Glycosaminoglycan Binding Properties of Annexin IV, V, and VI
1998; Elsevier BV; Volume: 273; Issue: 16 Linguagem: Inglês
10.1074/jbc.273.16.9935
ISSN1083-351X
AutoresReiko Ishitsuka, Kyoko Kojima, Hideko Utsumi, Haruko Ogawa, Isamu Matsumoto,
Tópico(s)Computational Drug Discovery Methods
ResumoWe have previously demonstrated that annexin IV, one of the calcium/phospholipid-binding annexin family proteins, binds to glycosaminoglycans (GAGs) in a calcium-dependent manner (Kojima, K., Yamamoto, K., Irimura, T., Osawa, T., Ogawa, H., and Matsumoto, I. (1996) J. Biol. Chem. 271, 7679–7685). In this study, we investigated the GAG binding specificities of annexins IV, V, and VI by affinity chromatography and solid phase assays. Annexin IV was found to bind in a calcium-dependent manner to all the GAG columns tested. Annexin V bound to heparin and heparan sulfate columns but not to chondroitin sulfate columns. Annexin VI was adsorbed to heparin and heparan sulfate columns in a calcium-independent manner, and to chondroitin sulfate columns in a calcium-dependent manner. An N-terminal half fragment (A6NH) and a C-terminal half fragment (A6CH) of annexin VI, each containing four units, were prepared by digestion with V8 protease and examined for GAG binding activities. A6NH bound to heparin in the presence of calcium but not to chondroitin sulfate C, whereas A6CH bound to heparin calcium-independently and to chondroitin sulfate C calcium-dependently. The results showed that annexin IV, V, and VI have different GAG binding properties. Some annexins have been reported to be detected not only in the cytoplasm but also on the cell surface or in extracellular components. The findings suggest that the some annexins function as recognition elements for GAGs in extracellular space. We have previously demonstrated that annexin IV, one of the calcium/phospholipid-binding annexin family proteins, binds to glycosaminoglycans (GAGs) in a calcium-dependent manner (Kojima, K., Yamamoto, K., Irimura, T., Osawa, T., Ogawa, H., and Matsumoto, I. (1996) J. Biol. Chem. 271, 7679–7685). In this study, we investigated the GAG binding specificities of annexins IV, V, and VI by affinity chromatography and solid phase assays. Annexin IV was found to bind in a calcium-dependent manner to all the GAG columns tested. Annexin V bound to heparin and heparan sulfate columns but not to chondroitin sulfate columns. Annexin VI was adsorbed to heparin and heparan sulfate columns in a calcium-independent manner, and to chondroitin sulfate columns in a calcium-dependent manner. An N-terminal half fragment (A6NH) and a C-terminal half fragment (A6CH) of annexin VI, each containing four units, were prepared by digestion with V8 protease and examined for GAG binding activities. A6NH bound to heparin in the presence of calcium but not to chondroitin sulfate C, whereas A6CH bound to heparin calcium-independently and to chondroitin sulfate C calcium-dependently. The results showed that annexin IV, V, and VI have different GAG binding properties. Some annexins have been reported to be detected not only in the cytoplasm but also on the cell surface or in extracellular components. The findings suggest that the some annexins function as recognition elements for GAGs in extracellular space. Annexins are a family of about 13 structurally related calcium-dependent phospholipid-binding proteins. They consist of four or eight conserved repeating structures of approximately 70 amino acid residues and with an N-terminal domain that is highly variable in both sequence and length, which distinguishes different family members. The N-terminal domain is thought to confer functional diversity, whereas this family's common calcium and