- Open Access
Enhanced expression of a 35 kDa fragment of inter-alpha-trypsin inhibitor H4 in sera of healthy pregnant women and patients with hydatidiform mole
© Mohamed et al.; licensee BioMed Central Ltd. 2013
- Received: 8 March 2013
- Accepted: 8 May 2013
- Published: 15 May 2013
Accumulated data from previous studies appear to suggest a link between the overexpression of a 35 kDa fragment of serum inter-alpha-trypsin inhibitor H4 (ITIH4) with cancers that are associated with up-regulated levels of oestrogens. The truncated fragment was postulated to be a product of oestrogen-induced action of kallikrein on native ITIH4. The present lectin-based proteomic analyses were performed to assess the specificity of the 35 kDa fragment of ITIH4 as a potential cancer biomarker and determine whether it was also overexpressed in the sera of cancer-negative pregnant women who are known to have high levels of plasma oestrogens.
Our results demonstrated that the 35 kDa fragment of ITIH4 was overexpressed in healthy pregnant women and patients with hydatidiform mole, relative to the controls. The serum oestradiol levels of both groups of pregnant subjects were also confirmed to be higher than those of the control women who were not pregnant.
Overexpression of the 35 kDa fragment of ITIH4 was not restrictive to patients with cancers but also occurred in women who were pregnant and those diagnosed with hydatidiform mole. Our data implicate the limitation of the 35 kDa ITIH4 fragment as a cancer biomarker and its correlation with serum oestrogen levels.
- Endometrial Cancer
- Epithelial Ovarian Cancer
- Healthy Pregnant Woman
- Hydatidiform Mole
- Serum Oestrogen
Inter-alpha-trypsin inhibitor heavy chain 4 (ITIH4) is a member of the inter-alpha-trypsin inhibitor (ITI) family of hepatic origin [1, 2]. It is the only member of the ITI family which harbours a kallikrein-released bradykinin-like domain in its C-terminal sequence , making it plasma kallikrein sensitive [4–6]. Trace amounts of plasma kallikrein have been shown to cleave ITIH4 to yield two fragments, i.e., a 35 kDa C-terminal polypeptide and an 85 kDa N-terminal fragment . The 35 kDa ITIH4 fragment, which is O-glycosylated [3, 8], is assumed to remain intact. However, the 85 kDa ITIH4 fragment is further cleaved to produce an N-terminal 57 kDa fragment and a putative 28 kDa fragment. The latter is believed to be further processed by protease(s) to generate smaller fragments .
We have previously analyzed the expression of the 35 kDa ITIH4 fragment in groups of patients with nine different types/subtypes of cancers using the gel-based proteomics approach and a lectin that binds to O-glycosylated proteins [9–12]. Whilst the serum ITIH4 fragment was demonstrated to be overexpressed in patients with endometrial cancer, ovarian cancer (germ-line and epithelial ovarian carcinoma) and breast cancer compared to the control subjects, its levels were not significantly different in sera of patients with nasopharyngeal carcinoma, osteosarcoma (localized disease), cervical cancer (squamous cell cervical carcinoma and cervical adenocarcinoma) and prostate cancer [9–11]. In the latter cancer, however, significantly enhanced levels of a similar ITIH4 fragment was later detected in the urine of the patients .
One of the obvious differences between the types of cancers that are associated with overexpression of the 35 kDa ITIH4 fragment with those that did not is that the former also appears to be associated with the up-regulated levels of serum oestrogens [13, 14]. This is suggestive of the potential use of the ITIH4 fragment as a complementary biomarker for oestrogen-related cancers. However, enhanced blood oestrogen levels are not restrictive to patients with cancer but also occur in healthy women who are pregnant as well as those detected with benign tumours. Hence, the present study was carried out to assess the specificity of the 35 kDa ITIH4 fragment as a potential biomarker for cancer and whether it was also overexpressed in two groups of cancer-negative women who were pregnant and are known to have high levels of plasma oestrogens. The first group of subjects comprised healthy pregnant women, whilst the second were those who were diagnosed with hydatidiform mole, a type of gestational trophoblastic disease that is associated with a rare mass or growth that forms inside the uterus at the beginning of a pregnancy.
Determination of serum oestradiol levels
Two-dimensional electrophoretic profiles of serum O-glycosylated proteins
In this study, the expression of a 35 kDa ITIH4 fragment, which is O-glycosylated, was analysed using an earlier established method involving two-dimensional gel electrophoresis (2-DE), western blotting and the use of champedak galactose binding (CGB) lectin to detect O-glycosylated proteins [9–12]. The CGB lectin was chosen on the basis of its specific interaction with O-glycans [15, 16].
