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Proteomic Analysis of RBP4/Vitamin A in Children with Cleft Lip and/or Palate J. Zhang, S. Zhou, Q. Zhang, S. Feng, Y. Chen, H. Zheng, X. Wang, W. Zhao, T. Zhang, Y. Zhou, H. Deng, J. Lin and F. Chen J DENT RES 2014 93: 547 originally published online 2 April 2014 DOI: 10.1177/0022034514530397 The online version of this article can be found at: http://jdr.sagepub.com/content/93/6/547
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research-article2014
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93610.1177/0022034514530397
Research Reports Clinical
Proteomic Analysis of RBP4/ Vitamin A in Children with Cleft Lip and/or Palate
J. Zhang1, S. Zhou1, Q. Zhang2, S. Feng3, Y. Chen3, H. Zheng1, X. Wang1, W. Zhao4, T. Zhang5, Y. Zhou1, H. Deng3, J. Lin1*, and F. Chen2* 1
The Department of Orthodontics, Peking University School and Hospital of Stomatology, Beijing, China; 2The Center of Laboratory, Peking University School and Hospital of Stomatology, Beijing, China; 3The School of Life Sciences, Tsinghua University, Beijing, China; 4The Department of Orthodontics, Nanfang Hospital, Southern Medical University, Guangdong, China; and 5Yantai Stomatology Hospital, Shandong, China; *corresponding authors, [email protected], [email protected]. J Dent Res 93(6):547-552, 2014
Abstract
Cleft of the lip and/or palate (CLP) is one of the most common congenital craniofacial defects. Non-syndromic CLP (NSCLP) is a multifactorial disease influenced by the interaction of genetic and environmental factors. However, there are few studies reporting on the developmental or metabolic status of babies with NSCLP after birth. In our study, we sought to identify and evaluate the differential expression of serum protein profiles in NSCLP children and unaffected babies. Thus, a ‘shotgun proteomics’ approach was first used to analyze the plasma proteome of 13 children with NSCLP and 10 control children, aged 2 to 3.5 years. In total, more than 300 proteins were identified in the serum sample. With gene ontology (GO) analysis, we detected many differentially expressed proteins that could be related to NSCLP, including those involved in lipoprotein metabolism, insulin-like growth-factor-related processes, and so on, especially the proteins involved in retinol transport. Retinol binding protein 4 (RBP4), one protein of the retinol transport category, was significantly decreased in the NSCLP group. Thus, serum vitamin A levels were further determined by high-performance liquid chromatography (HPLC). A significant difference (p < .01) was also found in vitamin A concentrations, consistent with the trend of RBP4. Our results indicated that reduced levels of RBP4 and vitamin A were related to newborns with NSCLP and should thus receive more attention. These results also suggest that vitamin A supplementation might be necessary at an early stage.
KEY WORDS:
retinol-binding proteins, vitamins, proteomics, craniofacial abnormalities, congenital, nonsyndromic.
DOI: 10.1177/0022034514530397 Received November 21, 2013; Last revision March 4, 2014; Accepted March 12, 2014 A supplemental appendix to this article is published electronically only at http://jdr.sagepub.com/supplemental. © International & American Associations for Dental Research
Introduction
C
left of the lip and/or palate (CLP) is one of the most common congenital craniofacial defects, occurring in approximately 1 out of 700 live births, with wide variability according to geographic and ethnic origins (Dixon et al., 2011). CLP can cause many adverse effects. Severe CLP can lead to birth morbidity. Children born with less severe CLP can experience problems with appearance, swallowing, speaking, and social communication. Families with CLP babies can suffer substantial burdens in terms of finances and psychological problems (Mossey et al., 2009). Given the effects of CLP, much research has focused on etiological exploration, aimed at finding specific, related genes. Since the advent of the genomics era, various studies of CLP have been performed to identify genes differentially expressed between CLP and non-CLP (Beaty et al., 2010; Mangold et al., 2010). Furthermore, recent developments have focused on syndromic CLP (Dixon et al., 2011). Basic biologists have focused mainly on etiological research, while clinicians have focused more on improving surgical and/or orthodontic treatment plans (Williams et al., 1999). For children with CLP, the associated structural defects mean that food intake might be inhibited compared with that in an unaffected child (Ize-Iyamu and Saheeb, 2011). However, few data are available regarding the basic biology of post-birth babies with CLP, such as metabolic defects or associated developmental conditions. Given these limitations, research on differences in post-birth babies with CLP vs. unaffected babies is important. Furthermore, the cleft defect arises because multiple factors interact early in embryological development. Some such differences remain after birth (Johansson and Ringsberg, 2004). Thus, we should be concerned not only with etiological aspects, but also with the differential expression of certain factors in post-birth individuals. Fortunately for babies ages between 2 and 3.5 yr, the primary dentition has been established, and the teeth can basically function, which could assist eating and chewing even with the existence of facial clefting. Over the past decade, there has been dramatic progress in proteomics research. Today, the analysis of proteins by mass spectrometry enables potential disease biomarkers to be systematically identified (Heller et al., 2007). These developments have resulted in the emergence of shotgun proteomics, a
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J Dent Res 93(6) 2014
Materials & Methods Serum Sample Collection and Preparation
Figure 1. Differential proteomic profiles from CLP babies and control babies. (A) Flow chart of the shotgun-based proteomics strategy showing the general procedures, including abundant protein filtering, enzymatic digestion and desalting, 2D-Nano-LC-ESI-MS/MS analysis, and final database searching with statistical validation. (B) GO analysis based on the shotgun proteomics results, indicating the classification of the proteins significantly differentially expressed between the two groups. It showed that many categories of proteins were related to CLP, including those involved in lipoprotein metabolism and lipid transport processes, the phosphatidylcholine catabolic process, retinol transport, and the insulin-like growth-factorrelated process. (C) Comparison of the numbers of significantly differentially expressed proteins detected by the 2 algorithms. Most proteins identified by the first algorithm were also detected by the second algorithm, indicating the reliability of both algorithms. Blue indicates searching against the database ipi human 3.84, orange indicates searching against the Swiss Prot database, and purple indicates both.
strategy based on a combination of protein digestion, liquid chromatography and tandem mass spectrometry (LC-MS/MS), peptide sequencing, and sequence database searching (Shteynberg et al., 2011). The shotgun approach permits hundreds to thousands of proteins to be identified (Dowell et al., 2008). The efficiency and sensitivity of shotgun proteomics have made it a useful technology for comparative proteomics (Nesvizhskii et al., 2006). Thus, to explore the developmental differences between babies with NSCLP and unaffected babies, we used a shotgun proteomics approach in this study to identify differences in the plasma protein profiles of post-birth babies with NSCLP and control (unaffected) babies, and, further, to provide information, based on these differences, that might lead to treatment recommendations.
