Proteolytic Digestion of Band 3 from Bovine Erythrocyte Membranes in Membrane-Bound and Solnbilized States 1

Transcription

1 /. Biochem. 95, (1984) Proteolytic Digestion of Band 3 from Bovine Erythrocyte Membranes in Membrane-Bound and Solnbilized States 1 Shio MAKINO, Ryuichi MORIYAMA, Takashi KITAHARA, and Shozo KOGA Department of Food Science and Technology, Faculty of Agriculture, Nagoya University, Chikusa-ku, Nagoya, Aichi 464 Received for publication, November 1, 1983 Bovine band 3 in membrane-bound and solubilized states was digested with chymotrypsin, trypsin, and papain. Bovine band 3 in red blood cells was fragmented by the proteases in a 5 mm NaH,PO 4 -Na,HPO 4 buffer containing 0.3 M glucose, ph 8.0, but not in a 5 mm NaH,PO 1 -Na 2 HPO 1 buffer containing 0.15 M NaCl, ph 8.0, in which human band 3 is cleaved by chymotrypsin and papain. When compared with the known data for human band 3, however, major fragments of bovine band 3 derived from intact cells, inside-out vesicles and unsealed ghosts were similar to those of human band 3, except that tryptic fragments were formed on the extracellular attack. The results suggest that bovine band 3 adopts a quite similar molecular arrangement in the membrane to in the human case. However, it was strongly suggested by molecular weight evaluation of fragments that the only detectable water-soluble 38,000-39,000 dalton fragment does not account for the entire hydrophilic pole of the band 3 molecule exposed in the cytoplasmic region of the membrane. When isolated band 3 was treated with the enzymes in a 2 % solution of nonaethyleneglycol /z-dodecyl ether, the major product was indistinguishable on sodium dodecyl sulfate-gel from the water-soluble fragment of the cytoplasmic domain origin of band 3. This fragment lost its resistance to further enzymatic degradation when treated with dimethylmaleic anhydride, thus band 3 oligomers were converted into their monomers. The chymotryptic 38,000 dalton water-soluble fragment obtained in nonaethyleneglycol /j-dodecyl ether solution was a subfragment of a 50,000 dalton piece which was produced in a 2% solution of deoxycholate after chymotrypsin treatment of band 3. Band 3 J from erythrocyte membranes is a major intrinsic membrane protein having a molecular weight of about 100,000 and functions as an anion transporter (for reviews, see Refs. 1 and 2). The 1 This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan, Erythrocyte membrane protein band 3 is named according to Fairbanks et al. ((1971) Biochemistry 10, ). Abbreviations: CnE,, nonaethyleneglycol n-dodecyl ether; DLDS, 4,4'-diisothiocyano-2,2'-stilbene sulfonic acid; SDS, sodium dodecyl sulfate; PAGE; polyacrylamide gel electrophoresis. Vol. 95, No. 4,

2 1020 S. MAKINO, R. MORIYAMA, T. KITAHARA, and S. KOGA molecular architectural features of human band 3 in membranes have been extensively studied by the proteolytic and chemical fragmentation of the protein (3-12). The results show that the protein consists of two distinct domains; the 40,000 dalton amino-terminal part of the protein is a hydrophilic domain on the cytoplasmic side of the membrane, while the carboxyl-terminal portion of the protein is a hydrophobic membrane-bound domain that probably spans the membrane several times. Band 3 is present universally in mammalian and avian red blood cell membranes (13-15) and so far most studies on band 3 have been limited to that of human origin. As to mammalian erythrocytes, there is a remarkable difference in the phospholipid composition between bovine and human erythrocyte membranes (16, 17). Such a difference may have some effects on the interaction of band 3 with lipids and on the protein structure and function in membranes. We therefore selected bovine band 3 for our comparative studies. In this paper, the fragmentation of bovine band 3 with chymotrypsin, trypsin, and papain is described. Bovine band 3 in intact cells was cleaved by the proteases in a medium of low ionic strength containing 0.3 M glucose, but not in an iso-osmotic solution of high ionic strength. The possible ordering of bovine band 3 fragments produced by the digestion of intact cells, inside-out vesicles and unsealed ghosts indicates that the molecular arrangement of bovine band 3 in the membrane is quite similar to that of human band 3. When isolated band 3 was hydrolyzed by the proteases in detergent solutions, the major fragment was suggested to be a product of the cytoplasmic domain origin. The portion existing in the membrane domain was rather sensitive to the degradation in the solubilized state. However, the site susceptible to chymotrypsin in the solubilized state was affected by the detergents used. EXPERIMENTAL PROCEDURES Materials a-chymotrypsin and papain were obtained from Miles and Sigma, respectively, L- 1 - Tosylamido phenylethyl chloromethylketonetreated trypsin was supplied through the courtesy of Dr. T. Sasaki of our laboratory. Polyoxyethyleneglycol /j-dodecyl ether with an average of 9 polyoxyethylene chains per molecule (CuE,) 1 was obtained from Nikko Chemicals. Dimethylmaleic anhydride and aminoethyl-sepharose 4B were prepared according to the procedures reported previously (18). All the other reagents used were