Identification of ITIH4 fragment spot cluster
Confirmation of the identity of serum ITIH4 by MS/MS
Spot/ Cluster ID
Matched protein identity
Swiss-prot accession number
Theore-tical Mass (Da)
No. of peptides matched
Sequence coverage (%)
Inter-alpha-trypsin inhibitor heavy chain H4 (a)
Image analysis of O-glycosylated ITIH4 fragment
Our previous accumulated studies have demonstrated the overexpression of a 35 kDa ITIH4 fragment selectively in cancers associated with elevated oestrogen levels, including cancers of the breast, endometrium, ovary and prostate but not in nasopharyngeal carcinoma, osteosarcoma and cervical cancer. To investigate the possibility that the 35 kDa ITIH4 fragment may also be enhanced in non-cancer conditions that are associated with up-regulated levels of oestrogens, analysis was extended to include two groups of non-cancer patients with similar hormonal dysregulation. In the present study, two groups of women with different pregnancies and with increased levels of oestrogens were chosen. The first group comprised healthy women who were pregnant, which represents a normal condition, while the second involved patients with hydatidiform mole and represents a benign condition.
The maternal levels of circulating oestrogens increase continuously throughout a normal pregnancy, as it is required to support foetal development [17–19]. This is also seen when the healthy women subjects who were pregnant were analysed for their serum oestrogens in the present study. Similarly, women with hydatidiform mole have also been reported to have plasma oestrogen levels as high as those with normal pregnancy  and this is also reflected from their serum oestradiol values that were determined in this study.
The marked difference in the expression of the 35 kDa ITIH4 cleavage fragment detected in the various subjects with enhanced oestrogen levels may be attributed to the increased cleavage of ITIH4 by elevated levels of circulating kallikreins in the serum. The idea that the abundance of the ITIH4 fragment is linked to high amounts of serum kallikreins is derived from previous reports demonstrating overexpression of members of the kallikrein family in cancers of the breast, ovary and endometrium [21–23]. This is not surprising as kallikreins are known to be expressed in hormone-dependant tissues such as the breast and ovary . In addition, the expression of the kallikrein genes have been shown to be regulated by steroid hormones (including oestrogens) in cancer cell lines [22, 25]. As the ITIH4 protein is kallikrein-sensitive, there is likelihood that the overexpression of kallikrein may lead to increased cleavage of serum ITIH4 which in turn led to the enhanced liberation of its 35 kDa C-terminal fragment. In support of this correlation is a study conducted by Gangadharan et al. , which showed that the down-regulation of kallikrein in patients with hepatic cirrhosis resulted in low abundance of the ITIH4 fragments including the 35 kDa fragment.
The data of the present study, when taken together with those of our previous reports, suggest that overexpression of the 35 kDa fragment of ITIH4 is oestrogen-related and occurs in patients with selective cancers, hydatidiform mole as well as healthy women who are pregnant. This implicates the limitation of the ITIH4 fragment as a biomarker for cancer.
Collection of serum samples
Serum samples were collected with patients’ consent at the Obstetrics and Gynaecology ward, University of Malaya Medical Centre (UMMC), Kuala Lumpur in accordance to a protocol that was approved by the Medical Ethics Committee of the centre. Samples from groups of women who were pregnant (n = 20) and patients with hydatidiform mole (n = 20) were collected in their first trimester of pregnancy. For comparison, sera from normal healthy non-pregnant women (n = 20) were obtained from age-matched volunteers (range of 21–45 years). Blood samples were collected in fresh 1.5 ml BD vacutainers (Becton, Dickinson & Co, Franklin Lakes, New Jersey, USA) and were centrifuged at 3000 g for 10 min (Centrifuge 5403, Eppendorf, Hamburg, Germany). Serum was collected and stored in aliquots of 100 μl at −80° until used.
Quantitative measurements of oestradiol in the subjects’ serum samples were performed according to the manufacturer’s instructions using the ADVIA Centaur and ADVIA Centaur XP Systems (Siemens Medical Solutions Diagnostics, Tarrytown, USA). Values of serum oestradiol are expressed in mean ± SEM.