phospholipid binding activities probably reside in each repeating unit. Several functions proposed for annexins based on their membrane and calcium binding activities include inhibition of phospholipase A2 and blood coagulation, regulation of membrane traffic and exocytosis, binding to cytoskeletal proteins, transmembrane channel activity, and intracellular signaling as a kinase substrate (1Raynal P. Pollard H.B. Biochim. Biophys. Acta. 1994; 1197: 63-93Crossref PubMed Scopus (1030) Google Scholar, 2Swairjo M.A. Seaton B.A. Annu. Rev. Biophys. Biomol. Struct. 1994; 23: 193-213Crossref PubMed Scopus (190) Google Scholar, 3Edwards H.C. Moss S.E. Mol. Cell. Biochem. 1995; 149/150: 293-299Crossref Scopus (30) Google Scholar). However, the complete range of their physiological functions has not been elucidated. We have previously purified a calcium-dependent carbohydrate-binding protein, p33/41, from bovine kidney by two-step affinity chromatography on heparin and fetuin columns (4Kojima K. Ogawa H.K. Seno N. Matsumoto I. J. Chromatogr. 1992; 597: 323-330Crossref PubMed Scopus (16) Google Scholar). Amino acid sequence analyses and cDNA cloning (5Kojima K. Ogawa H.K. Seno N. Yamamoto K. Irimura T. Osawa T. Matsumoto I. J. Biol. Chem. 1992; 267: 20536-20539Abstract Full Text PDF PubMed Google Scholar, 6Kojima K. Yamamoto K. Irimura T. Osawa T. Ogawa H. Matsumoto I. J. Biol. Chem. 1996; 271: 7679-7685Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar) revealed that p33/41 is identical to annexin IV and is a newly identified carbohydrate-binding protein having neither known conserved amino acid sequences in carbohydrate recognition domains (CRDs) 1The abbreviations used are: CRD, carbohydrate recognition domain; GAG, glycosaminoglycan; PAGE, polyacrylamide gel electrophoresis; TBS, Tris-buffered saline; ELISA, enzyme-linked immunosorbent assay; HRP, horseradish peroxidase; PVDF, polyvinylidene difluoride; AN, annexin; GST, glutathione S-transferase; HPLC, high performance liquid chromatography; UTI, urinary trypsin inhibitor; MTBS, Tris-buffered saline containing 4 mm2-mercaptoethanol; BSA, bovine serum albumin. of animal lectins (7Drickamer K. Taylor M. Annu. Rev. Cell Biol. 1993; 9: 237-264Crossref PubMed Scopus (710) Google Scholar,8Gabius H.J. Eur. J. Biochem. 1997; 243: 543-576Crossref PubMed Scopus (484) Google Scholar) nor heparin-binding motifs reported for a number of heparin-binding proteins such as growth factors and extracellular matrix molecules (9Cardin A.D. Weintraub H.J.R. Arteriosclerosis. 1989; 9: 21-32Crossref PubMed Google Scholar,10Guo N.H. Krutzsch H.C. Negre E. Zabrenetzky V.S. Roberts D.D. J. Biol. Chem. 1992; 267: 19349-19355Abstract Full Text PDF PubMed Google Scholar). Proteoglycans (macromolecules consisting of a protein core and GAG side chains, which are found in the extracellular matrix, on the cell surface, and in secretory granules) are involved in a broad range of activities. Some of their functions are likely to depend on the direct interactions between GAGs and the other molecules (11Ruoslahti E. J. Biol. Chem. 1989; 264: 13369-13372Abstract Full Text PDF PubMed Google Scholar, 12Kjellen L. Lindahl U. Annu. Rev. Biochem. 1991; 60: 443-475Crossref PubMed Scopus (1678) Google Scholar, 13Iozzo R.V. Murdoch A.D. FASEB J. 1996; 10: 598-614Crossref PubMed Scopus (550) Google Scholar). Therefore, annexin IV was assumed to be one kind of lectin that recognizes these GAGs of proteoglycans. In this study, we investigated the GAG binding activities of annexin IV, V, and VI and their binding specificities to GAGs. Bovine brain and liver were obtained from a local slaughterhouse and used fresh or stored at −80 °C. Heparin (porcine intestinal mucosa) was purchased from Wako Pure Chemicals (Osaka, Japan). Heparan sulfate was prepared from porcine kidney in our laboratory (14Akiyama F. Seno N. Natl. Sci. Rep. Ochanomizu Univ. Tokyo. 