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Keywords:

  • histidine-rich glycoprotein (HRG);
  • plasma protein;
  • protein purification;
  • metal affinity chromatography;
  • phosphatidic acid

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Experimental
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. REFERENCES
  10. Supporting Information

Histidine-rich glycoprotein (HRG) is a relatively abundant plasma protein that has been implicated in multiple biological processes including immunity, tumor progression, and vascular biology. However, current protocols for purifying HRG from plasma result in the copurification of contaminating proteins and raise questions over the validity of biological activities ascribed to HRG. In this study, we describe a two-step protocol for the large-scale purification of HRG from human plasma using a combination of metal affinity and ion exchange chromatography. The protocol employs a rapid and simple strategy to isolate highly purified HRG that minimizes proteolytic cleavage of the protein. The purification of HRG was assessed at each stage by measuring the amount of HRG immunoreactive protein using a specific monoclonal antibody against total protein, and demonstrated ∼1,000-fold purification with an overall yield of ∼32%. Mass spectrometry analysis demonstrated that plasma-derived HRG was free of contaminating proteins and gel electrophoresis showed it to have minimal proteolytic degradation. Characterization of protein by physical method showed that the protein exists as a single, monodisperse species. In contrast to the previous studies of HRG purified by different methods, HRG purified using the new procedure demonstrated a reduced profile of functions. Although the HRG retained binding to heparin and phosphatidic acid, it did not interact with necrotic cells or other cellular lipids. These data demonstrate that HRG does not exhibit the broad interactive properties that have been reported previously, suggesting that copurification of HRG-binding partners or other impurities are responsible for some of the reported functional properties. The findings in this study demonstrate that the new purification procedure can provide a ready source of pure HRG to assess ligand specificity and biological function of this important plasma protein. ©2013 IUBMB Life, 65(6):550–563, 2013


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Experimental
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. REFERENCES
  10. Supporting Information

Histidine-rich glycoprotein (HRG), also known as histidine-proline-rich glycoprotein, was first isolated in 1972 by Heimburger etal. (1) as a heparin-binding plasma protein. HRG is approximately a 65-kDa protein, abundant in the blood of all vertebrates as well as some invertebrates including marine bivalves (1, 2). In mammals, the majority of HRG is synthesized by the parenchyma cells of the liver, providing an approximately 2 mM basal level of HRG circulating in the blood. The protein has a relatively short half-life of approximately 3 days (3). More recently, HRG expression has been suggested to occur also in brain (4). A small proportion of HRG has been reported in the α-granules of platelets and megakaryocytes (5) as well as on the surface of monocytes and macrophages (6), indicating that some circulating HRG may be associated with cell surfaces, such as bound to receptors or produced locally by hematopoietic cells.

HRG is a multidomain protein composed of two cystatin-like regions at the N-terminus, a central histidine-proline-rich region, and a C-terminal domain (7). HRG has been classified as a type 3 cystatin and to share homology with other abundant plasma proteins including Fetuin A, Fetuin B, and kininogen (7). Although the precise physiological function of HRG is unclear, it has been reported to interact with numerous soluble and cell-associated ligands including metal ions, heparin, IgG, C1q, plasminogen, fibrinogen, factor XIIa, heparanase, heparan sulfate, and phospholipids (8, 9). Thus, HRG has been proposed to act as an adaptor molecule by interacting with several ligands simultaneously via its modular domains (8). Recently, HRG deficient (HRG−/−) mice have become available to address its role in vivo. Although HRG−/− mice are viable and fertile with no gross abnormalities, they showed a shorter plasma prothrombin time and bleeding time, indicating that HRG may exhibit anticoagulation properties in vivo (10). Fibrin clots are also dissolved more rapidly in HRG−/− mice when compared to wild-type mice, suggesting that HRG can function in vivo as an antifibrinolytic molecule (10). Furthermore, consistent with the previous studies demonstrating that HRG can mediate the lysis of microbes in vitro (11), HRG−/− mice are more susceptible to both bacterial and fungal infections (12, 13). Recent studies have also suggested a role for HRG in tumor progression. The overexpression of HRG in allograft tumors resulted in a reduction of necrotic areas and skewed tumor-associated macrophages toward a proinflammatory phenotype (14). Consistent with these observations, HRG−/− mice when compared to wild-type mice, grow larger tumors with increased necrotic areas, and show increased infiltration of M2 macrophages (15).

Although the in vivo significance of HRG is emerging based on the characterization of HRG−/− mice, most studies, to date, on HRG have been performed in vitro using plasma-derived rabbit or human HRG purified via a number of different methods (Supplementary Table 1). These HRG purification methods generally result in copurification of several contaminants including known HRG-binding partners and possibly other nonassociating proteins. For example, isolation of HRG using the phosphocellulose method, one of the most commonly used procedures, can lead to the copurification of IgG (9). Similarly, HRG purification utilizing PEG 4000 and ion exchange chromatography resulted in the copurification of IgG, plasminogen, and fibrinogen (16). These purification procedures often require a number of downstream affinity chromatography steps to remove various contaminants, which can reduce the yield of HRG and generate a mixture of intact and partially cleaved HRG species. In this study, we describe a simple and robust method for the purification of HRG from human plasma based on its binding properties to the metal ion cobalt and carried out a comparative assessment of functional differences with the phosphocellulose method. Significantly, HRG purified by the new purification method had more restricted functional characteristics from those previously reported. These data highlight that the method of purification can profoundly influence the purity, integrity, and perceived function of the isolated HRG.

