The primary structure of porcine colipase II. II. The disulfide bridges

Biochimica et Biophysica Acta, 359 (1974) 198-203

© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands BBA 36762 T H E P R I M A R Y S T R U C T U R E OF PORCINE COLIPASE II. II. T H E D I S U L F I D E BRIDGES

C. ERLANSON*, M. CHARLES, M. ASTIER and P. DESNUELLE Centre de Biochimie et de Biologie Mol~eulaire du C.N.R.S. 31, Chemin Joseph-Aiguier, 13274 Marseille Cedex 2 (France)

(Received January 11th, 1974)

SUMMARY Three disulfide bridges out of a total of five were identified in porcine pancreatic colipase II by peptic or thermolysin digestion of the intact protein followed by separation of the cystine peptides by column chromatography and resolution of the cysteic acid fragments by diagonal electrophoresis. The position of the two other bridges could not be definitely ascertained because of the inability of the enzymes employed to cleave, to a detectable extent, the chain between the adjacent half-cystines 22 and 23. The two possible versions of the bidimensional structure of colipase suggest that the molecule is formed of two loosely bound "tails" and a heavily cross-linked central "core" probably containing the structure responsible for the colipase effect.

INTRODUCTION The complete sequence of the 84 residues composing porcine colipase II is given in the preceding paper. The present paper deals with the position of the disulfide bridges linking the ten half-cystine residues of the protein molecule. The classical procedure employed for this investigation includes: (a) Hydrolysis of native colipase by pepsin or thermolysin. (b) Isolation of the resulting cystine pet~ tides by Sephadex G 50 filtration followed by SP-Sephadex chromatography or high voltage electrophoresis. (c) Separation and identification of the cysteic acid fragments arising from performic acid oxidation of the cystine peptides. METHODS Only methods not specifically described in the first paper of this series are presented.

* Present address: Department of Physiological Chemistry, University of Lund, P.O. Box 750, S-220 07 Lund, Sweden.

199

Enzymatic digestion of intact colipase by pepsin or thermolysin For peptic digestions, 50 mg of colipase with intact disulfide bridges were incubated for 24 h at 37 °C with 5 mg of pepsin in 5 ml of 5 % formic acid. According to a recently published technique [2], colipase (50 mg) and thermolysin (1 mg) were maintained for 48 h at 37 °C in 5 ml of a 0.1 M N-ethylmorpholine buffer (pH 6.5) containing 2 mM CaCI~. The reaction was stopped by adjusting the pH to 3.0 by glacial acetic acid. In both cases the cystine-containing peptides separated by column chromatography were localized by application of aliquots of the effluent on paper followed by revelation with the nitroprusside reagent of Toennies and Kolb [3]. After high voltage electrophoresis the reagent was directly applied to the paper sheet.

Diagnonal electrophoresis [4~ The peptide mixtures were applied to 3 MM Whatman paper sheets and submitted to high voltage electrophoresis in one direction at either pH 3.5 or 6.5. The cysteic acid peptide pairs resulting from exposure to performic acid vapors were resolved into their two constituents by a second electrophoresis in the other direction under identical conditions. Net chargescarried by the oxidized peptides were calculated from their electrophoretic migration [5]. RESULTS

Identification of Bridge VI-IX in a peptic digest A typical elution profile obtained by filtration through Sephadex G 25 of peptic digests of colipase II is reproduced in Fig. 1. The cystine-positive Fraction I was further purified by passage through a Sephadex G 50 column (1.5 cm × 200 cm) in 1% acetic acid. The second peak emerging from this type of column was submitted to diagonal electrophoresis at pH 3.5 and 53 V/cm for 45 min. A major cystine peptide with Asx and Thr at the amino end was separated in the first direction. This peptide yielded, in this second direction, two cysteic acid fragments with the following properties;

1.0

A230nm

A 2 8 0 nm

(~)

( ....

)

• ,

0.5

0.2 /~ • ~/

:

; -//

1 ,

~

~ .......... ~ Breaktrough volumes Z , JL

0,1

3 :

~r

,

Fig. 1. Elution profile of a peptic digest of colipas¢ (50 mg) on Sephadex G 25 fine. The column (1.5 cm × 200 cm) was equilibrated and eluted by 1% acetic acid. Breakthrough volume of the column, ! 15 ml. Fraction size, 2 ml. Flow rate, 12 ml/h.

200 (1) N-terminal Asx. Amino acid composition; Ala (0.6 mole/mole), Asx (0.6), Cy(SOaH) (1.2), Glx (0.9), Phe (1.0), Ser (1.0). (2) N-terminal Thr. Composition; Asx (1.0), Cy(SO3H) (1.2), Glx (1.3), Gly (1.4), Leu (0.6), Lys (0.8), Ser (0.7), Thr (0.8). These data are consistent with Sequence 40-45 and 61-68 in colipase and consequently they demonstrate the existence of a bridge between half-cystines VI and IX (see Fig. 4 in the previous paper for the numbering of residues in colipase). The structure of the protein in the corresponding region of the protein is: 40 41 42 43 44 45 Asn-Ser-Glu-Cys-Ala-Phe Thr-Cys-G lu-Gly-Asp-Lys-Ser-Leu 61 62 63 64 65 66 67 68 Fragment 4 0 4 5 was designated Pl in the preceding paper and served for the reconstitution of the tryptic peptide Ts. Peptides P2 and P3 employed for the same purpose were purified here from Fractions II and III by chromatography on SP-Sephadex and QAE-Sephadex.

