Identification of the amniotic fluid insulin-like growth factor binding protein-1 phosphorylation sites and propensity to proteolysis of the isoforms ` Lorenzo Dolcini1, Alberto Sala1, Monica Campagnoli1, Sara Labo1, Maurizia Valli1, Livia Visai1,2, Lorenzo Minchiotti1, Hugo L Monaco3 and Monica Galliano1 Department of Biochemistry ‘A Castellani’, University of Pavia, Italy Center for Tissue Engineering (C I T), University of Pavia, Italy Biocrystallography Laboratory, Department of Biotechnology, University of Verona, Italy Keywords IGFBP; insulin-like growth factor binding protein-1; mass spectrometry; phosphorylation; proteolysis Correspondence M Galliano, Department of Biochemistry ‘A Castellani’, University of Pavia, viale Taramelli 3b, 27100 Pavia, Italy Fax: +39 0382 423108 Tel: +39 0382 987724 E-mail: galliano@unipv.it (Received 16 March 2009, revised 27 July 2009, accepted 19 August 2009) doi:10.1111/j.1742-4658.2009.07318.x Insulin-like growth factor binding protein-1 (IGFBP-1) is the major secreted protein of human decidual cells during gestation and, as a modulator of insulin-like growth factors or by independent mechanisms, regulates embryonic implantation and growth The protein is phosphorylated and this post-translational modification is regulated in pregnancy and represents an important determinant of its biological activity We have isolated, from human normal amniotic fluid collected in the weeks 16–18, the intact nonphosphorylated IGFBP-1 and five electrophoretically distinct phosphoisoforms and have determined their in vivo phosphorylation state The unmodified protein was the most abundant component and mono-, bi-, triand tetraphosphorylated forms were present in decreasing amounts The phosphorylation sites of IGFBP-1 were identified by liquid chromatography–tandem mass spectrometry analysis of the peptides generated with trypsin, chymotrypsin and Staphylococcus aureus V8 protease Five serines were found to be phosphorylated and, of these, four are localized in the central, weakly conserved, region, at positions 95, 98, 101 and 119, whereas one, Ser169, is in the C-terminal domain The post-translational modification predominantly involves the hydrophilic stretch of amino acids representing a potential PEST sequence (proline, glutamic acid, serine, threonine) and our results show that the phosphorylation state influences the propensity of IGFBP-1 to proteolysis Introduction The insulin-like growth factor binding proteins (IGFBP) are six homologous molecules (IGFBP-1–6) that play critical roles in a wide variety of important physiological processes Upon binding they regulate the availability of both insulin-like growth factors I and II (IGF-I and -II) and their affinity for these ligands is modulated by several mechanisms: attachment to the extracellular matrix and post-translational modifications, such as proteolysis, phosphorylation and glycosylation Additionally, several studies have revealed that IGFBPs, as well as their proteolytic fragments, have IGF-independent biological activities in cell adhesion and migration and in the regulation of the cell cycle and apoptosis [1–4] Furthermore, although these Abbreviations IGF, insulin-like growth factor; IGFBP, insulin-like growth factor binding protein; LC-ESI-MS, liquid chromatography-electrospray ionizationmass spectrometry FEBS Journal 276 (2009) 6033–6046 ª 2009 The Authors Journal compilation ª 2009 FEBS 6033 Phosphorylation of amniotic fluid human IGFBP-1 L Dolcini et al proteins are all secreted in the bloodstream, the functional role of IGFBP-1 [5], IGFBP-3 [2] and IGFBP-5 [2] inside the cells has been well documented IGFBP-1, the subject of the present study, is predominantly expressed in the liver and, in adult mammals, the synthesis of the protein is upregulated in a number of catabolic or stressful conditions, such as fasting and diabetes [6] Additionally, it has recently been shown that a portion of intracellular IGFBP-1 localizes to mitochondria, where it acts as a prosurvival factor and protects the liver from apoptosis [5] IGFBP-1 represents a minor IGF binding protein in the circulation of nonpregnant adults, but it is regarded as the most relevant member of the IGFBP family during gestation [7–10] Several studies indicate that IGFBP-1 regulates embryonic growth as a local modulator of IGF bioavailability and stimulates trophoblast migration through binding of its C-terminal domain, which contains an Arg–Gly–Asp sequence recognized by the integrin family of cell surface receptors [11–14] Furthermore, the protein has also been shown to play crucial roles in ovarian, endometrial, trophoblast and fetal–placental physiology and pathology and its level in maternal as well as in fetal circulation and in the liver increases under hypoxic conditions in the uterus, resulting in intrauterine growth retardation [15,16] Its mature polypeptide chain consists of 234 amino acids and, as all other IGFBPs, contains an N-terminal and a C-terminal domain linked by a mid-region, which has a less ordered structure [1,17] This central domain is more variable in the different members of the family and contains most of the sites involved in post-translational modifications IGFBP-1 is subject to phosphorylation on serine residues and backbone cleavage [1,17] In the serum of nonpregnant adults, the protein occurs as a highly phosphorylated single species [18], whereas differently phosphorylated IGFBP-1 isoforms are present in the amniotic fluid [19] Earlier reports have indicated the presence of a variable number of isomers [19], probably reflecting sampling at different gestational periods We have recently shown that normal amniotic fluid, collected in the weeks 16–18 of gestation, contains the unmodified protein and five electrophoretically distinct phosphoisoforms [20] A previous study has shown that IGFBP-1 expressed in Chinese hamster ovary cells is phosphorylated at Ser101, 119 and 169 [21,22] and a recent paper described the modification of Ser98, which was revealed in the highly phosphorylated protein isoforms found in