Journal of Radiation and Cancer Research

ORIGINAL ARTICLE
Year
: 2021  |  Volume : 12  |  Issue : 4  |  Page : 147--158

Phospho zinc finger protein: A promising serum biomolecule as noninvasive diagnostic marker of chronic Hepatitis B related liver diseases including liver cancer


Yeshika Bhatia1, Gautam Mondal2, Saimul Islam3, Rishila Ghosh3, Sankhadeep Dutta3, Sudip K Ghosh4, Ajay Duseja1, Chinmay Kumar Panda3, Bishnu Pada Chatterjee3,  
1 Department of Hepatology, Post Graduate Institute of Medical Education and Research, Chandigarh, India
2 Department of Natural Science, Maulana Abul Kalam Azad University and Technology, Kolkata, India
3 Department of Oncogene Regulation, Chittaranjan National Cancer Institute, Kolkata, India
4 Department of Biotechnology, Indian Institute of Technology, Kharagpur, West Bengal, India

Correspondence Address:
Dr. Bishnu Pada Chatterjee
Department of Oncogene Regulation, Chittaranjan National Cancer Institute, Kolkata
India

Abstract

Context: Liver cancer or hepatocellular carcinoma (HCC) is a dreadful complex disease generally occurring from chronic hepatitis B (HBV-CH) due to its latency, which leads to liver cirrhosis and ultimately liver cancer. To prevent cancer at root level, diagnosis of HBV-CH is highly necessary which based on clinical presentation, serum biochemistry, and viral markers. Aim: The aim of the present study was to detect and identify phosphorylated proteins in HBV-CH patients' sera, among chronic hepatitis B-induced liver cirrhosis (HBV-LC) and HCC by developing antibody against this targeted phosphoprotein by enzyme-linked immunosorbent assay (ELISA). This candidate phosphoprotein in patients' sera can be a noninvasive biomarker of HBV-CH. Setting and Design: Our experimental approach was to detect phosphoproteins in HBV-CH, HBV-LC, and HCC, their quantification by ELISA and Western blot. Identification of highly expressed targeted phosphoproteins was done by two-dimensional (2D) gel electrophoresis followed by MALDI-ToF-MS analysis. Antibody is to be developed against synthesized peptide of targeted phosphoprotein of HBV-CH to use by ELISA. This will be a non-invasive approach to identify candidate phosphoprotein as biomarker of HBV-CH. Methodology: Our experimental approach consisted of three steps: (1) detection of serum phosphoproteins by Pro-Q diamond dye in HBV-CH, HBV-LC and HCC patients' groups as well as control subjects; (2) quantification of serum phosphoproteins using different phospho-specific monoclonal antibodies viz., antiphosphoserine (pSer), antiphosphothreonine (pThr), and antiphosphotyrosine (pTyr) antibodies by ELISA and Western blot; (3)identification of differentially expressed phosphorylated proteins in HBV-CH, HBV-LC and HCC by 2D electrophoresis (2DE) followed by in gel trypsin digestion and subsequently by MALDI-ToF-MS analysis. Statistical Analysis Used: Student's t-test and ANOVA was applied for statistical analysis. Results: There were four phosphoprotein bands namely at 25, 50, 70, 75 kDa in HBV-CH, HBV-LC, HCC and control subjects detected by ProQ diamond dye. Besides there appeared one more band at 60 kDa in HCC. The phosphorylation level at serine and threonine residues was highest in HCC patient groups among HBV-CH, HBV-LC and control groups whereas no phosphorylation level of tyrosine was observed among liver disease patient and control groups. Serum phosphorylated proteins were detected and quantified by Western blot. The results were corroborated to those obtained by ELISA. The differential expression of seven phosphoprotein spots was detected in HBV-CH, HBV-LC, HCC patients and control subjects by 2DE and were identified by MALDI-ToF-MS analysis. Conclusion: Thus circulating phosphoproteins could represent important disease biomarkers because of their differential expression in liver diseases.



