The presence of citrulline in epidermal proteins

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Biomedical

11 1

121

Press

BBA 3~311

T H E P R E S E N C E O F ( T | ' R U I A A N E IN E P I D E R M A L P R O T E I N S

JOSEPHKUBILU,q,

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l ) e p a , ' l m e n l o f l)ermattd~>~25", llart.'ard M e d i c a l S c h o o l . M a s s a c h u s e t t s (;ct~,'r(21 Ih~.spilal. lh)stt~n, M A 0 2 1 1 I ( l " N .4 ) (Receiw~d February

12th. 1979)

K e y u , - r d s : E p i d e r m ( t l p r o t e i n : ( ' i l r u l l i n e : . l r g i n i n e c~>nt,crsi<.~: ¢ S l r a t u m (',~rm'um,

Summary ('itrulline is p r e s e n t in the s t r a t u m c o r n e u m p r o t e i n s o f tu~man, c o w snoul. pig s n o u t and guinea pig e p i d e r m i s b u t is at)sent f r o m the s t r a t u m c o r n e u m proteins o f frog, m o u s e , turtle, rat and h a m s t e r e p i d e r m i s . T h e a m i n o acid is released by aci(t hy(trolysis and ranges f r o m 1.7 to 5.5 residues per t h o u s a n d residues o f p r o t e i n a m i n o acid. Protein derived citrulline c o - c h r o m a t o g r a p h s with a u t h e n t i c L-('itrulline on an a m i n o acid a n a l y z e r , on l ) o w e x - 5 0 , on Dowex-2 and on t h i n - l a y e r c h r o m a t o g r a p h y . D a n s y l a t e d material c o - c h r o m a t o g r a p h e d with a u t h e n t i c dansyl-I,-citrulline in t w o t h i n - l a y e r c h r o m a t o g r a p h y systems. I,abelling e x p e r i m e n t s have s h o w n t h a t the p r o t e i n b o u n d citrulline is derived f r o m p r o t e i n b o u n d arginine and p r o b a b l y results f r o m e n z y m a t i c conversion o f the g u a n i d o g r o u p to the ureido group.

Introduc[,ion A l t h o u g h p r o t e i n s as a rule (to n o t c o n t a i n the a m i n o acM citrulline, its presence in certain p r o t e i n s o f t h e hair follicle has been f i r m l y establishe(t [ 1,2,31. Citrulline is n o t i n c o r p o r a t e d at the t i m e o f p r o t e i n s y n t h e s i s as no k n o w n t r a n s c r i p t i o n a l ('()de for citrulline has b e e n r e p o r t e d n o r is t h e r e a n y evidence for citrulline p a r t i c i p a t i n g in a m i n o acyl t r a n s f e r a s e r e a c t i o n s [41. [t a p p e a r s , h o w e v e r , t h a t citrulline exists in the p r o t e i n s o f the hair follicle as the result o f p o s t - t r a n s l a t i o n a l m o d i f i c a t i o n o f p r o t e i n b o u n d arginine residues [ 5 ] . Since e p i d e r m a l cells are p r e c u r s o r s to hair follicle cells, and since a similar c o u r s e o f g r o w t h and k e r a t i n i z a t i o n o c c u r s in b o t h hair and e p i d e r m i s , it was d e c i d e d to * To w h o m r e p r i n t r e q u e s t s sh,>ldd be a(hlre';sed.

