259
Biochem. J. (1991) 275, 259-262 (Printed in Great Britain)
Identification of complexes between the tyrosine-O-sulphatebinding protein and tyrosine-sulphated proteins in bovine liver membrane lysates Ming-Cheh LIU,*§ Ruiliang LU,* Jian Rong HAN,* Xibai TANG,* Masahito SUIKOt and Chau-Ching LIU: *Department of Chemistry and Biochemistry, The University of Oklahoma, 620 Parrington Oval, Norman, OK 73019, U.S.A.,
tDepartment of Agricultural Chemistry, Miyazaki University, Miyazaki, Japan, and tThe Rockefeller University, 1230 York Avenue, New York, NY 10021, U.S.A.
Rabbit antiserum against electrophoretically purified bovine liver tyrosine-O-sulphate (TyrS)-binding protein was prepared. Affinity-purified antibodies from the antiserum were found to be capable of immunoprecipitating the TyrSbinding protein from the sodium choleate extract of a bovine liver microsomal membrane fraction. Using purified specific antibodies as the probe, Western blot analysis for the presence of TyrS-binding protein/tyrosine-sulphated protein complexes in bovine liver membrane lysates was performed. It was found that the TyrS-binding protein co-precipitated with three tyrosine-sulphated proteins (fibronectin, fibrinogen and complement C4) immunoprecipitated by their respective antibodies. In contrast, for the two non-tyrosine-sulphated proteins (haptoglobin and transferrin) tested, coprecipitation of the TyrS-binding protein was not observed. On employing an affinity gel fractionation technique, it was shown that partially purified TyrS-binding protein exhibited binding affinity towards Sepharose gels covalently bonded to fibronectin or fibrinogen, but not towards Sepharose gels bonded to albumin or transferrin. These results indicate that the TyrS-binding protein formed complexes with tyrosine-sulphated proteins both in vivo and in vitro, and thus provide support for the putative role of the former being the receptor of the latter.
INTRODUCTION
Sulphation on tyrosine residues, first identified in bovine fibrinogen [1], is now known to occur widely among proteins of multicellular eukaryotic organisms [2]. Its functional importance is in part reflected by the fact that as many as 1 % of the total proteins of an organism may be tyrosine-sulphated [2]. Despite the considerable volume of phenomenological findings made in the past few years, the functional implication of this unique posttranslational protein modification still remains poorly understood. Considering that the vast majority of tyrosine-sulphated proteins identified are secretory proteins, a linkage between tyrosine sulphation and protein secretion had been proposed [3]. It was hypothesized that tyrosine 0-sulphate (TyrS) residues may serve as a signal for the targeting and intracellular transport of tyrosine-sulphated proteins. For this to be the case, a receptor capable of recognizing specifically the protein-bound TyrS residues would seem to be a requirement. Indeed, we have recently identified a 175 kDa TyrS-binding protein extracted from the bovine liver microsomal membrane fraction [4]. The protein appears to exhibit specificity for TyrS, as it binds neither the unmodified tyrosine nor the structurally similar tyrosine 0phosphate. While it remains a possibility that this TyrS-binding protein might play a role in the internalization of exogenous tyrosine-sulphated proteins, it is also likely that it may in fact be involved in the biosynthetic transport of newly synthesized tyrosine-sulphated proteins. In the present study, we have prepared an antiserum against electrophoretically purified 175 kDa TyrS-binding protein, and used the affinity-purified antibodies in examining the presence of complexes between the TyrS-binding protein and several tyrosine-sulphated proteins in bovine liver membrane lysates. Partially purified TyrS-binding protein was prepared and shown to
be capable of binding to representative tyrosine-sulphated teins covalently bonded to Sepharose gel. MATERIALS AND METHODS Materials
Protein molecular mass markers, 6-aminohexanoic-acidactivated Sepharose-4B, sodium choleate, aprotinin, phenylmethanesulphonyl fluoride (PMSF), Protein A-Sepharose CL4B and three bovine plasma proteins (fibrinogen, transferrin and albumin) were products of Sigma. TyrS was synthesized according to the procedure of Jevons [5]. Bovine plasma fibronectin was purified by gelatin-Sepharose column chromatography [6]. 125I-labelled Protein A (7.2 1sCi/ug) was purchased from ICN Biomedicals. Antibodies specific for fibronectin, fibrinogen, complement C4, transferrin, haptoglobin and albumin were from DAKO Corp. Affinity Sepharose gels bonded to TyrS, fibronectin, fibrinogen, transferrin or albumin were prepared according to the procedure recommended by the manufacturer. Preparation of rabbit antiserum against electrophoretically purified 175 kDa bovine liver TyrS-binding protein The TyrS-binding protein was fractionated from the sodium choleate extract of bovine liver membranes using Sepharose gel covalently bonded to TyrS [4]. Fractionated protein(s) were solubilized in Laemmli sample buffer, followed by SDS/PAGE [7]. After electrophoresis, the 175 kDa protein band was located by Coomassie Blue staining, and the gel band containing the protein was cut off. The excised gel band was sliced into pieces and subjected to electroelution using an ISCO model 1750 Electrophoretic Sample Concentrator, based on the procedure of Hunkapillar et al. [8]. The eluted TyrS-binding protein, migrating
Abbreviations used: TyrS, tyrosine 0-sulphate; PBS, phosphate-buffered saline; PMSF, phenylmethanesulphonyl fluoride. § To whom correspondence should be addressed.
