Plasma Hyaluronan-binding Protein Is a Serine Protease
2000; Elsevier BV; Volume: 275; Issue: 30 Linguagem: Inglês
10.1074/jbc.m904640199
ISSN1083-351X
AutoresAlexander A. Vostrov, Wolfgang Quitschke,
Tópico(s)Enzyme Production and Characterization
ResumoCTCF is an essential factor for optimal transcription from the amyloid β-protein precursor promoter. A proteolytic activity detected in bovine, rabbit, horse, and human serum cleaves CTCF at three major sites, resulting in a modified mobility shift pattern of the fragments that retain DNA binding ability. The protease was purified to electrophoretic homogeneity, partially sequenced, and identified as the plasma hyaluronan-binding protein. The proteolytic activity was selectively abolished by various serine protease inhibitors, including the Kunitz-type protease inhibitor domain of amyloid β-protein precursor. Reduction with β-mercaptoethanol showed that the 70-kDa protein consists of two polypeptides with apparent molecular masses of 44 and 30 kDa. The serine protease domain was localized to the 30-kDa polypeptide as determined by [3H]diisopropylfluorophosphate binding. CTCF is an essential factor for optimal transcription from the amyloid β-protein precursor promoter. A proteolytic activity detected in bovine, rabbit, horse, and human serum cleaves CTCF at three major sites, resulting in a modified mobility shift pattern of the fragments that retain DNA binding ability. The protease was purified to electrophoretic homogeneity, partially sequenced, and identified as the plasma hyaluronan-binding protein. The proteolytic activity was selectively abolished by various serine protease inhibitors, including the Kunitz-type protease inhibitor domain of amyloid β-protein precursor. Reduction with β-mercaptoethanol showed that the 70-kDa protein consists of two polypeptides with apparent molecular masses of 44 and 30 kDa. The serine protease domain was localized to the 30-kDa polypeptide as determined by [3H]diisopropylfluorophosphate binding. amyloid β-protein precursor 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate fetal calf serum plasma hyaluronan-binding protein polyacrylamide gel electrophoresis diethylaminoethyl diisopropylfluorophosphate Kunitz-type protease inhibitor The promoter of the amyloid β-protein precursor (APP)1 gene is a necessary element in the regulation of APP transcription and it has been shown to confer cell type-specific expression in transgenic mice (1Wirak D.O. Bayney R. Kundel C.A. Lee A. Scangos G.A. Trapp B.D. Unterbeck A.J. EMBO J. 1991; 10: 289-296Crossref PubMed Scopus (61) Google Scholar, 2Fox N.W. Johnstone E.M. Ward K.E. Schrementi J. Little S.P. Biochem. Biophys. Res. Commun. 1997; 240: 759-762Crossref PubMed Scopus (20) Google Scholar). The proximal APP promoter is devoid of CCAAT and TATA boxes but contains a prominent initiator element associated with the main transcriptional start site (+1) (3Quitschke W.W. Matthews J.P. Kraus R.J. Vostrov A.A. J. Biol. Chem. 1996; 271: 22231-22239Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). The integrity of the initiator element is essential for both start site selection and optimal transcriptional activity (3Quitschke W.W. Matthews J.P. Kraus R.J. Vostrov A.A. J. Biol. Chem. 1996; 271: 22231-22239Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). In addition, transcription from the APP promoter is critically dependent on the presence of an intact nuclear factor binding site designated APBβ (3Quitschke W.W. Matthews J.P. Kraus R.J. Vostrov A.A. J. Biol. Chem. 1996; 271: 22231-22239Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, 4Quitschke W.W. J. Biol. Chem. 1994; 269: 21229-21233Abstract Full Text PDF PubMed Google Scholar). The core recognition sequence for this binding site is located between positions −82 and −93 and its elimination reduces transcriptional activity by ∼70–90% (3Quitschke W.W. Matthews J.P. Kraus R.J. Vostrov A.A. J. Biol. Chem. 1996; 271: 22231-22239Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, 4Quitschke W.W. J. Biol. Chem. 1994; 269: 21229-21233Abstract Full Text PDF PubMed Google Scholar). The nuclear factor that activates transcription from APBβ was identified as CTCF (5Vostrov A.V. Quitschke W.W. J. Biol. Chem. 1997; 