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Analysis of plasma proteins that bind to glycosaminoglycans
Biochimica et Biophysica Acta 1770 (2007) 241 – 246 www.elsevier.com/locate/bbagen
Analysis of plasma proteins that bind to glycosaminoglycans Akio Saito ⁎, Hiroshi Munakata Department of Biochemistry, School of Medicine, Kinki University, 377-2 Ohno-Higashi, Osaka-Sayama, Osaka 589-8511, Japan Received 15 July 2006; received in revised form 3 October 2006; accepted 6 October 2006 Available online 7 November 2006
Abstract Glycosaminoglycan-binding proteins, with specific emphasis on dermatan sulfate, have been investigated in human plasma by affinity chromatography, mass spectrometry and Western blotting. Diluted plasma was applied to affinity columns and bound protein was eluted with 500 mM NaCl. Dermatan sulfate and heparan sulfate bound 7% of the total protein. Heparin bound 22% of the total protein, but chondroitin sulfate A bound only 0.23%. Mass spectrometric analysis identified 20 proteins as dermatan-sulfate-binding proteins, most of which were confirmed by Western blotting. Some of these binding proteins, such as fibrinogen, fibronectin, apolipoprotein B, LMW kininogen, inter-α-trypsin inhibitor, and factor H, were degraded to various extents during the chromatography step, but this degradation could be prevented by the inclusion of a serine protease inhibitor. The protein fraction binding to the dermatan sulfate column showed amidase activity, whereas that binding to the heparan sulfate and heparin columns showed 1/2 and 1/20, respectively, of the activity of the dermatan sulfate binding fraction. Dermatan sulfate was similar to heparan sulfate with respect to its capacity to bind plasma proteins and its activation of protease, but differed from chondroitin sulfate and heparin in these properties. © 2006 Elsevier B.V. All rights reserved. Keywords: Glycosaminoglycan; Dermatan sulfate; Affinity column; Mass spectrometry
1. Introduction Glycosaminoglycan (GAG) is a polysaccharide consisting of uronic acid and galactosamine and/or glucosamine and is present at the cell surface, in the extracellular matrix and in secretory granules [1–3]. Except for heparin, sulfated GAGs constitute proteoglycans and participate in various biological and pathological pathways, including blood coagulation, cell growth, cell division, wound repair, infection and tumorigenesis, in addition to providing mechanical support functions [4–6]. Endothelial cells in blood vessels are rich in GAGs such as heparan sulfate (HS), chondroitin sulfate (CS) and dermatan sulfate (DS), which contribute to the anticoagulant activity of the cells [7]. Both CS and DS have N-acetyl galactosamine in common, whereas iduronic acid is present in DS and HS as well as in heparin [1]. Heparin has been recognized as a potential anticoagulant reagent and has been used in various clinical settings; however, the molecules that interact with antithrombin III and heparin cofactor II under physiological conditions are not ⁎ Corresponding author. Tel.: +81 72 366 0221; fax: +81 72 366 0245. E-mail address: [email protected] (A. Saito). 0304-4165/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.bbagen.2006.10.015
heparin but HS and DS. Thus, studies on the interaction of plasma proteins with GAGs have been focused on the coagulation system, but other biological events may be mediated by the interaction of plasma proteins with various GAGs. We previously found that complement protein factor H (FH) binds to DS and that FH is selectively cleaved by a serine protease activated by DS [8]. Moreover, amidase activity is detected in the fraction binding to a DS affinity column, and this activity is diminished by pretreating the plasma with DS [8]. In this report, we have compared the capacity of chondroitin sulfate A (CSA), DS, HS and heparin affinity columns to bind plasma proteins, and investigated the amidase activity of binding fractions from the respective columns. The DS-binding proteins have been further analyzed by mass spectrometry: 20 proteins have been identified, most of which have not been previously recognized as DS-binding proteins. 2. Materials and methods 2.1. Materials Chondroitin sulfate from whale cartilage, dermatan sulfate from pig skin, and heparan sulfate from bovine kidney were obtained from Seikagaku
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Corporation (Tokyo, Japan). EZ-Link Sulfo-NHS-LC-Biotin and Immuno Pure HABA were the products of Pierce (Rockford, IL, USA). Nitroblue Tetrazolium (NBT), 5-bromo-4-chloro-3-indolyl-phosphate p-toluidine salt (BCIP), (p-amidinophenyl)methanesulfonyl fluoride (APMSF), and DTT were obtained from Wako (Osaka, Japan). Immobiline P (PVDF), Ultrafree and ZipTip μ-C18 were the products of Millipore Corporation (Bedford, MA, USA). The silver staining kit was the product of Atto Corporation (Tokyo, Japan). Chromogenic substrate S-2302 (prolyl-phenylalanyl-arginine p-nitroanilide) was obtained from Chromogenix Instrumentation Laboratory SpA (Milano, Italy). α-Cyano4-hydroxycinnamic acid (CHCA) and standard peptides (angiotensin 1 and the fragments of adrenocorticotrophic hormone) for the calibration of protein mass fingerprinting measurements were purchased from Applied Biosystems (Foster City, CA, USA). HiTrap Streptavidin HP (1 ml), HiTrap Heparin HP (1 ml), Immobiline DryStrip and DeStreak Rehydration Solution were obtained from GE Healthcare Bio-Sciences Corp. (Piscataway, NJ, USA). We used the following antibodies against human plasma proteins: Complement component 4 (C4), factor B (FB), factor H (FH), factor I (FI), factor XII (FXII), C1 inactivator, prothrombin and HMW kininogen were from The Binding Site Limited (Birmingham, UK); Complement component 3 (C3), gelsolin and fibronectin were from Sigma (St. Louis, MO, USA); heparin cofactor II (HC II) was from BioPur AG (Bubendorf, Switzerland); fibrinogen was from MBL (Nagoya, Japan); inter-α-trypsin inhibitor (ITI) was from DAKO (Glostrup, Denmark); Protein C inhibitor was from Cedarlane Laboratories LTD (Hornby, Canada); and pigment epithelium-derived factor (PEDF) was from Upstate (Lake Placid, NY, USA). Antibodies against human apolipoproteins B and E were kindly supplied by H. Ogawa, Department of Hygiene, Kinki University. Alkaline phosphatase conjugate anti-sheep and anti-goat IgG were obtained from Sigma, and alkaline phosphatase conjugate anti-rabbit IgG was from New England Biolabs, Inc. (Beverly, MA, USA).
2.4. SDS-PAGE and Western blotting An aliquot of the GAG column eluate was separated by electrophoresis on a 10% polyacrylamide gel under reducing conditions. The gel was stained with silver or CBBR. For Western blotting, the gel was overlaid with a PVDF membrane and the membrane was then blocked with 3% BSA, treated with primary antibodies followed by alkaline phosphatase-conjugated secondary antibodies, and visualized with BCIP and NBT. Some bands of DS-bound proteins that stained intensely with CBBR were excised, destained, digested ingel with trypsin, and subjected to mass spectrometry [10].
