Hypo-Phosphorylation of Salivary Peptidome as a Clue to the Molecular Pathogenesis of Autism Spectrum Disorders Massimo Castagnola,*,†,⊥ Irene Messana,‡ Rosanna Inzitari,†,⊥ Chiara Fanali,†,⊥ Tiziana Cabras,‡ Alessandra Morelli,§ Anna Maria Pecoraro,§ Giovanni Neri,# Maria Giulia Torrioli,§ and Fiorella Gurrieri# Istituto di Biochimica e Biochimica Clinica, Istituto di Genetica Medica, Istituto di Neuropsichiatria Infantile, Facolta` di Medicina, Universita` Cattolica, Rome, Italy, Istituto Scientifico Internazionale “Paolo VI” and Istituto per la Chimica del Riconoscimento Molecolare, Consiglio Nazionale delle Ricerche (C.N.R.), Rome, Italy, and Dipartimento di Scienze Applicate ai Biosistemi, Sezione di Biochimica e Biologia Molecolare, Universita` di Cagliari, Cagliari, Italy Received June 4, 2008
Abstract: RP-HPLC-ESI-MS profile of naturally occurring salivary peptides of subjects with autistic spectrum disorder [ASD; N ) 27: 12 with diagnosis of autism, 1 with diagnosis of Asperger, 14 with diagnosis of pervasive developmental disorders not otherwise specified (PDD-NOS)] was compared to that of age-matched controls with the goal of identifying differences that could turn out to become hallmarks of at least a subgroup of ASD individuals. Phosphorylation level of four specific salivary phospho-peptides, namely statherin, histatin 1 (both, p < 0.0001) and acidic proline-rich proteins (both entire and truncated isoforms) (p < 0.005) was found significantly lower in autistic patients, with hypo-phosphorylation of at least one peptide observed in 18 ASD subjects (66%). Developmental scale assessment (Griffith or WISC-R) carried out on 14 ASD subjects highlighted a normal to borderline cognitive development in 10 of them, all included in the hypo-phosphorylated group. Phosphorylation of salivary peptides involves a Golgi casein kinase common to many organs and tissues, CNS included, whose expression seems to be synchronized during fetal development. Hypo-phosphorylation of salivary peptides suggests potential asynchronies in the phosphorylation of other secretory proteins, which could be relevant in CNS development either during embryonic development or in early infancy. These results suggest that analysis of salivary phospho-peptides might help to discriminate a considerable subgroup of ASD patients.
* To whom correspondence should be addressed. Prof. Massimo Castagnola, Istituto di Biochimica e Biochimica Clinica, Facolta` di Medicina, Universita` Cattolica, Largo F. Vito, 00168, Roma, Italy. Tel. and/or Fax: ++3906-3053598. E-mail:
[email protected]. † Istituto di Biochimica e Biochimica Clinica, Universita` Cattolica. ⊥ Istituto per la Chimica del Riconoscimento Molecolare, Consiglio Nazionale delle Ricerche (C.N.R.). ‡ Dipartimento di Scienze Applicate ai Biosistemi, Universita` di Cagliari. § Istituto di Neuropsichiatria Infantile, Universita` Cattolica. # Istituto di Genetica Medica, Universita` Cattolica. 10.1021/pr8004088 CCC: $40.75
2008 American Chemical Society
Keywords: human • saliva • peptide • phosphorylation • autism • autistic spectrum disorders • statherin • histatin 1 • acidic proline-rich proteins
