Zinc-alpha2-glycoprotein

Is a secreted glycoprotein of 43kD molecular mass.
date: 7. 1. 2008, author: BioVendor
Name Zinc-Alpha-2-Glycoprotein Human ELISA
Cat. No. RD191093100R RUO
Other names ZA2G, ZAG, AZGP1, Zn-alpha-2-glycoprotein, Zn-alpha-2-GP, ZNGP1
Product category Energy metabolism and body weight regulation, Oncology
Assay format Sandwich ELISA, Biotin-labelled antibody
Calibration range 3-100 ng/ml
Limit of detection Analytical Limit of Detection is calculated from the real Zinc-Alpha-2-Glycoprotein values in wells and is 0.673 ng/ml
Applications Plasma-Citrate, Plasma-EDTA, Plasma-Heparin, Serum
Sample requirements 10 µl/well
Storage/Shipping Store the kit at 2-8°C. Under these conditions, the kit is stable until the expiration date (see label on the box).

Zinc-alpha2-glycoprotein structure

Its gene spans over 9.7 kb, and its overall organization and nucleotide sequence are very similar to those of the first four exons of class I MHC genes. However, the ZAG gene differs from these genes in several significant ways. It lacks the coding information for the transmembrane and cytoplasmic domains typical of MHC genes, which is consistent with its presence as a soluble protein in different physiological and pathological fluids.

Zinc-alpha2-glycoprotein ligand

The ZAG structure includes a large groove analogous to class I MHC peptide binding grooves. Instead of a peptide, the ZAG groove contains a nonpeptidic compound that may be implicated in lipid catabolism under normal or pathological conditions. The natural ligand may be a polyunsaturated fatty acid. Arg-73 projects into one end of the binding groove and is the sole charged amino acid adjacent to the ligand. Replacing this amino acid with alanine abolished ligand binding and closed the groove to solvent. Arg-73 may therefore have an unexpected dual role in binding site access and anchor for an amphiphilic ligand.

Zinc-alpha2-glycoprotein regulation

Steroids

Dexamethasone (66 microg kg(-1)) administration to normal mice produced a six-fold increase in ZAG expression in both white and brown, which was attenuated by the glucocorticoid antagonist RU38486. In vitro studies using 3T3L1 adipocytes showed dexamethasone (1.68 microM) to stimulate lipolysis and increase ZAG expression, and both were attenuated by RU38486 (10 microM), anti-ZAG antibody (1 microg ml(-1)), and the beta3-adrenoreceptor (beta3-AR) antagonist SR59230A (10 microM). Zinc-alpha2-glycoprotein also increased its own expression and this was attenuated by SR59230A, suggesting that it was mediated through the beta3-AR. This suggests that glucocorticoids stimulate lipolysis through an increase in ZAG expression, and that they are responsible for the increase in ZAG expression seen in adipose tissue of cachectic mice.

In mice bearing the MAC16 tumour, serum cortisol concentrations increased in parallel with weight loss, and the glucocorticoid receptor antagonist RU38486 (25 mg kg(-1)) attenuated both the loss of body weight and ZAG expression in WAT.

Prostaglandis

The anti-cachectic agent eicosapentaenoic acid (EPA) (0.5 g/kg) attenuated the loss of body weight in mice bearing the MAC16 tumour, and this was accompanied by downregulation of ZAG expression in both white and brown adipose tissue, as determined by Western blotting. Dexamethasone (1.68 microM) induced a two-fold increase in ZAG expression in both cells and media, and that this was attenuated by EPA (50 microM). These results suggest that EPA may preserve adipose tissue in cachectic mice by downregulation of ZAG expression through interference with glucocorticoid signalling.

PPAR-y agonists

PPARgamma agonist rosiglitazone induced a 3-fold increase in ZAG mRNA level, while TNF-alpha led to a 4-fold decrease.