Families with unaffected babies (n = 10) and those seeking treatment for babies with NSCLP (n = 13) were recruited at the Peking University School and Hospital of Stomatology in November 2011. The cleft at the lip in the NSCLP babies showed partial splits in the upper lip with an intact nasal floor. The cleft at the palate showed a complete cleft of the soft palate accompanied by a partial cleft of the hard palate. The babies in the control group came to the hospital because of oral mucosa trauma, condylar fracture, or presence of an esophageal foreign body. Basic biochemistry tests of the blood samples were performed to confirm the basic health of the control babies. All 23 babies were divided into two groups (NSCLP group and control group, ages 2-3.5 yr). The mean age and sex distribution in each group were matched as far as possible and showed no significant difference (Appendix Table 1). This study was approved by the Biomedical Ethics Committee of Peking University. Parents of the babies signed an informed consent form before their children participated in the study. Blood samples were obtained and serum protein concentration was determined by the Bradford assay. Serum samples were stored at -80°C until further use.
Shotgun-based Proteomics Strategy
Serum samples from the 13 babies with NSCLP and the 10 control infants were subjected to the proteomics analysis. The proteins obtained were digested enzymatically, desalted, and then eluted. Peptide mixtures were enriched and then separated by reverse-phase (RP) chromatography. Eluted peptides were ionized directly by nano-ESI ionization and transferred into the orifice of a LTQ-XL Orbitrap hybrid mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). Mass spectrometry was performed in a data-dependent mode with one full scan in the Orbitrap (m/z 300-1800, resolution of 60,000, AGC target value of 1×106). Dynamic collision time for the selected precursor ions was 30 sec. All samples were analyzed in triplicate, resulting in 36 LC-MS/MS runs. A GO analysis was conducted to classify the proteins significantly differentially expressed between the two groups in the shotgun analysis.
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J Dent Res 93(6) 2014 549 Proteomic Analysis of RBP4/Vitamin A in Children with CLP Table. Significantly Different Protein-discriminating Samples from the Two Groups Protein ID
Protein Name
Down-regulated Proteins IPI01017938.1 Ig lambda-6 chain C region IPI00473011.3 Hemoglobin subunit delta IPI00909594.1 Complement component C7 IPI00021856.3 Apolipoprotein C-II IPI00984754.1 Protein IPI00021855.1 Apolipoprotein C-I IPI00794777.1 Uncharacterized protein IPI00328609.3 Kallistatin IPI00643437.1 Complement component 4 binding protein, beta IPI00019372.2 Serglycin IPI00645213.1 Apolipoprotein M, isoform CRA_a IPI01015433.1 Glutamate dehydrogenase IPI00884981.2 Isoform 2 of pregnancy zone protein IPI00925358.2 Insulin-like growth factor binding protein 3 IPI00216588.2 Uncharacterized protein IPI00004373.1 Mannose-binding protein C IPI00293057.6 Isoform 2 of carboxypeptidase B2 IPI00607725.6 Uncharacterized protein IPI00030790.9 Uncharacterized protein KIAA0556 IPI00478809.4 Coagulation factor V IPI00783390.3 Isoform 1 of neural cell adhesion molecule L1-like protein IPI00296176.2 Coagulation factor IX IPI00027482.1 Corticosteroid-binding globulin IPI00980674.1 Uncharacterized protein IPI00019576.1 Coagulation factor X IPI00166729.4 Zinc-alpha-2-glycoprotein IPI00552578.2 Serum amyloid A protein IPI00878282.1 23-kDa protein IPI00029717.1 Isoform 2 of Fibrinogen alpha chain IPI00480192.1 Retinol binding protein 4, plasma IPI00883722.1 Complement factor H-related 1 IPI00010295.1 Carboxypeptidase N catalytic chain IPI00007240.2 Coagulation factor XIII B chain IPI00879231.1 Alpha-2-antiplasmin IPI00879915.1 Uncharacterized protein IPI00967087.1 Protein IPI00743766.2 Fetuin-B IPI00019581.2 Coagulation factor XII IPI00925177.1 Uncharacterized protein IPI00926737.2 Uncharacterized protein IPI00395488.2 Vasorin IPI00218795.1 L-selectin precursor IPI00027410.1 Platelet glycoprotein V IPI00910923.1 cDNA FLJ60404, highly similar to mitogen-activated protein kinase 7 IPI00023014.3 von Willebrand factor Up-regulated Proteins IPI00945517.1 59 kDa protein IPI00299503.2 Phosphatidylinositol-glycan-specific phospholipase D IPI00656111.1 Isoform E of Proteoglycan 4 IPI00940493.1 Vitamin K-dependent protein C IPI00894486.1 66 kDa protein
ΣCoverage
ΣPSM
Ratio
p Value
2 2.4 2.4 5.1 4.4 3.2 4.2 3.4 2.2 3.2 2.1 3.1 2.5 2.4 2.4 2.6 3.2 3 2.1 2.7 3.7 2.6 2.3 3.4 2.4 3.3 3 3 3 2.6 2.5 2.5 2.3 2.25 2 2 2 2 2 2 2 2 2 2
.031 .02 .041 .035 .037 .028 .023 .048 .026 .049 .038 .031 .031 .003 .007 .012 .002 .043 .035 .017 .039 .026 .017 .006 .004 .024 .046 .004 .023 .012 .039 .04 .016 .016 .015 .011 .049 .018 .049 .021 .036 .018 .026 .009
32.08 28.57 26.54 23.76 21.52 13.25 13.24 9.84 9.39 8.86 8.62 8.17 7.02 6.3 5.06 4.84 4.17 1.94 1.3 1.3 0.99 9.76 8.4 24.43 6.35 20.81 27.87 14.21 2.48 25.13 32.47 14.19 13.62 9.98 46.62 20.34 12.83 12.03 7.63 5.74 4.75 4.68 2.32 1.59
2 9 24 1 4 2 2 4 1 1 1 1 45 4 4 2 3 1 1 2 1 7 7 10 5 13 4 4 4 25 7 7 10 13 18 3 6 21 3 6 3 3 3 6
0.85
3
2
.015
4 2.02 1.71 11.64 2.28
3 3 3 4 1
0.5 0.5 0.5 0.3 0.4
.042 .022 .009 .043 .043
Down-regulated proteins, the levels of which were lower in the CLP group than in the control; up-regulated proteins, the levels of which were higher in the CLP group than in the control. Ratio, the PSM value of Control:CLP. The fold-change cut-off was two-fold.