1978; 29: 147-153Google Scholar). Other GAGs (chondroitin, chondroitin sulfate A (whale cartilage, super-special grade), chondroitin sulfate C (shark cartilage, super-special grade), and dermatan sulfate (porcine skin, super-special grade)) were purchased from Seikagaku Kogyo (Tokyo, Japan). Desulfated heparin was prepared by solvolysis of heparin according to Inoue (15Inoue Y. Nagasawa K. Carbohydr. Res. 1976; 46: 87-95Crossref PubMed Scopus (184) Google Scholar) and Ogamo (16Ogamo A. Metori A. Uchiyama H. Nagasawa K. Carbohydr. Res. 1989; 193: 165-172Crossref Scopus (24) Google Scholar), and desulfation was confirmed by measuring the sulfate content as reported previously (17Dodgson K.S. Price R.G. Biochem. J. 1962; 84: 106-110Crossref PubMed Scopus (1296) Google Scholar). Affinity gels coupled with GAGs were prepared by use of epoxy-activated Sepharose 4B (18Matsumoto I. Seno N. Golovtchenko-Matsumoto A.M. Osawa T. J. Biochem. (Tokyo). 1980; 85: 1091-1098Crossref Scopus (143) Google Scholar, 19Kitagaki-Ogawa H. Yathogo T Izumi M. Hayashi M Kashiwagi H. Matsumoto I. Seno N. Biochim. Biophys. Acta. 1990; 1033: 49-56Crossref PubMed Scopus (52) Google Scholar). Proteases, endoproteinase Lys-C (Lysobacter enzymogenes, sequencing grade), and V8 protease were purchased from Boehringer Mannheim (Mannheim, Germany). Rabbit polyclonal antibodies to annexin IV, V, and VI were prepared in our laboratory (20Harlow E. Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1988Google Scholar). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG antibody was purchased from Kirkegaard & Perry Laboratories, Inc. (Gaithersburg, MD). Bovine annexin IV and V cDNA were inserted into plasmid pGEX-3X (Amersham Pharmacia Biotech, Uppsala, Sweden), and recombinant proteins were produced as GST fusion proteins as described previously (6Kojima K. Yamamoto K. Irimura T. Osawa T. Ogawa H. Matsumoto I. J. Biol. Chem. 1996; 271: 7679-7685Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Annexin V was purified from bovine brain extract. Bovine brain was homogenized in 4.5 volumes (v/w) of 2 mm EDTA in MTBS (150 mm NaCl, 4 mm 2-mercaptoethanol, 0.5 mmphenylmethanesulfonyl fluoride, 10 mm Tris-HCl, pH 7.5). The homogenate was shaken for 30 min and centrifuged at 40,000 ×g for 30 min. CaCl2 was added to the supernatant to a final concentration of 7 mm. After 15 min on ice, the fraction was centrifuged at 40,000 × g for 30 min. The pellet was washed twice with 5 mm CaCl2-MTBS. The pellet was then resuspended in 2 mm EDTA-MTBS and centrifuged at 100,000 × g for 1 h. The final supernatant was applied to a heparin column which had been equilibrated with 2 mm EDTA-MTBS, and the flow-through fraction was recovered; CaCl2 was added to a final concentration of 5 mm excess. After 30 min on ice, it was applied to another heparin column equilibrated with 5 mmCaCl2-MTBS. After the column had been washed with 5 mm CaCl2-MTBS, proteins bound to the column were eluted with 2 mm EDTA-MTBS. The two forms of annexin V were separated from one another by anion-exchange high performance liquid chromatography (HPLC), according to Donato et al.(21Donato R. Giambanco I. Pula G. Bianchi R. FEBS Lett. 1990; 262: 72-76Crossref PubMed Scopus (17) Google Scholar). Briefly, the protein fractions were desalted, resuspended in 10 mm Tris-HCl (pH 7.6), and applied to a column of DEAE (TSK DEAE 5pw, inner diameter 7.5 × 0.75 cm; Tosoh, Tokyo, Japan) equilibrated in the same buffer. The column was eluted using a linear NaCl gradient (0–1 m). Annexin VI was purified from bovine liver extract by the method of Creutz et al. (22Creutz C.E. Zaks W.J. Hamman H.C. Crane S. Martin W.H. Gould K.L. Oddie K.M. Parsons S.J. J. Biol. Chem. 1987; 262: 1860-1868Abstract Full Text PDF PubMed Google Scholar) with some modifications. 