Disulfide Bond Pattern of Transforming Growth Factor β-Induced

Sep 8, 2016 - Mutations in the gene cause corneal dystrophies, a group of debilitating protein misfolding diseases that lead to severe visual impairme...
0 downloads 0 Views 4MB Size
Download PDF
Article pubs.acs.org/biochemistry

Disulfide Bond Pattern of Transforming Growth Factor β‑Induced Protein Marie V. Lukassen,† Carsten Scavenius,‡ Ida B. Thøgersen,†,‡ and Jan J. Enghild*,†,‡ †

Interdisciplinary Nanoscience Center (iNANO) and ‡Department of Molecular Biology and Genetics, Aarhus University, DK-8000 Aarhus, Denmark ABSTRACT: Transforming growth factor β-induced protein (TGFBIp) is an extracellular matrix protein composed of an NH2-terminal cysteine-rich domain (CRD) annotated as an emilin (EMI) domain and four fasciclin-1 (FAS1-1−FAS1-4) domains. Mutations in the gene cause corneal dystrophies, a group of debilitating protein misfolding diseases that lead to severe visual impairment. Previous studies have shown that TGFBIp in the cornea is cross-linked to type XII collagen through a reducible bond. TGFBIp contains 11 cysteine residues and is thus able to form five intramolecule disulfide bonds, leaving a single cysteine residue available for the collagen cross-link. The structures of TGFBIp and its homologues are unknown. We here present the disulfide bridge pattern of TGFBIp, which was determined by generating specific peptides. These were separated by ion exchange followed by reversed-phase high-performance liquid chromatography and analyzed by mass spectrometry and Edman degradation. The NH2-terminal CRD contains six cysteine residues, and one of these (Cys65) was identified as the candidate for the reducible cross-link between TGFBIp and type XII collagen. In addition, the CRD contained two intradomain disulfide bridges (Cys49−Cys85 and Cys84−Cys97) and one interdomain disulfide bridge to FAS1-2 (Cys74−Cys339). Significantly, this arrangement violates the predicted disulfide bridge pattern of an EMI domain. The cysteine residues in FAS1-3 (Cys473 and Cys478) were shown to form an intradomain disulfide bridge. Finally, an interdomain disulfide bridge between FAS1-1 and FAS1-2 (Cys214−Cys317) was identified. The interdomain disulfide bonds indicate that the NH2 terminus of TGFBIp (CRD, FAS1-1, and FAS1-2) adopts a compact globular fold, leaving FAS1-3 and FAS1-4 exposed.

ransforming growth factor β-induced protein (TGFBIp) also known as keratoepithelin and βig-h3 is a 72 kDa extracellular matrix (ECM) protein found in several tissues.1−6 TGFBIp belongs to a group of homologous proteins that includes TGFBIp (Uniprot entry Q15582), Periostin (Uniprot entry Q15063),7 and the drosophila protein Midline fasciclin (Uniprot entry O61457).8 They are all predicted to be composed of a putative NH2-terminal emilin (EMI)9 or cysteine-rich domain (CRD) and four fasciclin-1 domains.10 In addition, TGFBIp contains a COOH-terminal Arg-Gly-Asp (RGD) sequence. The three-dimensional structure of the intact protein and the disulfide bonding are not known. The function of TGFBIp is unknown, but it has been suggested that the protein serves as an adhesion protein in the ECM and as a ligand for integrin receptors through the FAS1 domains11−17 or through the RGD integrin-binding motif at the COOH terminus.18,19 Furthermore, TGFBIp can bind ECM macromolecules such as collagens, fibronectin, decorin, and biglycan.20−23 Of particular interest is the role of TGFBIp in the human cornea where it is the second most abundant protein.24 Mutations in the TGFBIp (TGFBI) gene are associated with several types of corneal dystrophies, a group of progressive protein misfolding diseases that cause severe visual impairment.25−27 The aggregation of TGFBIp in these dystrophies appears to be caused by misfolding correlated with changes in the thermodynamic stability of the protein.28−30 A key component in the stability and folding of proteins containing cysteine residues is the pairing of the disulfides. To date, only an NMR structure of

T

© XXXX American Chemical Society

the human FAS1-4 domain is available, and structures of fulllength TGFBIp or the homologous proteins have not been determined.30 TGFBIp contains 11 cysteine residues. Six are located in the NH2-terminal CRD; one is located in the FAS1-1 domain, and a pair are located in each of the FAS1-2 and FAS1-3 domains. Additionally, one of the most common mutations in TGFBI R124C introduces an extra cysteine residue into the FAS1-1 domain.31 In this study, we have determined the disulfide bond pattern of TGFBIp. Previous studies have shown that 60% of TGFBIp is associated with the SDS-insoluble fraction of homogenized human corneal tissue and that corneal TGFBIp binds to type XII collagen through a reducible bond.32,33 Moreover, it has been shown that bovine TGFBIp binds to type VI collagen in nuchal ligament through a reducible bond.34 The data presented here reveal that Cys65 in the NH2-terminal CRD was not bound to another TGFBIp cysteine residue but protected by a cysteinylation. This reducible modification can take part in a disulfide bridge exchange with other cysteine residues and may thus be able to form the reducible intermolecular bond observed between human TGFBIp and type XII collagen. In addition, the disulfide bridge arrangement determined in this study was inconsistent with the existence of an NH2-terminal EMI domain. Alternatively, the data suggest the NH2-terminal CRD, FAS1-1, Received: July 8, 2016 Revised: September 6, 2016

A

DOI: 10.1021/acs.biochem.6b00694 Biochemistry XXXX, XXX, XXX−XXX

Article

Biochemistry

Figure 1. Isolation and separation of disulfide-bonded peptides by SCX and RP-HPLC. TGFBIp was sequentially digested with trypsin and Glu-C. (A) The SCX run was divided into seven pools as indicated, and the presence of Cys-containing peptides was assayed after reduction and alkylation of the samples. (B) Pool 5, (C) pool 6, and (D) pool 7 contained Cys-containing peptides and were further purified by RP-HPLC (Aquapore RP-300 C8 column). The major fractions (numbered) were analyzed by LC−MS/MS and N-terminal sequencing. Fractions marked with asterisks contained Cys-containing peptides as shown by LC−MS/MS in Table 1.

before being applied to a 5 mL HiTrap heparin column (GE Healthcare). Proteins were eluted by employing a linear gradient of 1% buffer B [20 mM Tris-HCl and 1 M NaCl (pH 7.5)] per minute. Fractions containing TGFBIp were pooled and dialyzed into buffer A. The dialysate was then applied to a 5 mL HiTrap Q anion exchange column (GE Healthcare). Proteins were eluted by employing a gradient 1% buffer B per minute. The flow rates in both purification steps were 2 mL/min, and the eluates were monitored at 280 nm. The columns were connected to an AKTA Purifier LC system (GE Healthcare) and were operated at RT. Purified TGFBIp was dialyzed against 20 mM Tris-HCl and 150 mM NaCl (pH 7.5) and frozen at −20 °C. The yield for 1 L of expression was approximately 2 mg of protein for the WT and 200 μg for the R124C mutant. The purity was 99% for the WT and 95% for the R124C mutant based on visual inspection of sodium dodecyl sulfate−polyacrylamide gel electrophoresis (SDS−PAGE) gels. Proteolytic Digest of rTGFBIp. Before proteolytic digestion, 2 mg of rTGFBIp was denatured in 100 mM ammonium bicarbonate containing 6 M urea and free cysteine residues were alkylated with 25 mM iodoacetamide (IAA) for 30 min at RT in the dark. The sample was diluted to a final urea concentration of 1 M, and 40 μg of trypsin was added. The digestion was continued for 5 h at 37 °C before the solution was lyophilized and solubilized in 50 mM Tris-HCl (pH 8) to a final urea concentration of 1 M. The tryptic digest was further digested with 35 μg of endoproteinase Glu-C (Glu-C) at RT for 16 h. Some purified peptides were subdigested with 0.4 μg of Glu-C in 50 mM Tris-HCl (pH 8) overnight at 37 °C. For the analysis of the rTGFBIp R124C mutant, 5 μg of protein was denatured and alkylated by the same procedure described above. In brief, the protein was digested with 0.2 μg of trypsin at 37 °C for 5 h, followed by a 16 h digestion using 0.2 μg of Glu-C. Purification of Disulfide-Bonded rTGFBIp Peptides. The samples were kept under nonreducing conditions at all times unless otherwise stated. Trypsin and Glu-C peptides were separated