115

investigate the possibility that citrulline might also be present in epidermal proteins. Methods The epidermis was isolated from cow and pig snouts by cutting slices parallel to the surface, while in hairy animals, the skin was epilated using the wax sheet method. The freshly epilated skin was removed from the animal immediately after sacrifice. Whole skin was exposed to water at 60°C for 1 min, ('hilled in an ice bath, and the epidermis was removed with forceps. Epidermis was homogenized in 0.15 M saline using a ground glass homogenizer and the homogenate centrifuged at 15 000 ~ g for 20 min. Prekeratin was prepared from the pellet by extraction with 0.1 M citrate buffer, pH 2.6 [6]. After centrifugation of the acid extract, the pH of the clear supernatant was adjusted to 4 and the resulting precipitate was collected by centrifugation at 600 ×g. The pellet was then redissolved in citrate buffer and dialyzed exhaustively against distilled water. Fibrous proteins from stratum corneum were prepared from the citrate buffer extracted pellet by overnight stirring in a solution of 6 M urea containing 0.1 M Tris, pH 9.0 and 0.1 M ~-mercaptoethanol [7]. These proteins were precipitated by exhaustive dialysis against distilled water and the pellets redissolved in the Tris/urea/mercaptoethanol buffer and passed down 1.5 ~ 30 cm columns of Sephadex G-50. Material eluting in the void volume was pooled and again exhaustively dialyzed vs distilled water. Prekeratin and straum corneum proteins were hydrolyzed for 24 h in 6 N HC1 and the amino acid contents were determined using a Beckman model 116 amino acid analyzer. A large sample of human stratum corneum was treated as above and yielded about 100 mg of protein. This sample was hydrolyzed and to the hydrolyzate was added 0.5 pc of L-[ureido-'4C]citrulline (specific activity 5--10 Ci/mol). The hydrolyzate was evaporated to dryness and the residue redissolved in 3 ml of 0.25 M pyridine acetate buffer, pH 4.25. The sample was chromatographed on a 3.5 × 80 cm column of Dowex 50 X-8 200--400 mesh which had been equilibrated with the same buffer. The column was developed and fractions which contained radioactive label were pooled. After drying, the material was applied to a Beckman model 116 amino acid analyzer and the eluate collected from the column. The radioactive peak was rechromatographed on the analyzer and the a m o u n t of citrulline and other amino acids determined by the usual reaction with ninhydrin. Dansyl L-citrulline was prepared both from purchased L-citrulline {Sigma) and citrulline purified from human stratum corneum protein [8]. The systhesized dansylated materials were compared to each other and to commercial dansyl-L-citrulline by chromatography and co-chromatography on polyamide layers.

In vivo synthesis of citrulline-containing proteins The backs of adult guinea pigs of about 150 g were epilated and 24 h later injected intradermally with 160/JCi of L-[guanido-~4C]ar~nine. Three days later the animals were sacrificed and the whole skin was removed from a 2 cm 2 area around each of 6 injection sites. The epidermis was heat separated and