150 mg of bovine liver was homogenized in 400 ml of buffer A (150 mm NaCl, 5 mm EGTA, 4 mm 2-mercaptoethanol, 0.5 mmphenylmethanesulfonyl fluoride, 50 mm HEPES-NaOH, pH 7.3). After the homogenate was centrifuged at 500 × g for 10 min, the pellet fraction was discarded, and CaCl2 was added to the supernatant to a final concentration of 7 mm. The extract was centrifuged at 27,000 × g for 30 min. The supernatant was discarded, and the pellet fraction resuspended in 200 ml of buffer B (150 mm NaCl, 2 mmCaCl2, 4 mm 2-mercaptoethanol, 0.5 mm phenylmethanesulfonyl fluoride, 50 mmHEPES-NaOH, pH 7.3). This fraction was then centrifuged at 27,000 × g for 30 min, resuspended in 200 ml of buffer B, and centrifuged again at 27,000 × g for 30 min to complete the washing of the particulate fraction in Ca2+-containing buffer. The final pellet was resuspended in 60 ml of buffer A and centrifuged at 27,000 × g for 30 min. The supernatant was recentrifuged at 100,000 × g for 60 min. The supernatant containing the proteins extracted in EGTA was applied directly to a 1.6 × 20-cm column of phenyl-Sepharose (Amersham Pharmacia Biotech) equilibrated with buffer A. Ammonium sulfate was added to the flow-through fractions (66%) to concentrate the proteins by precipitation. The protein precipitate was collected by centrifugation at 27,000 × g for 45 min, resuspended in 5 ml of 0.3 m sucrose, 25 mm HEPES-NaOH, pH 7.3, and then desalted by passage through a Sephadex G-25 column (Amersham Pharmacia Biotech) equilibrated in the same buffer. The protein fraction was then applied to a DEAE-cellulose column equilibrated in the same buffer, and adsorbed proteins were eluted with a salt gradient formed by mixing with 1 m KCl, 0.3m sucrose, 25 mm HEPES-NaCl, pH 7.3. In the final purification step, the fraction eluted with 240 mmKCl from a DEAE-cellulose column was applied to a heparin-Sepharose 4B column (inner diameter 0.8 × 6 cm) equilibrated with 150 mm NaCl, 5 mm CaCl2, and 10 mm Tris-HCl (pH 7.5). The column was washed with the equilibration buffer, and the adsorbed protein was eluted with 2 mm EDTA and 300 mm NaCl by stepwise elution. Annexin VI was digested with V8 protease at a ratio of 5:1 (w/w) for 1.5 h at 37 °C in 200 mm NaCl, 5 mm CaCl2, 1 mm dithiothreitol, and 20 mm Tris-HCl (pH 7.5) (23Hazarika P. Sheldon A. Kaetzel M.A. Diaz-Monoz M. Hamilton S.L. Dedman J.R. J. Cell. Biochem. 1991; 46: 86-93Crossref PubMed Scopus (25) Google Scholar). The size of the fragments obtained was determined by gel filtration HPLC using a Shodex Protein KW-803 column (inner diameter 0.8 × 6 cm; Showa Denko, Tokyo, Japan). One-dimensional SDS-PAGE was carried out by the method of Laemmli (24Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207208) Google Scholar). The electrophoresed proteins were transferred to a polyvinylidene difluoride (PVDF) membrane and then probed with rabbit polyclonal antibodies, and the bound antibodies were detected with HRP-conjugated anti-rabbit IgG antibody and 4-chloro-1-naphthol. Annexin V and VI were digested with proteinases, and peptide fragments produced were purified by reverse-phase HPLC using a C18 column (Waters, Burlington, MA) or were subjected to SDS-PAGE and successive electroblotting onto a PVDF membrane. The PVDF membrane containing peptide fragments was cut into small pieces for direct sequencing. Amino acid sequencing was performed with a pulse-liquid protein sequencer 476A (Applied Biosystems Japan, Tokyo, Japan). All GAG-Sepharose gels were prepared by the methods described previously (18Matsumoto I. Seno N. Golovtchenko-Matsumoto A.M. Osawa T. J. Biochem. (Tokyo). 