The Role of Alpha-1-acid Glycoprotein 2 Protein and the Underlying Orosomucoid 2 Gene in Different Diseases

Alpha-1-Acid glycoprotein 2 (AGP2) or orosomucoid 2 (ORM2) is a major plasma protein. AGP2 is a member of the immunocalin family, a lipocalin subfamily expressed mainly in hepatocytes and also in astrocytes and endothelial cells. The detailed physiological role of this protein has not yet been known, but some physiological activates have been related to this protein, including drug binding and immunomodulating effects. As it belongs to the positive acute phase proteins (APP), AGP2 increases during acute phase reactions and under certain pathological conditions such as unspecific inflammatory stimulation. AGP2 play a critical role in many diseases throughout its critical biological pathways. This literature review evaluates and discusses the contribution of AGP2 in different diseases, including several types of cancer, Rheumatoid Arthritis and Coronavirus Disease 2019 (COVID-19). Systematic Review Article Alotaibi et al.; JPRI, 34(20A): 15-29, 2022; Article no.JPRI.81891 16


INTRODUCTION
Alpha-1-acid glycoprotein (AGP), or Orosomucoid (ORM), is one of the leading and vital plasma proteins. It has been more than 70 years since discovering this glycosylated single chain protein and its isoforms. Since then, thousands of publications have been published investigating its structure and functions. AGP is considered a transport protein with immunomodulation roles. However, the molecular function of AGP is still not fully understood.
AGP have two main isoforms, AGP1 and AGP2. Most of the published studies were about AGP in the big picture as protein family and their general functions. This study aims to overview AGP2 and focuses on its physiological roles in different diseases. According to our awareness, no review article published focusing on the ORM2 gene and its AGP2 protein yet.

STUDY DESIGN
Information about the protein and the underlying gene in the literature were obtained from virtual databases such as NCBI, Medline, and PubMed. Articles were chosen according to selected keywords from 1950 until 2021. Keywords of protein/gene were looked at which either scientific names or scientific symbols. The preferred name of this protein is alpha-1-acid glycoprotein 2, and the other names are AGP2 and AAGP2. The gene's scientific terms are; orosomucoid 2 whereas scientific symbols for this gene are ORM2; AGP2; AGP-B; AGP-B'.

ORM genes
ORM gene is synthesized in leukocytes but mainly in the liver. Moreover, Orosomucoid is composed of two variants: ORM1 and ORM2, both genes are positioned on the long arm of chromosome 9 region 3 bands 2 (9q32) with 6 exons (Fig. 1).
Moreover, ORM1 encoded by AGP-A, with three common alleles (AGP S, AGP F1, and AGP F2) generally referred to as F1*S variants, while ORM2 encoded by AGP-B; AGP-B', also referred to as the A variant. AGP-B and AGP-B' genes are identical due to a recent duplication [1]. They differ by some features, such as the number of base pairs, as shown in Table 1. However, ORM1 has 764 base pairs while ORM2 has 759 base pairs with 201 amino acids linear for both. ORM is mainly found as a combination of these variants where the F1*S and A variants molar ratio in the blood from 2 to 3:1, and it changes depending on the acute phase reactions. However, the ORM1 concentration is five times higher than ORM2. ORM1 is the predominant subtype and the major isoform in circulation, while ORM2 is the major isoform in the brain.

AGP2 protein
AGP2 (alpha-1-acid glycoprotein 2) is an acutephase protein produced mostly by the liver, and also excreted in other extrahepatic tissues [1]. AGP2 molecular weight is 23.6 kDa. Its carbohydrate content is high, 45% of the total protein mass, representing one of the most extensively glycosylated proteins with an isoelectric point (pI) of 5.03 [2]. AGP2 considers a member of the immunocalin family and a lipocalin subfamily [3]. The precursor product of AGP genes is a single polypeptide chain of 201 residues, and a secretory N-terminal signal peptide of 18 residues is cleaved off, resulting in a protein with a single polypeptide chain of 183 amino acids [1,4].
The difference between AGP variants (AGP1 and AGP2) is 22 amino acid substitutions out of a total of 183 amino acids [2]. X-ray diffraction was used to identify the crystal structure of AGP2 with a resolution of 2.1.Å. As shown in Fig. 2, AGP2 is composed of four α-helices in addition to an eight-stranded β -barrel [5]. These eight βstrands represents a typical lipocalin fold forming the common drug-binding pocket feature in the lipocalin family and flanked by an α-helix [6]. Two lobes make up the AGP2 binding pocket, I and II, and its entrance is positioned at the open end of the β -barrel, unlike AGP1 where the pocket is deep, wide and contains three lobes (I-III). This indicates that the AGP2 variant binding pocket is narrower than the AGP1 variant, making AGP2 a distinctive ligand selectivity feature [5].In addition, AGP2 displays two disulfide bounds that are located between cysteines 5-147 and 72-164 [7]. AGP2 carries five sites of N-glycosylation attached to asparagine residues at Asn15, Asn38, Asn54, Asn75 and Asn85 [8]. The structural heterogeneity of AGP2 is mostly due to these glycans, depending on the branching degree and the terminating sugars attached [7]. AGP2 can show various levels of branching such as bi-, tri-and tetra-antennary along with great diversity of the terminating sugars like sialic acid and fucose [4].

