Golgi protein 73 and its diagnostic value in liver diseases

Abstract Golgi protein 73 (GP73, also referred to as Golph 2) with 400 amino acids is a 73 kDa transmembrane glycoprotein typically found in the cis‐Golg complex. It is primarily expressed in epithelial cells, which has been found upregulated in hepatocytes in patients suffering from both viral and non‐viral liver diseases. GP73 has drawn increasing attention for its potential application in the diagnosis of liver diseases such as hepatitis, liver cirrhosis and liver cancer. Herein, we reviewed the discovery history of GP73 and summarized studies by many groups around the world, aiming at understanding its structure, expression, function, detection methods and the relationship between GP73 and liver diseases in various settings.


| Structural features of GP73
The GP73 gene is located in the short arm of chromosome 9 at position 9q21. 33. It has a total length of 3042 bp, containing 9 introns and 10 exons. It also contains a unique open-reading frame (1200-1430 bp) that encodes 400 amino acids. [80][81][82][83][84] The 3′ untranslated region of GP73 gene contains three polyadenylation sites (3003, 2950 and 1448 bp) and a stop codon (bp 1351). The encoded protein contains more acidic amino acids, such as aspartic acid and glutamic acid, and the isoelectric point for the amino acid is 4.72, containing single transmembrane region and signal peptidase cleavage site aa28-aa29 and N-terminal hydrophobic ( Figure 1). [85][86][87][88][89][90][91][92][93] The amino acid encoded by C-terminus is located in the extracellular region and contains 5 glycosylation sites and 14 cellulose acylated sequences. [94][95][96] Several coiled coils immediately after the transmembrane region can participate in the interaction between proteins, and their characteristics indicate that the GP73 protein can interact with other proteins through the extracellular region.

| Expression and distribution of GP73
The GP73 promoter −2618 bp/−19 bp has specificity for epithelial cells, so GP73 is widely expressed in almost all epithelial cells. 14,95,97-103 Results from immunohistochemistry have also shown that the GP73 protein can be expressed in many types of cells in normal human tissues, but most important is epithelial cells, where the expression levels are very different. The difference may be up to 20 F I G U R E 1 A, GP73 expression levels in hepatocytes in the course of acute and chronic liver disease. Normal hepatocyte GP73 expression is minimal. Marked and reversible increases in the percentage of GP73-positive hepatocytes, and their cellular GP73 levels occur in acute hepatitis. Progressive up-regulation is also observed in chronic liver disease and can be reversed by treatment of underlying disease aetiology (ie, steroid treatment of autoimmune hepatitis) and resolution of chronic hepatitis. GP73 expression in hepatocellular cancer cells is further increased (2to 3-fold) in comparison with patients with cirrhosis. B, Structural features of GP73 and sGP73. GP73 consists of a short cytoplasmic N-terminus with a myristoylation domain followed by a single TM and large C-terminal ectodomain. The N-terminus contains two coiled-coil domains and is N-glycosylated. The C-terminus is highly acidic of unknown function. Furin cleavage after aa 55 results in the extracellular release of sGP73. aa, amino acid; ELISA, enzyme-linked immunosorbent assay; GP73, Golgi protein 73; HCC, hepatocellular carcinoma; sGP73, serum form of Golgi protein 73; TM, transmembrane domain 103 times. 9,77,104-108 GP73 mRNA is expressed at higher levels in bronchial, gastric, colonic and prostate tissues, but with lower or no expression in muscle, white blood cells, lymphoid tissues and hearts. 72,[109][110][111][112][113][114][115][116][117][118] The most densely expressed GP73 site is the digestive tract. It is also expressed in bile duct epithelial cells, tubular monolayer epithelial cells, pulmonary bronchioles, islets and hippocampus.
The high expression of GP73 belongs to epithelial cells, and these parts have well-developed Golgi apparatus whose cells have strong secretory ability. 9,119-124 GP73 is mainly expressed in normal liver tissue by bile duct epithelial cells in the portal area. This site is typical for the Golgi apparatus in epithelial cells but it has less or no expression in normal liver cells. [125][126][127][128][129] However, in the presence of adenovirus infection, HBV or cancer, the expression level of GP73 in the bile duct cells is not changed significantly, but it is consistently high in liver cells, which increases the expression of GP73. These results indicate that hepatocytes undergo some transformation when liver diseases occur, altering some of the factors that regulate the expression of GP73. Studying these regulatory mechanisms will thus help to understand pathogenesis of liver diseases. [130][131][132][133][134][135][136]