and FAS1-2 adopt a more compact structure, most likely leaving FAS1-3 and FAS1-4 exposed. The disulfide bond pattern determined using recombinant human TGFBIp was verified by analyzing the disulfide connectivity of authentic TGFBIp extracted from human cornea. Furthermore, it was shown that Cys124 in the R124C mutant did not bind to another cysteine but was cysteinylated like Cys65 in wild-type TGFBIp. This structural information provides clues about the role of TGFBIp in the homeostasis of the healthy cornea and the structural effect of the mutations that cause corneal dystrophies.

■

EXPERIMENTAL PROCEDURES Materials. All reagents were purchased from Sigma-Aldrich (St. Louis, MO), unless otherwise stated. Healthy human corneas were obtained post mortem at the Department of Forensic Medicine of Aarhus University Hospital (Aarhus, Denmark) as described previously.35 The corneas were dried in a vacuum desiccator overnight and homogenized in liquid nitrogen. The resulting powder was stored at −20 °C until it was used. Expression and Purification of Recombinant TGFBIp (rTGFBIp). A cDNA clone of human TGFBI (Invitrogen) was obtained from a human placenta cDNA library and cloned into the pCMV-SPORT6 vector.36 An identical cDNA sequence with a mutation at residue 124 from an arginine to a cysteine (R124C) was also cloned into a pCMV-SPORT6 vector. Expression was induced in human cell line FreeStyle 293-F (Invitrogen) using the polyethylenimine (PEI) transfection method.37,38 Plasmid and PEI were mixed in a 1:3 ratio and preincubated for 20 min at room temperature (RT) in expression medium (Gibco FreeStyle 293, Invitrogen) without penicillin/streptomycin (P/S). Before being transfected, the cells were grown to a density of 1.0 × 106 cells/mL in medium with P/S, and the preincubated plasmid was added. For 1 L of expression medium, 1 mg of plasmid was used. Medium was harvested after expression for 3 days, and proteases were inhibited with 1 mM PMSF and 4.8 mM EDTA. The medium was dialyzed into buffer A [20 mM Tris-HCl (pH 7.5)] B

DOI: 10.1021/acs.biochem.6b00694 Biochemistry XXXX, XXX, XXX−XXX

Article

Biochemistry Table 1. Identification of Peptides in RP-HPLC (C8) Fractions by LC−MS/MS and NH2-Terminal Sequencing fraction

LC−MS/MSa

5.1 5.2 5.3 5.4

R.YHMVGR.R R.NHIIKDQLASK.Y R.NSLCIENSCIAAHDKR.G

6.1

E.VGCSGDMLTINGK.A K.ADHHATNGVVHLIDK.V

6.2

R.QHGPNVCAVQK.V K.STVISYECCPGYEK.V K.VPGEKGCPAALPLSNLYE.T

6.3

K.STVISYECCPGYEK.V R.QHGPNVCAVQK.V K.GCPAALPLSNLYE.T

6.4

R.QHGPNVCAVQK.V

7.1 7.2

E.VGCSGDMLTINGK.A

7.3 7.4 7.5 7.6

NH2-terminal sequencingb YHMVGR NHIIxDQLAS NSLxIENSxIAAHD STVISYExxPc VPGExGxPAAc VGxSGDMLTING ADHHATNGVVHLID KIxGK QHGPNVxAVQK STVISYExxPGYE VPGEKGxPAALPLS GxPAALPLSNLYE xxPGYEK STVISYExxPGYE QxGPNVxAVQ GxPAALPLSNLYE KGxPAAxPLSNLYE VPGExGxPAALPLSN HGMTLTSMYQNSNI SAMxAE QxGPNVxAVQ STVISYExxPGYE GxPAALPLxNLYE HGMTLTSMYQNSNI SAMxAE YHMVGc VGxSGDMLTING KIxGK HGMTLTSxYQc SAMxAEc HGMTLTSMYQc SAMxAEc HGMTLTSMYQc SAMxAEc

R.QHGPNVCAVQK.V HGMTLTSMYQc SAMxAEc

theoretical peptide sequence

Cys number

YHMVGR 437 NHIIKDQLASK 470 NSLCIENSCIAAHDKR 77 STVISYECCPGYEK 91 VPGEKGCPAALPLSNLYE 337 VGCSGDMLTINGK 220 ADHHATNGVVHLIDK 72 KICGK 43 QHGPNVCAVQK 77 STVISYECCPGYEK 91 VPGEKGCPAALPLSNLYE 96 GCPAALPLSNLYE 84 CCPGYEK 77 STVISYECCPGYEK 43 QHGPNVCAVQK 96 GCPAALPLSNLYE 95 KGCPAALPLSNLYE 91 VPGEKGCPAALPLSNLYE 187 HGMTLTSMYQNSNIQIHHYPNGIVTNCAR 314 SAMCAE 43 QHGPNVCAVQK 77 STVISYECCPGYEK 96 GCPAALPLSNLYE 187 HGMTLTSMYQNSNIQIHHYPNGIVTNCAR 314 SAMCAE 173 YHMVGR 337 VGCSGDMLTINGK 72 KICGK 187 HGMTLTSMYQNSNIQIHHYPNGIVTNCAR 314 SAMCAE 187 HGMTLTSMYQNSNIQIHHYPNGIVTNCAR 314 SAMCAE 187 HGMTLTSMYQNSNIQIHHYPNGIVTNCAR 314 SAMCAE 43 QHGPNVCAVQK 187 HGMTLTSMYQNSNIQIHHYPNGIVTNCAR 314 SAMCAE

− − 473, 478 84, 85 97 339 − 74 49 84, 85 97 97 84, 85 84, 85 49 97 97 97 214 317 49 84, 85 97 214 317 − 339 74 214 317 214 317 214 317 49 214 317

173

a Peptides observed more than 20 times; samples are reduced and alkylated. bx indicates that the amino acid residue was not detected. cTen cycles instead of 15.