2.5. 2D-PAGE and mass spectrometry An aliquot of the bDS-N eluate was diluted tenfold with H2O and then concentrated with an Ultrafree to 20 μl. The sample was mixed with 100 μl of Destreak solution and applied to an Immobiline dry strip (pH 3–10 NL, 7 cm). The gel strip was subjected to electrophoresis for 15000 Vh at 20 °C and then equilibrated in SDS-PAGE buffer containing DTT (50 mg/10 ml). The gel strip was then applied to the top of a 10% SDS-PAGE gel and subjected to electrophoresis, followed by silver staining. Several of the spots were excised, destained, digested in-gel with trypsin, and subjected to mass spectrometry. Each digest was extracted with 50% acetonitrile containing 0.1% trifluoro acetic acid, concentrated, and deposited on a sample plate with an equal volume of 5% CHCA. Mass spectrometry (MALDI-TOF MS) was performed with a Voyager STR (Applied Biosystems) in reflector mode, and the fragment ions were matched to proteins by searching against peptide mass fingerprints using Mascot [8].
3. Results 2.2. Preparation of GAG affinity columns A DS affinity column (bDS-N) was prepared by applying biotinylated DS to the HiTrap streptavidin column (1 ml in bed volume), as described previously [8,9]. CSA (4.43 mg) and HS (1.75 mg) were dissolved in 1 ml of 50 mM sodium bicarbonate overnight at 4 °C, and 1.0 mg of Sulfo-NHS-LC Biotin in 1 ml of H2O was added to the CSA and HS solutions. Each mixture was rotated end-over-end for 6 h at room temperature and then subjected to dialysis at 4 °C and lyophilization. The yields of biotinylated CSA and HS were 4.35 mg and 1.80 mg, respectively, and these were dissolved in PBS to 5 mg/ml. Affinity columns of CSA and HS were prepared according to the method used for bDS-N. Briefly, a HiTrap Streptavidin column (1 ml) was washed with 10 ml of PBS and the ligand solution was applied at a flow rate of less than 1 ml/min. After 30 min at room temperature, unbound ligand was removed with PBS. The amount of CSA and HS bound to the column was estimated from the recovered biotinylated samples as 1.0 and 0.7 mg, respectively.
2.3. Application of human plasma to GAG affinity columns Human plasma (0.5 ml) was diluted with 1.5 ml of 20 mM Tris–HCl pH 7.5 (TB) to reduce the salt concentration of the sample. The GAG affinity columns, bCSA-N, bDS-N, bHS-N and Heparin HP, were equilibrated with 50 mM NaCl in TB and the diluted plasma (2 ml) was loaded. The column was eluted with 6 ml of 50 mM NaCl in TB and the bound proteins were subsequently eluted with 1.5 ml of 500 mM NaCl in TB. The column was washed with 1 ml of 2 M NaCl and regenerated with 5 ml of 50 mM NaCl in TB. The eluate obtained with 50 mM NaCl (8 ml) was concentrated to 2 ml with an Ultrafree (5 kDa molecular cut) and subjected to a second passage through the affinity column. 2.3.1. Amidase activity of the affinity column eluate Chromogenic substrate S-2302 was dissolved in N-methyl-2-pyrrolidone to 20 mM. An aliquot of the 500 mM NaCl eluate was mixed with 20 mM Tris– HCl pH 7.5 containing 150 mM NaCl (TBS) and the volume was adjusted to 500 μl with TBS. Next, 5 μl of the S-2302 solution was added and the reaction was started at 37 °C. The activity was expressed by the increase in absorbance at 410 nm/mg of protein/min.
3.1. GAG-binding human plasma proteins Except for heparin, GAG affinity columns were prepared from a streptavidin column and the biotinylated GAG, which was made by introducing biotin to the amino group of the remaining core peptide. We previously prepared two different types of biotinylated DS and found that attachment of biotin to the amino group showed better binding capacity than its attachment to the uronic acid [9]. Therefore, we introduced biotin to the amino group of CSA and HS. Diluted human plasma was loaded onto the column and the nonbinding fraction was washed with 50 mM NaCl in TB. Next, the bound fraction was eluted with 500 mM NaCl. The amount of protein that bound to the affinity columns is shown in Table 1A. The DS and HS columns showed a similar binding capacity. Plasma proteins did not bind to a streptavidin column free of any GAGs in 50 mM NaCl. The heparin column bound three times more protein than the DS column, whereas the CSA column bound only a small amount of protein. In order to clarify the specificity of the DS column, the nonbinding fractions from the CSA, HS and heparin columns were loaded onto the DS column. As shown in Table 1B, the CSA-nonbinding fraction showed binding to the DS column comparable to that of plasma. Table 1A Amount of plasma protein bound to GAG affinity columns GAG column
CSA
DS
HS
Heparin
Bound protein (%)
0.25
7.34
7.54
22.31
The amount of protein was estimated from the absorbance of the eluate at 280 nm.
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Table 1B Amount of DS-binding protein in CSA-, HS- or heparin-nonbinding fractions First GAG column
CSA
HS
Heparin
DS-bound protein (%)
6.37
1.94
0.73
Bound protein was calculated on the basis of the protein loaded on the first column, and the amount of protein was estimated from the absorbance of the eluate at 280 nm.
By contrast, the heparin-nonbinding fraction showed only one tenth of the binding of plasma proteins to DS, indicating that few plasma proteins were bound specifically to DS and that 90% of the DS-binding proteins were also heparin-binding proteins. 3.2. Amidase activity of GAG column eluate We previously found that the protein fraction binding to a DS column showed amidase activity associated with the cleavage of FH [8]; we therefore measured the amidase activity of the binding fraction from other GAG columns using a chromogenic substrate. The binding fraction from the DS column showed the highest activity, followed by that from the HS and heparin columns, as shown in Table 2. The nonbinding fractions did not show any amidase activity, irrespective of the GAG column (data not shown). The HS-binding fraction showed half of the activity of the DS fraction, whereas the heparin-binding fraction showed one tenth of the activity of the DS fraction. The CSAbinding fraction showed no activity, but activity was recovered in the DS-binding fraction obtained from the CSA-nonbinding fraction. 3.3. Effect of protease inhibitors on DS-binding proteins We have shown that when the serine protease inhibitor APMSF is included during DS column chromatography, cleavage of FH is prevented due to inactivation of the DSregulated protease [8]. We therefore considered that other DSbinding proteins may be affected by protease inhibitors and determined the electrophoretic profile of DS-binding proteins in the presence and absence of APMSF. Pretreatment of diluted plasma with DS prior to loading it onto the column also prevents the cleavage of FH, as the activated protease is promptly inactivated by protease inhibitors in the plasma [8]; we therefore also compared the electrophoretic profile of DS-pretreated plasma. As shown in Fig. 1, the intensities of high molecular weight bands were diminished in the untreated plasma (lane 1) and an N-terminal 30 kDa band of FH was evident. The DS-pretreated sample and the APMSF-treated sample showed similar profiles
Table 2 Amidase activity of proteins bound to GAG affinity columns GAG column
CSA
Activity 0 (Δ 410 nm/mg/min)
DS
HS
Heparin
CSA/DS
Heparin/DS
0.586
0.334
0.032
0.520
0
CSA/D and Heparin/DS indicate the DS-binding protein from the CSA- and heparin-nonbinding columns, respectively.
