,25℃,4℃and -20℃respectively in first two days.At -80℃,ALT2 activity dropped 20%in first two days but remained stable thereafter through day 20.6,Effect of glycerol on ALT activityThe preservation of ALT1 activity was noted with the increase of glycerol concentration to 25%,at which 99%and 95%of the activity was remained during 24 and 48 hours,respectively,and further increases of glycerol concentration to 30%did not preserve more ALT1 activity.In contrast,the protective effect of glycerol was more pronounced for ALT2 with increasing concentration of glycerol up to 25%,at which about 94%and 89%of the ALT2 activity was preserved during 24 and 48 hours.This experiment indicated that storage condition of 25%of glycerol preserved most of the ALT1 and ALT2 activity.7,Effect of pH on ALT activityThe specific activities of recombinant hALT1 increased in the range of pH 5.0 to 6.5, then flatted in the range of pH 6.5 to 8.0,but decreased from pH 8.0 and above.For hALT2,the specific activities increased from pH 5.0 to 6.5 and then remained active over a broader pH range than hALT1 from pH 6.5 to 8.5.Hence,both hALT1 and hALT2 protein remained near fully active at pH 6.5 to 8.0.8,Antibody purificationAntibody purification is successful,and perhaps 1 to 5,000 dilution is the optimal concentration.9,Serum ALT isoenzyme protein expression in healthy voluteers and liver injury subjects Both ALT1 and ALT2 can be detected in human sera with normal and high ALT activity and there appears higher ALT protein level in hepatic injury patients with high ALT activity than that in healthy volunteers with normal ALT activity.ALT1 and ALT2 protein contents are elevated,but not well correlated with the activities.Conclusions1,Both ALT1 and ALT2 are highly expressed in the liver and muscle in male and female rats.Striking difference in ALT isoenzyme expression is observed in the intestine where ALT1 is highly expressed in the small intestine versus ALT2 where there is minimal expression in the tissue.Sex-dependent difference was found in hepatic ALT2 expression.Male rats are higher than female rats.Hepatic ALT activity is higher in males than females.Serum ALT elevated in mice treated with CCl4.2,We can purify the His-tagged hALT1 and hALT2 fusion protein into homogeneity in one step.Compare to hALT1,hALT2 is less stable.It is difficult to differentiate ALT isoenzymes according to pH value.3,In current assay,ALT1 mainly contributes ALT activity if there are equal protein levels of ALT1 and ALT2 in tissues and sera.4,Both ALT1 and ALT2 can be detected in human sera with normal and high ALT activity and there appears higher ALT protein levels in hepatic injury patients with high ALT activity than that in healthy volunteers with normal ALT activity.ALT1 and ALT2 protein contents are elevated,but not well correlated with the activities.