How to cite this article:
Bhatia Y, Mondal G, Islam S, Ghosh R, Dutta S, Ghosh SK, Duseja A, Panda CK, Chatterjee BP. Phospho zinc finger protein: A promising serum biomolecule as noninvasive diagnostic marker of chronic Hepatitis B related liver diseases including liver cancer.J Radiat Cancer Res 2021;12:147-158


How to cite this URL:
Bhatia Y, Mondal G, Islam S, Ghosh R, Dutta S, Ghosh SK, Duseja A, Panda CK, Chatterjee BP. Phospho zinc finger protein: A promising serum biomolecule as noninvasive diagnostic marker of chronic Hepatitis B related liver diseases including liver cancer. J Radiat Cancer Res [serial online] 2021 [cited 2022 Aug 16 ];12:147-158
Available from: https://www.journalrcr.org/text.asp?2021/12/4/147/332108


Full Text



 Introduction



Reversible protein phosphorylation is a posttranslational modification that plays a critical role in the regulation of a wide spectrum of biological events and cellular functions including signal transduction, gene expression, cell proliferation, and apoptosis.[1] Phosphorylation and dephosphorylation of proteins are also intimately connected to the signalling pathways in the cell. Initial changes in phosphorylation of a receptor usually result in a large number of changes in protein signalling pathways typically associated with major alteration in cell function. Blood plasma/serum proteome is typically used as a source of biomarker, has been studied extensively over the past two decades using proteomics approaches.[2],[3],[4],[5] There have been only limited studies specifically on phosphorylated proteins in the body fluid.[6],[7] The specific phosphorylation state of a polypeptide may also facilitate a better understanding of disease mechanisms by the discovery of biomarkers. Whole sera are particularly attractive for biomarker studies due to its diagnostic potential and non-invasive nature of sample collection.[8],[9],[10] Phosphorylation of serum protein occurs on serine (Ser), threonine (Thr) and tyrosine (Tyr) residue and the level of phosphorylation is <1% of the total protein.[11]

Circulating phosphoproteins could represent important disease biomarkers because of their well-known importance in cellular function. The analysis of phosphoproteins or free phosphopeptides in biological fluids such as saliva, cerebrospinal fluid or human serum has recently been reported.[12],[13],[14] Recently antibodies have been used to study phosphoproteins that selectively recognize phosphorylated amino acid residue, thus enabling a broader search of phosphoproteins.[15] A variety of experimental strategies for the enrichment and detection of phosphorylated proteins has been developed. Newly available sensitive methods such as mass-spectrophotometry coupled with phosphopeptides enrichment using immobilized metal ion affinity chromatography has facilitated these studies. Recently, detection of small amounts of phosphorylated proteins has been achieved using phosphosensor dye technology.[16],[17] This phosphosensitive dye was capable of detecting phosphorylated proteins quantitatively on a global scale. It has been used directly on 1D, two-dimensional (2D) gels and also in a microarray format. Additionally, this technology is appropriate for the determination of protein kinase and phosphatase activity. Therefore, phosphorylation of proteins may be important targets for diagnostic/therapeutic intervention and differential phosphorylation of proteins might be used as potential biomarkers.[18] Mass spectrometry has emerged as the technique of choice for identification of proteins, as well as for analysis of phosphorylation.

