Characterisation of calmodulin from Drosophila heads

228

Biochimica et Biophvsic'a Acta 832 (1985) 228 232 Elsevier

BBA 30116

BBA Report

Characterisation of calmodulin from

Drosophila

heads

M a t t h i a s G 6 r l a c h ~, P e t e r D i e t e r ~,h,., H a n s H. S e y d e w i t z c, C l e m e n s K a i s e r ~ I r e n e W i t t c a n d D i e t e r M a r i n e ,,d "linstitut fur Biologie IlL Universit{it Freiburg, Schiinzlestrasse 1. bBiochernisches lnstitut, UniversitFtt Freiburg, Hermann-Herder-Strasse 7. ' Biochemisches Labor der Kinderklinik, Universit~tt Freiburg, Mathildenstrasse 1 and dGOdecke Forschungs-lnstitut, Mooswaldallee 1-9, 7800 Freiburg, ( F. R. G) (Received June 28th, 1985)

Key words: Calmodulin; Tryptic digest; Peptide map: Amino acid composition: ( Drosophila )

Calmodulin from Drosophila heads has been purified to apparent electrophoretic homogeneity, it has the same characteristics as bovine brain calmodulin with respect to the migration upon polyacrylamide gel electrophoresis and maximal activation of a caimodulin-deficient cAMP phosphodiesterase. The amino acid composition resembles bovine brain calmodulin with the exception that trimethyllysine is absent and that it contains only one tyrosine. The tryptic peptide map of Drosophila calmodulin suggests some differences in the amino acid sequence as compared to bovine brain calmodulin. These proposed differences in the primary structure may explain why Drosophila calmodulin is less potent that bovine brain calmodulin in the activation of a cAMP phosphodiesterase from bovine brain.

Calmodulin, which modulates a large number of Ca2+-dependent biochemical reactions inside the cell (for review see Ref. 1), has been isolated from a variety of eucaryotic organisms including plants, invertebrates and vertebrates (for review see Ref. 2 and 3). A comparison of the physical and biochemical properties including amino acid sequence analysis of some calmodulins shows that this protein has been well conserved through the phylogenic scale. As for the invertebrate phylum of Arthropoda, its presence has been demonstrated in crustaceae [4] and insects [5-8]. Recently, Yamanaka and Kelly [9] presented some evidence for the presence of calmodulin in Drosophila heads. Heads of Drosophila melanogaster (wild-type strain 'Oregon R') were harvested and homoge-

* To whom correspondence should be addressed at Biochemisches Institutr Hermann-Herder-Strasse 7, D-7800 Freiburg, F.R.G..

D

B +Ca

D

B -Ca

Fig. 1. 12.5% SDS polyacrylamide gel electrophoresis [14] of purified calmodulin from bovine brain (B) and Drosophila heads (D). Samples contained either 5 mM CaC12 (left lanes) or 5 mM EGTA (right lanes).

0167-4838/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)

229

nized according to Jiminez and Rudloff [10]. A clear supernatant was obtained by centrifugation of the crude homogenate at 50 000 × g for 30 rain and applied to 200 g DEAE-Sephacel on a glass funnel. The Sephacel was washed with buffer a containing 1 mM EDTA and 0.1 M NaC1. For the elution of calmodulin the NaCI concentration was raised to 0.5 M NaC1. After addition of 1 mM Ca 2+ the eluate was applied to a chlorpromazine (CAPP)-Sepharose column. The CAPP (10-(3aminopropyl)-2-chlorophenathiazine)-Sepharose was prepared as described by Jamieson and Vanaman [11]. The column was washed with buffer containing 1 mM Ca 2+ and 0.5 mM NaC1 until the absorption at 280 nm was nearly zero. Calmodulin was eluted from the CAPP-Sepharose with buffer A containing 10 mM E G T A and 0.5 M NaC1. After addition of 15 mM CaC12 the eluate was extensively dialyzed against 1 mM N H 4 H C O 3 / 1 mM 2-mercaptoethanol. The solution was heated for 2 min at 95°C and centrifuged for 20 min at 50 000 × g. The obtained supernatant was lyophilized. The lyophilized material was dissolved in 1 mM N H 4 H C O 3 / 1 mM 2-mercaptoethanol and loaded on a Sephadex G-100 column. The column was washed with 10 mM N H 4 H C O 3 and the fractions containing calmodulin were lyophilized and stored at - 2 0 ° C . Calmodulin from bovine brain was purified as described earlier [12]. The amounts of calmodulin were determined by weight. i