Fig. 1. SDS-PAGE of the DS-bound fraction. The DS-bound fraction was eluted with 500 mM NaCl in the absence (lanes 1, 3 and 4) and the presence (lanes 2 and 5) of 1 mM APMSF, and an aliquot (2.6 μg for lanes 1 to 3, 18 μg for lanes 4 and 5) of the eluate was subjected to electrophoresis on a 10% gel. The sample in lane 3 was pretreated with DS (50 μg/ml) prior to chromatography, as described in the text. The gel was stained with silver (lanes 1 to 3) and CBBR (lanes 4 and 5). Major bands in the CBBR-stained gel were subjected to mass spectrometry as described in the text, and the following proteins were identified: Factor H (a, i and l), apoB (b and k), factor B (c), gelsolin (d), C4 (e and j), albumin (f and m), and fibrinogen (g, h and n). Molecular weight standards (kDa) are indicated at the right.
(lanes 2 and 3), confirming our previous observation that samples pretreated with DS exhibit no amidase activity [8]. To identify the proteins in the bands, scaled-up samples from SDSPAGE were stained with CBBR and distinct bands were subjected to mass spectrometry (lanes 4 and 5). Complement proteins including FH, factor B (FB) and C4, apolipoprotein B, fibrinogen, gelsolin and albumin were identified by peptide mass fingerprinting (see below); however, the presence of albumin was thought to due to nonspecific binding owing to its high concentration in the plasma. 3.4. Identification of DS-binding proteins by mass spectrometry DS-binding proteins in the absence of APMSF were analyzed by 2D-PAGE and the intense spots were subjected to MALDITOF mass spectrometry. Identification of the proteins was conducted by peptide mass fingerprinting: Table 3 lists proteins with a Mascot score of more than 50. Protease inhibitors were the largest group of DS-binding proteins, followed by complement proteins and apolipoproteins. 2D-PAGE and mass spectrometry were carried out several times under various conditions and the complement proteins listed in Table 3, as well as PEDF, protein C inhibitor and gelsolin, were constantly identified with high probability. FB, PEDF and gelsolin represent several horizontally aligned spots on the 2D-PAGE gel, indicating that they were posttranscriptionally modified to some extent It is reported that PEDF was heterogeneously glycosylated [11]. DS-binding
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Table 3 DS-binding proteins identified by MALDI-TOF MS after SDS- and 2D-PAGE MW
Coverage (%)
Mascot score
Complement proteins C3 C4 FB FH
187,030 192,741 85,479 139,034
14 16 33 39
147 183 102 86
Protease inhibitors α-1-AT ATIII PEDF Protein C inhibitor C1 inhibitor ITI LMW kininogen
46,709 44,330 46,313 45,702 55,119 103,321 47,853
48 24 47 29 20 15 25
137 86 282 127 111 69 76
Apo proteins ApoB ApoE ApoA1
187,126 36,132 28,061
33 41 65
102 51 168
Others Gelsolin Fibrinogen Fibronectin Serum amyloid P HRGP
85,644 37,625 246,513 23,244 59,541
35 31 22 36 16
193 52 204 85 121
Mascot score represents the probability based on the Mowse score [29]. Coverage indicates the percentage of the amino acid sequence recovered in the mass spectra.
proteins identified by mass spectrometry were further confirmed by Western blotting (see below) and, except for α-1-AT, ATIII, apolipoprotein A, serum amyloid P and histidine-rich glycoprotein (HRGP), other plasma proteins in the Table 3 were detected in the DS-binding fraction. 3.5. Identification of DS-binding proteins by Western blotting Because we did not identify proteins present in minor amounts by mass spectrometry and had not detected HCII, which is known to bind to DS in addition to heparin [7], we carried out Western blotting using antibodies for 43 plasma proteins. As expected, we detected HCII as a band of 72 kDa in the 500 mM NaCl eluate; however, this band was also detected in the 50 mM NaCl nonbinding eluate. By contrast, the bands due to FH appeared exclusively in the 500 mM NaCl eluate. Other DS-binding proteins recovered in the 500 mM NaCl eluate in an intact form were protein C inhibitor, FB, apolipoprotein E, gelsolin, PEDF, C1 inhibitor, and complement components C3 and C4, whereas LMW kininogen, fibrinogen apolipoprotein B, fibronectin and inter-α-trypsin inhibitor, in addition to FH, were detected as forms cleaved to various extents in the 500 mM NaCl eluate. When APMSF was included during DS column chromatography, these proteins were recovered in an intact form. Furthermore, FI, FXII and prothrombin were detected in the 500 mM NaCl eluate only in the presence of APMSF. Moreover, in the absence of APMSF most of the DS-binding
proteins decomposed during prolonged storage (1–2 weeks) at 4°C and the bands due to fibrinogen, fibronectin and inter-αtrypsin inhibitor completely disappeared. 4. Discussion The key to the success in the affinity chromatography depends on the accessibility of the target proteins to the ligand on the column. Previously we prepared DS affinity columns with different types of linkage between DS and the matrix of the column, and found that conjugating the residual amino group of DS to the matrix through a biotin–avidin bond showed the highest binding capacity for plasma proteins [9]. We therefore prepared CSA and HS affinity columns by generating a biotin– avidin bond between their residual amino groups and the streptavidin column, and compared the binding capacity of the resulting CSA, DS, HS and heparin affinity columns for diluted human plasma. Stepwise elution was used to compare the group of proteins that bound to the respective affinity columns. The ionic strength of the eluent is critical in affinity chromatography. We used 50 mM NaCl in TB as a washing buffer following sample loading. Because human plasma contains 150 mM NaCl, washing the columns with 150 mM NaCl in TB may be considered reasonable for analyzing GAG-binding proteins; however, most proteins were washed out and did not bind to DS or HS in 150 mM NaCl buffer (data not shown). Moreover, heparin cofactor II, which is confirmed to bind to DS and exhibits anticoagulant activity in physiological conditions [7,12], did not bind to the DS column at 150 mM NaCl in TB (data not shown). Therefore, we reduced the buffer to an ionic strength of 50 mM NaCl in order to equilibrate the column and wash out the nonbinding proteins, and subsequently eluted the bound fraction with 500 mM NaCl. It has been reported that plasma contains GAGs, mainly chondroitin sulfate [13,14]. None of the GAGs in plasma are covalently linked to plasma proteins and less than 0.5% of the proteins were associated with GAG [14]. We have treated plasma sample with 1 M NaCl to dissociate any GAGs prior to DS column application, but significant differences in the profile of the protein band on the SDS-PAGE and amidase activity in the bound fraction between the treated and untreated samples were not observed. Based on structural similarity, it was expected that DS and CSA would have similar binding capacity and that HS would bind many proteins comparable to heparin [1]. As shown in Table 1A, DS and HS bound 7% of plasma proteins and heparin bound three times more, whereas the binding capacity of CSA was considerably smaller. The binding capacity of the heparin column was so high that more than half of the albumin-free plasma proteins were bound to the column at 50 mM NaCl. The similarity between the DS and HS columns was observed not only in their capacity to bind plasma proteins but also in their activation of a protease in the plasma, as shown in Table 2. When the amidase activity of the 500 mM NaCl eluate was measured with a chromogenic substrate, the DS and HS column eluates showed, respectively, 18.3 and 10.5 times the activity of the heparin column fraction. Although heparin
