..),)

AcTA BtocHIMIcA PoLoNIcAVol. 35

THIS PAPER

l988 No2

IS DEDICATED TO THE MEMORY OF PROFF.SSORw. MEJBALIM_KATZENELLENBOGEN

ALEKSANDER F. SIKORSKI**, ARKADIUSZ KOZUBEK and JAN SZOPA

PROTEOLYSIS OF SPECTRIN BY TRYPSIN AND PRONASEIN TI{E PRESENCE OF PHOSPHOLIPID SUSPENSIONS*

Institute of Biochemistry, Wrocław University,Tamka 2; 50-137 l|roclaw, Poland

Received 15 June, 1987; Revised 30 September, 1987

The effect of phospholipid suspensions on the proteolysis of isolated spectrinwas examined by SDS-polyacrylamide gradient gel electrophoresis. Proteolysis ofspechin in the membranes by trypsin and pronase was al§o sfudied. It was foundthat electrophoretic patterns of spectrin fragments were influenced by the plesenceofthe suspension prepared from phosphatidylethanolamine:phosphatidylseńne (60:40)mixture and of phosphatidylcholine. Qualitative changes in the proteolytic patternsobtained after proteolysis of spectrin by pronase in the presence of phosphatidyl-choline suspension were observed. The changes in the sensitiviĘ of spectrin towardsproteases result probably from changes in the accessibi|iĘ of some peptide bondsupon the interaction of this extrinsic protein with phospholipids.

The erythrocyte membrane skeleton is composed mainly of spectrin andactin, and also of band 4.1 protein (for a review see e.g. refs. l1,2]).Spectrin, a high molecular weight protein, consists of two nonidenticalsubunits,.,a (M,:240000) and l](M.:220000) which associate with eachother to form (r/) l00 nm long heterodimer. Two heterodimers associatehead to head to form a 200 nm long heterotetramer which is supposedto be the functional form of this protein in the membrane [3, 4]. Frag-mentation of spectrin molecule into intermediate molecular weight pro-teolytic fragments called "chemical domains" of spectrin facilitated extensivestudies on the primary structure of this protein [5, 6].

Hereditary changes in the region of rI domain of spectrin seem to beresponsible for the altered shape and deformability of erythrocytes, Thisdefect was also reported to cause impaired association of spectrin hetero-dimers and instability of erythrocyte membrane skeletons ([| and referencestherein).

* Supported by the Polish Academy of Sciences under the Proiect CPBP 04.0l.** Author to whom correspondence should be addressed.

A F. SIKORSKI and OTHERS 1 988

Ankyrin (band 2.1 protein) was found to be the main receptor site forspectrin [8]. This protein forms a complex ofl - 5 x l07 M ' 1S1; wittr spectrin and also wband 3 protein (affinity constant 1.3 - 2 x l07

membrane skeleton to the intrinsic membrane domain.There are also indications that spectrin interacts with phospholipids of

the inner leaflet of membrane bilayer, playing an essential role in main-

Il4, l5].T,he aim of the present study was to compare the polypeptide patterns

resul rin with trypsin and pronase in the absence

or p lipid suspónsions in order to test'whether

thę be changed. The proteolysis of spectrin

in erythrocyte membrane was also analysed.

MATERIALS AND METHODS

on Sepharose CL 48 column (1.8 x 50 cm) equilibrated lł,ith 5 mM phosphatt

buffer, pH 7,5, containing 1 mM NaN3, 0.1 mM EDTA and 0.1 mM 2

-mercaptoethanol. The second peak containing pure spectrin dimer was usec

for the experiments,Phospholipid suspensions were prepared by evaporating chloroform fron

an appropriate volume of phospholipid solution and then shaking the conten

with 0.1 mM sodium phosphate buffer, pH 7.5 (the concentration of phos

pholipid was 2,].). The suspension was heated to 60 C for 0.5 min anc

Śhuk* for 15 min at room temperature. An appropriate volume of sucl

a suspension (large multilammellar vesicles) was added to the incubatior

mixture to obtain the desired lipid to spectrin ratios.Proteolysis of spectrin with TPCKl-treated trypsin and pronase wa|

l Abbrevitrtions used:phosphatidylethanolarn i ne :

methyl ketone.

