Serum Amyloid P Component (SAP) Interactome in Human Plasma

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The Serum Amyloid p Component (SAP) Interactome in Human Plasma Containing Physiological Calcium Levels Ebbe Toftgaard Poulsen, Kata Wolff Pedersen, Anna Maria Marzeda, and Jan Johannes Enghild Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.6b01027 • Publication Date (Web): 18 Jan 2017 Downloaded from http://pubs.acs.org on January 19, 2017

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The Serum Amyloid p Component (SAP) Interactome in Human Plasma Containing Physiological Calcium Levels

Ebbe Toftgaard Poulsen1, #, Kata Wolff Pedersen1, #, Anna Maria Marzeda1 and Jan J. Enghild1, *

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Department of Molecular Biology and Genetics and Interdisciplinary Nanoscience Center, Aarhus University, Gustav Wieds Vej 10, Aarhus, Denmark

Running title: Interactome of SAP in plasma

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Contributed equally to this study.

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To whom correspondence should be addressed: Tel.: +45-2338 2262; E-mail: [email protected]

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Abbreviations SAP, serum amyloid p component; Ca2+, calcium ion; CRP, C-reactive protein; α1PI, α1-antitrypsin; α2M, α2-macroglobulin; IαI, inter-α-trypsin inhibitor; ACT, antichymotrypsin; TBS, tris-buffered saline; Ambic, ammonium bicarbonate; XIC, extracted ion chromatogram

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Abstract The pentraxin serum amyloid p component (SAP) is secreted by the liver and found in plasma at a concentration of approximately 30 mg/L. SAP is a 25 kDa homo-pentamer known to bind both protein and non-protein ligands, all in a calcium-dependent manner. The function of SAP is unclear, but likely involves the humoral innate immune system spanning the complement system, inflammation, and coagulation. Also, SAP is known to binding to the generic structure of amyloid deposits and possibly to protect these against proteolysis. In this study, we have characterized the SAP interactome in human plasma containing the physiological Ca2+ concentration using SAP affinity pull-down and co-immunoprecipitation experiments followed by mass spectrometry analyses. The analyses resulted in the identification of 33 proteins of which 24 were direct or indirect integration partners not previously reported. The SAP interactome can be divided into categories including apolipoproteins, complement system, coagulation, and proteolytic regulation.

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Introduction Serum amyloid P component (SAP, also referred to as pentraxin-2) belongs to the group of short pentraxins in the evolutionary conserved pentraxin family. SAP is a pentameric glycoprotein consisting of five identical 25 kDa subunits arranged non-covalently in a flat disc-like configuration where each subunit contains two calcium ion (Ca2+) binding sites essential for ligand binding.(1) SAP shares 51% amino acid sequence identity with the acute-phase protein C-reactive protein (CRP), another member of the short pentraxins.(2) SAP is secreted by the liver and the plasma concentration of SAP is approximately 30 mg/L.(3) Besides plasma, SAP is also associated with the extracellular matrix in many tissues and is a normal component of many basement membranes.(4) SAP has been shown to bind in vivo and/or in vitro in a Ca2+-dependent manner to DNA,(5) type IV collagen,(6) laminin,(7) fibronectin, C4-binding protein,(8) C1q,(9) glycosaminoglycans,(10) ApoAI(11) and it associates to high density and very low-density lipoproteins.(12) In addition, SAP also binds to the generic cross-beta-sheet secondary structure of amyloid fibrils and accumulates in pathological deposits of all types of amyloidosis(13-15) where it has been suggested to stabilize and protect the amyloid structure against proteolysis.(16) In the presence of Ca2+, SAP itself is highly resistant to proteolytic degradation,(17) whereas in the absence of Ca2+, SAP becomes susceptible to cleavage in the Ca2+ binding region, leading to loss of its ligand binding ability.(17) Purified SAP aggregates in the presence of Ca2+ (18) and it has previously been debated whether SAP exists as a pentamer or decamer in vivo, of which the pentamer has been shown to exist in serum or in vitro in presence of physiological albumin concentrations.(19) The biological functions of SAP are still unclear, but the protein is known to be involved the humoral innate immune system spanning the complement system, inflammation, and coagulation.(20) In this study, we characterized the protein interactome of SAP in human plasma using immobilized SAP affinity pull-down and co-immunoprecipitation experiments combined with liquid chromatography mass spectrometry (LC-MS/MS) analysis. The analyses resulted in identification of 33 protein SAP ligands of which 24 were unknown binding partners. Western blotting against 4 protein ligands was performed to validate the findings of the LC-MS/MS analysis. SAP was shown to interact with a group of apolipoproteins as well as with proteins of the complement- and