obtained from Wako Chemical Industries. Membrane Preparation Bovine blood was collected in 3.8% trisodium citrate solution at a slaughterhouse and used within 2 h. All procedures were performed at 0-4 C and washing of the cells was carried out by centrifugation (3,000 rpm for 15 min). Cells were washed three times with a 5 mm NaH a PO 4 -NatHPO 4 buffer containing 0.15 M NaCl, ph8.0, or a 5 mm NaH,PO 4 - NajHPO 4 buffer containing 0.3 M glucose, ph 8.0. When cells were treated with a protease, the cells were washed four times with the 5 mm NaH,PO 4 - Na,,HPO 4 buffer containing 0.15 M NaCl, ph 8.0, after termination of the enzymatic reaction. Then, unsealed ghosts were prepared from intact and enzyme-treated cells according to the procedure described previously (18). Sealed inside-out membrane vesicles were prepared from freshly obtained unsealed ghosts by the method of Steck (19) and the vesicles were used without further purification by density gradient ultracentrifugation. All membranes prepared were stored in 5 mm NaH,PO 4 - Na,HPO 4 buffer, ph 8.0, and used within 3 days for enzymatic degradation experiments. Stripped membranes were prepared by mixing ghosts or vesicles with 10 volumes of ice-cold 0.1 M-NaOH for 1 min and immediate centrifugation at 15,000 rpm for 30 min to collect the membranes at 4 C. The alkali-stripped membranes were further washed twice with 5 mm NaH,PO 4 - NajHPO 4 buffer, ph 8.0, at 15,000 rpm for 30 min. 4,4'-Diisothiocyano-2,2'-Stilbene Sulfonic Acid (DIDS) Treatment of Bovine Blood Cells Cells at a 25% hematocrit were labeled with 10^g/ml of DIDS for 1 h at 37 C (12). The unreacted DIDS was removed by washing the cells twice with the 5 mm NaH,PO 4 -Na,HPO 4 buffer containing 0.15 M NaCl, ph 8.0, with 0.5 % of bovine serum albumin and twice in normal buffer. Unsealed ghosts from intact cells and cells treated with chymotrypsin were prepared by the procedure described above. Solubilization of Unsealed Ghosts and Purification of Band 3 One volume of packed ghosts was mixed with one volume of 5 mm NaH,PO 4 - Na,HPO 4 buffer, ph 8.0, and detergents were added to the suspension to a final detergent con- /. Biochem.

3 FRAGMENTATION OF BOVINE BAND 3 BY PROTEOLYTIC ENZYMES digestion of band 3 in intact cells was attempted in two buffer systems; iso-osmotic solutions of low ionic strength using a 5 mm NaH2PO4-Na,HPO4 buffer containing 0.3 M glucose, ph 8.0, and of high ionic strength using a 5 mm NaH,PO4Na2HPO4 buffer containing 0.15 M NaCl, ph 8.0. Enzymatic treatments in the latter buffer virtually did not cause any detectable fragmentation of band 3, while band 3 was cleaved extracellularly in the low ionic strength buffer containing 0.3 M glucose. Figure 1 shows SDS-PAGE profiles of alkalistripped membranes obtained from cells treated with the proteases in the two buffer systems. In the 5 mm NaH,PO4-Na,HPO4 buffer containing 0.3 M glucose, ph 8.0, chymotrypsin and trypsin produced two band 3 fragments (molecular weights of 67,000 and 41,000) which were still membrane-embedded. Predominant papain fragments of molecular weights of 67,000 and 38,000 M l" I Band3-( 0 { «67K- ' Polyacrylamide Gel Electrophoresis in the Pres- Band 3 - I ence of Sodium Dodecyl Sulfate (SDS-PAGE) 41K67K38KSDS-PAGE was carried out at a gel concentration of 6% for cylindrical gels (0.6 x 8 cm) or 7.5 % for slab gels (0.2 x 14 x 14 cm) in the presence of 0.1 % 41KSDS, using the buffer system of Weber-Osborn (20). Protein samples of fa containing 2560 /ig protein, 5% SDS, and 0.1% 2-mercaptoethanol were heated at 90 C for 5 min and then loaded on the gel. Protein was stained with Co1 2 3 I omassie brilliant blue. Standard proteins (phosfig. 1. Effect of enzymes on band 3 from the external phorylase a, bovine serum albumin, catalase, aldoside of the membrane. Intact red blood cells were lase, chymotrypsinogen, and cytochrome c) were shaken gently for 16 h at room temperature in a 5 mm electrophoresed with samples in each experiment. NaH PO -Na,HPO buffer containing 0.15 M NaCl, t 4 4 The fluorescence of DIDS-labeled bands in ph 8.0 (gels 1, 3,4, and 5) or a 5 mm NaH,POj-Na,HPO 4 the gels was visualized under UV illumination after buffer containing 0.3 M glucose, ph 8.0 (gels 2, 6, 7, and removal of SDS from the unstained gel by soaking 8), with 100 /ig/ml of chymotrypsin (gels 1, 2, 3, and 6), the gel in aqueous 5% methanol-7% acetic acid trypsin (gels 4 and 7) or papain (gels 5 and 8). After solution. The fluorescence bands were marked the enzyme was inhibited, the cells were washed and the with black ink and the gels were then stained for enzyme-treated ghosts were prepared. Ghosts prepared from chymotrypsin-treated cells were electrophoresed on proteins. I cylindrical gels without alkali treatment (gels 1 and 2). Protease-treated ghosts were stripped of their peripheral proteins with 1 M-NaOH and then electrophoresed on RESULTS slab gels (gels from 3 to 8). Proteins derived from Enzymatic Digestion of Band 3 in Membrane- 50 /<1 (gels 1 and 2) or 25 /il (gels from 3 to 8) of packed Bound State (a) Red blood cells: Proteolytic membranes were run on each gel. Vol. 95, No. 4, 1984 centration of 2 %. The mixture stirred at 37 C for 30 min was slightly cloudy. Band 3 was purified in 0. 