hypoxia-treated cells [23] Post-translational modification influences the interaction of IGFBP-1 with IGF-I [21,22,24] and has been 6034 associated with gestational and fetal abnormalities [24– 26] However, neither the phosphorylation state of the protein isolated from normal human amniotic fluid nor the biological properties of the homogeneous IGFBP-1 phosphoisoforms have been examined so far It has been shown that, in vitro, several metalloproteases recognize IGFBP-1 as a substrate [27] and a modulating effect of phosphorylation on the backbone cleavage of the protein has been recognized [8] Recently, a specific IGFBP-1 protease activity has been described in a patient with multiple myeloma and identified as azurocidin [28] Here we describe the purification on a preparative scale of the six isoforms present in human normal amniotic fluid collected in the weeks 16–18 The availability of sufficient amounts of the pure protein allowed a detailed identification of the amino acid residues involved in the IGFBP-1 post-translational modifications occurring in vivo An important result is that three of the five phosphorylation sites identified in the present work are in a region enriched in proline, glutamic acid, serine and threonine, as it is known that phosphate addition represents a mechanism for activating a latent PEST sequence [29] Amniotic fluid contains a specific IGFBP-1 protease activity, yielding a stable, functional and well-structured C-terminal domain [30] and we have examined the propensity of the different isoforms of IGFBP-1 to backbone cleavage in the presence of this partially purified specific protease Results Purification of IGFBP-1 isoforms The amniotic fluid proteins were separated by gel filtration and the low molecular mass components, eluting after the albumin peak, were pooled and further purified by anion exchange chromatography In a previous study [30], we have shown that amniotic fluid contains a metalloprotease that cleaves the majority of IGFBP-1, yielding a stable C-terminal fragment Thus, to prevent the proteolytic process, 10 mm EDTA was added during storage of the fluid and in all buffers used throughout the gel filtration separation The proteins were then subjected to anion exchange chromatography (Fig 1) and the fractions obtained, resolved by SDS gel electrophoresis, were transferred to poly(vinylidene difluoride) membranes by electroblotting and probed with anti-IGFBP-1 IgGs (data not shown) Immunoblotting showed that IGFBP-1 eluted in six peaks (I–VI) as a single positive band with identical molecular mass of  30 kDa and that no fragmentation of the protein had occurred during the FEBS Journal 276 (2009) 6033–6046 ª 2009 The Authors Journal compilation ª 2009 FEBS L Dolcini et al Phosphorylation of amniotic fluid human IGFBP-1 The six proteins were submitted to treatment with alkaline phosphatase and, when analysed by isoelectric focusing, all acquired the same isoelectric point of the polypeptide chain eluting under peak I (data not shown) This form thus represents the unmodified protein, whereas the other fractions contain distinct phosphoisoforms These results showed that the nonphosphorylated protein, as well as the five distinct phosphoisoforms, were obtained in homogeneous form by anion exchange chromatography Furthermore, the addition of the chelating agent was effective in preventing proteolytic cleavage, as only the intact proteins were obtained Fig Purification of IGFBP-1 isoforms The low molecular mass fraction obtained from human amniotic fluid was resolved by Q-Sepharose ion exchange chromatography The elution was carried out with a linear gradient from to 100% using 6.25 mM Bistris-propane, pH 7.5 as buffer A and 6.25 mM Bistris-propane, pH 9.5, 0.35 M NaCl as buffer B The peaks I–VI, positive when probed with anti-IGFBP-1 IgGs, were pooled and further purified by gel filtration purification procedure The peaks were pooled and further purified by gel filtration chromatography Six homogeneous polypeptide chains with the same N-terminal sequence, APWQCAPCSA, corresponding to the first 10 residues of intact mature IGFBP-1, were obtained Nondenaturing gel electrophoresis (Fig 2A) and immunoblotting (Fig 2B) showed that the electrophoretic mobilities of the IGFBP-1 isoforms increased, but not progressively, as expected on the basis of the anion exchange chromatography elution profile Peak IV migrated more anodically than the preceding chromatographic fraction and the last eluting fraction, VI, displayed a very similar mobility to that of fraction V A 5 B Mass determination of the IGFBP-1 isoforms The molecular mass of the six IGFBP-1 isoforms was probed by liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) (Table 1) The mass value determined deconvoluting the multiple charged ions of the protein eluting in peak I was 25 252 Da and, following reduction, 25 270 Da These values match the theoretical molecular mass of the intact, unmodified IGFBP-1 and the difference (+18 Da) measured for the reduced sample accounts for the presence of nine disulfide bonds involving the 18 cysteine residues of the polypeptide chain Analysis of peak II yielded a molecular mass of 25 332 Da and the increase of 80 Da over the previous component is consistent with the presence of one phosphate group Peaks III and IV displayed the same molecular mass increase of 160 Da over the unmodified protein, corresponding to the presence of two phosphates Peaks V (+240 Da) and VI (+320 Da) contain the tri- and tetraphosphorylated protein, respectively (Fig 3) The relative amount of each form was determined by measuring the corresponding peak area in the chromatographic profile and were in good agreement with the absolute amounts obtained from the absorbance at 280 nm of the pooled fractions (assuming an absorbance value of 1.42 for a mgỈmL)1 solution of IGFBP-1 as calculated on the basis of the amino acid Table Molecular mass of the IGFBP-1 peaks isolated by anion exchange chromatography IGFBP-1 isoforms Fig Nondenaturing gel electrophoresis and western blot analysis of IGFBP-1 isoforms