Table 1. Overview of buffers used in the purification of HRG from human plasma
Column usedPurification stepSolution constituents/pHMinimum volume required (mL)
  1. All buffers are supplemented with EDTA-free protease inhibitor tablets and kept ice cold.

Cobalt affinity sepharose chromatographyWash buffer 150 mM NaH2PO4; 50 mM NaCl; 20 mM imidazole; 2 μg/mL aprotinin; pH 7.5250–500
 Wash buffer 250 mM NaH2PO4; 500 mM NaCl; 80 mM imidazole; 2 μg/mL aprotinin; pH 7.5250–500
 Wash buffer 350 mM NaH2PO4; 150 mM NaCl; 80 mM imidazole; 2 μg/mL aprotinin; pH 7.5250–500
 Elution buffer50 mM NaH2PO4; 150 mM NaCl; 500 mM imidazole; 2 μg/mL aprotinin; pH 7.540–60
 Column storage solution20% Ethanol 
 Buffer A20 mM Tris HCl; pH 8.5Variable
 Buffer B20 mM Tris HCl; 500 mM NaCl; pH 8.5Variable
 Column storage solution20% Ethanol 

Experimental

  1. Top of page
  2. Abstract
  3. Introduction
  4. Experimental
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. REFERENCES
  10. Supporting Information

Purification of Human Plasma-derived HRG by Cobalt/Anion Exchange Method

Frozen fresh human plasma (Australian Red Cross Blood Service) was thawed and divided into 45-mL aliquots. To avoid proteolysis of HRG, all steps were carried out at 4 °C. The plasma was gently rocked for 1–2 h at 4 °C with 3 mL of cobalt sepharose beads (TALON Metal affinity resin, Clontech, Palo Alto, CA) in the presence of 4 μg/mL of aprotinin (Sigma-Aldrich, St Louis, MO), 20 mM imidazole, and an ethylenediaminetetraacetic acid (EDTA)-free protease inhibitor cocktail (Roche, Mannheim, Germany). The cobalt sepharose was then recovered by centrifugation and the resin was sequentially washed with wash buffers 1–3 (Table 1), followed by the elution of bound material with elution buffer (Table 1).

To isolate an isoelectrically pure protein, the eluted material was further purified using the Anion chromatography (Hi Trap Q FF, GE Healthcare, Uppsala, Sweden). This procedure was also used as a buffer exchange step to reduce the length of the purification procedure as this was crucial in preventing proteolytic degradation of the HRG. The eluted material from the cobalt affinity column was diluted 15-fold with 20 mM Tris HCl, pH 8.5 (buffer A) and the sample loaded onto the column (flow rate, 3 mL/min). The column was washed with buffer A (flow rate, 3 mL/min) and HRG eluted with 20 mM Tris HCl, 500 mM NaCl, pH 8.5 (buffer B) (flow rate, 0.8 mL/min), employing a linear salt gradient. Fractions of 3 mL were collected and the elution profile monitored by measuring the absorbance at 280 nm (Fig. 1). Fractions 14–20 were pooled and the purity of HRG was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting, and mass spectrometry (MS) analysis. The protein concentration was measured using the Bradford reagent (Bio-Rad, Hercules, CA), with bovine serum albumin (BSA) as a standard.

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Figure 1. Anion exchange elution profile for human plasma-purified HRG. The anion exchange chromatographic elution pattern of human HRG on HiTrap QFF column. The diluted elution obtained from cobalt affinity chromatography was loaded on the column at a flow rate of 3 mL/min. The column was washed with buffer A and HRG eluted with buffer B (NaCl, 500 mM) at a flow rate of 0.8 mL/min, utilizing a linear salt gradient with fraction of 3 mL being collected (fractions, 14–20). Elution was monitored by measuring the absorbance at 280 nm.

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Purification of Human Plasma-derived HRG Using Phosphocellulose

Plasma-derived HRG was isolated from frozen fresh human plasma (Australian Red Cross Blood Service) using P-11 phosphocellulose (Whatman, Kent, United Kingdom) according to a previously described method (17–19).

SDS-PAGE and Protein Staining

Protein samples were electrophoresed on 4–12% NuPAGE® Novex® Bis-Tris Mini gels (Invitrogen, Carlsbad, CA) in NuPAGE® MES SDS Running buffer (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. Subsequently, protein gels were stained either with Coomassie Brilliant Blue or with silver using the Silver Quest staining kit (Invitrogen, Carlsbad, CA).