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coagulation systems including proteases. Also, binding of proteinase inhibitors suggests that SAP is involved in proteolytic regulation. Our results add yet other roles to SAP as being a potential shuttle protein in human plasma and as being a regulator of enzymatic activity.

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Experimental procedures Plasma collection – Human blood was collected from three healthy individuals (two men and one woman, age 30-37). The blood was collected in blood collections tubes without additives (Ref: 367614, BD Vacutainer, New Jersey, US) and the coagulation was inhibited with a direct thrombin inhibitor (7 µM), Bivalirudin(21) (Lot: 064M4731V, Sigma Aldrich, Missouri, US). The tubes were spun at 1,200 x g for 15 min before the plasma fraction was aliquoted and stored at -20 oC.

Purification of serum amyloid P component – SAP was purified from frozen outdated citrate treated plasma from Aarhus University Hospital, Denmark. For a typical purification polyethylene glycol 8000 (PEG) was added to 50 mL of citrated plasma to a final concentration of 4% ,the pellet was collected by centrifugation and discarded before the supernatant was adjusted to a total of 20% PEG. The precipitate was collected by centrifugation and resuspended in tris-buffered saline (TBS) (20 mM Tris-HCl, 150 mM NaCl, pH 7.4) overnight. The resuspended 4% to 20% PEG cut were adjusted to 2 mM CaCl2 and filtered through a 0.45 µm syringe filter (Q-Max, Frisenette, Kenbel, Denamark). The solution was then applied to a 1 mL HiTrap-heparin column (GE Healthcare Life Science, Little Chalfont, UK) connected to a ÄKTA protein purification system (GE Healthcare Life Science), equilibrated in TBS containing 2 mM CaCl2. After thorough washing, the column was eluted at 1 mL min-1 using a gradient of 1% B min-1 from 0 to 10 mM EDTA. The SAP-containing fractions were pooled, diluted 10 times in 10 mM Tris-HCl, 10 mM EDTA, pH 7.4 and applied to an anion exchange chromatography column (1 mL HiTrap Q, GE Healthcare Life Science, Little Chalfont, UK), equilibrated in 10 mM Tris-HCl, pH 7.4 containing 10 mM EDTA. The column was developed at 1 mL min-1 using a gradient of 1% B min-1 from 0 to 1 M NaCl. The fractions containing purified SAP were pooled, dialyzed into 20 mM sodium phosphate, 137 mM NaCl, pH 7.4 and stored at -80 oC.

Dynabeads epoxy ligand coupling – Dynabeads® M-270 Epoxy (Invitrogen, California, US) used for the co-immunoprecipitation were incubated with mouse monoclonal IgG1 raised against human SAP

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(ab51085, Abcam, Cambridge, UK) or control mouse IgG1 (Cat.no 02-6100, Lot: QF217975, Thermo Scientific, Massachusetts, US) for 16 h with slow end-over-end rotation in the presence of 0.8 M ammonium sulfate at 37 oC. Beads (Dynabeads® M-270 Epoxy) for the affinity pull-down experiment were incubated with purified SAP or PBS (blank beads) in the presence of 1 M ammonium sulfate for 16 h at 37 oC with slow end-over-end rotation. To block unreacted Epoxy groups, 1 M Tris-HCl, pH 7.5 was added to a final concentration of 100 mM and incubated for two additional hours slowly rotating at 37 oC.