272: 33353-33359Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar), a nuclear regulatory protein comprising 727 amino acids (6Filippova G.N. Fagerlie S. Klenova E.M. Myers C. Dehner Y. Goodwin G. Neiman P.E. Collins S.J. Lobanenkov V.V. Mol. Cell. Biol. 1996; 16: 2802-2813Crossref PubMed Scopus (413) Google Scholar). It contains a centrally located DNA binding domain with 11 zinc finger motifs that are flanked by 267 amino acids on the N-terminal side and 151 amino acids on the C-terminal side. This protein was first identified as a factor that binds to the chicken c-myc promoter (7Lobanenkov V.V. Nicolas R.H. Adler V.V. Paterson H. Klenova E.M. Polotskaja A.V. Goodwin G.H. Oncogene. 1990; 5: 1743-1753PubMed Google Scholar) and to the silencer element of the chicken lysozyme gene (8Baniahmad A. Steiner C. Köhne A.C. Renkawitz R. Cell. 1990; 61: 505-514Abstract Full Text PDF PubMed Scopus (366) Google Scholar, 9Burcin M. Arnold R. Lutz M. Kaiser B. Runge D., F. Lottspeich F. Filippova G.N., V.V. Lobanenkov V.V. Renkawitz R. Mol. Cell. Biol. 1997; 17: 1281-1288Crossref PubMed Scopus (120) Google Scholar). A functional role for CTCF in both positive and negative transcriptional regulation has been documented (5Vostrov A.V. Quitschke W.W. J. Biol. Chem. 1997; 272: 33353-33359Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar, 6Filippova G.N. Fagerlie S. Klenova E.M. Myers C. Dehner Y. Goodwin G. Neiman P.E. Collins S.J. Lobanenkov V.V. Mol. Cell. Biol. 1996; 16: 2802-2813Crossref PubMed Scopus (413) Google Scholar, 10Köhne A.C. Baniahmad A. Renkawitz R. J. Mol. Biol. 1993; 232: 747-755Crossref PubMed Scopus (35) Google Scholar, 11Klenova E.M. Nicolas R.H. Paterson H.F. Carne A.F. Heath C.M. Goodwin G.H. Neiman P.E. Lobanenkov V.V. Mol. Cell. Biol. 1993; 13: 7612-7624Crossref PubMed Scopus (226) Google Scholar). While purifying CTCF, we observed that binding to the APBβ sequence became increasingly unstable as the level of purity increased. However, this instability could be overcome by supplementing the incubation mixture with the zwitterionic detergent CHAPS and large amounts of nonspecific protein (5Vostrov A.V. Quitschke W.W. J. Biol. Chem. 1997; 272: 33353-33359Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). Routinely, fetal calf serum (FCS) was used as a source of such protein. While optimizing the conditions for the binding reaction, we noticed that if crude nuclear extract was preincubated with an excess of FCS prior to the binding reaction, substantial changes in the electrophoretic mobility shift pattern occurred, resulting in binding complexes with higher electrophoretic mobilities. We have here isolated the factor responsible for this altered mobility shift and identified it as a protease activity associated with the plasma hyaluronan-binding protein PHBP (12Choi-Miura N.-H. Tobe T. Sumiya J.-I. Nakano Y. Sano Y. Mazda T. Tomita M. J. Biochem. 1996; 119: 1157-1165Crossref PubMed Scopus (121) Google Scholar). The cDNA for this protein was previously cloned, and sequence analysis indicated the presence of a serine protease consensus domain. However, in the original preparations, proteolytic activity of PHBP was not demonstrated (12Choi-Miura N.-H. Tobe T. Sumiya J.-I. Nakano Y. Sano Y. Mazda T. Tomita M. J. Biochem. 1996; 119: 1157-1165Crossref PubMed Scopus (121) Google Scholar). Nuclear extracts were prepared from HeLa cells grown in suspension to a density of 5–8 × 105 cells/ml (3Quitschke W.W. Matthews J.P. Kraus R.J. Vostrov A.A. J. Biol. Chem. 1996; 271: 22231-22239Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar,13Heberlein U. Tjian R. Nature. 1988; 331: 410-415Crossref PubMed Scopus (74) Google Scholar). The final protein concentration in extracts was 10–15 mg/ml in buffer D containing 25 mm Hepes, pH 7.6, 100 mmKCl, 2 mm MgCl2, 0.2 mm EDTA, 1 mm dithiothreitol, 0.1 mm phenylmethylsulfonyl fluoride, and 10% glycerol. Extract preparations were aliquoted and stored at −80 °C. Double-stranded oligonucleotide APBβ-WT (5Vostrov A.V. Quitschke W.W. J. Biol. Chem. 1997; 272: 33353-33359Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar) containing the CTCF recognition sequence was 5′-end-labeled with [γ-32P]ATP (14Maniatis T. Fritsch E.F. Sambrook J. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). 10 ng of labeled oligonucleotide (50,000–500,000 cpm) were incubated for 30 min at 25 °C with 10–20 μg of protein from nuclear extract in buffer D supplemented with 2 μg of poly(dI-dC), 5 μg of yeast tRNA, 2.5% CHAPS, and 1 ml of FCS in a total reaction volume of 30 μl. The incubation mixture was electrophoresed in 1% agarose or 6% polyacrylamide gels with 0.5× Tris-borate-EDTA (14Maniatis T. Fritsch E.F. Sambrook J. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) at 180 V constant voltage for 45 min. Gels were dried and autoradiographed for 2–4 h at −80 °C. Proteolytic activity was monitored by incubating 2 μl of nuclear extract aliquots prior to the binding reaction either with whole serum or with purified serum protein fractions in 5–10 μl of buffer D at 25 °C for 1 h. When needed, concentrated purified material was prediluted 10–100-fold with buffer D. Human serum (male) derived from whole clotted blood was purchased from Sigma. Solid ammonium sulfate was added to 500 ml of serum to 25% saturation. After centrifugation at 10,000 × g for 15 min, the supernatant was collected, and proteins were further precipitated by increasing the ammonium sulfate concentration to 50% saturation. The centrifugation was repeated, and the resulting pellet containing the bulk of the proteolytic activity was resuspended in 100 ml of buffer T (20 mm Tris-HCl, pH 7.5, 2 mmMgSO4, 0.1 mm EDTA, and 200 mmKCl). The solution was dialyzed overnight against 2 liters of buffer T with one change of buffer. The dialyzed material was loaded on a 10-ml DEAE Sepharose Fast Flow (Amersham Pharmacia Biotech) column preequilibrated with buffer T. The column was subsequently washed with three 20-ml portions of buffer T containing 300, 350, and 400 mm KCl. The bulk of the proteolytic activity was eluted with 30 ml of buffer T containing 700 mm KCl. Proteins were precipitated in ammonium sulfate at 60% saturation. After centrifugation, the pellet was resuspended in 5 ml of buffer D and dialyzed against 500 ml of the same buffer. The material was then loaded on a 1-ml HiTrap heparin-agarose column (Amersham Pharmacia Biotech) preequilibrated with buffer D. The column was washed with 10 ml of buffer D, and the proteins were eluted with 18 ml of a linear KCl gradient (100–700 mm). Fractions of 1 ml were collected, and the proteolytic activity was monitored as described above. One milliliter of HeLa cell nuclear extract was incubated with 3 μl of heparin-agarose-purified protease for 1 h at 25 °C. The reaction was stopped by the addition of 15 μg of a peptide containing the KPI domain of APP (15Wagner S.L. Siegel R.S. Vedvick T.S. Raschke W.C. Van Nostrand W.E. Biochem. Biophys. Res. Commun. 1992; 186: 1138-1145Crossref PubMed Scopus (59) Google Scholar). The KCl concentration in the reaction mixture was adjusted to 200 mm, and the reaction products were loaded on a 1-ml HiTrap SP Sepharose column preequilibrated with buffer D containing 200 mm KCl. The column was washed with 5 ml of buffer D containing 200 mm KCl and 4 ml of the same buffer containing 300 mm KCl. Proteins were eluted with 20 ml of a 300–700 mm linear KCl concentration gradient. Fractions of 1 ml were collected and analyzed by mobility shift electrophoresis. An Amersham Pharmacia Biotech Superose 6HR 10/30 gel filtration column was equilibrated with buffer D containing 500 mm KCl, 2.5% CHAPS and calibrated with a set of globular proteins (Amersham Pharmacia Biotech HMW Calibration kit). SP Sepharose chromatography fractions 19–23 containing mobility shift activity were combined, supplemented with 2.5% CHAPS, concentrated on a Centricon-10 device (Amicon), and loaded on the gel filtration column. The gel filtration was performed at a 0.4 ml/min flow rate. Fractions of 0.5 ml were collected and analyzed by either mobility shift electrophoresis or Western blotting. Antibodies against the N- and C-terminal CTCF sequences were described elsewhere (5Vostrov A.V. Quitschke W.W. J. Biol. Chem. 1997; 272: 33353-33359Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). They were affinity purified with a Pierce Sulfo-link kit and used as primary antibodies in the Western blotting ECL procedure (Amersham Pharmacia Biotech), which was carried out according to manufacturer's instructions. SDS-PAGE of proteins was performed with the Laemmli Tris-glycine system (14Maniatis T. Fritsch E.F. Sambrook J. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). For some applications, 4–20% precast gradient Ready Gels (Bio-Rad) were used. When indicated, reducing agent (β-mercaptoethanol) was omitted in the sample buffer. Gels were stained with Coomassie Brilliant Blue R-250 and photographed. Proteins were extracted from gels and renatured as described (16Wang J. Nishiyama K. Araki K. Kitamura D. Watanabe T Nucleic Acids Res. 1987; 15: 10105-10116Crossref PubMed Scopus (15) Google Scholar) with some modifications. After electrophoresis under nonreducing conditions, the gel was stained with cold 0.25M KCl, and the protein bands were excised. The gel slices were weighed, rinsed with nonreducing sample buffer, minced, and incubated with an equal volume of nonreducing sample buffer at 75 °C for 20 min. Extracted protein samples were collected with an Amicon Micropure .22 spin device. The gel extraction was repeated, and the samples were combined. Aliquots of the extracted proteins were analyzed by SDS-PAGE. For renaturing purposes, the remaining extracted samples were supplemented with 9 volumes of 50 mm Tris-HCl, pH 7.6, 0.1 mm EDTA, 0.15m NaCl, and 0.1% SDS. To each sample was added as a carrier 5 μl of DEAE chromatography (see above) flow-through fraction. This fraction exhibited no protelytic activity and was thus preferred over bovine serum albumin, which could carry traces of endogenous serum-derived proteolytic activity. The proteins were precipitated with 5 volumes of acetone at −20 °C overnight. Protein precipitates were centrifuged at 14,000 × gfor 10 min, air dried, resuspended in 20 μl of 6 mguanidine hydrochloride, and incubated at 25 °C for 1 h. Subsequently, the concentration of guanidine hydrochloride in the samples was reduced to ∼5, 4, 3, 2, and 1.2 m by the addition of aliquots of buffer D at 10-min intervals. Residual guanidine hydrochloride was removed from the samples by desalting on Amersham Pharmacia Biotech G-25 spin columns preequilibrated with buffer D. Proteolytic activity was analyzed in the renatured samples as described above. For protein sequencing, samples were separated by SDS-PAGE in the presence of β-mercaptoethanol, transferred to polyvinylidene difluoride membrane, and stained with amido black. Bands corresponding to 44- and 30-kDa proteins were excised. N-terminal sequencing was performed by Harvard Microchem (Cambridge, MA) Ten microliters of heparin-agarose-purified protease was incubated with 10 μCi of [3H]diisopropylfluorophosphate ([3H]DFP) [NEN] at 37 °C for 1 h. The reaction products were analyzed by SDS-PAGE both under reducing and nonreducing conditions. The gel was stained with Coomassie Brilliant Blue R-250, impregnated with [3H]Enchance (NEN Life Science Products), dried, and exposed to x-ray film at −80 °C for 72 h. The protease was purified to electrophoretic homogeneity by cation exchange chromatography in the presence of 6 m urea. Specifically, heparin-agarose-purified protease was diluted with 4 volumes of buffer D containing 6 m urea and was concentrated with a Centriplus-10 device (Amicon) to the original volume. The resulting material was diluted with an additional three volumes of buffer D containing 6 m urea, and the concentration procedure was repeated. The final concentrate was loaded on an UnoS 0.12-ml polishing column (Bio-Rad) preequilibrated with buffer D containing 6 m urea. Proteins were eluted with 5 ml of a 100–600 mm linear KCl gradient in buffer D containing 6 m urea. 200-μl fractions were collected, aliquoted, and immediately frozen at −80 °C. 0.05 μl of heparin-agarose-purified protease was incubated in 4 μl of buffer D with the indicated amounts of the protease inhibitors (Fig. 7) for 10 min at 25 °C. Subsequently, the protease assay was performed as described above. A recombinant KPI domain peptide comprising amino acids 285–345 of the APP-751 protein (15Wagner S.L. Siegel R.S. Vedvick T.S. Raschke W.C. Van Nostrand W.E. Biochem. Biophys. Res. Commun. 