The preparation and characterization of water-soluble proteolipid protein from bovine brain white matter

BIOCHIMICA ET BIOPHYSICA ACTA

141

BBA 2 5 4 5 4

T H E PREPARATION AND CHARACTERIZATION OF WATER-SOLUBLE P R O T E O L I P I D P R O T E I N FROM BOVINE BRAIN W H I T E MATTER* D A V E D A T E N E N B A U M AND J O R D I F O L C H - P I

McLean Hospital Research Laboratory, Belmont, Mass., and Harvard Medical School, Boston, Mass. (U.S.A.) (Received May 3Ist, 1965)

SUMMARY

Proteolipid proteins, essentially lipid-free, have been prepared and solubilized in aqueous solutions. Earlier attempts to isolate this protein moiety usually resulted in an insoluble product; though, chromatography on silicic acid did give proteins which were soluble in chloroform-methanol mixtures. Both types, however, contained about 5 % tightly-bound phospholipid. The present method utilizes dialysis against acidified chloroform-methanol followed by dialysis against a succession of solvent mixtures of slowly increasing water content. The final product is water soluble while still retaining solubility in chloroform-methanol mixtures. Chemical analyses show it to be above 95 % protein, and to contain 1 4 - 1 6 % nitrogen, no inositol, and practically no phosphorus or neutral sugars. Upon gel filtration, the protein is excluded from Sephadex G-Ioo and shows one broad peak on G-2oo. The previously reported amino acid pattern for these proteolipid proteins is unchanged by the procedure, and like other proteolipid preparations from the same tissue, this protein is neither attacked by trypsin (EC 3.4.4.4) nor pepsin (EC 3.4.4.1).

INTRODUCTION

In a study of the nature of a protein-lipid interaction, a method was developed for the preparation of water-soluble proteolipid protein from bovine central white matter. White matter proteolipids 1 show a variation in lipid components dependent upon the method of isolation. Lipid contents range from 35-45 % in emulsion-centrifugation proteolipids 2, to 20 % in the products obtained by dialysis against organic solvents 3, and down to 5 % when prepared by silicic acid chromatography 4. Several earlier attempts have been made to obtain pure proteolipid protein. FOLCH AND LEES1 first obtained it as an insoluble residue by vacuum distillation of total lipid extracts in the presence of water. This residue contained about IO % lipid. WEBSTER AND FOLCH5 obtained a similar residue, though with approx. 5 % tightlyA b b r e v i a t i o n : ACM-solution, c h l o r o f o r m - m e t h a n o l - 12 N HCI ( 200 : i oo: i). * The d a t a in this p a p e r is t a k e n f r o m a thesis to be s u b m i t t e d to the Division of Medical Sciences of H a r v a r d U n i v e r s i t y in p a r t i a l fulfillment of the r e q u i r e m e n t s for the degree of D o c t o r of Philosophy,

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bound lipid, by treating total lipid extracts with salts at an alkaline p H in a biphasic c h l o r o f o r m - m e t h a n o l - w a t e r system. The same results were obtained in completely aqueous media in which total lipids were suspended by emulsification (TENENBAUM AND FOLCH, unpublished results). The work of MATSUMOTO et al. 4, chromatography on silicic acid, yielded soluble proteolipids having the highest protein content. The largest protein fraction, representing 4 ° % of the total protein, could be removed from the column only by elution with slightly acidified organic solvents. When the solvent concentrations of this fraction were adjusted to chloroform- m e t h a n o l - w a t e r (8:4:3), 20 % of the protein in the fraction (about 8 % of the total protein) became soluble in the upper or aqueous methanolic phase. All other fractions went quantitatively into the lower phase. This observation led to the idea that it might be possible to obtain proteolipid protein with very little lipid which would also be soluble in aqueous solvents. Such a preparation would have few or possibly just one type of lipid-protein linkage and would further be suitable for enzymatic and physical chemical studies of the protein moiety itself, studies which were previously unfeasible. The upper phase, as described above, was dialyzed against successive solvent mixtures of gradually increasing water content. The final retentate was an aqueous solution of protein with the same amino acid composition as that of the starting material. Although this was the desired product, the yield was small. Further attempts to find an alternative method led to the present report. MATERIALS AND METHODS Pronase, trypsin (EC 3.4.4.4), and pepsin (EC 3.4.4.1) were purchased from Calbiochem. The following analytical methods were used: total neutral sugars by a modification of the SORENSEN AND HAUGAARD method6; free amino groups by a modification of the method of STEIN A~,'D MOORET; total phosphorus b y a modification of the SPERRY methodS; total nitrogen by de Arnold-Gunning methodg; and protein by the method of LOWRY et al. 1°. Amino acid analyses were performed on the automatic analyzer according to SPACKMAN, STEIN AND MOORE11. Thin-layer chromatography was done on silica gel-G, neutral plates, according to SKIPSKI et al. ~2. Extent of enzymatic proteolysis was determined with a ninhydrin reagenff. RESULTS Preparation of water-soluble proteol@id protein LE]3ARON et al. 13 showed that tightly-bound phosphoinositides 14 could be removed from insoluble proteolipid proteins by extraction with slightly acidified solvents at room temperature. We used the same solvent, 2 0 0 : I 0 0 : I of chloroformm e t h a n o l - I 2 N HC1 (ACM-solution) on soluble proteolipids in a dialysis system in order to keep the protein in solution while separating free lipids and to allow the use of various solvents. Thus, the present method represents a simple extension of the method of LEES et al. 3. Biochim. Biophys. Acts, 115 (1966) 141-147