Vol. 275
pro-
M.-C. Liu and others
260 as a single protein band upon subsequent SDS/PAGE, was used as the antigen for the preparation of rabbit antiserum according to the procedure previously described [6]. Antibodies were purified from the antiserum using Protein A-Sepharose column chromatography [9]. Immunoprecipitation of the TyrS-binding protein Affinity-purified antibodies (15 1d; 7.5 mg/ml) were added to 1 ml of a sodium choleate extract of bovine liver microsomal membranes [4] in a 1.5 ml Eppendorf microcentrifuge tube, and the mixture was incubated at room temperature for 1 h. After incubation, 30 ,ul of Protein A-Sepharose suspension [in a 1: I ratio of packed beads to phosphate-buffered saline (PBS; NaCl, 8 g/l; KCI, 0.2 g/l; Na2HPO4,2H20, 1.15 g/l; KH2PO4, 0.2 g/l, pH 7.4)] was added. Following a 20 min agitation, Protein A-Sepharose beads bound to antigen-antibody complexes were pelleted by centrifugation, washed three times with PBS and placed in 50 ,u of Laemmli SDS sample buffer. After heating at 100 °C for 1 min, the solubilized proteins were subjected to SDS/PAGE. After electrophoresis, the polyacrylamide gel was stained with Coomassie Blue, destained and dried.
Preparation of bovine liver membrane lysates A crude homogenate of bovine liver prepared as previously described [4] was centrifuged at 140000 g for 2 h at 4 'C. The pelleted membrane fraction was subjected to four cycles of washing with 1O mM-Tris/HCI, pH 7.4, plus 5 mM-PMSF to remove the soluble proteins. Each washing was followed by centrifugation at 140000 g for 1 h. The pelleted membrane obtained following the last washing was placed in 3 vol. of a modified radioimmune precipitation (RIPA) buffer [10] containing 0.05 M-Tris/HCl (pH 7.4), 0.15 M-NaCl, 1 % Triton X100, 1 % sodium choleate and 5 pg of aprotinin/ml. The suspension was homogenized in a Dounce tissue grinder to facilitate solubilization of the membrane. The crude membrane lysate thus prepared was centrifuged at 10000 g for 20 min at 4 'C. The clear supernatant separated was used in the Western blot experiments. Western blot analysis for the presence of TyrS-binding protein/tyrosine-sulphated protein complexes in bovine liver membrane lysates Immunoprecipitations of individual proteins present in bovine liver membrane lysate were carried out in 1.5 ml microcentrifuge tubes based on the above-mentioned procedure using antibodies against (i) the TyrS-binding protein, (ii) fibronectin, (iii) fibrinogen, (iv) complement C4, (v) haptoglobin and (vi) transferrin. Immunoprecipitated samples were subjected to SDS/PAGE, then the polyacrylamide gel was stained with Coomassie Blue, destained and dried. For the Western blot analysis, an identical set of immunoprecipitated samples was prepared and subjected to SDS/PAGE; proteins separated in the polyacrylamide gel were then electrotransferred on to a Millipore Immobilon-P membrane using a Bio-Rad Transblot apparatus. The blotting was performed at a constant 300 mA for 24 h in a buffer solution containing 25 mM-Trizma base and 192 mM-glycine [11]. The blotted Immobilon-P membrane was subsequently blocked with 1 % BSA in PBS for 1 h and probed with 20 ,1 of anti-(TyrSbinding protein) antibodies (7.5 mg/ml). After a 30 min incubation, the membrane was washed five times with PBS to remove unbound antibodies. The washed membrane was treated for 30 min with PBS containing 0.9 1sCi of 125I-labelled Protein A plus 1 % BSA, washed five times with PBS to remove unbound '25I-Protein A, and dried. Autoradiography was then performed on the dried membrane.
Partial purification of the TyrS-binding protein and its affinity fractionation using Sepharose gels covalently bonded to representative tyrosine-sulphated proteins Unless otherwise pointed out, buffer solutions used in the following were all at pH 7.4 and contained 0.15 mM-Triton X-100. A sodium choleate (4 mg/ml) extract of bovine liver membrane (prepared from 300 g of freshly removed bovine liver) was applied to a TyrS-Sepharose column (2.5 cm x 15 cm) preequilibrated with 10 mM-Tris/HCl. Unbound proteins were washed away using 500 ml of 10 mM-Tris/HCl, and the TyrSbinding protein was eluted with 100 ml of 15 mM-sodium deoxycholate in 10 mM-Tris/HCl. The eluted TyrS-binding protein fraction was adsorbed on to a Bio-Gel HTP hydroxyapatite column (1.5 cm x 2 cm) pre-equilibrated with 10 mM-Tris/HCl and eluted with 3 ml of 200 mM-potassium phosphate buffer. The eluted TyrS-binding protein preparation was dialysed extensively against PBS at 4 'C. In the affinity fractionation experiment, 1 ml portions of the partially purified TyrS-binding protein (approx. 10 /pg/ml) in PBS were placed in 1.5 ml microcentrifuge tubes. To individual tubes, 100 pl of a suspension [in a 1: 1 (v/v) ratio with PBS] of fibronectin-Sepharose, fibrinogen-Sepharose, transferrin-Sepharose or albumin-Sepharose was added. The tubes containing the mixtures were agitated for 15 min at room temperature and centrifuged. The pelleted gel beads in individual tubes were washed with 4 x 1 ml of PBS. The washed gel beads were placed in Laemmli SDS sample buffer, heated at 100 'C for 2 min to solubilize the adsorbed protein(s), and subjected to SDS/PAGE; the polyacrylamide gel was then stained with Coomassie Blue, destained and dried.
