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Mutation-Induced Deamidation of Corneal DystrophyRelated Transforming Growth Factor #-Induced Protein. Nadia Sukusu Nielsen, Dennis Wilkens Juhl, Ebbe Toftgaard Poulsen, Marie V. Lukassen, Emil Christian Poulsen, Michael W Risør, Carsten Scavenius, and Jan Johannes Enghild Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b00668 • Publication Date (Web): 15 Nov 2017 Downloaded from http://pubs.acs.org on November 17, 2017

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Biochemistry

Mutation-Induced Deamidation of Corneal Dystrophy-Related Transforming Growth Factor β-Induced Protein Nadia Sukusu Nielsen, Dennis Wilkens Juhl, Ebbe Toftgaard Poulsen, Marie V. Lukassen, Emil Christian Poulsen, Michael W. Risør, Carsten Scavenius and Jan J. Enghild* Interdisciplinary Nanoscience Center (iNANO) and Department of Molecular Biology and Genetics, Aarhus University, DK-8000 Aarhus, Denmark KEYWORDS: TGFBIp, deamidation, NMR, LC-MS/MS, FAS1-4, corneal dystrophies

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ABSTRACT: Mutations in the transforming growth factor β-induced protein (TGFBIp) cause phenotypically diverse corneal dystrophies, where protein aggregation in the cornea leads to severe visual impairment. Previous studies have shown a relationship between mutant-specific corneal dystrophy phenotypes and the thermodynamic stability of TGFBIp. Using LC-MS/MS and NMR, we investigated correlations between the structural integrity of disease-related mutants of the fourth FAS1 domain (FAS1-4) and deamidation of TGFBIp residue Asn622. We observed a high rate of Asn622 deamidation in the A546D and A546D/P551Q FAS1-4

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mutants that were both largely unstructured by NMR. Conversely, the more structurally organized A546T and V624M FAS1-4 mutants had reduced deamidation rates suggesting that a folded and stable FAS1-4 domain precludes Asn622 deamidation. Wild-type (WT), R555Q and R555W FAS1-4 mutants displayed very slow deamidation, which agrees with their similar and ordered NMR structures, where Asn622 is in a locked conformation. We confirmed the FAS1-4 mutational effect on deamidation rates in full-length TGFBIp mutants and found a similar ranking compared to the FAS1-4 domain alone. Consequently, the deamidation rate of Asn622 can be used to predict the structural effect of the many destabilizing/stabilizing mutations reported for TGFBIp. In addition, the deamidation of Asn622 may influence the pathophysiology of TGFBIp-induced corneal dystrophies.


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Biochemistry

INTRODUCTION Transforming growth factor β-induced protein (TGFBIp, UniProt entry Q15582) is a 72 kDa extracellular matrix protein encoded by the TGFBIp (TGFBI) gene.1 In addition to being expressed in a wide array of human tissues2-6, TGFBIp is a major constituent of the human cornea and is found throughout its various layers.7, 8 The protein consists of an N-terminal cysteine-rich domain (CRD), four consecutive fasciclin-1 (FAS1) domains, and an integrinbinding motif near its C-terminus.1 Recent analyses of the TGFBIp disulfide bond pattern have revealed disulfide bridges between the first FAS1 domain and both the CRD domain and the second FAS1 domain, whereas the third and fourth FAS1 domain (FAS1-4) appear to be less restrained.9 Mutations in the TGFBI gene cause TGFBIp aggregation within one or more corneal layers leading to visual impairment. Until now, close to 70 mutations in the TGFBI gene have been identified and categorized into five distinct phenotypes of corneal dystrophies.10, 11 The most prevalent TGFBIp-linked corneal dystrophies arise from mutations at residues Arg124 and Arg555 in the first and fourth FAS1 domains, respectively. The remaining dystrophic mutations are primarily located in the FAS1-4 domain.11 The thermodynamic stability of isolated FAS1-4 domain mutants mimics the stability of full-length (FL) TGFBIp mutants exemplified by the stabilizing and destabilizing effects of the R555W and A546T mutations, respectively.12 The differential influence of mutations on the stability of the FAS1-4 domain has been correlated to the mutant-specific phenotypes observed for TGFBIp.12-14 Therefore, further structural characterization of TGFBIp mutants is important for understanding the mechanism(s) of corneal dystrophies. In this study, we investigate the time-dependent deamidation of Asn residues in wild-type (WT) and mutant FAS1-4 domains of TGFBIp using mass spectrometry. We correlate the mutant-specific deamidation rates with the structural integrity of the FAS1-4 domain using


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NMR spectroscopy and show that the deamidation pattern of full-length TGFBIp mutants is similar to the isolated FAS1-4 domains. Our biochemical characterization of FAS1-4 deamidation was inspired by the appearance of a slower migrating FAS1-4 band over time that contained a clear Asn622 deamidation. We discuss the applicability of deamidation as a proxy for compromised structural integrity in disease mutants of TGFBIp.