1 % C ^ E, solution at 4 C by aminoethyl-sepharose 4B ion-exchange column chromatography of a 4 % CnE^-extract from unsealed ghosts, as described previously (18). Enzymatic Treatment of Membrane and Band 3 Intact red blood cells in the 5 ITIM NaH,PO 4 N a t H P O 4 buffer containing M NaCl, ph 8.0, or the 5 ITIM NaHjPO 4 -Na,HPO 4 buffer containing 0.3 M glucose, ph 8.0 (hematocrit of 50%), were incubated for 16 h at room temperature with the proteases at the concentration of 100/<g/ml. Packed ghosts and vesicles were diluted with one volume of 5 mm NaH,PO 4 -Na,HPO 4 buffer, p H 8.0, and enzymatic treatment of these membranes was carried out at an enzyme concentration of 50,ug/ml for 16 h at room temperature. Proteolysis of isolated band 3 and ghosts solubilized by detergent solutions was performed at an enzyme concentration of 20,ug/ml for 2 h at room temperature. The hydrolysis was terminated by the addition of diisopropylfluorophosphate for chymotrypsin and trypsin, and sodium monoiodoacetate for papain. 1021

4 1022 S. MAKINO, R. MORIYAMA, T. KITAHARA, and S. KOGA with chymotrypsin or papain, and 39,000 when treated with trypsin, as indicated by the absence of the polypeptides in the alkali-stripped membranes. (c) Unsealed ghosts: Fragmentation of unsealed ghosts by the enzymes produced three membrane-intercalating fragments and a water-soluble fragment, as shown in Fig. 3. The water-soluble fragment and the membrane-bound fragment of molecular weight ~ 40,000 were electrophoretically indistinguishable from those obtained from insideout vesicles and intact cells, respectively. Other fragments residing in the about 25,000 and 17,000 dalton regions were not freed from the membrane by NaOH. Though all enzymes generated the 17,000 dalton fragment, the formation of the former fragment(s) depended on the proteases used: a sharp, single 27,000 dalton band with chymotrypsin; 27,000, 23,000, and 20,000 dalton bands with trypsin; and a 20,000 dalton band with papain. Unsealed ghosts were proteolyzed with a low concentration of chymotrypsin (10 /«g/ml) and the time dependence of their degradation was examined (Fig. 4). With the hydrolysis of band 3, the first Band3- a**3" g 1 38K-»= 27K- 23K20K- 17K3 U K-H f t 8 Fig. 2. Enzymatic digestion of inside-out vesicles. Inside-out vesicles were incubated for 16 h at room temperature in a 5 ITIM NaH,PO.,-Na,HPO4 buffer, ph 8.0 (gels 1 and 2) or the same buffer containing 50 /(g/ml of enzymes (gels from 3 to 8). After the enzyme was inhibited, electrophoresis was carried out on slab gels. Inside-out vesicles (gel 1) and the same alkali-stripped vesicles (gel 2), chymotrypsin-treated vesicles (gel 3), and the same alkali-stripped vesicles (gel 4), trypsintreated vesicles (gel 5) and the same alkali-stripped vesicles (gel 6), and papain-treated vesicles (gel 7) and the same alkali-stripped vesicles (gel 8). Proteins derived from 30 /i\ of vesicles were run on each gel. Fig. 3. Enzymatic digestion of unsealed ghosts. Enzymatic treatment and electrophoresis were performed under the same conditions as in Fig. 2. Unsealed ghosts (gel 1) and the same alkali-stripped membrane (gel 2), chymotrypsin-treated ghosts (gel 3) and the same alkali-stripped membranes (gel 4), trypsin-treated ghosts (gel 5) and the same alkali-stripped membranes (gel 6). and papain-treated ghosts (gel 7) and the same alkali-stripped membranes (gel 8). Proteins obtained from 30 /<1 of packed membranes were run on slab gels. J. Biochem. were not eluted from the membrane by NaOH. These 67,000 dalton bands were indistinguishable from each other in the SDS-PAGE system employed here. The results suggest the existence of at least two extracellular sites on the band 3 polypeptide which are sensitive to papain and one of them is located close to the extrafacial chymotrypsin and trypsin cleavage site. (b) Inside-out vesicles: Figure 2 shows the patterns of SDS-PAGE of sealed inside-out vesicles proteolyzed by the enzymes and their alkalistripped membranes. Though unwanted fragments might be derived from contaminated right-side out vesicles and unsealed vesicles because of the use of unpurified inside-out vesicles, the protease treatment produced three novel fragments which were not observed on extracellular digestion. Two of these were 58,000 and 47,000 dalton fragments, both of which were equally produced by all enzymes used here. These were membrane-bound and the latter showed a diffuse band on SDS-gel. Another fragment was a water-soluble polypeptide whose molecular weight was 38,000 when treated