IGFBP-1 isoforms isolated by ion exchange chromatography from peaks I–VI were (A) resolved on a 17% nondenaturing polyacrylamide gel and stained with Coomassie Brilliant Blue (lanes 1–6) and (B) following western blot were probed with anti-IGFBP-1 IgGs (lanes 1–6) Measured mass (Da) Theoretical mass (Da) Dmass (Da) Peak Peak Peak Peak Peak Peak 25 25 25 25 25 25 25 25 25 25 25 25 +1.2 +1.3 +1.4 +2.1 )0.7 +0.1 I II III IV V VI FEBS Journal 276 (2009) 6033–6046 ª 2009 The Authors Journal compilation ª 2009 FEBS 251.2 331.1 411.0 410.3 493.1 572.2 252.4 332.4 412.4 412.4 492.4 572.3 6035 Phosphorylation of amniotic fluid human IGFBP-1 L Dolcini et al 23+ 22+ 21+ 20+ 24+ 19+ 25+ 27+ 18+ 17+ 16+ 26+ E 25572 Da 21+ 20+ 23+ 22+ 19+ 18+ 25+ 24+ 27+ 17+ 26+ 16+ 15+ 13+ 14+ D 25493 Da 21+ 20+ 22+ 19+ 18+ 23+ 17+ 16+ 25+ 24+ 22+ 13+ 14+ C 25410 Da 21+ 20+ 19+ 18+ 23+ 25+ 15+ 17+ 16+ 15+ 13+ 14+ 24+ B 21+ 20+ 19+ 22+ 16+ 18+ 23+ 25+ 17+ 15+ 24+ 14+ 13+ A 1000 1200 25331 Da 1400 1600 1800 25251 Da 2000 m/z Fig Multicharged mass spectra of the IGFBP-1 phosphoisoform isolated from human amniotic fluid The mass values reported on the y-axis were obtained by deconvoluting the multiply charged pattern obtained for (A) nonphosphorylated protein eluting as peak I; (B) the mono-1-phosphorylated form, peak II; (C) the biphosphorylated chains eluting in peaks III and IV; (D) the triphosphorylated protein, peak V and (E) the tetraphosphorylated molecule eluting as peak VI composition) The most abundant nonphosphorylated form, eluting as peak I, accounted for 69.5% of the total protein; the monophosphorylated isoform, eluting in peak II, was 14.9%; the doubly modified forms, eluting as peaks III and IV, accounted for 6.5 and 5.5%, respectively; the triphosphorylated protein, peak V, represented 2.7%, and peak VI, corresponding to the form with four phosphate groups, was 0.9% These results show that amniotic fluid IGFBP-1 is modified by up to four phosphate groups that produce five chromatographically distinct isoforms The different chromatographic behaviour of the doubly modified protein is probably due to the alternative positions of the charged groups, resulting in different isoelectric points Identification of IGFBP-1 phosphorylation sites The examination of the amino acidic sequence of human IGFBP-1 with the protein pattern database 6036 NetPhos [31] predicted 12 serines, four threonines and one tyrosine, which could potentially serve as phosphoacceptor sites Thus, the assumption that the phosphate groups can have alternative distribution within the polypeptide chain suggested that a low level of modification at each site could be expected Furthermore, it is known that the ionization efficiency of phosphopeptides is generally slightly lower than that of their unmodified counterparts and only relatively large fragments, between six and 25 amino acids, are suitable for mass spectral analysis [32] Therefore, to enhance sequence coverage and ensure that each phosphorylation site was contained in at least one peptide of suitable size and hydrophobicity, the IGFBP-1 isoforms were cleaved with three different proteolytic enzymes Reduction of disulfide bonds was carried out before each enzymatic digestion and the resulting peptide mixtures were immediately submitted to LC-ESI-MS ⁄ MS analysis The HPLC separation FEBS Journal 276 (2009) 6033–6046 ª 2009 The Authors Journal compilation ª 2009 FEBS L Dolcini et al Phosphorylation of amniotic fluid human IGFBP-1 Table List of the phosphopeptides revealed by LC-ESI-MS ⁄ MS Modified residues are underlined Residues Chymotryptic peptides 70-113 +P 70-113 +2P 114-127 +P 157-175 +P Glu C peptides 83-108 +2P 93-100 +P 93-103 +P 93-103 +2P 93-108 +P 100-111 +P 109-121 +P 109-122 +P 112-121 +P 113-121 +P 161-172 +P Tryptic peptides 165-175 +P 165-183 +P Sequence Calculated mass (Da) Measured mass (Da) Dmass (Da) HALTRGQGACVQESDASAPHAAEAGSPESPESTEITEEELLDNF HALTRGQGACVQESDASAPHAAEAGSPESPESTEITEEELLDNF HLMAPSEEDHSILW RVVESLAKAQETSGEEISKF 4660.1 4741.0 1743.7 2287.1 4660.8 4741.4 1743.7 2287.4 +0.7 +0.4 +0.3 SDASAPHAAEAGSPESPESTEITEEE AGSPESPE AGSPESPESTE AGSPESPESTE AGSPESPESTEITEEE STEITEEELLD LLDNFHLMAPSEE LLDNFHLMAPSEED NFHLMAPSEE FHLMAPSEE SLAKAQETSGEE 2787.1 853.4 1169.4 1249.4 1770.7 1357.6 1594.7 1708.7 1253.5 1139.4 1330.0 2787.8 853.2 1169.3 1249.2 1771.4 1357.6 1595.2 1708.6 1253.9 1139.9 1330.3 +0.7 )0.2 )0.1 )0.2 )0.3 )0.5 )0.1 +0.4 +0.5 +0.3 AQETSGEEISK AQETSGEEISKFYLPNCNK 1258.5 2237.0 1258.6 2239.0 +0.1 +2.0 was performed under acidic conditions in order to maximize positive charge ionization and the elution gradient was chosen in order to reduce the loss of small and hydrophilic peptides during the desalting step Phosphoric acid was added to the sample solution because it significantly enhances the detection of mono- and multiphosphorylated peptides following RP-HPLC [33] The identity of the fragments was assessed by manual inspection and by comparing the experimental fragmentation pattern with the theoretical values obtained using the protein prospector (http://prospector.ucsf.edu/prospector/mshome.htm) and peaks studio software (Bioinformatic Solution Inc., Waterloo, Canada) In addition, as phosphorylation can reduce the efficiency of cleavage [34], the data were also examined for incompletely digested fragments The phosphorylated peptides were identified by the presence of species with a theoretical peptide mass increased by 80 Da (plus one phosphate group) or multiples thereof, and in all cases, ions corresponding to the loss of H3PO4 ()98 for single charged ions and )49 for doubly charged ions) dominated the fragmentation pattern A complete sequence coverage of the protein was obtained (data not shown) and Table lists the phosphorylated peptides observed The V8 fragments spanning residues 93–100 (AGSPESPE) from the unmodified protein, at m ⁄ z 773.3, and from the monophosphorylated one, at m ⁄ z 853.2, displayed a mass difference of 80 Da, which is consistent with the presence of one phosphate group Fragmentation in the ion trap showed that this peptide was