Western Blotting

SDS-PAGE-separated protein samples were transferred onto a nitrocellulose membrane (Hybond ECL, GE Healthcare, Buckinghamshire, United Kingdom) using the TE77 PWR Semi-Dry Transfer Unit (GE Healthcare, Buckinghamshire, United Kingdom). Membranes were blocked with 3% BSA/phosphate-buffered saline (PBS) for 1 h at room temperature or overnight at 4 °C and immunoprobed as described elsewhere (20, 21). The following antibodies were used; mouse antihuman HRG monoclonal antibody (1:2,000) followed by horseradish peroxidase (HRP)-conjugated sheep antimouse immunoglobulin (Ig) antibody (1:3,000; Sigma-Aldrich, St Louis, MO), domain-specific rabbit antibodies 0116, 0119, and 0115 (1:3,000) followed by HRP-conjugated antirabbit Ig antibody (1:3,000; Sigma-Aldrich, St Louis, MO).

For the detection of human IgG and IgM, a HRP-conjugated rabbit antihuman IgG antibody (γ-chain specific; 1:1,000; Dako) and a HRP-conjugated rabbit antihuman IgM antibody (μ-chain specific; 1:1,000; Dako, Glostrup, Denmark) were used. Human plasminogen was detected by a rabbit antihuman plasminogen antibody (1:500; Dako, Glostrup, Denmark), followed by a HRP-conjugated donkey antirabbit Ig antibody (1:3,000; Sigma-Aldrich, St Louis, MO).

Membranes were then visualized by chemiluminescence using the SuperSignal® West Pico Chemiluminescent Super Substrate (Thermo Scientific, Rockford, IL).

Blue Native Electrophoresis

Blue Native electrophoresis was performed as described previously (22). Purified HRG from cobalt affinity/anion exchange chromatography and phosphocellulose chromatography (protein, 0.5 μg) or fresh serum (1 μL) was analyzed on a 4–16% large gel using 20 μg of thyroglobulin (669 kDa), ferritin (440 kDa), and BSA (134 and 67 kDa) as molecular weight markers (Sigma-Aldrich, St Louis, MO, USA). After Blue native electrophoresis, gels were transferred to polyvinylidene difluoride membrane. For the detection of HRG, rabbit antihuman HRG polyclonal antibody (1:3,000 in 1%BSA/PBS; kindly provided by Prof. Christopher Parish, ANU, Australia) was used followed by appropriate HRP conjugate antibody.

Mass Spectrometry

The mass of HRG was analyzed using HPLC-ESI-MS (HPLC: Ultimate 3000; Thermo Fisher, USA; MS: MicrOTOF-Q; Bruker-Daltonics, Bremen, Germany). The resulting spectra were analyzed using the Data Analysis program (Bruker-Daltonics, Bremen, Germany). To further establish the integrity and purity of the HRG preparation, the sample was reduced (10 mM dithiothreitol, 60°C, 1 h), alkylated (40 mM iodoacetamide, room temperature, 20 min), and digested with trypsin (1 μg) at 37 °C for 2 h. The digested protein preparation was dried in a speedy vac and analyzed by electrospray MS/MS (Micro-TOF-Q-MS; Bruker-Daltonics, Bremen, Germany). The recorded masses and the corresponding MS/MS spectra were analyzed by Data Analysis (Bruker-Daltonics, Bremen, Germany) and the identity of the protein(s) determined using the Bio tools program (Bruker-Daltonics, Bremen, Germany).

Circular Dichroism Spectroscopy

Circular dichroism (CD) spectra were analyzed using an AVIV Model 410-SF CD spectrometer. Wavelength scans of human HRG (105 μg/mL in PBS, pH 7.4) were performed between 190 and 260 nm in 1-mm quartz cuvettes at 20 °C. All data collected below 197 nm were removed from the analysis owing to poor signal to noise. Data were analyzed using the CDPro software package (23, 24).

Quantification of HRG by Enzyme-linked Immunosorbent Assay

HRG was quantitated in samples from different stages of the purification using a sandwich enzyme-linked immunosorbent assay (ELISA). F96 Maxisorp Immuno plates (Nunc, Roskilde, Denmark) were coated with a rabbit antihuman HRG polyclonal antibody (1:2,000) in carbonate coating buffer (pH 9.6) and incubated at 4 °C overnight. Plates were washed three times with PBS and incubated for 1 h at room temperature with 3% BSA/PBS to block nonspecific binding. Purified plasma-derived HRG was used to generate a standard curve. HRG was detected by a mouse antihuman HRG monoclonal antibody (1:2,000 in 1% BSA/PBS), followed by a HRP-conjugated sheep antimouse Ig antibody (1:3,000 in 1% BSA/PBS). Bound HRP-conjugated antibody was detected using the chromogenic substrate 3,3′,5,5′-tetramethylbenzidine (Sigma-Aldrich, St Louis, MO). The reaction was stopped with 2 M sulfuric acid and the absorbance was measured at 450 nm using a ThermoMax Microplate Reader (Molecular Devices, Sunnyvale, CA). The data were analyzed by SoftMaxPro 4.0 software (Molecular Devices, Sunnyvale, CA) (Fig. 2).