SDS. sodiun dodecyl sulphate; PC. phosphatidylcholine; PE

PS. phosphatidylserine; TPCK. p-tosyl-L-phenylalanine chloro

]

rt3

n

Ą

t-

l\

te

)_

d

n1t

Vol. 35 PROTEOLYSIS OF SPECTR|N

carried out at 25 C for 60 min. Samples containing dimeric spectrin in0.1 M phosphate buffer, pH 7.5, with or without 1.0 M NaCl,0.1 mM 2--mercaptoethanol and l mM CaCl2 were pre-incubated with or withoutliposomes for 15 min. Then l0 itl of TPCK-treated trypsin (Worthington)dissolved just before use in 0.1 M sodium phosphate buffer was added toobtain trypsin to spectrin ratio of l: l00. The reaction was completed bythe addition of phenylmethylsulphonyl fluoride to the final concentrationof l00 pg/ml. Then 20 pl of the reducing reagent (50 mM Tris/HCl, pH 8.0,

-§ mM EDTA, l2.5')ź SDS, 25']; 2-mercaptoethanol) was added and thesample was boiled for 5 min, When proteolysis was carried out in thepresence of l M NaCl the sample was diluted with 4 volumes of waterprior to the electrophoresis and the increased volume (about 20 - 60 pgprotein) was applied directly onto the gel. The treatment of spectrin withpronase (Sigma) was performed only at high salt concentration withoutthe addition of CaCl2. The pronase to spectrin ratio was also .1: l00. Thetreatment of erythrocyte ghosts with TPCK-trypsin and pronase was alsoperformed at 25'C for 60 min at the protease to membrane protein ratiosfrom l:20 to l:l00 in 0.1 M NaCl and 0.05 M sodium phosphate buffer,pH 7.5. Electrophoresis was performed in the discontinuous Laemmli [1|system, in gradient of the polyacrylamide gel concentration of 6 - 20')| or4 - 2Vź. Staining of polyacrylamide gels following electrophoresis was per-formed after the gels had been fixed in 5'1.; sulphosalicylic acid containing10"i, trichloroacetic acid and washed with 30l'." metbanol in l0'lj aceticacid. Freshly prepared 0.25'),; Coomassie Brilliant Blue R-250 in 30'l." methanoland l0']" acetic acid was used. The quantitation of fractions was performedby weighing the peaks cut off from the densitograms recorded at 600 nm(Pye-Unicam SP-l800). Each scanning was performed at least twice. Thefollowing molecular weight standards (pharmacia Fine chem.) were used:phosphorylase B - 94 000. bovine serum albumin - 67 000. ovalbumin --,13000, carbonic anhydrase - 30000, soybean trypsin inhibitor - 20000, u-lact-albumin - 14 400. The proteolysis of spectrin in the membranes was visualizedafter transfer of the proteins separated in polyacrylamide gel electrophoresisonto nitrocellulose filter paper (Schleicher and Schuell 0.45 !m) accordingto Towbin et al. [l8]. The reaction with rabbit anti-spectrin antibodies(serum of rabbits immunized with spectrin, diluted l:200) and stainingwith goat antirabbit IgG (Bio-Rad) coniugated to horseradish peroxidasewith the use of 4-chloro-1-naphtol (Bio-Rad) was performed as describedpreviously Il9].

Rabbit antispectrin antiserum was obtained by consecutive subcutaneousinjections of spectrin emulsified with complete Freund's adjuvant followedby spectrin emulsified with incomplete Freund's adjuvant at multiple sitesinto rabbits.

phosphatidylserine and phosphatidylethanolamine from bovine were from

73

I

s-

Ld

:h

)n

Et]_

l 98874 A F. SIKORSKI and OTHERS

Koch-Light and gave single spots in thin-layer chromatography on silicagel plates (Kieselgel 60, Merck) in chloroform: methanol: water; 65:25:4.Phosphatidylcholine (Fluka) was additionally purified according to Banghamet al. |20l and tested for purity by thin-layer chromatography as aboveusing dimirystoylphosphatidylcholine (Avanti) as a standard.