1992; 186: 1138-1145Crossref PubMed Scopus (59) Google Scholar) and a mutated version of the recombinant peptide with a single substitution of arginine with isoleucine at position 301 were kindly provided by W. Van Nostrand. When nuclear extract from HeLa cells was incubated with an 80-mer oligonucleotide containing the APBβ domain of the APP promoter, a characteristic mobility shift complex b was formed (Fig. 1, lane 2). This complex is the result of transcription factor CTCF binding to the APBβ recognition sequence (5Vostrov A.V. Quitschke W.W. J. Biol. Chem. 1997; 272: 33353-33359Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). However, upon incubating the nuclear extract for 1 h at 25 °C with FCS before assembling the binding reaction, a substantial change in the electrophoretic mobility shift pattern occurred (Fig. 1, lanes 3–6). With increasing amounts of FCS in the preincubation reaction, the original mobility shift complex b was gradually eliminated as two new complexes, b1 and b2,with higher electrophoretic mobilities emerged. Moreover, at the highest concentration of FCS, complex b1 was also eliminated and only b2, the complex with the highest mobility, remained. A similar change in the mobility shift pattern was observed with adult bovine, horse, or rabbit serum (not shown), as well as with an ∼5–10-fold lower concentration of human serum (Fig. 1,lanes 7–10). They also occurred when crude nuclear extract was replaced with purified CTCF. However, significantly lower amounts of serum were required to achieve comparable results (not shown). The most plausible explanation for this alteration in mobility shift behavior is a proteolytic cleavage of the CTCF protein by a serum enzyme. To isolate the putative proteolytic activity, human serum was initially fractionated by ammonium sulfate precipitation, and the proteolytic activity was recovered in fractions that precipitated between 30 and 50% saturation. The precipitated material was solubilized and loaded on a DEAE-Sepharose column. The active fraction, which eluted at a 700 mm KCl concentration, was again precipitated with ammonium sulfate, solubilized, and further purified by affinity chromatography on heparin-agarose. The proteolytic activity eluted over a broad range of KCl concentration from 250 to 500 mm (Fig.2 B), which coincided with the concentration profile of eluted protein as measured by A 280 (Fig. 2 A). In addition to numerous other proteins distributed over a wide molecular mass range, the elution fractions contained a major protein with an approximate molecular mass of 70 kDa (Fig. 2 C). Fractions 12–18 that contained the highest level of activity (Fig. 2) were combined and used for further experiments. Because the excess of serum distorts the bands on polyacrylamide gels during mobility shift electrophoresis, we were restricted to agarose gels in our initial experiments (Figs. 1 and 2). Employing heparin-agarose-purified material instead of whole serum for nuclear extract preincubation allowed us to switch to polyacrylamide gels in mobility shift assays. This enabled us to study the mobility shift patterns at a higher level of resolution (Fig.3 A). In addition to the three major complexes b, b1, and b2 observed in agarose gels (Fig. 2), two additional complexes b0 and b10 could be resolved (Fig. 3 A). As with whole serum, increasing amounts of partially purified active material in the preincubation reaction lead to a gradual increase in the amount of complex b2 at the expense of all other bands, which were progressively eliminated from the mobility shift pattern. At very high concentrations of material purified from human serum there was also a gradual decrease in complex b2 (Fig.3 A). To demonstrate that the changes in the mobility shift patterns were indeed because of proteolytic digestion of the CTCF protein, we proceeded with separation of the treated nuclear extract and analysis of the resulting fractions by mobility shift electrophoresis and Western blotting. Initially, nuclear extract in preparative quantities was treated with heparin-agarose-purified active material. This resulted in a mobility shift pattern where the prominent complexes b, b1, and b2, as well as minor complexes b0 and b10, were represented (Fig.3 B, lane 1). The treated extract was loaded on a cation exchange column, and all proteins generating the mobility shift