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Differential proteomics of urinary exovesicles from classical galactosemic patients reveals subclinical kidney insufficiency Simon Staubach, Murat Pekmez, and Franz-Georg Hanisch J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.5b00902 • Publication Date (Web): 22 Apr 2016 Downloaded from http://pubs.acs.org on April 23, 2016

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Journal of Proteome Research

Differential proteomics of urinary exovesicles from classical galactosemic patients reveals subclinical kidney insufficiency

Simon Staubach 1, Murat Pekmez 1, Franz-Georg Hanisch 1,2*

1

Institute of Biochemistry II, Medical Faculty, University of Cologne, Köln, Germany;

2

Center for Molecular Medicine Cologne, University of Cologne, Köln, Germany

*Corresponding address: Prof. Dr. Franz-Georg Hanisch, Institute of Biochemistry II, Medical Faculty, University of Cologne, Joseph-Stelzmann-Str. 52, 50931 Köln, Germany; Tel: +49 221 478 4493; Fax: +49 221 478 7788; e-mail: [email protected]

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ABSTRACT: Classical galactosemia is caused by a nearly complete deficiency of galactose1-phosphate uridyltransferase (GALT; EC 2.7.712) resulting in a severely impaired galactose metabolism with galactose-1-phosphate and/or galactitol accumulation. Even on a galactoserestricted diet, patients develop serious long-term complications of the central nervous system and ovaries, which may result from chronic cell toxic effects exerted by endogenous galactose. To address the question whether disease-associated cellular perturbations could affect kidney function of the patients, we performed differential proteomics of detergentresistant membranes from urinary exovesicles. Galactosemic samples (showing drastic shifts from high-mannose to complex-type N-glycosylation on exosomal N-glycoproteins) and healthy, sex-matched controls were analysed in quadruplex iTRAQ experiments performed in biological and technical replicates. Particularly in the female patient group the most striking finding was a drastic increase of abundant serum (glyco)proteins, like albumin, leucine-rich alpha-2-glycoprotein, fetuin, immunoglobulins, prostaglandin H2 D-isomerase, and alpha-1microglobulin protein (AMBP) pointing to a subclinical failure of kidney filter function in galactosemic patients and resulting in heavy overload of exosomal membranes with adsorbed serum (glyco)proteins. Several of these proteins are connected to TBMN/IgAN, proteinuria and renal damage. The impairment of renal protein filtration was also indicated by increased protein contents derived from extracellular matrices and lysosomes.

KEY WORDS classical galactosemia, differential proteomics, urinary exosomes, membrane raft protein, cubilin-megalin-complex, iTRAQ


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INTRODUCTION Classical galactosemia is caused by a mutation-induced deficiency of galactose-1-phosphate uridyltransferase (GALT; EC 2.7.712), resulting in a severely impaired galactose metabolism. In healthy individuals galactose is metabolized in the glycolytic pathway or activated to UDPgalactose, the cosubstrate involved in enzymatic galactosylation of glycoproteins and glycolipids. Newborn infants suffering from severe GALT deficiency develop a potentially lethal hepatotoxic syndrome induced by dietary (exogenous) galactose, which is usually reversible under a galactose-restricted diet. In contrast to rapid recovery from liver disease, long term outcome in classical galactosemia is characterized by complications. Despite strict adherence to the galactose-restricted diet, a great percentage of patients develop long-term syndroms caused by endogenously synthesized galactose. The long-term complications comprising cognitive impairment, speech defects, motor function disturbances and in most female patients hypergonadotropic hypogonadism are related to the CNS and ovary as target tissues. The developmental defects have their origin in prenatal life and show in general only weak progression1,2. Pathomechanisms could be related to excess of galactose-1-phosphate, galactitol or other galactose metabolites, or to defective glycosylation of proteins and lipids leading to direct and /or indirect cell or tissue dysfunction3. An increasing number of reports have demonstrated that GALT deficiency leads to aberrant protein glycosylation4-6. Most reports on aberrant glycosylation in galactosemic patients refer to secreted proteins, in particular to plasma glycoproteins such as transferrin and glycopeptide hormones such as FSH 3,6

. Under the influence of unrestricted exogenous galactose transferrin of galactosemic

patients expresses mostly truncated N-glycans lacking terminal sialic acid and galactose residues4. More importantly, plasma membrane-bound glycoproteins with aberrant 3 ACS Paragon Plus Environment