phosphorylated at Ser95 and, to a much lower extent, also at Ser98 The presence of the fragment ion at m ⁄ z 755.3 is due to the loss of phosphoric acid (98 Da), the ion at m ⁄ z 296.1 (b3+P) is consistent with the modification of Ser95 and the ions at m ⁄ z 638.2, (y5+P) and at m ⁄ z 442.2 (b5) are indicative of the modification at Ser98 (Fig 4B) Figure 4A shows the MS ⁄ MS spectrum obtained for the unmodified peptide The two peptides coelute and the doubly modified fragment was not observed The MS ⁄ MS spectra of the V8 fragment 93–103 (AGSPESPESTE) from the monophosphorylated isoform, at m ⁄ z 1172.1 (+80 Da shift) is shown in Fig 5A The loss of phosphoric acid (98 Da) from the precursor yielded a charged species at m ⁄ z 1072.1 and the presence of a product ion at m ⁄ z 642.0 (y5+P) is consistent with phosphorylation at Ser101 The fragment ions at m ⁄ z 562.2 and at m ⁄ z 649.0 correspond to unmodified y5 and y6, respectively, and indicate that Ser95 is phosphorylated as well The modification of Ser98 could not be distinguished in this peptide Analysis of the V8 digest of the triphosphorylated protein showed the presence of peptide 93–103 carrying two phosphate groups MS ⁄ MS spectra (Fig 5B) of the charged ion at m ⁄ z 1250.0 displayed fragment ions at m ⁄ z 1152.1 and at m ⁄ z 1054.1, consistent with the loss of one and two phosphate groups, respectively The ion at m ⁄ z 642.0 (y5+P) indicates the phosphorylation of Ser101 and the ion at m ⁄ z 955.0 (y8+P) indicates that in the FEBS Journal 276 (2009) 6033–6046 ª 2009 The Authors Journal compilation ª 2009 FEBS 6037 Phosphorylation of amniotic fluid human IGFBP-1 L Dolcini et al Fig LC-MS ⁄ MS ion spectra of IGFBP-1 peptide 93–100 (A) MS ⁄ MS spectrum of the singly charged 773.3 Da ion corresponding to the nonphosphorylated AGSPESPE fragment (B) MS ⁄ MS spectrum of the singly charged 853.2 Da ion corresponding to the monophosphorylated AGSPESPE sequence biphosphorylated fragment, Ser95 and Ser101 are modified The triphosphorylated peptide was not observed These results show that the sequence spanning residues 93–103 contains three serine residues whose adjacent amino acids form consensus sites for phosphorylation The V8 fragment spanning residues 109–121, at m ⁄ z 1515.3, was found in the unmodified protein and the fragmentation pattern of its phosphorylated counterpart, at m ⁄ z 1595.2, displayed ions accounting for the modification of Ser119 (data not shown) The modification of Ser169 was detected in the V8 fragment spanning residues 161–172, at m ⁄ z 1330.3, which showed the expected fragmentation ions (data not shown) The MS ⁄ MS spectrum of the V8 fragment TSMDGE, found in all the IGFBP-1 samples, and spanning residues 219–224 at m ⁄ z 654.9 (Fig 6), showed that the molecular mass increase of 15.9 Da over the theoretical value was due to the oxidation of methionine to sulfoxide, a common 6038 post-translational modification to proteins occurring in vivo The nonoxidized sequence was also present at m ⁄ z 639.1 These results lead to the conclusion that the IGFBP-1 phosphoisomers isolated from normal amniotic fluid contain five phosphoacceptor sites The phosphorylation of the five serine residues was detected in all the chromatographically distinct isomers A complete sequence coverage was obtained for each polypeptide chain and Table lists the molecular mass of the peptides with phosphorylated residues One novel phosphorylation site on Ser101 was revealed, and the phosphoacceptor sites Ser95, Ser98, Ser119 and Ser169, previously described in cultured cells [22,23], were confirmed in our natural samples of human amniotic fluid IGFBP-1 In agreement with previous data [22,35], we did not find any evidence of threonine and tyrosine phosphorylation For this reason, and because it is well FEBS Journal 276 (2009) 6033–6046 ª 2009 The Authors Journal compilation ª 2009 FEBS L Dolcini et al Phosphorylation of amniotic fluid human IGFBP-1 A B Fig LC-MS ⁄ MS ion spectra of IGFBP-1 peptide 93–103 (A) MS ⁄ MS spectrum of the singly charged 1172.1 Da ion corresponding to the monophosphorylated AGSPESPESTE fragment (B) MS ⁄ MS spectrum of the singly charged 1250.0 Da ion corresponding to the triphosphorylated AGSPESPESTE peptide Fig LC-MS ⁄ MS spectrum of the IGFBP1 peptide 219–224 The asterisk marks the oxidized methionine in the sequence and in the internal ions at m ⁄ z 234.5 and 262.9 known that the phosphopeptides are not eluted with high efficiency from RP-HPLC, a quantitative analysis of each phosphorylation site could not be performed Chromatographic separation of the V8 peptides In order to decide whether a major phosphoacceptor serine is present in the monophosphorylated protein, it FEBS Journal 276 (2009) 6033–6046 ª 2009 The Authors Journal compilation ª 2009 FEBS 6039 Phosphorylation of amniotic fluid human IGFBP-1 L Dolcini et al A B Fig HPLC elution profiles of the V8 peptides Trace A was obtained from the nonphosphorylated IGFBP-1 and trace B from the monophosphorylated protein The arrows indicate the major differences between the two chromatograms (peaks and 2) was digested with endopeptidase V8 from Staphylococcus aureus and compared with the unmodified form The peptide mixtures were resolved by RP-HPLC chromatography and Fig shows the superimposition of the UV traces monitored at 214 nm The peaks were manually collected and submitted to N-terminal sequencing and LC-ESI-MS ⁄ MS analysis The chromatographic profiles, as well as the peak area and shape, did not differ in a significant manner (Fig 7) The only major discrepancy was the presence of a peak, indicated as 1, in the trace of the unmodified protein, which was absent in the profile of the monophosphorylated molecule, where an additional one, indicated as peak 2, was present The N-terminal sequence analysis showed that peak contains the sequence AGSPESPE (93–100) and that peak corresponds to the fragment AGSPESPESTE (93–103) Mass analysis confirmed these data and showed that the latter was phosphorylated These results indicate that the presence of the phosphate group prevents the