Cytosolic Serine Hydroxymethyltransferase - The Journal of Biological ...

THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1993 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 268, No. 19, Issue of July 5, pp. 1378”13790,1993 Printed in U.S.A.

Cytosolic Serine Hydroxymethyltransferase DEAMIDATION OF ASPARAGINYL RESIDUES AND DEGRADATION IN XENOPUS LAEVIS

OOCYTES*

(Received for publication, February 22,1993)

Antonio Artigues, Helen Farrant, and Verne SchirchS From the Departmentof Biochemistry and Molecular Biophysics, Virginia Commonwealth University, Richmond, Virginia 23298

Rabbit liver cytosolic serine hydroxymethyltrans- Clarke, 1985; Wright, 1991). Evidence that these deamidation ferase exists as a set of subforms which exhibit differ- reactions in proteins are significant is supported by the obent isoelectric points. Previous studieshave shown that servation that the widely distributed enzyme protein isoasdeamidation of an asparagine residueat position 5 of partylmethyltransferase (PIMT)*catalyzes the transfer of a theaminoacidsequenceaccountedforsomeofthe methyl group from S-adenosylmethionine (AdoMet) to form charge heterogeneity (Artigues, A., Birkett, A., and the methyl ester of the a-carboxyl group (Aswad, 1984;MurSchirch, V. (1990) J. Biol. Chem. 265, 4853-4858). ray and Clarke, 1984; Diliberto and Axelrod, 1976; O’Connor The present study has also identified asparagine 220 as being partially deamidated. An estimated 25-30% and Clarke, 1985). of the purified enzyme contains an isoaspartyl residue Neither the function nor biological consequences of the at position 220. This suggests that deamidation ofas- deamidation of Asn residues in proteins is known. One proparagine 220 occurs by the @-aspartylshift mecha- posal is that the deamidation reactions serve as biological nism. Western blot analysis of purified cytosolicserine markers of aging of proteins and signals them for proteolytic hydroxymethyltransferase, after isoelectricfocusing degradation (Robinson and Rudd, 1974). The accumulation of deamidated proteins in aged organisms has been observed, underreducinganddenaturingconditions,showed four subforms of the individual subunitswith respect suggesting that the mechanism for degradation of proteins to isoelectric point.Extracts from 3-day- and 3.5-year- can no longer recognize the deamidated forms (Yuksel and old rabbit livers showed the presence of these same Gracy, 1986; Lowenson and Clarke, 1988; Ladino and O’Confour subunit subforms. Purified cytosolic serine hy- nor, 1990). A second proposal is that PIMTforms the methyl droxymethyltransferasewas found to be degraded in esters of isoAsp residues which can reform the succinimide 24 h after mechanical injection into Xenopus laevis intermediate. The succinimide intermediate then hydrolyzes oocytes. However, when the first 14 amino acid resi- to reform both Asp and isoAsp residues in a 3:l ratio. Repedues are removed from the enzyme by digestion with tition of this cycle of methylation followedby hydrolysis chymotrypsin, leaving a fully catalytically active en- results in repair of isoAsp residues to normal Asp residues. zyme, the rate and extent of degradation of the truncated enzyme in oocytes were significantly reduced. This repair has been shown to result in partial restoration of One of the deamidated asparagine residuesis at posi- biological activity (Johnson et al., 1987a, 1987b;George-Nastion 5, suggesting that this deamidation site may be a cimento et al., 1990). However, it is possible that deamidation of Asn residues in peptides and proteins exhibits some yet signal for degradation of the enzyme. undiscovered biological function. There is a need for careful documentation of the presence of deamidated proteins and thedegree to which these deamiIncreasing numbers of proteins are being found to contain dations have resulted in isoAsp residues. We have shown deamidated asparagine residues (Clarke, 1985; Lowenson and previously that two-thirds of Am‘ of purified rabbit liver Clarke, 1990; Johnson et al., 1989; Galletti, et al., 1989; cytosolic serine hydroxymethyltransferase(cSHMT) is deamWright, 1991;Teshima et al., 1991).During deamidation many idated, with equal amounts of Asn, Asp, and isoAsp residues of these residues have isomerized to isoAsp’ bonds in which at position 5 (Artigues et al., 1990). The isoAsp residue serves the peptide backbone is through the P-carboxyl group of Asp, as a substrate for PIMT in the native protein. However, we leaving the a-carboxyl group free (Johnson et al., 1987a, 1987b; concluded from this study that there was at least 1additional isoAsp residue in this protein that was not a substrate for * This work was supported by National Institutes of Health Grant PIMT in the native state. In this paper we locate a second GM 28143. The costs of publication of this article were defrayed in isoAsp residue and address the questions of whether the part by the payment of page charges. This article must therefore be 2 Asn residues in this enzyme are agehereby marked “advertisement” in accordance with 18 U.S.C. Section deamidations of the related and whether repair of isoAsp’ occurs in vitro. 1734 solely to indicate this fact. Studies on the possible role of deamidation ofAsn’ as a $ T o whom correspondence and reprint requests should be addressed. Tel.: 804-786-9482;Fax: 804-786-1473. signal for proteolytic degradation were also initiated. DegraThe abbreviations used are: isoAsp, a peptide linkage in which dation of cSHMT and aproteolytic form of cSHMT in which the amide bond between Asp and the next amino acid is through the thefirst 14 amino acids are removedwasfollowed after 4-carboxyl group of Asp; KBES, potassium N,N-bis(2-hydroxyethyl)2-aminoethanesulfonic acid cSHMT,rabbit liver cytosolic serine mechanical microinjection into Xenopus laevis oocytes. The results suggest that theproteolytic degradation of cSHMT in hydroxymethyltransferase; S”-cSHMT, serine hydroxymethyltransferase missing the first 14 residues with aserine atthe amino terminus; PIMT, protein isoaspartyl methyltransferase; HPLC, high performance liquid chromatography; PAGE, polyacrylamide gel electrophoresis; IEF, isoelectric focusing; TPCK, L-1-tosylamido-2-phenylethyl chloromethyl ketone.