(a)
(b) 2
X
3
e. "
5
4
.
6 Molecular mass (kDa)
7
8
9
Zs; !205 -
116
- 97.4 -
I
-
66 43
Fig. 1. Immunoprecipitation of the TyrS-binding protein from the sodium choleate extract of a bovine liver membrane fraction (a) and identification of representative tyrosine-sulphated proteins coprecipitated with immunoprecipitated TyrS-binding protein by Western blot analysis (b) In (a), prepared samples were subjected to SDS/PAGE, followed by Coomassie Blue staining. Lanes 1 and 2, sodium choleate extract before and after TyrS-Sepharose fractionation (the amounts loaded were 1/40 of that used for preparing each of the following samples); lane 3, TyrS-Sepharose-fractionated sample; lane 4, immuno-
precipitated sample using affinity-purified preimmune antibodies; lane 5, immunoprecipitated sample using affinity-purified anti-(TyrSbinding protein) antibodies; lane 6, affinity-purified antibody control (no sodium choleate extract added). In (b), samples immunoprecipitated by anti-(TyrS-binding protein) antibodies were subjected to SDS/PAGE, electrotransferred on to an Immobilon-P membrane, probed with antibodies against fibronectin (lane 7), fibrinogen (lane 8) or complement C4 (lane 9), treated with '25I-Protein A, and subjected to autoradiography.
1991
Tyrosine-O-sulphate-binding protein in complexes RESULTS AND DISCUSSION Immunoprecipitation of the 175 kDa TyrS-binding protein Although the rabbit antiserum was prepared against SDSdenatured TyrS-binding protein, it was found to be capable of immunoprecipitating the same protein present in the sodium choleate extract of bovine liver membrane. As shown in Fig. 1 (a), among a large number of proteins extracted from the membrane (lane 1), a major protein species (indicated by the arrow) was immunoprecipitated using affinity-purified antibodies prepared from the antiserum (lane 5). The protein exhibited the same electrophoretic mobility as that of the 175 kDa TyrS-binding protein fractionated on a TyrS-Sepharose gel (lane 3). Although a few other protein bands in addition to those corresponding to the antibody heavy and light chains were present in the immunoprecipitated sample (lane 5), they were also observed in either the preimmune (lane 4) or the immune antibody control (lane 6) sample, and therefore are not immunospecific. Other than these non-specific bands, a few bands, in addition to that of the TyrSbinding protein, were present in the immunoprecipitated sample (lane 5). They are thought to be tyrosine-sulphated proteins in complexes with the TyrS-binding protein, and thereby were coprecipitated. A Western blot technique was employed to examine the identities of some of these proteins. Immunoprecipitated samples, prepared identically to that electrophoresed on lane 5, were subjected to SDS/PAGE, electrotransferred on to an Immobilon-P membrane and probed with antibodies against fibronectin, fibrinogen or complement C4, followed by 12511 Protein A treatment. As shown in Fig. l(b), radioactive bands corresponding to fibronectin (220 kDa; lane 7), fibrinogen (64, 54 and 49 kDa; lane 8) and complement C4 (175 kDa; lane 9) were indeed observed on the autoradiograph taken from the dried Immobilon-P membrane after Western blot analysis. Presence of TyrS-binding protein/tyrosine-sulphated protein complexes in bovine liver membrane lysate To clarify the identity of the TyrS-binding protein as a putative receptor for tyrosine-sulphated proteins, it is important to demonstrate that TyrS-binding protein/tyrosine-sulphated protein complexes are present in vivo. Information of this kind could be obtained by employing the Western blot technique. The underlying assumption is that in freshly prepared membrane lysate the TyrS-binding protein may be bound with tyrosinesulphated proteins, and such complexes could be immuno-
precipitated using antibodies against tyrosine-sulphated proteins. Co-precipitation of the TyrS-binding protein would be indicative of its being in complex form with the tyrosine-sulphated protein tested. Immunoprecipitations of three representative tyrosinesulphated proteins (fibronectin, fibrinogen and complement C4) and two non-tyrosine-sulphated proteins (haptoglobin and transferrin) were performed. As shown in Fig. 2(b), of the five proteins tested, the TyrS-binding protein (indicated by the arrowheads) was found to co-precipitate with all three tyrosine-sulphated proteins (Fig. 2a), whereas for haptoglobin and transferrin, coprecipitation of the TyrS-binding protein was not found. These results strongly suggested that, in the cells, the TyrS-binding protein indeed forms complexes with tyrosine-sulphated proteins, and therefore provide support for the proposed role of the former as a receptor for the latter. It could, however, be argued that the complexes detected might have formed after solubilization of the membrane, which made possible the interaction between proteins originally segregated in the cell. To minimize such a possibility, efforts were made to remove unbound tyrosinesulphated proteins trapped in membrane vesicles by treating the isolated membrane with sodium choleate (1 mg/ml), in addition to the extensive washing with 10 mM-Tris/HCl as described in Vol. 275
261 (a) 1 2 3 4 5 6 7 Molecular mass (kDa) - 205 -
116
- 97.4
- 66
- 43
(b)
1
2 3 4 5
6
7
Fig. 2. Presence of TyrS-binding-protein/tyrosine-sulphated protein complexes in bovine liver membrane lysates (a) Protein band patterns revealed by Coomassie Blue staining; (b) autoradiograph taken from the Immobilon-P membrane after the Western blot analysis. For both (a) and (b), the antibodies used were: lane 1, preimmune serum; lane 2, anti-(TyrS-binding protein) antibody; lane 3, anti-fibronectin antibody; lane 4, anti-fibrinogen antibody; lane 5, anti-(complement C4) antibody; lane 6, antihaptoglobin antibody; lane 7, anti-transferrin antibody. Protein bands corresponding to the individual immunoprecipitated proteins are indicated by the arrowheads. In (a), the prepared samples were subjected to SDS/PAGE followed by Coomassie Blue staining. In (b), the samples were analysed by SDS/PAGE, blotted on to an Immobilon-P membrane and probed with anti-(TyrS-binding protein) antibody, followed by "25I-Protein A treatment.