The peptides were eluted using flow rates of 200 μL/min and a 0.5 or 1% B/min gradient from solvent A (0.1% TFA) to solvent B (0.07% TFA and 90% acetonitrile). The eluate was monitored at 220 nm, and the peptides were collected manually. Fractions containing Cys peptides were further purified using another RP-HPLC column (2.1 mm × 220 mm Vydac C18, Phenomenex). LC−MS/MS of Purified Peptides. The unreduced samples were lyophilized and rehydrated in 0.1% formic acid (solvent A) before being analyzed. Samples destined for reduction were dissolved in 50 mM ammonium bicarbonate (pH 8) containing 5 mM DTT for 30 min at RT before they were alkylated using 25 mM IAA for 20 min at RT. Both reduced and unreduced samples were then desalted on homemade reversed-phase micro columns containing small plugs of Octadecyl C18 Solid Phase Extraction disks (Empore, 3M) and dissolved in 0.1% formic acid before LC−MS/MS analysis.39 Nano-LC−MS/MS analyses were performed on an EASY-nLC II system (Thermo Scientific) connected to a TripleTOF 5600+ mass spectrometer

by strong cation exchange (SCX) using a 2.1 mm × 220 mm PolySulfoethyl column (The Nest Group) connected to an AKTA Ettan LC system (GE Healthcare). Before the sample was applied to the column, it was diluted in a 1:1 ratio with buffer A [10 mM KH2PO4 and 20% acetonitrile (pH 2.8)] and the pH was adjusted to 2 with HCl. The column was operated at RT using a flow rate of 100 μL/min and developed by gradients from buffer A to buffer B [10 mM KH2PO4, 20% acetonitrile, and 500 mM KCl (pH 2.8)] in the following manner: 0 to 15% B over 7.5 min, 15 to 50% B over 40 min, and 50 to 100% B over 25 min. The peptides were detected at 220 nm. The SCX fractions were combined into seven pools based on visual inspection of the elution profile. Pools containing Cys peptides (pools 5−7) were further separated by reversed-phase HPLC (RP-HPLC) using a 2.1 mm × 220 mm Aquapore RP-300 C8 column (Brownlee, Applied Biosystems) connected to an AKTA Ettan LC system (GE Healthcare). The SCX pools were lyophilized and rehydrated in 0.1% TFA before being loaded onto the RP-HPLC column. C

DOI: 10.1021/acs.biochem.6b00694 Biochemistry XXXX, XXX, XXX−XXX

Article

Biochemistry (Sciex, Framingham, MA) equipped with a NanoSpray III source (AB Sciex) operated under the control of Analyst TF version 1.5.1. The samples were injected and desalted on a trap column [2 cm × 100 μm (inner diameter)] and separated on an in-house pulled fused silica emitter [15 cm × 75 μm (inner diameter)]. Both columns were packed with PeproSil-Pur C18-AQ 3 μm resin (Dr. Marisch GmbH, Ammerbuch-Entringen, Germany). The peptides were electrosprayed into the mass spectrometer by gradients from 5% buffer A to 35% buffer B (90% acetonitrile in 0.1% formic acid) at a constant flow rate of 250 nL/min. The collected MS files were converted to Mascot generic format (MGF) using the SCIEX MS Data Converter beta 1.1 (Sciex) and the “proteinpilot MGF” parameters. Analysis of MS Data. The peptides present in the reduced samples were identified by generating peak lists (MGF). These were searched against an in-house database containing the TGFBIp sequence using the Mascot search engine (Matrix Science) and the following search parameters: MS tolerance of 10 ppm, MS/MS tolerance of 0.1 Da, trypsin and Glu-C with two missed cleavages, carbamidomethyl as the fixed modification, and oxidized methionine as the variable modification. Fractions containing Cys peptides were further analyzed by nonreduced LC−MS/MS. MS spectra of the unreduced samples were manually inspected for m/z values corresponding to the crosslinked Cys-containing peptides. Cross-links were verified by assigning the corresponding MS/MS spectra to the peptide sequence. MS/MS spectra were merged with a retention time gap tolerance of 0.2 min and a mass tolerance of 5.0 ppm. Automated Edman Degradation of Purified Peptides. Aliquots (1/25) of the RP-HPLC fractions were applied directly to micro TFA filters and sequenced on a Shimadzu PPSQ-31B protein sequencer (Shimadzu Corp.). The PTH-amino acids were detected online at 269 nm after separation on a reversedphase C18 column (4.6 mm × 2.5 mm) under isocratic conditions according to the manufacturer’s instructions. Analysis of the Disulfide-Bonded Triple Peptide. The disulfide bond pattern of the triple peptide (three peptides linked by two disulfide bridges) was analyzed as previously described.40 Briefly, the RP-HPLC fraction containing the triple peptide was subjected to one round of NH2-terminal sequencing as described above. The membrane was then rinsed in water and incubated in extraction buffer [50 mM Tris-HCl (pH 8.5) and 40% acetonitrile] for 3 h at 37 °C. The extracted peptides were lyophilized, dissolved in 0.1% formic acid, micropurified, and subjected to unreduced LC−MS/MS as described above. SDS−PAGE. SDS−PAGE was performed on 5 to 15% polyacrylamide gradient gels using an ammediol/glycine buffer system.41 The samples were boiled for 5 min with SDS (1%). For reduced samples, 50 mM DTT was also added before the samples were boiled. Gels were stained with Coomassie Brilliant Blue. Analysis of the Disulfide Bond Pattern of Authentic TGFBIp Derived from Human Cornea. Homogenized human cornea (0.4 mg) was boiled in nonreducing SDS sample buffer for 10 min, and then the supernatant was analyzed by unreduced SDS−PAGE. The band corresponding to TGFBIp was excised and subjected to in-gel digestion with trypsin.42 Glu-C was added after incubation with trypsin for 5 h, and digestion proceeded overnight. The sample was desalted and analyzed by LC−MS/MS. The MS spectra were manually inspected, and MS/MS spectra of cross-linked Cys-containing peptides were compared to the assigned MS/MS spectra from rTGFBIp. Analysis of the rTGFBIp R124C Mutant by LC−MS/MS. The digested protein was desalted and analyzed by nonreducing

Figure 2. Additional separation of disulfide-bonded peptides by RP-HPLC. Fractions enriched in Cys-containing peptides in the first RP-HPLC purification (Figure 1B−D) were further purified by another RP-HPLC column (Vydac, C18). Chromatograms of fractions 5.3 and 7.2 are not shown because they produced only one peak. Fractions (A) 6.1, (B) 6.2, and (D) 6.4 were all eluted with 1% B/min, while fraction 6.3 (C) was eluted with 0.5% B/min for better separation. Collected fractions (numbered) were analyzed by LC−MS/MS and NH2-terminal sequencing.

LC−MS/MS as previously described. The generated MGF file was subjected to an error tolerant search against an in-house database containing the R124C mutant. Multiple-Sequence Alignment of EMI Domains. Sequences of EMI domains were aligned by the T-Coffee program.43 Conservation of residues was based on chemical character: aromatic (F, Y, and W), hydrophobic (L, I, V, and M), acidic (E and D), basic (K, R, and H), polar (S and T), tiny (G and A), and amide (N and Q). Homology Modeling of FAS1 Domains. Homology modeling was conducted on the SWISS-MODEL server (http://swissmodel.expasy.org).44 The NMR structure of the human FAS1-4 domain [Protein Data Bank (PDB) entry 2LTB] was used as a template, and the sequences of the FAS1-1, FAS1-2, and FAS1-3 domains from UniProt were used for modeling. The structures were visualized in PyMol, and the structures of the FAS1-1 and FAS1-2 domains were individually aligned on the structure of the FAS1-3 and FAS1-4 domain pair from Drosophila fasciclin-1 (PDB entry 1O70).

■

RESULTS Isolation and Separation of Disulfide-Bonded Peptides from a Digest of rTGFBIp. Purified rTGFBIp was digested with trypsin and Glu-C. The digestion resulted in cleavages among all 11 cysteine residues except the two adjacent Cys84 and Cys85 residues. The generated peptides were separated by SCX, and the collected fractions were divided into seven pools based on visual inspection of the elution profile (Figure 1A). The pools were reduced with DTT and analyzed by LC−MS/MS, revealing D

DOI: 10.1021/acs.biochem.6b00694 Biochemistry XXXX, XXX, XXX−XXX

Article

Biochemistry

Figure 3. Assigned MS/MS spectra of the five disulfide bonds in rTGFBIp. Fraction 5.3 contained the Cys473−Cys478 cross-link (A, 11 merged spectra). Fraction 6.1.1 contained the Cys74−Cys339 cross-link (B, 10 merged spectra). Fraction 6.3.2 contained the Cys214−Cys317 cross-link (C, two merged spectra). Fraction 6.2.2 contained a triple peptide with Cys49, Cys84, Cys85, and Cys97 (D, two merged spectra). The peptide sequences indicating the disulfide bridges are shown at the right with the assigned b- and y-ion fragments. The theoretical and observed precursor masses of the cross-linked peptide (precursor ion m/z in parentheses) together with the error are also shown at the right. Fragment ions marked with asterisks indicate neutral loss of water. Fractions marked with number signs indicate tyrosine immonium ions.