RESULTS AND DISCUSSION

The electrophoretic patterns of polypeptides obtained after proteolysisof purified spectrin by trypsin in the presence and absence of phospholipidsuspensions are presented in Plate l. Table l shows the relative molecularweight values of spectrin fragments obtained in the absence (Plate l) andin the presence of 1.0M NaCl (Plate 2). The data for the fragmentsare in good agreement with the data for "chemical domains" of spectrinobtained by others [5].

Table l

Spectrin fragments obtained as a resull of limited proteolysis of spectrin by trypsin at 25'C

The nomenclature of "chemical {"?^!!r":':^rtaken from Speicher et al. |5].

FractionNo.

Proteolysis in0.1 M phosphate

buffer

Proteolysis in0.1 M phosphatebuffer containing

1.0 M NaClchemical domains

M, M,+sD

Illa2

2aJ

3a4

5

67

8

Ea

80 00074 00064 000

45 000

40 000

32 000 - 34 00030 00028 00022 0o0

l l00 00084 000+ l 20072 000+ 600ó4400+ 33059 500l 35048 l00+ l 300

43 800+ l 60040 l00+ l 200

33 800+2 l0030 300+ l 90025 800+ I 600l8 500+ 1 l0015 800+ l 200

r (T100). P (Tl l0)r I (T80)

P lV (T74). l | (T74)p II (T65)

p lv (T52)

P II (T52). a IV (T52)e I(T50)c III (T52)a II (T46)p Iv (T4l)a IV (T4l). a V (T4l)p II (T35)

B II (T33). B IV (T30)p IV (T28)p I (Tl7)B I (T|2)

Vol, 35 PROTEOLYS|S OF SPECTRIN 75

11a2,456?8

7ą *{n|

1a2av:'atł,6788a

edcbo

4*d§§f ffi

-" ,3ń

-Za

Plate 2. SDS-PolYacrYlamide ge| electrophoresis of spectrin fragments generated in the proteo-lysis of isolated spectrin by TPCK treated trypsin in the p."r"n." of 1 M NaCl. other

conditions and sample content as in plate l and in Materials and Methods

-9Ę-67

-Ą3-30

dcbo

A. F. slKoRsKl and oTHERs 1988

ThecontentofindividualpolypeptidefractionsdeterminedfromtheareaofscannedpeaksispresentedinTable2'Theircontentintherelation

in Plate 2 and Table 3, The pattern

from those obtained in the absence

Table 2

Conlenl of individual polvpeptide fractions r,,htoinetl on t, l/s

'r;r:;i;;;r"ir;; thi p,oi.,oiy,i, by trvpsin _in lhe presence or ns,

S,/Sr-the ratio or ttre'|artióubr'peak u,_"u': the are , i:§"/St -

tllg ląLlU Ur lrlv Pcr!rv9,-^ r- _ __^l l

t*o irrd.p.nd.nt experiments are shown, Each gel was scanned a! lgdJt

Proteolysis in the presence of

liposomes prepared from PCProteolysis in the presence of

liposomes pr.par.d f.om PE/PS

Protein:lipid molar ratio

l :1 900

. S,/S,l: l 900

]; S-/Sl39.8ż.5 0.063

5.1 0.13

6,8 0. l7l0.1 0,ż5

5.,7 0.1429 .| 0.,73

41.02.7 0.0664,6 0.11

6.1 0.15

1l .3 0.28

4.9 0.21

ż9,6 0.72

39. 1

2.7 0.069

5.4 0. 14

8.2 0,ż1

11.5 0.29

8.4 0.21

n.6 0.60

35,45.9 0.17

5.3 0.15

9.0 0.25

11.9 0,34

l0.0 0.28

25.I 0,7l

ż8.91.9 0.066

6.6 0,238,2 0.28

16.5 0.57

12.5 0.43

25.5 0.88

Vol, 35 PROTEOLYSIS OF SPECTRIN

Table 3

Content of individual polypeptide fractiotts obtained on eleclrophoresis of the spectrin fragmenlsresulting /rom the proteolysis of spectrin lrypsin in 1.0 M NaCl in the presence or absence

of phospholipid yesicles.S"/SI the ratio of the particular peak area to the area of ąI domain. other details in

Materials and Methods and in legends to Table 2 and l.