Molecular mass (kDa) 205 - #i *-
1
2
3
4
5
6
7
8
9
97.4-
43
-
29 -^
Fig. 3. Fractionation of partially purified TyrS-binding protein using Sepharose gels bonded to tyrosine-sulphated proteins Fractionated samples were subjected to SDS/PAGE, followed by Coomassie Blue staining. Samples analysed were fractionated on the following: lane 1, TyrS-Sepharose; lane 2, fibronectin-Sepharose; lane 4, fibrinogen-Sepharose; lane 6, transferrin-Sepharose; lane 8, albumin-Sepharose. Samples in lanes 3, 5, 7 and 9 were affinity Sepharose controls for samples in lanes 2, 4, 6 and 8. Arrowheads indicate the positions of the TyrS-binding protein fractionated by the affinity Sepharose gels.
the Materials and methods section. When the resulting membrane was again solubilized in RIPA buffer and subjected to Western blot analysis, band patterns identical to those shown in Fig. 2 were observed. These results again indicated co-precipitation of the TyrS-binding protein with tyrosine-sulphated proteins, but
M.-C. Liu and others
262 not with non-tyrosine-sulphated proteins. It therefore appears that the TyrS-binding protein/tyrosine-sulphated protein complexes detected in this study were the ones originally present in bovine liver cells.
Affinity fractionation of partially purified TyrS-binding protein using Sepharose gels covalently bonded to tyrosine-sulphated proteins To provide additional evidence for the binding of the TyrSbinding protein with tyrosine-sulphated proteins, affinity fractionation of partially purified TyrS-binding protein using Sepharose gels covalently bonded to tyrosine-sulphated proteins was performed. As shown in Fig. 3, partially purified TyrSbinding protein bound to a Sepharose gel bonded with either fibronectin (lane 2) or fibrinogen (lane 4) when the two were incubated in PBS containing a sub-critical-micellar concentration (0.15 mM) of Triton X-100. Arrowheads in these two lanes indicate the position of the TyrS-binding protein band. Protein bands deriving from fibronectin or fibrinogen released from the affinity Sepharose gels upon heating in SDS sample buffer were also discernible (doublet bands at approx. 220 kDa for fibronectin, and three subunit bands at 64, 54 and 49 kDa for fibrinogen). Other minor bands, also observed for the two affinity gel control samples (lanes 3 and 5), were apparently due to the slightly impure fibronectin and fibrinogen used for the preparation of the affinity gels. In contrast with these two affinity gels, binding of the TyrS-binding protein to Sepharose gels bonded to transferrin (77 kDa; lanes 6 and 7) or albumin (66 kDa; lanes 8 and 9) was not observed. Identical band patterns were found with tested and control samples for the Sepharose gels bonded with these two non-tyrosine-sulphated proteins. It awaits to be demonstrated whether the TyrS-binding
protein indeed shows indiscriminate binding affinity for all tyrosine-sulphated proteins. In summary, we have obtained evidence indicating that the 175 kDa TyrS-binding protein forms complexes with tyrosinesulphated proteins both in vivo and in vitro. The results appear to lend support to the hypothesis that the former is a receptor for the latter. Whether such a putative receptor functions in the biosynthetic transport of tyrosine-sulphated proteins or in the internalization of exogenous tyrosine-sulphated proteins remains to be clarified. This research was supported in part by funds from the American Cancer Society (grant no. BC-705) and the American Heart Association, Oklahoma Affiliate (grant no. OK-90-G-6).
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8.
9. 10. 11.
Bettelheim, F. R. (1954) J. Am. Chem. Soc. 76, 2838-2839 Huttner, W. B. (1988) Mod. Cell Biol. 6, 97-140 Hille, A., Rosa, P. & Huttner, W. B. (1984) FEBS Lett. 177, 129-134 Liu, M.-C., Suiko, M. & Tang, X. (1988) Biochem. Biophys. Res. Commun. 156, 964-969 Jevons, F. R. (1964) Biochem. J. 89, 621-624 Liu, M.-C. & Lipmann, F. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 34-37 Laemmli, U. K. (1970) Nature (London) 227, 680-685 Hunkapillar, M. W., Lujan, E., Ostrander, R. & Hood, L. E. (1983) Methods Enzymol. 91, 227-236 Ey, P. L., Prowse, S. J. & Jenkin, C. R. (1978) Biochemistry 15, 429-436 Gilead, Z., Jeng, Y.-H., Wold, W. S. M., Sugawara, K., Rho, H. M., Harter, M. L. & Green, M. (1976) Nature (London) 264, 263-266 Towbin, H., Staehelin, T. & Gordon, J. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 4350-4354
Received 5 September 1990/31 January 1991; accepted 6 February 1991
1991
Biochem. J. (1991) 275, 259-262 (Printed in Great Britain)
Identification of complexes between the tyrosine-O-sulphatebinding protein and tyrosine-sulphated proteins in bovine liver membrane lysates Ming-Cheh LIU,*§ Ruiliang LU,* Jian Rong HAN,* Xibai TANG,* Masahito SUIKOt and Chau-Ching LIU: *Department of Chemistry and Biochemistry, The University of Oklahoma, 620 Parrington Oval, Norman, OK 73019, U.S.A.,
tDepartment of Agricultural Chemistry, Miyazaki University, Miyazaki, Japan, and tThe Rockefeller University, 1230 York Avenue, New York, NY 10021, U.S.A.