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bound three times more proteins than DS, the activation of protease by heparin was 5% of that by DS. Interestingly, Okada et al. have reported that low molecular weight heparin enhances the amidase activity of human plasma and the activated protease cleaves plasminogen activators [15]. Amidase activity was detected in DS-binding fraction of the CSA-nonbinding fraction but not that of the heparin-nonbinding fraction, indicating that either the protease was eluted from the heparin column without activation or the protease was not eluted with 500 mM NaCl. Another explanation is that a specific protease inhibitor was bound to the heparin column and the activated protease was promptly inactivated by this inhibitor. Activation and inhibition of human plasma kallikrein by glycosaminoglycans including DS was studied by Gozzo et al. [16]. They found that DS increased 5.4 times the efficiency of kallikrein on FXII activation but heparin also activated 7.7 times the kallikrein activity. On the other hand, both DS and heparin increased 2–3 times the inactivation of plasma kallikrein by ATIII. Cardoso and Mourao have investigated the constitution of GAGs in human arteries and found that DS is present in comparable concentrations in all arteries [17]. They have reported that the relative contents of GAGs in abdominal aorta are hyaluronic acid (6.1%), HS (31.8%), DS (23.2%) and chondroitin 4/6-sulfate (38,9%). Plasma contains various kind of proteases, most of which are circulating in an inactive form, and these proteases are involved in cascade activation pathways such as coagulation, fibrinolysis and complement systems. Therefore, the protease that was activated by DS or HS was not necessarily the same as the one that cleaved FH or other proteins. It is of interest to consider whether the observed activation of protease by DS is physiologically relevant or simply an artifact. In order to bind proteins to DS and activate the protease, the ionic strength had to be lower than that in physiological conditions; however, we cannot reproduce the exact conditions of circulating plasma in the affinity column. Factors other than ionic strength that facilitate protein binding to DS may be present in the plasma. Identification of the protease that is activated by DS and HS will provide insight into the mechanism of DS-mediated activation of protease and the cryptic roles of GAGs in the plasma. Until now, known DS-binding proteins in the plasma have been limited to heparin cofactor II, apolipoprotein B and several cytokines [12,18,19], and more recently to FH [8,20]. Because mass spectrometry in conjunction with 2D-PAGE is one of the most sensitive techniques to identify proteins in a mixture, we used it to screen as many DS-binding proteins in the plasma as possible. Six proteins other than albumin were identified by SDS-PAGE, followed by mass spectrometry and peptide mass fingerprinting, and 19 proteins were detected by 2D-PAGE analysis. Albumin was detected by both SDS-PAGE and 2D PAGE experiments, but its binding seems likely to be nonspecific due to its high concentration in the plasma. In addition, α-1-AT and ATIII might be nonspecific binding proteins because a major portion of these proteins was detected in the nonbinding fraction from the DS column by Western blotting. Most other binding proteins identified by mass spectrometry were ascertained by Western blotting. We pre-
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viously reported that cleavage of FH was prevented by APMSF, and hence the 500 mM NaCl eluate from the DS column in the presence and absence of APMSF was investigated by Western blotting. Antibodies of plasma proteins involved in coagulation, complement reaction and apolipoproteins were used for blotting, and FXII, FI and prothrombin were detected only in the presence of APMSF. The DS-binding sites on these proteins may be severely degraded by the protease activated by DS. Most of the other DS-binding proteins detected in the absence of APMSF were also degraded to various degrees during prolonged storage. It is interesting that both FH and FI were bound to DS and were affected by the DS-mediated protease, because FH is a cofactor of FI and both molecules are responsible for the inactivation of C3b and downregulating the complement cascade alternative pathway [21,22]. Taking into consideration the fact that C3 and FB are also members of the alternative pathway, DS on the endothelial surface could be an essential modulator of the complement system. Moreover, four independent groups have reported that a common variant of the FH gene in the seventh domain is strongly associated with the onset of agerelated macular degeneration, a leading cause of visual impairment and blindness in the elderly [23–26]. The role of FH in this disease has not been investigated as yet but it may be involved in inflammation. Among the DS-binding proteins, PEDF is of interest in connection with age-related macular degeneration, because this protein was first found in cultured pigment epithelial cells of the fetal human retina [27] and has since been found to be a member of the serine protease inhibitors [28]. Although protease inhibitory activity has not been reported, these three molecules, PEDF, FH and FI, were able to bind to DS and therefore may have a role in the onset of this disease. In summary, we have found that the interaction of plasma proteins with DS is similar to their interaction with HS but different from their interaction with CSA or heparin in terms of binding capacity and protease activation. In addition, we have identified 24 plasma proteins as DS-binding proteins by mass spectrometry and Western blotting. The newly identified DSbinding proteins will shed light on unknown roles of DS and will provide insight into our understanding of the physiology and pathology of plasma proteins. References [1] H.E. Conrad, Structure of heparan sulfate and dermatan sulfate, Ann. N. Y. Acad. Sci. 556 (1989) 18–28. [2] R.L. Jackson, S.L. Busch, A.D. Cardin, Glycosaminoglycans: molecular properties, protein interactions and role in physiological processes, Physiol. Rev. 71 (1991) 481–539. [3] J.M. Trowbridge, R.L. Gallo, Dermatan sulfate: new functions from an old glycosaminoglycan, Glycobiology 12 (2002) 117R–125R. [4] L. Kjellen, U. Lindahl, Proteoglycans: structures and interactions, Annu. Rev. Biochem. 60 (1991) 443–475. [5] R.V. Iozzo, A.D. Murdoch, Proteoglycans of the extracellular environment: clues from the gene and protein side offer novel perspectives in molecular diversity and function, FASEB J. 10 (1996) 598–614. [6] R.V. Iozzo, Matrix proteoglycans: from molecular design to cellular function, Annu. Rev. Biochem. 67 (1998) 609–652.