143

WATER-SOLUBLE PROTEOLIPID PROTEIN

A washed total lipid extract 15 was diluted with one half its volume of chloroform and concentrated under vacuum to a final solute concentration of about 9 %, or emulsion-centrifugation proteolipids 2, with elimination of ether and ethanol extractions, were dissolved in chloroform-methanol (2: I) to a concentration of 2 %. 32

30 28 26 24 22

.~ 20

;~L6 z

o

14

8

I

2

3

4

5

6

7

8

g

10 II

12 13

DIFFUSATES

Fig. i. Amount of total solids and of phosphorus removed from crude emulsion-centrifugation proteolipids b y successive dialysis against chloroform-methanol (2: I) (No. 1-7) followed by dialysis against acidified c h l o r o f o r m - m e t h a n o l (No. 8-13): O - - O , % of initial phosphorus; & - - & , % of initial total solids. Starting material refers to crude emulsion-centrifugation proteolipids. Data is from a single experiment.

Either solution is dialyzed against chloroform-methanol (2:1) with daily changes of solvents 3. When the rate of lipid loss from the retentate approaches a constant (Fig. I), the sac is transferred to ACM-solution. This takes approx. 7 days if the outer to inner volume ratio is between 5 and IO. With the same volume ratios, dialysis against ACM-solution requires 6 days and removes the remaining phospholipid. From Fig. I it can be seen that if one starts the dialysis procedure with crude proteolipids, 58 % of the phosphorus and 34% of the dry weight are removed in the chloroform-methanol (2:1) dialysates. ACM-solution removes 39 % and 15 %, respectively. The material remaining in the sac contains 0.05-o.09 % phosphorus and is shown by chemical analysis to be almost identical to the final water-soluble product. Thin-layer chromatography of the dialysates (Fig. 2) shows a variety of lipids in the chloroform-methanol (2 : I) dialysates. Only one major spot and one faint spot are seen in the ACM-solution dialysates, presumably representing phosphoinositides and phosphatidylserine, respectively. The entire dialysis system is changed from ACM-solution to water by the succession of outer solvent mixtures shown in Table I. I t is extremely important that Biochim. Biophys. Acta, 115 (1966) I 4 I - I 4 7

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the minimal lengths of time given in the table be taken so as to permit the attainment of dialysis equilibrium. If equilibrium has not been reached before changing solvents, a two-phase system might form transiently, a development which favors precipitation of the protein.

Fig. 2. T h i n - l a y e r c h r o m a t o g r a p h y of s t a r t i n g material, diffusates, a n d p r o d u c t s : 4 ° 60 Itg- Developing s p r a y was a m m o n i u m n l o l y b d a t e - p e r c h l o r i c acid. I, total lipid e x t r a c t ; 2, crude e m u l s i o n c e n t r i f u g a t i o n proteolipids; 3-9, c h l o r o f o r n l - m e t h a n o l (2:1) diffusates; lO-15, ACM-solution diffusates; 16, water-soluble proteolipid. This s p o t s h o w e d no inobfle lipid a n d gave no blue color after spraying.

Dialysis tubing is always washed thoroughly with water and then with chlorof o r m - m e t h a n o l (2:1) before use. The cellulose membranes become quite brittle and weak in organic solvents and should be changed; twice is sufficient. This is conveniently done following dialysis against chloroform-methanol (2:I) and following ACM-solution. Under conditions given above, a clear protein solution in water is obtained with a final concentration of about o.I % which can easilv. be .concentrated . . to 0.5 % by simply allowing evaporation from the sac.