EXPERIMENTAL PROCEDURES Materials. Unless otherwise stated, chemicals were from Sigma-Aldrich Co. Cloning, Expression, and Purification of FAS1-4 and FL TGFBIp. WT and mutant FAS1-4 domains encoding amino acid residues 502–657 of human TGFBIp plus two additional N-terminal amino acid residues (denoted A500' and G501') were cloned, expressed and purified as previously described.12 The N622D mutant was constructed from the WT FAS1-4 clone using the following primers: 5’-CATCATGGCCACAGATGGCGTGGTCCATGTCATCAC 3-’ (forward primer) and 5’-GGACCACGCCATCTGTGGCCATGATGTCAGGCTC-3’ (reverse primer) to introduce the point mutation in a QuikChange reaction (Agilent Technologies). The mutation was confirmed by sequencing both strands of the construct and LC-MS/MS analysis of the protein product. For NMR, expression of the FAS1-4 domains was done in M9 minimal medium containing 1 g/L

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NH4Cl to obtain uniformly

15

N-labeled

protein. WT and mutant FL TGFBIp were cloned, expressed and purified as previously described.9 Expression of R555Q FL TGFBIp was unsuccessful. Time-Course Deamidation of FAS1-4 and FL TGFBIp. Triplicate samples of the FAS14 domains (5.9 µM) and FL TGFBIp variants (0.4 µM) were incubated at 37 °C in PBS [20 mM sodium phosphate, 137 mM NaCl (pH 7.4)] including proteinase inhibitors (cOmplete Proteinase inhibitor cocktail – EDTA-free, Roche Diagnostics, Basel Switzerland), NaN3 (0.02%) and EDTA (5 mM). Aliquots of each sample, taken at different time points for up to a


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Biochemistry

final incubation time of 14 days, were analyzed by LC-MS/MS and SDS-PAGE (FAS1-4 domains only). Sets of identical experiments for the FAS1-4 domains were carried out at unfolding conditions in 6 M guanidine hydrochloride (GdnHCl). For these experiments, the samples were desalted prior to SDS-PAGE analyses using POROS 50 R2 RP column material (Applied Biosystems) packed in GELoader Tips (Eppendorf).15 SDS-PAGE. Samples were boiled for 5 min in SDS (1%) and 35 mM dithiothreitol (DTT). SDS-PAGE was performed using 12% (w/v) acrylamide gels and the discontinuous ammediol/glycine buffer system.16 Gels were stained with Coomassie Brilliant Blue. Densitometric analysis of the gel bands for each time point was performed using ImageJ (v. 1.50i) 17. The deamidation ratio was calculated as the intensity of the deamidated upper band divided by the combined intensity of the deamidated and non-deamidated band. Sample Preparation and LC-MS/MS Analysis. The triplicate incubated samples of WT and mutant FL TGFBIp were subjected to reducing and denaturing conditions (boiling in 5 mM DTT for 10 min) before any reactive groups were alkylated in 15 mM iodoacetamide for 2 min. The samples were lyophilized, suspended in 40 µl 100 mM acetic acid, 50 mM NaCl (pH 2.8) and digested with 1:30 w/w porcine pepsin A for 16 h at 37 °C. Triplicate samples of FAS1-4 domains (not containing cysteine residues) were digested similarly without any prior reduction and alkylation. Gel bands at 0 and 14 days of incubation for A546D/P551Q FAS1-4 were excised and digested as described by Shevchenko et al.18 with the following modifications; pepsin was used instead of trypsin, and 100 mM acetic acid, 50 mM NaCl (pH 2.8) instead of ammonium bicarbonate. All samples were desalted using Empore™ SPE Disks of C18 octadecyl packed in 10 µl pipette tips. Samples were analyzed on an EASY-nLC II system (Thermo Fisher Scientific) connected to a TripleTOF 5600+ mass spectrometer (AB SCIEX) operated under Analyst TF 1.6.0 control and equipped with a NanoSpray III source (AB SCIEX).