5 1023 FRAGMENTATION OF BOVINE BAND 3 BY PROTEOLYTIC ENZYMES Band367K58K47K«1K38K- :i 1 I 8 w 67K41K- 41K38K- 41K39K- 27K- m t 23K- 27K- 17K- 4 w Fig. 4. Time dependence of chymotryptic digestion of unsealed ghosts. Unsealed ghosts were incubated at room temperature in a 5 ITIM NaH,PO 4 -Na,HPO 4 buffer, ph 8.0, containing 10/ig/ml of chymotrypsin. At the indicated times, aliquots of the reactant were taken out and the enzyme was inhibited. Membranes were alkali-stripped and then electrophoresed. The reaction times were 0 h (gel 1), 2 h (gel 2), 6 h (gel 3), and 16 h (gel 4). Gel 5 shows the patterns of unstripped membranes of 6 h-incubated ghosts. Proteins derived from 30/il of ghosts were run on slab gels. Fig. 5. Double digestion of unsealed ghosts prepared from chymotrypsin-treated cells. Unsealed ghosts were prepared from chymotrypsin-treated erythrocytes and the unsealed ghosts (gel 1) were further digested with chymotrypsin (gels 2 and 3), trypsin (gels 4 and 5), or papain (gels 6 and 7) under the same conditions as in Fig. 2. Gels 1, 2, 4, and 6 are the SDS-gel patterns of enzyme-treated ghosts and gels 2, 5, and 7 show the patterns of alkali-stripped membranes. Proteins derived from 30 /<! of ghosts were run on slab gels. of the smaller fragment survived without further degradation by chymotrypsin and trypsin. As expected, the patterns of SDS-PAGE were idendetectable and predominant fragments were mem- tical with those obtained for the unsealed ghosts brane-bound 58,000 and 47,000 dalton fragments after enzyme treatment. and a water-soluble 38,000 dalton fragment, which (e) Band 3 fragment labeled with D1DS: were observed on chymotryptic digestion of insidebovine erythrocytes were treated with DIDS, an out vesicles. The 67,000, 58,000, and 47,000 dalton anion transport inhibitor, under the same condibands were reduced in intensity during prolonged tions as in the human case. The fluorescence of chymotrypsin treatment, with the progressive apdids located at the band 3 region of the SDS-gel pearance of 41,000, 27,000, and 17,000 dalton pattern of the unsealed ghosts and purified band membrane-bound fragments. This suggests that 3 exhibited absorbance at 340 nm attributable to each cleavage site differs in its susceptibility to bound DIDS. From the values of absorbance at proteolysis. 280 and 340 nm of DIDS-labeled purified band (d) Digestion of unsealed ghosts derived from 3, it was suggested that DIDS bound to band 3 chymotrypsin-treated membrane: Unsealed ghosts in an approximate molar ratio of I to 1 (unpublished result). The DIDS fluorescence was visuderived from chymotrypsin-treated red blood cells alized at the 67,000 dalton band on SDS-PAGE were further digested with chymotrypsin, trypsin, of ghosts derived from chymotrypsin-treated cells. and papain. The results are shown in Fig. 5. Of After treatment of unsealed ghosts with chymothe 67,000 and 41,000 dalton fragments derived on the extracellular chymotryptic attack, the larger trypsin, DIDS was found to reside on the 17,000 dalton fragment. The 58,000 and diffuse 47,000 fragment was very sensitive to the enzymes. On dalton bands also showed fluorescence when unthe other hand, the results show that at least part Vol. 95, No. 4, K-

6 S. MAKINO, R. MORIYAMA, T. KITAHARA, and S. KOGA K50K- W <*) w - Fig. 6. Effect of enzymes on solubilized ghosts. (A) Unsealed ghosts were solubilized (protein concentration, ~ 2 mg/ml) in a 5 min NaH,PO4-Na,HPO4 buffer containing 1 /o of detergents, ph 8.0. The solubilized ghosts were incubated for 2 h in 2% Q.E, (gels 1, 2, and 3) and 2 /o deoxycholate (gels 4, 5, and 6) containing 20 /'g/ml of enzymes. After the reaction was terminated, each mixture was electrophoresed. Chymotrypsin digestion (gels 1 and 4), trypsin digestion (gels 2 and 5), and papain digestion (gels 3 and 6). Proteins derived from 40 fi\ of solutions were run on slab gels. (B) Unsealed ghosts prepared from chymotrypsin-treated cells were solubilized in 2% deoxycholate solution (protein concentration, ~ 2 mg/ml), followed by enzyme treatments in the same manner as in Fig. 6A. Chymotrypsin digestion (gel 1), trypsin digestion (gel 2), and papain digestion (gel 3). Proteins derived from 40 /JI of solutions were run on slab gels. sealed ghosts treated with 10/ig/ml of chymotrypsin for 2 h were electrophoresed. Enzymatic Cleavage of Band 3 in Detergent Solutions Unsealed ghosts were solubilized with 2% C1SE, or 2% deoxycholate solution followed by protease digestions. The results of SDS-PAGE are shown in Fig. 6A. Among several bands produced in C^E, solution, a characteristic band showed a mobility corresponding to that of the water-soluble fragment derived from unsealed ghosts and inside-out vesicles. Trypsin and papain treatment in deoxycholate solution produced eventually the same fragment as a major one in C12E, solution. However, a 50,000 dalton fragment as a predominant band was generated by chymotrypsin in deoxycholate solution, in place of the 38,000 dalton fragment in Ci,E, solution. Such characteristic bands were also observed when unsealed ghosts prepared from chymotrypsintreated cells were digested in the detergent solu- tions. Figure 6B shows SDS-PAGE profiles of the digested products in 2% deoxycholate solution and it is suggested from the results that the chymotryptic 50,000 dalton fragment obtained in deoxycholate solution must arise from the 67,000 dalton fragment, if the former is a degradation product of band 3. Figure 7 illustrates the effects of chymotrypsin on isolated band 3 in CnE, and deoxycholate solutions. The molecular weight of a fragment obtained in 0.1% QjE, solution coincided with that of the major fragment arising on chymotryptic digestion of 2% Q.Es-solubilized ghosts. Solid deoxycholate was added to the purified band 3 solution containing 0.1 % C^E, to a final deoxycholate concentration of above 0.2%, and then band 3 was exposed to chymotrypsin. Below 0.5 % deoxycholate, band 3 was cleaved to give products having molecular weights of 50,000 and 38,000. Only the 50,000 dalton band was produced above J. Biochem. i