Fraction 7.6 was not selected for further separation because of its low intensity. The repurification of fractions 5.3 and 7.2 is not shown because they produced only a single peak. The FAS1-3 Domain Contains an Intradomain Disulfide Bond. Fraction 5.3 from the first RP-HPLC separation was shown by LC−MS/MS and NH2-terminal sequencing to encompass a peptide containing both Cys473 and Cys478 and a Glu-C missed cleavage (Figure 1B and Table 1). These two cysteine residues are both located in the FAS1-3 domain. The fraction was analyzed by unreduced LC−MS/MS, and the MS/MS spectrum

Cys-containing peptides in pools 5−7 (data not shown). Each of these pools was separated by RP-HPLC using an Aquapore RP300 C8 column (Figure 1B−D). Aliquots of the collected fractions were reduced with DTT and analyzed by LC−MS/MS to identify the Cys-containing peptides. NH2-terminal sequencing of the unreduced peptides confirmed the identity and revealed the cysteine residues involved in the disulfide bonds as “blank” cycles. Cys-containing peptides were found in fractions 5.3, 6.1-4, 7.2, and 7.6 (Table 1). These fractions except 7.6 were further separated by RP-HPLC using a Vydac C18 column (Figure 2). E

DOI: 10.1021/acs.biochem.6b00694 Biochemistry XXXX, XXX, XXX−XXX

Article

Biochemistry

peptides containing Cys49, Cys97, and the two vicinal cysteine residues (Cys84 and Cys85) were identified as different cleavage variants. Some of these peptides were also observed in fractions 5.4, 6.3, 6.4, and 7.6 (Table 1). After further separation of fraction 6.2-4, the three cysteine residues were identified in fraction 6.2.1-3, fraction 6.3.1, and fraction 6.4.1-2 (Figure 2B−D). The unreduced MS/MS spectrum of fraction 6.2.1 showed that Cys49 and Cys97 were cross-linked to the two vicinal cysteine residues (Figure 3D). Different combinations of miscleavage variants in the triple peptide explained the separation into multiple fractions during RP-HPLC. To identify which of the pair of Cys49 and Cys97 was bound to Cys84 or Cys85, it was necessary to separate the two vicinal cysteine residues. The major part of fraction 6.2.2 contained a triple peptide that was cleaved just before Cys85, but a minor part contained a missed cleavage at this site. The fraction was therefore treated with Glu-C to ensure NH2-terminal homogeneity at Cys84. The triple peptide was then subjected to one round of NH2-terminal sequencing, which releases Cys84 from Cys85. Next the peptides were extracted from the membrane and analyzed by LC−MS/MS without prior reduction. Only the combination in which Cys49 was cross-linked to Cys85 was observed, indicating that Cys97 was bound to Cys84 (Figure 4). No fragments containing the crosslink were found, indicating fragmentation of the disulfide. All four cysteine residues are located in the NH2-terminal CRD. The Disulfide Bond Pattern of TGFBIp Was Verified in Human Cornea. The disulfide bond pattern of TGFBIp from human cornea was examined to verify the disulfide bond pattern found in rTGFBIp. The soluble fraction of the human cornea was extracted by boiling homogenized corneal powder in SDS sample buffer. The supernatant was separated by unreduced SDS−PAGE, and the band corresponding to TGFBIp was digested with trypsin and Glu-C (Figure 5A). The peptides were analyzed by LC−MS/MS. The MS/MS spectra of the cross-linked peptides were manually assigned and compared to the MS/MS spectra recorded during the analysis of the cross-linked peptides derived from rTGFBIp (Figure 5B−E). The triple peptide was observed with a missed cleavage before only Cys84. The MS/MS spectrum was therefore compared to a spectrum of the corresponding triple peptide from fraction 6.3.1. Two of the peptides were found to contain a deamidated Asn residue (Figure 5C,E). We were able to verify all five disulfide bridges found in rTGFBIp in corneal TGFBIp extracted from human corneas.

of the peptide corresponded to the sequence of an internal disulfide bond between Cys473 and Cys478 (Figure 3A). Attempts were made to cleave between the two cysteine residues to obtain the two cross-linked peptides, but it was not possible, probably because of steric hindrance in the tight loop between the cross-linked cysteine residues. The NH2-Terminal CRD and the FAS1-2 Domain Are Linked through a Disulfide Bond. Peptides containing Cys74 and Cys339 were identified in both fraction 6.1 and fraction 7.2, and they were not observed together with any other Cyscontaining peptides (Figure 1C,D and Table 1). Further separation of fraction 6.1 by RP-HPLC (C18) produced two fractions (Figure 2A). These were analyzed by LC−MS/MS prior to reduction with DTT, and major fraction 6.1.2 was identified as the peptide ADHHATNGVVHLIDK, while the MS/MS spectrum of minor fraction 6.1.1 corresponded to a cross-link between Cys74 and Cys339 (Figure 3B). The same cross-link was found in fraction 7.2, indicating that the crosslinked peptides eluted in the intersection between pools 6 and 7 from the SCX column. These results are consistent with a disulfide bond between Cys74 in the NH2-terminal CRD and Cys339 in the FAS1-2 domain. A Disulfide Bond Links the First Two FAS1 Domains. Peptides covering Cys214 and Cys317 were observed together in fraction 7.3-6. Fractions 6.3 and 6.4 also contained these two peptides. Neither peptide was detected during LC−MS/MS analyses of the reduced peptide fractions, but both were detected by NH2-terminal sequencing (Table 1). The reason for this is likely that peptide SAMCAE (Cys317) is too small to be detected by the mass spectrometer and the large peptide, containing Cys214, is not observed probably because of an unfavorable charge distribution caused by the three His residues, in addition to heterogeneity caused by partial oxidation of the two Met residues and deamidation of the Asn-Gly and possible Asn-Ser. Further separation of fraction 6.3 produced fraction 6.3.2, and when analyzed by LC−MS/MS before reduction, the MS/MS spectrum revealed a cross-link between the two peptides (Figure 3C). This cross-link was also found in the unreduced samples of the other fractions containing Cys214 and Cys317. The different retention times for the cross-linked peptide were due to heterogeneous modifications of the peptides. The NH2-Terminal CRD Contains Two Intradomain Disulfide Bonds Represented by a Triple Peptide. In fraction 6.2,

Figure 4. Determining the disulfide pattern in the Cys49, Cys84, Cys85, and Cys97 triple peptide. Fraction 6.2.2, which contained the triple peptide, was treated with Glu-C, and the fully cleaved triple peptide was purified by RP-HPLC. The triple peptide was subjected to one round of NH2-terminal Edman degradation and analyzed by LC−MS/MS without reduction of the disulfides. The single round of NH2-terminal sequencing removes the NH2-terminal Cys84 together with the disulfide-bound peptide. The MS/MS spectrum corresponded to a dipeptide with a Cys49−Cys85 cross-link. The peptide sequences are shown at the right with the assigned b- and y-ion fragments. The theoretical and observed precursor masses of the crosslinked peptide (precursor ion m/z in parentheses) together with the error are also shown at the right. Fragment ions marked with asterisks indicate neutral loss of water. The MS/MS spectrum is from 12 merged spectra. F