1980; 85: 1091-1098Crossref Scopus (143) Google Scholar, 19Kitagaki-Ogawa H. Yathogo T Izumi M. Hayashi M Kashiwagi H. Matsumoto I. Seno N. Biochim. Biophys. Acta. 1990; 1033: 49-56Crossref PubMed Scopus (52) Google Scholar). 100 mg of GAG was incubated with 5 g of amino-Sepharose and 100 mg ofN-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline/5 ml of 40% (v/v) ethanol at 40 °C overnight. Then the gel was acetylated to block the remaining amino groups. The amounts of immobilized GAGs were calculated from the hexosamine values of each GAG reported previously (14Akiyama F. Seno N. Natl. Sci. Rep. Ochanomizu Univ. Tokyo. 1978; 29: 147-153Google Scholar, 16Ogamo A. Metori A. Uchiyama H. Nagasawa K. Carbohydr. Res. 1989; 193: 165-172Crossref Scopus (24) Google Scholar) and from actual hexosamine contents of the gels obtained by the methods of Blix (25Blix G. Acta. Chem. Scand. 1948; 2: 467Crossref Google Scholar) and Gardell (26Gardell S. Acta. Chem. Scand. 1953; 7: 207-215Crossref Google Scholar). GAG binding activity was assayed by affinity column chromatography. Annexins were applied to a heparin-, heparan sulfate-, N-desulfated heparin-,N,O-desulfated heparin-, chondroitin sulfate A-, chondroitin sulfate B-, chondroitin sulfate C-, or chondroitin-Sepharose column (inner diameter, 0.3 × 7 cm) in the presence of 5 mmCaCl2, and the bound protein was eluted by stepwise elution with 2 mm EDTA and then 300 mm NaCl. The amount of protein in each fraction was monitored by enzyme-linked immunosorbent assay (ELISA) using rabbit anti-annexin polyclonal antibodies, HRP-conjugated anti-rabbit IgG antibodies, and o-phenylene diamine. GAG binding activity of peptide fragments produced from annexin VI were assayed using chondroitin sulfate C- and heparin-Sepharose 4B columns under the same conditions as those for intact annexin VI except for column size (inner diameter 0.5 × 7 cm). GST fusion annexin IV and annexin V, and native annexin VI were used. GAGs were coupled with the aid of N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (19Kitagaki-Ogawa H. Yathogo T Izumi M. Hayashi M Kashiwagi H. Matsumoto I. Seno N. Biochim. Biophys. Acta. 1990; 1033: 49-56Crossref PubMed Scopus (52) Google Scholar). A microtiter plate was coated with 100 μl of BSA-conjugated GAG in TBS (1 μg/ml heparin-BSA or 10 μg/ml chondroitin sulfate-BSA as protein concentration) at 4 °C overnight. The plate was blocked with 3% BSA in TBS for 2 h. Annexins at various concentration were added to each well and incubated for 1 h in the presence of 5 mm CaCl2. The amounts of GST-annexin IV or GST-annexin V, and annexin VI bound to BSA-GAG were monitored by ELISA using anti-GST antibodies and anti-annexin VI antibodies, respectively. On SDS-PAGE, proteins purified from bovine brain produced double bands corresponding to proteins with molecular masses of 34 and 35 kDa as was seen for annexin V (27Learmonth M.P. Howell S.A. Harris A.C.M. Amess B. Patel Y. Giambanco I. Bianchi R. Pula G. Ceccarelli P. Donato R. Green B.N. Aitken A. Biochim. Biophys. Acta. 1992; 1160: 76-83Crossref PubMed Scopus (26) Google Scholar) (data not shown). The partial amino acid sequences of eight peptide fragments obtained after digestion with endoproteinase Lys-C were shown to be the same as those reported for annexin V (27Learmonth M.P. Howell S.A. Harris A.C.M. Amess B. Patel Y. Giambanco I. Bianchi R. Pula G. Ceccarelli P. Donato R. Green B.N. Aitken A. Biochim. Biophys. Acta. 1992; 1160: 76-83Crossref PubMed Scopus (26) Google Scholar), i.e. 193FITIFGTRSVSHLRRVFDK211. The protein purified from bovine liver gave one band corresponding to a protein with a molecular mass of 66 kDa on SDS-PAGE (data not shown). The value obtained was equivalent to the values reported for annexin VI: 68, 70, and 73 kDa, respectively. The partial amino acid sequences of eight liver protein fragments were identical to those of annexin VI (28Sudhof T.C. Slaughter C.A. Leznicki I. Barjon P. Reynolds G.A. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 664-668Crossref PubMed Scopus (87) Google Scholar),i.e. 191WGTDEAOFIYILGNRSK217. Affinity chromatography technique was used to study the specific interactions between GAGs and annexins. GAGs have a negative charge derived from sulfate and carboxyl groups, and heparin is one of the most highly sulfated molecules among them. To examine the effect of sulfate groups of heparin on the interaction between annexins and heparin, desulfated heparins were also immobilized. For affinity chromatography, annexins were applied onto the GAG-columns in the presence of 5 mm CaCl2 and eluted with 2 mm EDTA. In the case of annexin VI, an additional elution with 300 mm NaCl was performed. The results of affinity chromatography are shown in Figs.Figure 1, Figure 2, Figure 3. Annexin IV bound to heparin and heparan sulfate in the presence of 5 mm CaCl2 and was eluted with 2 mmEDTA. Complete chemical desulfation of heparin had no effect on the binding (Fig. 1 a). Annexin IV bound to chondroitin sulfate A, B, and C, and chondroitin in a calcium-dependent manner (Fig. 1 b).Figure 2Affinity chromatography of annexin V on GAG-Sepharose gels. Annexin V was applied to heparin-related GAG columns of heparin (•), heparan sulfate (○),N-desulfated heparin (▪), and N,O-desulfated heparin (□) (a), and to chondroitin sulfate-related GAG columns of chondroitin sulfate A (•), B (○), C (▪), and chondroitin (□) (b) in the presence of 5 mmCaCl2 and eluted with 2 mm EDTA. The detection of annexin V was performed by ELISA with anti-annexin V antibodies and HRP-conjugated secondary antibodies.View Large Image Figure ViewerDownload (PPT)Figure 3Affinity chromatography of annexin VI on GAG-Sepharose gels. Annexin VI was applied to heparin-related GAG columns of heparin (•), heparan sulfate (○),N-desulfated heparin (▪), and N,O-desulfated heparin (□) (a), and to chondroitin sulfate-related GAG columns of chondroitin sulfate A (•), B (○), C (▪), and chondroitin (□) (b) in the presence of 5 mmCaCl2 and eluted with 2 mm EDTA and 300 mm NaCl. The detection of annexin VI was performed by ELISA with anti-annexin VI antibodies and HRP-conjugated secondary antibodies.View Large Image Figure ViewerDownload (PPT) As shown in Fig. 2 a, annexin V bound to heparin and heparan sulfate in a calcium-dependent manner. The desulfation of heparin significantly reduced its binding to annexin V, and the N-desulfation of heparin restored the binding only slightly, suggesting that the interaction between annexin V and heparin requires sulfate groups of heparin. In contrast, annexin V was not adsorbed onto chondroitin sulfate-related GAG-columns in the presence of 5 mm CaCl2 (Fig. 2 b). Annexin VI was found to bind to the heparin and heparan sulfate columns in the presence of 5 mm CaCl2, and the binding was not dissociated by 2 mm EDTA but was with 300 mm NaCl (Fig. 3 a). In addition, annexin VI bound to heparin even in the absence of calcium (data not shown). Therefore, annexin VI binds to heparin and heparan sulfate in a calcium-independent manner. Annexin VI bound to N-desulfated heparin in a calcium-independent manner, but the protein adsorbed toN, O-desulfated heparin was eluted with 2 mm EDTA. Annexin VI was adsorbed onto the chondroitin sulfate A, B, and C and chondroitin