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[20] S.J. Clark, V.A. Higman, B. Mulloy, S.J. Perkins, S.M. Lea, R.B. Sim, A.J. Day, His-384 allotypic variant of factor H associated with age-related macular degeneration has different heparin binding properties from the non-disease-associated form, J. Biol. Chem. 281 (2006) 24713–24720. [21] P.F. Zipfel, J. Hellwage, M.A. Friese, G. Hegasy, S.T. Jokiranta, S. Meri, Factor H and disease: a complement regulator affects vital body functions, Mol. Immunol. 36 (1999) 241–248. [22] P.F. Zipfel, Complement factor H: physiology and pathophysiology, Semin. Thromb. Hemost. 27 (2001) 191–199. [23] R.J. Klein, C. Zeiss, E.Y. Chew, J.-Y. Tsai, R.S. Sackler, C. Haynas, A.K. Henning, J.P. SanGiovanni, S.M. Mane, S.T. Mayne, M.B. Bracken, F.L. Ferris, J. Ott, C. Barnstable, J. Hoh, Complement factor H polymorphism in age-related macular degeneration, Science 308 (2005) 385–389. [24] J.L. Haines, M.A. Hauser, S. Schmidt, W.K. Scott, L.M. Olson, P. Gallins, K.L. Spencer, S.Y. Kwan, M. Noureddine, J.R. Gilbert, N. S-Boutaud, A. Agarwal, E.A. Postel, M.A. P-Vance, Complement factor H variant increases the risk of age-related macular degeneration, Science 308 (2005) 419–421. [25] A.O. Edwards, R. Ritter III, K.J. Abel, A. Manning, C. Panhuysen, L.A. Farrer, Complement factor H polymorphism and age-related macular degeneration, Science 308 (2005) 421–424. [26] G.S. Hageman, D.H. Anderson, L.V. Johnson, L.S. Hancox, A.J. Taiber, L.I. Hardisty, J.L. Hageman, H.A. Stockman, J.D. Borchardt, K.M. Gehrs, R.J.H. Smith, G. Silferstri, S.R. Russell, C.C.W. Klaver, I. Barbazetto, S. Chang, L.A. Yannuzzi, G.R. Barile, J.C. Merriam, R.T. Smith, A.K. Olsh, J. Bergeron, J. Zernant, J.E. Merriam, B. Gold, M. Dean, R. Allikmets, A common haplotype in the complement regulatory gene factor H (HF1/CFH) predisposes individuals to age-related macular degeneration, Proc. Natl. Acad. Sci. U. S. A. 102 (2005) 7227–7232. [27] J. Tombran-Tink, G.G. Chader, L.V. Johnson, PEDF: a pigment epithelium-derived factor with potent neuronal differentiative activity, Exp. Eye Res. 53 (1991) 411–414. [28] F.R. Steele, G.J. Chader, L.V. Johnson, J. Tombran-Tink, Pigment epithelium-derived factor: neurotrophic activity and identification as a member of the serine protease inhibitor gene family, Proc. Natl. Acad. Sci. U. S. A. 90 (1992) 1526–1530. [29] D.J. Pappin, P. Hojrup, A.J. Bleasby, Rapid identification of proteins by peptide-mass fingerprinting, Curr. Biol. 3 (1993) 327–332.
Analysis of plasma proteins that bind to glycosaminoglycans Akio Saito ⁎, Hiroshi Munakata Department of Biochemistry, School of Medicine, Kinki University, 377-2 Ohno-Higashi, Osaka-Sayama, Osaka 589-8511, Japan Received 15 July 2006; received in revised form 3 October 2006; accepted 6 October 2006 Available online 7 November 2006
Abstract Glycosaminoglycan-binding proteins, with specific emphasis on dermatan sulfate, have been investigated in human plasma by affinity chromatography, mass spectrometry and Western blotting. Diluted plasma was applied to affinity columns and bound protein was eluted with 500 mM NaCl. Dermatan sulfate and heparan sulfate bound 7% of the total protein. Heparin bound 22% of the total protein, but chondroitin sulfate A bound only 0.23%. Mass spectrometric analysis identified 20 proteins as dermatan-sulfate-binding proteins, most of which were confirmed by Western blotting. Some of these binding proteins, such as fibrinogen, fibronectin, apolipoprotein B, LMW kininogen, inter-α-trypsin inhibitor, and factor H, were degraded to various extents during the chromatography step, but this degradation could be prevented by the inclusion of a serine protease inhibitor. The protein fraction binding to the dermatan sulfate column showed amidase activity, whereas that binding to the heparan sulfate and heparin columns showed 1/2 and 1/20, respectively, of the activity of the dermatan sulfate binding fraction. Dermatan sulfate was similar to heparan sulfate with respect to its capacity to bind plasma proteins and its activation of protease, but differed from chondroitin sulfate and heparin in these properties. © 2006 Elsevier B.V. All rights reserved. Keywords: Glycosaminoglycan; Dermatan sulfate; Affinity column; Mass spectrometry
1. Introduction Glycosaminoglycan (GAG) is a polysaccharide consisting of uronic acid and galactosamine and/or glucosamine and is present at the cell surface, in the extracellular matrix and in secretory granules [1–3]. Except for heparin, sulfated GAGs constitute proteoglycans and participate in various biological and pathological pathways, including blood coagulation, cell growth, cell division, wound repair, infection and tumorigenesis, in addition to providing mechanical support functions [4–6]. Endothelial cells in blood vessels are rich in GAGs such as heparan sulfate (HS), chondroitin sulfate (CS) and dermatan sulfate (DS), which contribute to the anticoagulant activity of the cells [7]. Both CS and DS have N-acetyl galactosamine in common, whereas iduronic acid is present in DS and HS as well as in heparin [1]. Heparin has been recognized as a potential anticoagulant reagent and has been used in various clinical settings; however, the molecules that interact with antithrombin III and heparin cofactor II under physiological conditions are not ⁎ Corresponding author. Tel.: +81 72 366 0221; fax: +81 72 366 0245. E-mail address: [email protected] (A. Saito). 0304-4165/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.bbagen.2006.10.015
heparin but HS and DS. Thus, studies on the interaction of plasma proteins with GAGs have been focused on the coagulation system, but other biological events may be mediated by the interaction of plasma proteins with various GAGs. We previously found that complement protein factor H (FH) binds to DS and that FH is selectively cleaved by a serine protease activated by DS [8]. Moreover, amidase activity is detected in the fraction binding to a DS affinity column, and this activity is diminished by pretreating the plasma with DS [8]. In this report, we have compared the capacity of chondroitin sulfate A (CSA), DS, HS and heparin affinity columns to bind plasma proteins, and investigated the amidase activity of binding fractions from the respective columns. The DS-binding proteins have been further analyzed by mass spectrometry: 20 proteins have been identified, most of which have not been previously recognized as DS-binding proteins. 2. Materials and methods 2.1. Materials Chondroitin sulfate from whale cartilage, dermatan sulfate from pig skin, and heparan sulfate from bovine kidney were obtained from Seikagaku
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Corporation (Tokyo, Japan). EZ-Link Sulfo-NHS-LC-Biotin and Immuno Pure HABA were the products of Pierce (Rockford, IL, USA). Nitroblue Tetrazolium (NBT), 5-bromo-4-chloro-3-indolyl-phosphate p-toluidine salt (BCIP), (p-amidinophenyl)methanesulfonyl fluoride (APMSF), and DTT were obtained from Wako (Osaka, Japan). Immobiline P (PVDF), Ultrafree and ZipTip μ-C18 were the products of Millipore Corporation (Bedford, MA, USA). The silver staining kit was the product of Atto Corporation (Tokyo, Japan). Chromogenic substrate S-2302 (prolyl-phenylalanyl-arginine p-nitroanilide) was obtained from Chromogenix Instrumentation Laboratory SpA (Milano, Italy). α-Cyano4-hydroxycinnamic acid (CHCA) and standard peptides (angiotensin 1 and the fragments of adrenocorticotrophic hormone) for the calibration of protein mass fingerprinting measurements were purchased from Applied Biosystems (Foster City, CA, USA). HiTrap Streptavidin HP (1 ml), HiTrap Heparin HP (1 ml), Immobiline DryStrip and DeStreak Rehydration Solution were obtained from GE Healthcare Bio-Sciences Corp. (Piscataway, NJ, USA). We used the following antibodies against human plasma proteins: Complement component 4 (C4), factor B (FB), factor H (FH), factor I (FI), factor XII (FXII), C1 inactivator, prothrombin and HMW kininogen were from The Binding Site Limited (Birmingham, UK); Complement component 3 (C3), gelsolin and fibronectin were from Sigma (St. Louis, MO, USA); heparin cofactor II (HC II) was from BioPur AG (Bubendorf, Switzerland); fibrinogen was from MBL (Nagoya, Japan); inter-α-trypsin inhibitor (ITI) was from DAKO (Glostrup, Denmark); Protein C inhibitor was from Cedarlane Laboratories LTD (Hornby, Canada); and pigment epithelium-derived factor (PEDF) was from Upstate (Lake Placid, NY, USA). Antibodies against human apolipoproteins B and E were kindly supplied by H. Ogawa, Department of Hygiene, Kinki University. Alkaline phosphatase conjugate anti-sheep and anti-goat IgG were obtained from Sigma, and alkaline phosphatase conjugate anti-rabbit IgG was from New England Biolabs, Inc. (Beverly, MA, USA).