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The pepsin-digested samples were dissolved in 0.1% formic acid, injected and trapped on an in-house packed trap column [2 cm × 100 μm (inner diameter)] using RP ReproSil-Pur C18AQ 3 μm resin (Dr. Maisch GmbH). Peptides were eluted from the trap column and separated on a 15 cm analytical column (75 μm i.d.) produced in-house with RP ReproSil-Pur C18-AQ 3 μm resin (Dr. Maisch GmbH) and electrosprayed directly into the mass spectrometer. Peptides were eluted using a 20 min gradient from 5% to 35% phase B (0.1% formic acid in acetonitrile) with a flow rate of 250 nL/min. The acquisition method used for the area based extracted ion chromatogram (XIC) quantification was set up as an information-dependent acquisition experiment collecting up to 25 MS/MS spectra in each 1.6 s cycle using an exclusion window of 6 s. The collected MS files were converted to mascot generic format (MGF) using the AB SCIEX MS Data Converter beta 1.1 and the “proteinpilot MGF” parameters. Data Handling. The generated MGF files were searched in an in-house database (306 sequences) containing WT and mutant TGFBIp protein sequences using Mascot 2.5.1 (Matrix Science, London, UK) with the following search parameters: MS tolerance of 10 ppm, MS/MS tolerance of 0.2 Da, ESI-QUAD-TOF as the instrument, no enzyme specified and deamidation of Asn and Gln as variable modifications. Propionamide was selected as a fixed modification of cysteine residues for in-gel digest samples whereas carbamidomethyl was selected as a fixed modification of cysteine residues of in-solution digest samples. All data were imported with an ion score cut-off of 30 and a significant threshold of 0.01 (p A546D >> A546T > V624M. In support of the correlation between increased deamidation rates of Asn622 and decreased structural integrity, we found identical deamidation rates for all FAS1-4 variants under denaturing conditions (Figure 6).

FIGURE 6. Denaturation overrules mutant-specific Asn622 deamidation. FAS1-4 WT and mutants incubated at unfolding conditions (6M GdnHCl) for up to 14 days followed by desalting and SDS-PAGE analysis show similar rates of deamidation judged by the upper band build-up. Deamidation of FL TGFBIp Mutants Resembles the Isolated FAS1-4 Domain. We measured the time-resolved deamidation in FL TGFBIp variants to probe the structural integrity of the FAS1-4 domain in the context of the full-length protein. We determined the extent of deamidation by LC-MS/MS on recombinantly expressed WT FL TGFBIp, R555W,


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A546T, A546D, A546D/P551Q, and V624M FL TGFBIp mutants incubated for up to 14 days, (Figure 7). Despite an overall lower rate of deamidation in FL TGFBIp compared to FAS1-4, a similar mutation dependency was observed. The A546D and A546D/P551Q mutants showed the highest degree of deamidation with half-times determined to 56 ± 8 days and 94 ± 28 days, respectively (table 1). The evolutions for the other mutants could not be appropriately fitted, but the end-point values indicate a significantly higher deamidation rate for the V624M mutant compared to the A546T and R555W FL TGFBIp mutants. WT FL TGFBIp was not deamidated suggesting full stability of the FAS1-4 domain in the native protein.

FIGURE 7. Destabilized mutants of FL TGFBIp show higher rates of Asn622 deamidation. Time-incubated FL TGFBIp WT and mutants were analyzed for relative Asn622 deamidation by LC-MS/MS. The data for A546D and A546D/P551Q was fitted to eq. 1, while fitting of the data for the rest of the FL TGFBIp variants was unsuccessful.


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Biochemistry

DISCUSSION Mutations in the FAS1-4 domain of TGFBIp are strongly associated with the pathology of TGFBIp-related corneal dystrophies. Therefore, biochemical and biophysical differences between mutants of FAS1-4 can be important in understanding how the TGFBIp mutants cause disease. The mutants included in this study have all been identified in corneal dystrophies with R555W being associated with granular corneal dystrophy type 1, R555Q with Thiel-Behnke corneal dystrophy and the remaining four mutants being related to different variants of lattice corneal dystrophy (LCD). A correlation between genotype, phenotype and previously reported stability of these FAS1-4 mutants are depicted in table 2.

