SALIVARY CYSTATINS: EXPLORING NEW POST-TRANSLATIONAL

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SALIVARY CYSTATINS: EXPLORING NEW POSTTRANSLATIONAL MODIFICATIONS AND POLYMORPHISMS BY TOP-DOWN HIGH RESOLUTION MASS SPECTROMETRY Barbara Manconi, Barbara Liori, Tiziana Cabras, Federica Vincenzoni, Federica Iavarone, Massimo Castagnola, Irene Messana, and Alessandra Olianas J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.7b00567 • Publication Date (Web): 11 Oct 2017 Downloaded from http://pubs.acs.org on October 17, 2017

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Journal of Proteome Research

SALIVARY CYSTATINS: EXPLORING NEW POST-TRANSLATIONAL MODIFICATIONS AND POLYMORPHISMS BY TOP-DOWN HIGH RESOLUTION MASS SPECTROMETRY Barbara Manconia§*, Barbara Lioria§, Tiziana Cabrasa, Federica Vincenzonib, Federica Iavaroneb, Massimo Castagnolab,c, Irene Messanab,c, Alessandra Olianasa a

Department of Life and Environmental Sciences, Biomedical Section, University of Cagliari,

Monserrato Campus 09042, Monserrato, CA, Italy. bBiochemistry and Clinical Biochemistry Institute, Medicine Faculty, Catholic University of Rome, and cInstitute of Chemistry of the Molecular Recognition CNR, L.go F. Vito 1, 00168 Rome, Italy.

KEYWORDS Cystatin A, Cystatin B, Cystatin D, S-type Cystatins, human saliva, post-translational modifications, top-down high-resolution mass spectrometry.

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ABSTRACT

Cystatins are a complex family of cysteine peptidase inhibitors. In the present study, various proteoforms of cystatin A, cystatin B, cystatin S, cystatin SN, and cystatin SA were detected in the acidic soluble fraction of human saliva and characterized by a top-down HPLC-ESI-MS approach. Proteoforms of cystatin D were also detected and characterized by an integrated topdown and bottom-up strategy. The proteoforms derive from coding sequence polymorphisms and post-translational modifications, particularly phosphorylation, N-terminal processing and oxidation. This study increases the current knowledge on salivary cystatin proteoforms and provides the basis to evaluate possible qualitative/quantitative variations of these proteoforms in different pathological states and reveal new potential salivary biomarkers of disease.

Data are available via ProteomeXchange with identifier PXD007170. INTRODUCTION Cystatins are a superfamily of evolutionary related proteins whose main function in saliva is to provide protection of the oral cavity by inhibiting cysteine proteases.1 The mammalian superfamily includes type 1 cystatins (cystatins A and B); type 2 cystatins (C, D, E, F, S, SN, SA); type 3 cystatins, or kininogens. Human cystatins A (also named stefin A or acid cysteine protease inhibitor or epidermal SHprotease inhibitor) and B (also named stefin B or CPI-B or liver thiol proteinase inhibitor) are inhibitors of cathepsin L, cathepsin S and cathepsin H.2 They are both single chain proteins with 98 amino acid residues exhibiting 54% sequence identity, a molecular mass of about 11 kDa, lack of signal peptide, disulfide bonds and phosphorylations, and are generally expressed


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intracellularly.2 Recently, our group was able to establish that cystatin B in saliva is mainly present as S-modified derivatives, namely (Cys3) S-glutathionylated, S-cysteinylated and S-S 2mer.3 Cystatin A is expressed in the epidermis,4 oral mucosa,5 lymphocytes, neutrophils,6 lymphoid tissue,7 Hassall's corpuscles and in medullary cells from thymus.8 Cystatin B is a multifunctional protein widely expressed in different cells and tissues.9-11 Besides its well-known inhibitory role against cysteine proteinases, additional and/or alternative functions are being discovered for cystatin B, such as lysosome-associated function connected to the molecular pathogenesis of progressive myoclonus epilepsy of the Unverricht-Lundborg type,12 prevention of apoptosis,13,14 reduction of oxidative stress15 and neuroprotective role by a chaperone-like activity binding with Aβ.16 Type 2 cystatins comprise five major S-type (S, S1, S2, SA, SN) and two minor (C and D). They are composed of about 115-120 amino acid residues with a molecular mass ranging from 13.5 to 14.5 kDa and present signal peptide and disulfide bridges.17 Cystatins S may be either monophosphorylated on Ser3 (cystatin S1) or diphosphorylated on Ser1 and Ser3 (cystatin S2).18 Even though S-type cystatins exhibit a very high sequence identity (about 88%), cystatins SN and SA display greater inhibition toward papain-like cysteine proteinases with respect to cystatin S.1 Recently has been shown that the salivary levels of human S-type cystatins show an agerelated variation during the development.19 Human cystatin C consists of a single polypeptide chain of 120 amino acid residues containing four conserved cysteine residues, which can form two disulﬁde bonds.2,20 This protein has a