Fraction

Proteolysiswithout

liposomes

'(, S.,S;

Proteolysis in the presence ofliposomes prepared from PE/PS

Proteolysis in the presence ofliposomes prepared from PC

Protein:lipid molar ratio

l: l 900

" S,/Sll :950n;

S*/SI

l 1900( S*/S

l:950'/; s"/sl

l1a

2+2a3ł3a

45+ó

7

8+8a

12 87

8.64 0,67l8,30 |.42,7.90

0.ó0|2.20 0.95l3.20 l ,03

15.43 1.20

l 1 .51 0.89

9.978.79 1.07

21.78 2.4012.16 1.34l0.90 1.201 l .89 1.3ll5.4l 1.69

8.96 0.99

l 1.609.78 0.88

21.3I 1 .91

l0.9 1 0.9810.44 0.93l 1.59 1.04

16.43 1.479.57 0.86

11.178.12 0.76

20.53 1.69

l0.39 1.0612.5| 1.0,7

14,16 0.9ó16.44 L546.69 0.62

l 1.51

9.22 0.8020.30 1,769.30 0,819.93 0.8612.30 1.07

17.30 l ,6010. 18 0.88

domain is also reduced, which may be due to its cleavage to the fragmentof M,'14000 [24]. Quantitative changes observed after proteolysis of spictrinin the presence of phospholipid suspensions (plate 2 and Table 3) in thepresence of l M Nacl were less significant than at lower ionic strength(Plate 1 and Table 2). However, an increase in the content of fragmentsof intermediate relative molecular weights namely of M, 62 000. 59 500,48 000 and 44 000 was observed (Table 3). A rather unexpected resultwere the changes in the polypeptide patterns obtained aftei proteolysisin the presence of phosphatidylcholine which has been shown to interactrvith spectrin rather weakly as revealed by other techniques |14,15,25].The existence of such an interaction could also be inierred from theobsen,ation of polypeptide patterns following the proteolysis of spectrin bypronase in the presence of this phospholipid suspension (plate 3). whenproteolysis was carried out in the presence of the suspension preparedfrom the mirtltre of phosphatidylethanolamine and phosphatidylserine theamount of undigested spectrin was increased (Plate 3, lane d)..In thepresence of smaller amounts of this suspension or in the absence of phos-pholipids large amounts of low molecular weight polypeptides and poly-peptides of M, of about 65 000 were present (Plate 3, lane b, e). tn tńepresence of phosphatidylcholine suspension (plate 3, lanes a and c) a sub-stantial inhibition of proteolysis and generation of a series of fragmentsof intermediate molecular weight took place. The results similarly to thoseobtained after proteolysis of spectrin by trypsin would point to the restricted

l 98878 A, F SIKORSK| and OTHERS

9ą67ł33CIż0

edboplate 3. SDS-polyacrylamide gel electrophoresis ol spectrin liagments generated in the Proteo-lysis of isolated spectrin by pronase. Proteolysis was carried out in 0.1 M sodium phos_

phate buffer, pH 7.5 containing 1.0 M NaCl. Lanes (a) and (q) proteolysis in the presence

of phosphatidylcholine vesicles; (a), lipid to protein molar ratio 950; (c), lipid to protein

ratio l 900; (b), and (d). proteolysis carried out in the presence of vesicles prepared from

the mixture of phosphatidylethanolamine and phosphatidylserine; (b), lipid to protein molar

ratio 950; (d). lipid to protein molar ratio 1 900; (e), proteolysis carried out in the absence

of phospholipid vesicles

accessibility of certain peptide bonds in the presence of phosphatidylcholineand phosphatidylethanolamine/phosphatidylserine vesicles. The possibility ofinteraction of spectrin with phosphatidylcholine vesicles is a rather un-