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glycosylation should have considerable impact on cellular functions, since proper N- and Oglycosylation of glycoproteins is a precondition for proper sorting and trafficking of membrane glycoproteins, in order to reach their definite destination7. In this context we have recently shown that N-linked chains on urinary exosomes from galactosemic patients exhibit shifts in the relative proportions of complex-type to high-mannose-type glycans, the former being considerably increased8. Interstingly, this shift was not seen when analysing the Nglycoprofile of exosome-associated THP as a marker for urothelial membrane glycoproteins. THP is integrated into the plasma membrane via a GPI-anchor and hence represents an integral membrane protein. However, it is shed in large quantities into urine. As the previously described shift from high-mannose-type to complex-type N-glycans on exosomal raft proteins could result from an impairment of the kidney filter function and the massive absorption of complex-type N-glycosylated serum proteins, we performed a differential proteomic study of urinary exosomal rafts. The results reported in this paper reveal unequivocal evidence for a subclinical insufficiency of the kidney filter resulting in heavy overload of exosomal membranes with adsorbed serum/plasma (glyco)proteins. The female patient group showed more pronounced affection of kidney filter function compared to the two male patients included into this study.

MATERIALS AND METHODS Materials Urine samples (min. 200 ml) were obtained from healthy volunteers (samples N1 to N5: N1N4 male, N5 female) and from patients with classical galactosemia (samples G1 to G5: G1 and G2 male, G3-G5 female; approved by the Ethical Commision of the University Clinic of Cologne on November 23, 2010) and kept frozen until use. The galactosemic patients carried the c.A1466G (p.Q188R) mutation in a homozygous fashion and exhibited a very low residual 4 ACS Paragon Plus Environment

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GALT activiy characteristic of classical galactosemia. All galactosemic patients were on a severely galactose-restricted diet.

Preparation of lipid rafts from urinary exovesicles Urine samples were stored at -80°C. After thawing the urinary exovesicles were separated from 200 ml urine by differential centrifugation at 4°C. The urine samples were initially filtered through whatmann filters and then centrifuged at 17.000 x g for 45 min. In the next step the supernatant was ultracentrifuged at 114.000 x g for 90 min. The pelletted exovesicles were suspended and combined in cold PBS. Triton X-100 (10%) was added to a final concentration of 1%. The samples were incubated for 30 min. on a rotator at 4°C to extract lipid rafts from the vesicles and to release unspecific cytosolic content of the vesicles. Finally the lipid rafts were pelleted in 1.5 ml tubes by a final ultracentrifugation at 135.000 x g for 60 min. The mainly exosomal lipid rafts obtained by this centrifugation step were stored frozen at -20°C.

Protein quantitation in the context of i-TRAQ experiments To the stored pelleted samples 100 µl cold PBS was added the samples were sonicated on ice with three strokes at lowest energy level. The amount of protein was quantified using the standard Lowry DC assay. An aliquot equivalent to 50 µg protein was chloroform/methanol precipitated, the pellet air dried for 10 minutes and further processed by filter-aided sample preparation (FASP).

Chloroform / methanol-precipitation of protein Proteins were precipitated by methanol/chloroform precipitation as follows: One volume of sample was mixed with four volumes methanol, one volume of chloroform and 3 volumes of 5 ACS Paragon Plus Environment


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ddH2O. After centrifugation at 16.100 x g for 10 min. the aqueous phase was removed without disturbing the protein-containing interphase. Four volumes of methanol were added, the sample was mixed thoroughly and again centrifuged at 16.100 x g for 10 min. The supernatant was removed and the pellet air dried for 10 min.

Filter assisted sample preparation (FASP) A volume of 20 µl 125 mM Tris HCl pH 6.8, 4% SDS, was added to the air dried sample. To lyse proteins, 2 µl 1 M dithiothreitol were added, the samples were heated to 96°C for 10 min, cooled down to ambient temperature and sonicated 3 x 10 s. After centrifugation at 14.000 rcf for 5 min. at 20°C the supernatants were transferred to Amicon Ultra centrifugal filter units (0.5 ml, 10kD). Filter assisted sample preparation (FASP) with sequential Lys C (40 ng/µl) and trypsin (10 ng/µl) digestion was performed according to Wisniewski et al.9 with minor modifications: Briefly, apart from the initial lysis buffer, 0.1M TrisHCl was replaced by 0.1M triethylammonium bicarbonate (TEABC) pH 8.5 in all buffers used. After digestion the samples were centrifuged and filtrates were collected. 60 µl 10% acetonitril in water were added to the filter, mixed with the residual sample and the units were centrifuged once more. Combined filtrates were applied to C18 Pepclean spin columns (Thermo) activated with 80 % acetonitril and equilibrated to 0.1M TEABC. The absorbed sample was washed two times with 100µl 0.1M TEABC and desalted peptides were eluted with 20 µl 80% acetonitril in 0.1M TEABC.