Introduction The autistic spectrum disorders (ASDs) are a group of syndromes with a neuro-developmental impairment characterized by altered social, emotional and communicative skills and by stereotyped motor and mental process with an onset within three years from birth. Children with ASDs are not in contact with environment, partly or completely, and about 50% are affected by mental retardation. ASDs are the most common neuro-developmental disorders (0.6% of the population) and are of complex and unknown etiology.1 Genetic factors play a major role and include chromosomal abnormalities (in about 10% of patients) or mutations in single genes (probably less than 5%) involved in synaptic development.2 However, according to a general way of thinking, it has been proposed that a few strong acting genes or, alternatively, many weak genes work together to cause autism susceptibility.3 Given such multiple genes involvement, and the related phenotypic complexity, it has always been challenging to adopt an appropriate research method toward the identification of biological causes. Proteomic studies of body fluids can offer a high-throughput approach for the identification of potential biomarkers in multifactorial diseases. Among different body fluids, whole saliva is until now neglected for diagnostic and prognostic purposes, but still very attractive for different reasons, including the noninvasive specimen collection.4,5 An increasing amount of experimental work is pointing to the salivary peptidome as a potential source of biomarkers for complex diseases.5 We hypothesized that proteomic studies of ASD salivary samples might provide clues toward a better understanding of the molecular and biochemical processes involved in the development of the disease. Human saliva contains specific peptides and small proteins pertaining to the classes of proline-rich proteins (PRP, acidic, basic and glycosylated), histatins and statherin, whose derivatives and isoforms account for more than 60% in weight of the total salivary proteome.6 Salivary peptide secretion is ensured by three couples of major glands (parotid, submandibular and sublingual) and by a variable number of minor glands. The secretion of the majority of these Journal of Proteome Research 2008, 7, 5327–5332 5327 Published on Web 11/07/2008
technical notes
Castagnola et al.
Table 1. m/z Values Used for the XIC Searches of Differently Phosphorylated Salivary Peptides/Proteins M aver. Da (theor) exp.
elution time (min)
(5299.7) 5299.9 ( 0.5 (5379.7) 5379.9 ( 0.5 (4848.2) 4848.2 ( 0.5 (4928.2) 4928.2 ( 0.5 (15434-15435) 15435 ( 2 (15514-15515) 15515 ( 2 (15594-15595) 15595 ( 2 15548 ( 3 15628 ( 3 15708 ( 3 (11081-11082)11082 ( 1 (11161-11162)11162 ( 1 (11241-11242)11242 ( 2
27.9-28.3 28.1-28.5 23.0-23.3 23.0-23.3 23.2-23.6 22.4-22.8 22.0-22.4 23.2-23.6 22.4-22.8 22.0-22.4 23.5-24.0 22.8-23.3 22.3-22.8
peptide or protein
Statherin Statherin Histatin 1 Histatin 1 PRP-1 typea PRP-1 type PRP-1 type PRP-1 type PRP-1 type PRP-1 type PRP-3 typeb PRP-3 type PRP-3 type a
monophos. (diphos.) nonphos. (monophos.) monophos. (diphos.) triphos. monophos.(TFA adduct) (diphos.) (TFA adduct) triphos.(TFA adduct) monophos. (diphos.) triphos.c
PRP-1 type isoforms include: PRP-1, PRP-2, PIF-s.
b
(5) 1060.9 (5) 1076.9 (4) 1213.5 (4) 1233.5 (12) 1287.3 (12) 1293.9 (12) 1300.6 (12) 1296.7 (12) 1303.3 (12) 1310.3 (10) 1109.2 (10) 1117.2 (10) 1125.2
(4) 1325.9 (4) 1345.9 (3) 1617.4 (3) 1644.1 (11) 1404.2 (11) 1411.5 (11) 1418.7 (11) 1414.5 (11) 1421.7 (11) 1429.0 (9) 1232.3 (9) 1241.2 (9) 1250.0
(3) 1767.6 (3) 1794.2
(10) 1544.5 (10) 1552.5 (10) 1560.5 (10) 1555.8 (10) 1563.8 (10) 1571.8 (7) 1584.2 (7) 1595.6 (7) 1607.1
PRP-3 type isoforms include: PRP-3, PRP-4, PIF-f. c Often not detectable.