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J Dent Res 93(6) 2014 (BioyongTech, Beijing, China). A p value < .05 was considered to indicate statistical significance.
Results
Figure 2. RBP4 levels in the plasma samples from children with CLP children and control children. (A) Plasma RBP4 and GAPDH levels were detected by Western blotting. (B) Normalized RBP4 (RBP4 relative to GAPDH) band intensity differences between control children and children with CLP. **Significantly different at p < .01.
Western Blotting for Identification of the Selected Proteins Western blotting was used to detect RBP4 in plasma. Plasma proteins were separated by 15% acrylamide SDS-PAGE and were transferred to nitrocellulose membranes. Primary antibodies (anti-RBP4, purchased from Biological Inc. [Beijing, China] with catalog number 10354-MM01; anti-GAPDH, from Cell Signaling Technology Inc., Danvers, MA, USA, with catalog number #2118) were added at a ratio of 1:500 after blocking for 1 hr. After incubation with the primary antibody overnight, blots were washed with Tris-buffered saline and then treated with anti-IgG antibody conjugated to horseradish peroxidase for 1 hr. Signals indicating abundance of the protein were visualized with an enhanced chemiluminescent substrate (SuperSignal WestPico; Thermo Fisher Scientific) and autoradiographic film.
HPLC Assay of Vitamin A Plasma retinol (vitamin A) was determined by high-performance liquid chromatography (HPLC) (Gamble et al., 2001; Berggren Söderlund et al., 2005). The HPLC column was a reverse-phase “Waters-Spherisorb, ODS2” stainless steel column (25 cm × 4.6 mm I.D.; Waters Associates, Inc., Milford, MA, USA). In this system, 95% methanol was used as the mobile phase, and the flow rate was 2.5 mL/min. Vitamin A standard and plasma samples were first extracted with chloroform and then centrifuged (5000 × g, 5 min) to separate the layers. The top layer was removed, and the residue was dissolved in 1 mL chloroform/ methanol solution (1:1) for chromatographic analysis to determine the content of vitamin A in the plasma samples.
Statistical Analyses Raw data obtained via 2D-Nano-LC-ESI-MS/MS were submitted to the Trans Proteomic Pipeline (revision 4.0, Institute of Systems Biology, Seattle, WA, USA) for database searching. The data were searched against the human entries in the Swiss Prot protein database using the built-in decoy option. Trypsin was assigned as the proteolytic enzyme with a maximum of 2 missed cleavages. The independent t test and Pearson’s correlation analysis were used to identify differences between the two groups. Data were analyzed with the BioExplorer statistical software package
Protein Profiles Showed Differential Expression between the Children with NSCLP and Unaffected Control Children We determined plasma protein profiles using a shotgun-based proteomics method (Fig. 1A) to detect differences between the children with and those without NSCLP. There were more than 300 proteins identified in the serum sample. In particular, peptideto-spectrum matches (PSMs) were produced by automated database search engines and have been found to be useful for meaningful comparison of proteomics datasets. Unique proteins were credibly identified by shotgun proteomics, with a final protein-level false-discovery rate (FDR) < 1% in the plasma of the children. Among the identified proteins, 50 proteins showed significantly different expression between the NSCLP and control groups, based on the ipi human 3.84 database (Table). All of these proteins were identified with > 95% confidence.
Level of Serum RBP4 was Significantly Lower in the Children with NSCLP than in the Control Children A GO analysis was then conducted, depending on the shotgun analysis results (Fig. 1B). It classified the proteins significantly differentially expressed, and many biological processes or functions presented were found to present differences between the two groups (Appendix Table 2). We also analyzed the peak intensity of the mass spectra by searching and matching with another database using a different algorithm. Most significantly differentially expressed proteins overlapped (Fig. 1C). Retinoic acid, a kind of bioactive metabolite of retinol, is a known teratogenic effector in cleft palate (Emmanouil-Nikoloussi et al., 2000; Choi et al., 2011). Interestingly, of the 43 significantly different overlapping proteins, one was the retinol-transportrelated protein, with RBP4 being specifically identified by the shotgun proteomics. Expression of RBP4, 1 of the 4 kinds of retinol-binding proteins, was significantly almost three-fold lower in the NSCLP group than in the control group, and it was at the intersection of the 2 algorithms, which confirmed its importance. Thus, we focused on this small protein of molecular weight 21 kDa with 181 amino acids. We verified and compared the RBP4 levels in the plasma of each individual by Western blotting; the results confirmed those obtained with the shotgun proteomics (Fig. 2).