Hepatitis B virus (HBV) is an important cause of chronic liver disease in Asia and India leading on to chronic hepatitis B, cirrhosis liver and hepatocellular carcinoma (HCC). HCC is a lethal tumor of the liver and is a common cause of cancer related deaths the world over. Even though the recent data suggests both alcohol and nonalcoholic fatty liver disease as the emerging causes of HCC, HBV-CH still continues to be the commonest of HCC in Asia and India.[19] HCC incidence accounts for 4.7% of all cancer worldwide, affecting both sexes every year. Among the most common cancer occurrences HCC ranks first in the countries of Africa (Egypt) and in South-East Asia (Thailand, Lao, Vietnam and Mongolia). In India it is the 11th most common cancer, nearly 34,743 new cases were diagnosed with HCC in the year 2020.[20]

In the present study, we detected serum phosphoproteins using Pro-Q diamond dye in control and HBV related patients. The level of serum phosphoproteins and their detection was estimated using different phospho-specific antibodies by enzyme-linked immunosorbent assay (ELISA) and Western blot, respectively. Identification of serum phosphoproteins was done by 2D gel electrophoresis followed by MALDI-ToF-MS analysis.

 Methodology



Chemicals

Proteo-prep blue albumin depletion kit, proteomic grade trypsin, α-cyano-4-hydroxycinnamic acid, monoclonal antiphosphoserine antibody, monoclonal antiphosphothreonine antibody, monoclonal antiphosphotyrosine antibody, secondary antimouse immunoglobulin G (IgG) peroxidase were purchased from Sigma Aldrich (USA). Dithiothreitol and iodoacetamide were obtained from Fluka. Pro-Q diamond stain and SYPRO Ruby stain were procured from Invitrogen. All other materials and reagents used were of high analytical grade and obtained from local sources.

Sample collection

Ten microliters of phosphatase inhibitor-cocktail were mixed with 1 ml of collected blood samples and incubated for few minutes at room temperature. Afterwards, blood samples were centrifuged at 2500 × g for 15 min at 4°C and serum layer was collected and stored at-80°C till use. Written informed consent was taken from each individual before collecting samples.

Serum samples

Serum samples from twenty chronic hepatitis B (HBV-CH), twenty hepatitis B-induced liver cirrhosis (HBV-LC) and twenty HCC patients were collected from Liver Clinic, Post Graduate Institute of Medical Education and Research, (PGIMER), Chandigarh, India. The diagnosis of chronic hepatitis B was based on clinical examination and biochemical tests (measurement of serum ALT, AST, ALP, albumin, bilirubin, prothrombin time, and virological investigations (HBsAg, AntiHBe, HBeAg, and HBV DNA). HBV-related liver cirrhosis diagnosed on the basis of clinical, biochemical, and radiological evidence along with the presence of esophageal varices on endoscopy. HBV related HCC was diagnosed on the basis of European Association for the study of the liver criteria, i.e., the presence of hypervascular liver lesion on arterial phase and wash out on portal venous phase on triphagic computed tomography or contract-enhanced magnetic resonance imaging of abdomen with or without elevated alfafeto protein (AFP). Sera from twenty age- and sex-matched healthy individuals served as controls. All the serum samples were stored at −20°C till use. Informed consent was obtained from each patient and healthy individual. Ethical committee of PGIMER, Chandigarh, and Chittaranjan National Cancer Institute, Kolkata, India approved this study. The detailed clinicopathological parameters of the included patients and healthy volunteers are shown in [Table 1].{Table 1}

Depletion of albumin and immunoglobulin G from sera

Albumin and IgG were depleted from plasma using the Proteo-PrepTM blue Albumin depletion kit (Sigma, USA) as per the manufacturer's instruction. The protein concentration of depleted plasma was estimated by Bradford.[21]

Staining of gels with SYPRO Ruby and Pro-Q diamond dye

Identification of phosphoproteins was determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by staining with ProQ diamond fluorescent dye. Equal amount of protein (20 μg) from HBV-CH, HBV-LC, and HCC patients' sera as well as healthy control groups was loaded on 10% SDS-polyacrylamide gel.[22] After SDS-PAGE, gels were fixed in 50% methanol/10% acetic acid and sequentially washed with three changes of water for 10 min each. Then, Pro-Q diamond stain was added to gel and kept for 60 min with shaking in dark condition and destaining was performed by addition of destaining buffer containing 50 mM acetate (pH 4.0) containing 20% (v/v) acetonitrile for 30 min each of three times. Then, the same gel was stained with SYPRO Ruby dye to compare with the Pro-Q diamond stained gel for identification of phosphoproteins. The image of the gel was taken by ultraviolet-visible transilluminator (BIO RAD).

