2-Glycoprotein : A Multidisciplinary Protein α Zinc Updated

Zinc A2-glycoprotein (ZAG) is a protein of interest because of its ability to play many important functions in the human body, including fertilization and lipid mobilization. After the discovery of this molecule, during the last 5 decades, various studies have been documented on its structure and functions, but still, it is considered as a protein with an unknown function. Its expression is regulated by glucocorticoids. Due to its high sequence homology with lipid-mobilizing factor and high expression in cancer cachexia, it is considered as a novel adipokine. On the other hand, structural organization and fold is similar to MHC class I antigen-presenting molecule; hence, ZAG may have a role in the expression of the immune response. The function of ZAG under physiologic and cancerous conditions remains mysterious but is considered as a tumor biomarker for various carcinomas. There are several unrelated functions that are attributed to ZAG, such as RNase activity, regulation of melanin production, hindering tumor proliferation, and transport of nephritic by-products. This article deals with the discussion of the major aspects of ZAG from its gene structure to function and metabolism. (Mol Cancer Res 2008;6(6):892–906) Introduction Zinc a2-glycoprotein (ZAG) is a 40-kDa single-chain polypeptide (1), which is secreted in various body fluids (2). ZAG is known to stimulate lipolysis in murine epididymal adipocytes through stimulation of adenylate cyclase in a GTPdependent process (3) via binding through h3-adrenoreceptor (4). It is involved preferentially in depletion of fatty acids from adipose tissues, subsequently named as lipid-mobilizing factor (5). This factor, which is highly expressed in cancer cachexia (6), is characterized by the extensive reduction of fat in the human body. The protein assay (7) and mRNA expression (8) in the mammary tumor have shown that there is a relation between the ZAG levels at histologic grade of the breast cancer tumors. Moreover, many studies suggested that ZAG is also a potential serum marker of prostate cancer that may be elevated early in tumor growth (9). High degree of similarity between ZAG and class I MHC molecules has been evaluated both at sequence and structural levels (10-12). The crystal structure of ZAG consists of a large groove analogous to class I MHC peptide-binding grooves. The structure and environment of groove reflect its role in immunoregulation and in lipid catabolism (11). The alteration in residues of the peptide-binding groove clearly showed uniqueness of ZAG among MHC class I– like proteins (13, 14). These observations indicate that ZAG might be able to bind with different peptides, antigens, and ligands. Hence, it was suggested that function of ZAG has diverged from the peptide presentation and T-cell interaction functions of class I MHC molecules (15). There are various reports on different aspects of ZAG. The works, however, have never been reviewed. Here, we have compiled for the first time a detailed analysis of all the relevant information for ZAG that may be helpful in understanding of functional importance of ZAG in the body system. Site of Expression The ZAG was first reported in human serum and subsequently purified (1). The presence of ZAG in human seminal fluid was reported in 6-fold molar excess compared with human serum (16, 17), which suggested as a key element for fertilization without any experimental evidences. The serum ZAG is synthesized by the genes of liver. The seminal ZAG, however, is of prostatic origin (9, 16, 17). The presence of ZAG in normal human body fluids and kidney extract has been described (18). Jirka and Blanicky (19) have reported three isoforms of ZAG through immunoelectrophoresis and reported that concentration of each human serum ZAG isoform increases from its lowest value in the fetal and early newborn period to the highest ones in children and adults. Through immunohistochemical analysis, Mazoujian (20) studied normal skin and 41 benign sweat gland tumors and found that ZAG was expressed predominantly in tumors of apocrine differentiation. It was also, however, expressed in some tumors of eccrine differentiation (21). Abundant proteins expressed in human saliva are ZAG with some other proteins determined by proteomic analysis and mass fingerprinting. Recently, the gene expression of ZAG in normal human epidermal and buccal epithelia was reported (22). ZAG is also produced by adipocytes, where the mRNA of ZAG was detected by reverse transcription-PCR in the mouse Received 12/10/07; revised 1/17/08; accepted 1/23/08. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Requests for reprints: Faizan Ahmad, Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi 110025, India. Phone: 91-1126983409; Fax: 91-11-26983409. E-mail: faizan_ahmad@yahoo.com Copyright D 2008 American Association for Cancer Research. doi:10.1158/1541-7786.MCR-07-2195 Mol Cancer Res 2008;6(6). June 2008 892 Research. on September 27, 2014. © 2008 American Association for Cancer mcr.aacrjournals.org Downloaded from white adipose tissue and in the interscapular brown fat. Finally, ZAG is synthesized by epithelial cells of prostate gland and liver, secreted into various body fluids such as serum (1), semen (23), sweat (24), saliva (24), cerebrospinal fluid (24), milk (24), urine (25), and amniotic fluid (26). The concentration of ZAG has been reported to increase dramatically in carcinomas (3). Therefore, it is also considered as a good biomarker for prostate (27), breast (28), oral (22), and epidermal (29) carcinomas. Gene Structure and Regulation The gene for ZAG, assigned to the chromosome 7q22.1 through fluorescent hybridization karyotyping, comprised four exons and three introns (30). The first exon (exon 1) is for the region from the cap site to the 6th amino acid (Gly), the second exon (exon 2) is for the region from the 6th to the 93rd amino acid (Gly), the third exon (exon 3) is for the region from the 93rd to the 185th amino acid (Asp), and the fourth exon (exon 4) is for the region from the 185th amino acid to the end of the mRNA (31). The exon 4 also contains the entire 3¶-untranslated region, including a hexanucleotide AATAAA that probably represents the polyadenylation signal of the gene (32). The serum ZAG shows complete nucleotide sequence homology with that from the prostate, which includes the signal peptide (29). The full gene of ZAG was reported by Freije et al. (8). The gene sequence of ZAG includes an open reading frame encoding an 18-amino acid long hydrophobic signal peptide and the 278 residues of the mature protein. A comparison of the amino acid sequence deduced from the nucleotide sequence with that determined for the protein isolated from the serum through chemical methods (10) reveals some minor differences. There are two substitutions (i.e., Gln and Glu) present at positions 65 and 222 of the mature polypeptide chain, respectively, instead of the Glu and Gln residues at these positions as determined by the protein sequencing. In addition, the nucleotide sequencing of ZAG (8, 32) detected the insertion of an Ile-Phe pair between residues located at positions 75 and 76 of the previous sequence determined by chemical methods (10). The nucleotide sequence analysis of ZAG gene reported that there are some intervening sequences (32), and the length of intron is larger due to the presence of an unusually high density of Alu repetitive sequences within them. A total of nine Alu sequences were identified. The five are present in the first intron, and the remaining four in the second intron. This reflects that repetitive sequences are clearly overrepresented in the ZAG gene. Eight of these Alu sequences are oriented in the opposite direction of the ZAG, whereas the remaining one (Alu-6) is oriented in the same direction (32). In addition, two MER sequences belonging to subfamilies 12 and 14 (33) and one MIR element (34) were found in the first and second introns of the gene, respectively. The gene expression of ZAG is predominantly regulated by androgens and progestins (35, 36). Glucocorticoids are also responsible for the increased ZAG expression in adipose tissue (37). Russell and Tisdale (38) examined its 5¶-flanking region that could affect the transcription of the gene. They proposed that 5¶-flanking region of the gene containing several consensus sequences could be relevant in the transcription of the ZAG gene. Finally, they suggested that ZAG expression is likely to be mediated by the interaction of several transcription factors acting synergistically on different cisacting elements. In addition, the lipolytic action of dexamethasone was attenuated by anti-ZAG antibody, suggesting that the induction of lipolysis was mediated through an increase in ZAG expression. It was further proposed that expression of ZAG is mediated through the h3-adrenoreceptor present on the ZAG gene (35). ZAG as a Biomarker Although the exact mechanism by which ZAG actively participates in tumor proliferation is not known, a large body of data exists in favor of the expression of ZAG with respect to the stages of tumor (39-42). ZAG is designated as a potential biomarker of different types of carcinomas (6, 7, 27, 35, 38, 43-46). ZAG is synthesized in the prostate itself, and its high concentrations in prostatic tissue and prostatic secretion should facilitate its action in prostate and in other tissues (9). Furthermore, the increased concentration of ZAG in semen is directly linked with the prostate pathophysiology. The immunohistochemical analysis revealed an elevated concentration of ZAG in prostatic adenocarcinoma (47). In other analysis, it was observed that the majority of prostate cancer cells tested (i.e., 35 of 48 cells) have reacte