Serum Vitamin A Significantly Decreased in the Group with NSCLP Retinol binding proteins (RBP) have also been shown to be a useful biomarker for retinol because of the approximately 1:1 molar correlation between retinol and RBP in serum (Gamble et al., 2001). Thus, we further quantified the retinol (vitamin A) content in plasma samples using HPLC, since serum retinol concentrations are commonly used to evaluate vitamin A levels. The
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J Dent Res 93(6) 2014 551 Proteomic Analysis of RBP4/Vitamin A in Children with CLP vitamin A concentration was significantly lower in the group of children with NSCLP than in the unaffected control group, consistent with the trend of RBP4 (p = 1.52013E-08; Fig. 3).
Discussion Some studies showed that babies born with CLP could also display other congenital abnormalities or organ developmental defects, including those affecting the cardiovascular, musculoskeletal, nervous, digestive, respiratory, urinary, Figure 3. Plasma vitamin A levels. (A) Vitamin A levels in each group were determined by immune, and endocrine systems (Rittler HPLC. (B) Vitamin A levels were subjected to statistical analysis. ** Significantly different at et al., 2011). Thus, to explore the differp < .01. (C) Vitamin A concentration for each child with CLP and control children; the SD and ences between the post-birth babies with the exact p value are shown. NSCLP and control babies, we used a shotgun proteomics strategy as a pilot in our study to identify differentially problem in more than 100 countries, affecting an estimated 100 expressed factors in plasma samples from children with NSCLP million children (Hix et al., 2004). and unaffected control. We detected many significantly differenMoreover, many cellular functions, including those of bone tially expressed proteins, one of which was a retinol transport cells, are mediated by vitamin A; however, there has been confactor, RBP4. RBP are one of the 5 retinoids binding retinol troversy as to its specific function. Some studies have indicated proteins and the only category present in plasma. RBP has been that the primary skeletal effect of vitamin A was to increase shown to be a useful surrogate biomarker of retinol, indicating bone resorption, but others showed that vitamin A was involved that it may be used to assess and monitor vitamin A content in two-way regulation in which it could not only stimulate but (Craft, 2001; Gamble et al., 2001; Erikstrup et al., 2009). RBP4 also inhibit osteoclast formation (Kneissel et al., 2005). Both a itself, previously known as RBP (Wolf, 2007), plays various lack of and excess retinol result in vitamin A abnormalities, important roles and participates in the transport of vitamin A, leading to various defects, including congenital malformations lipid metabolism, and immune and inflammatory responses (Conaway et al., 2013). Thus, because of its various essential (Vaisbuch et al., 2010). RBP4 was also recently shown to play functions in humans, potential biomarkers of vitamin A are also a role as an early marker of fetal growth restriction in maternal diverse (Tanumihardjo, 2011). serum. Besides, RBP4 is reported to be expressed in chondroIn addition to retinol transport proteins, the MS and GO cytes and to be involved in bone growth. Some other studies identianalyses suggested that several other proteins, most of which fied RBP4 as a factor linked to obesity and its co-morbidities, had some connection with lipid metabolism processes, differed including insulin resistance, type 2 diabetes (T2D), and metabetween the groups. Proteins involved in lipid metabolism were bolic syndrome (Fernandez-Real et al., 2008; Kotnik et al., also significantly under-expressed at the protein level in the 2011). Moreover, all-trans retinoic acid (at RA) is a known teragroup with NSCLP, compared with those in the control group, togenic effector in cleft palate and has been shown to be able to including insulin-like growth factor and several lipoproteins. induce cleft palate in animal models (Okano et al., 2007). Also, Both of these types of factors also play vital functions in the in our previous study, we showed a relationship between retinol human body. Thus, the roles of these proteins related to lipid acid and CLP (Yu et al., 2005; Han et al., 2006). metabolism should be identified in our future studies. This background resulted in our hypothesis that vitamins, Our study had several limitations. First, the clinical sample including retinol, are associated with the condition of children size was small and, to some extent, could lead to objective indiwith NSCLP. Since some studies have shown that children with vidual differences, and data related to the diet of these children CLP exhibit a lower growth curve for height, nutrition might should be included to rule out the influence of extrinsic factors. differ between those with NSCLP and unaffected children. It is necessary to plan to confirm these preliminary results, Thus, we assessed the plasma retinol content and demonstrated including the comparison of the RBP4 and vitamin A levels in that vitamin A levels were significantly lower in children with the mothers and the newborn babies with and without NSCLP at NSCLP than in those without. Some studies have reported that different growth points. Conversely, in a future study, we could the risk of facial clefts increased among mothers with lower provide information about more downstream elements of the intakes of vitamin A around the time of conception. Vitamin A pathway, such as retinoic acid to establish the status of the is a crucial factor for diverse functions in the human body. metabolic process. Clinicians should monitor dynamic changes Vitamin A deficiency (VAD) can affect embryogenesis, growth, in serum protein levels—a longitudinal monitoring study would development, immunity, epithelial differentiation, and other help to make these results more credible. Further basic research aspects of human development (Tanumihardjo, 2011). More into the mechanisms underlying NSCLP is necessary, and both recent investigations have indicated that VAD is a public health
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oral clinicians and scientists should expend effort to improve the situation of babies with NSCLP. In conclusion, serum RBP4 and vitamin A levels exhibited statistically significant differences (p < .01) between post-birth babies with NSCLP and unaffected control babies. These results indicated that reduced levels of RBP4 and vitamin A were related to post-birth babies with NSCLP and thus should receive more attention.
Acknowledgments This work was supported by grant 81200762 from the National Natural Science Foundation of China and by funding from the Peking University School of Stomatology (PKUSS20110301). We gratefully acknowledge the assistance of Yan Li from BioyongTech for technical data analysis and her expertise in shotgun proteomics analysis. The authors declare no potential conflicts of interest with respect to the authorship and/or publication of this article.