2.4. SDS-PAGE and Western blotting An aliquot of the GAG column eluate was separated by electrophoresis on a 10% polyacrylamide gel under reducing conditions. The gel was stained with silver or CBBR. For Western blotting, the gel was overlaid with a PVDF membrane and the membrane was then blocked with 3% BSA, treated with primary antibodies followed by alkaline phosphatase-conjugated secondary antibodies, and visualized with BCIP and NBT. Some bands of DS-bound proteins that stained intensely with CBBR were excised, destained, digested ingel with trypsin, and subjected to mass spectrometry [10].
2.5. 2D-PAGE and mass spectrometry An aliquot of the bDS-N eluate was diluted tenfold with H2O and then concentrated with an Ultrafree to 20 μl. The sample was mixed with 100 μl of Destreak solution and applied to an Immobiline dry strip (pH 3–10 NL, 7 cm). The gel strip was subjected to electrophoresis for 15000 Vh at 20 °C and then equilibrated in SDS-PAGE buffer containing DTT (50 mg/10 ml). The gel strip was then applied to the top of a 10% SDS-PAGE gel and subjected to electrophoresis, followed by silver staining. Several of the spots were excised, destained, digested in-gel with trypsin, and subjected to mass spectrometry. Each digest was extracted with 50% acetonitrile containing 0.1% trifluoro acetic acid, concentrated, and deposited on a sample plate with an equal volume of 5% CHCA. Mass spectrometry (MALDI-TOF MS) was performed with a Voyager STR (Applied Biosystems) in reflector mode, and the fragment ions were matched to proteins by searching against peptide mass fingerprints using Mascot [8].
3. Results 2.2. Preparation of GAG affinity columns A DS affinity column (bDS-N) was prepared by applying biotinylated DS to the HiTrap streptavidin column (1 ml in bed volume), as described previously [8,9]. CSA (4.43 mg) and HS (1.75 mg) were dissolved in 1 ml of 50 mM sodium bicarbonate overnight at 4 °C, and 1.0 mg of Sulfo-NHS-LC Biotin in 1 ml of H2O was added to the CSA and HS solutions. Each mixture was rotated end-over-end for 6 h at room temperature and then subjected to dialysis at 4 °C and lyophilization. The yields of biotinylated CSA and HS were 4.35 mg and 1.80 mg, respectively, and these were dissolved in PBS to 5 mg/ml. Affinity columns of CSA and HS were prepared according to the method used for bDS-N. Briefly, a HiTrap Streptavidin column (1 ml) was washed with 10 ml of PBS and the ligand solution was applied at a flow rate of less than 1 ml/min. After 30 min at room temperature, unbound ligand was removed with PBS. The amount of CSA and HS bound to the column was estimated from the recovered biotinylated samples as 1.0 and 0.7 mg, respectively.
2.3. Application of human plasma to GAG affinity columns Human plasma (0.5 ml) was diluted with 1.5 ml of 20 mM Tris–HCl pH 7.5 (TB) to reduce the salt concentration of the sample. The GAG affinity columns, bCSA-N, bDS-N, bHS-N and Heparin HP, were equilibrated with 50 mM NaCl in TB and the diluted plasma (2 ml) was loaded. The column was eluted with 6 ml of 50 mM NaCl in TB and the bound proteins were subsequently eluted with 1.5 ml of 500 mM NaCl in TB. The column was washed with 1 ml of 2 M NaCl and regenerated with 5 ml of 50 mM NaCl in TB. The eluate obtained with 50 mM NaCl (8 ml) was concentrated to 2 ml with an Ultrafree (5 kDa molecular cut) and subjected to a second passage through the affinity column. 2.3.1. Amidase activity of the affinity column eluate Chromogenic substrate S-2302 was dissolved in N-methyl-2-pyrrolidone to 20 mM. An aliquot of the 500 mM NaCl eluate was mixed with 20 mM Tris– HCl pH 7.5 containing 150 mM NaCl (TBS) and the volume was adjusted to 500 μl with TBS. Next, 5 μl of the S-2302 solution was added and the reaction was started at 37 °C. The activity was expressed by the increase in absorbance at 410 nm/mg of protein/min.
3.1. GAG-binding human plasma proteins Except for heparin, GAG affinity columns were prepared from a streptavidin column and the biotinylated GAG, which was made by introducing biotin to the amino group of the remaining core peptide. We previously prepared two different types of biotinylated DS and found that attachment of biotin to the amino group showed better binding capacity than its attachment to the uronic acid [9]. Therefore, we introduced biotin to the amino group of CSA and HS. Diluted human plasma was loaded onto the column and the nonbinding fraction was washed with 50 mM NaCl in TB. Next, the bound fraction was eluted with 500 mM NaCl. The amount of protein that bound to the affinity columns is shown in Table 1A. The DS and HS columns showed a similar binding capacity. Plasma proteins did not bind to a streptavidin column free of any GAGs in 50 mM NaCl. The heparin column bound three times more protein than the DS column, whereas the CSA column bound only a small amount of protein. In order to clarify the specificity of the DS column, the nonbinding fractions from the CSA, HS and heparin columns were loaded onto the DS column. As shown in Table 1B, the CSA-nonbinding fraction showed binding to the DS column comparable to that of plasma. Table 1A Amount of plasma protein bound to GAG affinity columns GAG column
CSA
DS
HS
Heparin
Bound protein (%)
0.25
7.34
7.54
22.31
The amount of protein was estimated from the absorbance of the eluate at 280 nm.
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Table 1B Amount of DS-binding protein in CSA-, HS- or heparin-nonbinding fractions First GAG column
CSA
HS
Heparin
DS-bound protein (%)
6.37
1.94
0.73
Bound protein was calculated on the basis of the protein loaded on the first column, and the amount of protein was estimated from the absorbance of the eluate at 280 nm.