DOI: 10.1021/acs.biochem.6b00694 Biochemistry XXXX, XXX, XXX−XXX

Article

Biochemistry

Figure 5. Verification of the disulfide bond pattern of TGFBIp in human cornea. (A) Proteins from 0.4 mg of homogenized human corneal powder were extracted by being boiled in SDS sample buffer for 10 min. The extracted proteins were separated by unreduced SDS−PAGE, and the band containing TGFBIp was excised and subjected to in-gel digestion with trypsin and Glu-C prior to LC−MS/MS analysis. MS/MS spectra of the disulfide-bonded peptides representing the five disulfide bonds in TGFBIp: (B) Cys474−Cys478, (C) Cys74−Cys339, (D) Cys49−Cys85, (D) Cys84−Cys97, and (E) Cys214−Cys317. The top spectra are from TGFBIp extracted from human cornea and the inverted spectra from rTGFBIp. All the inverted spectra are identical with the spectra shown previously (Figure 3) except that of the triple peptide (D). The peptide sequences are shown atop the spectra with the assigned b- and y-ion fragments, and deaminations are indicated with “dea”. Fragment ions marked with asterisks indicate neutral loss of water. Peaks marked with number signs indicate tyrosine immonium ion. Noise was removed from the data by thresholding at 2% (D, corneal TGFBIp), 0.1% (D, rTGFBIp), 28% (E, corneal TGFBIp), or 5% (E, rTGFBIp).

Figure 6. Schematic overview of the disulfide bond pattern of TGFBIp. The identified disulfide bridges were three intradomain disulfide bridges between Cys49 and Cys85 (CRD), Cys84 and Cys97 (CRD), and Cys473 and Cys478 (FAS1-3) and two intradomain disulfide bridges between Cys74 and Cys339 (CRD−FAS1-2) and Cys214 and Cys317 (FAS1-1−FAS1-2). Cys65 was not engaged in any disulfide bridge.

Cys65 Is Modified with a Cysteinylation. We found the disulfide bond pattern of TGFBIp to consist of the following cross-links: Cys49−Cys85, Cys74−Cys339, Cys84−Cys97,

Cys214−Cys317, and Cys473−Cys478 (Figure 6). The only cysteine residue that was not disulfide-bonded to another peptide was Cys65. We were not able to identify Cys65 as an G

DOI: 10.1021/acs.biochem.6b00694 Biochemistry XXXX, XXX, XXX−XXX

Article

Biochemistry

tolerant search in which only TGFBIp is used as a target by the search algorithm. This allows the Mascot search engine to consider all the possible modifications in the Unimod database (http://www.unimod.org). The results showed that Cys65 was cysteinylated in both rTGFBIp and corneal TGFBIp (Figure 7). The Disulfide Bond Pattern Is Not Altered in the rTGFBIp R124C Mutant. Purified rTGFBIp R124C was digested with trypsin and Glu-C, and the peptides were analyzed by LC−MS/MS under nonreducing conditions. An error tolerant Mascot search of the generated MGF file found Cys124 in the mutant to be cysteinylated (Figure 8). All the disulfide bridges identified in WT rTGFBIp were also present in the R124C mutant, and we were not able to identify any disulfide bonds between Cys124 and other TGFBIp peptides. These data indicate that the disulfide bond pattern of the R124C mutant is the same as in WT rTGFBIp.

■

DISCUSSION The structure and function of TGFBIp are largely unknown even though this protein is the second most abundant protein in the human cornea.24 In this study, we have characterized the disulfide bond pattern of TGFBIp to obtain structural information and to identify the cysteine residue that is free to interact with type XII collagen through a reducible bond. We found three intradomain disulfide bonds, one in the FAS1-3 domain and two in the CRD. A disulfide bond links the CRD to the FAS1-2 domain. The FAS1-1 domain and the FAS1-2 domain are also linked through an interdomain disulfide bond. The interdomain disulfide bonds indicate that the NH2 terminus of TGFBIp (CRD, FAS1-1, and FAS1-2) adopts a compact globular fold. We suggest that FAS1-1 and FAS1-2 adopt a FAS1-like folding based on the homology modeling discussed below. Whether the CRD is folded as an individual domain cross-linked to FAS1-2 or is “wrapped” around the structures of the first two FAS1 domains remains to be determined. The identified disulfide bond pattern leaves Cys65 in the CRD free to interact with other molecules such as type XII collagen through a reducible bond.

Figure 7. Assigned MS/MS spectra of cysteinylated Cys65 from authentic TGFBIp and rTGFBIp. The top spectra are from authentic TGFBIp extracted from human cornea and the inverted spectra from rTGFBIp. The peptide sequence is shown atop the spectra with the assigned b- and y-ion fragments, and cysteinylations are indicated. The theoretical and observed precursor masses of the peptide (precursor ion m/z in parentheses) together with the errors are also shown. Fragment ions marked with asterisks indicate neutral loss of water. The peak marked with number signs indicate tyrosine immonium ion. Noise was removed from the corneal TGFBIp data by thresholding at 4%.

iodoacetaminde-alkylated variant without prior reduction, indicating that Cys65 was protected by a reducible modification. To determine the nature of the Cys65 modification, samples of both rTGFBIp and authentic TGFBIp were subjected to an error

Figure 8. Assigned MS/MS spectrum of cysteinylated Cys124 from R124C TGFBIp. The sequence of the peptide is shown with the assigned y- and b-ion fragments. The theoretical and observed precursor masses of the cross-linked peptide (precursor ion m/z in parentheses) together with the error are shown at the top right. Fragment ions marked with asterisks indicate neutral loss of water. The MS/MS spectrum is from four merged spectra. H

DOI: 10.1021/acs.biochem.6b00694 Biochemistry XXXX, XXX, XXX−XXX

Article

Biochemistry

(MMRN1), which lacks the second cysteine residue. We suggest, like others, that the CRD of TGFBIp and Periostin should not be regarded as an EMI domain but rather termed other entities because of a lack of EMI traits.48 In this study, we identified a disulfide bond between the two cysteine residues (Cys473−Cys478) in the FAS1-3 domain. Homology modeling of FAS1-3 to the NMR structure of the FAS1-4 domain (PDB entry 2LTB)30 locates these residues at a distance of 3.6 Å (Figure 10A).32,49 The two cysteine residues in the FAS1-2 domain (Cys317 and Cys339) were on the other hand found to form interdomain disulfide bonds. The homology model confirms that these residues are located farther apart (11.6 Å) and would require some adjustments if they were to form an intradomain disulfide bond (Figure 10B).49 Homology modeling of FAS1-1 and FAS1-2 to the structure of the FAS1-3 and FAS1-4 domain pair from Drosophila fasciclin-1 (PDB entry 1O70)50 shows that the two cysteine residues forming the interdomain cross-link are located in flexible loop structures close to the interface of the two domains (Figure 10C). TGFBIp binds to bovine type VI and human type XII collagens in nuchal ligament and cornea, respectively, through a reducible bond.32,34 In this study, we found that Cys65 in the CRD is the only cysteine residue that is not bound to another TGFBIp peptide. We found that Cys65 was modified with a cysteinylation both in rTGFBIp and in the soluble TGFBIp extracted from human cornea. This modification can, like a disulfide bond, take part in a disulfide bond exchange with a free cysteine residue in another protein and thereby form a new disulfide bridge between TGFBIp and the other protein. We, therefore, suggest that it is Cys65 in the CRD that mediates the reducible bond to type VI and type XII collagens. We did not observe the bond to collagen type XII in the cornea, because we analyzed only the soluble fraction of TGFBIp. Mutations in TGFBI are associated with corneal dystrophies,25−27 and one of the most common mutations is the R124C mutation, which has been shown to cause lattice corneal dystrophy type I (LCDI) in many populations.31 This mutation is located in the NH2 terminus of the FAS1-1 domain just after the CRD. It has been suggested that the substitution of an arginine with a cysteine in this position might increase the propensity for