Affinity pull-down experiment – Beads coated with purified SAP were divided in three, and incubated with plasma from three individuals. In addition, two control experiments were included consisting of (1), blank beads without coupled protein and (2), beads coupled to purified SAP. Each sample contained 1 mg beads. SAP beads and control 1 (blank beads) were incubated with 50 µL plasma diluted in TBS, pH 7.4 containing 2 mM CaCl2 and 7 µM Bivalirudin for 1 h slowly rotating at room temperature. Beads of the second control (beads coated with SAP) were incubated with 50 µL plasma diluted in TBS, pH 7.4 containing 10 mM EDTA and 7 µM Bivalirudin for 1 h slowly rotating at room temperature. The beads were washed 5 times with TBS, 7 µM Bivalirudin, pH 7.4 supplemented with either 2 mM CaCl2 or 10mM EDTA using a magnetic rack (Dynal MPC®-M, Oslo, Norway) discarding the supernatant in between. During the last wash, the beads were moved to a clean tube, and the supernatant was discarded. To the beads were added 200 µL elution buffer (TBS, 10mM EDTA, pH 7.4) followed by 5 min endover-end rotation at room temperature. The elution step was repeated once, and the two eluates were pooled and kept on ice until further use.

Co-immunoprecipitation experiment – Anti-SAP IgG1 coated beads and control IgG1 coated beads were divided in three and incubated with plasma from three individuals. Each sample contained 3 mg beads and was incubated with 50 µL plasma diluted in TBS, 2mM CaCl2, 7µM Bivalirudin, pH 7.4 for 15 min at room temperature. The beads were thoroughly washed 5 times with TBS-CaCl2, 7 µM Bivalirudin , pH 7.4 using a magnetic rack. During the last wash, the beads were moved to a clean tube. The beads were

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eluted with 200 µL TBS-EDTA, pH 7.4 followed by incubation for 5 min slowly rotating at room temperature. The elution step was repeated once and the two eluates were pooled and kept on ice until further use.

In-solution digest – The protein samples were lyophilized in a speedvac concentrator (Savant SPD121P, Thermo Scientific, Massachusetts United States) and resuspended in 8 M urea in 0.1 M ammonium bicarbonate (Ambic), pH 8.0. The samples were then reduced with a final concentration of 15 mM dithiothreitol (DTT) in 50 mM Ambic, pH 8.0 for 30 min and subsequently alkylated with a final concentration of 45mM iodoacetamide in 50 mM Ambic, pH 8.0 for 20 min. The samples were diluted 5 times in 50 mM Ambic, pH 8.0 and digested overnight with 1:50 w/w sequencing grade modified trypsin (Promega, Madison, WI, USA) at 37 oC.

LC-MS/MS sample preparation – The digested sample was desalted, concentrated and micro-purified using Octadecyl C18 Empore extractions disk (3M, Minnesota, US) packed in 0.1-10 µL pipet tips (VWR, Pennsylvania, US). The material was activated with 10 µL acetonitrile with 0.1% formic acid and washed with 10 µL 0.1% formic acid. The digested samples were acidified with formic acid, loaded on to the column and washed with 10 µL 0.1% formic acid. The peptides were eluted with 10 µL 70% acetonitrile in 0.1% formic acid and the acetonitrile was subsequently evaporated from the sample by vacuum concentration. The purified peptides were suspended in 0.1% formic acid and stored at -20 oC until LC-MS/MS analysis.

LC-MS/MS analysis – The mass spectrometry analyses were performed on an EASY-nLC II system (Thermo Fisher Scientific) connected to a TripleTOF 5600+ mass spectrometer (AB SCIEX, Massachusetts, US) equipped with a NanoSpray III source (AB SCIEX, Massachusetts, US) and operated under Analyst TF 1.6.0 control (AB SCIEX, Massachusetts, US). The trypsin cleaved samples were dissolved in 0.1% formic acid injected and trapped on a 2 cm trap column (id = 100 µm) packed in-house

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using RP ReproSil-Pur C18-AQ 3 µm resin (Dr. Maisch GmbH). Peptides were eluted from the trap column and separated on a 15 cm analytical column (id = 75µm) pulled and packed in-house with RP ReproSil-Pur C18-AQ 3 µm resin (Dr. Maisch GmbH). The peptides were eluted from the column with a flow rate of 250 nL min-1 using a 30 min gradient from 5% to 35% buffer B (acetonitrile with 0.1% formic acid) and sprayed directly into the mass spectrometer. The acquisition method used for the area based extracted ion chromatography (XIC) quantification was set up as an information-dependent acquisition experiment collecting up to 25 MS/MS spectra in each 1.6 sec cycle using an exclusion window of 6 sec.