peptides is granule mediated, and during the secretion pathway, the peptides undergo various post-translational modifications,7 such as phosphorylation, sulfation, glycosylation as well as different cleavages through a complex set of different proteinases, just like other peptides and proteins are processed in many endocrine and exocrine glands of other tissues.8 Recent studies of our group centered on RP-HPLC separations of the intact naturally occurring peptidome coupled with ESIMS allowed a better definition of the numerous salivary components of these classes and the detection of many components that can provide useful information on the activity of the enzymes responsible for peptide maturation.8 For instance, many mature salivary peptides are phosphorylated in the proximity of their N-terminus, such as acidic-PRPs, statherin and histatin 1. Peptidomic analysis of the acidic soluble fraction of human whole saliva allows the detection, together with the fully phosphorylated components, of either partially phosphorylated or nonphosphorylated forms and the evaluation of their relative amounts using the eXtracted Ion Current (XIC) area. Encouraged by the noninvasiveness of sample collection, we have analyzed by RP-HPLC-ESI-IT-MS profiles of naturally occurring whole salivary peptidome soluble in acidic solution of a group of ASD patients, and compared the results with those obtained from an age-matched control group, with the purpose of finding potential metabolic ASD hallmarks.
Materials and Methods Subjects under Study and Sample Collection. We enrolled 27 children (7 females and 20 males) with autistic spectrum diagnosis: 12 with autism, 1 with Asperger’s syndrome and 14 with pervasive developmental disorder not otherwise specified (PDD-NOS) according to DSMS IV criterions. All subjects were evaluated by the same team of child neuro-psychiatrists in order to obtain uniform evaluation. The diagnostic procedure included two interviews with parents and three play sessions with the children, with and without parents, to evaluate the communication and relational skills with parents and therapist. Stereotyped behaviors were also taken into account. Only in 14 children it was possible to carry out a developmental evaluation (Griffith or WISC-R); for the others, the evaluation was impossible because of lack of collaboration related to either mental retardation or relational impairment. Of these 14 subjects, 10 resulted with normal or borderline cognitive development, while 4 resulted with mental retardation. The age 5328
m/z values ((0.5) selected for the XIC searches (charge (z) in parenthesis)
Journal of Proteome Research • Vol. 7, No. 12, 2008
of 26 subjects was between 1.5 and 9 years, except for one subject (male) who was 15 years old. The control group comprised 23 healthy children with an age between 1.5 and 8.5 years. The age of the two groups was not statistically different (p > 0.4). Informed consent for specimen collection was obtained from the parents of the children of both groups. Materials and Apparatus. All general chemicals and reagents were of analytical grade and were purchased from FarmitaliaCarlo Erba (Milan, Italy), Merck (Damstadt, Germany), and Sigma Aldrich (St. Louis, MI). The HPLC-ESI-MS was a ThermoFinnigan (San Jose, CA) apparatus. The Surveyor HPLC system was equipped with a PDA (Photodiode Array) detector and connected by a T splitter to the electrospray ionization/ ion trap mass spectrometer LCQ Deca XP Plus (ThermoFinnigan). The chromatographic column was a Vydac (Hesperia, CA) C8 with 5 µm particle diameter (column dimensions 150 × 2.1 mm). Sample Collection. Whole human saliva was collected according to a standard protocol. The collection was performed around 11 a.m. to 1 p.m. and at least 30 min after any food or beverage intake and tooth brushing. Whole saliva was collected with small plastic aspirator at the basis of the tongue for less than 1 min and transferred to a plastic tube. An acidic solution (0.2% TFA, 2,2,2-trifluoroacetic acid) was immediately added to salivary samples in 1:1 (v/v) ratio in ice and the mix centrifuged at 8000g at 4 °C for 5 min. After precipitate removal (mainly mucins), the acidic solution was either immediately analyzed by HPLC-ESI-IT-MS or stored at -80 °C. HPLC-ESI-IT-MS. Separation and detection of salivary proteins and naturally occurring peptides was performed using RP-HPLC-ESI mass spectrometry. The following solutions were utilized for the reversed-phase chromatography: (eluent A) 0.056% (v/v) aqueous