By contrast, the heparin-nonbinding fraction showed only one tenth of the binding of plasma proteins to DS, indicating that few plasma proteins were bound specifically to DS and that 90% of the DS-binding proteins were also heparin-binding proteins. 3.2. Amidase activity of GAG column eluate We previously found that the protein fraction binding to a DS column showed amidase activity associated with the cleavage of FH [8]; we therefore measured the amidase activity of the binding fraction from other GAG columns using a chromogenic substrate. The binding fraction from the DS column showed the highest activity, followed by that from the HS and heparin columns, as shown in Table 2. The nonbinding fractions did not show any amidase activity, irrespective of the GAG column (data not shown). The HS-binding fraction showed half of the activity of the DS fraction, whereas the heparin-binding fraction showed one tenth of the activity of the DS fraction. The CSAbinding fraction showed no activity, but activity was recovered in the DS-binding fraction obtained from the CSA-nonbinding fraction. 3.3. Effect of protease inhibitors on DS-binding proteins We have shown that when the serine protease inhibitor APMSF is included during DS column chromatography, cleavage of FH is prevented due to inactivation of the DSregulated protease [8]. We therefore considered that other DSbinding proteins may be affected by protease inhibitors and determined the electrophoretic profile of DS-binding proteins in the presence and absence of APMSF. Pretreatment of diluted plasma with DS prior to loading it onto the column also prevents the cleavage of FH, as the activated protease is promptly inactivated by protease inhibitors in the plasma [8]; we therefore also compared the electrophoretic profile of DS-pretreated plasma. As shown in Fig. 1, the intensities of high molecular weight bands were diminished in the untreated plasma (lane 1) and an N-terminal 30 kDa band of FH was evident. The DS-pretreated sample and the APMSF-treated sample showed similar profiles
Table 2 Amidase activity of proteins bound to GAG affinity columns GAG column
CSA
Activity 0 (Δ 410 nm/mg/min)
DS
HS
Heparin
CSA/DS
Heparin/DS
0.586
0.334
0.032
0.520
0
CSA/D and Heparin/DS indicate the DS-binding protein from the CSA- and heparin-nonbinding columns, respectively.
Fig. 1. SDS-PAGE of the DS-bound fraction. The DS-bound fraction was eluted with 500 mM NaCl in the absence (lanes 1, 3 and 4) and the presence (lanes 2 and 5) of 1 mM APMSF, and an aliquot (2.6 μg for lanes 1 to 3, 18 μg for lanes 4 and 5) of the eluate was subjected to electrophoresis on a 10% gel. The sample in lane 3 was pretreated with DS (50 μg/ml) prior to chromatography, as described in the text. The gel was stained with silver (lanes 1 to 3) and CBBR (lanes 4 and 5). Major bands in the CBBR-stained gel were subjected to mass spectrometry as described in the text, and the following proteins were identified: Factor H (a, i and l), apoB (b and k), factor B (c), gelsolin (d), C4 (e and j), albumin (f and m), and fibrinogen (g, h and n). Molecular weight standards (kDa) are indicated at the right.
(lanes 2 and 3), confirming our previous observation that samples pretreated with DS exhibit no amidase activity [8]. To identify the proteins in the bands, scaled-up samples from SDSPAGE were stained with CBBR and distinct bands were subjected to mass spectrometry (lanes 4 and 5). Complement proteins including FH, factor B (FB) and C4, apolipoprotein B, fibrinogen, gelsolin and albumin were identified by peptide mass fingerprinting (see below); however, the presence of albumin was thought to due to nonspecific binding owing to its high concentration in the plasma. 3.4. Identification of DS-binding proteins by mass spectrometry DS-binding proteins in the absence of APMSF were analyzed by 2D-PAGE and the intense spots were subjected to MALDITOF mass spectrometry. Identification of the proteins was conducted by peptide mass fingerprinting: Table 3 lists proteins with a Mascot score of more than 50. Protease inhibitors were the largest group of DS-binding proteins, followed by complement proteins and apolipoproteins. 2D-PAGE and mass spectrometry were carried out several times under various conditions and the complement proteins listed in Table 3, as well as PEDF, protein C inhibitor and gelsolin, were constantly identified with high probability. FB, PEDF and gelsolin represent several horizontally aligned spots on the 2D-PAGE gel, indicating that they were posttranscriptionally modified to some extent It is reported that PEDF was heterogeneously glycosylated [11]. DS-binding
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Table 3 DS-binding proteins identified by MALDI-TOF MS after SDS- and 2D-PAGE MW
Coverage (%)
Mascot score
Complement proteins C3 C4 FB FH
187,030 192,741 85,479 139,034
14 16 33 39
147 183 102 86
Protease inhibitors α-1-AT ATIII PEDF Protein C inhibitor C1 inhibitor ITI LMW kininogen
46,709 44,330 46,313 45,702 55,119 103,321 47,853
48 24 47 29 20 15 25
137 86 282 127 111 69 76
Apo proteins ApoB ApoE ApoA1
187,126 36,132 28,061
33 41 65
102 51 168
Others Gelsolin Fibrinogen Fibronectin Serum amyloid P HRGP
85,644 37,625 246,513 23,244 59,541
35 31 22 36 16
193 52 204 85 121
Mascot score represents the probability based on the Mowse score [29]. Coverage indicates the percentage of the amino acid sequence recovered in the mass spectra.
proteins identified by mass spectrometry were further confirmed by Western blotting (see below) and, except for α-1-AT, ATIII, apolipoprotein A, serum amyloid P and histidine-rich glycoprotein (HRGP), other plasma proteins in the Table 3 were detected in the DS-binding fraction. 3.5. Identification of DS-binding proteins by Western blotting Because we did not identify proteins present in minor amounts by mass spectrometry and had not detected HCII, which is known to bind to DS in addition to heparin [7], we carried out Western blotting using antibodies for 43 plasma proteins. As expected, we detected HCII as a band of 72 kDa in the 500 mM NaCl eluate; however, this band was also detected in the 50 mM NaCl nonbinding eluate. By contrast, the bands due to FH appeared exclusively in the 500 mM NaCl eluate. Other DS-binding proteins recovered in the 500 mM NaCl eluate in an intact form were protein C inhibitor, FB, apolipoprotein E, gelsolin, PEDF, C1 inhibitor, and complement components C3 and C4, whereas LMW kininogen, fibrinogen apolipoprotein B, fibronectin and inter-α-trypsin inhibitor, in addition to FH, were detected as forms cleaved to various extents in the 500 mM NaCl eluate. When APMSF was included during DS column chromatography, these proteins were recovered in an intact form. Furthermore, FI, FXII and prothrombin were detected in the 500 mM NaCl eluate only in the presence of APMSF. Moreover, in the absence of APMSF most of the DS-binding
proteins decomposed during prolonged storage (1–2 weeks) at 4°C and the bands due to fibrinogen, fibronectin and inter-αtrypsin inhibitor completely disappeared. 4. Discussion The key to the success in the affinity chromatography depends on the accessibility of the target proteins to the ligand on the column. Previously we prepared DS affinity columns with different types of linkage between DS and the matrix of the column, and found that conjugating the residual amino group of DS to the matrix through a biotin–avidin bond showed the highest binding capacity for plasma proteins [9]. We therefore prepared CSA and HS affinity columns by generating a biotin– avidin bond between their residual amino groups and the streptavidin column, and compared the binding capacity of the resulting CSA, DS, HS and heparin affinity columns for diluted human plasma. Stepwise elution was used to compare the group of proteins that bound to the respective affinity columns. The ionic strength of the eluent is critical in affinity chromatography. We used 50 mM NaCl in TB as a washing buffer following sample loading. Because human plasma contains 150 mM NaCl, washing the columns with 150 mM NaCl in TB may be considered reasonable for analyzing GAG-binding proteins; however, most proteins were washed out and did not bind to DS or HS in 150 mM NaCl buffer (data not shown). Moreover, heparin cofactor II, which is confirmed to bind to DS and exhibits anticoagulant activity in physiological conditions [7,12], did not bind to the DS column at 150 mM NaCl