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broad distribution being found in most bodily fluid,21and under pathological conditions it may form amyloid deposit in brain arteries of young adults, leading to cerebral hemorrhage.22 Human cystatin D consists of a single polypeptide chain of 122 amino acid residues found in two natural forms with Cys/Arg at position 26 due to gene polymorphism.23 Cystatin D exhibits antiproteinase activity against cathepsin S, H and L but, unlike the other cystatins, not for cathepsin B.23,24 Unexpectedly, cystatin D is a multifunctional protein and shows several activities not related to the antiproteinase activity, such as inhibition of proliferation, migration and invasion of colon carcinoma cells,25 regulation of antigen presenting cells activity26 and modulation of gene expression related to its nuclear activity localization.27 It displays a narrow tissue distribution with respect to other cystatins because it has been originally found only in saliva23 and only recently in colon cancer cells.27 Moreover, cystatin D may undergo internalization into antigen-presenting cells originated from parotid glands, which are responsible for secretion of cystatin D in other body fluids than saliva.26 The broad spectrum of functions displayed by cystatins explains the reason why variation in their level and structure may affect human health in numerous ways, and indeed they are recently being considered as possible biomarkers in various pathologies not only confined to the oral cavity.21,28,29 In particular, the elucidation of new post-translational modifications, and natural variants of salivary cystatins could offer information useful to evidence possible diagnostic and prognostic markers in different oral and systemic diseases. The two main proteomic approaches used to characterize proteins are the so called “bottom-up” and “top-down” methodologies: topdown proteomics explores intact naturally-occurring proteome including proteoforms deriving from post-translational modifications, mutations and alternative splicing; bottom-up proteomics


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refers to the characterization of the proteome by the analysis of the peptides released from the proteins by proteolysis. During a large survey of the acidic soluble fraction of adult human saliva carried out with an integrated top-down/bottom-up HPLC-ESI-MS/MS platform30, we were able to characterize several naturally occurring proteoforms belonging to members of the cystatin family deriving from polymorphisms of the coding sequence and different post-translational modifications. EXPERIMENTAL SECTION Reagents Chemicals and reagents, all of LC–MS grade, were purchased from Merck (Darmstadt, Germany), Waters Corporation (MA, USA), Thermo Fischer Scientific (IL, USA), Bio-Rad (Hercules, CA, USA), GE Healthcare (LC, United Kingdom), Santa Cruz Biotechnology (TX, USA) and Sigma Aldrich (St. Louis, MO, USA). Ethics Statements and Subjects under Study The study protocol and written consent form were approved by the Ethical Committee of the University Hospital of Cagliari, and has therefore been performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki. All rules were respected and written consent forms were obtained by the donors. Unstimulated whole saliva (WS) was collected from 54 adult healthy donors (40 ± 10 years old, males n = 23, females n = 31), recruited from the general population. Sample Collection


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Unstimulated WS was collected according to a standardized protocol optimized to preserve saliva proteins from proteolytic degradation. Donors did not eat or drink at least 2h before the collection, which was performed in the morning between 10:00 A.M. and 12:00 A.M. with a soft plastic aspirator. Saliva was transferred into a plastic tube in ice bath, and 0.2% 2,2,2trifluoroacetic acid (TFA) was immediately added in 1:1 v/v ratio. The solution was then centrifuged at 10000 x g for 10 min at 4 °C. The acidic supernatant was separated from the precipitate, and either immediately analyzed by HPLC-ESI-MS (33 µl, corresponding to 16.5 µl of saliva) or stored at -80° C until the analysis. HPLC Low-resolution ESI-IT-MS Experiments Search of peptides and proteins was made by reversed phase (RP)-HPLC low-resolution ESI-MS analysis of the acidic soluble fraction of WS samples. The measurements were carried out by a Surveyor HPLC system connected to an LCQ Advantage mass spectrometer (Thermo Fisher Scientific, San Jose, CA) equipped with an ESI source. The chromatographic column was a Vydac (Hesperia, CA) C8 column with 5 µm particle diameter (150 x 2.1 mm). The following eluents were used: (eluent A) 0.056% (v/v) aqueous TFA, and (eluent B) 0.05% (v/v) TFA in acetonitrile-water 80/20. The gradient applied was linear from 0 to 55% of B in 40 min, and from 55% to 100% of B in 10 min, at a flow rate of 0.10 ml/min toward the ESI source. During the first 5 min of separation, eluate was diverted to waste to avoid instrument damage because of the high salt concentration. Mass spectra were collected every 3 ms in the m/z range 300-2000 in positive ion mode. The MS spray voltage was 5.0 kV, and the capillary temperature was 260 °C. MS resolution was 6000. Deconvolution of averaged ESI-MS spectra was performed by MagTran 1.0 software.31 Average experimental mass value (Mav) were compared with average