i

' 0.1

Ii 0

i

b

Z ~I00

~ so uJ

~ 6o o Ill

~ 20

E ,

I£

0

' I'0 100 calmodulin, InM]

1000

Fig. 2. Activation of a calmodulin-deficient cAMP phosphodiesterase [16] by various amounts of Drosophila (D) and bovine brain (B) calmodulin (CAM). The basal activity of the cAMP phosphodiesterase was 2.4 mU. At saturating amounts of calmodulin, EGTA (5 mM) was added. The arrows indicate half-maximal activation of the enzyme.

Calmodulin from Drosophila heads was purified utilizing mainly its calcium-dependent binding to chlorpromazine (CAPP)-Sepharose. The yield of calmodulin was found to be about 150 ~ag per g of Drosophila heads. The purified calmodulin appeared as a single band on SDS-polyacrylamide gels when the gel electrophoresis was performed in the absence of Ca 2+ (Fig. 1). When the electrophoresis is performed in the presence of Ca 2+, Drosophila calmodulin and bovine brain calmodulin show another band with an apparent higher molecular mass. Such an appearance of additional bands has already been described for purified bovine brain calmodulin and seems to be due to storage of the protein [17]. The purified Drosophila calmodulin shows the characteristic calcium-dependent shift in its electrophoretic mobility (Fig. 1) and has the same apparent molecular mass as bovine brain calmodulin of 16 000 and 19000 Da in the presence and absence of Ca 2+, respectively. These values are higher than those reported by Yamanaka and Kelly [9], who calculated molecular masses of 14300 and 16500 Da. These authors, however, already suggested that the molecular mass of Drosophila calmodulin is higher than the calculated values because it migrated with a mobility identical to that of porcine calmodulin, which is known to have an apparent molecular mass of 16 500 Da. Yamanaka and Kelly [9] showed that the purified Drosophila calmodulin activates a calmodulin-deficient rat brain phosphodiesterase to the same extent as the porcine calmodulin. In order to investigate whether the insect and vertebrate calmodulins are equally effective, we determined the activation of a calmodulin-deficient phosphodiesterase with different amounts of both calmodulins. Fig. 2 shows the activation of the phosphodiesterase as a function of various a m o u n t s of Drosophila and bovine brain calmodulin. It can be seen that the activation totally depends on the presence of Ca 2+ and that Drosophila calmodulin activates the phosphodiesterase to the same extent but at concentrations about 5-times higher as compared to bovine brain calmodulin. It is not quite clear at present what the reason for this discrepancy is. It may be that the lower potency of Drosophila calmodulin is due to suggested differences in the protein structure between insect and vertebrate calmodulin (see be-

230 TABLE I AMINO ACID COMPOSITION OF CALMODULIN FROM DROSOPHILA, BOVINE BRAIN; ZUCCHINI AND NEURO-

SPORA The absence of tryptophan was deduced from absorption spectrum. Amino acid analyses were performed with a KONTRON Liquimat Ill amino acid analyzer (Kontron Technik, Eching, F.R.G.) using a standard sodium citrate hydrolysate program. For the determination of N~-trimethyl-L-lysineseparate runs were performed in which the running time of buffer C (pH 4.1) was shortened by 9 rain. Under these conditions N-trimethyl-L-lysineelutes 2.8 rain before the ammonia peak. As a further control, ammonia was eliminated by pretreating the samples with 2 M NaOH at 95°C for 30 rain and neutralizing thereafter with 2 M HCI. The samples were hydrolyzed in 5.7 M HC1 for 24, 48 and 72 h at ll0°C in sealed glass tubes that had been thoroughly flushed with nitrogen and evacuated. Residue Asx Thr Ser Glx Pro Gly Ala Cys Val Met lie Leu Tyr Phe His Lys(Me3) Lys Arg Trp