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Proteomic Analysis of RBP4/Vitamin A in Children with Cleft Lip and/or Palate J. Zhang, S. Zhou, Q. Zhang, S. Feng, Y. Chen, H. Zheng, X. Wang, W. Zhao, T. Zhang, Y. Zhou, H. Deng, J. Lin and F. Chen J DENT RES 2014 93: 547 originally published online 2 April 2014 DOI: 10.1177/0022034514530397 The online version of this article can be found at: http://jdr.sagepub.com/content/93/6/547
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research-article2014
JDR
93610.1177/0022034514530397
Research Reports Clinical
Proteomic Analysis of RBP4/ Vitamin A in Children with Cleft Lip and/or Palate
J. Zhang1, S. Zhou1, Q. Zhang2, S. Feng3, Y. Chen3, H. Zheng1, X. Wang1, W. Zhao4, T. Zhang5, Y. Zhou1, H. Deng3, J. Lin1*, and F. Chen2* 1
The Department of Orthodontics, Peking University School and Hospital of Stomatology, Beijing, China; 2The Center of Laboratory, Peking University School and Hospital of Stomatology, Beijing, China; 3The School of Life Sciences, Tsinghua University, Beijing, China; 4The Department of Orthodontics, Nanfang Hospital, Southern Medical University, Guangdong, China; and 5Yantai Stomatology Hospital, Shandong, China; *corresponding authors, [email protected], [email protected]. J Dent Res 93(6):547-552, 2014
Abstract
Cleft of the lip and/or palate (CLP) is one of the most common congenital craniofacial defects. Non-syndromic CLP (NSCLP) is a multifactorial disease influenced by the interaction of genetic and environmental factors. However, there are few studies reporting on the developmental or metabolic status of babies with NSCLP after birth. In our study, we sought to identify and evaluate the differential expression of serum protein profiles in NSCLP children and unaffected babies. Thus, a ‘shotgun proteomics’ approach was first used to analyze the plasma proteome of 13 children with NSCLP and 10 control children, aged 2 to 3.5 years. In total, more than 300 proteins were identified in the serum sample. With gene ontology (GO) analysis, we detected many differentially expressed proteins that could be related to NSCLP, including those involved in lipoprotein metabolism, insulin-like growth-factor-related processes, and so on, especially the proteins involved in retinol transport. Retinol binding protein 4 (RBP4), one protein of the retinol transport category, was significantly decreased in the NSCLP group. Thus, serum vitamin A levels were further determined by high-performance liquid chromatography (HPLC). A significant difference (p < .01) was also found in vitamin A concentrations, consistent with the trend of RBP4. Our results indicated that reduced levels of RBP4 and vitamin A were related to newborns with NSCLP and should thus receive more attention. These results also suggest that vitamin A supplementation might be necessary at an early stage.
KEY WORDS:
retinol-binding proteins, vitamins, proteomics, craniofacial abnormalities, congenital, nonsyndromic.
DOI: 10.1177/0022034514530397 Received November 21, 2013; Last revision March 4, 2014; Accepted March 12, 2014 A supplemental appendix to this article is published electronically only at http://jdr.sagepub.com/supplemental. © International & American Associations for Dental Research
Introduction
C
left of the lip and/or palate (CLP) is one of the most common congenital craniofacial defects, occurring in approximately 1 out of 700 live births, with wide variability according to geographic and ethnic origins (Dixon et al., 2011). CLP can cause many adverse effects. Severe CLP can lead to birth morbidity. Children born with less severe CLP can experience problems with appearance, swallowing, speaking, and social communication. Families with CLP babies can suffer substantial burdens in terms of finances and psychological problems (Mossey et al., 2009). Given the effects of CLP, much research has focused on etiological exploration, aimed at finding specific, related genes. Since the advent of the genomics era, various studies of CLP have been performed to identify genes differentially expressed between CLP and non-CLP (Beaty et al., 2010; Mangold et al., 2010). Furthermore, recent developments have focused on syndromic CLP (Dixon et al., 2011). Basic biologists have focused mainly on etiological research, while clinicians have focused more on improving surgical and/or orthodontic treatment plans (Williams et al., 1999). For children with CLP, the associated structural defects mean that food intake might be inhibited compared with that in an unaffected child (Ize-Iyamu and Saheeb, 2011). However, few data are available regarding the basic biology of post-birth babies with CLP, such as metabolic defects or associated developmental conditions. Given these limitations, research on differences in post-birth babies with CLP vs. unaffected babies is important. Furthermore, the cleft defect arises because multiple factors interact early in embryological development. Some such differences remain after birth (Johansson and Ringsberg, 2004). Thus, we should be concerned not only with etiological aspects, but also with the differential expression of certain factors in post-birth individuals. Fortunately for babies ages between 2 and 3.5 yr, the primary dentition has been established, and the teeth can basically function, which could assist eating and chewing even with the existence of facial clefting. Over the past decade, there has been dramatic progress in proteomics research. Today, the analysis of proteins by mass spectrometry enables potential disease biomarkers to be systematically identified (Heller et al., 2007). These developments have resulted in the emergence of shotgun proteomics, a
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J Dent Res 93(6) 2014
Materials & Methods Serum Sample Collection and Preparation
Figure 1. Differential proteomic profiles from CLP babies and control babies. (A) Flow chart of the shotgun-based proteomics strategy showing the general procedures, including abundant protein filtering, enzymatic digestion and desalting, 2D-Nano-LC-ESI-MS/MS analysis, and final database searching with statistical validation. (B) GO analysis based on the shotgun proteomics results, indicating the classification of the proteins significantly differentially expressed between the two groups. It showed that many categories of proteins were related to CLP, including those involved in lipoprotein metabolism and lipid transport processes, the phosphatidylcholine catabolic process, retinol transport, and the insulin-like growth-factorrelated process. (C) Comparison of the numbers of significantly differentially expressed proteins detected by the 2 algorithms. Most proteins identified by the first algorithm were also detected by the second algorithm, indicating the reliability of both algorithms. Blue indicates searching against the database ipi human 3.84, orange indicates searching against the Swiss Prot database, and purple indicates both.
strategy based on a combination of protein digestion, liquid chromatography and tandem mass spectrometry (LC-MS/MS), peptide sequencing, and sequence database searching (Shteynberg et al., 2011). The shotgun approach permits hundreds to thousands of proteins to be identified (Dowell et al., 2008). The efficiency and sensitivity of shotgun proteomics have made it a useful technology for comparative proteomics (Nesvizhskii et al., 2006). Thus, to explore the developmental differences between babies with NSCLP and unaffected babies, we used a shotgun proteomics approach in this study to identify differences in the plasma protein profiles of post-birth babies with NSCLP and control (unaffected) babies, and, further, to provide information, based on these differences, that might lead to treatment recommendations.