Determination of phosphoprotein level by enzyme-linked immunosorbent assay

Each well of a 96 well microtiter plate (NUNC) was coated with 100 μl monoclonal antiphosphoserine, monoclonal antiphosphothreonine, and monoclonal antiphosphotyrosine antibodies (1 μg/well) in buffer (0.01 M Na2CO3 and 0.035 M NaHCO3, pH 9.6). The plate was left for 24 h at 4°C, washed with 100 μl 0.01 M TBS, pH 7.4, containing 0.005% NP-40, and incubated 1% BSA at 37°C for 1 h. The plate was washed as before. Diluted sera (1:50) of HBV-CH, HBV-LC, and HCC patient groups and control group were added to each well and incubated at room temperature for 1.5 h. After washing, 100 μl of secondary antibody (IgG- Horse reddish peroxidase (HRP), 1:10,000) was added to each well followed by incubation at room temperature for 1.5 h. After washing, 100 ul of 0.1% O-phenylenediamine dihydrochloride and 0.05% H2O2 in 0.05 M citrate phosphate buffer (pH 5.0) was added to wells and plate was left for 30 min at room temperature in dark. The absorbance of each well was measured at 450 nm after stopping the reaction by adding 3 (N) H2SO4. All experiments were done in triplicate and data presented are their mean values.

Western blot analysis for phosphoproteins detection

For Western blotting experiments, equal amount of albumin and IgG depleted proteins (20 μg), from pooled sera of control and patient groups, were separated on 12% SDS-PAGE. After separation, proteins were electroblotted onto nitrocellulose membrane with a constant current 80 mA for 2 h at room temperature.[23] The membranes were blocked with 5% BSA prepared in washing buffer (10 mM TBS containing 0.05% NP-40) for 1 h at room temperature, washed, and were separately incubated with monoclonal antiphosphoserine antibody (pSer), monoclonal antiphosphothreonine antibody (pThre), and monoclonal antiphosphotyrosine antibody (pTyr) (1:2000) in blocking buffer. After washing, the membranes were subsequently incubated with secondary antibody (anti-IgG-HRP, 1:5000) prepared in 0.1% Tris-buffered saline with 20% Tween® 20 (TBST). Reactive protein bands were visualized by addition of diaminobenzidine and 0.01% H2O2 in sodium acetate buffer (pH 5), the intensity of differentially expressed phosphoprotein bands were analyzed using ImageJ software.

Two-dimensional gel electrophoresis

The protein gel corresponding to Western blot picture has cut by sterile surgical blade and gel pieces kept in four individual tubes representing four samples. The interest band pieces have collected from total of four gels by similar manner. Likewise, target band pieces corresponding to phosphoserine and phosphothreonine Western blot was collected from another four 10% SDS polyacrylamide gel. Next, 400 ul of elution buffer (50 mM Tris-HCl, 150 mM NaCl, 0.1 mM EDTA, pH 7.5) was added into each tube, so that gel pieces were completely immersed. In consequence, the gel pieces were crushed by pestle and incubated on a rotary shaker at 30°C for overnight. Next day, all the tubes centrifuged at 10,000 × g for 10 min and carefully pipetted the supernatant into a new microfuge tube. Ultimately, the protein concentration of each sample was determined by Bradford protein estimation method. 2D gel electrophoresis was run after pull down of the equal amount of protein from phosphoserine and phosphothreonine; samples were rehydrated using 180 μg each and run for IEF separation subsequently SDS gel electrophoresis.