Experimental setup and i-TRAQ labeling Two independent quadruplex iTRAQ experiments (A and B) were designed as follows: A, samples from galactosemic patients G1 and G2 (both male) were run against two normal 6 ACS Paragon Plus Environment

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controls (N1 and N2, male). In Experiment B samples from patients G3 to G5 (female) were run against a pooled control sample from male and female volunteers (N3+N4+N5). The iTRAQ labeling pattern was in Experiment A: N1 (114), N2 (115), G1 (116), G2 (117), and in Experiment B: N3-5 (114), G3 (115), G4 (116), G5 (117). Sample volumes were reduced to approximately 10 µl in a centrifugal evaporator, 15 µl of 1M TEABC were added and volumes were adjusted to 30 µl with bidest. water. iTRAQ reagents (ABSciex) were dissolved in 70 µl ethanol and mixed with the corresponding samples immediately. After 2h incubation at ambient temperature in the dark, labeled samples were combined and desalted using a PepClean spin column as described above. However, 0.1M TEABC was replaced by 0.1% formic acid in all steps. After salt removal, samples were dried in a centrifugal evaporator and resuspended in 50 µl of 40% acetonitril in 0.1% formic acid. SCX tips were prepared with 10 mg BioBasic 5 µm SCX resin (Thermo) packed into a 200 µl C8 StageTip. Samples were loaded onto a SCX tip equilibrated in 40% acetonitril in 0.1% formic acid. After washing with 2 x 20 µl equilibration buffer, peptides were eluted with 20 µl 0.5M sodium chloride, 40% acetonitril in 0.1% formic acid. Acetonitril was removed by brief vacuum centrifugation and samples volumes were adjusted to 20 µl with 0.1% TFA.

MALDI target spotting Peptides were separated by reversed phase HPLC on an Eksigent nanoLC 1D plus system (Axel Semrau GmbH, Sprockhövel, Germany) using a vented column setup comprising a 0.1mm-by-20-mm trapping column and a 0.075-by-200-mm analytical column, both packed with ReproSil-Pur C18-AQ, 5 µm (Dr. Maisch, Ammerbuch, Germany) and operated at 40°C. 18 µl sample were aspirated into the sample loop and a total of 30 µl was loaded onto the trap column using a flow rate of 6 µl/min. Loading pump buffer was 0.1% TFA. Peptides were eluted with a gradient of 5% to 35% acetonitril in 0.1% TFA over 70min. and a column flow 7 ACS Paragon Plus Environment


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rate of 300 nl/min. 0.7 mg/ml alpha-cyano-4-hydroxycinnamic acid (HCCA) in 95% acetonitril in 0.1% TFA, 1mM ammonium phosphate were fed in using a syringe pump operated at 150 µl/h and a post column T-union. 384 fractions (10 seconds) were deposited onto a MTB AnchorChip 384-800 MALDI target (Bruker Daltonics, Bremen, Germany) using a Probot (Dionex, Idstein, Germany) fraction collector.

MALDI MS/MS analysis MALDI-MS and -MS/MS analysis were carried out on an Ultraflextreme MALDITOF/TOF mass spectrometer (Bruker Daltonics) operated with a laser repetition rate of 1 GHz. The process of data acquisition was controlled by Flexcontrol 3.0 and WarpLC. MALDI MS spectra were acquired over a mass range from 700 – 4.000 Da. Spectra were calibrated externally using the Peptide Calibration Standard II (all Bruker Daltonics) on the designated target calibration spots. The laser was used with a fixed energy setting and 3.000 shots / spectrum were collected from random raster points. Precursor ions with a signal to noise ratio equal or better than 10, were chosen for MS/MS analysis. Identical peaks in adjacent spots were measured only once, generally from the spot with maximum peak intensity. The software was programmed possibly not to take more than 20 MS/MS spectra from one spot. However, this is a “soft” value that was exceeded if no alternative positions were available. Polymer signals and peaks appearing in more than 40 % of all spots were filtered out. MS/MS spectra (3.500 shots) were acquired with the instrument calibration and iTRAQ reporter ions as well as peptide immonium ions were used for internal recalibration.