Rabbit antiserum against electrophoretically purified bovine liver tyrosine-O-sulphate (TyrS)-binding protein was prepared. Affinity-purified antibodies from the antiserum were found to be capable of immunoprecipitating the TyrSbinding protein from the sodium choleate extract of a bovine liver microsomal membrane fraction. Using purified specific antibodies as the probe, Western blot analysis for the presence of TyrS-binding protein/tyrosine-sulphated protein complexes in bovine liver membrane lysates was performed. It was found that the TyrS-binding protein co-precipitated with three tyrosine-sulphated proteins (fibronectin, fibrinogen and complement C4) immunoprecipitated by their respective antibodies. In contrast, for the two non-tyrosine-sulphated proteins (haptoglobin and transferrin) tested, coprecipitation of the TyrS-binding protein was not observed. On employing an affinity gel fractionation technique, it was shown that partially purified TyrS-binding protein exhibited binding affinity towards Sepharose gels covalently bonded to fibronectin or fibrinogen, but not towards Sepharose gels bonded to albumin or transferrin. These results indicate that the TyrS-binding protein formed complexes with tyrosine-sulphated proteins both in vivo and in vitro, and thus provide support for the putative role of the former being the receptor of the latter.
INTRODUCTION
Sulphation on tyrosine residues, first identified in bovine fibrinogen [1], is now known to occur widely among proteins of multicellular eukaryotic organisms [2]. Its functional importance is in part reflected by the fact that as many as 1 % of the total proteins of an organism may be tyrosine-sulphated [2]. Despite the considerable volume of phenomenological findings made in the past few years, the functional implication of this unique posttranslational protein modification still remains poorly understood. Considering that the vast majority of tyrosine-sulphated proteins identified are secretory proteins, a linkage between tyrosine sulphation and protein secretion had been proposed [3]. It was hypothesized that tyrosine 0-sulphate (TyrS) residues may serve as a signal for the targeting and intracellular transport of tyrosine-sulphated proteins. For this to be the case, a receptor capable of recognizing specifically the protein-bound TyrS residues would seem to be a requirement. Indeed, we have recently identified a 175 kDa TyrS-binding protein extracted from the bovine liver microsomal membrane fraction [4]. The protein appears to exhibit specificity for TyrS, as it binds neither the unmodified tyrosine nor the structurally similar tyrosine 0phosphate. While it remains a possibility that this TyrS-binding protein might play a role in the internalization of exogenous tyrosine-sulphated proteins, it is also likely that it may in fact be involved in the biosynthetic transport of newly synthesized tyrosine-sulphated proteins. In the present study, we have prepared an antiserum against electrophoretically purified 175 kDa TyrS-binding protein, and used the affinity-purified antibodies in examining the presence of complexes between the TyrS-binding protein and several tyrosine-sulphated proteins in bovine liver membrane lysates. Partially purified TyrS-binding protein was prepared and shown to
be capable of binding to representative tyrosine-sulphated teins covalently bonded to Sepharose gel. MATERIALS AND METHODS Materials
Protein molecular mass markers, 6-aminohexanoic-acidactivated Sepharose-4B, sodium choleate, aprotinin, phenylmethanesulphonyl fluoride (PMSF), Protein A-Sepharose CL4B and three bovine plasma proteins (fibrinogen, transferrin and albumin) were products of Sigma. TyrS was synthesized according to the procedure of Jevons [5]. Bovine plasma fibronectin was purified by gelatin-Sepharose column chromatography [6]. 125I-labelled Protein A (7.2 1sCi/ug) was purchased from ICN Biomedicals. Antibodies specific for fibronectin, fibrinogen, complement C4, transferrin, haptoglobin and albumin were from DAKO Corp. Affinity Sepharose gels bonded to TyrS, fibronectin, fibrinogen, transferrin or albumin were prepared according to the procedure recommended by the manufacturer. Preparation of rabbit antiserum against electrophoretically purified 175 kDa bovine liver TyrS-binding protein The TyrS-binding protein was fractionated from the sodium choleate extract of bovine liver membranes using Sepharose gel covalently bonded to TyrS [4]. Fractionated protein(s) were solubilized in Laemmli sample buffer, followed by SDS/PAGE [7]. After electrophoresis, the 175 kDa protein band was located by Coomassie Blue staining, and the gel band containing the protein was cut off. The excised gel band was sliced into pieces and subjected to electroelution using an ISCO model 1750 Electrophoretic Sample Concentrator, based on the procedure of Hunkapillar et al. [8]. The eluted TyrS-binding protein, migrating
Abbreviations used: TyrS, tyrosine 0-sulphate; PBS, phosphate-buffered saline; PMSF, phenylmethanesulphonyl fluoride. § To whom correspondence should be addressed.