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Figure 2. Standard curve of the purified human HRG generated by sandwich ELISA. Microtiter plates were coated with a rabbit antihuman HRG antibody, followed by addition of serially diluted HRG from 2 to 0.005 μg/mL in duplicates. Bound HRG was detected using a mouse anti-HRG monoclonal antibody, followed by a HRP-conjugated antimouse Ig antibody. The concentration of HRG at each stage of the purification process was calculated using the equation indicated.

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Heparin Binding Assay

HRG binding to heparin was measured by ELISA which was performed according to a previously described method (20, 21). Bound HRG was detected using a mouse antihuman HRG monoclonal antibody and a HRP-conjugated sheep antimouse Ig antibody as described above.

Lipid binding assay

HRG binding to various phospholipids was performed as described previously (9) Chemiluminescence was detected as detailed for the Western blotting procedure.

Necrotic Cell-binding Assay

HRG binding to necrotic cells was determined by immunofluorescence flow cytometry as described previously (9, 17). Samples were analyzed immediately by flow cytometry using FACS Canto II Flow Cytometer and FACS Diva software (BD Biosciences, San Jose, CA).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Experimental
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. REFERENCES
  10. Supporting Information

Purification of HRG from Human Plasma

Human HRG contains a high proportion of histidine residues in the histidine-rich region (HRR) that account for almost 13% of the total amino acids present. This property of HRG was exploited in the initial purification step from human plasma, as histidine-rich sequences have been well characterized in the efficient binding of immobilized transition metal ions (e.g., cobalt, nickel and copper, and zinc) (25–27). The HRG purification strategy used cobalt-immobilized metal affinity chromatography (TALON resin) as the initial step of purification. To prevent proteolysis of HRG during the purification process, all solutions were kept ice-cold and supplemented with protease inhibitors and the entire procedure was completed in a single day. Initially, imidazole at 20 mM was present in binding buffer and wash buffer 1 to reduce nonspecific interactions, and subsequently increased to 80 mM in wash buffers 2 and 3 to remove nonspecifically bound proteins (Table 1). The salt concentration was also increased from 50 to 500 mM in wash buffer 2 to dissociate any ionic interactions. Finally, HRG was eluted from the cobalt column with 500 mM imidazole and 150 mM NaCl (Table 1). It is worth noting that a lower concentration of imidazole of approximately 250 mM could also be used to elute HRG (data not shown). As shown in Fig. 3, most of the proteins in human plasma were not retained on the cobalt-immobilized TALON resin and passed through the column in the unbound fraction. Furthermore, the cobalt-eluted material was found to contain predominately intact HRG that migrated as a single band at approximately 65 kDa under reducing condition although a low level of proteolytically cleaved forms of HRG and a large molecular weight (>180 kDa) contaminant were present (Fig. 3).

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Figure 3. Reducing SDS-PAGE analysis of proteins in fractions collected at different steps of HRG purification from human plasma. Protein samples obtained from the purification of HRG from human plasma were separated on 4–12% Bis-Tris SDS-PAGE under reducing conditions and stained with Coomassie Brilliant Blue (left panel) or analyzed by Western blot using a monoclonal antibody specific for HRG (right panel). Lane 1, molecular weight marker (kDa); lane 2, human plasma (0.0001%); lane 3, unbound plasma through cobalt affinity column (0.0001%); lane 4, wash fraction 1 (0.004%); lane 5, wash fraction 2 (0.004%); lane 6, wash fraction 3 (0.004%); lane 7, eluted HRG after cobalt affinity chromatography (0.04%, 7 μg); lanes 8, 9 and 10, pooled fractions 1, 2, and 3, respectively, after anion exchange chromatography (0.1%, 5.6, 1.8, and 0.3 μg, respectively). The percentage value represents the proportion of volume loaded onto each lane to the total volume for each fraction.

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To further purify human plasma-derived HRG by anion exchange chromatography, the cobalt-eluted material was immediately diluted with ice-cold buffer A (to reduce the salt concentration by 15-fold) before binding to an anion HiTrap Q FF column and subsequent elution with a salt gradient. Eluted fractions were analyzed by SDS-PAGE and HRG was shown to elute at approximately 200 mM NaCl (i.e., 40% buffer B) (Fig. 1). Fractions 14–20, containing HRG, were pooled and found to be of a higher purity compared with cobalt-eluted material based on SDS-PAGE and Western blotting (Fig. 3, lanes 8 and 9). The anion exchange chromatography step was included not only to further purify HRG, but also to concentrate HRG to avoid filter-based centrifugation techniques that were found to result in considerable degradation of HRG (data not shown). As HRG does not possess any enzymatic activity and there is no known quantitative assay, verification of the amount of HRG purified to obtain the specific activity values at different stages of the purification process was carried out by sandwich ELISA (Fig. 2) as described in the EXPERIMENTAL section. Thus, specific activity (units) for HRG is defined as the total amount of immunoreactive HRG divided by the total protein in the sample. However, as plasma at neat concentration contained material that significantly interfered with the immunoassay, the starting levels were based on the known concentration of HRG in plasma (28). A summary of the purification process is provided in Table 2. The purification was ∼1,000-fold and the yield was >30%.