7 FRAGMENTATION OF BOVINE BAND 3 BY PROTEOLYT1C ENZYMES H J025 The monomeric band 3 in C lt E, solution, which was prepared by the reaction of band 3 with dimethylmaleic anhydride (21), was completely degraded by the enzymes used in the present experiment into small peptides and no band was detected on SDS-PAGE. This was also the case for the enzymatic treatment in detergent solutions of alkali-stripped membrane derived from unsealed ghosts. ff Band3-50K38K- Possible Ordering of Bovine Band 3 Fragments For proteolytic digestion of membranes, in general, there is some doubt as to whether fragments produced are derived from the target protein. Fortunately, since band 3 is a predominant red blood cell membrane polypeptide, its major cleavage products might be detected more clearly than Fig. 7. Chymotryptic digestion of purified band 3. those of other polypeptides. Fragments labeled The band 3 solution (~1 mg/ml) purified in 0.1% C,,E, (gel 1) was treated for 2h with 20/ig/ml of with DIDS, which binds only to the bovine band chymotrypsin (gel 2). Band 3 solution (~1 mg/ml) 3 molecule in a 1 to 1 molar ratio as in the human was adjusted to 2% of deoxycholate by adding the solid case (22), allow the visual identification of the detergent, and then treated with 20 fig/m\ chymotrypsin cleavage products derived from band 3. for 2 h (gel 3). Then, deoxycholate was removed by Possible ordering of chymotryptic and papain dialysis followed by further digestion with chymotrypsin fragments of bovine band 3 is illustrated in Fig. (gel 4). Purified band 3 (~1 mg/ml) was treated with 8, based on the discussion below. Molecular dimethylmaleic anhydride and then treated with chymoweights of the fragments estimated by SDS-PAGE trypsin in 0.1% Cj,E, (gel 5). Forty //I of each solution are included in the figure. Integral membrane was run on slab gels. proteins often migrate abnormally on SDS-gels, leading to inaccurate molecular weight estimation 1 % concentration of deoxycholate. After the re- (23). However, bovine band 3 and its extracelmoval of dexoycholate, further digestion of the lular chymotryptic fragments are known to show fragment with chymotrypsin generated the 38,000 normal behavior in migration on SDS-gel as waterdalton piece, indicating that the latter is a sub- soluble proteins do (21). It seems, therefore, not unreasonable to place reliance on the molecular fragment of the 50,000 dalton fragment. weights estimated by SDS-PAGE in the present We have observed that antisera prepared in case. rabbit against the 38,000 dalton fragment obtained (a) Chymotrypsin: Based on the molecular from unsealed ghosts in C18E, solution react against both intact band 3 and the 50,000 dalton fragment weight and amino acid composition data, it has obtained from unsealed ghosts in deoxycholate been indicated that chymotrypsin extracellularly solution. This indicates that these fragments are attacks one peptide bond on bovine band 3, producing two membrane-anchoring polypeptides, apparently generated from band 3. Treatment of purified band 3 with trypsin and 67,000 and 41,000 dalton fragments (21, 24). The papain produced 39,000 dalton and 38,000 dalton present results suggest that the membrane-bound fragments in both CltE, and deoxycholate solu- 17,000 and water-soluble 38,000 dalton fragments tions, respectively. These fragments again showed are subfragments of the 67,000 dalton fragment, the same mobility on SDS-PAGE as the water- since the 38,000 dalton fragment emerged with soluble fragments produced from the cytoplasmic disappearance of the 67,000 dalton fragment as shown in Fig. 5 and DIDS fluorescence resided pole of band 3. Vol. 95, No. 4, 1984 t DISCUSSION

8 1026 S. MAKINO, R. MORIYAMA, T. KJTAHARA, and S. KOGA Chymotrypsin H*. 38 K millbh-v K- >67K«58K«Papain 36 K Fig. 8. Possible ordering of chymotrypsin and papain fragments of bovine band 3. It is assumed that the water-soluble 38,000 dalton fragment contains the amino-terminal end of the band 3 molecule, as indicated in the case of human band 3. The locations of cleavage sites with respect to the outside ( ) or inside (I) faces of the membrane are indicated. Observed fragments are shown by solid lines and undetected polypeptides by hatched lines. The effect of trypsin on bovine band 3 differs from that of chymotrypsin in that the former enzyme produces a water-soluble 39,000 dalton fragment instead of the 38,000 dalton fragment obtained with chymotrypsin. on both the 67,000 and 17,000 dalton fragments. Judging from the occurrence of the 38,000 dalton fragment on proteolysis of inside-out vesicles, it is apparent that the fragment is located on the cytoplasmic side. The sum of the molecular weights of these two fragments does not account for that of the parent 67,000 dalton fragment, suggesting that at least two peptide bonds must be cleaved on the cytoplasmic side of the membrane, though the excised peptide(s) was not detected in the present experiment. It is not clear at present whether the water-soluble 38,000 