‘This enzyme has also been shown to methylate the 4-carboxyl group of D-aSpartyl residues in peptides and proteins and is referred toas protein carboxyl methyltransferase (Lowenson and Clarke, 1992).

13784

Serine Hydroxymethyltramferase

13785

bated with the first antibody, which was a 12300 dilution of guinea pig anti-cSHMT serum, for 2 h a t 37 "C. After washing, the filters were treated with rabbit anti-guinea pig IgG linked to horseradish peroxidase for 1 h a t 37 "C. After extensive washing, substrate was EXPERIMENTALPROCEDURES added and incubated until the color had developed. The gels were Materials-Pyridoxal phosphate, chymotrypsin, trypsin-TPCK, then scanned in a Shimadzu CS-9000 scanning densitometer at 600 and S-adenosyl-L-methionine chloride were purchased from Sigma. nm. Preparation of S'5-cSHMT-Native enzyme, 10 mg in 1 ml of 20 Reagents for amino acid analysis and peptide sequencing were purchased from Pierce Chemical Co. Peroxidase substrates were from mM potassium phosphate,pH 7.3,was digested with 100 pgof Kirkegaard and Perry Laboratories, Inc., Gaithersburg, MD. [rnethyl- chymotrypsin for 30 min at 30 "C. Chymotrypsin cleaves the enzyme 3H]S-Adenosyl-~-methionine(15 Ci/mmol) and [rnethyl-'4C]S-aden- between Trp14and Ser15releasing a 14-mer peptide and a fully active osyl-L-methionine (60 mCi/mmol) were purchased from Amersham truncated enzyme (Schirch et al., 1986). The released 14-mer peptide Corp. Rabbit livers were obtained frozen from Pel-Freeze Biologicals, and chymotrypsin were removed from the S"-cSHMT by filtration Rogers, AK. Adult female, oocyte-positive, Xenopus lueuis frogs were through a Sephadex (2-50 column (1 X 20 cm) equilibrated with 20 purchased from North Carolina Biological Co. Chemicals for main- mM potassium phosphate, pH 7.3. The decrease in the size of the taining the frogs were purchased from Sigma. [3H]Sodium cyanobo- chymotrypsin modified enzyme was verified by SDS-PAGE. This rohydride (120 mCi/mg) was purchased from Amersham. The pep- enzyme contains an amino-terminal Ser residue and is referred to as and Ile-Ala-Asp-Glu-isoAsp- S"-cSHMT. tides Ile-Ala-Asp-Glu-Asn-Gly-Ala-Tyr Repair of IsoAsp to Asp-The repair of isoAsp5to Asp5 wasinvesGly-Ala-Tyr were synthesized on a Milligen 9600 solid phase peptide synthesizer using t-butoxycarbonyl chemistry protocols and purified tigated both in cSHMT and the 14-mer peptide released by chymotrypsin digestion. To a 1.5-ml plastic centrifuge tube was added 0.2 by HPLC chromatography on a C-18 column. cSHMT was purified from rabbit liver as described previously mM [rnethyl-3H]AdoMet(5 Ci/mmol), 50 pg of PIMT, 5 mM L-serine, (Schirch and Peterson, 1980). Its concentration was determined using in 75 mM KBES, pH 7.2, and the reaction started by the addition of an A Z n mof7.2 (Gavilanes et ai., 1982). PIMT' was purified from either 0.62 nmol of the 14-mer peptide, corresponding to residues 1bovine erythrocytes according to the modified method previously 14 of cSHMT or 1 nmol of cSHMT. The tubes were placed in 20-ml described for the human enzyme (Kim et al., 1983; Artigues et al,. counting vials containing 1 mlof toluene-based scintillation fluid. 1990). SDS-PAGE analysis suggested the enzyme was a mixture of The vials were capped and placed in the scintillation counter. As the reaction proceeded the reforming of the succinimide intermediate the two isoenzymes known to exist in erythrocytes. Enzyme Assays-cSHMT activity was determined using alloth- from the isoAsp methyl ester releases methanol which diffuses out o€ reonine as a substrate as described previously (Schirch and Gross, the 1.5-ml centrifuge tube and into the scintillation fluid. The reac1968). The methylation of isoaspartyl residues by PIMT was per- tions were counted every hour for 28 h. During this period there was formed according to the methods of Murray and Clarke (1984) and an increase in the number of counts for about the first 18 h. Addition of more PIMT and [rneth~l-~HIAdoMet to reaction the tube did not Johnson and Aswad (1991), at 37 "C in sodium citrate buffer, pH 6.06.2, in a final volume of 100 pl. Reactions done at higher pH values result in an increase in counts during an additional 2-h incubation. used 50 mM KBES as the buffer. Total isoAsp residues in a peptide At the end of the reaction the 1.5-ml centrifuge tube containing the or protein were determined by adding sufficient PIMT and AdoMet reaction solution was removed and the vial counted again to assure until the number of counts in the assay became independent of time that all the counts were in the counting fluid. Repair of isoAsp5to Asp' was also investigated by incubating a 5 of incubation, the concentration of PIMT and AdoMet, and was linear with peptide substrate concentration. The specific activity of mg/ml solution of cSHMT in the pH 7.2 KBES buffer for periods of the correct isomer of radioactive AdoMet was determined by incu- up to 24 h. Additional PIMT and AdoMet were added after every 8 bating a known concentration of the tetrapeptide acetyl-Val-isoAsp- h.At intervals 100-p1 aliquots were removed and treated with 1% Gly-Ala with an excess of AdoMet and PIMT and determining the chymotrypsin. After 15 min at 23 "C the solution was placed in a Centricon-30 filter tube and centrifuged at 5000 rpm for 15 min. The incorporation of counts/min as the methyl ester. This tetrapeptide has been shown to be an excellent substrate for PIMT and is rapidly eluate was collected and the released 14-mer peptides analyzed for methylated to 100% in this system (Lura and Schirch, 1988). the distribution of Am5, Asp5, and isoAsp5 containing peptides by NaCNBH3 Reduction of cSHMT-Prior to performing either SDS- HPLC asdescribed previously (Artigues et al., 1990). PAGE or IEF the pyridoxal-P bound as a Schiff base to cSHMTwas Analysis of cSHMT and S"-cSHMT Peptides for Total IsoAsp reduced to a stablesecondary amine with NaCNBH3. For experiments Residues-Ten mg (0.72 pmol) of either cSHMT or S"-cSHMT in 1 not requiring incorporation of a tritium atom duringreduction, about ml of 20 mM potassium phosphate, pH 7.6, were denatured by the 2 mg of cSHMT was reacted with 5 pl of a 5mg/ml methanol solution addition of urea to a final concentration of 6 M. These solutions were of NaCNBH3. Following incubation on ice for 20 min, the enzyme dialyzed against 1 liter of0.1 M ammonium bicarbonate for 12 h was dialyzed against the appropriate buffer for at least 8 h. For those followed by addition of 100 pg of both trypsin-TPCK andchymotrypreactions requiring incorporation of a tritium atom during reduction, sin to the precipitated enzymes. These solutions were incubated at about 5 mgof cSHMT was reacted with 10 pl of [3H]NaCNBH3 37 "C for 2.5 h and the resulting clear solution of soluble peptides dissolved in methanol. After 20 min an additional 5 pl of unlabeled lyophilized to dryness. The tryptic-chymotryptic peptides were disNaCNBH3 was added and theenzyme treated as described above. solved in the sodium citrate buffer, pH 6.2, used in the methylation Electrophoresis, Isoelectric Focusing, and Western Blotting-SDSassay for determination of total isoAsp residues. PAGE was performed ina 12% slab gel with a3%stacking gel. Purification of Peptides Containing IsoAsp Residue-Ten mg (0.72 Electrophoretic conditions and reagents were those of O'Farrell pmol) of S"-cSHMT, previously reacted with [3H]NaCNBH3,was (1975). Isoelectric focusing under nondenaturing conditions was per- denatured in 6 M urea. The denatured enzymewasreduced and formed as described previously (Artigues et al., 1990). Isoelectric alkylated with 2-mercaptoethanol and iodoacetamide (Martini et al., focusing under denaturing conditions was performed as for nonde1987). The reduced and alkylated enzyme was digested with trypsin naturing conditions, but with the addition of 6 M urea to the gel as described above and analiquot used to determine the total concenmixture, andthe focusing was done at 23"C. The enzyme was tration of isoAsp residues with PIMT. The tryptic peptides were denatured by incubation for 2 h in 6 M urea a t 23 "C. The buffers dissolved in 5% acetic acid and fractionated bygel filtration on a containing urea, used for gel electrophoresis and isoelectric focusing, Sephadex G-25 fine column (30 X 1 cm) equilibrated with 5% acetic were made immediately before use with ultrapure urea and contained acid. Elution of peptides was monitored at 254 and 325nm and Tris buffer, 50 mM, to react with cyanates formed during electropho- collected in 1-ml fractions. One-hundred p1 of each fraction was resis. removed and dried under vacuum. These aliquots were then assayed Western blotting was performed on protein which had been sepa- for isoAsp residues using the PIMT assay. The fractions containing rated previously by IEF in urea gels. The developed gels were incu- isoAsp residues were pooled and lyophilized to dryness. bated with 5% perchloric acid for 30 min. After removal of the The isoAsp-containing fractions were dissolved in 0.1% trifluoroperchloric acid, the gels wereincubated with a 25% isopropyl alcohol, acetic acid and fractionated by HPLC on a C-18 column. The peptides 10% acetic acid solution for another 30 min. This procedure was were eluted with a linear gradient of10-70% buffer B in 70 min repeated three times to fix the protein and remove all the urea. The (bufferA was0.1% trifluoroacetic acid, and buffer B was0.1% gels were incubated in 5% 2-mercaptoethanol and 0.3% SDS buffer trifluoroacetic acid in 70% acetonitrile) with a flow rate of 1 ml/min. and then transferred to nitrocellulose according to the method of Elution of peptides was monitored at 254 and 325 nm and collected Burnette (1981). The blotted nitrocellulose filters were then incu- as I-ml fractions. One-hundred pl of each fraction was assayed for