Fig. 3. Timeline of ORM events and discoveries and the association with some diseases
The 3D structure consists of N-terminal, Cterminal, eight β-strands (red), and four α-helices (light blue) connected by four loops (light magenta)

Functions of AGP2
AGP2 is a member of the immunocalin family, a lipocalin subfamily. Immunocalin functions as immunomodulatory and anti-inflammation, where the lipocalin can bind and transport a range of hydrophobic ligand and drugs [9,4]. Although the biological function of AGP2 is still poorly understood, AGP2 has been shown to have a key role in drug binding and immunomodulating effects, as well as a variety of biochemical processes with physiological implications [10]. Furthermore, AGP2 is considered as the most critical drug-binding and drug-transporting protein along with Albumin and lipoproteins, which directly affects pharmacodynamics and pharmacokinetics [10]. Changes in AGP2 plasma levels during the acute-phase reactions (APR) and other pathological conditions, were reported to alter the concentration of free drugs without affecting the total drug concentration [2]. Depending on AGP physical-chemical properties, due to the presence of neuraminic acid residues on the glycoprotein's surface, this protein binds mostly to basic and neutral medicines from both endogenous and exogenous sources [1]. Drug binding properties have been reported to be affected by the differences between AGP variants, where drugs such as binedaline, dipyridamole and warfarin were shown to bind more strongly to AGP1. While other drugs such as imipramine, methadone and disopyridamole show a higher affinity for AGP2 [10]. The different binding selectivity between the two variants could be explained due to the different conformation of the binding sites; binding pocket lobes I and II are common in both variants, while a unique lobe III is only found in AGP1 [11]. As a result, AGP2 has a higher drug binding selectivity than AGP1. Therefore, AGP drug binding selectivity depends on AGP2 selectivity since AGP1 binds a wider range of drugs [4].
On the other hand, many drugs such as propranolol, progesterone and chlorpromazine bind equally to both variants due to their similarity. These drugs are highly hydrophobic and neutral drugs that bind to the hydrophobic lobe I that are present in AGP1 as well as AGP2 [10]. Several factors influence AGP binding capacity, including concentration and protein conformational change, temperature, pH, the concentration and polarity of the ligand, the relative abundance of the AGP variants, and other amino acid residues that exist near AGP hydrophobic domains [2]. One of the unique properties found in AGP2 while binding to drugs is preventing the transferring of drugs from the bloodstream to the brain, limiting the effecting on the central nervous system due to therapeutic effects of drugs [12].

Timeline of the ORM
ORM was first discovered by scientist K. Schmid in 1950, later Schmid clarified the α1-Acid Glycoprotein amino acid sequence (linear) in 1973 [13,14]. Previous genetic evidence of a second orosomucoid structural locus (ORM2) in human blood (plasma) was discovered in 1987 [15]. Rojo-Domnguez and Hernández-Arana calculated the first 3D modeling of human 1-acid glycoprotein in 1993. Furthermore, the crystal structure of the recombinant F1*S variant (AGP1) of α1-acid glycoprotein had been determined in 2008 by using UV radiation-damage-induced phasing (UV RIP) [6]. Then, in 2011, a second crystal structure was determined using X-ray diffraction as the A variant (AGP2) [5], identifying variations in drug-binding selectivity between two AGP genetic variants. In order to determine the ORM biological function, a previous study in 1977 by Chiu et al., indicated the interactions of the immune system with α1acid glycoprotein specially on lymphocytes [6]. However, many studies indicated the connection of AGP with many different diseases. For instance, in Neoplastic Disease patients, an abnormal expression of ORM was detected [16], and a high concentration of AGP plasma was detected in tumors such as lung cancer, ovarian cancer, and breast cancer [17]. ORM1 and ORM2 plasma levels were found to be higher in varying severity COVID-19 situations in a recent investigation [18]. The Summarized timeline of ORM events and its association with some diseases were represented in Fig. 3.