In-gel digestion of proteins

The differentially expressed protein spots from 2D gel were excised from the corresponding gels and placed in separate microcentifuge tubes. Gel pieces were washed with milli Q water followed by addition of destaining solution containing K3 [Fe (CN) 6], Na2S2O7 and Na2CO3. Then, 100 mM NH4HCO3 and 100% acetonitrile (ACN) were added into the gel pieces. Finally, gel pieces were dehydrated by addition of 100% ACN and completely dried in speed vac. Then, the gel pieces were rehydrated by the addition of 50 mM NH4HCO3 and 100% ACN followed by addition of proteomics grade trypsin (20 ug/ml). The samples were digested at 37°C for 12 h. After digestion the reaction was stopped by addition of 1% TFA and digested peptides were collected after centrifugation and transferred to a fresh microcentifuge tube. Further, peptides were extracted by the addition of two types of peptides extraction buffer: 50% ACN/0.5% TFA and 1% TFA followed by vortex and centrifugation. The extracted peptides were dried completely by speed vac.

Matrix-assisted laser desorption ionization- time of flight mass spectrometry and database search for protein identification

The peptides were purified with C18 reversed-phase minicolumn filled in a micropipette tip, ZipTip C18 (Sigma). Purified peptides (0.5 μl) were co-crystallized with α-cyano-4-hydroxy cinnamic acid matrix (0.5 μl) (Applied Biosystems) on a matrix-assisted laser desorption ionization (MALDI) target plate. Both mass spectrometry (MS) and MS/MS spectra were acquired by MALDI-time of flight (MALDI-ToF/ToF) Mass Spectrometer (Applied Biosystems 4800 Proteomics Analyzer). It has the GPS Explorer™ software that provides features to remotely submit acquisition jobs to the 4800 Proteomics Analyzer. All spectra were collected in the reflector mode. Spectra were externally calibrated using peptide calibration standard. Protein identification was performed by sending trypsin digested peptide masses to the human databases of National Centre for Biotechnology Information using MASCOT (Matrix Science) peptide mass fingerprinting search engine. The search parameters were as follows: (1) trypsin digestion with one missed cleavage (2) carbamidomethyl modification of cysteine as a fixed modification (3) oxidation of methionine and phosphorylation at serine, threonine and tyrosine residues as a variable modification and peptide mass tolerance range was ± 0.6 Da. The prediction of phosphorylation site was performed using NetPhos 2.0 data base (http://www.cbs.dtu.dk/services/NetPhos/).

Peptide synthesis

Keyhole limpet hemocyanin conjugation

Since the peptide is a small molecule and does not elicit any immune response on its own, it is important to conjugate the peptide with a carrier molecule. Keyhole limpet hemocyanin (KLH) is the most popular and immunogenic carrier protein for the preparation of peptide antigens used for immunization and antibody production. KLH is conjugated with the peptide by using Sulfo-Succinimidyl-4-(N-maleimidomethyl), cyclohexane-1-carboxylate (Sulfo-SMCC). Peptide and the KLH are bound with a di-sulfide bond (covalent binding) between them. The peptide has been chosen on the basis of the phospholyrated site on the full length protein. A Cys residue has been added to the N-terminal of the peptide to facilitate the KLH conjugation. The peptide has been synthesized by the peptide synthesizer. A control (non-phosphorylated) peptide has also been synthesized. Peptides are (1) CLKGFRNSPSALTKHQ, (2) CLKGFRNSSALTKHQ (Control).

 Results



Clinicopathological details of the HBV related patients and healthy volunteers are shown in [Table 1].