Table 2. Summary of purification process of HRG from human plasma
 ABCDEFGH
  • a

    Total protein as determined by Braford reagent.

  • b

    Concentration of HRG in human plasma as reported in the literature (28).

  • c

    Concentration of HRG as measured by sandwich ELISA.

 Total proteina (mg)Volume (mL)conc. of HRG (mg/mL)Total amount of HRG (mg) (B × C)Specific activity (units) (D/A)Overall yield (%)Step yield (%)Purity (fold)
Crude human plasma27 × 1032400.105b25.20.000941001
Cobalt affinity chromatography19600.235c14.10.74255.9555.95789
Anion exchange chromatography9210.385c8.10.91032.1457.44968

Analysis of the Purity of Human Plasma-derived HRG

To evaluate the purity of HRG, silver staining of human plasma-derived HRG was initially performed and indicated the presence of a dominant band of approximately 65 kDa and few minor bands of lower molecular weight (Fig. 4A). Domain-specific rabbit antihuman HRG antibodies were also used in Western blotting and showed that these minor bands were proteolytic fragments originating from the N-terminus of HRG (Fig. 4B). To further examine the purity of the HRG preparation, the presence of abundant plasma proteins reported to interact with HRG was assessed by Western blot analysis. This indicated no detectable levels of human IgG, IgM, and plasminogen in the purified HRG (Fig. 4C).

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Figure 4. Determination of the integrity and purity of human HRG by silver staining and Western blotting. (A) The integrity and purity of purified HRG (400, 200, 100, 50, and 25 ng) was verified by silver staining following SDS-PAGE under reducing conditions. (B) Assessment of the integrity of purified HRG by Western blot analysis. Purified HRG (750 ng) was separated by 4–12% Bis-Tris SDS-PAGE under nonreducing or reducing conditions prior to Western blot analysis using domain-specific rabbit antihuman HRG antibodies. The domain specificity of the antibodies (0115, 0116, and 0119) is shown diagrammatically. (C) Western blot analysis to assess the human IgG, IgM, and plasminogen content of purified HRG (1.5 μg) following SDS-PAGE under reducing conditions using antibodies specific for these proteins. Human IgG (100 ng), IgM (200 ng), and plasminogen (200 ng) are included as positive controls.

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To assess the purity of plasma-derived HRG, MS analysis was performed using Micro-TOF-Q-MS. The data showed three predominant masses for the purified protein of 64,632, 64,924, and 65,292 Da (Fig. 5A), which correlated well with the predicted molecular weight of human HRG and are consistent with known differential glycosylation states (1, 29). It is also interesting to note that the MS data also revealed the possibility of phosphorylation of HRG, with masses at 64,711 and 65,003 Da (Fig. 5A), representing an increment of 80 Da and which corresponds to the expected mass increase for phosphorylation. The authenticity of the purified protein was confirmed by sequence identity. For this reason, the protein sample was reduced and alkylated and then digested with trypsin, with the resulting peptides analyzed by Micro-TOF-Q-MS/MS. The peptides obtained were assigned to HRG and sequence coverage of almost 65% was obtained (Fig. 5B). Consistent with the Western blot results using domain-specific antibodies as shown in Fig. 4B, the MS data on the intact protein identified peptides corresponding to sequences located in the N-terminal, histidine-rich, and C-terminal regions of human HRG (Fig. 5B), indicating the protein to be intact and not truncated. The only impurity found by MS analysis using Micro-TOF-Q-MS was an extremely low amount of fibrinogen. Collectively, these results demonstrate that the purified HRG was predominately intact and substantially free of impurities. Similar studies were undertaken on HRG purified using the alternate phosphocellulose method and showed that only 55% of the sample was HRG, whereas the remaining 45% was unknown material (data not shown).

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Figure 5. Characterization of purified HRG by MS analysis. (A) The deconvoluted electrospray micro-TOF-Q-MS spectrum of HRG. The spectrum shows at least three glycosylation states of HRG at 64,632, 64,924, and 65,292 Da. The peaks at 64,711 and 65,003 Da correspond to an increment of 80 Da, which is the expected mass increase for phosphorylation (within error). (B) Purified human HRG by cobalt affinity chromatography/anion exchange chromatography was reduced and alkylated and further digested with trypsin. The digest was analyzed by micro-TOF-Q-MS. The measured masses were assigned to HRG sequence and coverage of 65% was achieved. The bars represent the identified peptides and the small boxes indicate the amino acids identified. Peptides from both the N- and C-terminal were identified. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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Structural Analysis of Purified Human Plasma-derived HRG