Data processing – The data analysis was performed as previously described.(22) Briefly, data obtained from LC-MS/MS analysis were searched against the Swiss-prot homo sapiens database (v. 2016_3) using the Mascot search engine 2.5.1 (Matrix science, London, UK) and imported to MS Data Miner v.1.3.0.(23) Trypsin was specified as digestion enzyme and a maximum of 1 miss cleavage was allowed. Carbamidomethyl modification of cysteines and oxidation of methionine were selected as fixed and variable modifications, respectively. Mass tolerance of the precursor and product ions was specified as 10 ppm and 0.2 Da using ESI-QUAD-TOF as the instrument settings. The significant threshold used was 0.01 and the expected value was set to 0.005. Mascot DAT files were imported in Skyline (MacCoss Lab, University of Washington)(24) for quantification using the XIC (MS1) of selected precursor ions. All cytokeratins were considered contamination and along with proteins identified by less than three peptides they were excluded from the dataset. Proteins not represented by three unique peptides in all three samples (SAP or anti-SAP IgG) were excluded as well, independent of the representation in the controls. Quantitative ratios between samples and controls were calculated based on XIC quantification, and only proteins upregulated five fold or more were reported.

SDS PAGE – Proteins were separated on 5-15% (w/v) gradient polyacrylamide sodium dodecyl sulfate gels, cast in house.(25) Protein samples and a board range protein marker (BIORAD, California, US) were

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added DTT to a final concentration of 25 mM, sample buffer and TBS (50 mM Tris-HCl, 150 mM NaCl, pH 7.5) to adjust the volume. The samples were boiled for 5 min, prior to loading and the gels were run for 60-90 min at 20 mA.

Western blotting – The SDS-PAGE gels were washed twice in blotting buffer (200 mL CAPS, pH 11), 200 mL methanol (VWR chemicals) and 1,800 mL H2O) for 10 min. Polyvinylidene difluoride (PVDF) membranes (Immobilon-P, Millipore, Billerica, MA) were activated with ethanol for 2 min and washed with water and blotting buffer. The proteins were transferred to the membranes by electroblotting at 0.5 A for 15 min and the membranes were blocked using 5% milk (Milex 240) in TBS-T (20 mM Tris-HCl, pH 7.6, 137 mM NaCl and 0.1 % tween) for an hour at room temperature. The membranes were incubated overnight at 4 oC with primary antibodies against vitronectin (polyclonal rabbit Ig with specificity for human vitronectin, Lot: 08302, Sigma Aldrich, Missouri, US), factor B (polyclonal rabbit Ig against human complement factor B, Lot: 110, DAKO, Glostrup, Denmark), α1-antitrypsin (α1PI), (polyclonal goat Ig against human 1-antitrypsin, Cat: No 003-03 Lot: 024, ATAB, Scarborough, UK) or C1q (polyclonal rabbit Ig against human C1q, Lot: 094(011), DAKO, Glostrup, Denmark), all diluted 1:5000 in TBS-T with 2% skim-milk powder. The next day, the membrane was washed three times in TBS-T before incubation with secondary antibodies against rabbit (peroxidase conjugated anti-rabbit IgG, lot: SLBM7730v, Sigma) or goat (peroxidase conjugated anti-goat IgG, lot: 78H9200, Sigma Aldrich, Missouri, US) diluted 1:25000 in 2% skim milk TBS-T for two hours at room temperature. Finally, the membrane was washed three times with TBS-T and developed using Amersham Hyperfilm (GE Healthcare Life Science, Little Chalfont, UK) and the enhanced chemiluminescence (ELC) kit (GE healthcare Life Sciences, Chalfont, UK). The membranes were stained with Coomassie blue (AMRESCO) to visualize the standard markers.