Detection of phosphoproteins and total proteins

To obtain an initial profile of whole serum phosphoprotein, serum protein was loaded in SDS-PAGE and stained with fluorescent Pro-Q Diamond dye for detection of phosphoproteins in polyacrylamide gel. This dye permits phosphoproteins identification in a complex protein mixture with sensitivity in the nanogram range. Although this sensitivity is significantly good, it is however not sufficient for comprehensive analysis of the phosphoproteome. For this reason it is important to visualize total protein profile which was done using SYPRO Ruby dye. [Figure 1] shows SDS-polyacrylamide gel stained with ProQ Diamond dye and SYPRO Ruby dye [Figure 1]a and [Figure 1]b, respectively. After ProQ diamond stain, only four phosphoprotein bands around 25, 50, 70, and 75 kDa were identified in all patients' groups and control subjects whereas another one phosphoprotein band around 60 kDa was observed only in HCC patients. On the other hand, many protein bands were observed after SYPRO Ruby staining.{Figure 1}

Determination of phosphorylation level (serine and threonine residue) of serum proteins by enzyme-linked immunosorbent assay

In ELISA, the level of serine-phosphorylated proteins were significantly higher in HCC patient groups (0.142 ± 0.021, P < 0.0002) than control groups (0.119 ± 0.011) [Figure 2], whereas this phosphoprotein level was not significantly higher in HBV-CH (0.122 ± 0.015, P < 0.214) and HBV-LC (0.134 ± 0.017, P < 0.0013) patients' groups. On the other hand, threonine-phosphorylated protein level was also significantly higher in HCC patient groups (0.128 ± 0.021, P < 0.0002) than HBV-CH (0.115 + 0.014, P < 0.064), HBV-LC (0.121 + 0.017, P < 0.013), and control groups (0.109 ± 0.011). No binding was observed with tyrosine-phosphorylated serum proteins when ELISA was performed with pTyr specific monoclonal antibody.{Figure 2}

Detection of serum phosphoproteins by immunoblot analysis

To validate the detection of serum phosphoproteins by ProQ diamond stain, Western blot analysis was performed to identify serum phosphorylated proteins using two types of monoclonal antibodies viz., monoclonal antiphosphoserine and antiphosphothreonine. Three serine-phosphorylated proteins were identified around at 25, 50 and 75 kDa in all patient groups as well as healthy controls when immunoblot was performed using pSer specific monoclonal antibody [Figure 3]a. The intensity level of these three phosphoproteins was analysed by image J software. The expression level of pSer immunoreactive bands at 50 and 25 kDa was significantly higher in HCC patient group then HBV-CH and HBV-LC as compared to control, whereas no such difference in the expression level of 75 kDa band was observed among control and other three patients group HBV-CH, HBV-LCand HCC [Figure 3]b. On the other hand, two threonine-phosphorylated proteins were detected around at 25 and 50 kDa in control as well as all patient groups using pThre specific monoclonal antibody [Figure 4]a. The expression level of two threonine-phosphorylated 25 and 50 kDa proteins were significantly higher in HCC than healthy control, HBV-CH and HBV-LC [Figure 4]b. The total expression of phosphorylation level was corroborated to ELISA results.{Figure 3}{Figure 4}

Two-dimensional gel electrophoresis, protein identification, and expression level

Comparison of the differential protein expression profiles in the sera of HBV-CH, HBV-LC, and HCC patients' group and control subjects was performed by 2-D gel electrophoresis. The protein expression level in HBV-CH, HBV-LC, and HCC patients' group as well as control shows six differentially expressed protein spots as identified by SwissPort database. In addition, a hypothetical protein of 25 kDa was observed in HBV-CH, HBV-LC and HCC. The six differentially expressed proteins [Figure 5] and [Table 2] were identified as alpha-1 antitrypsin (AAT, spot 1), apolipoprotein (A-V, spot-2), haptoglobin beta chain (Hp-β, spot-3), albumin (spot-4), zinc finger protein (spot-5), and Zn-alpha2-glycoprotein (spot-6). Besides spot-7 was marked as hypothetical protein. The expression level of six protein spots is shown in [Figure 6]. The expression level of zinc finger protein (spot-5) in HBV-CH and Zn-alpha2-glycoprotein (spot-6) in HBV-LC were mostly higher in patients' group than control subjects. Hypothetical protein (spot-7) showed gradual increase in patients' group than control subjects [Figure 6].{Figure 5}{Figure 6}{Table 2}