Table 2. Correlation between corneal dystrophy genotype, phenotype and stability of TGFBIp Stability1 compared to WT protein

Age of onset (decade)

Penetrance

Ref.

+

Decreased

4.-5.

Anterior stroma

(26)

LCD I

+

Decreased

3.-4.

Mid-stroma

(26)

A546T

LCD IIIA

+ +

+

Decreased Decreased

3.-4.

Mid-stroma

(12) (27)

R555Q

TBCD

+

+

Unchanged

1.-2.

Bowman’s layer

(12)

R555W

GCD1

+ +

+

Increased Increased Decreased Increased Increased

1.-2.

Anterior stroma

(12) (14) (28) (29) (30)

Genotype

Phenotype

FAS1-4

A546D

Atypical LCD

A546D/ P551Q

TGFBIp

+ + + 1

The stability has been determined by different methods including urea unfolding, thermal

unfolding, far and near UV CD and limited proteolysis.

We conclude that several FAS1-4 mutations render the domain prone to Asn622 deamidation that causes a migration change by SDS-PAGE. In the main reaction pathway of Asn


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deamidation, the first step includes the formation of a succinimide intermediate by a nucleophilic attack from the amide nitrogen of the succeeding residue. Asparagine residues succeeded by glycine like Asn622 are more prone to this nucleophilic attack31, 32, explaining the elevated deamidation rates at this particular site compared to other asparagine residues in the sequence. Hydrolysis of the succinimide intermediate leads to the conversion of Asn to either aspartate or isoaspartate. Consequently, an additional negative charge is introduced that might cause a migrational change by affecting the stoichiometry of protein-SDS complexes.25 In the amino acid sequence Asn622 is located between β-sheet β6 (residues 611-619) and β7 (residues 624-628) and the NMR WT FAS1-4 structure places the Asn622 side chain in a hydrophobic pocket constituted by Val505 (helix αL) and Leu531 (helix α2) (figure 8). We hypothesize that the decreased structural integrity caused by specific mutations leads to FAS14 domain unfolding and backbone flexibility that exposes Asn622 to the nucleophilic attack leading to deamidation. However, it is also possible that deamidation of Asn622 in turn destabilize the entire domain structure because of its location at the interface between the helix and β-sheet elements of the structure.


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Biochemistry

FIGURE 8. Localization of Asn622 and FAS1-4 mutation sites in the sequence and structure of WT FAS1-4. (A) The deamidation site Asn622 (Cyan) is highlighted in the sequence of FAS1-4 along with the investigated FAS1-4 mutation sites (red). Secondary structural elements are underlined and named according to Underhaug et al, 2013. (B) Depiction of the mutation sites Ala546, Pro551 and Arg555 in the NMR structure of the WT FAS1-4 domain. (C) Depiction of the mutation site Val624 and the deamidation site Asn622. The V624M mutation is located in the β-sheet rich part (β7) of the FAS1-4 sequence unlike the other mutations included here and is near the deamidation site. Chemical shift differences introduced by the V624M mutation were most severe in the two sheets β1 and β6 located on each side of β7 and for Leu578 positioned right above the mutation site in the FAS1-4 WT


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structure. T2 values were decreased for most residues in the V624M FAS1-4 mutant (Figure 5D), indicating a slower tumbling induced by a less compact structure of the protein. A less compact structure is likely caused by increased flexibility locally or globally, which may explain the increased deamidation rates. In FAS1-4 WT Ala546 is located in the α3 helix (residues 544-549) with the side chain placed in a hydrophobic pocket constituted by Pro542 (β1) and Val631. The A546D mutation introduces a charged residue within the interior of the protein and causes complete unfolding of the protein. Both mutants containing the A546D mutations showed highly accelerated deamidation half-times of 3 days which is close to the 1 day deamidation half-time determined for a non-structured pentapeptide containing a similar deamidation site Thr-Asn-Gly.33 The A546T mutation introduces a polar residue within the hydrophobic pocket of FAS1-4, which seems to destabilize the protein without causing complete denaturation. Residues in or in close proximity to the hydrophobic pocket showed the biggest changes in chemical shifts although changes were detected throughout the sequence. Small structural changes in the α3 helix could propagate to the adjacent β1 sheet (538-542) and thereby alter the β-sheet rich part of the protein where the deamidation site is located. The apparent changes in structure did not have any significant impact on the overall size of the protein, as the relaxation properties are similar to the WT for most residues (Figure 5C). Arg555 is located in helix α3´ (residues 555-558) with the side chain exposed to the solvent. The previously determined structure of the R555W FAS1-4 mutant reveals how the mutation induces a slight rotation of the helix such that the Trp side chain points towards the protein interior where it stabilizes a slightly more compact structure. Similar but less substantial structural effects can be assigned to the R555Q mutation. The R555 mutations did not directly influence the deamidation site, and no significant differences in Asn622 deamidation rates were observed when compared to FAS1-4 WT.