enzymatic cleavage Furthermore, as no other evident peak shift was appreciable, it can be argued that the monophosphorylated IGFBP-1 form is predominantly modified in the sequence spanning residues 93–103 Proteolysis In a previous paper [30], we described the isolation of the C-terminal domain of IGFBP-1 and suggested that the proteolytic cleavage could be ascribed to a specific amniotic fluid metalloprotease Therefore, prior to examining the effect of phosphorylation on the proteolytic process, we prefractionated the amniotic fluid proteins by gel filtration and analysed the fractions 6040 C Fig IGFBP-1 proteolysis (A) Aliquots of the nonphosphorylated protein (1 lg in 20 lL of 20 mM Tris pH 7.5 containing 20 mM CaCl2) were incubated overnight at 37 °C with lL of the amniotic fluid fractions containing proteins eluting ahead (lane 1) and after (lane 2) the albumin peak in gel filtration chromatography; as a control, the isolated C-terminal fragment and the untreated protein are shown in lanes and 4, respectively Following SDS gel electrophoresis, the proteins were electroblotted and probed with anti-IGFBP-1 IgGs (B) Aliquots of the nonphosphorylated IGFBP-1 (1 lg in 20 mM Tris pH 7.5 containing 20 mM CaCl2) were incubated overnight with no addition (lane 1), and with the addition of increasing amounts of the amniotic fluid fraction displaying proteolytic activity: lL (lane 2); lL (lane 3); lL (lane 4); and 16 lL (lane 5) Sample volumes were adjusted to 30 lL and incubations were terminated after 48 h by the addition of 30 lL of sample buffer The samples were then submitted to SDS gel electrophoresis The proteins were electroblotted and probed with anti-IGFBP-1 IgGs (C) Substrate in gel electrophoresis of the amniotic fluid proteasecontaining fraction Gelatin (lane 1) and IGFBP-1 (lane 2) were added to a 10% polyacrylamide solution before gel casting at a concentration of mgỈmL)1 The asterisk indicates the lysis area observed in IGFBP-1 zymography obtained for their IGFBP-1 protease activity As shown in Fig 8A, unmodified IGFBP-1 incubated with the fraction containing components with molecular mass above 50 kDa, remained almost unchanged, whereas in FEBS Journal 276 (2009) 6033–6046 ª 2009 The Authors Journal compilation ª 2009 FEBS L Dolcini et al A B Fig Proteolysis of IGFBP-1 isoforms (A) Aliquots of each IGFBP-1 isoform (1 lg in 20 lL of 20 mM Tris pH 7.5 containing 20 mM CaCl2) were submitted to SDS electrophoresis and immunoblotting following incubation at 37 °C overnight with the addition of a lL aliquot of the amniotic fluid fraction containing IGFBP-1 protease activity As a control, identical aliquots of the six isoforms diluted to the same concentration with the buffer containing 20 mM CaCl2 were incubated separately at 37 °C overnight and analysed For each isoform, indicated with the peak number, the left lane contains the untreated protein and the right lane the sample treated with the protease-containing fraction (B) Histogram representation of the susceptibility to proteolytic degradation of IGFBP-1 isoforms The y-axis shows the percentage of the ratio between the spot area of the degraded and that of the untreated protein measured using IMAGE J 1.37V software the presence of proteins eluting after the albumin peak, IGFBP-1 was partially cleaved, yielding two major bands, one corresponding to the C-terminal domain and a second with an apparent molecular mass of 20 kDa No fragmentation was observed when the incubation was performed in the presence of 10 mm EDTA (data not shown) The fraction containing the IGFBP-1 protease activity was then used to assess the dose dependence of the proteolytic process Figure 8B shows that by increasing the amount added to the protein the intensity of the 30 kDa band is progressively reduced The protease-containing fraction was then submitted to zymography using gelatine and IGFBP-1 as substrates in order to examine the specificity of the IGFBP-1 degrading activity The gelatine substrate zymogram showed the presence of several areas of lysis, representing gelatine-degrading proteinase activity in the sample On the contrary, only one area was evidenced in the IGFBP-1 substrate zymogram (Fig 8C) These results indicate that a specific protease is involved in the prote- Phosphorylation of amniotic fluid human IGFBP-1 olytic cleavage occurring in amniotic fluid We then examined the effect of phosphorylation on this process Each isoform was incubated with the proteinasecontaining fraction and the samples were then submitted to western blot analysis, together with identical aliquots of the untreated proteins (Fig 9A) The degradation process was evaluated on the basis of the ratio between the integrated areas of the intact protein in the treated and untreated samples This value was determined for each isoform and the results (Fig 9B) show that the monophosphorylated IGFBP-1 was cleaved almost to the same extent as the unmodified protein and that the susceptibility to proteolytic degradation of the isoforms increased with the number of phosphates linked to the polypeptide chains The different position of the groups bound to the biphosphorylated protein also played a role in the cleavage process Discussion The purification procedure used in this study permitted the isolation of the unmodified as well as of the five phosphoisoforms of IGFBP-1 previously detected by proteomic analysis in human amniotic fluid collected in weeks 16–18 of gestation [20] To obtain a sufficient amount of the biological fluid, several hundred individual samples were pooled and, therefore, the preparation was assumed to be normal as every individual difference would be below the detectability score The molecular mass of the nonphosphorylated protein was determined under both reducing and nonreducing conditions The values obtained matched the molecular mass deduced from the amino acid sequence and were consistent with the presence of nine disulfide bonds In addition, the molecular mass increments of the other isoforms defined the exact number of phosphate groups linked to the