The CD spectrum of the cobalt/anion exchange-purified HRG displayed a single minimum at approximately 205 nm (Fig. 6A) and the CD spectrum fitted by nonlinear least squares regression using the CDPro software package against relevant protein databases (23). The best fit for the purified HRG was determined using the CONTINLL algorithm against the SP22x database and had a root-mean-square deviation (r.m.s.d.) value of 0.057. It predicted that the purified protein has 45% unordered structure in combination with 55% defined secondary structure with substantial amounts of β-structure, β-turn, and polyproline 2 (PP2) helix and very little of α-helix (approximately, 1%) (Fig. 6C). In contrast, HRG purified by the phosphocellulose method was difficult to fit to the CONTINLL algorithm and had an r.m.s.d value of 0.104 (Fig. 6B), indicative of the presence of multiple species in the sample. The secondary structure predictions for phosphocellulose purified HRG are shown in Fig. 6C.

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Figure 6. Comparative CD spectrum of purified human HRG. (A) CD spectrum for cobalt affinity/anion exchange-purified HRG shows wavelength scan performed between 190 and 260 nm, with data below 197 nm being excluded from analysis owing to low signal to noise. The scan was performed at a protein concentration of 105 μg/mL. The final spectrum (open circles) is the average result of three scans measured at 20 °C. The CONTINLL algorithm from the CD Pro software package was used to generate the nonlinear best fit (solid line) against the SP22x protein database, with an r.m.s.d. value = 0.057. (B) CD spectrum for phosphocellulose chromatography-purified HRG same as performed above with the data (closed circles) and best fit (solid line) having an r.m.s.d value = 0.104. (C) Secondary structure proportions of purified human HRG from CONTINLL best fit analysis.

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Functional Analysis of Purified Human Plasma-derived HRG

It was of interest to determine whether the purified HRG via the mentioned purification method has the same interaction profile as previously determined for the phosphocellulose-purified HRG.

HRG, like antithrombin III, can bind heparin with high affinity and regulate coagulation through this interaction (30). The binding of the purified HRG to immobilized biotinylated heparin was tested by ELISA using a mouse antihuman HRG monoclonal antibody. Consistent with the previous studies (8, 30, 31), HRG bound to heparin in a concentration-dependent manner (Fig. 7A), indicating that the purified HRG retains its heparin binding function. Besides heparin, phosphocellulose-purified HRG has been shown recently to specifically recognize negatively charged phospholipids including phosphatidic acid (PA), sulfatide, phosphatidylinositol 3-phosphate, and phosphatidylinositol 4-phosphate (9) (Fig. 7B(ii)). To investigate the interaction of the purified HRG with cellular phospholipids, a protein–lipid overlay assay was carried out using commercially available membrane strips spotted with 100 pmol of various biologically active lipids. Cobalt/anion exchange-purified HRG (1 μg/mL) was found to bind exclusively to PA and not the other lipids previously implicated in HRG binding (9) (Fig. 7B(i)).

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Figure 7. Analysis of the interaction of cobalt/anion exchange-purified HRG with heparin, cellular lipids, and necrotic cells. (A) Ability of cobalt/anion exchange-purified human HRG to bind immobilized heparin by ELISA well precoated with ExtraAvidin (10 μg/mL) and biotinylated heparin (10 μg/mL). Heparin-bound HRG was detected using a mouse antihuman HRG monoclonal antibody followed by a HRP-conjugated antimouse Ig antibody. (B) Analysis of cobalt/anion exchange (i) or phoshocellulose (ii) purified human HRG binding to an array of cellular lipids spotted on either Membrane Lipid Strips™ or PIP Strips™. HRG was incubated with membrane strips overnight and bound HRG was detected using a mouse antihuman HRG monoclonal antibody followed by a HRP-conjugated antimouse Ig antibody. Membranes were then visualized by chemiluminescence. (C) Analysis of the ability of cobalt/anion exchange purified or phosphocellulose-purified human HRG (100 μg/mL) to bind to viable and heat-killed (56 °C, 30 min) necrotic Jurkat T cells, as measured by flow cytometry. Representative flow cytometry histograms are shown, with filled histograms representing antibody only control and open histograms representing HRG binding detected using a mouse antihuman HRG monoclonal antibody and a phycoerythrin-conjugated sheep F(ab')2 antimouse Ig antibody.

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Furthermore, a number of studies have shown that HRG as well as recombinant domains of HRG can bind strongly to late apoptotic and necrotic cells (9, 17, 20, 32). The binding of HRG to these membrane-permeabilized cells was postulated to aid their recognition and removal by phagocytes, possibly through recruiting IgG and complement components to the cell surface (9, 20, 33). To examine whether cobalt/anion exchange-purified HRG could bind strongly to membrane-permeabilized cells, a flow cytometry-based cell-binding assay on necrotic cells was performed. Jurkat T cells were made necrotic by heat treatment at 56 °C for 30 min and incubated with 100 μg/mL of purified HRG. Cell-bound HRG was detected using a mouse antihuman HRG monoclonal antibody. Surprisingly, the cobalt/anion exchange-purified HRG bound relatively poorly to heat-killed necrotic cells compared to phosphocellulose-purified HRG (Fig. 7C). Collectively, these results suggest that the binding function attributed to HRG is influenced by the presence of impurities in the sample.