Zinc-alpha2-glycoprotein action

ZAG caused a stimulation of adenylate cyclase in murine adipocyte plasma membranes in a GTP-dependent process, with maximum stimulation at 0.1 microM GTP and with saturation at protein concentrations of >5 microg/assay. ZAG induced a concentration-dependent increase in the expression of Uncoupling Protein-1 (UCP-1) in primary cultures of brown, but not white, adipose tissue, and this effect was attenuated by the beta3-adrenergic receptor (beta3-AR) antagonist SR59230A. A 6.5-fold increase in UCP-1 expression was found in brown adipose tissue after incubation with 0.58 microM ZAG. ZAG also increased UCP-2 expression 3.5-fold in C2C12 murine myotubes, and this effect was also attenuated by SR59230A and potentiated by isobutylmethyl­xanthine, suggesting a cyclic AMP-mediated process through interaction with a beta3-AR. ZAG also produced a dose-dependent increase in UCP-3 in murine myotubes with a 2.5-fold increase at 0.58 microM ZAG. This effect was not mediated through the beta3-AR, but instead appeared to require mitogen activated protein kinase. These results confirm the ability of ZAG to directly influence UCP expression, which may play an important role in lipid utilization during cancer cachexia.

Zinc-alpha2-glycoprotein and fat mass reduction

Administration of ZAG to exbreeder male mice over a 89-h period produced a decrease in body weight without a change in food and water intake. Body composition analysis showed a 42% reduction in carcass lipid when compared with controls. Treatment of ob/ob mice with human ZAG over a 160-h period also produced a decrease in body weight, with a 19% reduction in carcass fat, without a change in body water or nonfat mass. Serum levels of glycerol and 3-hydroxybutyrate were significantly increased, as was oxygen uptake by interscapular brown adipose tissue, providing evidence of increased lipid mobilization and utilization. Human white adipocytes responded to both ZAG and isoprenaline to the same extent, although the maximal response was lower than that for murine white adipocytes.

Zinc-alpha2-glycoprotein in body fluids and tissues

ZAG is present at a high concentration in blood, seminal plasma and at significant levels in other human body fluids. Localization of the reactive product to anti-human plasma ZAG antibody was demonstrated in the following cells: luminal and basal cells of the prostate gland, luminal epithelial cells of the acini and of some ducts of the mammary glands, luminal cells of the secretory portion of the eccrine and apocrine sweat glands, serous cells of the salivary, tracheal, and bronchial glands, acinar cells of the esophageal glands, exocrine acinar cells of the pancreas, hepatocytes of the liver, and epithelial cells of the proximal and distal tubules in the kidney. The present results suggest that ZAG exerts some unknown but fairly widespread exocrine function and may be produced in the various epithelial cells tested. Hepatocytes are suggested to be a major source of the protein in the blood plasma.

Zinc-alpha2-glycoprotein expression in adipose tissue

ZAG mRNA was detected by RT-PCR in the mouse WAT depots examined (epididymal, perirenal, s.c., and mammary gland) and in interscapular brown fat. In WAT, ZAG gene expression was evident in mature adipocytes and in stromal-vascular cells. Using a ZAG antibody, ZAG protein was located in WAT by Western blotting and immunohistoche­mistry. Mice bearing the MAC16– tumor displayed substantial losses of body weight and fat mass, which was accompanied by major increases in ZAG mRNA and protein levels in WAT and brown fat. ZAG mRNA was detected in 3T3-L1 cells, before and after the induction of differentiation, with the level increasing progressively after differentiation with a peak at days 8–10. Both dexamethasone and a beta3 agonist, BRL 37344, increased ZAG mRNA levels in 3T3-L1 adipocytes. Overexpression in WAT of tumor-bearing mice suggests a local role for adipocytederived ZAG in the substantial reduction of adiposity of cancer cachexia. ZAG gene expression and protein were also detected in human adipose tissue (visceral and s.c.). ZAG mRNA was detected by RT-PCR in mature human adipocytes and in SGBS cells post-, but not pre-, differentiation to adipocytes. Relative ZAG mRNA levels increased rapidly after differentiation of SGBS cells, peaking at day 8 post-induction. ZAG protein was evident in differentiated adipocytes (by day 3) and also detected in the culture medium (by day 6) post-induction. Human adipocytes express and secrete ZAG, with ZAG expression being regulated particularly through TNF-alpha and the PPARgamma nuclear receptor. ZAG is a novel adipokine, which may be involved in the local regulation of adipose tissue.