dalton fragment involves the amino-terminal end of band 3. Jn view of the demonstration that the human tryptic water-soluble fragment contains the amino-terminal end of band 3 (5, 6, 25), it is reasonably postulated that the excised peptide(s) must reside between the 17,000 and 38,000 dalton pieces on the primary structure of the 67,000 dalton fragment. Two membrane-associated 58,000 and 47,000 dalton fragments were produced on treating the internal membrane surface with chymotrypsin. These fragments showed fluorescence with DIDS, indicating that the 17,000 dalton fragment is a product from these fragments. The appearance of these membrane-bound fragments suggests the presence of two different species of band 3 molecules, since the protein has merely one binding site for DTDS. One of the molecular species must have another site attacked by chymotrypsin from the inner surface of the membrane, in addition to the cleavage sites in the cytoplasmic region which lead to the release of the water-soluble fragment and smaller peptide(s). This may reflect either the heterogeneity of the polypeptide chain itself or of the protein conformation such as the state of association. Though there is no direct evidence for the polypeptide heterogeneity of bovine band 3, the possibility has been suggested that bovine band 3 molecules exist in different aggregate states in the membrane (21, 26). Jt must be again emphasized that the molecular weight of bovine band 3, 107,000±5,000 (21), is not accounted for by the sum of the two observable fragments, membrane-bound 58,000 and water-soluble 38,000 dalton fragments, obtained from inside-out vesicles. The origin of another membrane-intercalating 27,000 dalton fragment is not so clear as those of the others. Judging from the molecular weight, however, it may be reasonable to assume that this fragment is a subfragment of the 47,000 dalton /. Biochem.

9 FRAGMENTATION OF BOVINE BAND 3 BY PROTEOLYTIC ENZYMES 1027 piece produced by the second digestion at the intracellular surface, with concomitant release of the 17,000 dalton fragment. (b) Trypsin: Trypsin generates fragments that are virtually the same as the chymotryptic ones. However, the tryptic water-soluble fragment has a slightly higher molecular weight than the corresponding chymotryptic fragment. (c) Papain: Cleavage sites on the cytoplasmic region for papain are located close to those for chymotrypsin, because the fragments produced by both enzymes show nearly the same mobility on SDS-gel. However, papain differs from the other two enzymes in cleaving more than one peptide bond on the external surface of the membrane. A membrane-bound 20,000 dalton band may be generated from the membrane-bound 47,000 dalton piece, as in the chymotrypsin case. Fragmentation of Bovine Band 3 in Solubilized State Bovine band 3 in C ls Et, solution was cleaved by the enzymes in a different manner from that in the membrane-bound state and the product from isolated band 3 was the 38,000 dalton fragment with chymotrypsin and papain or the 39,000 dalton fragment with trypsin. Such a fragment was indistinguishable electrophoretically from the watersoluble fragment generated by each protease from the cytoplasmic pole of band 3 in the membranebound state. Since nonionic detergents virtually do not interact with the hydrophilic region(s) of proteins, it may be expected that the fragment generated from the cytoplasmic pole of band 3 in the membrane-bound state is also produced by proteolysis in a medium containing QJEj. Furthermore, we have observed that the chymotryptic 38,000 dalton fragment obtained in CuE e solution is soluble in an aqueous solution without detergents and is denatured completely in 6 M guanidine hydrochloride, a typical denaturant for water-soluble proteins. On the other hand, the chymotryptic 67,000 dalton fragment is resistant to denaruration in the medium because of the existence of the hydrophobic 17,000 dalton portion. In view of the fact that the chymotryptic 38,000 dalton fragment is a subfragment of the chymotryptic 67,000 dalton fragment (Figs. 6B and 7), the above observation strongly suggests that at least the chymotryptic 38,000 dalton fragment is a product of the cytoplasmic domain origin. (Details of molecular characteristics of the 38,000 dalton fragment including the immunoelectrophoretic property will be published elsewhere) On the other hand, as typically demonstrated by no or little appearance of the membrane-bound 41,000 and 17,000 dalton fragments from band 3 when treated with chymotrypsin in C 1S E, solution (Fig. 7), the present results indicated that most of the peptide bonds of band 3 which showed resistance to proteolysis in the membrane-bound state become accessible to proteases in CHEJ solution. It is generally accepted that the membraneembedded portions of integral membrane proteins are covered by a micellar-like structure when solubilized by nonionic detergents, reproducing an environment which resembles the membrane lipid bilayer, and that the native structure of the membrane-associated domain of proteins is preserved in such detergent solutions (23, 27). However, there might be differences in the thickness of the hydrophobic core and the hydrophilic region