Families with unaffected babies (n = 10) and those seeking treatment for babies with NSCLP (n = 13) were recruited at the Peking University School and Hospital of Stomatology in November 2011. The cleft at the lip in the NSCLP babies showed partial splits in the upper lip with an intact nasal floor. The cleft at the palate showed a complete cleft of the soft palate accompanied by a partial cleft of the hard palate. The babies in the control group came to the hospital because of oral mucosa trauma, condylar fracture, or presence of an esophageal foreign body. Basic biochemistry tests of the blood samples were performed to confirm the basic health of the control babies. All 23 babies were divided into two groups (NSCLP group and control group, ages 2-3.5 yr). The mean age and sex distribution in each group were matched as far as possible and showed no significant difference (Appendix Table 1). This study was approved by the Biomedical Ethics Committee of Peking University. Parents of the babies signed an informed consent form before their children participated in the study. Blood samples were obtained and serum protein concentration was determined by the Bradford assay. Serum samples were stored at -80°C until further use.
Shotgun-based Proteomics Strategy
Serum samples from the 13 babies with NSCLP and the 10 control infants were subjected to the proteomics analysis. The proteins obtained were digested enzymatically, desalted, and then eluted. Peptide mixtures were enriched and then separated by reverse-phase (RP) chromatography. Eluted peptides were ionized directly by nano-ESI ionization and transferred into the orifice of a LTQ-XL Orbitrap hybrid mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). Mass spectrometry was performed in a data-dependent mode with one full scan in the Orbitrap (m/z 300-1800, resolution of 60,000, AGC target value of 1×106). Dynamic collision time for the selected precursor ions was 30 sec. All samples were analyzed in triplicate, resulting in 36 LC-MS/MS runs. A GO analysis was conducted to classify the proteins significantly differentially expressed between the two groups in the shotgun analysis.
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J Dent Res 93(6) 2014 549 Proteomic Analysis of RBP4/Vitamin A in Children with CLP Table. Significantly Different Protein-discriminating Samples from the Two Groups Protein ID
Protein Name
Down-regulated Proteins IPI01017938.1 Ig lambda-6 chain C region IPI00473011.3 Hemoglobin subunit delta IPI00909594.1 Complement component C7 IPI00021856.3 Apolipoprotein C-II IPI00984754.1 Protein IPI00021855.1 Apolipoprotein C-I IPI00794777.1 Uncharacterized protein IPI00328609.3 Kallistatin IPI00643437.1 Complement component 4 binding protein, beta IPI00019372.2 Serglycin IPI00645213.1 Apolipoprotein M, isoform CRA_a IPI01015433.1 Glutamate dehydrogenase IPI00884981.2 Isoform 2 of pregnancy zone protein IPI00925358.2 Insulin-like growth factor binding protein 3 IPI00216588.2 Uncharacterized protein IPI00004373.1 Mannose-binding protein C IPI00293057.6 Isoform 2 of carboxypeptidase B2 IPI00607725.6 Uncharacterized protein IPI00030790.9 Uncharacterized protein KIAA0556 IPI00478809.4 Coagulation factor V IPI00783390.3 Isoform 1 of neural cell adhesion molecule L1-like protein IPI00296176.2 Coagulation factor IX IPI00027482.1 Corticosteroid-binding globulin IPI00980674.1 Uncharacterized protein IPI00019576.1 Coagulation factor X IPI00166729.4 Zinc-alpha-2-glycoprotein IPI00552578.2 Serum amyloid A protein IPI00878282.1 23-kDa protein IPI00029717.1 Isoform 2 of Fibrinogen alpha chain IPI00480192.1 Retinol binding protein 4, plasma IPI00883722.1 Complement factor H-related 1 IPI00010295.1 Carboxypeptidase N catalytic chain IPI00007240.2 Coagulation factor XIII B chain IPI00879231.1 Alpha-2-antiplasmin IPI00879915.1 Uncharacterized protein IPI00967087.1 Protein IPI00743766.2 Fetuin-B IPI00019581.2 Coagulation factor XII IPI00925177.1 Uncharacterized protein IPI00926737.2 Uncharacterized protein IPI00395488.2 Vasorin IPI00218795.1 L-selectin precursor IPI00027410.1 Platelet glycoprotein V IPI00910923.1 cDNA FLJ60404, highly similar to mitogen-activated protein kinase 7 IPI00023014.3 von Willebrand factor Up-regulated Proteins IPI00945517.1 59 kDa protein IPI00299503.2 Phosphatidylinositol-glycan-specific phospholipase D IPI00656111.1 Isoform E of Proteoglycan 4 IPI00940493.1 Vitamin K-dependent protein C IPI00894486.1 66 kDa protein
ΣCoverage
ΣPSM
Ratio
p Value
2 2.4 2.4 5.1 4.4 3.2 4.2 3.4 2.2 3.2 2.1 3.1 2.5 2.4 2.4 2.6 3.2 3 2.1 2.7 3.7 2.6 2.3 3.4 2.4 3.3 3 3 3 2.6 2.5 2.5 2.3 2.25 2 2 2 2 2 2 2 2 2 2
.031 .02 .041 .035 .037 .028 .023 .048 .026 .049 .038 .031 .031 .003 .007 .012 .002 .043 .035 .017 .039 .026 .017 .006 .004 .024 .046 .004 .023 .012 .039 .04 .016 .016 .015 .011 .049 .018 .049 .021 .036 .018 .026 .009
32.08 28.57 26.54 23.76 21.52 13.25 13.24 9.84 9.39 8.86 8.62 8.17 7.02 6.3 5.06 4.84 4.17 1.94 1.3 1.3 0.99 9.76 8.4 24.43 6.35 20.81 27.87 14.21 2.48 25.13 32.47 14.19 13.62 9.98 46.62 20.34 12.83 12.03 7.63 5.74 4.75 4.68 2.32 1.59
2 9 24 1 4 2 2 4 1 1 1 1 45 4 4 2 3 1 1 2 1 7 7 10 5 13 4 4 4 25 7 7 10 13 18 3 6 21 3 6 3 3 3 6
0.85
3
2
.015
4 2.02 1.71 11.64 2.28
3 3 3 4 1
0.5 0.5 0.5 0.3 0.4
.042 .022 .009 .043 .043
Down-regulated proteins, the levels of which were lower in the CLP group than in the control; up-regulated proteins, the levels of which were higher in the CLP group than in the control. Ratio, the PSM value of Control:CLP. The fold-change cut-off was two-fold.