Peptide synthesis

The complete peptide sequence of Zinc finger protein (135) in UniPort was searched based on selected peptide of Phospho Zinc finger protein as obtained from MALDI-MS/MS in the study. The peptide2: CLKGFRNSSALTKHQ was purified. Purification of peptide 2 was shown in [Figure 7] and [Figure 8].{Figure 7}{Figure 8}

 Discussion



Phosphorylation is one of the post translation modifications (PTMs) of protein in which phosphate group (PO4-) is attached to amino acid residues of a particular protein. More than one-third of the protein phosphorylation events occurs on serine (Ser or S), threonine (Thr or T), and tyrosine residues (Tyr or Y) (O-phosphorylation). Protein phosphorylation regulates many important cellular processes such as protein synthesis, cell division, signal transduction, cell growth, development and aging as many enzymes and receptors are activated and deactivated via phosphorylation/dephosphorylation events due to specific kinases and phosphatases. In fact, the phosphorylation of protein is rigidly controlled by interplay of two types of enzymes viz., protein kinases and phosphatases through sequential phosphorylation and dephosphorylation.[24] Recently, it has been reported that aberrant phosphorylation is associated with deregulation of different pathophysiological processes. Frequent aberration of phosphoprotein was found to occur in tissue, serum, saliva and urine of the patients of the different diseases including cancer.[25],[26]

Cancer is the most complex and aggressive disease worldwide as well as in India. Despite advanced diagnosis and treatment, cancers show very high incident of recurrence with worst prognosis of the patient.[27] Thus, effective, novel and precise biomarkers are in the need for early diagnosis and treatment in order to increase the life expectancy of the patients. The biomarker indicates the change in expression of biomolecule which associates with susceptibility and progression of a particular disease including cancer.[28] In order to use biomarker for diagnosis, clinical specimens such as blood, saliva, urine, and other biological fluid of the patients are needed. It is logical to target protein in clinical research for diagnosis as biomarker because the proteins are direct participant of oncogenes. Thus, targeting the serum protein phosphorylation will be effective biomarker of cancer patients.

Serum protein markers are critically important candidates for detecting and predicting liver injury, inflammation and carcinogenesis in chronic HBV patients and are valuable tool to direct antiviral treatment. The parameters ALT, AST, ALP, and prothrombin time as well as liver fibrosis serum markers can indicate the extent of liver injury and degree of inflammation in evaluating, HBV-CH, HBV-LC and HCC infection. However, the current biochemical markers for liver injury are not sufficient to meet clinical needs because sometimes normal ALT levels are observed when serious liver injury and inflammation occur in these patients. Even though liver biopsy is still the gold standard for determining a hepatic inflammation and fibrosis, an invasive procedure, has risk of complications and discomfort to patients. Thus, to minimize the need for biopsy, it is important to explore non-invasive biomarkers that can compensate the deficiencies of current ones. Moreover, the current advanced cancer treatment with antiangiogenesis agents and protein kinase inhibitors showed profound impact on phosphorylation/dephosphorylation of proteins involved in cell proliferation, apoptosis, angiogenesis, tumor progression and metastasis in laboratory and in preclinical and clinical settings. These phosphorylated proteins are secreted from cells into the circulation system and are easily available in the serum.

The commonly used biomarkers for the diagnosis of HCC impute to AFP and des-gamma carboxy protombin which may not be positive in all patients with HCC. In fact, the largest study of HCC from India has recently shown the positivity of AFP (>10 ng) in 74% of patient. Serum phosphoprotein may thus be useful not only in the diagnosis of HBV-CH or HBV-cirrhosis but also for HBV related HCC.[19]

The results of the present study show four phosphoproteins of Mw 25, 50, 70, and 75 kDa, respectively in the sera of HBV-CH, HBV-LC, and HBV-HCC patients as well as healthy volunteers by Pro Q diamond dye besides a number of protein bands appeared by SYPRO Ruby dye. Their presence was confirmed by Western blot analysis and subsequently quantified by ELISA using phosphospecific monoclonal antibodies.

