N-epsilon-acetylation of porcine mature erythrocytes ubiquitin

Int. J. Biochem. Vol. 17, No. 6, pp. 719 721, 1985

0020-711X/85 $3.00+0.00 Copyright (c) 1985 Pergamon Press Ltd

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N-EPSILON-ACETYLATION OF PORCINE MATURE ERYTHROCYTES UBIQUITIN DE-Xu ZHU t, LIE-XIAN Xu t, NIAN-ZHou ZHU t, GILBERT BRIAND2 and KIA-K! HAN2. ~Department of Biochemistry, University of Nanjing, Hankou Road II, Nanjing, People's Republic of China 2Unit6 INSERM No. 16, Biochimie des Prot6ines, Place de Verdun, 59045 Lille C6dex, France [Tel. (20) 97-26-15] (Receired 1 November 1984) A b s t r a c t - - l . Highly purified of porcine mature erythrocytes ubiquitin were obtained according to the experimental procedure reported by Jabusch and Deustch (1983). 2. N-epsilon-acetylation in vitro of internal lysyl residues of ubiquitin by p-nitro-phenyl-acetate at pH 8.0 was performed. The extent of acetylation of ubiquitin was determined: about 4-5 residues (4.5 residues) of N-epsilon-lysine groups of ubiquitin were acetylated. 3. We have assigned by Edman degradation the sites of acetylation and the sites of remaining free internal N-epsilon-lysine residues in the sequence: fully acetylated: Lys-6, Lys-I 1 and Lys-33. Partially free N-epsilon-lysine: Lys-27 and Lys-29 and probably Lys-48 and Lys-63. 4. 50 cycles Edman degradation were performed on porcine ubiquitin and the first 45 N-terminal residues were identified. 5. We have partially determined that the molecular conservation of 45 amino acid sequence of ubiquitin between cattle, man and swine since the 45 amino acid sequence out of 76 residues are identical. The amino acid composition between human and porcine ubiquitin are also identical.

INTRODUCTION The small polypeptides, ubiquitin (Ub) has a molecular weight of 8623 daltons and is constituted by 76 amino acid residues. The amino acid sequence was firstly determined by Schlesinger et al. (1975). It is widely distributed in plant, animal, yeast and bacterial cells (Goldstein et al., 1975). The amino acid sequence was also confirmed by Low and Goldstein (1979). The relationship between the primary structure and the tertiary structure of Ub is that this molecule is extremely resistant to tryptic digestion despite the high content of basic amino acids (7 lysines and 4 arginines residues) out 76 residues (Schlesinger et al., 1975). It is reasonable to believe that this structure is tightly coiled in such a way that lysine and arginine residues are unavailable to the active sites of trypsin and other endopeptidases. The nuclear magnetic resonance investigations have revealed that Ub has a highly globular, compact, pH and temperature resistant properties conformation requiring 7 M guanidine, HC1 to denature it (Lenkinski et al., 1979). Studies with circular dichroism (CD) on the basis of prediction of protein conformation by Zhu and Deustch (1984), the N H 2 terminal 1 15 were suggested to be a fl-pleated sheet: 16-19 random coiled; 2(~23: fl-turn; 24~31 : ~-helix; 32~,5: fl-pleated sheet and 46-54 was suggested to be a helical region: 55-64: fl-pleated sheet; 65-68: fl-turn and 69 76: fl-sheet. It is established by Y o e m a n et al. (1973) that Ub was covalently linked to the histone-2A, especially on

*Author to whom correspondence should be addressed. 719

the e-amine of Lys-119 to form a " Y - s h a p e " with 2-N terminals and 1-C terminal amino acid ( G o l d k n o p f and Busch, 1977). Therefore, the Ub-histone conjugates were firstly found by Yoeman et al. (1973). Enzymic acetylation is a post-synthetic modification which mainly occurs in the N-terminal and basic region of 4 nucleosomal core histones (WoutersTyrou et al., 1981). Also, it has been shown by these authors that the degree of histone acetylation is related to the transciptional activity of the cell. Recently, M a t s u m o t o et al. (1984) have found that Ub possesses CO2 hydratase and limited esterase activity. In its reaction with aromatic acetate esters, it is converted to more anodic forms (Matsumoto et al., 1984) (Deutsch et al., personnal communication). Therefore, it will be interested to assign the sites and the number of acetylation of Ub. Since the increased interest in Ub has been stimulated by studies indicating that a number of proteins contains it as all or a part of their structures.