l t(; h o m o g e n i z e d in 0.2 M Tris, flit 9.5. with 8 M urea. A.fter ()w,rmght ext.raction. the s a m p l e was c e n t r i f u g e d aL l ( 1 0 0 0 ' g and lh~, resulting I)~qlet was washed wilh the s a m e b u f f e r . "['he wash and s u p e r n a t a n t fraction w(,r(, ,'o)nhined an(l d e s i g n a t e d as Extract I. T h e pellet was exlract(,d m 0.2 M'l'ris. p]! 9.5, with S M urea and 0.2 M / 3 - m e r e a p t o e t h a n o l (wernight. The suspensi,m was ,'~,ntrifu~e(I at 10 000 g. T h e pellet was washe(t with b u f f e r and the stlt~erllatalll t'ra('tion and wash were c o m b i n e d and d e s i g n a t e d E x t r a c t 11. E x t r a c t s I and II wt,re ultrafiltered t h r o u g h an A m i c o n I'M 10 ( l ( ) 0 0 0 dalton c u t o f f ) m ( , m b r a n e and washed with the a p p r o p r i a t e e x t r a c t i o n b u f f e r ['ho (:(m('entrated sanq)les were applie(I to a S e p h a d e x G - 2 0 0 c o l u m n (0.9 5.1 ,'mJ and material with an a p p a r e n t m o l e c u l a r w~,ight o f 50 00O ()r ~r~,ater wep o o l e d and di~dyze(1 e x h a u s t i v e l y against distilled water. Samples were h y d r o l y z e d in g N HC1 for 24 h and then purified by thin layer , . ' h r o m a t o g r a p h y ('I'L(') on silica gel and cellulose. Samples wer(. sTr(,aked in a 1 5 ('m line on a 20 . 20 cm sheet o f Bakerflex [B-2. (:old ('arrier was a d d e d and the sheets were (teveloped with c h l o r o f o r m / m e t h a n o l / 1 7 " ~ ammonium h y d r o x i d e q2:2:1, v:'v). A 3 c m strip was cul f r o m the ( ' h r ( ) m a t o g r a m anti s p r a y e d with p - d i m e l . h y l a m i n o c i n n a m a l d e h y d e 191 m o r d e r to (teI(,~'t citrulline. T h e citrulline z o n e was sliced f r o m the rest o f the c h r o m a t o g r a m , the slica gel was scraped f r o m th(, backing, and the citrulline was e x t r a c t e d hy washing the, silica gel t h r e e times with water. Citrulline was f u r t h e r purified hy 'I'I,(' on cellulose. T h e s a m p l e was applied as in the silica gel c h r o m a t o g r a t ) h y hut develol)e~t in i s o p r o p y l ah'()hol/5 N a m m o n i u m h y d r o x i d e (7:3. v:vL D e t e c t i o n and solubilization o f th(, ,'itrulline was similar to th(, t)rocedur(' used m T I , C ()n silica ~(,l. Final p u r i f i c a t i o n (:)f citrulline was accomplish(,d by ( ' h r o m a t o ) z r a p h y ,)n a B e c k m a n m o d e l 116 a m i n o acid a n a l y z e r . Material eluting at the c i t r u l l m e position was c o l l e c t e d and )zave a positive r e a c t i o n with p - d i m e t h y l a m i n o c i n n a m a l d e h y d e . Material was placed m a scintillation vial and c o u n t e d in the present(, o f 10 ml o f aquasol II. Bovine s n o u t e p i d e r m a l slice e x p e r i m e n t s were carried o u t for ;:; or 24 h in t t a n k ' s Balan('ed Salt s o l u t i o n c o n t a i n i n g 0.50 ~Ci of t,-[guanido-'4C]',u'ginine 50 ~g/ml s t r e p t o m y c i n , 50 u n i t s / m l penicillin, 50 Hg/ml g e n t a m y c i n , and 2.5 i~g/ml a m p h o t e r i c i n . Following i n c u b a t i o n , the slices were r e m o v e d f r o m th¢, m e d i a and h o m o g e n i z e d in distilled water. In s o m e e x p e r i m e n t s , s n o u t epidermal slices were i n c u h a t e d with L-[ ureido-~4C]citrulline, l t o m o g e n a t e s were c(,ntrifug, ed at 10 000 "..¢ and the (:lear s u p e r n a t a n t s o l u t i o n as well as the in(:uba.lion m e d i u m were m a d e 0.25 M in p e r c h l o r i c acid. After standing <~vernight m 4 C, the s a m p l e s were c e n t r i f u g e d at 10 0 0 0 "4 g. T h e pellets were washed ()n('e with 0.25 M p e r c h l o r i c acid and 3 times with a b s o l u t e e t h a n o l . Ethanol wa.s r e m o v e d u n d e r v a c u u m and the pellets h y d r o l y z e d in 6 N t t C L for 24 h at I I 0"C. A f t e r drying, residues were dissolved in I I , O , ('old arginine and c i t r u l l m e were a d d e d and the pll adjusted to 8 to 9. Samples to pr(,pared were passed d o w n c o l u m n s o f Dow(,x-2 (X-8), [ O t i ] form. C o l u m n s were washed with distilled w a t e r and t h e n d e v e l o p e d with 0.2 M a m m o n i u m f o r m a t e , plI 5.0. T h e aliquots were s p o t t e d on filter l)aper, dried and s p r a y e d with b o t h n i n h y d r i n and p - d i m e t h y l a m m o c i n n a m a l d e h y d e . Th(, p o s i t i o n s o f a r g i n m e , citrulline and urea were d e t e r m i n e d as b o t h a m i n o acids

117 reacted with ninhydrin while urea and citrulline reacted with p-dimethylaminocinnamaldehyde. Under the conditions of c h r o m a t o g r a p h y , arginine and urea eluted in the void volume while citrulline eluted some 20 fractions later. The purity of citrulline eluted from Dowex 2 was established by c h r o m a t o g r a p h y on the amino acid analyzer. Results Fig. 1 shows a typical amino acid analysis of stratum corneum protein. A small peak is visible at the citrulline position. Although this peak was barely distinguishable from baseline, it was observed that as sample size increased, this apparent citrulline peak also increased in size. Quantitation of the material as shown in Table I was based on several analyzer runs of different sized samples using a color value determined for L-citrulline. Stratum co r n eu m proteins from a num ber of species were analyzed for amino acid content. Citrulline was found to be present in the stratum corneum proteins o f human, cow snout, pig snout, and guinea pig epidermis, but not in frog, mouse, turtle, rat, or hamster epidermis. In no species was citrulline present in the acid soluble prekeratin. Table I shows that the a m o u n t of citrulline present ranges from about 1.9 to 5.5 residues per thousand residues. It is notew o r t h y that citrulline was found in non-hair bearing areas (snout of cow and pig, palm and sole of human) of several species so its presence was not due to hair contamination. In order to determine if the ninhydrin positive material which eluted at the citrulline position of the analyzer was indeed citrulline, it was purified in quantity and identified by chemical and physical methods. Dialyzed human stratum co r n eu m protein was h y d r o l y z e d in 6 N HC1, trace labelled with L-[ureido-14C]citrulline and chromatographed on Dowex 50. Labelled fractions were combined and chromatographed on the amino acid analyzer. Fig. 2 shows the analysis of the Dowex 50 purified material. Traces of serine, glycine, aspartic acid and alanine were present as well as the bulk of the material which co~chromatographed with labelled citrulline..Using the extinction coefficient for citrulline, the a m o u n t of material in the initial extract was calculated as 1.7 residues per thousand residues, the same n u m b e r determined in the animal sur-