oocytes is determined in part by information on the first 14 residues ofthe enzyme.

13786

Serine Hydroxymethyltrunsferase

total isoAsp residues as described above. Those fractions containing isoAsp residues were pooled, lyophilized to dryness, and chromatographed on a second analytical C-18 column with a gradient from 30 to 40% buffer B of the solvent system described above. The absorbance profile showed two peaks absorbing at 254 nm. The eluate absorbing a t 254 nm wascollected as two separate fractions, and each was shown to both absorb at 325 nm and tocontain isoAsp residues. The two fractions were lyophilized to dryness. Each of the fractions from the second C-18 column was dissolved in 0.2 ml of 0.1 M NH4HC03 anddigested with 10 pg of chymotrypsin at 37 "C for 2 h. The resulting peptides were separated on an HPLC C-18 column using either the solvent system described above with a gradient of 0-30% buffer B in 30 min or a solvent system containing ammonium acetate (solvent A,20 mM ammonium acetate, pH 5.0, and solvent B, 80% acetonitrile). Each absorption peak was collected as a separate fraction, dried, and analyzed for isoAsp groups and amino acid composition. The fractions containingisoAsp groups were sequenced on an Applied Biosystems model 470 gas phase sequencer. Preparation of Oocytes-Frogs were maintained in an aquarium containing artificial pond water at 20 "C and twice a week fed dried food pellets from North Carolina Biological Co. Donor animals were anesthetized by immersion in a solution of 1:7500 tricaine methanesulfonate. A piece of ovary was removed through a small incision in the body cavity under sterile conditions and placed into a solution of OR-2 medium. Oocytes in developmental stage 5 and measuring 1.00 k 0.05 mm in diameter were manually dissected and maintained overnight ineither Dulbecco's modified Eagle's medium or OR-2 solutions. These solutions contained 0.125 pg/ml of gentamicin, 50 pg/ml penicillin, 0.125 pg/ml of fungizone, and 50 pg/ml streptomycin. Microinjection of Oocytes-Microinjection of oocytes was performed as described in Hitchcock et al. (1987). Oocytes were injected with 20-30 nl of solutions of cSHMT (10-12 mg/ml) resulting in injection of about 0.3pgof enzyme/oocyte. Microinjected oocytes were cultured individually in 200 p1of either OR-2 or Dulbecco's modified Eagle's medium solutions in a 96-well tissue culture dish at room temperatureinawater-saturated chamber. The cells were routinely observed with a microscope during the incubation period to check for damaged oocytes which were discarded. Oocyte Fractionation-For determination of [3H]cSHMT and its degradation products, oocytes were separated from the medium a t various time periods. Each oocyte was washed with another 200 p1 of medium which was added to the incubation medium. The oocytes were homogenized in 20 mM potassium phosphate buffer, pH 7.3, and centrifuged at 13,000 X g for 10 min. The pellet was dissolved in 2 ml of scintillation fluid and counted for radioactivity. To the homogenized oocyte supernatant an equal volume of 10% trichloric acetic acid was added and after a 10-minincubation the solution centrifuged at 13,000 X g for 10 min. After separating the soluble and insoluble fractions, each was dissolved in 2 ml of scintillation fluid and counted. For analysis of counts appearing in the medium, an equal volume of 10% trichloric acetic acid was added to themedium and wash and after 10 min centrifuged for 10 min a t 13,000 X g. After separation of the soluble and insoluble fractions, each fraction was dissolved in 2 mlof scintillation fluid and counted. For degradation studies of unlabeled cSHMT, groups of five microinjected oocytes were homogenized in 20mM Tris chloride buffer, pH 8.0, containing 10%sodium dodecyl sulfate and 3 mM 2-mercaptoethanol. The soluble proteins were separated on a 12% gel by SDS-PAGE as described previously.