Protein aggregation can adversely affect or alter protein function and activity. To determine whether aggregate formation was responsible for the functional differences observed, HRG purified via cobalt/anion exchange or phosphocellulose was analyzed under native conditions (Fig. 8A) on a native gradient gel and Western transfers probed with an antihuman HRG polyclonal antibody. It was observed that cobalt/anion exchange-purified HRG ran as a single species, whereas the phosphocellulose-purified HRG formed higher molecular weight species similar to the serum sample, suggesting that phosphocellulose-purified HRG formed complexes with other proteins that copurified with HRG. In addition, HRG purified using phosphocellulose contains additional proteins or is substantially degraded when analyzed on SDS-PAGE under reducing and nonreducing conditions (Fig. 8B).

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Figure 8. Physical comparative analysis of cobalt/anion exchange and phosphocellulose-purified HRG. (A) Purified HRG (0.5 μg) via different purification methods was separated on 4–16% gel under native conditions and analyzed by Western blot using an antihuman HRG rabbit polyclonal antibody with human serum as a positive control. Lane 1, human serum; lane 2, phosphocellulose-purified plasma HRG; and lane 3 cobalt/anion exchange-purified HRG, along with native molecular weight marker. (B) Purified HRG (4 μg) was separated on 4–12% Bis-Tris SDS-PAGE gel under reducing and nonreducing conditions. Following the electrophoresis, the gel was stained with Coomassie stain for protein identification. Lanes 1 and 2, cobalt/anion exchange-purified HRG and phosphocellulose-purified HRG, respectively, under reducing conditions; lane 3, molecular weight marker; lanes 4 and 5, cobalt/anion exchange-purified HRG and phosphocellulose-purified HRG, respectively, under nonreducing conditions.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Experimental
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. REFERENCES
  10. Supporting Information

Human plasma is a complex biological system that contains an array of different proteins, making the isolation of a pure preparation of a specific protein from plasma challenging. HRG is an abundant multifunctional protein in human plasma that was first identified in 1972 when it was copurified with the complement factor C1q (1). Since its discovery, it has been described as a protein with multiple functions. A significant issue in considering these data is whether these functions truly reside in HRG, or in proteins that copurify with HRG as a result of the purification procedure used. Although a number of methods have been described (Supplementary Table 1), phosphocellulose and nickel nitrilotriacetic acid (Ni-NTA) chromatography remain the most frequently used methods for obtaining native HRG. There is also the possibility to obtain pure protein by expression of the protein in bacteria although in the case of HRG, the protein has multiple glycosylation sites and it is not clear how essential glycosylation is for the function of the protein. Expression systems using mammalian cells typically yield very low HRG. As mentioned above, most of the purification methods result in preparations that are contaminated to varying degrees with plasma proteins. For example, plasminogen is a significant contaminant, capable of stimulating angiogenesis (34), and IgG (9) could enhance necrotic cell clearance.

We therefore designed a simple two-step purification method to isolate a highly purified preparation of HRG from human plasma. Two widely used chromatography techniques were applied to purify HRG, the first being immobilized metal affinity chromatography (IMAC), followed by anion exchange chromatography. Initially, exploiting the ability of HRG to bind metal ions with high affinity via its HRR (35, 36), resins with covalently linked nickel (Ni-NTA, Qiagen, Valencia, CA) or cobalt (TALON, Clontech) were compared for use in the purification of HRG from human plasma. Nickel and cobalt immobilized resins are the most widely used IMAC resins, with each metal providing six metal coordination sites that mediate specific affinity for histidine residues (25–27). In particular, Ni-NTA is commonly used for HRG purification and most protocols described are single step (11, 13, 33, 37). However, Ni-NTA-purified HRG was substantially degraded and contain a number of impurities (data not shown). In contrast, relatively pure and intact HRG was isolated from human plasma following the initial step of purification using cobalt (TALON) resin, possibly owing to a reduced binding of nonspecific plasma protein to cobalt resin that may exhibit proteolytic activities against HRG. It was to avoid proteolytic cleavage of HRG during the purification process, as the previous studies have shown that the cleaved fragments of HRG generated by proteases like plasmin may have modified biological functions in regulating angiogenesis and host defense (11, 21, 38, 39).

Although cobalt-purified HRG was relatively pure as judged by SDS-PAGE, additional anion exchange chromatography was performed to further enhance the purity of plasma-derived HRG. The advantage of this step was that it combined purification with buffer exchange to remove the imidazole and concentration of the protein. Utilizing these procedures, HRG was purified from human plasma almost 1,000-fold (Table 2). Furthermore, the step yield in both purification steps was comparable, indicating that there was no substantial loss of HRG during these steps, with an overall yield being 32%. The purification method we describe obtained reproducible results with different plasma preparations from multiple healthy donors.