Drosophila 23.4 12.1 5.8 26.3 2.2 11.7 10.5 0 6.7 7.1 7.6 9.8 0.9 8.6 1.0 0 8.6 5.9 0

Neurospora

Bovine Brain (tool/tool)

Zucchini [131

[20]

24.0 12.1 4.3 28.4 1.6 11.3 11.3 0 7.0 7.8 7.6 9.2 1.5 7.9 1.2 1.2 7.5 6.1 0

25.9 8.9 5.2 26.1 2.2 10.4 10.5 1.1 7.3 6.7 7.3 11.8 0.95 8.9 1.3 1.0 9.9 5.0 0

25 9 11 26 3 10 8 0 6-7 7 7 9 1 8 1 0 7 8 6-7 0

low). The a m i n o acid composition of Drosophila c a l m o d u l i n is shown in Table I. In general, the a m i n o acid composition is similar to c a l m o d u l i n from a vertebrate (bovine brain), a fungus (Neurospora crassa) and a higher plant (zucchini). These similarities include the relative high a m o u n t of the negatively charged a m i n o acids, aspartic acid a n d glutamic acid, a n d the lack of tryptophan. With respect to the c o n t e n t of tyrosine, it differes from the vertebrate calmoluin and resembles the plant a n d fungi calmodulin. The insect and p l a n t c a l m o d u l i n have a relatively low A277/A26o ratio (0.84) compared to m a m m a l i a n c a l m o d u l i n (1.1) indicating also the lower tyrosine content. Recently Morishima and Bodnaryk [8] reported that two other insects, Mamestra configurata Wlk. and Bombyx mori L. c o n t a i n also only one tyrosine residue per mole of calmodulin. Drosophila c a l m o d u l i n exhibits another characteristic feature,

the lack of Lysine (Me3), which has been found in all vertebrate a n d p l a n t calmodulins investigated so far. The absence of this particular a m i n o acid has been reported only for c a h n o d u l i n from microorganisms, e.g., Dictyostelium [18], Chlamydomonas [19] a n d N e u r o s p o r a [20] a n d from the insects M. configurata Wlk. a n d B. mori L. [8]. It is k n o w n that the enzymatic modification of the lysine residue occurs post-translationally [21], suggesting that the methylating enzyme may not be present in the insect cell. In order to o b t a i n some more i n f o r m a t i o n on the p r i m a r y sequence relationship of the c a l m o d u l i n s from Drosophila and b o v i n e brain, the peptide maps of their tryptic digests were compared. The H P L C m a p of the tryptic digest of bovine b r a i n c a l m o d u l i n without a d d i t i o n of E D T A shows a b o u t 12 peak (Fig. 3A u p p e r part), which is in accordance with the theoretically expected 12 peptides (bovine b r a i n