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J Dent Res 93(6) 2014 (BioyongTech, Beijing, China). A p value < .05 was considered to indicate statistical significance.
Results
Figure 2. RBP4 levels in the plasma samples from children with CLP children and control children. (A) Plasma RBP4 and GAPDH levels were detected by Western blotting. (B) Normalized RBP4 (RBP4 relative to GAPDH) band intensity differences between control children and children with CLP. **Significantly different at p < .01.
Western Blotting for Identification of the Selected Proteins Western blotting was used to detect RBP4 in plasma. Plasma proteins were separated by 15% acrylamide SDS-PAGE and were transferred to nitrocellulose membranes. Primary antibodies (anti-RBP4, purchased from Biological Inc. [Beijing, China] with catalog number 10354-MM01; anti-GAPDH, from Cell Signaling Technology Inc., Danvers, MA, USA, with catalog number #2118) were added at a ratio of 1:500 after blocking for 1 hr. After incubation with the primary antibody overnight, blots were washed with Tris-buffered saline and then treated with anti-IgG antibody conjugated to horseradish peroxidase for 1 hr. Signals indicating abundance of the protein were visualized with an enhanced chemiluminescent substrate (SuperSignal WestPico; Thermo Fisher Scientific) and autoradiographic film.
HPLC Assay of Vitamin A Plasma retinol (vitamin A) was determined by high-performance liquid chromatography (HPLC) (Gamble et al., 2001; Berggren Söderlund et al., 2005). The HPLC column was a reverse-phase “Waters-Spherisorb, ODS2” stainless steel column (25 cm × 4.6 mm I.D.; Waters Associates, Inc., Milford, MA, USA). In this system, 95% methanol was used as the mobile phase, and the flow rate was 2.5 mL/min. Vitamin A standard and plasma samples were first extracted with chloroform and then centrifuged (5000 × g, 5 min) to separate the layers. The top layer was removed, and the residue was dissolved in 1 mL chloroform/ methanol solution (1:1) for chromatographic analysis to determine the content of vitamin A in the plasma samples.
Statistical Analyses Raw data obtained via 2D-Nano-LC-ESI-MS/MS were submitted to the Trans Proteomic Pipeline (revision 4.0, Institute of Systems Biology, Seattle, WA, USA) for database searching. The data were searched against the human entries in the Swiss Prot protein database using the built-in decoy option. Trypsin was assigned as the proteolytic enzyme with a maximum of 2 missed cleavages. The independent t test and Pearson’s correlation analysis were used to identify differences between the two groups. Data were analyzed with the BioExplorer statistical software package
Protein Profiles Showed Differential Expression between the Children with NSCLP and Unaffected Control Children We determined plasma protein profiles using a shotgun-based proteomics method (Fig. 1A) to detect differences between the children with and those without NSCLP. There were more than 300 proteins identified in the serum sample. In particular, peptideto-spectrum matches (PSMs) were produced by automated database search engines and have been found to be useful for meaningful comparison of proteomics datasets. Unique proteins were credibly identified by shotgun proteomics, with a final protein-level false-discovery rate (FDR) < 1% in the plasma of the children. Among the identified proteins, 50 proteins showed significantly different expression between the NSCLP and control groups, based on the ipi human 3.84 database (Table). All of these proteins were identified with > 95% confidence.
Level of Serum RBP4 was Significantly Lower in the Children with NSCLP than in the Control Children A GO analysis was then conducted, depending on the shotgun analysis results (Fig. 1B). It classified the proteins significantly differentially expressed, and many biological processes or functions presented were found to present differences between the two groups (Appendix Table 2). We also analyzed the peak intensity of the mass spectra by searching and matching with another database using a different algorithm. Most significantly differentially expressed proteins overlapped (Fig. 1C). Retinoic acid, a kind of bioactive metabolite of retinol, is a known teratogenic effector in cleft palate (Emmanouil-Nikoloussi et al., 2000; Choi et al., 2011). Interestingly, of the 43 significantly different overlapping proteins, one was the retinol-transportrelated protein, with RBP4 being specifically identified by the shotgun proteomics. Expression of RBP4, 1 of the 4 kinds of retinol-binding proteins, was significantly almost three-fold lower in the NSCLP group than in the control group, and it was at the intersection of the 2 algorithms, which confirmed its importance. Thus, we focused on this small protein of molecular weight 21 kDa with 181 amino acids. We verified and compared the RBP4 levels in the plasma of each individual by Western blotting; the results confirmed those obtained with the shotgun proteomics (Fig. 2).