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Results Purification of SAP from plasma using heparin Sepharose – SAP was purified from human plasma as opposed to serum otherwise widely used for SAP purification.(26) The citrated plasma was subjected to a 4-20% PEG precipitation step. The obtained precipitate was resuspended in a calcium chloride containing buffer and applied to a heparin-Sepharose column. Bound SAP was eluted using an EDTA gradient. The SAP containing fractions were pooled and directly applied to an anion exchange column without prior dialysis and eluting using a linear NaCl gradient. SAP containing fractions were pooled, dialyzed into 20 o mM Tris-HCl, pH 7.4 and frozen at -80 C. The homogeneity was assessed by SDS-PAGE showing a

high degree of purity (Figure 2). Approximately 0.7 mg SAP was purified from 50 mL of human plasma equivalent to a 50% yield according to the reported SAP concentration of 30 mg/L.(3)

Plasma preparation for SAP affinity pull-down and co-immunoprecipitation – In this study, we have deliberately characterized the plasma SAP interactome to avoid adventitious proteolysis of putative SAP ligands by activated proteases involved in the coagulation or fibrinolysis. Moreover, classical anticoagulant methods using sodium citrate, EDTA, or heparin have been avoided as these either chelate Ca2+ (citrate and EDTA) or bind directly to SAP (heparin) thereby possibly perturbing the binding profile of the SAP ligands. Instead, freshly drawn blood was immediately mixed with bivalirudin, a specific reversible thrombin inhibitor,(27) using a 5-fold molar excess of inhibitor to enzyme. However, as the halflife of bivalirudin is short(28) due to proteolytic degradation, fresh inhibitor was added every 30 min during the experiments to prevent clotting. Plasma was obtained from 3 healthy individuals and all of the following experiments were conducted in biological triplicates including negative controls.

SAP affinity pull-down and co-immunoprecipitation – Because SAP binds to Sepharose in the presence of Ca2+ all the affinity pull-down and co-immunoprecipitation experiments were conducted using polystyrene magnetic beads (Figure 1). In addition, to outcompete the endogenous plasma SAP, all pull-

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down experiments were performed using a 3-fold molar excess of SAP bound to the beads. In another set of experiments endogenous SAP with bound ligands was purified using a co-immunoprecipitation approach. Eluted proteins from affinity pull-down and co-immunoprecipitation experiments were identified by LC-MS/MS. Proteins identified with 3 or more peptides in all 3 biological experiments and with 5-fold upregulation or higher compared to the control beads were considered genuine direct or indirect SAP ligands. The LC-MS/MS analyses of pull-down experiments resulted in identification of 13 protein ligands, whereas co-immunoprecipitation experiments resulted in identification of 28 ligands. The two methods showed an overlap of 8 protein ligands, resulting in a total number of 33 protein ligands identified to interact directly or indirectly with SAP in plasma (Table 1). Of the total number of ligands identified, 24 have not previously been reported as SAP interaction partners.

Validation of SAP ligands using Western blotting – To corroborate the affinity pull-down and coimmunoprecipitation experiments, Western blotting against selected SAP ligands including vitronectin, complement C1q, complement factor B, and α1PI were performed on independent pull-down samples (Figure 3). The 4 antibodies chosen for validation represented one known and 3 previously undescribed SAP ligands. The antibodies against the 3 previously undescribed ligands each represented one of the major groups in Table 1. All 4 proteins identified by LC-MS/MS analyses were also detected by Western blotting, thereby confirming the results and validity of the experimental approach. Of note, complement C1q was only identified by LC-MS/MS analyses of co-immunoprecipitates (Table 1), but could likewise be verified in SAP affinity pull-down experiments using Western blotting (Figure 3B).

SAP affinity pull-down of purified ligands – SAP has previously been associated with the complement system, coagulation system, and apolipoproteins,(20) whose members were similarly strongly represented among the identified SAP protein ligands. In addition, five proteinase inhibitors were identified as SAP interaction partners (Table 1). Protease inhibitors have not previously been described as SAP ligands. To support our LC-MS/MS data and to clarify if protease inhibitor-SAP interactions are direct or indirect, we

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performed SAP affinity pull-downs of purified α1PI, α2-macroglobulin (α2M), inter-α-trypsin inhibitor (IαI) and vitronectin as well as of fibronectin, a known SAP ligand(8) not identified in this study. However, none of these five proteins were able to bind SAP in the presence of Ca2+ (data not shown). Because SAP could interact with these proteins in plasma, we suggest that plasma components required for these indirect interactions to SAP were absent in the purified system.