231 ~2 ¸

[3

A

-EOTA

035

-EDTA

0.10 0.1

L

005-

~

o-7"-

t

0.2g

* EDTA

i o,15-

+EOTA

0.100.1

0.05-

0

,

o

~b

so

~b

i'0

5b t cm,',l

0, 0,

1'0

~'0

- -30~

~0

, 5'0 t ¢,.~.1

Fig. 3. HPLC tryptic peptide maps of bovine brain (B) (A) and Drosophila (D) (B) calmodulin. Calmodulin (0.5 rag) was digested in the absence (upper part) and presence (lower part) of 1 mM EDTA. 0.5 mg of protein was dissolved in 0.5 ml 0.l M NH,~HCO 3 buffer (pH 8.3) and incubated at 37°C. 50#1 of a freshly prepared 0.01% solution of trypsin were added in two portions, at the beginning of and after 3 h of incubation. The reaction was stopped after 6 h by addition of 150 /~| of 3% acetic acid. The reaction mixture was centrifuged at 13000× g and the supernatant was lyophilized. Separations were performed at room temperature on a RP-18 column (4.0 × 250 mm, 5 # m particle size, Hibar, Merck, Darmstadt, F.R.G. ) protected by a 30 mm guard column of Perisorb RP-18, 30-40/~m (Merck). A flow-rate of 1 m l / m i n resulted in a pressure of about 2000 psi = 140 bar. The primary aqueous solvent A was 0.05 M NaH2PO4-H3PO 4 buffer (pH 2,3, total phosphate concentration 0.1 M), filtered through a glass fritte G4 (Schott, Mainz, F.R.G.). After addition of acetonitrile (3%, v/v) the solution was held under helium atmosphere. The secondary solvent B was acetonitrile (Lichrosolv, Merck). The peptides were dissolved at a concentration of 3-6 m g / m l in buffer A. The gradient from 0 to 40% B in 40 rain and from 40 to 60% in 5 rain. The level of 60% was maintained for 5 min and then returned to 0% within 5 min.

calmodulin contains 11 cleavage points from 6 arginine and 7 lysine residues (see Table I), one arginine, 74, being followed immediately by a lysine , 75, and one lysine being located at the carboxy-terminus [2]). If EDTA is included in the cleavage mixture, the resulting HPLC map (Fig. 3B, lower part) is qualitatively the same up to a retention time of 35 rain. Then two major differences are observed: the large peak complex between 40 and 41 rain disappears almost corn-

pletely as well as the peak at 44.5 rain and in their place two new peaks, at 37 and 3 min, arise. This suggests that some structural domains are protected by the binding of Ca 2+ against the proteolytic attack by trypsin, and the removal of Ca ~ + leads to a conformational change which makes some new cleavage points accessible to trypsin. This is in agreement with other data indicating that upon removal of Ca 2+ bovine brain calmodulin undergoes structural changes which result in a

232 less c o m p a c t structure due to a lower helix c o n t e n t [22,23]. C o m p a r i n g the H P L C tryptic maps of b o v i n e brain and Drosophila c a l m o d u l i n w it h o u t addition of E D T A (Figs. 3a and b, upper parts), it is seen thal all peaks of b o v i n e brain c a l m o d u l i n besides that at 24.8 rain are present in Drosophila c a l m o d u l i n too, b u t Drosophila c a l m o d u l i n contains three additional peaks at 11.2, 16.2 and 26.4 min which are not present in the bovine brain calmodulin. The fact that the tryptic p e p ti d e maps of Drosophila c a l m o d u l i n show two peaks or spots m o r e than those of b o v i n e brain c a l m o d u l i n is consistent with the fact that it contains two more lysine residues (Table I). T h e effect of addition of E D T A to the cleavage mixture of Drosophila c a l m o d u l i n is qualitatively the same as in b o v i n e brain calmodulin, though quantitatively not so impressive: the d i s a p p e a r a n c e of two peaks at 40 and 44.5 min and the a p p e a r a n c e of two new peaks, at 37 and 44.5 rain (Fig. 3b), suggesting that the c o n f o r m a t i o n a l change induced by removal of C a 2 + takes place in d o m a i n s which are very similar to those of bovine brain calmodulin. T a k e n together, these results show that the two c a l m o d u l in s from these species so distant in evolution show a very high degree of h o m o lo g y . This work was s up p o r te d by the D e u t s c h e F o r sc hun g s g em ei n s ch af t (SFB 206, SFB 46), by the Stiftung V o l k s w a g e n w e r k and by the Bundesminister f~r F o r s c h u n g und T e c h n o l o g i e (01 QV 3 1 8 - Z A / V F / W R K 027515).

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