Serum Vitamin A Significantly Decreased in the Group with NSCLP Retinol binding proteins (RBP) have also been shown to be a useful biomarker for retinol because of the approximately 1:1 molar correlation between retinol and RBP in serum (Gamble et al., 2001). Thus, we further quantified the retinol (vitamin A) content in plasma samples using HPLC, since serum retinol concentrations are commonly used to evaluate vitamin A levels. The
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J Dent Res 93(6) 2014 551 Proteomic Analysis of RBP4/Vitamin A in Children with CLP vitamin A concentration was significantly lower in the group of children with NSCLP than in the unaffected control group, consistent with the trend of RBP4 (p = 1.52013E-08; Fig. 3).
Discussion Some studies showed that babies born with CLP could also display other congenital abnormalities or organ developmental defects, including those affecting the cardiovascular, musculoskeletal, nervous, digestive, respiratory, urinary, Figure 3. Plasma vitamin A levels. (A) Vitamin A levels in each group were determined by immune, and endocrine systems (Rittler HPLC. (B) Vitamin A levels were subjected to statistical analysis. ** Significantly different at et al., 2011). Thus, to explore the differp < .01. (C) Vitamin A concentration for each child with CLP and control children; the SD and ences between the post-birth babies with the exact p value are shown. NSCLP and control babies, we used a shotgun proteomics strategy as a pilot in our study to identify differentially problem in more than 100 countries, affecting an estimated 100 expressed factors in plasma samples from children with NSCLP million children (Hix et al., 2004). and unaffected control. We detected many significantly differenMoreover, many cellular functions, including those of bone tially expressed proteins, one of which was a retinol transport cells, are mediated by vitamin A; however, there has been confactor, RBP4. RBP are one of the 5 retinoids binding retinol troversy as to its specific function. Some studies have indicated proteins and the only category present in plasma. RBP has been that the primary skeletal effect of vitamin A was to increase shown to be a useful surrogate biomarker of retinol, indicating bone resorption, but others showed that vitamin A was involved that it may be used to assess and monitor vitamin A content in two-way regulation in which it could not only stimulate but (Craft, 2001; Gamble et al., 2001; Erikstrup et al., 2009). RBP4 also inhibit osteoclast formation (Kneissel et al., 2005). Both a itself, previously known as RBP (Wolf, 2007), plays various lack of and excess retinol result in vitamin A abnormalities, important roles and participates in the transport of vitamin A, leading to various defects, including congenital malformations lipid metabolism, and immune and inflammatory responses (Conaway et al., 2013). Thus, because of its various essential (Vaisbuch et al., 2010). RBP4 was also recently shown to play functions in humans, potential biomarkers of vitamin A are also a role as an early marker of fetal growth restriction in maternal diverse (Tanumihardjo, 2011). serum. Besides, RBP4 is reported to be expressed in chondroIn addition to retinol transport proteins, the MS and GO cytes and to be involved in bone growth. Some other studies identianalyses suggested that several other proteins, most of which fied RBP4 as a factor linked to obesity and its co-morbidities, had some connection with lipid metabolism processes, differed including insulin resistance, type 2 diabetes (T2D), and metabetween the groups. Proteins involved in lipid metabolism were bolic syndrome (Fernandez-Real et al., 2008; Kotnik et al., also significantly under-expressed at the protein level in the 2011). Moreover, all-trans retinoic acid (at RA) is a known teragroup with NSCLP, compared with those in the control group, togenic effector in cleft palate and has been shown to be able to including insulin-like growth factor and several lipoproteins. induce cleft palate in animal models (Okano et al., 2007). Also, Both of these types of factors also play vital functions in the in our previous study, we showed a relationship between retinol human body. Thus, the roles of these proteins related to lipid acid and CLP (Yu et al., 2005; Han et al., 2006). metabolism should be identified in our future studies. This background resulted in our hypothesis that vitamins, Our study had several limitations. First, the clinical sample including retinol, are associated with the condition of children size was small and, to some extent, could lead to objective indiwith NSCLP. Since some studies have shown that children with vidual differences, and data related to the diet of these children CLP exhibit a lower growth curve for height, nutrition might should be included to rule out the influence of extrinsic factors. differ between those with NSCLP and unaffected children. It is necessary to plan to confirm these preliminary results, Thus, we assessed the plasma retinol content and demonstrated including the comparison of the RBP4 and vitamin A levels in that vitamin A levels were significantly lower in children with the mothers and the newborn babies with and without NSCLP at NSCLP than in those without. Some studies have reported that different growth points. Conversely, in a future study, we could the risk of facial clefts increased among mothers with lower provide information about more downstream elements of the intakes of vitamin A around the time of conception. Vitamin A pathway, such as retinoic acid to establish the status of the is a crucial factor for diverse functions in the human body. metabolic process. Clinicians should monitor dynamic changes Vitamin A deficiency (VAD) can affect embryogenesis, growth, in serum protein levels—a longitudinal monitoring study would development, immunity, epithelial differentiation, and other help to make these results more credible. Further basic research aspects of human development (Tanumihardjo, 2011). More into the mechanisms underlying NSCLP is necessary, and both recent investigations have indicated that VAD is a public health
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oral clinicians and scientists should expend effort to improve the situation of babies with NSCLP. In conclusion, serum RBP4 and vitamin A levels exhibited statistically significant differences (p < .01) between post-birth babies with NSCLP and unaffected control babies. These results indicated that reduced levels of RBP4 and vitamin A were related to post-birth babies with NSCLP and thus should receive more attention.
Acknowledgments This work was supported by grant 81200762 from the National Natural Science Foundation of China and by funding from the Peking University School of Stomatology (PKUSS20110301). We gratefully acknowledge the assistance of Yan Li from BioyongTech for technical data analysis and her expertise in shotgun proteomics analysis. The authors declare no potential conflicts of interest with respect to the authorship and/or publication of this article.
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