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vey experiment. The a m o u n t of purified material waa ahout 200 i,g. ]'his matt,rial and authent.ic L-citrulline were dansylated and the products were compared to purchasect dansyl L-citrulline. ]'able II shows that the dansylatcd material recovered from h y d r o l y z e d human stratum corneunL co-chromatographs both with commercial dansyl-I,-citrulline and with dansyl-l,-citrulline prepared m the laboratory. Rogers et al. [5] haw+• proposed that citrulline may be present in proteins as the result of enzymatic modification of arginyl resictues. In order to test this hypothesis, epidermal proteins were isolated from guinea pigs, which had been injected with I,-[guanido-~C]arginine. A summary of the isolation procedure is shown in Table III. Isolated epidermal proteins were hydrolyzed in 6 N !lCl and citrulline was isolated by TLC and purification on the amino acid analyzer. Final analysis showed that 0.95~ of the counts in the Tris/urea soluble proteins and l'~f of the counts in the Tris/urea//3-mercaptoethanol soluble pro-

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119

TABLE

II

COMPARISON CITRULLINE FIED ON THE

OF R F VALUES AND AND THE DANSYLATED AMINO ACID ANALYZER

CO-CHROMATOGRAPHY OF PURCHASED DANSYI.-LPRODUCTS OF L-CITRULLINE AND MATERIAL PURI-

C h r o m a t o g r a m s were run on polyamide layers (Schleicher and Schuell, F-1700). Solvent I: ndleptane/ n-butanol/acetic acid (3:3:1, v/v). Solvent 2: water/formic acid (200: 3, v/v). R F values .........................

Compound

Solvent 1 .................................. C o m m e r c i a l d a n s y l - L - c i t r ttUine Synthesized dansyl-L-citrunine Purified material from human stratum corueum hydrolyzate Mixture of purified material and commercial dansyl-L-citrulline

0.33 0.33 0.36 0.36 .........................

Solvent 2 0.60 0.60 0.64 0.64

TABLE Ill ISOLATION

OF 14C-LABELLED

CITRULLINE

FROM GUINEA PIG EPIDERMAL

PROTEINS

P e r c e n t l a b e l as c i t r u l l i n e is t h e r a t i o o f c i t r u l l i n e c o u n t s f r o m t h e a m i n o a c i d a n a l y z e r o v e r t o t a l c o u n t s after hydrolysis.

Extract PM-IO (concn.) G-200 Hydrolyzate T L C I (silica gel) TLC 2 (cellulose) Amino acid analyzer

Tris-urea (cpm)

Tris/urea/EtSH

1 400 690 350 225 11 3 2

87 60 23 17 1

% l a b e l as c i t r u l l i n e

000 000 000 000 000 000 000 0.89

* (cpm)

000 000 000 000 400 176 165 0.97

* ~-Mercaptoethanol.

TABLE

VI

PURIFICATION OF LABELLED CITRULLINE FROM PROTEINS OF COW SNOUT EPIDERMAL SLICES

L-[GUANIDO-14C]ARGININE

Citrulline to arginine ratios axe ratios of citrulline counts purified on D o w e x perchloric acid pellets after hydrolysis. Incubation time (h)

Supernatant counts (cpm)

Perchloric acid pellets (cpm)

Citrulline from D o w e x (cpm)

24 24 24 24 3

1 170 1 118 1 134 1 393 1 220

66 000 52 000 62 000

380 3 4 0 ** 400

*

000 000 000 000 000

0

--

41 0 0 0

0

2

LABELLED

2 to total counts in the

Citrulline axginine (%) 0.57 0.64 0.65 ---

* L-[Guanido 14C]arginin e added after the incubation time. ** W h e n this sample was chromatographed on the a m i n o acid analyzer, 9 0 % of the counts eluted at the citrulline position.