Zinc-alpha2-glycoprotein expression in the Liver

The rate of incorporation of amino acids from ingested protein labeled with stable isotope into individual plasma proteins was measured. This approach involves three steps: 1) production of stable isotope-labeled milk whey protein, oral administration of this intrinsically labeled protein, and subsequent collection of blood samples; 2) fractionation of the plasma and separation of the individual plasma proteins by a combination of anion exchange high-pressure liquid chromatography and gel electrophoresis; and 3) identification of individual plasma proteins by tandem mass spectrometry and measurement of stable isotopic enrichment of these proteins by gas chromatography-mass spectrometry. This method allowed the measurement of the fractional synthesis rate (FSR) of 29 different plasma proteins by using the same precursor pool. A 30-fold difference in FSR of different plasma proteins was found. The protein with the highest FSR in the postprandial state is ZAG (33%/hr).

Zinc-alpha2-glycoprotein knock-out

Both ZAG alleles were inactivated by gene targeting in mice. Subjecting these ZAG deficient animals to standard or lipid rich food regimens led to increased body weight in comparison to identically treated wild-type mice. This phenotype appeared to correlate with a significant decrease in adipocytic lipolysis that could not be rescued by several pharmacological agents including beta(3)-adrenoreceptor agonists. Furthermore, in contrast to previously reported data, ZAG was found to be ubiquitously and constitutively expressed, with an especially high level in the mouse liver. No overt immunological phenotype was identified in these animals.

Zinc-alpha2-glycoprotein and cancer cachexia

Cancer patients with weight loss showed urinary excretion of a lipid-mobilizing factor (LMF), determined by the ability to stimulate lipolysis in isolated murine epididymal adipocytes. Such bioactivity was not detectable in the urine of cancer patients without weight loss or in normal subjects. The LMF was purified using a combination of ion exchange, exclusion, and hydrophobic interaction chromatographies to give a single component of apparent Mr 43,000, which showed homology in amino acid sequence with human plasma ZAG. Both substances showed the same mobility on denaturing and nondenaturing gels and the same chymotrypsin digestion pattern, both stained heavily for carbohydrate, and they showed similar immunoreactivity. Polyclonal antisera to human plasma ZAG was also capable of neutralization of the bioactivity of human LMF in vitro. Using competitive PCR to quantify expression of ZAG, it was found that only those tumors that were capable of producing a decrease in carcass lipid expressed mRNA for ZAG. These results provide strong evidence to suggest that tumor production of ZAG is responsible for the lipid catabolism seen in cancer patients.

Zinc-alpha2-glycoprotein and obesity-associated type 2 diabetes

The KK/Ta strain serves as a suitable polygenic mouse model for the common form of type 2 diabetes associated with obesity in humans. The susceptibility loci contributing to type 2 diabetes and related phenotypes in KK/Ta mice were reported. In the kidneys and liver of KK/Ta and BALB/c mice using differential display (DD) PCR, ZAG mRNA levels were found increased in the kidneys and liver in KK/Ta mice, and sequence analysis revealed a missense mutation. ZAG is a possible candidate gene for regulation of body weight, elucidation of polygenic inheritance, and age-dependent changes in the genetic control of obesity.

Zinc-alpha2-glycoprotein and cancer

Adding ZAG to the culture medium and by stably transfecting SiHa cells with ZAG cDNA into SiHa tumor cells reduces proliferation. In this setting, only cdc2 expression responds to ZAG, with a reduction of up to over a factor of two. Cdc2 is the only cyclin-dependent kinase regulating the G(2)/M transition without redundancy and is required as a rate-limiting step in the cell cycle. Its increased expression has been directly linked to increased proliferation and decreased differentiation of advanced tumors; conversely, its downregulation by ZAG might hinder tumor progression.