complexes eluted in fractions 19–24 (Fig. 3 B, lanes 3–8). This purification step considerably reduced total protein concentration in the preparation, and the combined fractions 19–23 (Fig. 3 B, lanes 3–7) were used as starting material (Fig. 4 A, lane 1) for subsequent gel filtration. Fast protein liquid chromatography gel filtration was performed using a Amersham Pharmacia Biotech Superose 6 column. Mobility shift analysis of the loading material (Fig. 4 A, lane 1) revealed the presence of all binding complexes that were observed prior to the cation exchange chromatography (Fig. 3 B, lane 1). Meanwhile, Western blot analysis of the cation exchange-purified material with antibodies against either the N- (Fig.4 B) or C-terminal (Fig. 4 C) end of CTCF recognized a protein with an apparent molecular mass of 140 kDa (band p) corresponding to native CTCF (Fig. 4, B and C,lanes 1 and 2). Antibodies against the N-terminal part of the protein also reacted with a 120-kDa band [p0], as well as with a much less pronounced band at 130 kDa [p01] (Fig. 4 B, lane 2). In contrast, antibodies against the C-terminal end of CTCF recognized a new 70-kDa (p1) immunoreactive band (Fig. 4 C,lane 2). The emergence of these lower molecular weight bands that are differentially recognized by antibodies either against the C-terminal or the N-terminal end proves that CTCF indeed undergoes proteolytic cleavage during incubation with the purified fraction of serum. Gel filtration of the fragments allowed an estimation of the number and relative positions of the cleavage sites. Both antibodies recognized the peak of intact CTCF that eluted in fractions 13 and 14 (Fig. 4,B and C, lanes 4 and 5). This represented an apparent molecular mass of 400 kDa as determined by calibration with globular protein standards. The corresponding binding complex b was observed in the same fractions by mobility shift electrophoresis (Fig. 4 A, lanes 2 and 3). Fragment p0 eluted in fractions 15 and 16 (Fig. 4 B, lanes 6 and 7), generating the corresponding binding complex b0 (Fig. 4 A,lanes 4 and 5). Furthermore, fragment p1 eluted in fractions 17 and 18 (Fig. 4 C,lanes 8 and 9) where the matching complex b1 was observed (Fig. 4 A, lanes 6 and 7). The CTCF fragments corresponding to the complexes b10 and b2, which eluted in fractions 19–21, could not be recognized by either antibody (Fig. 4 A,lanes 8–10; B and C, lanes 9–11). According to both SDS-PAGE and gel filtration, the CTCF fragment producing the faint band p01 migrated to a position between the full-length CTCF and fragment p0 (Fig.4 B, lanes 5 and 6). However, no binding complex has been identified that could be assigned to this fragment, presumably because its low prevalence does not allow detection. Alternatively, the hypothetical binding complex formed by p01 may not be separable from binding complexes b and b0 under the applied mobility shift electrophoresis conditions. Assuming that CTCF binds to DNA as a monomer (10Köhne A.C. Baniahmad A. Renkawitz R. J. Mol. Biol. 1993; 232: 747-755Crossref PubMed Scopus (35) Google Scholar), the results of the gel filtration suggest that there are three prominent proteolytic cleavage sites on the CTCF molecule. One site is located between the N terminus and the zinc finger DNA binding domain, and two are located between the zinc finger domain and the C terminus (Fig. 4 D,arrows). Incomplete cleavage at these sites would produce numerous protein fragments. Among them, the fragments p0, p01, p1, p10, and p2, which are schematically shown in Fig. 4 D, would contain the zinc finger domain and produce the corresponding binding complexes. Fragments p0 and p01 would be recognized by the N-terminal but not the C-terminal antibody. Similarly, protein p1 would be only recognized by the C-terminal antibody. Finally, neither antibody would recognize the proteins p10and p2 that produce binding complexes b10 and b2. Depending on the conditions of the cleavage reaction, additional minor binding complexes could be observed on the mobility shift gel between the b1 and b2 bands. An arrowhead in Fig. 4 A (fraction 18) indicates an example of such a weak binding complex, designated b3. Incidentally, an exceedingly weak band (p31) that reacted with the C-terminal antibody was