Disulfide bridges identified in thermolysin digests A closer investigation of the other cystine peptides resulting from peptic digestion was not undertaken because of their poor migration on paper. Thermolysin peptides were generally easier to handle. As shown by Fig. 2, the filtration ofa thermolysin digest through Sephadex G 50 resulted in the clean separation of four cystinepositive fractions (I-IV) and of one peak (Fraction V) containing no cystine. The n-a1.5

A230 nm

(~)

1.0

3.4

'/

't;

0.2

"

~¢ ~

~ •

•~

~'~

~'/ 2 '

z

'

~

~

x~]

/ ~

I I

%<1

~'"

I

B~akt~gh ~ s

3

~

~

'

~

~

Fig. 2. Filtration through Sephadex G 50 of a thermolysin digest from 50 mg of native colipase. Conditions as in Fig. 1. Horizontal bars indicate the fractions pooled for further investigation (see text).

201 terial in Fraction I did not move on paper and it was discarded. The material in Fraction III was chromatographed in an SP-Sephadex column (1 cm × 20 cm) equilibrated with 1 ~ acetic acid. Elution of the column by a pH gradient from 3-5 yielded a cystine peptide which turned out later to correspond to the following region in colipase: 37

43 60--62

--67

Hence, the investigation of Fraction III merely confirmed the existence of Bridge VIIX. By contrast, a diagonal electrophoresis of Fraction IV (pH 6.5; 48 V/cm; 45 min) readily separated two cystine peptides A and B which yielded upon performic oxidation the following cysteic acid fragments: AI: N-terminal Leu; amino acid composition; Cy(SOaH) (1.2), Leu (0.8); mobility, -- 1. A2: N-terminal lie; amino acid composition; Asx (1.3), Cy(SOaH) (1.1), His (0.9), Ile (1.0); mobility, --1. BI: N-terminal Ala; amino acid composition; Ala (0.9), Cy(SO3H) (1.1), Glx (1.0), Lys (0.9); mobility, 0. B2: N-terminal Cys; amino acid composition; Ala (1.0), Cy(SO3H) (1.0); mobility, -- 1. These peptides were obviously generated by the following regions of colipase: 11 12 Leu-Cys

I Ile-Cys-His-Asn 79 80 81 82

16 17 18 19 Ala-Gln-Cys-Lys

I Cys-Ala 33 34

Thus, the existence of Bridges I-X and II-V was indicated. At this stage, three bridges out of five had been readily identified and it could reasonably be hoped that the other two were present in the still unexplored Fraction II. However, some difficulties had to be expected due to the inclusion of the four corresponding half-cystines in special sequences (Cys2z-Cys23 and Cys54-Pross-Cys~6) likely to be resistant towards most proteolytic enzymes. Fraction II was charged into a SP-Sephadex column (1 cm × 20 cm) equilibrated with a 1 ~o acetic acid. Elution of the column by varying the pH from 3-5 yielded four cystine-positive peaks. The last one could effectively be shown by analysis to contain the four expected half-cystine residues. Diagonal electrophoresis (pH 6.5, 48 V/cm, 60 min) gave three cysteic acid fragments with the following characteristics: 1. N-terminal Ser; amino acid composition; Asx (2.1), Cy(SO3H) (1.9), Glx (1.0), Gly (0.3), His (1.I), lie (0.5), Ser (0.7), Thr (0.5). 2. N-terminal Tyr; amino acid composition; Cy(SOaH) (1.3), Lys (0.9), Pro (1.0), Wyr (0.7). 3. N-terminal Cys; amino acid composition; Arg (1.0), Asx (1.0), Cy(SOaH) (1.6), Glx (1.2), Gly (1.5), lie (1.1).

202 52 53 54 55 Fragment 2 was identified to the sequence T y r - L y s - C y s - P r o . Fragments 1 and 3 were visibly still impure, but a major constituent could be discerned in each case and tentatively identified to the following sequences in colipase: 20 21 22 23 24 25 26 27 28 Ser-Asn-Cys-Cys-Gln-His-Asp-Thr-Ile and 56 57 58 59 Cys-Glu-Arg-Gly. These observations suggest that thermolysin had split the Pross-Cyss6 bond to a detectable extent, but apparently not the one linking the adjacent cystine residues Cys22 and Cys23. As a consequence, the two bridges were found to be still attached to the same fragments and their respective position ( I I I - V I I and IV-Vlll or I l l - V I I I and IV-VII) could not be definitely ascertained. Fig. 3 indicates the two bidimensional structures of colipase consistent with our present results. DISCUSSION Early investigations on the disulfide bridges in proteins made use of peptic digestion at acidic p H values in order to avoid disulfide interchanges shown to occur in the alkaline range. However, native colipase ll was found to be poorly attacked by pepsin. Thermolysin at pH 6.5, reported to yield correct results in other cases [2],