Vol. 275
pro-
M.-C. Liu and others
260 as a single protein band upon subsequent SDS/PAGE, was used as the antigen for the preparation of rabbit antiserum according to the procedure previously described [6]. Antibodies were purified from the antiserum using Protein A-Sepharose column chromatography [9]. Immunoprecipitation of the TyrS-binding protein Affinity-purified antibodies (15 1d; 7.5 mg/ml) were added to 1 ml of a sodium choleate extract of bovine liver microsomal membranes [4] in a 1.5 ml Eppendorf microcentrifuge tube, and the mixture was incubated at room temperature for 1 h. After incubation, 30 ,ul of Protein A-Sepharose suspension [in a 1: I ratio of packed beads to phosphate-buffered saline (PBS; NaCl, 8 g/l; KCI, 0.2 g/l; Na2HPO4,2H20, 1.15 g/l; KH2PO4, 0.2 g/l, pH 7.4)] was added. Following a 20 min agitation, Protein A-Sepharose beads bound to antigen-antibody complexes were pelleted by centrifugation, washed three times with PBS and placed in 50 ,u of Laemmli SDS sample buffer. After heating at 100 °C for 1 min, the solubilized proteins were subjected to SDS/PAGE. After electrophoresis, the polyacrylamide gel was stained with Coomassie Blue, destained and dried.
Preparation of bovine liver membrane lysates A crude homogenate of bovine liver prepared as previously described [4] was centrifuged at 140000 g for 2 h at 4 'C. The pelleted membrane fraction was subjected to four cycles of washing with 1O mM-Tris/HCI, pH 7.4, plus 5 mM-PMSF to remove the soluble proteins. Each washing was followed by centrifugation at 140000 g for 1 h. The pelleted membrane obtained following the last washing was placed in 3 vol. of a modified radioimmune precipitation (RIPA) buffer [10] containing 0.05 M-Tris/HCl (pH 7.4), 0.15 M-NaCl, 1 % Triton X100, 1 % sodium choleate and 5 pg of aprotinin/ml. The suspension was homogenized in a Dounce tissue grinder to facilitate solubilization of the membrane. The crude membrane lysate thus prepared was centrifuged at 10000 g for 20 min at 4 'C. The clear supernatant separated was used in the Western blot experiments. Western blot analysis for the presence of TyrS-binding protein/tyrosine-sulphated protein complexes in bovine liver membrane lysates Immunoprecipitations of individual proteins present in bovine liver membrane lysate were carried out in 1.5 ml microcentrifuge tubes based on the above-mentioned procedure using antibodies against (i) the TyrS-binding protein, (ii) fibronectin, (iii) fibrinogen, (iv) complement C4, (v) haptoglobin and (vi) transferrin. Immunoprecipitated samples were subjected to SDS/PAGE, then the polyacrylamide gel was stained with Coomassie Blue, destained and dried. For the Western blot analysis, an identical set of immunoprecipitated samples was prepared and subjected to SDS/PAGE; proteins separated in the polyacrylamide gel were then electrotransferred on to a Millipore Immobilon-P membrane using a Bio-Rad Transblot apparatus. The blotting was performed at a constant 300 mA for 24 h in a buffer solution containing 25 mM-Trizma base and 192 mM-glycine [11]. The blotted Immobilon-P membrane was subsequently blocked with 1 % BSA in PBS for 1 h and probed with 20 ,1 of anti-(TyrSbinding protein) antibodies (7.5 mg/ml). After a 30 min incubation, the membrane was washed five times with PBS to remove unbound antibodies. The washed membrane was treated for 30 min with PBS containing 0.9 1sCi of 125I-labelled Protein A plus 1 % BSA, washed five times with PBS to remove unbound '25I-Protein A, and dried. Autoradiography was then performed on the dried membrane.
Partial purification of the TyrS-binding protein and its affinity fractionation using Sepharose gels covalently bonded to representative tyrosine-sulphated proteins Unless otherwise pointed out, buffer solutions used in the following were all at pH 7.4 and contained 0.15 mM-Triton X-100. A sodium choleate (4 mg/ml) extract of bovine liver membrane (prepared from 300 g of freshly removed bovine liver) was applied to a TyrS-Sepharose column (2.5 cm x 15 cm) preequilibrated with 10 mM-Tris/HCl. Unbound proteins were washed away using 500 ml of 10 mM-Tris/HCl, and the TyrSbinding protein was eluted with 100 ml of 15 mM-sodium deoxycholate in 10 mM-Tris/HCl. The eluted TyrS-binding protein fraction was adsorbed on to a Bio-Gel HTP hydroxyapatite column (1.5 cm x 2 cm) pre-equilibrated with 10 mM-Tris/HCl and eluted with 3 ml of 200 mM-potassium phosphate buffer. The eluted TyrS-binding protein preparation was dialysed extensively against PBS at 4 'C. In the affinity fractionation experiment, 1 ml portions of the partially purified TyrS-binding protein (approx. 10 /pg/ml) in PBS were placed in 1.5 ml microcentrifuge tubes. To individual tubes, 100 pl of a suspension [in a 1: 1 (v/v) ratio with PBS] of fibronectin-Sepharose, fibrinogen-Sepharose, transferrin-Sepharose or albumin-Sepharose was added. The tubes containing the mixtures were agitated for 15 min at room temperature and centrifuged. The pelleted gel beads in individual tubes were washed with 4 x 1 ml of PBS. The washed gel beads were placed in Laemmli SDS sample buffer, heated at 100 'C for 2 min to solubilize the adsorbed protein(s), and subjected to SDS/PAGE; the polyacrylamide gel was then stained with Coomassie Blue, destained and dried.