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theoretical values using PeptideMass program available on the Swiss-Prot data bank (http://us.expasy.org/tools). Characterization of salivary cystatins The new proteoforms of cystatins were characterized by a top-down proteomic approach either in the acidic supernatant of WS or in enriched fractions obtained by preparative RP-HPLC. A bottom-up approach was also applied to characterize cystatin D proteoforms. Enriched fraction preparation and bottom-up proteomic experiments Enriched fractions were obtained by preparative RP-HPLC (Dionex Ultimate 3000 instrument, Thermo Fisher Scientific, Sunnyvale, CA) of selected saliva samples. The chromatographic column was a reversed phase Vydac (Hesperia, CA) C8 column with 5 µm particle diameter (250 x 10 mm). The eluents for preparative RP-HPLC were the same utilized for analytical HPLC low-resolution ESI-MS experiments. The step gradient was from 0 to 50% B in 30 minutes, from 50 to 65% B in 20 minutes, and from 65 to 100% B in 1 minute with a flow rate of 2.8 ml/min. Fractions corresponding to peaks eluting between 29 and 37 minutes were collected separately and lyophilized. Each fraction was solubilized in 100 µl of ultrapure H2O, and 1/3 of the solution was acidified with 0.2% TFA (1:1 v/v ratio) to be checked by HPLC low-resolution ESI-MS. The remaining sample was lyophilized for HPLC high-resolution-ESI-MS/MS experiments. For the structural characterization of cystatin D proteoforms, an aliquot of the lyophilized sample was submitted to reduction of disulfide bonds at 100 °C for 5 min followed by an incubation at 50 °C for 15 min in 100 mM ammonium bicarbonate buffer pH 8.0 containing 10 mM dithiotreitol (DTT) in a final volume of 35 µl. The reaction sample was alkylated in the dark at 30 °C for 45 min using 55 mM iodoacetamide (IAM) and subsequently


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submitted to enzyme digestion. The trypsin digestion was performed by the kit “Trypsin Singles Proteomic Grade” (Sigma-Aldrich) according to the manufacturer’s instructions. Digestion was stopped after 8 h by acidification with 0.1% formic acid (FA), the sample was lyophilized, and stored at -80° C until the analysis by HPLC high-resolution ESI-MS/MS. HPLC High-resolution ESI-MS/MS experiments The experiments were carried out by an Ultimate 3000 RSLC Nano System HPLC apparatus (Thermo Fisher Scientific, Sunnyvale, CA)coupled to a LTQ Orbitrap Elite apparatus (Thermo Fisher Scientific). The columns were a Zorbax 300SB-C8 column (3.5 µm particle diameter; 1.0 x 150 mm) for the top-down analysis, and a Zorbax 300SB-C18 column(3.5 µm particle diameter; 1.0 x 150 mm) for the bottom-up. Eluents were: (eluent A) 0.1% (v/v) aqueous FA and (eluent B) 0.1% (v/v) FA in acetonitrile-water 80/20. For both top-down and bottom-up analyses the gradient was: 0-2 min 5% B, 2-40 min from 5% to 55% B (linear), 40-45 min from 70% to 99% B, at a flow rate of 50 µL/min. MS and MS/MS spectra were collected in positive mode with the resolution of 60000. The acquisition range was from 350 to 2000 m/z for the top-down and the bottom-up experiments. Tuning parameters: capillary temperature was 300 °C, and the source voltage 4.0 kV, S-Lens RF level 60% in both experiments. In data-dependent acquisition mode the five most abundant ions were selected and fragmented by using collision-induced dissociation (CID) or higher energy collision dissociation (HCD), with 35% normalized collision energy for 30 ms, isolation width of 5 m/z, activation q of 0.25. The inject volume was 20 µL. HPLC-ESI-MS and MS/MS data were generated by Xcalibur2.2 SP1.48 (Thermo Fisher Scientific) using default parameters of the Xtract program for the deconvolution. In top-down experiments protein sequences and sites of covalent modifications were validated by manual inspection of the experimental fragmentation spectra against the theoretical ones generated by


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MS-Product software available at the Protein Prospector website (http://prospector.ucsf.edu/prospector/mshome.htm). In bottom-up experiments, MS/MS data were analyzed by the Proteome Discoverer 1.4 program, based on SEQUEST HT cluster as a search engine (University of Washington, licensed to Thermo Electron Corporation, San Jose, CA) against the Swiss-Prot Homo Sapiens proteome (UniProtKB, Swiss-Prot, release 2017_02). The settings were: trypsin enzyme with a maximum of two missed cleavage sites, precursor mass search tolerance was 10 ppm and fragment mass tolerance 0.5 Da. Target FDR was: 0.01 (strict), 0.05 (relaxed). The following modifications were selected: in dynamic mode oxidation of methionine and carbamidomethylation of cysteine in static mode. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (http://www.ebi.ac.uk/pride) via the PRIDE32 partner repository with the dataset identifier PXD007170. Reviewer account details: Username: [email protected] Password: zNWgzZi0 Data analysis The potential impact of the amino acid substitution on cystatin A function was predicted by SIFT Human Protein Prediction software available at the SIFT website (http://sift.jcvi.org/). SIFT (Sorting Intolerant From Tolerant) algorithm is based on the degree of conservation of amino acid residues in sequence alignments derived from closely related sequences, collected through PSI-BLAST.33 SIFT results with score

SALIVARY CYSTATINS: EXPLORING NEW POST-TRANSLATIONAL

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