Database searches MASCOT 2.4 (Matrix Science Ltd, London, UK) was used to search a composite decoy database (1,096,908 sequences; 390,818,894 residues) which was derived from Uniprot KB (downloaded 2015 05.13.) using the Perl script "makeDecoyDB" (Bruker Daltonics, Bremen, 8 ACS Paragon Plus Environment

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Germany). For each genuine entry the script added shuffled sequences and tagged accession numbers, which were used for the calculation of false positive rates in Proteinscape 3.0. Searches were submitted via Proteinscape (Bruker Daltonics, Bremen, Germany) and the following parameter settings: enzyme “trypsin” with 1 missed cleavage; species “human”; fixed modifications “carbamidomethyl”, “iTRAQ4plex (K)”, “iTRAQ4plex (N-term)”; optional modifications “Methionine oxidation”, “iTRAQ4plex (Y)”. The mass tolerance was set to 20 ppm Da for MS and 0.8 Da for MS/MS spectra. Protein lists were compiled in Proteinscape. Peptide hits were accepted when the ion score was equal or better than the mascot identity score, corresponding to a significance of better than p = 0.05 or a confidence of 95 % respectively. On the protein level, the decoy hits were used to keep the false positive rate below 2% 10.

Calculation of iTRAQ ratios After protein identification in Proteinscape the WARPLC™ software module (Bruker Daltonics, Bremen, Germany) was used to extract reporter ion intensities from MS/MS spectra and to calculate corresponding iTRAQ ratios. Briefly, ions in a defined low mass range of MS/MS (m/z 100 – 125) were picked without deisotoping. Within this range iTRAQ reporter ions were detected with a mass tolerance of 0.2 Da. Peptide iTRAQ ratios were calculated from peak heights that were corrected for isotope impurities, using the factors provided by the vendor (Sciex, Darmstadt, Germany). The medians of the peptide ratios were used to calculate corresponding protein ratios.

Statistics and calculation of thresholds Total protein lists containing accessions, scores and iTRAQ ratios were exported to Excel™. Standard Excel™ functions were used for basic statistical analysis and further processing of the protein list. ITRAQ ratios were transformed to log2 values which were normalised by subtraction of the median of the log2 values of 9 (Experiment A) or 11 (Experiment B) selected genuine lipid raft proteins. Standard deviations were calculated from normalised log2 values. Values above or below 2 x standard deviation (2xSD in Experiment A: 1.14, in Experiment B: 1.88) were considered to be significantly changed.



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Validation of differential protein contents in urinary exosomal rafts by Western blot Exosomal lipid rafts were prepared as described above by gradient centrifugation at 114.000 x g and a final washing step. SDS-buffer was added to dissolve the proteins from rafts and the sample was heated to 70°C for 15 min. The samples were centrifuged at 13.000 rpm at 4°C for 5 min. Supernatant was stored at -20°C or subsequently loaded onto SDS-PAGE (4% stacking gel, 5–20% gradient running gel) in a Mini Protean 3 cell (BioRad, Munich, Germany). The gel was equilibrated in transfer buffer (20 M glycine, 24 mM Trizma-base, 20% methanol) before it was wet-blotted (BioRad) onto a nitrocellulose membrane (Schleicher and Schuell, Einbeck, Germany) at 90 mA overnight. Thereafter, the membrane was blocked in TBS containing 5% non-fat dried milk and 0.1% Tween 20 for 1h at room temperature (RT) before incubation with the primary antibody (overnight at 4°C). Immunocomplexes were labeled with HRP-conjugated secondary antibody and detected using a Lumilight Kit (Roche). Between each incubation step, the membrane was washed three times with TBS (20 mM Tris-HCl, 137 mM NaCl, pH 7.6).