| Influencing factors for GP73 expression
GP73 expression is affected by many factors, such as viral infections, for example, the GP73 expression is increased in adenovirusinfected HepG2 cells. The GP73 protein and mRNA expression are also increased after adenovirus-infected Hep3B liver cancer cells.
The expression level of GP73 is no longer increased under lack of adenovirus E1A domain, indicating that the GP73 expression is associated with adenovirus infection. [137][138][139][140] The expression of GP73 is also increased in HBV-replicated cells, whereas the expression of GP73 does not increase in cells transfected with HBV but without HBV replication, indicating that HBV replication may stimulate the expression of GP73, and hence, the GP73 expression is associated with HBV replication. 141 Moreover, studies have shown that the

| Functions of GP73
At present, the biological function of GP73 is not fully understood.
The Golgi apparatus is a complex processing centre in the cell, and its abnormal function is closely related to occurrence and development of many diseases, such as muscular dystrophy and defects of innate glycoprotein glycosylation. 145,146 GP73 is located in the Golgi apparatus, so GP73 is very likely to be closely related to the structure and functions of Golgi apparatus, which are intracellular transport, protein modification and signal transduction. Studies have shown that the acidic and coiled-coil domains in the C-terminal domain are the main domains for GP73 protein expression; truncation of C-terminal acidic domain of GP73 can lead to significant reduction of survival rates in mice. 147 Various degrees of liver disease and kidney disease also be happened; for example, the GP73 C-terminal coiled-coil domain can bind sCLU to assist its post-translational modification and delivery functions, and it can also bind apolipoprotein E to promote hepatitis C virus secretion. 148,149 The −2618 bp/−19 bp promoter at the GP73 gene level is closely related to biological function of GP73, and the GC box in its promoter can regulate the transactivation of adenovirus E1A, providing a basis for activation of adenovirus E1A.
The core promoter contains a CpG island in the promoter region of housekeeping gene, suggesting that the GP73 may have functional part in the housekeeping genes. 51 According to distribution of GP73, its phenotype and physiological functions are mainly concentrated in the regulation of digestive system, hippocampus and blood glucose. GP73 is lowly expressed in normal hepatocytes, and its high expression in liver diseases indicates that GP73 levels may be associated with liver stress conditions. Monitoring of GP73 expression in patients with liver disease may indicate liver abnormalities and can determine the patient's liver repair status and repair capacity. 150

| DE TEC TI ON ME THODS FOR G P73
At present, the detection methods for biomarkers are attracting more and more attention, [151][152][153]  There is therefore still a need to find a simple, fast and inexpensive GP73 detection method suitable for clinical large sample detection.
Gu et al 156 used ELISA to detect expression level of GP73 in the serum with 82% sensitivity and 80% specificity, and the ratio of GP73 in healthy people and liver disease patients was 1:3. However, there was no significant difference in GP73 levels in patients with hepatocellular carcinoma (HCC), cirrhosis and hepatitis, so the ELISA method could not meet the detection for liver cancer. Beijing Hotview Company invented a kit for the detection of serum GP73 using ELISA method. It has since been used in clinical practice in China. As a tool for judging liver fibrosis and cirrhosis, its sensitivity is 62.8% and its specificity is 80.5%.
In addition, nanomaterials have represented perfectly suitable materials for a variety of biomedical and biotechnological applications. [157][158][159][160][161][162][163][164][165][166][167][168][169][170][171][172] Our research group also established a variety of methodological detection methods. We used magnetic nanoparticle enzyme-linked immunosorbent assay to detect serum GP73 levels in 79 cases of liver cancer and 64 healthy people. The sensitivity from our method was 78.43%, and specificity was 91.47%. 173 In addition, the GP73 content in serum from 80 cases of liver cancer and 80 healthy people was detected by latex-enhanced immunoturbidimetric method based on polyclonal antibody, with 94.6% sensitivity and 72.4% specificity. The use of three monoclonal antibody-based latex-enhanced immunoturbidimetric assays has a 96.7% sensitivity and 93.3% specificity. 2 These two methods can be more easily automated, and they are faster than ELISA therefore have very good clinical application prospects.