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Discussion SAP is constitutively expressed, and the concentration in plasma is relatively constant independent of infections. This is in contrast to the homologue acute-phase protein CRP, which is found to increase in plasma upon infection. This implies that the function of SAP stretches beyond a role in the innate immune system. To investigate this hypothesis, we determined the Ca2+ dependent plasma interactome of SAP in three healthy individuals by performing affinity pull-down experiments using purified SAP coupled to beads as well as co-immunoprecipitation of endogenous SAP in plasma (Figure 1). The following LC-MS/MS analyses resulted in 33 different protein ligands (Table 1), of which only about one third could be directly linked to the coagulation and complement systems, thus supporting that SAP has more diverse roles in plasma.

Correlation with known SAP protein ligands – The data reduction approach used in this study was based on the following two stringent criteria; (i) a protein ligand had to be identified with at least 3 peptides in all 3 biological plasma samples and, (ii) display a minimum of 5-fold increase when compared to LCMS/MS analyses of eluates from control beads. Therefore, we cannot rule out that some genuine SAP ligands failed to be identified using this approach. A previous report have showed that fibronectin and C4 binding protein interact with purified and immobilized (aggregated) SAP(8) whereas endogenous binding to SAP has not been found. Fibronectin and C4 binding protein were not identified as SAP interaction partners neither in the SAP affinity pull-down experiment nor during the co-immunoprecipitation against endogenous SAP, in this study. This suggests that the SAP-fibronectin and SAP-C4 binding protein interactions are observed as a consequence of using aggregated SAP as opposed to endogenous nonaggregated SAP in plasma as used in this study or that interactions are below the limit of detection in our experimental setup. As expected, none of the already known extracellular SAP ligands, type IV collagen(6) and laminin(7) were observed as they are not plasma proteins. Contrary to this, complement C1q and eight of the apolipoproteins were identified as SAP ligands in this study, which correlates well with previous observations(9, 12). In the case of the apolipoproteins, SAP may only interact directly with one or more of

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the apolipoproteins situated within the HDL and VLDL particles, whereas the remaining apolipoproteins identified may be observed as a consequence of indirect interactions to SAP.

SAP as a regulator of proteolysis – The proposed role of SAP in relation to proteolysis is presently restricted to its protection of amyloid fibril against degradation by proteases.(16) Furthermore, SAP has been suggested to suppress porcine pancreatic elastase activity and has therefore been believed to be a proteinase inhibitor of elastase(29), although that result has been disputed by others.(16) Our data adds new insights to the suggestion that SAP may function as a regulator of proteolysis, as the five major plasma inhibitors antithrombin-III, α1PI, α1-antichymotrypsin (ACT), α2M, and IαI inhibitor were identified as SAP ligands (Table 1) most likely in an indirect manner, as in vitro SAP affinity pull downs of purified inhibitors failed (data not shown). A role for SAP in proteolysis was further supported by its interaction with both C3 and factor B of the complement systems capable of forming the C3 convertase as well as its interaction with prothrombin, which upon activation cleaves fibrinogen during coagulation. Taken together, this suggests that SAP plays a role in regulation of proteolysis in plasma.

Acute-phase proteins – The characteristic of acute-phase proteins is their relatively dramatic change in plasma concentration upon infection and inflammation. Even though sometimes described as an acutephase protein, human SAP itself cannot be categorized as being an acute-phase protein as its plasma concentration remains stable during infections. However, searching the protein ligands identified in this study (Table 1) for known acute-phase proteins, 9 of the 33 proteins ligands were found to be acute-phase proteins. The positive acute-phase protein ligands (increasing in plasma concentration upon infection and inflammation) interacting with SAP are fibrinogen, prothrombin, haptoglobin, α1PI, ACT, α2M, IαI, and ceruloplasmin, whereas the negative acute-phase protein ligands (decreasing in plasma concentration upon infection and inflammation) count antithrombin-III and transthyretin (Table 1). This suggests that SAP also plays an important role in the regulation of the acute-phase response during non-infectious

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times, while this role may be less significant after activation of the acute-phase response, as concentrations of the acute-phase proteins then will exceed SAP concentration greatly.