formed by Q.Ej micelles and by the membrane lipid bilayer. Therefore, exact simulation will not be attained with CuE, micelles and this may lead to degradation of membrane-bound portions into small peptides. The results of the proteolytic cleavage in deoxycholate solution again indicate that the membrane-bound domain of band 3 is sensitive to the proteases when solubilized. However, it is certain that deoxycholate interacts with band 3 in a different manner from that of C 18 Es, since the cleavage of band 3 by chymotrypsin depended on the detergents used for solubilization. As deoxycholate resembles nonionic detergents in its inability to cause protein denaturation (23, 27), it appears that the discrepancy observed in the enzyme attack does not result from conformational differences of the band 3 polypeptide in the two detergent solutions. Although the reason for the discrepancy cannot be fully explained at the present time, it is likely that the interaction of the carboxylic group of deoxycholate with the positively charged amino acid residues of band 3 may lead to the formation of the chymotryptic fragment which differs from the fragment produced in C 1S E, solution. The chymotryptic 38,000 dalton fragment which is resistant to further degradation by chymotrypsin loses its resistance on treatment of band 3 with dimethylmaleic anhydride. Since the treatment converts band 3 oligomers into their mono- Vol. 95, No. 4, 1984

10 1028 S. MAKINO, R. MORJYAMA, T. KITAHARA, and S. KOGA mers, showing a 40% decrease in helical content (21), it is suggested that the secondary or tertiary structure of band 3 is responsible for the resistance to the enzymatic digestion in the cytoplasmic domain of band 3. Comparison of Band 3 Fragments between the Bovine and Human Cases Studies on enzymatic digestions of human band 3 have been extensively carried out in many laboratories. Table I compares several proteolytic fragments generated from bovine and human band 3. Though the molecular weights assigned to the fragments in the human case differ from laboratory to laboratory and there is no evidence showing that the corresponding fragments derived from the different species have the same molecular weight, inspection of the results summarized in Table I indicates that there is a fundamental agreement in primary sites cleaved by the proteases between the human and bovine cases. The formation of similar band 3 fragment from different origins allows us to conclude that bovine band 3 might adopt a quite similar disposition in the membrane to in the human case. TABLE I. Protease Chymotrypsin Despite the similarities between human and bovine band 3 in their molecular orientation in the membranes and in the fragments produced, bovine band 3 in red blood cells was virtually not proteolyzed at all in a 5 mm NaHjPC^-NajHPO* buffer containing 0.15 M NaCl, ph 8.0, which is in remarkable contrast to the human case. This suggests that bovine band 3 adopts a more tightly folded conformation as compared with human band 3 in a medium of high ionic strength. Furthermore, successful cleavage of bovine band 3 in intact cells by the proteases in a 5 mm NaH,PCV Na,HPOi buffer containing 0.3 M glucose, ph 8.0, probably reflects an ionic strength-dependence alteration in the structure of the extracellular region. So far as the present experiment is concerned, trypsin has easy access to the extracellular region of bovine band 3, which is again in contrast to the suggestion that the exofacial region of human band 3 is resistant to trypsin. These discrepancies between the two species observed in the membrane-bound state may result from a specific phospholipid-band 3 interaction due to the remarkable Comparison of proteolytic fragments generated from bovine and human band 3.* Bovine band 3 Membrane-bound 67,000 dalton fragment Membrane-bound 41,000 dalton fragment Membrane-bound 17,000 dalton fragment Water-soluble 38,000 dalton fragment Trypsin Membrane-bound 47,000 and 58,000 dalton fragments Papain Water-soluble 39,000 dalton fragment Membrane-bound 67,000 dalton fragment Membrane-bound 38,000 dalton fragment Fragment Human band 3 Membrane-bound 55,000 (3, 6), 58,000 (7), 60,000 (11, 28-30), 65,000 (31), or 70,000 dalton fragment (52) Membrane-bound 35,000 (12, 28, 29), 34,000-45,000 (11), or 38,000 dalton fragment (3, 6) Membrane-bound 17,000 dalton fragment (3, 12, 30) Water-soluble 40,000 dalton fragment (33) Membrane-bound 49,000 (7), 50,000 (28), 52,000 (3), 55,000 (29, 30), 48,000-62,000 dalton fragment (34), or 48,000 and 58,000 dalton fragments (35) b Water-soluble 40,000 (7, 30) or 41,000 dalton fragment (3, 29) Membrane-bound 60,000 dalton fragment (28) Membrane-bound 30,000 dalton fragment (28)» 1 Molecular weights were taken from the indicated references. b The membrane-bound tryptic fragment often shows a very diffase band on SDS-PAGE (34) and this band has been clearly resolved into double peaks by Lepke and Passow (35).» It is indicated that the papain 60,000 dalton fragment is slightly smaller than the chymotryptic 60,000 dalton fragment and that the papain 30,000 dalton fragment is produced from the chymotryptic 35,000 dalton fragment when the latter is treated with papain (28). J. Biochem.