The CRD is in UniProt assigned as an EMI domain, and the three predicted disulfides are annotated as follows: Cys49− Cys85, Cys65−Cys74, and Cys84−Cys97 (http://www.uniprot. org/uniprot/Q15582). This is based on the “prosite-Prorule:PRU00384” associated with the EMI domain (http://prosite. expasy.org/unirule/PRU00384). The prediction is based on sequence analysis45 but has to the best of our knowledge not been confirmed experimentally. The analogy with the EGF domain has led to the speculation that two EMI subdomains exist.45,46 On this basis, a disulfide bridge is predicted between the fifth and seventh cysteine residues in the COOH-terminal EMI subdomain. This corresponds to the disulfide bridge between Cys84 and Cys97 that we have experimentally verified. Our data also agree with the UniProt Cys49−Cys85 annotation. However, the remaining proposed disulfide bridge between Cys65 and Cys74 did not exist as Cys65 is free or bound to collagen type XII and Cys74 is forming a disulfide with Cys339. A multiple-sequence alignment of the CRD from TGFBIp and its homologue Periostin with the EMI domains of the “emilin domain endowed” (EDEN) superfamily47 revealed that, of the six cysteine residues in the CRD of TGFBIp, only the first and last three cysteine residues aligned with EMI cysteine residues (Figure 9). The four TGFBIp cysteine residues that aligned with the cysteine residues of the EDEN superfamily (Cys49, Cys84, Cys85, and Cys97) were those in which the disulfide bonding was predicted by sequence analysis. However, the two cysteine residues that did not align with the EDEN superfamily (Cys65 and Cys74) did not follow the predicted pattern and either formed an interdomain disulfide bond (Cys74) or were free to interact with other molecules (Cys65). Additional major differences between the CRD and the EMI domains are also revealed by multiple-sequence alignment (Figure 9). The CRDs are composed of only 55 residues, while the EMI domains are from 74 to 79 residues in length. The lacking residues in TGFBIp are located between the first and second cysteine residues. EMI domains contain the highly preserved unique consensus sequence WRCCPG(Y/F)xGGxxC toward the COOH terminus,46,48 and TGFBIp contains only a small part of this sequence (CCPGY). Furthermore, EMI domains contain seven cysteine residues except Multimerin1

Figure 9. Multiple-sequence alignment of CRD and EMI domains. Multiple-sequence alignment of EMI domains was performed with T-Coffee.43 The sequences are derived from TGFBIp, Periostin, and the seven proteins of the EDEN superfamily (boxed sequences). Protein names, accession numbers, and residue numbers (according to Uniprot) are given at the left. Cysteine residues are shown in bold and are numbered (CRD atop and EMI below the alignment). Disulfide bonds identified in this study are indicated with solid lines, and the UniProt annotated disulfide bonds are shown with dashed lines. The suggested NH2- and COOH-terminal subdomains of the EMI domain are indicated with arrows. Residues are colored according to conservation of chemical character: aromatic (F, Y, and W), hydrophobic (L, I, V, and M), acidic (E and D), basic (K, R, and H), polar (S and T), tiny (G and A), and amide (N and Q). Residues are shown in reverse font for 100% conservation, highlighted in dark gray for >70% conservation, and highlighted in light gray for >50% conservation. Underlined residues correspond to the EMI consensus motif WRCCPGaxGxxC, where a is either Y or F. I

DOI: 10.1021/acs.biochem.6b00694 Biochemistry XXXX, XXX, XXX−XXX

Article

Biochemistry

Figure 10. Homology models of the three first FAS1 domains of TGFBIp. The NMR structure of the human FAS1-4 domain (cyan) was used as a template for homology modeling in SWISS-MODEL. The sequences of the FAS1-3 (A, orange), FAS1-2 (B and C, green), and FAS1-1 (C, blue) domains from Uniprot were modeled to the template structure. The levels of sequence identity to the sequence of the FAS1-4 domain are indicated together with the QMEAN of the homology model. The structures were visualized in PyMol, and cysteine residues are shown as sticks. The distances are indicated with black lines. The structures of the FAS1-1 and FAS1-2 domains were individually aligned on the structure of the FAS1-3 and FAS1-4 domain pair from Drosophila fasciclin-1 (magenta), and the root-mean-square values of the alignments are indicated (C).

■

this region to form amyloid deposits.51,52 Another possible consequence of this mutation is a change in the TGFBIp disulfide bond pattern and/or the interaction with the ECM by the introduction of an extra cysteine residue. We did not observe a change in the disulfide bond pattern of the recombinant R124C mutant compared to that of WT rTGFBIp. We suggest that the propensity of the R124C mutant to form amyloid aggregates is not caused by an altered disulfide bond pattern. The extra free cysteine residue might have the ability to alter the protein’s ability to interact with other molecules such as ECM proteins.

■

ACKNOWLEDGMENTS We thank Nadia Sukusu Nielsen for providing the rTGFBIp R124C protein and Dr. Henrik Vorum for providing human corneal tissue.

■

ABBREVIATIONS TGFBIp, transforming growth factor β-induced protein; ECM, extracellular matrix; EMI, emilin; CRD, cysteine-rich domain; FAS1, fasciclin-1; TGFBI, transforming growth factor β-induced protein gene; LC−MS/MS, liquid chromatography and tandem mass spectrometry; rTGFBIp, recombinant transforming growth factor β-induced protein; PEI, polyethylenimine; RT, room temperature; P/S, penicillin/streptomycin; IAA, iodoacetamide; Glu-C, endoproteinase Glu-C; SCX, strong cation exchange; RP-HPLC, reversed-phase HPLC; TFA, trifluoroacetic acid.

AUTHOR INFORMATION

Corresponding Author

*Gustav Wieds Vej 10C, DK-8000 Aarhus C, Denmark. E-mail: [email protected]. Telephone: +45 87155449.

■

Funding

This work was supported by The Danish Council for Independent Research, the Carlsberg Foundation, and the Lundbeck Foundation.

REFERENCES

(1) Skonier, J., Neubauer, M., Madisen, L., Bennett, K., Plowman, G. D., and Purchio, A. F. (1992) cDNA cloning and sequence analysis of beta ig-h3, a novel gene induced in a human adenocarcinoma cell line after treatment with transforming growth factor-beta. DNA Cell Biol. 11, 511−522.