chromatographically distinct IGFBP-1 phosphoisomers isolated The characterization of the phosphorylation sites of IGFBP-I isoforms revealed that the post-translational modification involves five serine residues (numbered as Ser95, Ser98, Ser101, Ser119 and Ser169 in the mature polypeptide chain) In a previous study, we examined the phosphorylation of IGFBP-1 isolated from amniotic fluid following the selective extraction of phosphopeptides on a titanium dioxide (TiO2) cartridge and only three modified serines were located [36] Enrichment methods are well suited when only minute amounts of the phosphoprotein are available, but a detailed analysis of phosphoptides demands relatively high quantities of the homogeneous molecules All the residues identified in the present study were recognized by the protein FEBS Journal 276 (2009) 6033–6046 ª 2009 The Authors Journal compilation ª 2009 FEBS 6041 Phosphorylation of amniotic fluid human IGFBP-1 L Dolcini et al pattern database NetPhos [31] as phosphoacceptor sites, with a score always higher than 0.99 and, in agreement with previous data [22,35], no modification occurring at tyrosine and threonine residues was found Although a previous study, using the protein expressed in Chinese hamster ovary cells, identified three serines (Ser101, Ser119 and Ser169) as the only modified amino acids and excluded the involvement of other residues [22], our results, showing the presence of the tetraphosphorylated isoform, revealed a more extensive post-translational modification of the amniotic fluid protein Because the process can be the consequence of a combination of biological variables, including different activity levels of both the intracellular protein kinases and the extracellular phosphatases, this discrepancy is probably due to the different source of the examined molecules Moreover, it has been shown that the phosphorylation of IGFBP-1 is controlled by placental steroid hormones [37] and by IGFII [38] Both factors are probably regulated differently in Chinese hamster ovary cells and in human decidual cells A recent paper revealed the modification of Ser98 in the hyperphosphorylated IGFBP-1 expressed in hypoxia exposed cells and the finding was suggested to be associated with the condition [23] However, our data show that this phosphoacceptor site is also modified in protein purified from normal amniotic fluid The kinases responsible for in vivo phosphorylation of IGFBP-1 are as yet unknown, but it has been shown that IGFBP-1 is a substrate for several protein kinases [31,35] and the residues adjacent to all the modified serines form consensus sequences for post-translational modification by casein kinase II A quantitative determination of the modification occurring at each site could not be achieved because of the poor ionization efficiency and fast degradation of phosphopeptides In particular, the detection of multiphosphorylated peptides is reduced owing to their high propensity to adsorption to exposed surfaces [34] Furthermore, nonspecific and partial enzymatic cleavages caused the presence of several fragments containing the same phosphorylation site However, the examination of the peptides obtained following V8 proteolytic cleavage indicated that the monophosphorylated IGFBP-1 form is predominantly modified within the AGSPESPESTE sequence containing three of the five phosphorylated residues identified in the present study It is interesting to note that this polypeptide sequence is rich in proline, glutamic acid, serine and threonine, a so-called PEST region, which is typical of rapidly metabolized proteins Briefly, the PEST hypothesis suggests that protein regions containing a high local concentration of the amino acids proline, glutamic acid, 6042 serine, threonine, and, to a lesser extent, aspartic acid, are suitable targets for proteolytic degradation [29] Examination of the amino acid sequence of IGFBP-1 with the protein pattern database PESTfind (available at http://www.at.embnet.org/toolbox/pestfind/), a computational tool designed to predict potential PEST sequences, confirmed that the region spanning residues 89–114 (HAAEAGSPESPESTEITEEELLDNFH) is potentially one of those This region is located 27 residues far from the proteolytic site that leads to the formation of the C-terminal fragment previously isolated and characterized from amniotic fluid [30] Proteolysis of IGFBPs is an important mechanism that controls IGF bioavailability [39,40] and previous studies have focused on the cleavage of IGFBP-1 by matrix metalloproteases present in amniotic fluid and conditioned medium from decidualized endometrial cells [8,25] The effect of IGFBP-1 phosphorylation on its susceptibility to enzymatic cleavage has been examined in vitro and Gibson et al [8] showed that the highly phosphorylated protein is resistant to the protease activity in decidual conditioned medium and to plasmin, whereas Kabir-Salmani et al [37] suggested that matrix metalloproteinase-9 selectively degrades phosphorylated IGFBP-1 Recently, it has been reported that the IGFBP-1 specific protease activity identified as azurocidin cleaves both phosphorylated and nonphosphorylated IGFBP-1 [28] These discrepancies probably reflect the specificity of any enzymatic activity and, although the protein has been recognized as a potential physiological substrate for several matrix metalloproteases, little is known about the susceptibility of IGFBP-1 to enzymatic cleavage Because the only proteolytic degradation of IGFBP-1 occurring without the addition of endogenous reactants so far described under normal conditions is that observed in the amniotic fluid [41], we performed the assays using a partially purified fraction of amniotic fluid displaying IGFBP-1 specific protease activity Our data clearly show that the post-translational modification increases the susceptibility to cleavage by the amniotic fluid protease and suggest that not only the degree, but also the position of the phosphate groups influence the process As for the other IGFBPs, the proteolytic cleavage of IGFBP-1 occurs within the central