2-DE was performed as previously described  using approximately 800 μg protein. Neat serum samples were initially incubated in 2% v/v IPG sample buffer pH 4–7, containing 9 M urea, 60 mM DTT, and 0.5% v/v Triton X-100 at room temperature for 30 min. They were then incubated in a rehydration solution containing 8 M urea, 0.5% v/v IPG buffer, 0.5% v/v Triton X-100 for another 30 min before incubating with rehydrated IPG Immobiline Drystrips pH 4–7, 11 cm (GE Healthcare, Uppsala, Sweden) overnight. The strips were subjected to isoelectric focusing using the Multiphor Flatbed electrophoresis system (GE Healthcare, Uppsala, Sweden) for a total duration of 15 kV/h (Phase 1: 300 V, 2 mA, 5 W, 30 min; Phase 2: 3500 V, 2 mA, 5 W, ∼4–4.5 h). Focused strips were equilibrated in 1.5 M Tris–HCl (pH 8.8) solution containing 6 M urea, 2% w/v SDS, 30% v/v glycerol, and 0.06 M DTT for 15 min on a UNIMAX 2010 platform shaker and further incubated in a similar equilibration solution but containing 4.5% v/v iodoacetamide instead of DTT for another 15 min. The equilibrated strips were overlaid onto 8-18% gradient polyacrylamide gels and electrophoresis was performed following an optimized protocol (Phase 1: 50 V, 40 mA, 25 W for 30 min; Phase 2: 600 V, 40 mA, 25 W for 1–2 h) using the SE 600 Ruby Electrophoresis System and Power Supply-EPS601 (GE Healthcare, Uppsala, Sweden).
Western blotting and detection of O-glycosylated proteins
The 2-DE-separated proteins were transferred electrophoretically onto nitrocellulose (NC) membranes (0.45 mM; Whatman, Dassel, Germany) using the NovaBlot Kit of the Multiphor™ II Flatbed System (GE Healthcare, Uppsala, Sweden) for 2 h at a constant current of 0.8 mA/cm2 gel. Detection of transferred O-glycosylated serum proteins was performed using the CGB lectin that was affinity purified and characterized for its specificity to O-glycans using methods that were previously reported . The lectin was then conjugated to horseradish peroxidase before being used to probe for O-glycopeptides on the NC membranes. The membranes were finally developed by means of a colorimetric reaction using diamino-benzoic acid as substrates.
Protein spots of interest were carefully excised from the blot for the subsequent on-membrane trypsin digestion according to method that was previously described . The MS/MS analysis was performed using the 4800 Plus MALDI ToF/ToF analyzer (Applied Biosystems, Foster City, CA, USA).
Identification of proteins was performed using the MASCOT search engine . The MS data obtained was searched against Homo sapien entries in the Swiss-Prot database (Last update: February 15, 2012, containing 535248 sequences) according to the following selection parameters: enzyme - trypsin, missed cleavage - 1, variable modification - 2; i) carbamidomethylation of cysteine and ii) oxidation of methionine, MS precursor ion mass tolerance - 100 ppm, MS/MS fragment ion mass tolerance - 0.2 Da, and inclusion of monoisotopic masses only.
CGB lectin-probed NC blots were scanned using Imaging Densitometer GS690 (Bio-Rad Laboratories, Hercules, California, USA). Expression of ITIH4 fragment was analysed in terms of the percentage of volume contribution, which refers to the spot volume of the glycoprotein expressed as a percentage of the total spot volume of all detected serum glycoproteins, using the Image Master 2D Platinum software, version 7.0 (GE Healthcare Biosciences, Uppsala, Sweden). Cut-off parameters were: Smooth – 2; Saliency – 1; Min area – 5. Data expressed in this manner are independent of variations attributed to protein loading and staining.
This work was funded by the HIR-MOHE H-20001-00-E000009 research grant from the University of Malaya.