Conclusion – Radiolabelled SAP is used clinically during imaging of amyloid depositions(30) and in ongoing clinical trials small molecule SAP ligands are used to deplete systemic SAP to reduce amyloid deposits.(31) The Ca2+ dependent plasma interactome of SAP presented in this study indicates that systemic depletion of SAP by administration of small molecule ligands most likely influences a larger number of plasma proteins, which may cause unwanted side effects including changes in the regulation of proteolytic activity.

Acknowledgements We thank Connie Jenning Melchjorsen for assistance in obtaining fresh plasma and Carsten Scavenius for assisting with LC-MS/MS.

Funding Source Information This work was supported by The Danish Council for Independent Research − Medical Sciences (DFF4004-00471).

Conflict of interest The authors declare no conflict of interest.

Author contributions ETP, KWP and JJE conceived the study; ETP, KWP and AMM performed experiments, ETP, KWP and JJE wrote the paper.

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Bottazzi, B., Inforzato, A., Messa, M., Barbagallo, M., Magrini, E., Garlanda, C., and Mantovani, A. (2016) The pentraxins PTX3 and SAP in innate immunity, regulation of inflammation and tissue remodelling, Journal of hepatology.

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Biochemistry

Table 1. SAP protein ligands identified by affinity pull-down and co-immunoprecipitation

#

1

2 3 4 5 6 7 8 9 10 11 12 13 14 15

Accession no.

Accession Name

Albumin gene family P02774 VTDB_HUMAN Apolipoproteins P02647 APOA1_HUMAN P02652 APOA2_HUMAN P06727 APOA4_HUMAN P04114 APOB_HUMAN P02654 APOC1_HUMAN P02656 APOC3_HUMAN P05090 APOD_HUMAN P02649 APOE_HUMAN Coagulation P01008 ANT3_HUMAN P00742 FA10_HUMAN P02671 FIBA_HUMAN P04196 HRG_HUMAN

23 24

P00734 THRB_HUMAN P04004 VTNC_HUMAN Complement system P02746 C1QB_HUMAN P01024 CO3_HUMAN P00751 CFAB_HUMAN Extracellular chaperones P00738 HPT_HUMAN P10909 CLUS_HUMAN Heme P69905 HBA_HUMAN P68871 HBB_HUMAN P02790 HEMO_HUMAN Inhibitors P01009 A1AT_HUMAN P01011 AACT_HUMAN

25 26

P01023 P19827

A2MG_HUMAN ITIH1_HUMAN

27

P19823

ITIH2_HUMAN

16 17 18 19 20 21 22

Name

Table 1. Proteins interacting with SAP in plasma Mw Mascot # of (kDa) score unique peptides

Coverage (%)

Pulldown

Co-IP

Vitamin D-binding protein

52.964

125

7

16.0%

X

X

Apolipoprotein A-I Apolipoprotein A-II Apolipoprotein A-IV Apolipoprotein B-100 Apolipoprotein C-I Apolipoprotein C-III Apolipoprotein D Apolipoprotein E

33.778 11.175 45.399 515.605 9.332 10.852 21.276 36.154

5657 891 2213 1532 537 1088 1072 1981

36 8 31 71 5 7 13 23

81.6% 69.0% 63.1% 16.5% 37.3% 58.6% 41.3% 68.8%

X -

X X X X X X X X

Antithrombin-III Coagulation Factor X Fibrinogen Histidine-rich glycoprotein Prothrombin Vitronectin

52.602 54.732 94.973 59.578

182 461 237 103

15 9 6 3

36.6% 20.1% 9.2% 5.7%

X X -

X X X

70.037 54.306

684 676

22 12

40.7% 26.2%

X -

X X

Complement C1q Complement C3 Complement factor B

26.722 187.148 85.533

130 3504 293

3 80 12

14.2% 50.4 17.5%

X X

X X X

Haptoglobin Clusterin

45.205 52.495

918 3018

11 25

52.5% 45.4%

X -

X X

Hemoglobin

15.258 15.998 51.676

287 368 899

5 8 15

35.9% 67.3% 31.2%

X

-

X

-

46.737 47.651

1582 241

23 10

48.6% 23.9%

X X

-

163.291 101.389

2022 316

42 10

34.1% 12.5%

-

X X

106.463

457

14

19.5%

-

X

Hemopexin Alpha-1-antitrypsin Alpha-1antichymotrypsin Alpha-2-macroglobulin Inter-alpha-trypsin inhibitor heavy chain H1 Inter-alpha-trypsin inhibitor heavy chain H2

