Discussion
HE-4 is upregulated in ovarian carcinoma and also expressed in a variety of lung tumors. There are several reports in the literature which suggest important functions of HE-4: over-expression in ovarian carcinoma, over-expression in prostate cancer model mice with PTEN inactivation, interaction with pleiotrophin (PTN) which regulates angiogenesis and is involved in tumor formation as well as the upregulation of HE-4 during the expected window of receptivity in the endometrium under the control of progesterone in primates [20,30?2]. All of these evidences point towards an important role of HE-4 in human physiology but to date no study has tried to investigate the role or structure of HE-4 in normal or pathological conditions. We have developed a rapid and efficient method for purification of native HE-4 from human seminal fluid, which will help study the function and structure of HE-4.
HE-4 is glycosylated and highly stable protein
HE-4 exists as a disulfide bonded trimer in human seminal fluid as inferred from SDS-PAGE in reducing and non-reducing conditions. This is not unheard of in proteins and many human and some viral proteins employ intermolecular disulfide linkages to form tertiary structure [33?4]. HE-4 has eight predicted disulfide bonds per monomer of protein as it is a small protein, these bonds and intermolecular disulfide linkages would be expected to give it a compact structure resistant to denaturing agents. Accordingly, it found that HE-4 is resistant to pH, heat and even SDS in protease inhibition assay taking trypsin as a model protease. Seminal fluid has a high concentration of zinc and it regulates the function of several seminal fluid proteins like PSA and sememnogelin [35].Therefore, we measured Rh value and activity of HE-4 in
Figure 3. Multiple sequence alignment of HE-4 with other WAP protein and HE-4 of different species. At the end of each line of amino acid sequence, amino acid residue numbers are given and after that % homology is written. (A) Sequence alignment of HE-4 WAP 1 domain with other antiproteases like elafin and WAP-2 domain of SLPI. (B) Sequence alignment of HE-4 WAP2 domain with other antiproteases like elafin and SLPI. (C) Multiple sequence alignment of HE-4 protein of human with HE-4 protein of different species.
the presence of zinc and we observed lower Rh than that of purified native protein in Tris buffer. Introduction of EDTA in twice the molar ratio of zinc increased the Rh value a little bringing it closer to native HE-4. Trypsin inhibition activity of HE-4 reduced slightly in the presence of zinc (mean: 83%) while the addition of EDTA (twice the molar ratio) to the Zn supplemented HE-4 recovered the activity a little (mean: 85.6%) as shown in figure 4D. The activity lost by 2 mM ZnCl2 could be completely rescued by addition of increasing concentrations of EDTA (Figure 4E). At this stage evidence for mechanism of effect of zinc on activity and structure of HE-4 is inconclusive. However, this idea is being pursued further in our laboratory. Disulfide bonds play an important role in maintaining the native conformation of a protein, which in turn provide stability/ resistance towards pH and temperature treatments. HE-4 contains 8 disulfide bonds. Therefore, it was of interest to evaluate the effect of DTT reduction on the trypsin inhibitory activity of HE-4. Even at .05 mM there was a significant reduction in trypsin inhibitory activity of HE-4 and at 1 mM HE-4 lost all its activity against trypsin (Figure 4F). Disulfide bond reduced HE-4 was oxidized and refolded (as described in methods) but HE-4 was unable to restore the activity and at 16 hr. time-point only 3% activity was restored. PNGase F treatment of HE-4 produced a shift of approximately 2.5 kDa which is considerable given the molecular weight of the monomer (Figure 2B). This also partly explains the observed heat and pH resistance of the protein. Asn44 has been reported to be glcyosylated in salivary HE-4 previously [36], and no other glycosylation site has been either predicted or reported.
This implies that Asn44 which lies in the N-terminal WAP domain is heavily glycosylated. One previous study has reported HE-4 to be a secreted glycosylated protein of approximately 25 kDa in two ovarian carcinoma cell lines [32]. This difference with seminal fluid form of protein could be cell line specific, or it could be another isoform of the protein resulting from alternative splicing [36]. HE-4 can undergo alternative splicing to yield four different isofroms other than full length protein. Some of these isoforms has only N-terminal WAP domain and some C-terminal WAP domain while three of these isoforms own unique sequence not found in others. So this 25 kDa isoform found in ovarian cancer lines could be different from the presumably full length (based on molecular weight from SDS-PAGE) protein we have in seminal fluid, and it might even lack trimerization property, or have a different glycosylation pattern. Importance of glycosylation in functions of HE-4 (seminal fluid or other sites) requires further studies which are underway in the laboratory.
HE-4 is a disulfide-bonded trimer and bioinformatic analysis alone does not correctly predict its function
A consensus sequence was obtained by aligning HE-4 sequences form different species (Fig. 3C). Though the high degree of similar conservation of residues and spacing between them points towards a similarity in functional aspects of both domains, but we cannot deny the fact that length difference between first and second cysteine residues may contribute to the different functions of these domains or the ability to covalently oligomerise, which requires inter-molecular disulfide bridges. WFDC domain of elafin and oneFigure 4. Antiprotease activity of HE-4 at different concentration and at different condition against various serine, cystine and aspartic proteases. (A) Percentage inhibition of various proteases like: trypsin, chymottrypsin, PSA, proteinase K, pepsin and papain by HE-4. X-axis is the HE-4 concentration in mg/ml and Y-axis is the % inhibition of respective proteases. (B) Effect of temperature (25uC?00uC) on the protease inhibition activity of HE-4 against trypsin. (C) Effect of pH (2?0) on the protease inhibition activity of HE-4 against trypsin. (D) Effect of different chemicals like: ZnCl2 SDS and b-mercaptoethanol on the protease inhibition activity of HE-4 using trypsin as model protease. EDTA was added to ZnCl2 pre-treated HE-4 to check whether it can rescue the decrease in activity induced by ZnCl2. SDS control is the % inhibition of trypsin by 5% SDS and 5% SDS point is the activity of SDS-pretreated HE-4 against trypsin. +ve control is HE-4 inhibition of trypsin without any treatment and 2ve control is without HE-4, therefore 0% inhibition. (E) Effect of varying concentrations of EDTA on rescue of trypsin % inhibition decrease by ZnCl2 treatment of HE-4. (F) Effect of varying concentration of DTT on trypsin inhibitory activity of HE-4. of the two WFDC domains of SLPI is known to impart antiproteinase activity to both of these proteins. Therefore, it is a widely held notion that all of the members of the family will have anti-proteinase activity. Sequence analysis by Bingle et al showed that only these two members have the same spacing between cysteines essential for protease inhibition [19]. Authors of the paper argue that no other member of the WFDC family contains the same spacing, so it is not necessary that they will have antiproteinase activity. A separate study by Hu et al show that both wild type and WAP mutated KAL-1 enhanced amidolytic activity of uPA (urokinase-type plasminogen activator) [37] which are consistent with the prediction made by Bingle et al.