v,,~s~

09 7."~ <"~, i~..x 7;;A ~

~

~' 7,. ~<~<

<,,,

°<° ~~'~,~<~] ~# ~] <~;M ~ 2_7~7J J~~O' 7 I ~u ~

:'-j

~<,,~ I... ~

"",o Fig. 3. Bidimensional structure of porcine colipase I1 under the assumption that Residue Cys22is linked to Cys54and Residue Cys23to Cys56.On the right, the same structure under the reverse assumption (22-56 and 23-54).

203 must be also employed. A total of three bridges (I-X; II-V; VI-IX) were readily characterized in this manner, but the two remaining ones linking half-cystines III, IV, VII and VIII could not be definitely identified due to the difficult cleavage of the Cysz2-Cys23 bond by any of the enzymes employed. The possibility that this cleavage could be achieved by other enzymes under other conditions or by acid hydrolysis of the fragment containing the two bridges, was not explored. Chemical investigations in this respect will be resumed if the crystallographic studies now in progress on porcine colipase II fail to give the final answer. Moreover, the significance of the already mentioned accumulation of eight half-cystine residues in the central part of the colipase chain including about 45 residues (see previous paper) was considerably reinforced by the finding that these residues are linked together. Even before relevant information is available about the tridimensional structure of the molecule, colipase appears to be formed of two "tails" loosely bound by a single bridge (I-X) and of a heavily cross-linked central "core". This core is probably responsible for the unusual stability of colipase towards pH, temperature and water-miscible solvents. It may, for reasons given earlier, also be assumed to include the structure responsible for the colipase effect. ACKNOWLEDGEMENTS One of us (C.E.) acknowledges with thanks the provision, for one year, of a fellowship by the French Institut National de la Sant6 et de la Recherche M6dicale (INSERM). REFERENCES 1 Mayli6, M. F., Charles, M., Gache, C. and Desnuelle, P. (1971) Biochim. Biophys. Acta 229, 286-289 2 Guy, O., Shapanka, R. and Greene, L. J. (1971) J. Biol. Chem. 246, 7740-7747 3 Toennies, G. and Kolb, J. J. (1951) Anal. Chem. 23, 823-826 4 Brown, J. R. and Hartley, B. S. (1963) Biochem. J. 89, 59-60P 50fford, R. E. (1966) Nature 211, 591-593

204 VOLUME C O D I N G FOR BBA-PROTEIN STRUCTURE BBA is published according to a volume-numbering scheme that embraces all sections of the j o u r n a l : for 1974 the scheme (covering the volumes 332-374) is to be found on the inside cover of this issue. The seven individual sections are distinguished by a colour code. In addition to the colour code, each section is given its own sequential volume number. This system runs parallel to the overall BBA scheme: for the P R O T E I N S T R U C T U R E section the correspondence is indicated in the Table below. This issue is, therefore, P R O T E I N S T R U C T U R E , Vol. 359/1 or B B A - P R O T E I N S T R U C T U R E P33/1.

Parallel volume coding for BBA-Protein Structure Biochimica et Biophysica Acta Volume No.

Protein Structure Vohtme No.

Biochimica et Biophysica Acta Volume No.

Protein Structure Volume No.

Vol. Vol. Vol. Vol. Vol. Vol. Vol. Vol. Vo[. Vol. Vol. Vol. Vol. Vol. Vol. Vol. Vol.

P I (1967) P 2 (1967) P 3 (1967) P 4 (1968) P 5 (1968) P 6 (1968) P 7 (1969) P 8 (1969) P 9 (1969) P10 (1969) P I I (1970) PI2 (1970) PI3 (1970) P14 (1970) PI5 (1971) PI6 (1971) P17 (1971)

Vol. 251 Vol. 257 Vol. 263 Vol. 271 Vol. 278 Vol. 285 Vol. 295 Vol. 303 Vol. 310 Vol. 317 Vol. 322 Vol. 328 Vol. 336 Vol. 342 Vol. 351 Vol. 359 Vol. 365 Vol. 371

P18(1971) PI9 (1972) P20 (1972) P21 (1972) P22 (1972) P23 (1972) P24 (1973) P25 (1973) P26 (1973) P27 (1973) P28 (1973) P29 (1973) P30 (1974) P31 (1974) P32 (1974) P33 (1974) P34 (1974) P35 (1974)

133 140 147 154 160 168 175 181 188 194 200 207 214 221 229 236 243

--, = -: -~-: --~

~ ~ : -: ~: -~

--, :

A subscription to the P R O T E I N S T R U C T U R E section of BBA for 1974 (6 volumes) is Dfl. 480.00 plus Dfl. 42.00 postage. Back volumes (according to their P numbers) are available: rates will be supplied on request.