MS analysis of the purified protein indicated the presence of a minor amount of fibrinogen. It also revealed both glycosylation and phosphorylation as potential post-translational modification. Although glycosylation of HRG has been previously identified (1, 29), these data represent the first evidence that HRG may also be phosphorylated. Further analysis is required to identify the type and location of residue(s) in HRG that may be phosphorylated. It is interesting to note that both protease digestion and post-translational modifications such as phosphorylation may occur preferentially within regions of disorder (40, 41). As HRG is an intrinsically unstructured protein with a predicted disordered region located between residues 300 and 500 (42), it could be potentially phosphorylated within this region. Recent studies have suggested that post-translational modifications, in particular phosphorylation, may contribute to age-related changes in protein levels in plasma (43, 44). Ignjatovic etal. (43) demonstrated an age-related increase in protein phosphorylation in adult plasma compared to neonatal plasma, suggesting that this difference in phosphorylation could be a function of age. Based on these observations, phosphorylation of HRG could potentially regulate its level and function in human plasma.

Consistent with the previous studies on HRG (35, 45) together with bioinformatic predictions (42, 46, 47), the secondary structure analysis of human HRG by CD spectroscopy confirmed the intrinsically disordered nature of the protein, with a significant proportion of PP2 helix owing to a high proline content. Although it is currently unclear how this localized region of disordered structure could influence HRG functions, it is possible that, upon association with certain ligands, such as partner proteins, the structure of HRG may change to a more ordered state. Consistent with this notion, the presence of Zn2+ has been shown to promote HRG binding to soluble heparin and heparan sulfate on cell surfaces (17).

HRG has been proposed to interact with a diverse array of molecules including proteins, sulfated polysaccharides, phospholipids as well as metal ions, based on the direct and indirect evidence from in vitro studies (Supplementary Table 1). However, whether these suggested ligands are genuine interacting partners of HRG has not been extensively validated by multiple experimental approaches or using HRG preparations isolated by different methods. Although cobalt/anion exchange-purified HRG is functional in its ability to bind immobilized heparin, it exhibited properties that are strikingly different to HRG purified by the phosphocellulose procedure. Phosphocellulose-purified HRG was found recently to interact with a number of phospholipids including specific phosphatidylinositols, PA and sulfatide, with these phospholipids suggested to mediate HRG binding to membrane permeabilized cells (9). Surprisingly, HRG purified as described in this article bound solely to PA and did not bind to membrane permeabilized necrotic cells. These data suggest that the binding to exposed PA on necrotic cells by HRG is not important in the proposed mechanism of HRG-mediated necrotic cell clearance (9). Furthermore, these data clearly demonstrate that HRG purified by different methods can exhibit different properties in the same experimental system. A reason for such variability is likely owing to the differences in the presence and/or activity of copurified molecules that modify HRG function. As proposed previously, such molecules could be genuine components of a “HRG complex” that are required for specific HRG functions, for example, HRG–IgG for clearance of necrotic cells (9). Alternatively, depending on the purification methods, HRG preparations may contain impurities of other plasma proteins that have various functions independent of HRG. It should also be considered that the purification method can also have a major effect on the integrity of HRG, with proteolytic cleavage of HRG known to influence its function as mentioned previously. It is worth noting that as the binding partners of HRG could be a protein, polysaccharide, phospholipid, or metal ion, it may be difficult to determine whether a proportion of HRG molecules are already in complex with certain ligands during the purification process. The availability of HRG purified with high purity will now help to resolve the discrepancies of HRG function reported in the literature (9, 16, 33, 48) by enabling experiments to confirm the authenticity of known ligands as well as the identification of novel ligands and their roles in regulating HRG function.

Conclusions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Experimental
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. REFERENCES
  10. Supporting Information

In conclusion, the proposed biological properties of HRG, to date, have been largely based on in vitro studies using eithernative/recombinant HRG or synthetic peptides (8, 49). Although HRG−/− mice will provide a powerful tool to define the role(s) of HRG in vivo, the elucidation of the precise molecular mechanisms that underpin HRG function will be aided by the use of purified protein. The ability to isolate highly pure native plasma-derived HRG as described herein represents an important advance toward defining the true ligands and functions of HRG.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Experimental
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. REFERENCES
  10. Supporting Information

The authors thank Dr. Chris Adda and Fiona Durand (Department of Biochemistry, La Trobe University, Australia) for technical assistance with the ion exchange chromatography and mass spectrometry analysis, respectively. This study was funded by Australian National Health and Medical Research Council (NHMRC) Research Grants (418008) and CRC for Biomarker Translation Grants (1147/751225).

REFERENCES

  1. Top of page
  2. Abstract
  3. Introduction
  4. Experimental
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. REFERENCES
  10. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Experimental
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. REFERENCES
  10. Supporting Information

Additional Supporting Information may be found in the online version of this article.

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IUB_1168_sm_SuppTab1.pdf1863KSupporting Information Table 1.

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