Notes

The authors declare no competing financial interest. J

DOI: 10.1021/acs.biochem.6b00694 Biochemistry XXXX, XXX, XXX−XXX

Article

Biochemistry (2) Schorderet, D. F., Menasche, M., Morand, S., Bonnel, S., Buchillier, V., Marchant, D., Auderset, K., Bonny, C., Abitbol, M., and Munier, F. L. (2000) Genomic characterization and embryonic expression of the mouse Bigh3 (Tgfbi) gene. Biochem. Biophys. Res. Commun. 274, 267− 274. (3) Kitahama, S., Gibson, M. A., Hatzinikolas, G., Hay, S., Kuliwaba, J. L., Evdokiou, A., Atkins, G. J., and Findlay, D. M. (2000) Expression of fibrillins and other microfibril-associated proteins in human bone and osteoblast-like cells. Bone 27, 61−67. (4) Ferguson, J. W., Mikesh, M. F., Wheeler, E. F., and LeBaron, R. G. (2003) Developmental expression patterns of Beta-ig (betaIG-H3) and its function as a cell adhesion protein. Mech. Dev. 120, 851−864. (5) Norris, R. A., Kern, C. B., Wessels, A., Wirrig, E. E., Markwald, R. R., and Mjaatvedt, C. H. (2005) Detection of betaig-H3, a TGFbeta induced gene, during cardiac development and its complementary pattern with periostin. Anat. Embryol. 210, 13−23. (6) Sciandra, F., Morlacchi, S., Allamand, V., De Benedetti, G., Macchia, G., Petrucci, T. C., Bozzi, M., and Brancaccio, A. (2008) First molecular characterization and immunolocalization of keratoepithelin in adult human skeletal muscle. Matrix Biol. 27, 360−370. (7) Takeshita, S., Kikuno, R., Tezuka, K., and Amann, E. (1993) Osteoblast-specific factor 2: cloning of a putative bone adhesion protein with homology with the insect protein fasciclin I. Biochem. J. 294 (Part1), 271−278. (8) Hu, S., Sonnenfeld, M., Stahl, S., and Crews, S. T. (1998) Midline Fasciclin: a Drosophila Fasciclin-I-related membrane protein localized to the CNS midline cells and trachea. J. Neurobiol. 35, 77−93. (9) Doliana, R., Mongiat, M., Bucciotti, F., Giacomello, E., Deutzmann, R., Volpin, D., Bressan, G. M., and Colombatti, A. (1999) EMILIN, a component of the elastic fiber and a new member of the C1q/tumor necrosis factor superfamily of proteins. J. Biol. Chem. 274, 16773−16781. (10) Zinn, K., McAllister, L., and Goodman, C. S. (1988) Sequence analysis and neuronal expression of fasciclin I in grasshopper and Drosophila. Cell 53, 577−587. (11) Kim, J. E., Kim, S. J., Lee, B. H., Park, R. W., Kim, K. S., and Kim, I. S. (2000) Identification of motifs for cell adhesion within the repeated domains of transforming growth factor-beta-induced gene, betaig-h3. J. Biol. Chem. 275, 30907−30915. (12) Kim, J. E., Jeong, H. W., Nam, J. O., Lee, B. H., Choi, J. Y., Park, R. W., Park, J. Y., and Kim, I. S. (2002) Identification of motifs in the fasciclin domains of the transforming growth factor-beta-induced matrix protein betaig-h3 that interact with the alphavbeta5 integrin. J. Biol. Chem. 277, 46159−46165. (13) Nam, J. O., Kim, J. E., Jeong, H. W., Lee, S. J., Lee, B. H., Choi, J. Y., Park, R. W., Park, J. Y., and Kim, I. S. (2003) Identification of the alphavbeta3 integrin-interacting motif of betaig-h3 and its antiangiogenic effect. J. Biol. Chem. 278, 25902−25909. (14) Ohno, S., Noshiro, M., Makihira, S., Kawamoto, T., Shen, M., Yan, W., Kawashima-Ohya, Y., Fujimoto, K., Tanne, K., and Kato, Y. (1999) RGD-CAP ((beta)ig-h3) enhances the spreading of chondrocytes and fibroblasts via integrin alpha(1)beta(1). Biochim. Biophys. Acta, Mol. Cell Res. 1451, 196−205. (15) Bae, J. S., Lee, S. H., Kim, J. E., Choi, J. Y., Park, R. W., Yong Park, J., Park, H. S., Sohn, Y. S., Lee, D. S., Bae Lee, E., and Kim, I. S. (2002) Betaig-h3 supports keratinocyte adhesion, migration, and proliferation through alpha3beta1 integrin. Biochem. Biophys. Res. Commun. 294, 940−948. (16) Kim, M. O., Yun, S. J., Kim, I. S., Sohn, S., and Lee, E. H. (2003) Transforming growth factor-beta-inducible gene-h3 (beta(ig)-h3) promotes cell adhesion of human astrocytoma cells in vitro: implication of alpha6beta4 integrin. Neurosci. Lett. 336, 93−96. (17) Kim, H. J., and Kim, I. S. (2008) Transforming growth factor-betainduced gene product, as a novel ligand of integrin alphaMbeta2, promotes monocytes adhesion, migration and chemotaxis. Int. J. Biochem. Cell Biol. 40, 991−1004. (18) Pierschbacher, M. D., and Ruoslahti, E. (1984) Cell attachment activity of fibronectin can be duplicated by small synthetic fragments of the molecule. Nature 309, 30−33.

(19) D’Souza, S. E., Ginsberg, M. H., and Plow, E. F. (1991) Arginylglycyl-aspartic acid (RGD): a cell adhesion motif. Trends Biochem. Sci. 16, 246−250. (20) Hashimoto, K., Noshiro, M., Ohno, S., Kawamoto, T., Satakeda, H., Akagawa, Y., Nakashima, K., Okimura, A., Ishida, H., Okamoto, T., Pan, H., Shen, M., Yan, W. Q., and Kato, Y. (1997) Characterization of a cartilage-derived 66-kDa protein (RGD-CAP/beta ig-h3) that binds to collagen. Biochim. Biophys. Acta, Mol. Cell Res. 1355, 303−314. (21) Billings, P. C., Whitbeck, J. C., Adams, C. S., Abrams, W. R., Cohen, A. J., Engelsberg, B. N., Howard, P. S., and Rosenbloom, J. (2002) The transforming growth factor-beta-inducibie matrix protein beta ig-h3 interacts with fibronectin. J. Biol. Chem. 277, 28003−28009. (22) Kim, J. E., Park, R. W., Choi, J. Y., Bae, Y. C., Kim, K. S., Joo, C. K., and Kim, I. S. (2002) Molecular properties of wild-type and mutant beta IG-H3 proteins. Invest. Ophthalmo. Visual Sci. 43, 656−661. (23) Reinboth, B., Thomas, J., Hanssen, E., and Gibson, M. A. (2006) beta ig-h3 interacts directly with biglycan and decorin, promotes collagen VI aggregation, and participates in ternary complexing with these macromolecules. J. Biol. Chem. 281, 7816−7824. (24) Dyrlund, T. F., Poulsen, E. T., Scavenius, C., Nikolajsen, C. L., Thogersen, I. B., Vorum, H., and Enghild, J. J. (2012) Human cornea proteome: identification and quantitation of the proteins of the three main layers including epithelium, stroma, and endothelium. J. Proteome Res. 11, 4231−4239. (25) Klintworth, G. K., Valnickova, Z., and Enghild, J. J. (1998) Accumulation of beta ig-h3 gene product in corneas with granular dystrophy. Am. J. Pathol. 152, 743−748. (26) Munier, F. L., Korvatska, E., Djemai, A., Le Paslier, D., Zografos, L., Pescia, G., and Schorderet, D. F. (1997) Kerato-epithelin mutations in four 5q31-linked corneal dystrophies. Nat. Genet. 15, 247−251. (27) Streeten, B. W., Qi, Y., Klintworth, G. K., Eagle, R. C., Jr., Strauss, J. A., and Bennett, K. (1999) Immunolocalization of beta ig-h3 protein in 5q31-linked corneal dystrophies and normal corneas. Arch. Ophthalmol. 117, 67−75. (28) Karring, H., Runager, K., Thogersen, I. B., Klintworth, G. K., Hojrup, P., and Enghild, J. J. (2012) Composition and proteolytic processing of corneal deposits associated with mutations in the TGFBI gene. Exp. Eye Res. 96, 163−170. (29) Runager, K., Basaiawmoit, R. V., Deva, T., Andreasen, M., Valnickova, Z., Sorensen, C. S., Karring, H., Thogersen, I. B., Christiansen, G., Underhaug, J., Kristensen, T., Nielsen, N. C., Klintworth, G. K., Otzen, D. E., and Enghild, J. J. (2011) Human phenotypically distinct TGFBI corneal dystrophies are linked to the stability of the fourth FAS1 domain of TGFBIp. J. Biol. Chem. 286, 4951−4958. (30) Underhaug, J., Koldso, H., Runager, K., Nielsen, J. T., Sorensen, C. S., Kristensen, T., Otzen, D. E., Karring, H., Malmendal, A., Schiott, B., Enghild, J. J., and Nielsen, N. C. (2013) Mutation in transforming growth factor beta induced protein associated with granular corneal dystrophy type 1 reduces the proteolytic susceptibility through local structural stabilization. Biochim. Biophys. Acta, Proteins Proteomics 1834, 2812−2822. (31) Lakshminarayanan, R., Chaurasia, S. S., Anandalakshmi, V., Chai, S. M., Murugan, E., Vithana, E. N., Beuerman, R. W., and Mehta, J. S. (2014) Clinical and genetic aspects of the TGFBI-associated corneal dystrophies. ocular surface 12, 234−251. (32) Runager, K., Klintworth, G. K., Karring, H., and Enghild, J. J. (2013) The insoluble TGFBIp fraction of the cornea is covalently linked via a disulfide bond to type XII collagen. Biochemistry 52, 2821−2827. (33) Andersen, R. B., Karring, H., Møller-Pedersen, T., Valnickova, Z., Thogersen, I. B., Hedegaard, C. J., Kristensen, T., Klintworth, G. K., and Enghild, J. J. (2004) Purification and structural characterization of transforming growth factor beta induced protein (TGFBIp) from porcine and human corneas. Biochemistry 43, 16374−16384. (34) Hanssen, E., Reinboth, B., and Gibson, M. A. (2003) Covalent and non-covalent interactions of betaig-h3 with collagen VI. Beta ig-h3 is covalently attached to the amino-terminal region of collagen VI in tissue microfibrils. J. Biol. Chem. 278, 24334−24341. K