expected result, as there are no data in the literature concerning the specilrcityof binding of this lipid to spectrin [l]. May be certain regions of spectrin

bind to the vesicles without inducing large changes in spectrin confolmationwhich would be manifested e,g. in changes of intrinsic fluorescence ofspectrin. In the case of both protease preparations the immobilization ofthe enzyme on the surface of phospholipid vesicle by ionic interaction can

be excluded because of the high salt concentration. However, the behaviourof pronase should be studied in more detail. It should be added that ourpreliminary results suggests a possible interaction of isolated erythrocytemembrane cytoskeletons with sonicated vesicles prepared from different phos-pholipids including phosphatidylcholine (Sikorski & Zieliński, unpublished).

The above presented experiments suggest that proteolytic patterns becomealtered in the presence of phospholipid suspensions. Thus it seemed ofinterest to compare the results with those obtained by proteolysis of spectrinbound in a natural membrane, i.e. the erythrocyte ghosts. The polypeptidesderived from erythrocyte ghost treated with proteases following gel electro-phoresis were blotted onto nitrocellulose paper and visualized by stainingwith anti-spectrin antibodies (Plate 4). When red cell męmbranes were treated

Vol, 35 PROTEOLYSIS OF SPECTRIN

with trypsin. a considerable amount of undigested spectrin remained (Plate 4,lanes d and e), Also a higher molecular weight fragments of M, of l00 000 -- l20 000 were present in substantial amounts, All three bands of this regionexhibit a similar intensity of the stain in contrast to Coomassie Blue stainedpatterns obtained after proteolysis of purified spectrin by trypsin in thepresence of high salt concentration (Plate 2 lane a). It should be notedthat fragments of M, be|ow 25 000 reacting with antispectrin antibodieswere absent in the electrophoregrams obtained after treatment of erythrocytemembranes with both protease preparations, although such fragments derivedfrom other membrane proteins were present in rather large quantities(Plate 4 lanes f and h).

The patterns obtained after the digestion of spectrin in the membranesby pronase are presented in Plate 4 (lanes a - c). No undigested spectrinwas found and about twenty polypeptides of large to intermediate molecularweight were present. The accumulation of polypeptides of M, about 60 000 -

- 65 000 was observed similarly as in the case of treatment of purifiedspectrin with pronase. These fragments seem to be the most resistant toproteolysis by pronase (Plate 4. lane a).

When the reactivity of the antiserum was tested with erythrocyte membraneproteins separated in the SDS-polyacrylamide gradient gel no other poly-

Plate 4. Proteolysis of spectrin in erythrocyte ghosts by pronase at enzyne to lnetnbraneprotein ratio of (a) l:20. (b) 1:50. (c) and (0 l:l00; and by trypsin at enzyme tomembrane protein ratio of (d) and (h) l:50, (e) l:l00. About 30 Fg of membrane proteinwas subjected to SDS-polyacrylamide gel electrophoresis. Proteins were translerred electro-PhoreticallY onto nitrocellulose filter paper and incubated with antispectrin rabbit serumfo|lowed by goat anti-rabbit lgG coniugated to horseradish peroxidase (lanes a - e). Lanes (f)and (h) stained with Ponceau S. (g) stained with Ponceau S molecular weight standards

79

rl9f8dtbo

80A. F. SIKORSK| and OTHERS 1988

peptides except spectrin bands (a and P) were found to react with anti-].p.",.in antibodiós. No reaction of non_immune rabbit Serum with erythro_

cyte membrane polypeptides was observed (not shown). The efficiency of

tńe transfer of the fragments was checked by staining the gel with Coomassie

Blue; only little of undigested spectrin might have been left in the gel.

Diverse reactivity of antibodies with particular fragments would also affect

the results. In the case of antibodies used in this study, no differences

in the reactivity toward individual fragments of isolated spectrin digested

by trypsin were observed (not shown),

The data discussed above indicate that the interaction of spectrin with

phospholipid vesicles and with the męmbrane affects the accessibility of

i".tuio peptide bonds to proteases, It should be noted that the possibility

of interaciion of spectrirl with phosphatidylcholine vesicles has not been

reported earlier. Thi nature of this interaction should be further explored,

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l. Haest, c. w. M, (1982) Interaction between membrane skeleton protein and the intrinsic

domain of the erythrocyte membranę. Biochim. Biophys. Acta, 694,33l - 360.