(a)
(b) 2
X
3
e. "
5
4
.
6 Molecular mass (kDa)
7
8
9
Zs; !205 -
116
- 97.4 -
I
-
66 43
Fig. 1. Immunoprecipitation of the TyrS-binding protein from the sodium choleate extract of a bovine liver membrane fraction (a) and identification of representative tyrosine-sulphated proteins coprecipitated with immunoprecipitated TyrS-binding protein by Western blot analysis (b) In (a), prepared samples were subjected to SDS/PAGE, followed by Coomassie Blue staining. Lanes 1 and 2, sodium choleate extract before and after TyrS-Sepharose fractionation (the amounts loaded were 1/40 of that used for preparing each of the following samples); lane 3, TyrS-Sepharose-fractionated sample; lane 4, immuno-
precipitated sample using affinity-purified preimmune antibodies; lane 5, immunoprecipitated sample using affinity-purified anti-(TyrSbinding protein) antibodies; lane 6, affinity-purified antibody control (no sodium choleate extract added). In (b), samples immunoprecipitated by anti-(TyrS-binding protein) antibodies were subjected to SDS/PAGE, electrotransferred on to an Immobilon-P membrane, probed with antibodies against fibronectin (lane 7), fibrinogen (lane 8) or complement C4 (lane 9), treated with '25I-Protein A, and subjected to autoradiography.
1991
Tyrosine-O-sulphate-binding protein in complexes RESULTS AND DISCUSSION Immunoprecipitation of the 175 kDa TyrS-binding protein Although the rabbit antiserum was prepared against SDSdenatured TyrS-binding protein, it was found to be capable of immunoprecipitating the same protein present in the sodium choleate extract of bovine liver membrane. As shown in Fig. 1 (a), among a large number of proteins extracted from the membrane (lane 1), a major protein species (indicated by the arrow) was immunoprecipitated using affinity-purified antibodies prepared from the antiserum (lane 5). The protein exhibited the same electrophoretic mobility as that of the 175 kDa TyrS-binding protein fractionated on a TyrS-Sepharose gel (lane 3). Although a few other protein bands in addition to those corresponding to the antibody heavy and light chains were present in the immunoprecipitated sample (lane 5), they were also observed in either the preimmune (lane 4) or the immune antibody control (lane 6) sample, and therefore are not immunospecific. Other than these non-specific bands, a few bands, in addition to that of the TyrSbinding protein, were present in the immunoprecipitated sample (lane 5). They are thought to be tyrosine-sulphated proteins in complexes with the TyrS-binding protein, and thereby were coprecipitated. A Western blot technique was employed to examine the identities of some of these proteins. Immunoprecipitated samples, prepared identically to that electrophoresed on lane 5, were subjected to SDS/PAGE, electrotransferred on to an Immobilon-P membrane and probed with antibodies against fibronectin, fibrinogen or complement C4, followed by 12511 Protein A treatment. As shown in Fig. l(b), radioactive bands corresponding to fibronectin (220 kDa; lane 7), fibrinogen (64, 54 and 49 kDa; lane 8) and complement C4 (175 kDa; lane 9) were indeed observed on the autoradiograph taken from the dried Immobilon-P membrane after Western blot analysis. Presence of TyrS-binding protein/tyrosine-sulphated protein complexes in bovine liver membrane lysate To clarify the identity of the TyrS-binding protein as a putative receptor for tyrosine-sulphated proteins, it is important to demonstrate that TyrS-binding protein/tyrosine-sulphated protein complexes are present in vivo. Information of this kind could be obtained by employing the Western blot technique. The underlying assumption is that in freshly prepared membrane lysate the TyrS-binding protein may be bound with tyrosinesulphated proteins, and such complexes could be immuno-
precipitated using antibodies against tyrosine-sulphated proteins. Co-precipitation of the TyrS-binding protein would be indicative of its being in complex form with the tyrosine-sulphated protein tested. Immunoprecipitations of three representative tyrosinesulphated proteins (fibronectin, fibrinogen and complement C4) and two non-tyrosine-sulphated proteins (haptoglobin and transferrin) were performed. As shown in Fig. 2(b), of the five proteins tested, the TyrS-binding protein (indicated by the arrowheads) was found to co-precipitate with all three tyrosine-sulphated proteins (Fig. 2a), whereas for haptoglobin and transferrin, coprecipitation of the TyrS-binding protein was not found. These results strongly suggested that, in the cells, the TyrS-binding protein indeed forms complexes with tyrosine-sulphated proteins, and therefore provide support for the proposed role of the former as a receptor for the latter. It could, however, be argued that the complexes detected might have formed after solubilization of the membrane, which made possible the interaction between proteins originally segregated in the cell. To minimize such a possibility, efforts were made to remove unbound tyrosinesulphated proteins trapped in membrane vesicles by treating the isolated membrane with sodium choleate (1 mg/ml), in addition to the extensive washing with 10 mM-Tris/HCl as described in Vol. 275
261 (a) 1 2 3 4 5 6 7 Molecular mass (kDa) - 205 -
116
- 97.4
- 66
- 43
(b)
1
2 3 4 5
6
7
Fig. 2. Presence of TyrS-binding-protein/tyrosine-sulphated protein complexes in bovine liver membrane lysates (a) Protein band patterns revealed by Coomassie Blue staining; (b) autoradiograph taken from the Immobilon-P membrane after the Western blot analysis. For both (a) and (b), the antibodies used were: lane 1, preimmune serum; lane 2, anti-(TyrS-binding protein) antibody; lane 3, anti-fibronectin antibody; lane 4, anti-fibrinogen antibody; lane 5, anti-(complement C4) antibody; lane 6, antihaptoglobin antibody; lane 7, anti-transferrin antibody. Protein bands corresponding to the individual immunoprecipitated proteins are indicated by the arrowheads. In (a), the prepared samples were subjected to SDS/PAGE followed by Coomassie Blue staining. In (b), the samples were analysed by SDS/PAGE, blotted on to an Immobilon-P membrane and probed with anti-(TyrS-binding protein) antibody, followed by "25I-Protein A treatment.