TFA and (eluent B) 0.050% (v/v) TFA in acetonitrile-water 80/20. The proteins were eluted using a linear gradient from 0 to 55% of B in 40 min, at a flow rate of 0.30 mL/min. A T splitter addressed a flow rate of 0.20 mL/ min toward the diode array detector and a flow rate of 0.10 mL/min toward the ESI source. Usually, during the first 5 min of RP-HPLC separation, the eluate was not addressed toward the ion trap MS apparatus in order to avoid instrument damage deriving from the high salt content. Mass spectra, in the positive ion mode, were collected every 3 ms. MS spray voltage was 4.50 KV and the capillary temperature was 220 °C. By this method, more than 120 proteins and peptides, namely, aPRPs, bPRPs, histatins, statherin and P-B peptide, and naturally
technical notes
Hypo-Phosphorylation of Salivary Peptides in ASDs
Figure 1. RP-HPLC-ESI-MS profile of the acidic soluble fraction of a whole saliva sample from an ASD patient and procedure carried out for the measurement of statherin phosphorylation level. (Graphs A and B) RP-HPLC revealed at 214 nm and total ion current (TIC) profiles, respectively. (Graph C) ESI spectrum recorded in the 27.75-28.60 min elution range (average of 31 acquisitions), whose deconvolution (Graph D) provided the average masses of statherin (5379.7 Da, diphosphorylated) and monophosphorylated statherin (5299.8 Da). Graph E reports an enlargement of the TIC profile in the 27.52-29.91 min elution range. Graphs F and G show the XIC (eXtracted ion current) area of statherin and monophosphorylated statherin peaks, respectively. RT, retention time; NL, normalization level; AV, average; MA, measured area.
occurring fragments and derivatives deriving from them were already identified in previous studies.8 Data Analysis. Deconvolution of averaged ESI-MS spectra was automatically performed either by using the Bioworks Browser software provided with the Deca XP instrument or by MagTran 1.0 software.9 XIC strategy was employed to selectively reveal salivary peptides described in this study. The m/z values used in the multiple XIC strategy were carefully selected in order to exclude values common to other closely eluting proteins ((0.5 m/z; see Table 1). The area of the XIC peaks, considered when the S/N ratio was at least 5, was used for estimation of relative peptide/protein amounts. Statistical Analysis. Results are presented as mean ( standard deviation. Pearson r was used to evaluate linear correlation between the separate peptides. Hotellings T-squared generalized means test was used for multivariate simultaneous comparison of peptide levels between the ASD group and healthy controls. The Student’s t test was applied to investigate differences between the separate peptides between groups. A two tailed p < 0.05 was considered significant. Data were analyzed with Microsoft Excel and Stata Statistical Software: Release 10 (StataCorp LP, College Station, TX).”
Results HPLC-ESI-MS analysis of naturally occurring peptides (peptidome) of whole human saliva allowed the detection of more than 120 salivary components.8 Since the statistical analysis revealed significant differences between ASD patients and controls only for the phosphorylation level of statherin, histatin 1 and acidic-PRPs, we will restrict the description of the results to these specific salivary components. More detailed description of the salivary peptidome can be found in previous studies and in the literature herein reported.8 Statherin is a specific salivary peptide of 43 amino acid residues diphosphorylated on Ser-2 and Ser-3 (Swiss Prot code: P02808).10 It elutes at about 28.1-28.5 min under the specific HPLC-ESI-MS conditions described in Materials and Methods. A monophosphorylated statherin derivative is always detectable in whole saliva and its chromatographic elution slightly anticipates that one of statherin peak (Table 1, Figure 1). The principal features of the RP-HPLC-ESI-MS analysis are reported in Figure 1 and the phosphorylation level of Statherin was established on the basis of the following ratio of the XIC peak area: Ratio ) 100 × statherin monophos./(statherin monophos. + 2 × statherin diphos.). Journal of Proteome Research • Vol. 7, No. 12, 2008 5329
technical notes
Castagnola et al.
Table 2. Phosphorylation Levels of Specific Salivary PhosPho-Peptides subjects
age (y)
Autistic group(N ) 27)
a
r 1.5-8.5 m 4.65 r 1.5-9.0 m 5.00 n.s.(>0.4)
Controls(N ) 23) P (Student’s t test) a
Statherin
Histatin 1
aPRP (tot)
PRP-1
PRP-3
m 2.23 sd 1.31 m 0.95 sd 0.32