| Relationship between GP73 and hepatitis
When liver cells develop autoimmune hepatitis or are infected with hepatitis B virus or hepatitis C virus, their GP73 expression levels will increase. When HBV and HCV are infected, the expression of GP73 in hepatocytes is increased significantly and do not change in the bile duct cells, suggesting that the active replication of hepatitis virus may be an important factor in the expression of GP73 in hepatocytes. 174 Kladney et al 175  expressed GP73, suggesting that GP73 may be derived from activated hepatic stellate cells. In summary, they speculated that the secondary injury of chronic liver disease and the trigger mechanism for acute liver injury may lead to the expression of GP73. 140

| Relationship between GP73 and cirrhosis
Liver cirrhosis is generally an end-stage manifestation that occurs on the basis of diffuse liver damage, generally consisting of two mechanisms, namely, I: hepatocyte regeneration and proliferation of nodules; II: fibrous tissue proliferation and fibrous nodule formation, reconstruction of the original liver tissue and blood vessels. From hepatitis to liver fibrosis, liver cirrhosis and liver cancer are long and difficult to find process, and even if the alpha-fetoprotein (AFP) detection abnormalities, but the imaging confirmation takes months or longer. Clinically, it is considered that 3 cm of HCC is an important demarcation point that is related to the prognosis. It takes at least 5 months for the tumour to grow from 1 cm to 3 cm, which means that the tumour can be detected after 5 months. The early diagnosis has important value for patient's treatment and prognosis. 92 The study found that the expression level of GP73 in the liver cells increased significantly in patients with liver cirrhosis. Some scholars have studied 229 healthy people, liver cancer, liver cirrhosis and hepatitis patients and found GP73 expression levels in liver cancer and liver cirrhosis patients were significantly higher than in the healthy control group and hepatitis group. 34 However, in another 535 studies, the GP73 expression levels in patients with cirrhosis were significantly higher than those in patients with liver cancer and hepatitis, and in the Child-Pugh classification of cirrhosis, the GP73 levels were significantly higher in patients with grade B and grade C than those with grade A patients. 92 Kladney et al used Western blot to analyse differences in GP73 content between cirrhosis and healthy subjects caused by different causes, including 17 healthy subjects, seven cases of autoimmune cirrhosis, nine cases of alcoholic cirrhosis and 14 patients with chronic hepatitis B cirrhosis and 23 patients with chronic hepatitis C cirrhosis. Their results found that the GP73 expression was elevated in patients with cirrhosis caused by different causes, but GP73 expression was not increased in healthy controls. The expression of GP73 in patients with cirrhosis caused by HBV was significantly increased by about 70 times. The level of GP73 in patients with cirrhosis, autoimmune cirrhosis and alcoholic cirrhosis caused by HCV was also elevated, but the level was not obvious. Their results suggested that the GP73 may play a crucial role in the pathogenesis of liver disease, but the specific mechanism is not yet clear. 177