residues). The results show that for each nanomole of enzyme there are about 0.65 nmol of isoAsp residues (Fig. 1).Methylation of S1'-cSHMT, treated under the same conditions, resulted in about0.3 eq of isoAsp residues/subunit of enzyme, suggesting that 0.35 eq of isoAsp residues had been removed by loss of the first 14 amino acids (Fig. 1).This result is in agreement with our previous study which showed that 0.32 eq of Asn' is present as an isoAsp residue. The results, recorded in Fig. 1, of the methylation of the tryptic-chymotryptic peptides of denatured cSHMT show that there is another 0.3 eq of isoAsp residues/subunit which can be methylated only after unfolding of the enzyme. Evidence for Repair of ZsoAsp5-During methylation of the native enzyme we also attempted to determine if there was repair of isoAsp6to Asp6. Repair involves a cycle of methylation of isoAsp residues which then reforms the succinimide intermediate with release of methanol (Johnson et al., 1987a, 198713). In small peptides the succinimide intermediate then hydrolyzes to isoAsp and Asp residues in a 3:l ratio. In each cycle about 25% of the isoAsp residues are converted to Asp residues. Total repair of isoAsp to Asp with a hydrolysis ratio of 3:l would result in 4 eq of methanol being released per isoAsp residue. Hydrolysis of the succinimide intermediate is slow at pH values below 6.5. Repair reactions are usually observed at pHvalues above pH values of 7. This is important with cSHMT since the native enzyme is unstable at pHvalues below pH 6.5 and slowly denatures. In these studies the pH was maintained at 7.2, where cSHMT is stable for several days. A synthetic 14-mer peptide containing 100% isoAsp', corresponding to the amino-terminal peptide of cSHMT, was incubated with PIMT and AdoMet in an Eppendorf tube placed inascintillationcounting vial containing 1 mlof scintillation fluid. As each cycle of methylation-demethylation occurs, the released methanol will diffuse out of the Eppendorf reaction vial and dissolve in the scintillation fluid. Therefore, the increase in counts reflects the rate at which the isoAsp methyl ester is hydrolyzed to form methanol and the succinimide intermediate. With the 14-mer peptide the number of equivalents of methanol generated in a 24-h period was 3.3 times the concentration of isoAsp residues. This would correspond to the hydrolysis of the succinimide intermediate

RESULTS

e

Evidence for a Second IsoAsp Residue in cSHMT-We have

shown previously that homotetrameric cSHMT can be resolved into five overlapping fractions of activity on a TSKDEAE-HPLC column and at least 10 separate forms on nondenaturing isoelectric focusing gels (Artigues et al., 1990). The deamidation of Asn5 to Asp and isoAsp residues accounted for some of the subforms, but not all of them. We also provided evidence that theenzyme contained at least one additional isoAsp residue which was exposed to methylation by PIMT only after denaturation andproteolytic digestion of the enzyme. The amount of these buried isoAsp residues can be determined by methylating the native enzyme after denaturation anddigestion with trypsin andchymotrypsin to form a solution of soluble peptides (this gives the total isoAsp

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MINUTES FIG. 1. Number of isoAsp residues methylated by PIMT in a tryptic and chymotryptic digest of cSHMT and S''-cSHMT. Equal concentrations of cSHMT and S15-cSHMTwere denatured in 6 M urea and after removal of the urea digested with both trypsin and chymotrypsin. Aliquots were then assayed with PIMT for increasing periods of time and the incorporation of methyl groups into cSHMT (m) and S15-cSHMT(0)peptides determined.