ZAG expression was analyzed in malignant prostate epithelium in prostatectomy specimens from 228 prostate cancer patients. Low (i.e., absent or weak) ZAG expression was associated with clinical recurrence (defined as confirmed localized recurrence, metastasis, or death from prostate cancer; hazard ratio [HR] = 4.8, 95% confidence interval [CI] = 2.2 to 10.7, P<.001) and with bony metastases or death from prostate cancer (HR = 8.0, 95% CI = 2.6 to 24.3, P<.001). Among the 17 patients in the cohort in whom clinical recurrence was associated with short PSADT, absent or weak ZAG expression was observed in 13 patients. If these preliminary findings are validated in independent cohorts, the measurement of ZAG levels in radical prostatectomy specimens may permit an accurate and timely assessment of risk of metastatic progression after radical prostatectomy.

Zinc-alpha2-glycoprotein and cell adhesion

ZAG functions as a matrix protein for the Tu-138 oral squamous cell carcinoma cell line. Cell attachment to ZAG is comparable to that for fibronectin and is inhibited by the synthetic RGD peptides RGD, RGDV, and RGDS. The proliferation of Tu-138 cells is inhibited on a ZAG matrix, as compared with other matrix proteins (fibronectin, vitronectin, laminin, and collagens I and IV) on which growth resembles that on the BSA control. The role of ZAG in differentiation and its RNase activity are two likely suspects as agents of the inhibition of proliferation.

Zinc-alpha2-glycoprotein and skin

IFN-gamma strongly up-regulated the expression of cathepsin D and ZAG in normal keratinocytes but down-regulated them in psoriatic ones; the reverse was true of the apoptotic suppressor bcl-2. It is believed that the aberrant response to IFN-gamma plays a central role in the pathophysiology of psoriasis, particularly the disruption of apoptosis and desquamation

ZAG inhibits melanin production by B16 melanoma cells via post-transcriptional effects on tyrosinase protein. As ZAG is normally produced by epidermal keratinocytes, these studies raise the possibility that epidermal-derived ZAG may play a part in normal regulation of melanin production in vivo, in addition to its previously described role in cancer cachexia.

Zinc-alpha2-glycoprotein in frontotemporal dementia (FTD)

Prefractionated 2-DE analysis of FTD and control CSF showed that 26 protein spots were changed at least two fold. Using mass spectrometry, 13 of these protein spots were identified, including retinol-binding protein, ZAG, proapolipopro­teinA1, beta-2-microglobulin, transthyretin, albumin and alloalbumin.

Zinc-alpha2-glycoprotein in proliferative diabetic retinopathy (PDR)

Eight proteins were highly produced in PDR patients in comparison with nondiabetic subjects: zinc-alpha(2)-glycoprotein (ZAG), apolipoprotein (apo) A1, apoH, fibrinogen A, and the complement factors C3, C4b, C9 and factor B).

Zinc-alpha2-glycoprotein and spermatozoa

ZAG protein is localized on the spermatozoal pre-equatorial region covering the acrosome, neck, and middle piece of the flagellum. Using computerassisted sperm analysis, it was found that anti-human ZAG antibodies could significantly reduce the motility of human swim-up spermatozoa after 90– or 120-min incubation (P<0.05 and P<0.01 respec­tively), together with the decreasing of intracellular cAMP and PKA levels. These data suggest that ZAG is present in human spermatozoa and may be involved in the regulation of sperm motility via the cAMP/PKA signaling pathway. Forward motility index of bovine spermatozoa, incubated with ZAG and theophylline, was significantly different from that of spermatozoa incubated with theophylline alone.

Catalog NumberSpeciesAnalyteAssayRegulatoryFormat
RD191093100R Human Zinc-Alpha-2-Glycoprotein Sandwich ELISA, HRP-labelled antibody RUO 96 wells (1 kit)
categories: Energy metabolism and body weight regulation, Oncology