Molecular mass (kDa) 205 - #i *-
1
2
3
4
5
6
7
8
9
97.4-
43
-
29 -^
Fig. 3. Fractionation of partially purified TyrS-binding protein using Sepharose gels bonded to tyrosine-sulphated proteins Fractionated samples were subjected to SDS/PAGE, followed by Coomassie Blue staining. Samples analysed were fractionated on the following: lane 1, TyrS-Sepharose; lane 2, fibronectin-Sepharose; lane 4, fibrinogen-Sepharose; lane 6, transferrin-Sepharose; lane 8, albumin-Sepharose. Samples in lanes 3, 5, 7 and 9 were affinity Sepharose controls for samples in lanes 2, 4, 6 and 8. Arrowheads indicate the positions of the TyrS-binding protein fractionated by the affinity Sepharose gels.
the Materials and methods section. When the resulting membrane was again solubilized in RIPA buffer and subjected to Western blot analysis, band patterns identical to those shown in Fig. 2 were observed. These results again indicated co-precipitation of the TyrS-binding protein with tyrosine-sulphated proteins, but
M.-C. Liu and others
262 not with non-tyrosine-sulphated proteins. It therefore appears that the TyrS-binding protein/tyrosine-sulphated protein complexes detected in this study were the ones originally present in bovine liver cells.
Affinity fractionation of partially purified TyrS-binding protein using Sepharose gels covalently bonded to tyrosine-sulphated proteins To provide additional evidence for the binding of the TyrSbinding protein with tyrosine-sulphated proteins, affinity fractionation of partially purified TyrS-binding protein using Sepharose gels covalently bonded to tyrosine-sulphated proteins was performed. As shown in Fig. 3, partially purified TyrSbinding protein bound to a Sepharose gel bonded with either fibronectin (lane 2) or fibrinogen (lane 4) when the two were incubated in PBS containing a sub-critical-micellar concentration (0.15 mM) of Triton X-100. Arrowheads in these two lanes indicate the position of the TyrS-binding protein band. Protein bands deriving from fibronectin or fibrinogen released from the affinity Sepharose gels upon heating in SDS sample buffer were also discernible (doublet bands at approx. 220 kDa for fibronectin, and three subunit bands at 64, 54 and 49 kDa for fibrinogen). Other minor bands, also observed for the two affinity gel control samples (lanes 3 and 5), were apparently due to the slightly impure fibronectin and fibrinogen used for the preparation of the affinity gels. In contrast with these two affinity gels, binding of the TyrS-binding protein to Sepharose gels bonded to transferrin (77 kDa; lanes 6 and 7) or albumin (66 kDa; lanes 8 and 9) was not observed. Identical band patterns were found with tested and control samples for the Sepharose gels bonded with these two non-tyrosine-sulphated proteins. It awaits to be demonstrated whether the TyrS-binding
protein indeed shows indiscriminate binding affinity for all tyrosine-sulphated proteins. In summary, we have obtained evidence indicating that the 175 kDa TyrS-binding protein forms complexes with tyrosinesulphated proteins both in vivo and in vitro. The results appear to lend support to the hypothesis that the former is a receptor for the latter. Whether such a putative receptor functions in the biosynthetic transport of tyrosine-sulphated proteins or in the internalization of exogenous tyrosine-sulphated proteins remains to be clarified. This research was supported in part by funds from the American Cancer Society (grant no. BC-705) and the American Heart Association, Oklahoma Affiliate (grant no. OK-90-G-6).
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8.
9. 10. 11.
Bettelheim, F. R. (1954) J. Am. Chem. Soc. 76, 2838-2839 Huttner, W. B. (1988) Mod. Cell Biol. 6, 97-140 Hille, A., Rosa, P. & Huttner, W. B. (1984) FEBS Lett. 177, 129-134 Liu, M.-C., Suiko, M. & Tang, X. (1988) Biochem. Biophys. Res. Commun. 156, 964-969 Jevons, F. R. (1964) Biochem. J. 89, 621-624 Liu, M.-C. & Lipmann, F. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 34-37 Laemmli, U. K. (1970) Nature (London) 227, 680-685 Hunkapillar, M. W., Lujan, E., Ostrander, R. & Hood, L. E. (1983) Methods Enzymol. 91, 227-236 Ey, P. L., Prowse, S. J. & Jenkin, C. R. (1978) Biochemistry 15, 429-436 Gilead, Z., Jeng, Y.-H., Wold, W. S. M., Sugawara, K., Rho, H. M., Harter, M. L. & Green, M. (1976) Nature (London) 264, 263-266 Towbin, H., Staehelin, T. & Gordon, J. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 4350-4354
Received 5 September 1990/31 January 1991; accepted 6 February 1991
1991