11 FRAGMENTATION OF BOVINE BAND 3 BY PROTEOLYTIC ENZYMES 1029 differences in phospholipid components betwen the respective membranes. The present results suggested the existence of more than two cleavage sites on the cytoplasmic region of the 67,000 dalton fragment of bovine band 3, though there is no such suggestion for the human case. This suggestion is supported, to some extent, by the appearance of the 50,000 dalton fragment from the 67,000 dalton fragment with chymotrypsin in 2% deoxycholate solution and further digestion of the 50,000 dalton fragment into the 38,000 dalton piece in 2% d,e, solution. Our preliminary data show that the 50,000 dalton fragment isolated consists of two distinct domains and the unextractable ~ 10,000 dalton portion is more hydrophilic than the complementary 38,000 dalton fragment. The authors wish to thank Dr. Takuji Sasaki of this department for valuable discussions. REFERENCES 1. Cabantchik, Z.I., Knauf, P.A., & Rothstein, A. (1978) Biochim. Biophys. Acta 515, Steck, T.L. (1978) /. Supramol. Struct. 8, Steck, T.L., Ramos, B., & Strapazon, E. (1976) Biochemistry 15, Jenkins, R.E. & Tanner, M.J.A. (1977) Biochem. J. 161, Drickamer, L.K. (1977) /. Bio!. Chem. 252, Steck, T.L.. Koziarz, J.J., Singh, M.K., Reddy, G., & Kohler, H. (1978) Biochemistry 17, Fukuda, M., Eshdat, Y., Tarone, G., & Marchesi, V.T. (1978) /. Biol. Chem. 253, Drickamer, L.K. (1978) /. Biol. Chem. 253, Rao, A. & Reithmeier, R.A.F. (1979) /. Biol. Chem. 254, Williams, D.G., Jenkins, R.E., & Tanner, M.J.A. (1979) Biochem. J. 181, 477^ Markowitz, S. & Marchesi, V.T. (1981) /. Biol. Chem. 256, Ramjeesingh, M., Gaarn, A., & Rothstein, A. (1983) Biochim. Biophys. Acta 729, Kobylka, D., Khettry, A., Shin, B.C., & Caraway, K.L. (1972) Arch. Biochem. Biophys. 148, Weis, M.J. & Ingram, V.M. (1976) /. Biol. Chem. 251, Jay, D.G. (1983) /. Biol. Chem. 258, Nelson, G.J. (1967) Biochim. Biophys. Acta 144, Turner, J.D. & Rouser, G. (1970) Anal. Biochem. 38, Nakashima, H. & Makino, S. (1980) /. Biochem. 87, Steck, T.L. (1974) in Methods in Membrane Biology (Korn, E.D., ed.) Vol. 2, pp , Plenum Press, New York and London 20. Weber, K. & Osborn, M. (1969) /. Biol. Chem. 244, Nakashima, H. & Makino, S. (1980) J. Biochem. 88, Ramjeesingh, M., Gaarn, A., & Rothstein, A. (1981) Biochim. Biophys. Acta 641, Makino, S. (1979) in Advances in Biophysics (Kotani, M., ed.) Vol. 12, pp , Japan Scientific Societies Press, Tokyo, and University Park Press, Baltimore 24. Makino, S., Nakashima, H., & Shibagaki, K. (1981) /. Biochem. 89, Kaul, R.K., Murthy, S.N.P., Reddy, A.G., Steck, T.L., & Kohler, H. (1983) /. Biol. Chem. 258, Nakashima, H., Nakagawa, Y., & Makino, S. (1981) Biochim. Biophys. Acta 643, Tanford, C. & Reynolds, J.A. (1976) Biochim. Biophys. Acta 457, Jennings, M.L. & Passow, H. (1979) Biochim. Biophys. Acta 554, Reithmeier, R.A.F. (1979) /. Biol. Chem. 254, Grinstein, S., Ship, S., & Rothstein, A. (1978) Biochim. Biophys. Acta 507, Drickamer, L.K. (1976) /. Biol. Chem. 251, Passow, H. & Zaki, L. (1978) in Molecular Specialization and Symmetry in Membrane Function (Solomon, A.K. & Karnovsky, M., eds.) pp , Harvard University Press, Cambridge and London 33. Appell, K.C. & Low, P.S. (1981) /. Biol. Chem. 256, Jenkins, R.E. & Tanner, M.J.A. (1977) Biochem. J. 161, Lepke, S. & Passow, H. (1976) Biochim. Biophys. Acta 455, Vol. 95, No. 4, 1984

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