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Biochemistry

The Asn622 deamidation rate was significantly slower in FL TGFBIp when compared to the FAS1-4 domain alone. This could be explained by a stabilization of the mutant FAS1-4 domain when placed in contact with the other domains of TGFBIp. However, the ranking based on deamidation rates was similar for corresponding FAS1-4 and TGFBIp mutants. Deamidation rates provide three-dimensional structure information about the Asn environment33, 34, and we propose that the Asn622 deamidation serves as a measure of structural stability differences between mutants of TGFBIp, both in the isolated FAS1-4 domain and the full-length protein involved in disease. However, we cannot rule out that the structural stability changes are amplified by the deamidation that could destabilize the protein further as reported for some of the crystallins.35-37 Deamidation has been shown to impact protein aggregation by several proteins involved in human disease, including amyloid-β from Alzheimer’s38-41 and the crystallins from cataract.4244

Deamidation also increases the fibrillation rate of several amyloidogenic peptides.45, 46 Any

non-enzymatic modifications like Met oxidation or Asn and Gln deamidation of corneal TGFBIp have been assigned to results of sample handling before analysis.47 However, as presented here, mutations in TGFBIp can increase deamidation rates and introduce a modification to Asn622, which potentially could affect the fibrillation of TGFBIp in the cornea. Proteomic studies of amyloid deposits from patients suffering from corneal dystrophy have revealed specific regions of TGFBIp to accumulate in the cornea.13, 26, 48, 49 Recently, the 611642 polypeptide region containing Asn622 was shown to accumulate in amyloid deposits of LCD patients and this fragment also had the ability to form amyloid fibrils in vitro.50 The importance of changes in the properties of the Asn at position 622 is underlined by the mutations N622H and N622K that both lead to LCD.51, 52 One of the hypotheses for the development of corneal dystrophies links altered TGFBIp turnover to mutations in the protein, which may be contributing to the release of amyloidogenic


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peptides responsible for amyloid formation in the cornea.53 Because deamidation can work as a molecular clock for biological processes like protein turnover, development, and aging31, 33, 54-58

, a theory first coined by Drs. Art and Noah Robinson, we speculate that the increased

deamidation of mutant TGFBIp may contribute to the hypothesized disease-causing alteration of TGFBIp turnover. In conclusion, our study highlights a new connection between Asn622 deamidation and disease-causing mutations in the FAS1-4 TGFBIp domain that may help decipher the pathophysiology of TGFBIp-induced corneal dystrophies.

AUTHOR INFORMATION Corresponding Author *Gustav Wieds Vej 10C, DK-8000 Aarhus C, Denmark. E-mail: [email protected]. Telephone: +45 2338 2262. Funding This work was supported by The Danish Council for Independent Research − Medical Sciences (DFF-4004-00471), The Lundbeck Foundation (R164-2013-15912), VELUX FOUNDATION and Fight for Sight, Denmark. Notes The authors declare no competing financial interest.


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Biochemistry

ABBREVIATIONS TGFBIp, transforming growth factor β-induced protein; TGFBI, transforming growth factor β-induced gene; CRD, cysteine-rich domain; FAS1, fasciclin-1; FAS1-4, fourth FAS1 domain; FL, full-length; WT, wild-type; LC-MS/MS, liquid chromatography and tandem mass spectrometry; GdnHCl, guanidine hydrochloride; DTT, dithiothreitol; XIC, extracted ion chromatogram; DSS, 2,2-dimethyl-2-silapentane-5-sulfonate; HSQC, Heteronuclear single quantum coherence; LCD, lattice corneal dystrophy.


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