domain and we have shown that the process generates a C-terminal domain (residues 141–234) carrying a phosphate group linked to Ser169 [30] It is possible that the charge repulsion between the phosphate groups in the midregion and the one located in the C-terminal portion of the protein may cause a conformational change that makes the cleavage site more exposed to the protease Thus, the post-translational modification might act as FEBS Journal 276 (2009) 6033–6046 ª 2009 The Authors Journal compilation ª 2009 FEBS L Dolcini et al a mechanism for increasing the bioavailability of IGFs by enhancing the proteolytic cleavage as well as for producing the C-terminal domain We have examined the amino acid sequences of the other protein family members using the PESTfind database and found that a high score region as a PEST sequence is present only in the IGFBP-5 polypeptide chain, spanning residues 130–146, and, with a lower score, in IGFBP-6 between residues 150 and 175 The in vivo post-translational modification of IGFBP-5 has recently been examined in detail and mono- and biphosphorylated isoforms were shown to be secreted by human breast carcinoma cells [42] The major phosphorylation site of the protein was found to involve Ser96, in the central domain of IGFBP-5, and a minor one was localized very close to the C-terminus, at Ser248 Ser96 on IGFBP-5 shares the same proximity to the N-terminal domain as Ser95, Ser98 and Ser101 on IGFBP-1 (our results), Ser111 and Ser113 on IGFBP-3, and Ser106 on IGFBP-2 [42,43] IGFBP-1 only seems to have phosphoacceptor sites located in a PEST region, but the presence of phosphorylation at conserved serine residues points to a pivotal role of this post-translational modification in the regulation of biological activities of IGFBPs The present study identified five phosphorylated serine residues in the polypeptide chain of amniotic fluid IGFBP-1 and showed that the modification does not take place at the same extent at each of the potential sites, leading to heterogeneity Compared with previous studies, based on selective enrichment by affinity techniques, the availability of relatively high amounts of pure protein allowed us to carry out a more complete and detailed characterization of the phosphoacceptor sites Four of the five phosphorylated residues identified are located in the weakly conserved and poorly structured sequence composed of  65 amino acids connecting the N- and the C-terminal domains The prevalent phosphoacceptor sites of the monophosphorylated protein are located in a PEST region present in proteins that are a target for phosphorylated mediated degradation and our data show that this post-translational modification increases the propensity of IGFBP-1 to proteolytic cleavage Experimental procedures Chemicals Immobilon-P poly(vinylidene) difluoride transfer membranes were obtained from Millipore (Bedford, MA, USA) Sephadex G-100, Supedex-75 and Q-Sepharose resins, as well as the enhanced chemiluminescence detection system Phosphorylation of amniotic fluid human IGFBP-1 were purchased from Amersham Biosciences (Piscataway, NY, USA) Gelatin, type A from porcine skin, and alkaline phosphatase were purchased from Sigma (St Louis, MO, USA) A MilliQ system (Millipore) was used for water purification Laboratory chemicals were purchased from Sigma, unless otherwise specified Isolation of IGFBP-1 isoforms from human amniotic fluid Human amniotic fluid was obtained from discarded amniocentesis samples collected in weeks 16–18, pooled and stored frozen until used EDTA (10 mm) was added to avoid unwanted proteolysis The fluid (2 L) was saturated to 90% with ammonium sulfate and after centrifugation at 10 000 g for h, the pellet was dissolved in 50 mm Tris ⁄ HCl, pH 8.0, 150 mm NaCl, 10 mm EDTA, dialysed against the same buffer and fractioned twice by gel filtration on a Sephadex G-100 column (4 · 100 cm) Proteins eluting after the albumin peak were pooled, equilibrated in 6.25 mm Bistris-propane, pH 7.5, and separated on a Q-Sepharose (75 mL) column equilibrated with the same buffer (buffer A) and connected to an ¨ Akta Prime system (Amersham Biosciences) The elution was carried out with a linear gradient from 0% to 100% of 6.25 mm Bistris-propane, pH 9.5, 350 mm NaCl (buffer B) and monitored at 280 nm The fractions containing the IGFBP-1 isoforms were pooled, concentrated and submitted to gel filtration on a Superdex G-75 column (1.6 · 60 cm) equilibrated and eluted with 20 mm Tris ⁄ HCl buffer, pH 8.0, 150 mm NaCl The homogeneity of the proteins was checked by SDS PAGE analysis and by N-terminal sequencing performed in a Hewlett-Packard model G 1000 A sequencer (Centro Grandi Strumenti, University of Pavia) Gel electrophoresis and western blot analysis Isoelectric focusing was carried out on laboratory made gels, cast on GelBond with a 4–10 nonlinear immobilized pH gradient obtained with Acrylamido buffer solutions (Fluka, Sigma-Aldrich Schweiz, Buchs, Switzerland) SDS PAGE was carried out using a Mini PROTEAN II cell (Bio-Rad, San Diego, CA, USA) and the gels were stained with Coomassie Blue Nondenaturing PAGE was carried out on a discontinuous polyacrylamide gel system, using pH 6.8 in the 4% stacking gel and pH 7.5 in the 10% resolving gel Polyclonal antibodies were produced as previously described [30] and antibody titres were assayed either by ELISA or immunoblotting The specific IgGs were purified by affinity chromatography on a Protein A-Sepharose column according to the manufacturer’s recommendations (Amersham Biosciences) For western blot analyses, after electrophoresis, the proteins were transferred by electroblotting to Immobilon-P poly(vinylidene difluoride) membranes FEBS Journal 276 (2009) 6033–6046 ª 2009 The Authors Journal compilation ª 2009 FEBS 6043 Phosphorylation of amniotic fluid human IGFBP-1 L Dolcini et al using a Mini Protean II apparatus (Bio-Rad) The membrane was blocked with 5% w ⁄ v skim milk and probed with the mouse antiserum diluted : 2000 Immunoreactive spots were detected with horseradish peroxidase-conjugated anti-mouse