13787

Serine Hydroxymethyltransferase to isoAsp:Asp in about a 70:30 ratio during each cycleof methylation and hydrolysis. With nativecSHMT slightly less than 1eq of methanol/isoAsp5 residue was generated in a 24h incubation, suggesting that little or no repair had taken place. Longer incubations did result in more than 1 eq of methanol being produced per isoAsp5 residue, but we could not rule out that the enzyme was denaturing and exposing isoAsp220to methylation, Also, L-serine had to be included in the reaction to stabilize cSHMT against denaturation during the long incubation period. One possibility with cSHMT is thathydrolysis of the SUCcinimide intermediate does not occur with a product isoAsp:Asp ratio of 3:l as found in small peptides. If the hydrolysis of the succinimide intermediate occurred with the formation of nearly all Asp residues, then repair would have occurred with only a single equivalent of methanol being formed. To test this hypothesis aliquots of the incubation mixture of cSHMT, PIMT, and AdoMet were treated with chymotrypsin to release the 14-mer peptide from the amino terminus. The peptides were then analyzed for the presence of Asn, Asp, and isoAsp residues at position 5 by HPLC as described previously (Artigues et al., 1990). We found that no significant change had occurred during a 24-h incubation on the isoAsp:Asp ratio. We had shown previously that further deamidation of the Am5-containing peptide also does not occur in a 24-h incubation at pH 7.2 (Artigues et al., 1990). We conclude from these studies that the repair of isoAsp to Asp residues observed in small peptides does not readily occur for isoAsp' in cSHMT. Location of the Second Deamidation Site-To determine the number and location of the buried isoAsp residues in cSHMT, the native enzyme was digested with chymotrypsin to remove the amino-terminal 14 residues containing the exposed isoAsp5 giving S"-cSHMT. The truncated enzyme was then reacted with NaCNBH3 to reduce the pyridoxal-P internal aldimine on the enzyme to a secondary amine that wouldbe stable to proteolysis and chromatography procedures. This was done because a second Asn-Gly sequence at positions 220-221 would beon the same tryptic peptide as the pyridoxyl-P lysyl residue at position 256 (Martini etal., 1987). The reduced pyridoxyl-P residue absorbs at 325 nm. After reduction and alkylation of sulfhydryl groups, the enzyme was digested with trypsin. Trypsin will cleave the enzyme either at Arg214 or Lys215and Arg5' generating a 45-mer peptide containing the second Asn220-G1$21sequence and Lys256with the reduced pyridoxyl-P. The trypticpeptides were then fractionated on a Sephadex G-25 column. Fig. 2A shows the elution profile monitored at 254 nm (peptides) and 325 nm (pyridoxyl peptide). Each1-mlfraction was analyzed for isoAsp residues using the PIMTassay. The results show that the first eluting peptide(s) absorbs at 325 nm and contains isoAsp residues. The fractions containing the isoAsp residues were pooled and chromatographed on a C-18 HPLC column. The eluate was monitored at both 215 and 325 nm. Again, each 1-ml fraction was assayed with PIMT. The HPLC elution profile showed the presence of two peptides absorbing at 325 nm with each fraction also having isoAsp residues. Each of these fractions was homogeneous, and amino acid analysis showed that they were peptides corresponding to either residues 215-259 or 216-259, respectively. Apparently, trypsin cleaves some of the enzyme after Arg214 and some of the enzyme after Lys215.The amino acid analysis also confirmed that thereis only a single peptide containing the buried isoAsp residue(s). The yields of isoAsp residues for the purification steps on Sephadex G-25 and the C-18 HPLC column were 90 and 85%, respectively. This high recovery further suggests

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Minutes FIG. 2. Purification of the peptide containing the isoAsp residue which is methylated by PIMT. A , S"-cSHMT was reduced with NaCNBH3, denatured, and digested with trypsin and the resulting peptides separated on a Sephadex G-25 column in 5% acetic acid. Absorbance was monitored at 254 nm (peptides) and 325 nm (pyridoxyl-Lys) and an aliquot of each 1-ml fraction assayed with PIMT and ~-[methyl-"C]AdoMet (shown as a bar graph). B, the peptides containing the isoAsp residues from the G-25 column purified by HPLC on a C-18 column. The methods of assay were the same as in A . C, the major fraction which was methylated by PIMT, recorded in B , was digested with chymotrypsin and chromatographed on HPLC with a C-18 column. Peptides werefollowedby 215 nm absorbance and each fraction containing a peptide was assayed with PIMT (bar graph).

thatthereare no other peptides containing significant amounts of isoAsp residues in S15-cSHMT. Each of the peptides corresponding to the two fractions absorbing at 325 nm (Fig. 1B) were further digested with chymotrypsin and subjected to HPLC chromatography on a C-18 column (Fig. 1C). Only a single fraction contained an isoAsp residue with each peptide. Amino acid analysis showed that the isoAsp residue was in the peptide corresponding to residues Ile216to TyrZz3.This peptide was sequenced, and the

Serine Hydroxymethyltransferase

13788

results of each cycle are shown in Table I. After cycle 5 of sequencing, Asn was observed with little formation of Asp. However, the yields for each cycle of sequencing were 90% or greater except for cycle 5. The yield for this cycle was only 74%. This is compared with cycle 3 which gave 93% recovery for Asp. isoAsp residues do not yield phenylthiohydantoinderivatives during sequencing, suggesting that the poor yield at cycle 5 is the result of about 25% of the peptide containing an isoAsp residue. This would account for most of the buried 0.3 eq of isoAsp residues observed in the results shown in Fig. 1. A small amount of Aspwas also observed at cycle 5 , suggesting that some Asp-containingpeptide was also present. However, in our experience sequencing peptides with Asn residues usually resultina small amount of Asp,which probably occurs by deamidation of Asn residues under the acidic sequencing conditions. To confirm that both the Asn-Gly and the isoAsp-Glycontaining peptides coeluted in this solvent system the two octomers, which weredetermined from the sequence as shown in Table I, were made synthetically. Each of these peptides were chromatographed under a variety of conditions, including the solvent system described above, and a solvent system at pH 5.0 in ammonium acetate. In each solvent system the Asn-containing peptide coeluted with the isoAsp containing peptide. In the ammonium acetate system we also found that the chymotryptic peptides, used in the experiment recorded in Table I, also resulted in a single isoAsp fraction which eluted at thesame position as thesynthetic isoAsp containing peptide. Number of Charge Forms of c S H M T Subunits-After having determined that there are at least 2 Asn residues in cSHMT whichhave undergone deamidation, the question arises as to whether these are enough to account for the multiple forms of the tetrameric cSHMT observed on nondenaturing IEF gels. To determine the number of different charge forms of cSHMT subunits, the purified enzyme was denatured in 6 M urea and subjected to IEF ina urea gel. The first experiment was with purecSHMTand showed the presence of four distinct bands of about equal intensity when stained with Coomassie Blue. Two deamidation sites could only account for three bands, i.e. subunits with either no, one, or two deamidation sites. Separate bands should not be observed for peptides containing either Asp or isoAsp residues, since the PI values of the protein subforms are all above pH 6.2. A t this pH theAsp and isoAsp carboxyl groups would be fully ionized and thus not impart different PI values. Since ion exchange chromatography is used during purification, it is possible that either the number or relative concentration of subunit charge forms is different in the cell. The number and relative concentration of charge forms of cSHMT subunits present in liver cells was addressed by IEF of liver