DOI: 10.1021/acs.biochem.6b00694 Biochemistry XXXX, XXX, XXX−XXX

Article

Biochemistry (35) Karring, H., Runager, K., Valnickova, Z., Thogersen, I. B., MøllerPedersen, T., Klintworth, G. K., and Enghild, J. J. (2010) Differential expression and processing of transforming growth factor beta induced protein (TGFBIp) in the normal human cornea during postnatal development and aging. Exp. Eye Res. 90, 57−62. (36) Runager, K., Garcia-Castellanos, R., Valnickova, Z., Kristensen, T., Nielsen, N. C., Klintworth, G. K., Gomis-Ruth, F. X., and Enghild, J. J. (2009) Purification, crystallization and preliminary X-ray diffraction of wild-type and mutant recombinant human transforming growth factor beta-induced protein (TGFBIp). Acta Crystallogr., Sect. F: Struct. Biol. Cryst. Commun. 65, 299−303. (37) Reed, S. E., Staley, E. M., Mayginnes, J. P., Pintel, D. J., and Tullis, G. E. (2006) Transfection of mammalian cells using linear polyethylenimine is a simple and effective means of producing recombinant adeno-associated virus vectors. J. Virol. Methods 138, 85− 98. (38) Sorensen, C. S., Runager, K., Scavenius, C., Jensen, M. M., Nielsen, N. S., Christiansen, G., Petersen, S. V., Karring, H., Sanggaard, K. W., and Enghild, J. J. (2015) Fibril Core of Transforming Growth Factor Beta-Induced Protein (TGFBIp) Facilitates Aggregation of Corneal TGFBIp. Biochemistry 54, 2943−2956. (39) Rappsilber, J., Mann, M., and Ishihama, Y. (2007) Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips. Nat. Protoc. 2, 1896−1906. (40) Petersen, S. V., Due, A. V., Valnickova, Z., Oury, T. D., Crapo, J. D., and Enghild, J. J. (2004) The structure of rabbit extracellular superoxide dismutase differs from the human protein. Biochemistry 43, 14275−14281. (41) Bury, A. F. (1981) Analysis of Protein and Peptide Mixtures Evaluation of 3 Sodium Dodecyl Sulfate-Polyacrylamide Gel-Electrophoresis Buffer Systems. J. Chromatogr. 213, 491−500. (42) Shevchenko, A., Tomas, H., Havlis, J., Olsen, J. V., and Mann, M. (2007) In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nat. Protoc 1, 2856−2860. (43) Magis, C., Taly, J. F., Bussotti, G., Chang, J. M., Di Tommaso, P., Erb, I., Espinosa-Carrasco, J., and Notredame, C. (2014) T-Coffee: Tree-based consistency objective function for alignment evaluation. Methods Mol. Biol. 1079, 117−129. (44) Arnold, K., Bordoli, L., Kopp, J., and Schwede, T. (2006) The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling. Bioinformatics 22, 195−201. (45) Callebaut, I., Mignotte, V., Souchet, M., and Mornon, J. P. (2003) EMI domains are widespread and reveal the probable orthologs of the Caenorhabditis elegans CED-1 protein. Biochem. Biophys. Res. Commun. 300, 619−623. (46) Doliana, R., Bot, S., Bonaldo, P., and Colombatti, A. (2000) EMI, a novel cysteine-rich domain of EMILINs and other extracellular proteins, interacts with the gC1q domains and participates in multimerization. FEBS Lett. 484, 164−168. (47) Braghetta, P., Ferrari, A., De Gemmis, P., Zanetti, M., Volpin, D., Bonaldo, P., and Bressan, G. M. (2004) Overlapping, complementary and site-specific expression pattern of genes of the EMILIN/Multimerin family. Matrix Biol. 22, 549−556. (48) Colombatti, A., Spessotto, P., Doliana, R., Mongiat, M., Bressan, G. M., and Esposito, G. (2012) The EMILIN/Multimerin family. Front. Immunol. 2, 93. (49) Mosher, D. F., Johansson, M. W., Gillis, M. E., and Annis, D. S. (2015) Periostin and TGF-beta-induced protein: Two peas in a pod? Crit. Rev. Biochem. Mol. Biol. 50, 427−439. (50) Clout, N. J., Tisi, D., and Hohenester, E. (2003) Novel fold revealed by the structure of a FAS1 domain pair from the insect cell adhesion molecule fasciclin I. Structure 11, 197−203. (51) Munier, F. L., Frueh, B. E., Othenin-Girard, P., Uffer, S., Cousin, P., Wang, M. X., Heon, E., Black, G. C., Blasi, M. A., Balestrazzi, E., Lorenz, B., Escoto, R., Barraquer, R., Hoeltzenbein, M., Gloor, B., Fossarello, M., Singh, A. D., Arsenijevic, Y., Zografos, L., and Schorderet, D. F. (2002) BIGH3 mutation spectrum in corneal dystrophies. Invest. Ophthalmol. Visual Sci. 43, 949−954.

(52) Schmitt-Bernard, C. F., Chavanieu, A., Derancourt, J., Arnaud, B., Demaille, J. G., Calas, B., and Argiles, A. (2000) In vitro creation of amyloid fibrils from native and Arg124Cys mutated betaIGH3((110− 131)) peptides, and its relevance for lattice corneal amyloid dystrophy type I. Biochem. Biophys. Res. Commun. 273, 649−653.

L

DOI: 10.1021/acs.biochem.6b00694 Biochemistry XXXX, XXX, XXX−XXX