Characteristics of the product Chemical analyses on several preparations gave the following results: 95-97 To protein, 0.04-0.09% phosphorus, 14-16 % nitrogen, 0.08% galactose, and o-o.ol 4 % inositol. Relative ratios of amino acids found after acid hydrolysis are unchanged in these preparations as compared with other white matter preparations, excepting Biochim. Biophys. Acla, 115 (1966) 141-147

WATER-SOLUBLE

PROTEOLIPID

145

PROTEIN

serine whose decrease is to be anticipated due to the loss of phosphatidylserine % (Table II). The E i..... at 278.5 mt~ ranges from 16. 9 to 22.8. Gel filtration on Sephadex indicates heterogeneity. The material is completely excluded from G-Ioo and a large percentage comes out in the void volume of G-2oo TABLE

I

SOLVENT

CHANGES

NECESSARY

TO

TRANSFER

CHaOH

62 52 41 31 24 17 ii 6 2

33 38 45 5° 52 54 54 54 53 5° 4° 25

H~O 5 io 14 19 24 29 35 4° 45 5° 6o 75 IOO

I[

AMINO

ACID

COVERED

COMPOSITIONS

PER

IOO

No determinations

Amino acid

Arginine Histidine Lysine Aspartic acid Glutamic acid Serine Threonine Half cystine Methionine Proline Glycine Alanine Valine Leucine Isoleucine Phenylalanine Tyrosine

MOLES

ACM-sOLUTION

TO

WATER

12 12 12 12 12 12 12 12 I2 12 9 9 48 (with = six changes)

Inner volulne :outer volume TABLE

FROM

Equilibration time (h) *

Solvents (parts per hundred) CHCI a

PROTEIN

OF OF

=

i : 5 ; temperature

VARIOUS TOTAL

PROTEOLIPID

AMINO

w e r e m a d e of t r y p t o p h a n

Water-soluble proteolipid protein 2.6 i .9 4,3 4,2 6,o 5.4 8.5 4.2 1,7 2.9 lO. 3 12. 5 6.9 i i. i 4-9 7.9 4.7

: 2 3 - 2 5 °.

PREPARATIONS

EXPRESSED

AS

MOLES

RE-

ACID

or amide nitrogen,

Silicic acid chromatographed proteolipid* 3.0 2-4 4.9 4.5 6. 5 5 .0 7.7 3.4 1.5 3 .0 lO. 7 12.1 7.0 I i. i 4 .8 7.7 4.5

Emulsioncentrifugation proteolipid 2.6 2.3 4 .0 4 .2 5.8 7.4 8.3 2.5 1.6 3.o lo.8 I2.2 6.8 lo.8 4.9 8.o 5. i

* T h a t f r a c t i o n w h i c h is e l u t e d w i t h a c i d i f i e d s o l v e n t s a n d w h i c h p a r t i t i o n s phase in the chloroform-methanol-water (8 : 4 : 3) s y s t e m . ** L a r g e d i s c r e p a n c y is d u e t o t h e p r e s e n c e of p h o s p h a t i d y l s e r i n e .

into the upper

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Because there is only one peak even in this latter case, a continuous spectrum of micellar sizes is presumed, all above IOOOOOin molecular weight. Optical rotatory dispersion studies on emulsion-centrifugation proteolipids in chloroform-methanol show them to have a high helix content. The protein as prepared in the present report shows a large diminution in helical content (R. ZAND, unpublished results). However, it should be noted that these preliminary studies were carried out on lyophilized material redissolved in o.ooi N HC1, a solvent which generally favors unfolding of the polypeptide chain. The protein in a o.I % solution is neither precipitated by ethanol nor by any concentration of ammonium sulfate, a finding which suggests a low molecular weight for its monomeric species. However, this might be related more to its unusual amino acid composition than to its molecular size; it has a very high content of neutral amino acids. The material can be dried by lyophilization, and the dried protein is soluble in dilute acid for two to three days. Afterwards, it is completely soluble only in acidic urea or acidified chloroform-methanol mixtures (both p H 3 to paper), and retains these solubilities even after four months of storage at 4 ° . It is neither soluble in dilute salt solutions nor in 4 M guanidine hydrochloride even at low pH which is surprising because of its ready solubility in urea solutions. The protein is precipitated at alkaline p H ' s and can be redissolved by acidifying below p H 5. When allowed to remain in basic solutions, it gradually changes to a form which is not dissolved upon reacidification. Freezing also causes irreversible precipitation. In a two-phase system composed of c h l o r o f o r m - m e t h a n o l - w a t e r (S : 4:3), the water-soluble material goes quantitatively into the upper phase. The protein is attacked b y pronase. Previous insoluble preparations are, however, split by this enzyme to the same extent. (L. MOKRASCH,unpublished results). It is resistant to trypsin and pepsin. DISCUSSION