120

teins could be recovered as citrulline. When the same experiment was done using L-[ureido-~4C]citrulline labelling (if epidermal proteins also occurred, but over 99% o f the incorporated label could he recovered as arginine. When slices o f cow snout epidermis were incubated with I,-[guanido-'4C]arginine significant incorporation of label occurred in perchloric acid precipitated proteins (Tal)le IV) in l)oth 3 and 24 h incubations. In 24 h samples a small but reproducible a m o u n t of labelled citrulline (0.6% of the ineorl)orated arginine) could be positively identified in the h y d r o l y z a t e , while at 3 h no lahelled citrulline was detectable. When this experi m ent was repeated using L-[ureido-'4C]citrulline, it was found that, as in the in vivo guinea pig experiment, virtually all perchloric acid precipitable counts were recovered as argiIlino. l)iscussion The presence of citrulline covalently linked to proteins of hair follicle has been firmly established [1,2,3]. A mechanism by which citrulline is introduced into hair follicle protein is by enzymatic conversion of protein bound arginine to protein bound citrulline. These experiments d e m o n s t r a t e that covalently bound citrulline also occurs in certain proteins of the epidermis of some but not all species. The fact that citrulline was found in epidermal proteins from non-hair bearing areas of skin is significant in that it could not have arisen as a result of hair or hair follicle contamination. Both the in vivo labelling experiments with guinea pig and the in vitro labelling e x p e r i m e n t with cow snout epidermal slices show that, as in hair follicle, eitntlline is produced by post-translational modification of protein bound arginine residues. This observation strongly suggests that an epidermal enzym e similar to the arginine converting e n z y m e of hair follicle exists and indeed a recent report has confirmed the presence of this activity in cow snout epidermis [10]. The presentation of L-[ureido-'4C]citrulline to guinea pig in vivo and cow snout epidermal slices in vitro does result in labelling of epidermal protein, however, over 99~ of the label appears as arginine, suggesting direct incorporation of citrulline does not occur. It seems reasonable, t herefore, that the presem:e o f citrulline in epidermal proteins is due primarily, if not exclusively, to the action of an arginine converting enzym e on a suitable protein substrate. This mechanism is known to occur in hair follicle where it plays an u n k n o w n role m keratinization. It may play a similar role in epidermis, but this is y e t to be d e m o n s t r a t e d .

Acknowledgement This work was supported by Grant AM-06838 from N.I.H. References 1 R o g e r s , td.E. ( 1 9 6 2 ) N a t u r e 194. 1 1 4 9 - - 1 1 5 1 2 S t e i n e r t , P.M., H a r d i n g , H . W . J . a n d R o g e r s , G.E. ( 1 9 6 9 ) B i o c h i m . B i o p h y s . A c t a 1 7 5 , 1--9 3 S t e i n e r t , P.M. a n d R o g e r s , G.E. ( 1 9 7 3 ) B i o c h e m . J. 1 3 5 , 7 5 9 - 7 7 1

121 4 L o c k , R.A., Harding, H.W.J. and Rogers, G.E. ( 1 9 7 6 ) J. Invest. D e r m a t o l . 6 7 , 5 8 2 - - 5 8 6 5 Rogers, G.E., Harding, H.W.J. and L l e w e l l y n - S m i t h , I.J. ( 1 9 7 7 ) B i o c h i m . B i o p h y s . A c t a 4 9 5 , 1 5 9 - 175 6 Baden, H.P.° G o l d s m i t h , L,A. a n d F l e m i n g , B.F. ( 1 9 7 3 ) B i o c h i m . B i o p h y s . A c t a 3 1 7 , 3 0 3 - - 3 1 1 7 Baden, H.P., Lee, L.D. and Kubflus, J. ( 1 9 7 6 ) . J. Invest D e r m a t o l . 67, 573 8 Beale, D. ( 1 9 6 9 ) in: C h r o m a t o g r a p h i c and E l e c t r o p h o r e t i c T e c h n i q u e s ( S m i t h , l., ed.), 3rd edn., Vol. I, p. 198, P i t m a n Press, Bath 9 Harding. H.W.J. and Rogers, G.E. ( 1 9 7 6 ) Biochim. Biophys. A c t a 4 2 7 , 3 1 5 - - 3 2 4 10 Kubilus, J. and Baden, H.P. ( 1 9 7 8 ) Fed. Proc. 37, 2 7 9 5