It revealed from ELISA data that phosphoserine and phosphothreonine were found in highest quantity in HCC than HBV-CH, HBV-LC and normal subjects. It is obvious more chronic hepatitis B, HBV-CH advances toward HCC, more phosphorylation on serine and/or threonine residues of serum protein occurs. This result was corroborated to that of Western blot. Western blot analysis was performed to validate the results of serum phosphoproteins by ProQ diamond staining using monoclonal antiphosphoserine and antiphosphothreonine. Three serine-phosphorylated proteins were identified around at 25, 50 and 75 kDa in all patient groups as well as healthy controls. There was no protein of 70 kDa detected by Western blot, which was observed by ProQ diamond dye, because immunoblot analysis is more authentic compared to SDS-PAGE. The expression level of pSer immunoreactive bands at 50 and 25 kDa was significantly higher in HCC patients' group than HBV-CH and HBV-LC as compared to control, whereas no such difference in the expression level of 75 kDa band was observed among control and other three patients groups. Two threonine-phosphorylated proteins were detected around at 25 and 50 kDa in control as well as in all patient groups using pThr specific monoclonal antibody. The expression level of two threonine-phosphorylated 25 and 50 kDa proteins were significantly higher in HCC than healthy control, HBV-CH, and HBV-LC. It indicates phosphorylation occurs at both serine and threonine residues of 25 and 50 kDa proteins. The total expression of phosphorylation level was corroborated to ELISA results.

2D gel electrophoresis of the sera of three patients groups and control subjects produced seven differentially expressed spots of phosphoproteins of different molecular weight. In gel trypsin digestion followed by MALDI-ToF-MS analysis seven protein spots were identified. Of them high expression of Zinc finger protein (ZNF 135) of 70 kDa in HBV-CH, Zinc α2 glycoprotein of 32 kDa, in HBV-LC and one hypothetical protein of 25 kDa in HCC were observed. The change in expression of these three selective phosphoproteins could be considered as biomarkers of liver diseases including liver cancer as well as other cancers when tested by ELISA with their specific antibody to be prepared against the synthesized peptide CLKGFRNSpSALTKHQ (Phosphorylated). Raising antibody against the phosphopeptide and non-phosphopeptide followed by purification and subsequently ELISA will identify the phosphoproteins as biomarker in different liver diseases including liver cancer. The sensitivity and specificity as well as validation of the result will pinpoint the targeted phosphoproteins as biomarker. Thus ZNF (135) could be regarded as specific biomarker of chronic hepatitis B.

 Conclusion



Phosphorylation is important post-translational modification of proteins because phosphoproteins play critical role in the regulation of a broad spectrum of biological processes and cellular functions including signal transduction, gene expression, cell proliferation, and apoptosis. These phosphorylated proteins are secreted from cells in to the circulatory system and present in serum. In this study, seven phosphoproteins were identified and found to be differentially expressed in the serum of patients with chronic hepatitis B, chronic hepatitis B-related liver cirrhosis, and chronic hepatitis B-related HCC. Zinc finger protein (ZNF 135), Zinc α2 glycoprotein, and hypothetical protein were found to be highly expressed in HBV-CH, HBV-LC, and HCC. Peptide of ZNF protein was synthesized with a view to preparation of antibody to test by ELISA. This process will identify ZNF protein as biomarker of chronic hepatitis B.

Acknowledgements

The authors are thankful to Dr Hafiz Ahmed, Dr Abu Hena Hasanur Reja and Dr Anirban Roychowdhury for discussion in this study.

Financial support and sponsorship

This work is financially supported by ICMR.

Conflicts of interest

There are no conflicts of interest.

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