homogenates after minimal purification and detection of the cSHMT subunits by Western blot analysis of urea IEF gels. Several enzymes which undergo deamidation have beenshown to accumulate the deamidated subforms in aged tissue (Yuksel and Gracy, 1986; Ladino and O’Connor, 1990). To determine if this was also true for cSHMT, the distribution of the subunit subforms in two 3-day-old and four 3- to 3.5-year-old livers were investigated and compared with purified enzyme obtained from 6-8-month-old rabbit livers. To obtain clean results the liver extracts were heated in the presence of 50 mM L-serine, which has been shown to denature a protease which cleaves cSHMT at theamino terminus (Schirch et al., 1986).After centrifugation of the heated extract and desalting on a Sephadex G-25 column, the enzyme was absorbed to a CM-Sephadex column as described for the purification of this enzyme. About 90% of the proteins from liver extracts do not stick to this column, but cSHMT does bind and was eluted with 300 mM potassium phosphate,pH 7.3. ThecSHMT activity which eluted from the column wasdialyzed and concentrated and thenanalyzed by IEF in 6 M urea. The yield of activity for SHMT during the partial purification was 9095%, ensuring that littlealterationin the distribution of subunit charge forms had occurred. The results show that each of the liver extracts from a 3-day-old and a 3.5-year-old rabbit have the same four bands in about the same proportion as found for the purified enzyme (Fig. 3). However, in our experience the variation in the intensity of the bands of a Western Slot varied from gel to gel, and not all gels showed the same relative band intensities shown in Fig. 3. However, for any particulargel the band intensitiesof the old and young rabbit extracts were similar to those of the purified cSHMT. The results from another 3-day-old rabbit and three 3-yearold rabbits gave the same four bands as shown in Fig. 3. This suggests that during purification of cSHMT there is no significant loss of any of the subunit charge forms and thatthere may be no significant change in the distribution of charge forms occurring with age. Degradation of cSHMT in Oocytes-The ability to remove the first 14 amino acids, which contain isoAsp5,from cSHMT leaving a fully active enzyme provides a convenient test of whether information for in vivo degradation occurs in this part of the protein. We chose to use the oocyte as a cell to determine degradation rates of cSHMT. The enzyme was labeled by reducing the pyridoxal-P, bound as an aldimine to Lys256,with [3H]NaCNBH3,which gives a stable secondary amine. First, the incubation medium of the oocyte was separated from the cells and treated with trichloroacetic acid, forming the trichloroacetic acid-soluble and -insoluble frac-

““T

TABLEI Amino acid sequence of peptide containing an iso-Aspresidue Sequence: I-A-D-E-N-G-A-Y (-75%); I-A-D-E-iso-D-G-A-Y (-25%). Cycle

Amino acid

Nanomoles

Yield

1 2 3 4 5 6 7 8 9

Ile 90 Ala ASP 99 Glu Asn + Asp 94 G ~ Y Ala TYr TYr

3.02 2.71 2.50 2.47 1.71 + 0.12 = 1.83 1.70 1.80 1.06 0.23

100

%

93 74 106 59 22

6-0 months

3 days

3.5 y e a r s

FIG. 3. Densitometric scans of Western blot gels of cSHMT separated by isoelectric focusing in 6 M urea and 20 mM 2mercaptoethanol. The pattern on the left side is purified cSHMT from rabbits which were 6-8 months old. The middle patternand the pattern on the right side were cSHMT partially purified from 3-dayold and 3.5-year-old rabbits.

13789


27

tions of the medium. If injected cSHMT had leaked from the cell, it would showup in the insoluble fraction of the medium. Next, the cells were homogenized and separated into soluble and insoluble cell extract fractions. One would expect to find the injected cSHMT in the soluble cell extract. The soluble cell extract fraction was treated with trichloroacetic acid to form the soluble and insoluble trichloroacetic acid fractions. The injected cSHMT would be in the trichloroacetic acidinsoluble fraction, and any short peptides formed as theresult of proteolytic degradation would be in thetrichloroacetic acidsoluble fraction. Each fraction was analyzed for tritium and results calculated on the basis of the percent tritium injected into the oocytes. In general, each experimental time point used three to five oocytes. The results shown in Fig. 4A are the average of three experiments using oocytes from three 48 different frogs. The radioactivity from the reduced pyrid~xyl-P-Lys’~~ appeared in only two fractions, the trichloroacetic acid-soluble 72 fraction of the medium and the trichloroacetic acid-insoluble FIG.5 . Western blot analysis of cSHMT injected into oofraction of the cell soluble proteins. The sum of the two fractions accounts for more than 90% of the radioactivity cytes. About 0.3 fig of cSHMT in its active and native form was injected into the cells. The zero time point represents about injected into oocytes. The soluble cell extracts were run on SDSPAGE after indicated intervals of incubation in the oocyte. The a 30-min delay between injection of enzyme and homogeni- cSHMT was located by antibody stainingand scanned. The graph on zation of the five oocytes for that timepoint. The small the left shows the densitometric scans of the Western blots, and the amount of radioactivity in the trichloroacetic acid-soluble graph on the right is the area under each corresponding scan. fraction at zero time may indicate that some proteolytic degradation of the enzyme had occurred during this 30-min show that the full-length cSHMT is degraded more rapidly period. The results shown in Fig. 4A suggest that cSHMT is and to a greater extent then S15-cSHMT(Fig. 4B). degraded almost completely in 24 h to soluble peptides and The rapid degradation of cSHMT in oocytes may be the thatthe peptide (or residue) containing the pyridoxyl-P result of the inability of the enzyme to bind amino acid moiety is excreted rapidly into the medium. substrates, like serine, to form a more stable structure. PreThe next experiment was to determine if information on vious studies have shown that saturation of the enzyme with the first 14 amino acids would alter the rateof degradation of serine resultsin a conformational change to a more thermally cSHMT in oocytes. The S15-cSHMTwas prepared by diges- stableprotein(Schirch et al., 1991). This conformational tion with chymotrypsin. This enzyme was then reduced with change may alter the rate at which cSHMT is degraded in the [3H]NaCNBH3 aswas done with the full-length enzyme. the oocyte. To test this cSHMT was injected into oocytes Both native cSHMT andS“-cSHMT were then injected into without prior reduction with NaCNBH3. This enzyme was oocytes and their rates of degradation determined. The results fully active and could bind substrates. The injected protein was assayed as a function of time by doing Western blot analysis with anti-cSHMT antibodies on oocyte extracts sepn w arated on SDS-PAGE. The results show thatcSHMT is v degraded at essentially the same rateasthe NaCNBH3IZ reduced enzyme (Fig. 5 ) . The bands also migrated at thesame W v) distance as native cSHMT, suggesting that there wereno W lY shortened forms of the enzyme in the extract which react with a antibodies. This suggests that if cleavage of the 14 aminot terminal residues is an initial event in degradation, that the h iremainder of the protein is rapidly cleaved to smaller peptides V a and amino acids. 0