detected migrating slightly ahead of fragment p1 in fractions 17 and 18 (Fig. 4 C,arrowheads). This suggests the existence of an additional cleavage site on the CTCF molecule in close proximity to the major site that cleaves off the N terminus generating fragment p1(Fig. 4 D, arrowhead). Cleavage at that site would generate three additional hypothetical CTCF protein fragments containing the zinc finger DNA binding domain (Fig. 4 D, fragments p31, p32, and p33, indicated by a bracket). Fragment p31 would thus retain an intact C-terminal sequence of CTCF and therefore react with antibodies against the C terminus. The same fragment could conceivably account for the appearance of the minor binding complex b3observed in fraction 18 on the mobility shift gel. Because of the lower prevalence of CTCF protein cleavage at this site, it is possible for example that it only becomes accessible after cleavage at the N-terminal p1 site. However, we consider it to be a marginal cleavage site for the protease, and we therefore disregard it in the further discussion of the results. Initial attempts to further purify the protease under nondenaturing conditions were unsuccessful. Employing a variety of separation techniques, we observed the same major proteins were co-purified. Preliminary results indicated that the protease probably exists in serum as part of a high molecular weight multiprotein complex (data not shown). Therefore, we proceeded with the protease identification using preparative SDS-PAGE. Proteins eluted from the heparin-agarose column were separated by SDS-PAGE under nonreducing conditions (Fig.5 A, lane 1). Four major protein bands (pp1-pp4) were extracted from the gel and further analyzed by SDS-PAGE. Under nonreducing conditions the extracted proteins produced homogenous bands (Fig. 5 A, lanes 2–5). Treatment with β-mercaptoethanol differentially affected the electrophoretic behavior of proteins pp1-pp4 (Fig. 5 A,lanes 7–10). In particular, protein pp2 migrated as a single 70-kDa band under nonreducing conditions (Fig. 5 A,lane 3), whereas in the presence of β-mercaptoethanol it produced two bands with molecular masses of ∼44 and 30 kDa (Fig.5 A, lane 8). The extracted proteins were renatured and incubated with nuclear extract, followed by mobility shift electrophoresis (Fig.5 B, lanes 1–4). Proteolytic activity was detected only in the sample containing the pp2 protein (Fig.5 B, lane 2). As a control, the total heparin-agarose-purified protease fraction was denatured under reducing, as well as nonreducing, conditions and then renatured while omitting electrophoretic separation. Proteolytic activity could only be restored from the nonreduced sample (Fig. 5 B, lanes 5 and 6). From these experiments we conclude that the proteolytic activity is associated with the 70-kDa protein pp2. This protein comprises two polypeptide chains with apparent molecular masses of 44 and 30 kDa, which are connected via disulphide bonds. Disruption of the bonds irreversibly abolishes activity. To further characterize and identify the protease, the heparin-agarose-purified material was subjected to SDS-PAGE in the presence of β-mercaptoethanol. Proteins were subsequently transferred to polyvinylidene difluoride membrane and visualized by Amido Black staining. Both the 44- and 30-kDa proteins were excised, and the N-terminal sequences of the proteins were determined. The 44- and 30-kDa proteins contained the sequences SLLESLDPDTP and IYGGFKSTAGAKHP, respectively, and they displayed a perfect match with the sequence of the human PHBP protein described by Choi-Miura et al.(12Choi-Miura N.-H. Tobe T. Sumiya J.-I. Nakano Y. Sano Y. Mazda T. Tomita M. J. Biochem. 1996; 119: 1157-1165Crossref PubMed Scopus (121) Google Scholar). PHBP cDNA sequence data (12Choi-Miura N.-H. Tobe T. Sumiya J.-I. Nakano Y. Sano Y. Mazda T. Tomita M. J. Biochem. 1996; 119: 1157-1165Crossref PubMed Scopus (121) Google Scholar) suggested that the protein contained a putative serine protease domain. In such a case the active center of the protease might form a covalent bond with DFP. To provide additional evidence that the protease is PHBP, we performed [3H]DFP labeling of the protease. We incubated the
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