2. Sikorski, A. F. (1984) Erythrocyte spectrin and its analogues in nonerythroid cells. Posl.

Biochem.. §. 409 - 434.

3. Shotton. D, M.. Burke. B. E. & Branton. D. (1979) The molecular structure of human

erythrocyte spectrin. J. Mol. Biol.. 131.303-329.4. Byers, T. J, & Branton, D. (1985) Visualization of the protein associations in the erythro-

cyte membrane ske]eton. Proc. Natl. Acad. Sci. U.S.A.. 8ż. 6153,615'7.5. Speicher, D. W., Morrow, J. S.. Knowles, W. J. & Marchesi, v. T. (1982) A structural

model of human erythrocyte spectrin, Alignment of chemical and functional domains,

J. Biol. Chem., 257. 9093 - 910l.6. Speicher. D. W. & Marchesi. V, T. (1984) Erythrocyte spectrin is comprised ol many' homologous triple hetical segments. Nature (London),3ll. 177- 180.

,7. Lawler, J,, Palek, J., Liu, S_C.. Prchal, J. & Butler, W. M, (1983) Motecular hetero_

geneity of hereditary pyropoikilocytosis: Identification ol the second variant ol the

spectrin a-subunit. Blood, 62. 1l82 - 1198.

8. Bennett. V, & Stenbuck. P, J. (1979) Identification and partial purification ol ankyrin.the high affinity membrane attachment site for human erythrocyte spectrin. J. Biol.Chem.. 254, 2533 -254l.

9. Bennet. V. & Stenbuck. P. J. (l979) The membrane attachment protein lor spectrin

is associated with band 3 in human erythlocyte membranes. Nature (London). 2E0.

468 - 473.l0. Haest. C. W. M., Plasa. G.. Kamp. D. & Deuticke. B, (1978) Spectrin as a stabilizer

of the phospholipid asymmetry of the human erythrocyte membrane. Biochim. Biophys.Acta. 509. 2l - 32.

ll. Sikorski. A. F. & Kuczek. M. (1985) Labelling of erythrocyte spectrin "in situ" with

phenylisothiocyanate. Biochim. Biophys. Acta. 820, l4'7 - l53.12. Sikorski. A.F.. Kuczek, M,. Nyczka, Z. & Kubiak. Z. (1987) Hydrophobic labelling

ol spectrin in erythrocytes using arylisothiocyanates. Biomed. Biochim. Acta. 46.75 -82.

13. Sikorski. A, F, & Jezierski. A. (1986) Influence of spectrin on the fluidity of erythrocytemembrane. Stud. Biophys.. 1l3. l93-20l.

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14. Sftfr*L A- F., Michalak, K. & Bobrowska, M. (l98ó) Fluorescent quenching study ofttc inreraction of spectrin with phospholipid liposomes; il Biophysics of Membraneftnpłt, 8th School Proceedings (Kuczera, J. & Przestalski, S., eds.) p. 307. Agri-olmat University of Wrocław Press. Wrocław.

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22- London, Y., Demel, R. A., Geurts van Kessel, w. s. M., Vossenberg, F. G. A. & vanDeenen, L. L. M. (1973) The protection of A,1 myelin basic protein against the actionof proteolytic enzymes after the interaction with lipids at the air-water interface.Biochim. Biophys. Acta, 31|, 520 - 530.'23- Sidmowicz. A. & Michalak, K. (1985) Conformational changes of trypsin induced bylipid vesicles. An investigation using isoindole as a fluorescent probe. Stud. Biophys.,l08, l33 - l39.

24. Yurchenco, P. D., Speicher, D. W., Morrow, J. S. & Knowles, W. J. (1982) Monoclonalantibodies as probes of domain structure of the spectrin ł-subunit."/. Biol. Chem.,

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Ph. D. Thesis, University of Utrecht.

8l