- Pineiro M, Alava MA, Gonzalez-Ramon N, Osada J, Lasierra P, Larrad L, Pineiro A, Lampreave F: ITIH4 serum concentration increases during acute-phase processes in human patients and is up-regulated by interleukin-6 in hepatocarcinoma HepG2 cells. Biochem Biophys Res Commun 1999, 263: 224–229. 10.1006/bbrc.1999.1349PubMedView ArticleGoogle Scholar
- Salier JP, Rouet P, Raguenez G, Daveau M: The inter-alpha-inhibitor family: From structure to regulation. Biochem J 1996, 315: 1–9.PubMed CentralPubMedView ArticleGoogle Scholar
- Nishimura H, Kakizaki I, Muta T, Sasaki N, Pu PX, Yamashita T, Nagasawa S: cDNA and deduced amino acid sequence of human pk-120, a plasma kallikrein-sensitive glycoprotein. FEBS Lett 1995, 357: 207–211. 10.1016/0014-5793(94)01364-7PubMedView ArticleGoogle Scholar
- Cai T, Yu P, Monga SP, Mishra B, Mishra L: Identification of mouse ITIH-4 encoding a glycoprotein with two EF-hand motifs from early embryonic liver. Biochim Biophys Acta 1998, 1398: 32–37. 10.1016/S0167-4781(98)00049-9PubMedView ArticleGoogle Scholar
- Hashimoto K, Tobe T, Sumiya J, Sano Y, Choi-Miura NH, Ozawa A, Yasue H, Tomita M: Primary structure of the pig homologue of human IHRP: Inter-alpha-trypsin inhibitor family heavy chain-related protein. J Biochem 1996, 119: 577–584. 10.1093/oxfordjournals.jbchem.a021281PubMedView ArticleGoogle Scholar
- Saguchi K, Tobe T, Hashimoto K, Sano Y, Nakano Y, Miura NH, Tomita M: Cloning and characterization of cDNA for inter-alpha-trypsin inhibitor family heavy chain-related protein (IHRP), a novel human plasma glycoprotein. J Biochem 1995, 117: 14–18.PubMedGoogle Scholar
- Pu XP, Iwamoto A, Nishimura H, Nagasawa S: Purification and characterization of a novel substrate for plasma kallikrein (pk-120) in human plasma. Biochim Biophys Acta 1994, 1208: 338–343. 10.1016/0167-4838(94)90122-8PubMedView ArticleGoogle Scholar
- Song J, Patel M, Rosenzweig CN, Chan-Li Y, Sokoll LJ, Fung ET, Choi-Miura NH, Goggins M, Chan DW, Zhang Z: Quantification of fragments of human serum inter-alpha-trypsin inhibitor heavy chain 4 by a surface-enhanced laser desorption/ionization-based immunoassay. Clin Chem 2006, 52: 1045–1053. 10.1373/clinchem.2005.065722PubMedView ArticleGoogle Scholar
- Abdul-Rahman PS, Lim BK, Hashim OH: Expression of high-abundance proteins in sera of patients with endometrial and cervical cancers: Analysis using 2-DE with silver staining and lectin detection methods. Electrophoresis 2007, 28: 1989–1996. 10.1002/elps.200600629PubMedView ArticleGoogle Scholar
- Mohamed E, Abdul-Rahman PS, Doustjalali SR, Chen Y, Lim BK, Omar SZ, Bustam AZ, Singh VA, Mohd-Taib NA, Yip CH, Hashim OH: Lectin-based electrophoretic analysis of the expression of the 35 kDa inter-alpha-trypsin inhibitor heavy chain H4 fragment in sera of patients with five different malignancies. Electrophoresis 2008, 29: 2645–2650. 10.1002/elps.200700828PubMedView ArticleGoogle Scholar
- Jayapalan JJ, Ng KL, Razack AH, Hashim OH: Identification of potential complementary serum biomarkers to differentiate prostate cancer from benign prostatic hyperplasia using gel- and lectin-based proteomics analyses. Electrophoresis 2012, 33: 1855–1862. 10.1002/elps.201100608PubMedView ArticleGoogle Scholar
- Jayapalan JJ, Ng KL, Shuib AS, Razack AH, Hashim OH: Urine of patients with early prostate cancer contains lower levels of light chain fragments of inter-alpha-trypsin inhibitor and saposin B but increased expression of an inter-alpha-trypsin inhibitor heavy chain 4 fragment. Electrophoresis 2013. 10.1002/elps.201200583Google Scholar
- Key TJ, Verkasalo PK: Banks E:Epidemiology of breast cancer. Lancet Oncol 2001, 2: 133–140. 10.1016/S1470-2045(00)00254-0PubMedView ArticleGoogle Scholar
- Henderson BE, Feigelson HS: Hormonal carcinogenesis. Carcinogenesis 2000, 21: 427–433. 10.1093/carcin/21.3.427PubMedView ArticleGoogle Scholar