IgG and developed by the enhanced chemiluminescence method LC-MS analysis of IGFBP-1 isoforms The system used for LC-MS analyses was a Surveyor LC coupled with an ion trap mass spectrometer LCQ (Thermo Scientific, San Jose, CA, USA) equipped with an ESI source and controlled by xcalibur software 1.3 (Thermo Scientific) Experiments were carried out in positive ion mode under constant instrumental conditions: source voltage 4.5 kV, capillary voltage )20 V, capillary temperature 210 °C, tube lens voltage )5 V Mass spectra were acquired in the mass range of 200–2000 m ⁄ z by scanning the magnetic field in 200 ms The purified isoforms, 20 lL out of a 0.5 mgỈmL)1 solution in 100 mm ammonium carbonate, pH 8.5, were loaded onto a C18 column (Phenomenex Jupiter 300, lm, · 50 mm) and eluted with a linear gradient from solution A (5% acetonitrile) to solution B (95% acetonitrile) both containing 0.1% formic acid over 15 at a flow rate of 0.2 mLỈmin)1 An aliquot of the unmodified protein was reduced by adding 10% 1,4-dithiothreitol to the 100 mm ammonium carbonate solution Deconvolution of multiple charged ions was achieved using the mag tran 1.01 software [44] RP-HPLC separation of the V8 peptides Nonphosphorylated and monophosphorylated fractions (1 mg) were digested with the same protocol reported above and were loaded on to a Vydac C18 reverse phase column (4.6 · 250 mm) Elution was carried out with a linear gradient from 0% to 100% solvent B in 100 at a constant flow rate of mLỈmin)1 The solvents consisted of water (A) and acetonitrile (B), both containing 0.1% formic acid The elution profile was monitored, recording the UV trace at 214 nm Peaks were manually collected and subsequently analysed via LC-ESI-MS ⁄ MS and with N-terminal sequencing as described above Degradation of IGFBP-1 isoforms by amniotic fluid protease Preparation of proteolytic digests for LC-ESI-MS ⁄ MS Three aliquots of each IGFBP-1 isoform, 50 lg in 100 lL of 100 mm ammonium carbonate pH 8.5, were reduced for 30 at 60 °C with 10 lL of a 10 mm freshly prepared dithiothreitol solution in the same buffer Proteolysis was performed with either endopeptidase V8 from S aureus, trypsin or chymotrypsin The enzymes, in 100 mm ammonium bicarbonate at pH 8.5, were added at a ratio of : 50 (w ⁄ w), allowed to react at 37 °C for h and the digestions were stopped by the addition of phosphoric acid (final concentration 0.8% v ⁄ v) LC-ESI-MS ⁄ MS analysis of peptides The peptide solutions (20 lL) were resolved in an analytical C18 column (Phenomenex Jupiter 300, lm, 250 · 2mm) at a flow rate of mLỈmin)1 with a gradient 2–60% B in 120 min, 60–98% B in 30 and 98% B for 10 Solvents consisted of water (A) and 60% acetonitrile (B) both containing 0.1% trifluoroacetic acid The eluent was analysed online with an LCQ ion trap mass spectrometer (Thermo Scientific) with the ESI ion source controlled by the xcalibur software 1.4 (Thermo Scientific) Mass spectra were gener- 6044 ated in positive ion mode under constant instrumental conditions: source voltage 4.0 kV, capillary voltage 46 V, sheath gas flow 40 (arbitrary units), auxiliary gas flow 10 (arbitrary units), sweep gas flow (arbitrary units), capillary temperature 250 °C, tube lens voltage )105 V MS ⁄ MS spectra, obtained by collision-induced dissociation in the linear ion trap, were performed with an isolation width of Da (m ⁄ z), the activation amplitude was 35% of ejection radio frequency amplitude, which corresponds to 1.58 V MS ⁄ MS spectra were interpreted manually with the assistance of the prediction algorithm for peptide fragmentation proteinprospector [45] and were automatically analysed using peaks studio, version 4.2 (Bioinformatic Solution Inc., Waterloo, Canada) Fresh amniotic fluid (100 mL), without the addition of EDTA, was saturated to 90% with ammonium sulfate and the precipitated proteins were redissolved and fractioned by gel filtration chromatography as described above The components eluting ahead and after the albumin peak were pooled and concentrated separately to  mgỈmL)1 Aliquots of the amniotic fluid fractions were added to lg of either unmodified IGFPB-1 or phosphoisoforms in 20 lL of 20 mm Tris ⁄ HCl pH 7.5 containing 20 mm CaCl2 and incubated at 37 °C overnight The incubations were terminated by the addition of sample buffer, without reducing agents, and the degradation was analysed by western blot and quantified using the image j 1.37v software, freely available online at http://rsb.info.nih.gov/ij/ Each experiment was repeated three times in order to check the reproducibility of both the proteolytic process and the method of analysis For IGFBP substrate zymography, the unmodified protein was added to a 10% polyacrylamide solution before gel casting (1 mgỈmL)1) and an aliquot of the amniotic fluid fraction, diluted : in nonreducing sample buffer, was electrophoresed Only one lane, obtained with a spacer, was made using this substrate, whereas the remaining part of the cassette was filled with the resolving polyacrylamide gel FEBS Journal 276 (2009) 6033–6046 ª 2009 The 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⁄ MS and database searching Anal Chem 71, 2871–2882 46 Morodomi T, Ogata Y, Sasaguri Y, Morimatsu M & Nagase H (1992) Purification and characterization of matrix metalloproteinase from U937 monocytic leukaemia and HT1080 fibrosarcoma cells Biochem J 285, 603–611 FEBS Journal 276 (2009) 6033–6046 ª 2009 The Authors Journal compilation ª 2009 FEBS ... degradation of IGFBP-1 isoforms The y-axis shows the percentage of the ratio between the spot area of the degraded and that of the untreated protein measured using IMAGE J 1.37V software the presence of. .. almost to the same extent as the unmodified protein and that the susceptibility to proteolytic degradation of the isoforms increased with the number of phosphates linked to the polypeptide chains The. .. elevation of insulin-like growth factor (IGF) binding protein-1 decreases plasma free IGF-I and muscle protein synthesis Endocrinology 144, 3922–3933 Westwood M (1999) Role of insulin-like growth factor