columns in the presence of 5 mm CaCl2 and eluted with 2 mm EDTA (Fig. 3 b). The binding of annexin VI to chondroitin sulfate-related GAGs required calcium and differed from that to heparin/heparan sulfate GAGs. The results of affinity chromatography were summarized in Table I. It is noteworthy that there are two modes of binding, one calcium-dependent and the other calcium-independent, and that the three annexins showed different binding specificities toward GAGs.Table IGlycosaminoglycan binding properties of annexinsGlycosaminoglycanAmount of GAG1-aThe concentrations of immobilized GAGs were measured as described under "Experimental Procedures."AN IVAN VAN VImg/g of gelHeparin1.8+++*Heparan sulfate4.4+++*N-Desulfated heparin4.6+−+*N,O-Desulfated heparin1.9+−+Chondroitin sulfate A4.4+−+Chondroitin sulfate B3.2+−+Chondroitin sulfate C3.9+−+Chondroitin2.4+−+1-a The concentrations of immobilized GAGs were measured as described under "Experimental Procedures." Open table in a new tab Semi-quantitative binding assays on the annexins were also performed using heparin and chondroitin sulfate conjugated with BSA. As shown in Fig. 4, annexin IV, V, and VI bound to heparin immobilized on a microtiter plate dose-dependently. In the case of chondroitin sulfate, annexin IV and VI showed calcium-dependent specific binding. On the other hand, the interaction of annexin V with chondroitin sulfate was observed only at high concentrations of annexin. Annexin VI is unique structurally, containing eight conserved repeating units, in contrast to all other annexins, which have four repeating units. To investigate the domains of annexin VI responsible for the GAG binding activities, we prepared the annexin VI fragments by a limited proteolytic digestion and examined the binding activities of the fragments by affinity chromatography. Annexin VI was partially digested with V8 protease as described under "Experimental Procedures." The fragments produced were analyzed by SDS-PAGE and subsequent staining with Coomassie Brilliant Blue. As shown in Fig. 5, three peptide fragments with molecular masses of 33, 19, and 14 kDa were observed. The assignment of each peptide fragment was performed by N-terminal amino acid sequencing after separation by SDS-PAGE and subsequent electroblotting onto a PVDF membrane. The N-terminal amino acid sequence of the 19-kDa fragment was LKGTVRPAG...... The sequence is identical to an amino acid sequence of the C-terminal half of annexin VI starting from leucine 352 in the linker region between repeat 4 and 5 (28Sudhof T.C. Slaughter C.A. Leznicki I. Barjon P. Reynolds G.A. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 664-668Crossref PubMed Scopus (87) Google Scholar, 29Comera C. Creutz C.E. Moss S.E. The Annexins. Portland Press, London1992: 86-87Google Scholar) (Fig. 6). The 14-kDa fragment gave the N-terminal sequence IADTTSGD......, which is identical to that starting from isoleucine 531. Therefore, both low molecular mass fragments were derived from the C-terminal half of annexin VI. The resistance of the 33-kDa fragment to Edman degradation indicates that this fragment is an N-terminal half because the N-terminal residue of annexin VI is blocked (28Sudhof T.C. Slaughter C.A. Leznicki I. Barjon P. Reynolds G.A. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 664-668Crossref PubMed Scopus (87) Google Scholar, 30Crompton M.R. Owens R.J. Totty N.F. Moss S.E. Waterfield M.D. Crumpton M.J. EMBO J. 1988; 7: 21-27Crossref PubMed Scopus (122) Google Scholar). Gel filtration chromatography showed that the 14- and 19-kDa fragments of annexin VI exist as an associated form with an apparent molecular mass of 33 kDa (data not shown), which we hereafter call the C-terminal half.Figure 6The sequence alignment of the bovine annexin VI fragments with human annexin VI. N-terminal amino acid sequences of the bovine annexin VI fragments are shown under the full-length amino acid sequence of human annexin VI (28Sudhof T.C. Slaughter C.A. Leznicki I. Barjon P. Reynolds G.A. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 664-668Crossref PubMed Scopus (87) Google Scholar, 30Crompton M.R. Owens R.J. Totty N.F. Moss S.E. Waterfield M.D. Crumpton M.J. EMBO J. 1988; 7: 21-27Crossref PubMed Scopus (122) Google Scholar). The amino acid substitutions found in the bovine sequence reported partially by Comera et al. (29Comera C. Creutz C.E. Moss S.E. The Annexins. Portland Press, London1992: 86-87Google Scholar) are indicated above the human sequence. Asterisks denote identical amino acids between the full-length sequence and the fragments. The 19- and 14-kDa fragments start in the linker region and at the seventh repeat, respectively. The N-terminal amino acid of annexin VI was reported to be blocked, suggesting that the 33-kDa fragment is an N-terminal half.View Large Image Figure ViewerDownload (PPT) The digestion mixtures were directly applied onto the heparin-Sepharose column in the presence of 5 mm CaCl2 and eluted with 2 mm EDTA and 300 mm NaCl. The fractions were collected, and the amount of the annexin VI fragments in each fraction was monitored by ELISA using anti-annexin VI antibodies. Peaks were observed from 2 mm EDTA- and 300 mmNaCl-eluted fractions (Fig. 7 a). SDS-PAGE detected a single band corresponding to the 33-kDa fragment in the 2 mm EDTA-eluted fraction and two bands corresponding to the 19- and 14-kDa fragments in the 300 mm NaCl-eluted fraction (Fig. 8). In the end, the N- and C-terminal halves bound to the heparin column, and the N-terminal half was eluted with EDTA, whereas the C-terminal half was eluted with 300 mm NaCl.Figure 8SDS-PAGE analysis of the annexin VI fractions eluted from the GAG columns. The flow-through fraction (lane 1) and the 2 mm EDTA-eluted fraction (lane 2) from the chondroitin sulfate C-column, and the 2 mmEDTA-eluted fraction (lane 3) and the 300 mmNaCl-eluted fraction (lane 4) from the heparin-column were analyzed using 13% SDS-PAGE.View Large Image Figure ViewerDownload (PPT) The results of chondroitin sulfate C-Sepharose affinity chromatography of the annexin VI fragments are shown in Fig. 7 b. The N-terminal half (the 33-kDa fragment) passed through the column, and the C-terminal half (the 19- and 14-kDa fragments) was adsorbed onto the column and eluted with EDTA (Fig. 8). Western blotting analysis showed that the N-terminal half of annexin VI was more sensitive to the antibodies than the C-terminal half (data not shown). The results indicate that the two halves of annexin VI have completely different binding properties for heparin and chondroitin sulfate C and, therefore, annexin VI has at least two distinct GAG-binding sites located in the N- and C-terminal halves of the annexin VI molecule. The finding that each annexin has distinct binding specificities supports the theory that the GAG binding activities are involved in the functional diversities of each annexin. On solid phase binding assay, annexin IV, V, and VI were found to bind dose-dependently to heparin, and annexin IV and VI to chondroitin sulfate. These findings agree with those of affinity chromatography. Complete chemical desulfation of heparin significantly reduced its binding to annexin V but not to annexin IV. Therefore, sulfate groups of heparin appear to be important for the calcium-dependent interaction between annexin V and heparin, and the calcium-independent inte
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