I

6

n

a

IY

DISCUSSION

Rabbit liver cSHMT exists in several different forms with different PI values. Extensive studies have now been perT I M E (h) formed to determine the origin of the charge heterogeneity. FIG.4. Degradation of cSHMT microinjected into oocytes. The results in Fig. 3 show that there are significant amounts A, the pyridoxal-P on cSHMT was reduced with [3H]NaCNBH3.The of four different isoforms of subunits with respect to PI. Since enzyme was microinjected into oocytes, incubated for varying amounts of time, and theincubation medium and oocyte fractionated the PI values are all above 6.2, it is unlikely that any of these Asp and as described under“Experimental Procedures.” Each of the five bands distinguish between subunitscontaining fractions were counted, but only the trichloroacetic acid-soluble frac- isoAsp residues, whose pK, values will be below 4.7. Three of tion of the medium (0)and thetrichloroacetic acid-insoluble fraction these isoforms could result from the deamidation of Asn5 and of the soluble proteins of the oocyte (0) containeda significant AsnZz0(subunits containing either none, one, or two deaminumber of counts. The resultsare reported as the percentage of dation sites).Many attempts were made to purify in sufficient counts injected into the oocyte. Each point is the average of three experiments and plotted with 1 standard deviation. B , the protocol amounts the four charge forms of subunits from denatured shown in A was repeated with both cSHMT (lower curue) and S15- cSHMT to analyze each for isoAsp residues. Unfortunately cSHMT (upper curue). Only the radioactivity in the trichloroacetic we were not able to achieve this goal and cannot report the acid-insoluble fraction of the soluble oocyte proteins are reported. isoAsp concentration of the charge forms shown in Fig. 3.

13790


Recently, the cDNA of rabbit liver cSHMT was published (Byrne et al.,1992). This sequence confirmed the correctness of the published amino acid sequence and showed that there are no other Asn-Gly sequences in rabbit cSHMT.The fourth charged subform of cSHMT could be the result of either the deamidation of another Asn residue, occurring by a mechanism that does not form an isoAsp residue or some other modification resulting in the formation of a charge. Byrne et al. (1992) haveshown that rabbit cSHMT can be phosphorylated in vitro by a cyclic AMP-dependent protein kinase. We have not previously detected the presence of a phosphorylated enzyme, but this result would suggest that theisolated enzyme maybe partially phosphorylated, accounting for the additional fourth subform. A number of proteins has now been identified to contain deamidated Asn residues (Wright, 1991). However, the physiological roleof deamidation remains unexplained. This study, along with our previous work (Artigues et al., 1990) now defines the properties and location of the deamidation sites in rabbit cSHMT. We have shown that both sites contain isoAsp residues, but only isoAsp' is exposed for methylation by PIMT. We have shown that in the native enzyme further deamidation of isoAsp6 is extremely slow with a tllz of near 500 h. In this study we show that repair of this residue does not readily occur after methylation with PIMT. In fact, the results suggest that themethylated isoAsp' is stable and only slowly reforms the succinimide intermediate to release methanol. We have also shown that there is no major change in the degree of deamidation as a function of age of the rabbit. Although these studies do not define the physiological role of deamidation in cSHMT, they are necessary studies to proceed with additional work. This enzyme is now one of the best characterized proteins for which deamidation of Asn residues is known to occur, and the recent publication of the cloned cDNA by Byrne et al. (1992) provides the basis for future studies. A possible explanation for the apparentsimilarity in deamidation state of cSHMT from young and old liver is that deamidation leads to degradation and one is looking at a steady-state level of newly synthesized and old enzyme. However, others have found that old cells are deficient in degradation of proteins and that deamidated forms accumulate. This was first demonstratedby Yuksel and Gracy (1986) with triosephosphate isomerase. More recently Ladino and O'Connor (1990) have shown that thedeamidation state of the older fraction of red blood cells is greater than those of newly formed red blood cells. Previous studies with triosephosphate isomerase have suggested that deamidation leads to subunit dissociation with subsequent degradation by proteases (Yuksel and Gracy, 1986). Deamidation of cSHMT results in fully active subforms of the enzyme and does not lead to subunit dissociation. However, we want to determine if deamidation of Asn' and AsnZz0 to Asp and isoAsp residues might be a signal for degradation of cSHMT in vivo. We also want to clarify the roles played by PIMT and AdoMet in the protein degradation pathway. To do these studies requires a system where it has been established that an active PIMT and AdoMet are present, and a method to inhibit the invivo activity of PIMT is available. We chose the oocyte for these studies. Desrosiers et al. (1990) have shown that microinjected calmodulin is methylated by an endogenous PIMT in oocytes. However, they did not report on the degradation of this protein. This system offers an advantage for protein degradation studies over current systems, since you can inject not only a protein to be