The present preparation of proteolipid protein now makes it possible to study the protein moiety and its physical and chemical changes when brought into contact with lipids. It also enables the application of physical chemical techniques developed for aqueous protein solutions. The particular solubility characteristics of proteolipids had originally led to the assumption that the protein moiety was covered by a lipid shell16,17. The insolubility resulting from the removal of most of the lipids lent credence to this idea 16. When MATSUMOTO et al. 4 obtained, by chromatography, proteolipids containing less than 5 % lipids, this original model had to be discarded, and it became necessary to assume that the protein itself had a conformation that exposed a prevalence of nonpolar groups. The change to water solubility would therefore require an alteration in the conformation so that polar groups, that had been covered in the original proteolipid, are exposed. The removal of the tightly-bound polyphosphoinositides m a y play some part in this hypothetical conformational change. WEBSTER AND FOLCI-I5 reported that protein-lipid linkages in proteolipids were destroyed by treating them with various salts at an alkaline p H in a biphasic system. Biochim. Biophys. Acta,

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WATER-SOLUBLE PROTEOLIPID PROTEIN

147

The extent of "splitting" was computed from the weight of insoluble protein which separated at the interface. The present study demonstrates that proteolipid protein itself undergoes changes at alkaline pH which lead to its precipitation. In the "splitting" experiments the presence of a two-phase system is extremely important. Proteins have a tendency to accumulate at liquid-liquid interfaces, and this tendency is greatly increased by the presence of salt TM. Therefore it is possible that "splitting" is merely a reflection of a change in the protein conformation brought about by an increase in pH and taking place in an environment which favors irreversible protein precipitation with parallel release of associated lipids. Proteolipid protein obtained by earlier procedures had been found to be resistant to the action of trypsin, pepsin, papain (EC 3.4.4.~o), and erepsin TM. Indeed, the only enzyme found to attack it successfully thus far is pronase. The present product which is resistant to trypsin and to pepsin demonstrates that whatever change in conformation is at the basis of the change in solubility is not effective in changing susceptibility to enzymatic proteolysis. Attempts to shorten the preparative procedure while maintaining high yields and the integrity of the product have so far proved unsuccessful. ACKNOWLEDGEMENTS

This work has been aided by Grant NB oo13o from the National Institute of Neurological Diseases and Blindness, National Institutes of Health, One of the authors (D.T.) is a Predoctoral Fellow of the National Institutes of Health, U.S. Public Health Service, 1962-1965. REFERENCES i 2 3 4 5 6 7 8 9 io ii 12 13 14 15 16 17 18 19

J. FOLCH AND M. LEES, J. Biol. Chem., 191 (1951) 807. J. FOLCH, G. I~. WEBSTER AND M. LEES, Federation Proc., 18 (1959) 898. M. ]3. LEES, S. CARR AND J. FOLCH, Biochim. Biophys. Acta, 84 (1964) 464 . M. MATSUMOTO, R. MATSUMOTO AND J. FOLCH-PI, J. Neurochem., 11 (1964) 829. G. R. WEBSTER AND J. FOLCH, Biochim. Biophys. Acta, 49 (1961) 399. M. SORENSEN AND G. HAUGAARD, Biochem. Z., 260 (1933) 247. W. H. STEIN AND S. MOORE, J. Biol. Chem., 176 (1948) 367. W. SPERRY, Ind. Eng. Chem., Anal. Ed., 14 (1942) 88. J- P- PETERS AND D. D. VAN SLYKE, Quantitative Clinical Chemistry, Vol. 2, Williams and Wilkins, Baltimore, 1932, p. 518. O . H . LOWRY, N. J. I~OSEBROUGH, A. L. F&RR AND R. J. RANDALL, J. Biol. Chem., 193 (1951) 265. D. H. SPACKMAN, W. H. STEIN AND S. MOORE, Anal. Chem., 30 (1958) 119o. V. P. SKIPSKI, R. F. PETERSON AND M. BARCLAY, J. Lipid Res., 3 (1962) 467 . F. N. LEBARON, G. HAUSER AND E. E. l~UIZ, Biochim. Biophys. Aeta, 60 (1962) 338. E. T. PRITCHARD AND J. FOLCH-PI, Bioehim. Biophys. Acta, 7° (1963) 481. J. FOLCH, M. ]3. LEES AND G. H. SLOANE-STANLEY, J. Biol. Chem., 226 (1957) 497. J. ]3. FINEAN, J. N, HAWTHORNE AND J. D. E. PATTERSON, J. Neurochem., i (1957) 256. L. L. UZMAN, Arch. Biochem. Biophys., 76 (1958) 474R. ~ . PORTER, Biochem. J., 53 (1953) 32°. J. FOLCH-PI, Exposes Ann. Biochem. Med., 21 (1959) 81.

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