| Relationship between GP73 and liver cancer
At present, GP73 has attracted more and more attention as a serological marker for liver cancer. 178 Most of the studies suggest that GP73 can improve the diagnostic rate for liver cancer (Table 1), and its sensitivity and specificity are higher than that of AFP, a traditional hepatoma serum marker. However, there is also controversy. Block et al used glycoproteomics to find that GP73 serum levels in American cornfields with HCC in 2005 were significantly higher than those without HCC, and GP73 levels in patients with HCC were significantly higher than those in patients with colon metastases and HBV infection. 183 Marrero et al detected GP73 serum levels in 352 liver cancer patients, liver cirrhosis patients and healthy subjects using Western blot ( Figure 2). Their results showed that GP73 serum levels in liver cancer patients were significantly higher than those in patients with liver cirrhosis.
Compared with AFP, the area under the receiver operating characteristic (ROC) curve for GP73 was 0.79, which was significantly larger than the area under the ROC curve for AFP of 0.61. In the case of cut-off for 10 times relative units, the sensitivity of GP73 was 69% and specificity was 75%, while the sensitivity for AFP was 30% and specificity was 96%. And when the AFP level was 100 and 20 ng/mL, the patients who exceeded the optimal cut-off value were more than 71% and 62%, respectively, thus showing that the use of GP73 was more valuable in cases where the increased level of AFP is not significant or does not increase. 184 Hu et al 182   GP73-negative patients with small HCCs ( Table 2). The sensitivity of GP73 for detection of liver cancer was 74.6%, and specificity was 97.4%, while the sensitivity of AFP for detection of liver cancer was 58.2% and specificity was 85.35%. This indicated that the GP73 was superior than AFP in detecting liver cancer. However, the combined diagnosis of GP73 and AFP is more likely to increase the detection rate of liver cancer (Figure 4). The diagnostic accuracy of serum GP73 and AFP in the same patient population was assessed in Table 3. The results demonstrated that GP73 showed higher sensitivity than AFP for HCC diagnosis. However, several studies showed the specificity of GP73 was lower than AFP, which might be due to that GP73 can be expressed in many types of cells, but the maior cell type was epithelial cells.
Although GP73 has great potential as a diagnostic marker for liver cancer, there are still many differences. For example, most studies reported that the serum levels of GP73 in patients with liver cancer were higher than those in other liver diseases, but there were also some opposite results.  188 Exosomes have also been found in several types of body fluids, including plasma, malignant effusions, urine, saliva and amniotic fluid. 189 In addition to some common exosomal proteins such as TSG101, Alix, CD9, CD81, CD63 and cytoskeletal proteins including actin, and tubulin proteins, exosomes may carry some disease-associated proteins for they are a sub-part of originated cells. 190 Several researches have shown that the GP73 protein is present in exosome surface. [191][192][193][194] The advantages of exosomes as tumour markers are as follows: (a) a large quantity, the concentration of exosomes in the serum can reach 3 × 10 6 cells/μL. 195  can represent characteristics and state of its parental cell 197 ; (c) it is easy to preserve for a long time and remove interference; there is phospholipid bilayer to protect its degradation by proteases and nucleases, and as long as the separation of exosomes can remove the interference 198 ; and (d) non-invasive detection; exosomes can enter the circulatory system through endothelium and can therefore be detected in almost all body fluids. 199 Compared to tissue biopsy, exosomes are effective biomarkers for personalized medicine.
Therefore, analysis of GP73 protein in exosome surface will be valued in the diagnosis of liver cancer.

| CHALLENG E S AND S TR ATEG IE S
In summary, the serum levels of GP73 in patients with liver diseases, such as hepatitis, liver cirrhosis and liver cancer, have increased in varying degrees. Although accumulating studies indicate that abnormal GP73 expression is associated with tumour progression by interacting with the microenvironment, and GP73 has been regarded as a potential diagnostic marker for HCC, the diagnosis accuracy of GP73 in cirrhosis and HCC distinguishment is worthy discussed. 20,22,187 Although some studies found that ELISA result did not show a significant elevation of serum GP73 in HCC groups compared with that in liver cirrhosis groups. Recent reports described that GP73-specific serum autoantibodies might interfere with ELISA analysis. Researchers have found several isoforms of GP73 that correspond with different patterns or levels of glycosylation. [200][201][202] It still needs further research about the significance of HCC-specific GP73 isoform in diagnostic accuracy improvement.
Moreover, GP73 tends to be used as a useful biomarker to monitor HCC prognosis after primary tumour surgery and therapy in advanced HCC. 132,133 In terms of clinical application, it is still a new biomarker, and large-scale and multi-centred investigations are still needed. Moreover, more sensitive, specific and rapid diagnostic methods are also needed. Since exosomes have many advantages, and a new detection method for GP73 from the perspective of exosomes is imperative to enhance the role of GP73 in human health research.