studied, but also other small molecules whichmight affect the rate of protein degradation. One possibility to inhibit PIMT activity wouldbe homocysteine, which is a good product inhibitor of PIMT. With theselonger range goals in mind, we injected both cSHMT and S"-cSHMT into oocytes. As shown in Fig. 4 it is clear that the oocyte rapidly degrades cSHMT. The method of labeling was the very mildprocedure of reducing the active site pyridoxal-P with NaCNB3H. An interesting observation during the degradation of cSHMT in oocytes was that the radiolabel was exported from the cell and appeared in themedium. Apparently this exportwas very rapid relative to protein degradation, since essentially no pool of trichloroacetic acid-soluble radiolabeled products was observed in the cell. These studies document that the oocyte provides a valuable new method for measuring the rate and mechanism of protein degradation in vivo. Studies with S"-cSHMT consistently showed a slower and less extensive degradation compared with native enzyme. This suggests that the first 14 residues of cSHMT contain information involved in protein degradation in this system. There are a number of possibilities of what this information could be, but the fact that there is an isoAsp at position 5 provides one possibility. The most logical approach to determining if the deamidation of either Asn' or AsnZz0 is involved as asignal for degradation is to express mutant forms of this enzyme from a cloned cDNA which has other residues replacing Asn' and AsnZz0.The results recorded in Figs. 4 and5 demonstrate that further studies of cSHMT will provide important new information on the role of deamidation of Asn residues, isoAsp residues, and PIMT in the mechanism of protein degradation. REFERENCES Artigues, A,, Birkett, A. & Schirch, V. (1990) J. Biol. Chem. 265,4853-4858 Aswad. D. W. (1984) J. Biol. Chem. 259.10714-10721 Aswad; D. W. &Johnson, B. A. (1987) Trends Biochem. Sci. 1 2 , 155-158 Burnette, W. (1981) Anal. Biochem. 1 1 2 , 195-203 Bvrne. P. C.. Sanders. P. G.. and Snell. K. (1992) Biochem. J. 2 8 6 , 117-123 Ciarke, S. (1985) Annu. Reu: Biochem. '54,479-506 Desrosiers. R. R.. Romanik. E. A,. and OConnor. C. M. (1990) J . Bid. Chem. 265,21368-21374 Diliberto, E. J., Jr. & Axelrod, J. (1976) J. Neurochem. 26?1159-1165 Galletti, P., Iardino, P., Inmosso, Ingrosso, D., Manna, C. & Zappla, V. (1989) Int. J. Galletti. Pept. Protein 'ProteinRes.' Res. 3 3 , 397-402 Gavilanes., F... Peterson. D. & Schirch. L. (1982) J. Biol. Chem. 2 5 7 , 1143111436 Geor e Nascimento, C., Lowenson, J., Borissenko, M., Calderon, M., MedinoSe&, A., Kuo, J., Clarke, S. & Randolf, A. (1990) Biochemistry 2 9 , 95869591 Giulian G. G., Moss R. S. & Greaser, M. (1984) Anal. Biochem. 142,421-436 Hitchcdck. M. J. M.. Ginns. E. I. & Marcus-Sekura. C. J.(1987) Methods Enzymoi. 152,2761284 ' Johnson, B. A. & Aswad, D. W. (1991) Anal. Biochem. 192,384-391 Johnson, B. A.. Langmack, E. L. & Aswad, D. W. (1987a) J. Biol. Chem. 2 6 2 , 12283112287 Johnson, B. A,, Murray, E. D., Jr., Clarke, S., Glass, D. B. & Aswad, D. W. (198%) J. Biol. Chem. 262,5622-5629 Johnson, B. A., Shirokawa, J. M.,Hancock, W. S., Spellman, M. W., Louisette, L. J. & Aswad, D. W. (1989) J. Biol. Chem. 264,14262-14271 Kim, S., Choi, J. & Jun, G. (1983)J. Biochem. Biophys. Methods 8,9-14 Ladino, C. A. & O'Connor, C. M. (1990) Mech. Ageing Deu. 65,123-137 Lowenson, J. D. & Clarke, S. (1988) Bbod Cells 1 4 , 103-117 Lowenson, J. D. & Clarke, S. (1990) J. Biol. Chem. 265,3106-3110 Lowenson, J. D. & Clarke, S. (1992) J. Biol. Chem. 267,5985-5995 Lura, R. & Schirch, V. (1988) Biochemistry 2 7 , 7671-7677 Martini, F., Angelaccio, S., Pascarella, S., Barra, D., Bossa, F. & Schirch, V. (1987) J. Biol. Chem. 262,5499-5509 Murray, E. D., Jr. & Clarke, S. (1984) J..Biol. Chem. 259,10722-10732 O'Connor,C. M. & Clarke, S. (1985) Blochem. Bwphys. Res. Commun. 1 3 2 , 1144-1150 O'Farrell P. H. (1975) J. Biol. Chem. 250,4007-4021 Robinson', A. B. & Rudd, C. J. (1974) Curr. Top. Cell Regul. 8, 247-295 Schirch, L. & Gross, T. (1968) J. Siol. Chem. 243,5651-5655 Schirch, L. & Mason, M. (1963) J. Bml. Chem. 2 3 8 , 1032-1037 Schirch, L. & Peterson D. (1980) J. Biol. Chem. 2 5 5 , 7801-7806 Schirch, V., Schirch, D:, Martini, F. & Bossa, F. (1986) Eur. J. Biochem. 161, 45-49 Schirch, V., Shostak, K., Zamora, M. & Gautam-Basak, M. (1991) J. Biol. Chem. 2 6 6 , 759-764 Teshima, G., Porter, J., Yim, K., Ling, V. & Guzzetta, A. (1991) Biochemistry 30,3916-3922 Tyler-Cross, R. & Schirch, V. (1991) J. Biol. Chem. 266,22549-22556 Wright, H.T. (1991) Crit. Reu. Biochem. Mol. Biol. 2 6 , 1-52 Yuksel, K. U. & Gracy, R. W. (1986) Arch. Biochem. Biophys. 248,452-459 ~~~

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