- Hashim OH, Ng CL, Gendeh GS, Nik Jaafar MI: IgA binding lectins isolated from distinct Artocarpus species demonstrate differential specificity. Mol Immunol 1991, 28: 393–398. 10.1016/0161-5890(91)90152-APubMedView ArticleGoogle Scholar
- Hashim OH, Gendeh GS, Jaafar MI: Comparative analyses of IgA1 binding lectins from seeds of six distinct clones of Artocarpus integer . Biochem Mol Biol Int 1993, 29: 69–76.PubMedGoogle Scholar
- Takeyama J, Suzuki T, Inoue S, Kaneko C, Nagura H, Harada N, Sasano H: Expression and cellular localization of estrogen receptors alpha and beta in the human fetus. J Clin Endocrinol Metab 2001, 86: 2258–2262. 10.1210/jc.86.5.2258PubMedGoogle Scholar
- Loriaux DL, Ruder HJ, Knab DR, Lipsett MB: Estrone sulfate, estrone, estradiol and estriol plasma levels in human pregnancy. J Clin Endocrinol Metab 1972, 35: 887–891. 10.1210/jcem-35-6-887PubMedView ArticleGoogle Scholar
- Cunningham FG, Leveno KJ, Bloom SL, Hauth JC, Rouse DJ, Spong CY: Williams Obstetrics. McGraw-Hill Companies Inc: McGraw-Hill; 2010.Google Scholar
- Hegab HM, Schindler AE: The prognostic value of serum inhibin, 17 beta-estradiol and progesterone in cases of hydatidiform mole. Gynecol Endocrinol 2004, 18: 107–113. 10.1080/09513590310001652991PubMedView ArticleGoogle Scholar
- Borgono CA, Diamandis EP: The emerging roles of human tissue kallikreins in cancer. Nat Rev Cancer 2004, 4: 876–890. 10.1038/nrc1474PubMedView ArticleGoogle Scholar
- Myers SA, Clements JA: Kallikrein 4 (klk4), a new member of the human kallikrein gene family is up-regulated by estrogen and progesterone in the human endometrial cancer cell line, KLE. J Clin Endocrinol Metab 2001, 86: 2323–2326. 10.1210/jc.86.5.2323PubMedView ArticleGoogle Scholar
- Diamandis EP, Yousef GM, Soosaipillai AR, Bunting P: Human kallikrein 6 (zyme/protease m/neurosin): A new serum biomarker of ovarian carcinoma. Clin Biochem 2000, 33: 579–583. 10.1016/S0009-9120(00)00182-XPubMedView ArticleGoogle Scholar
- Diamandis EP, Yousef GM: Human tissue kallikreins: A family of new cancer biomarkers. Clin Chem 2002, 48: 1198–1205.PubMedGoogle Scholar
- Yousef GM, Kyriakopoulou LG, Scorilas A, Fracchioli S, Ghiringhello B, Zarghooni M, Chang A, Diamandis M, Giardina G, Hartwick WJ, Richiardi G, Massobrio M, Diamandis EP, Katsaros D: Quantitative expression of the human kallikrein gene 9 (klk9) in ovarian cancer: A new independent and favorable prognostic marker. Cancer Res 2001, 61: 7811–7818.PubMedGoogle Scholar
- Gangadharan B, Antrobus R, Dwek RA, Zitzmann N: Novel serum biomarker candidates for liver fibrosis in hepatitis C patients. Clin Chem 2007, 53: 1792–1799. 10.1373/clinchem.2007.089144PubMedView ArticleGoogle Scholar
- Doustjalali SR, Yusof R, Yip CH, Looi LM, Pillay B, Hashim OH: Aberrant expression of acute-phase reactant proteins in sera and breast lesions of patients with malignant and benign breast tumors. Electrophoresis 2004, 25: 2392–2401. 10.1002/elps.200305950PubMedView ArticleGoogle Scholar
- Abdul Rahman M, Anuar Karsani S, Othman I, Abdul Rahman PS, Hashim OH: Galactose-binding lectin from the seeds of champedak ( Artocarpus integer ): Sequences of its subunits and interactions with human serum O -glycosylated glycoproteins. Biochem Biophys Res Commun 2002, 295: 1007–1013. 10.1016/S0006-291X(02)00795-7PubMedView ArticleGoogle Scholar
- Luque-Garcia JL, Neubert TA: On-membrane tryptic digestion of proteins for mass spectrometry analysis. In Methods in molecular biology, protein blotting and detection. New Jersey: Humana Press; 2009:pp 331–341.View ArticleGoogle Scholar
- Perkins DN, Pappin DJ, Creasy DM, Cottrell JS: Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 1999, 20: 3551–3567. 10.1002/(SICI)1522-2683(19991201)20:18<3551::AID-ELPS3551>3.0.CO;2-2PubMedView ArticleGoogle Scholar
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