MATERIALS AND M E T H O D S

(a) Preparation o f Ub A relatively simple and reproductible procedure for the isolation of active form of porcine Ub has been performed according to the experimental procedure reported by Jabusch and Deutsch (1983). The amount of Ub polypeptide in mature porcine erythrocytes appears to be about 0.15 mg per g of hemoglobin and 60~o of material may be recovered according to these authors. The criteria of homogeneity of Ub was controlled by unicity of N-terminal residue obtained by dansylation: dansyl-Met (Gray and Hartley, 1963) and one band on

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DE-Xu ZntJ et al.

SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) in pH 8 according to Laemmli (1970) using 20° o acrylamide concentration. The amino acid composition was established on 24 hr total acidic hydrolysate on an autoanalyzer (Hitachi: Model type 835). (b) N-epsilon-acetylation in vitro q]" internal lysyl residues Ub ( 9 . 6 r a g - 1.2/~M) in a water solution 1.6ml and then dialyzed against borate buffer (0.1 M pH 8.0). The final volume and proteins concentration were 10ml of borate buffer containing about 10mg of protein. 50mg of pnitrophenylacetate was firstly dissolved in I ml of acetone (276/~M). 3 times addition of this reagent at 0, 5 and 10hr at room temperature (22 C) (each time 150/~1 of pNPA in acetone solution: 3 x 150111 = 450#1 containing 124/~M of pPNA). The reaction was allowed to perform during more 16hours and concentrated to a final volume of 2ml and then desalted on a column of Sephadex GI0 (1.2 x 57cm) with a flow rate of 1.5 ml/30 min. The elution buffer is a borate buffer 0.05 M at pH 8.0. (c) Determination of remaining non-N-epsihm-Acetylated internal Lysvl residues It was established by exhausted dinitrophenylation according to Sanger (1945) and the N-epsilon-DNP-Lys contents were quantified by amino acid autoanalyzer. (d) Automatic Edman de,~radation ~:~1 Uh It was performed in a liquid phase sequencer (Beckman 890C, USA). The following protocole according to Hunkapiller and Hood (1978) was performed: polybrene (5 rag) together with a dipeptide Gly-Gly (50-100 nM) are added to the cup. The preliminary performance of three cycles of degradation are necessary before the addition of Ub. These cycles of degradation serve to block any reactive groups in the polymer which might interfere during the degradation and to eliminate some artefacts during the identification of PTH-amino acids in HPEC. An then 120 nM of acetylated-Ub were introduced in the spinning cup and followed by the coupling buffer (quadrol D.I.M. pH 9.0). The classical program with double cleavages was applied. The samples (2 x 120 nM) were run twice. The tirst time 35 cycles were performed and the second time 50 cycles were performed. The PTH-amino acids (phenylthiohydantoin-amino acids) were analyzed and quantified by HPLC (High Pressure Liquid Chromatography) with a column of l~-Bondapak C18 and complemented by separation of PTH-amino acids on a column of tt-Bondapak phenyl. The standard N-epsilon-PTH-N-epsilon-acetyl-Lys was prepared by Wouters-Tyrou et al, (1981) in Lille under the