in TB (data not shown). Therefore, we reduced the buffer to an ionic strength of 50 mM NaCl in order to equilibrate the column and wash out the nonbinding proteins, and subsequently eluted the bound fraction with 500 mM NaCl. It has been reported that plasma contains GAGs, mainly chondroitin sulfate [13,14]. None of the GAGs in plasma are covalently linked to plasma proteins and less than 0.5% of the proteins were associated with GAG [14]. We have treated plasma sample with 1 M NaCl to dissociate any GAGs prior to DS column application, but significant differences in the profile of the protein band on the SDS-PAGE and amidase activity in the bound fraction between the treated and untreated samples were not observed. Based on structural similarity, it was expected that DS and CSA would have similar binding capacity and that HS would bind many proteins comparable to heparin [1]. As shown in Table 1A, DS and HS bound 7% of plasma proteins and heparin bound three times more, whereas the binding capacity of CSA was considerably smaller. The binding capacity of the heparin column was so high that more than half of the albumin-free plasma proteins were bound to the column at 50 mM NaCl. The similarity between the DS and HS columns was observed not only in their capacity to bind plasma proteins but also in their activation of a protease in the plasma, as shown in Table 2. When the amidase activity of the 500 mM NaCl eluate was measured with a chromogenic substrate, the DS and HS column eluates showed, respectively, 18.3 and 10.5 times the activity of the heparin column fraction. Although heparin
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bound three times more proteins than DS, the activation of protease by heparin was 5% of that by DS. Interestingly, Okada et al. have reported that low molecular weight heparin enhances the amidase activity of human plasma and the activated protease cleaves plasminogen activators [15]. Amidase activity was detected in DS-binding fraction of the CSA-nonbinding fraction but not that of the heparin-nonbinding fraction, indicating that either the protease was eluted from the heparin column without activation or the protease was not eluted with 500 mM NaCl. Another explanation is that a specific protease inhibitor was bound to the heparin column and the activated protease was promptly inactivated by this inhibitor. Activation and inhibition of human plasma kallikrein by glycosaminoglycans including DS was studied by Gozzo et al. [16]. They found that DS increased 5.4 times the efficiency of kallikrein on FXII activation but heparin also activated 7.7 times the kallikrein activity. On the other hand, both DS and heparin increased 2–3 times the inactivation of plasma kallikrein by ATIII. Cardoso and Mourao have investigated the constitution of GAGs in human arteries and found that DS is present in comparable concentrations in all arteries [17]. They have reported that the relative contents of GAGs in abdominal aorta are hyaluronic acid (6.1%), HS (31.8%), DS (23.2%) and chondroitin 4/6-sulfate (38,9%). Plasma contains various kind of proteases, most of which are circulating in an inactive form, and these proteases are involved in cascade activation pathways such as coagulation, fibrinolysis and complement systems. Therefore, the protease that was activated by DS or HS was not necessarily the same as the one that cleaved FH or other proteins. It is of interest to consider whether the observed activation of protease by DS is physiologically relevant or simply an artifact. In order to bind proteins to DS and activate the protease, the ionic strength had to be lower than that in physiological conditions; however, we cannot reproduce the exact conditions of circulating plasma in the affinity column. Factors other than ionic strength that facilitate protein binding to DS may be present in the plasma. Identification of the protease that is activated by DS and HS will provide insight into the mechanism of DS-mediated activation of protease and the cryptic roles of GAGs in the plasma. Until now, known DS-binding proteins in the plasma have been limited to heparin cofactor II, apolipoprotein B and several cytokines [12,18,19], and more recently to FH [8,20]. Because mass spectrometry in conjunction with 2D-PAGE is one of the most sensitive techniques to identify proteins in a mixture, we used it to screen as many DS-binding proteins in the plasma as possible. Six proteins other than albumin were identified by SDS-PAGE, followed by mass spectrometry and peptide mass fingerprinting, and 19 proteins were detected by 2D-PAGE analysis. Albumin was detected by both SDS-PAGE and 2D PAGE experiments, but its binding seems likely to be nonspecific due to its high concentration in the plasma. In addition, α-1-AT and ATIII might be nonspecific binding proteins because a major portion of these proteins was detected in the nonbinding fraction from the DS column by Western blotting. Most other binding proteins identified by mass spectrometry were ascertained by Western blotting. We pre-
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viously reported that cleavage of FH was prevented by APMSF, and hence the 500 mM NaCl eluate from the DS column in the presence and absence of APMSF was investigated by Western blotting. Antibodies of plasma proteins involved in coagulation, complement reaction and apolipoproteins were used for blotting, and FXII, FI and prothrombin were detected only in the presence of APMSF. The DS-binding sites on these proteins may be severely degraded by the protease activated by DS. Most of the other DS-binding proteins detected in the absence of APMSF were also degraded to various degrees during prolonged storage. It is interesting that both FH and FI were bound to DS and were affected by the DS-mediated protease, because FH is a cofactor of FI and both molecules are responsible for the inactivation of C3b and downregulating the complement cascade alternative pathway [21,22]. Taking into consideration the fact that C3 and FB are also members of the alternative pathway, DS on the endothelial surface could be an essential modulator of the complement system. Moreover, four independent groups have reported that a common variant of the FH gene in the seventh domain is strongly associated with the onset of agerelated macular degeneration, a leading cause of visual impairment and blindness in the elderly [23–26]. The role of FH in this disease has not been investigated as yet but it may be involved in inflammation. Among the DS-binding proteins, PEDF is of interest in connection with age-related macular degeneration, because this protein was first found in cultured pigment epithelial cells of the fetal human retina [27] and has since been found to be a member of the serine protease inhibitors [28]. Although protease inhibitory activity has not been reported, these three molecules, PEDF, FH and FI, were able to bind to DS and therefore may have a role in the onset of this disease. In summary, we have found that the interaction of plasma proteins with DS is similar to their interaction with HS but different from their interaction with CSA or heparin in terms of binding capacity and protease activation. In addition, we have identified 24 plasma proteins as DS-binding proteins by mass spectrometry and Western blotting. The newly identified DSbinding proteins will shed light on unknown roles of DS and will provide insight into our understanding of the physiology and pathology of plasma proteins. References [1] H.E. Conrad, Structure of heparan sulfate and dermatan sulfate, Ann. N. Y. Acad. Sci. 556 (1989) 18–28. [2] R.L. Jackson, S.L. Busch, A.D. Cardin, Glycosaminoglycans: molecular properties, protein interactions and role in physiological processes, Physiol. Rev. 71 (1991) 481–539. [3] J.M. Trowbridge, R.L. Gallo, Dermatan sulfate: new functions from an old glycosaminoglycan, Glycobiology 12 (2002) 117R–125R. [4] L. Kjellen, U. Lindahl, Proteoglycans: structures and interactions, Annu. Rev. Biochem. 60 (1991) 443–475. [5] R.V. Iozzo, A.D. Murdoch, Proteoglycans of the extracellular environment: clues from the gene and protein side offer novel perspectives in molecular diversity and function, FASEB J. 10 (1996) 598–614. [6] R.V. Iozzo, Matrix proteoglycans: from molecular design to cellular function, Annu. Rev. Biochem. 67 (1998) 609–652.
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