Antibodies Primary antibodies: anti-MUC1, C595 IgG (mouse monoclonal IgG), 1.1mg/mL, kindly provided by Dr. M. Price, Cancer Research Laboratory, University of Nottingham, Nottinghamshire, UK; anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH), MAB374, Chemicon (mouse monoclonal IgG); anti-alix, sc-166952, Santa Cruz Biotechnology (mouse monoclonal IgG); anti-LRG1, sc-163029 Santa Cruz Biotechnology (goat polyclonal IgG); anti-human IgG, P0214 Dako (polyclonal rabbit Ig-HRP); anti-CatD, sc-10725, Santa Cruz Biotechnology (mouse monoclonal antibody); anti-CD81, sc-7637 Santa Cruz Biotechnology (mouse monoclonal IgG); anti-Nid1, kindly provided by Dr. Roswita Nischt, University Clinic Cologne, Germany.; anti-LYAG, sc-373745, Santa Cruz Biotechnology (mouse 10 ACS Paragon Plus Environment

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monoclonal antibody); anti-TRFE, sc-365871, Santa Cruz Biotechnology (mouse monoclonal antibody). Secondary antibodies: Rabbit anti-mouse IgG, HRP (P0260/Dako, Hamburg, Germany); swine anti-rabbit IgG, HRP (P0399/Dako).



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RESULTS AND DISCUSSION

Overview of experimental performance and results of differential proteomics Five samples of exosomal lipid rafts from galactosemic patients (biological replicates G1 to G5) and five corresponding controls (N1 to N5) were analyzed by an in vitro label-based technique (iTRAQ). Two independent iTRAQ experiments were performed using the following settings: Experiment A (G1, G2 vs. N1, N2), and Experiment B (G3, G4, G5 vs. N3+N4+N5). Accordingly, in Experiment A two samples from galactosemic patients (male) were run against two individual sex-matched controls, whereas in Experiment B the three samples from female patients were run against a pooled control from male and female volunteers. Prior to analysis the samples were applied on SDS-PAGE (Fig. 1) to check for equalized protein contents. Remarkable was already at the level of coomassie-stained gels that the protein patterns of the galactosemia samples were similar to each other. In the same way the samples of healthy controls showed similar protein patterns, but strikingly there was a profound difference in the proteomes of healthy and galactosemic samples both on the qualitative and quantitative level. We used an offline nano-LC-MALDI-TOF-TOF setting without intermediate prefractionation, since the proteome under analysis was of low to medium complexity. Statistical evaluation of the data sets revealed high validity for both, the protein identification (Mascot scores) as well as the quantification (coefficient of variation). More than 200 proteins were identified in both experiments, among which only a minor fraction was assigned to be genuine exosomal proteins. In Experiment A a total number of 215 proteins was found, of which 152 were identified at the level of Mascot-Scores above 50. A proportion of these proteins was fluctuating in relative abundance in at least one of the two ratios considered (26 proteins, Tab. 1A). Similarly, in Experiment B among a total number of 245 proteins 182 were identified with Mascot scores above 50, of which about one third fluctuated and were found increased in 12 ACS Paragon Plus Environment

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at least one of three ratios (71 proteins, Tab. 1B). Of the total fold changed proteins in both experiments (with respect to at least one ratio) 10 overlapped, which corresponds to about 38.5% (Experiment A) or 14.1% (Experiment B) of the fold-changed proteins. Besides abundant serum proteins, like serum albumin and immunoglobulins, among these proteins overlapping in both experiments were kininogen-1, prostaglandin-H2 D-isomerase, pepsinA3, and proteins S100-A8 and A9, nidogen-1, and small proline-rich protein 3. A major portion of the increased proteins was either of serum origin (serum glycoproteins) or had a functional relationship to the extracellular matrix. A series of genuine exosomal proteins as well as exosome-associated proteins were detected (Tab. S-1 and S-2) in accordance with published data of urinary exosome proteomics11,12,13,14. However, neither genuine exosomal proteins nor proteins related to multivesicular bodies were found among significantly foldchanged proteins, which is in accordance with the mode of log2 ratio normalization (the median of log2 ratios for about ten selected genuine exosomal proteins was subtracted). Other non-genuine exosomal proteins found in both experiments were lysosomal proteins (Tab. 1A,B). Most, if not all lysosomal, extracellular matrix-associated and serum proteins showed consistently increased relative abundancies (Tab. 1A,B). GO Term Enrichment analysis (Cellular Component) at the statistical confidence level of p

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