following conditions: the complete digestion of N-epsilonacetyl-Lys containing peptides with amino peptidase M and then chemically coupled with phenyl-lso-thiocyanate (PITC) followed by cyclization and conversion reactions (Edman and Henschen, 1975: Han et el/., 1977, 1985). RESULTS AND DISCUSSION Ubiquitin (Ub) was converted to an acetylated form by reaction with p - n i t r o p h e n y l acetate in pH 8.0, essentially by the m e t h o d of Verpoorte et al. (1967). The extent of acetylation was determined by reacting the polypeptide, after it had been extensively dialyzed, with F D N B ( F l u o r o - D i - N i t r o - B e n z e n e ) to derivative in HC1 5 . 6 N at l l 0 C removed the N-epsilon-acetyl groups and the lysine contents of the acidic hydrolysate revealed the extent of the acetylation. We have obtained by this way the extent of acetylation of Ub: a b o u t 4~5 residues (4.5 residues) of N-epsilon-lysine groups of U b were acetylated. However it was shown by Z h u and Deutsch (1984), that the acetylation of 4~5 residues of N-epsilon-lysine groups in U b has little effect on its secondary structure as revealed by circular dichroism studies. Therefore, further investigations of the acetylated form of U b produced by its interaction with various a r o m a t i c esters will be of interest, since U b possesses limited esterase activity. F u r t h e r m o r e , since we have k n o w n by these studies that 4-5 (4.5) residues of N-epsilon-lysine groups of U b were acetylated, it will be of great interest to assign the sites of acetylation and the sites o f remaining free internal-N-epsilon-lysine residues in the sequence. Since the a m i n o acid sequence of porcine erythrocytes U b is still u n k n o w n , it will be also of interest to determine the N - t e r m i n a l residues simultaneous by stepwise E d m a n d e g r a d a t i o n and its a m i n o acid composition. We have run the U b samples twice, the tirst run in E d m a n protein's sequencer with classical program and the 35 residues of a m i n o acid sequence were determined. A n d the second run with double cleavage p r o g r a m s was performed a n d the first 45 N - t e r m i n a l residues were established over 50 E d m a n d e g r a d a t i o n cycles. The following a m i n o acid sequence of porcine U b was obtained:

l 5 NH2-Met-GIn-lle-Phe-Val-AcetyI-Lys Thr

10 Leu T h r Gly Acetyl Lys T h r lie T h r Leu 20 25 Glu Val Glu Pro S e r Asp T h r lie G l u Ash Acetyl Lys Val

Ala Lys

Acetyl Lys 30 -Ile-Gln gys

35 4O A s p - A c e t y l g y s - G l u Gly Ile Pro Pro Asp G i n Gln 45 Arg g e u Ile Phe

...

Amino acid structure of pig ubiquitin

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A n d the a m i n o acid c o m p o s i t i o n of porcine U b is determined as below: 7

Asx 7: T h r - 7 : Ser-3: Glx-13; Pro 3; Gly 6; Ala 2; Val-4; M e t - l ; lle 7: Leu 9; T y r - I : Phe-2; H i s - l : Lys 7; Argo4: total = 76 residues.

We have assigned the kys-6, Lys-l I a n d Lys-33 are fully acetylated whereas the Lys-27 and Lys-29 are only partially acetylated (30°~) and the r e m a i n i n g ~'o at A-epsilon-internal free lysyl residues are 7n...... kvs-27 and 701';; at Lys-29. The r e m a i n i n g two residues of lysine (7 5 determined = 2) must be b o t h partially acetylated. T h e p r e s u m e d position of 2 r e m a i n i n g lysyl residues could bc reasonably attributed to be Lys-48 and Lys-63 by sequence h o m o l o g y between porcine, h u m a n a n d bovine U b (Schlesinger et al., 1975; Schlesinger a n d Goldstein, 1975). It is of interest that we have assign the full acetylation site of Lys-33. Because the Lys-33 is positioned bctween Asp-32 a n d Glu-34. In spite of the negative charges of Asp-32 a n d Glu-34 the N-epsilon-Lys-33 is fully acetylated by p - P N A a n d these acetylations ~xill not interfere with the secondary structure. F u r t h e r m o r e , we have partially determined t h a t the molecular conservation of 45 a m i n o acid sequence of U b between m a n and swine, since the 45 a m i n o acid sequence out of 76 residues are identical and the a m i n o acids c o m p o s i t i o n between h u m a n a n d porcine U b are also identical. The reason of the high molecular c o n s e r v a t i o n of U b between cattle a n d swine a n d between m a n a n d swine m a y be attributed to t h a t a n u m b e r of proteins contain it as all or part of their structure a n d its p a r e n t gene is not evolved and this highly conservatives as histones. Acknowledgements--Our thanks are due to the helpful

guidance of Professor H. F. Deutsch in the University of Wisconsin, Madison, Wisconsin, U.S.A. and Dr P. Sauti6re for his advice and for the preparation of N-alpha-PTH-Nepsilon-acetyI-Lys standard. The technical assistance of Mme D. Bela'/che and Mme O. Moreau for thc quantitation of PTH-amino acids by HPLC.

REFERENCES

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