Reference

(11, 12) (12) (12) (12) (12) (12) (12) (12)

(9)

Immunoglobulin P01876 IGHA1_HUMAN Ig alpha-1 chain C region 37.655 971 9 30.3% X P01834 IGKC_HUMAN Ig kappa chain C region 11.609 1200 7 74.5% X Others 30 P02766 TTHY_HUMAN Transthyretin 15.887 344 8 73.5% X 31 P04217 A1BG_HUMAN Alpha-1B-glycoprotein 54.254 357 8 19.6% X X 32 P00450 CERU_HUMAN Ceruloplasmin 122.205 394 9 12.0% X 33 P02765 FETUA_HUMAN Alpha-2-HS-glycoprotein 39.325 527 8 28.9% X Proteins were accepted (X) as SAP ligands if identified by 3 or more peptides in all 3 biological replicates and with a minimum of 5 fold increase compared to blank control beads. Mascot search score, # of unique peptides and coverage (%) from the 1st experimental replicate of either CO-IP or pull-down are shown. Proteins reported by others as binding partner to SAP is marked with the reference in the column to the right. 28 29

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Biochemistry

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Page 22 of 27

Figure legends

Figure 1. Schematic overview of the experimental setup used for identifying protein ligands to SAP. Freshly drawn plasma was obtained from three healthy individuals and coagulation was prevented using the thrombin specific inhibitor bivalirudin. Plasma was either used for affinity pull-down experiments (left gray box) using beads coupled with plasma derived SAP or used for co-immunopricipitation using anti-SAP antibody (Ab) coupled beads (right gray box). Two controls were included for the affinity pulldown experiment including (I) blank non-protein coupled beads as well as (II) SAP coupled beads with no free calcium present. The control of the co-immunoprecipitation experiments consisted of IgG coupled beads. SAP binding protein ligands were eluted using EDTA. Eluates were digested by trypsin and analyzed by LC-MS/MS using a method allowing relative quantification afterwards. The list of identified SAP protein ligands is shown in Table 1.

Figure 2. Purification of SAP from plasma. A) Human citrated plasma was subjected to a 4% polyethylene glycol (PEG) precipitation followed by the addition of 16% PEG to the supernatant. The 20% PEG pellet was resuspended in TBS containing CaCl2, applied to a heparin column and eluted by using an EDTA gradient. The SAP fractions were pooled, diluted 10 times and applied to an anion exchange column, washed and developed with a NaCl gradient. The SAP fractions from the anion exchange column were pooled, dialyzed and stored at -80 ºC. B) Coomassie stained SDS-PAGE of purified SAP with size markers. The yield was approximately 50% based on a plasma concentration of 30 mg/L.

Figure 3. Western blotting against vitronectin (A), complement C1q (B), complement factor (C) and alpha-1-antitrypsin (D). Lanes 1-3 represents SAP affinity pull-down from 3 biological plasma samples and lanes 4-6 represent blank beads incubated with the 3 biological plasma samples. Proteins were separated on reduced 5-15% SDS-PAGE and subsequently immunoblotted using appropriate antibodies

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Biochemistry

against selected SAP ligands observed in Table 1. All western blots showed pull-down of the selected proteins in all three biological plasma samples, but not in any of the negative control samples, thereby supporting the findings of the LC-MS/MS analyses (Table 1). Panel A and C shows two bands representing intact and truncated isoforms of vitronectin and complement factor B, respectively.

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Biochemistry

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Figure 1

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Biochemistry

Figure 2

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Biochemistry

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Figure 3.

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Biochemistry

For Table of Contents Use Only

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Serum Amyloid P Component (SAP) Interactome in Human Plasma

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