THE ROLE OF AGP2 IN DIFFERENT MECHANISMS
AGP2 is involved in both pro-and antiinflammatory modulation along with being an immunomodulating molecule [2,10]. AGP2 has an inhibitory effect on platelet aggregation, lymphocyte proliferation and neutrophil chemotaxis via unknown mechanisms [1]. In addition, AGP2 reduces cytokine secretion as a protection mechanism in a way that has not been fully elucidated [19]. AGP2, on the other hand, is involved in monocyte activation and the production of pro-inflammatory cytokines such as IL-6 and IL-12 by monocytes [7]. In a study about AGP2 role in neuroinflammation, AGP2 was found to play an anti-inflammatory role in neuroinflammation. Astrocytes express AGP2 in the hippocampus and during neuroinflammation, AGP2 inhibits microglial migration and accumulation in the inflammatory site. In a way to decrease neuroinflammation throughout reducing the inflammatory cell activation and migration. Along with reducing the mRNA expression of IL-1β, IL-6, and TNF-α, resulted from neuroinflammation.
The hepatic production of many plasma proteins could be changed due to systemic injury such as inflammation called acute-phase reactants. Several pathogenic situations and stimulating agents, such as infection, inflammation, tumor, surgery, tissue injury, sepsis, and necrosis, cause AGP concentrations to rise tenfold [1]. In 2005, a proteomic analysis detected an increase in AGP2 in cerebrospinal fluid from chronic fatigue syndrome patients [20]. ORM gene and its protein have been used as a biomarker for many diseases as well as cancer. Moreover, ORM levels are used as a biomarker for an autoimmune disorder such as Rheumatoid arthritis (RA) which is an inflammatory joint disease [21], and also for early detection of type 2 diabetes nephropathy [22]. Besides, ORM could be used as a prognostic factor for the liver cancer [22].
Because of its ability to bind directly with endothelial cell pores and reduce plasma water filtration into the surrounding tissues, AGP2 is vital in the capillary barrier. Many studies showed that AGP2 is important for capillary charge selectivity, which may play a role in conserving capillary permeability and organ perfusions [1,23]. AGP2 is thought to be clinically helpful in avoiding microvascular permeability increases in a variety of disorders, including Alzheimer's and stroke. Also, a study found that AGP2 could enhance brain endothelial function in the rat by reinforcing the monolayer integrity [1,24]. Table  2 summarized several studies of AGP2 role in different biological mechanisms and pathways.  [25].
Active suppression of inflammation during mammary gland involution.
AGP2 role in mouse mammary gland involution.
AGP2 act as anti-inflammatory factors, where it can modulate the immune system activity in a negative way and protect the cells from endotoxic shock. [26].
Neutrophil migration inhibition. The role of AGP2 in polymicrobial sepsis susceptible diabetic mice.
AGP2 works as immunosuppressing by inhibiting neutrophil migration to the infection sites. [27]. Modulation of microvessel permeability.
AGP2 effects on the microvasculature of the brain.
The function of AGP2 in hepatocellular carcinoma.
Inducting of AGP2 expression throughout the CCAAT/enhancer binding protein (C/EBP) leads to repressing HCC cell migration and invasion. [9].
Modulating microglial activation and migration during neuroinflammation.
The expression and the function of AGP2 in the brain.
AGP2 inhibits microglial migration and activation by blocking the interaction of C-C chemokine ligand 4 (CCL4) with C-C chemokine receptor type 5. [21] Prognostic factor associated with cancer-promoting pathways.
The expression, significance, and assessment of the AGP2 prognostic value in liver cancer.
The expression of AGP2 was significantly decreased in liver tumor tissues and was positively related to cancer-promoting pathways. [28]. Involved in neuronal survival, and memory.
The AGP2 function during lipopolysaccharide (LPS)induced brain inflammation.
AGP2 reverses LPS-induced hippocampal-dependent spatial memory impairments after the α7 acetylcholine receptor treatment.

AGP2 INVOLVEMENT IN DISEASES
Many studies found and observed a relationship between AGP2 concentration and diseases. Upregulation or downregulation of ORM2 or AGP2 could be used as a biomarker, prognostic factor for cancer, or therapeutic response prediction for many diseases, while the specific function of AGP2 is not recognized yet. These studies may indicate ORM2 involvements and role in diseases, see Table 3.

AGP2 Contributions in Various Diseases
AGP2 mainly contributes to diseases throughout five modes of action; by inhibiting microglia activation, inflammation, glycosyl pattern modifications, capillary permeability and platelet and neutrophil aggregation inhibitory activity as summarized in Fig. 4.

Inhibit microglia activation
Autism, Parkinson's disease, and Alzheimer's disease (AD) are diseases that a strong neuroinflammatory component was proven. AGP2 expression in the brain was being studied in order to better understand its involvement in neuroinflammation. In this study, Lipopolysaccharide (LPS) -induced neuroinflammation model was created [9]. LPS is a gram-negative bacteria endotoxin known to incite inflammation by encouraging the cells to secret proinflammatory mediators. Therefore, LPS models were used to illustrate the neuroinflammatory mechanism in neuroinflammation and neurodegenerative diseases [42]. Systemic injection of LPS was used to induce the neuroinflammation condition in the brain. After the intraperitoneal injection of LPS, the mRNA expression of the AGP proteins family was examined. It revealed that AGP2 expression, not the other isoform, was strongly produced in the brain within 24 h after LPS injection. AGP2 mRNA showed a higher level in the hippocampus compared to other brain regions. The study also reported that AGP2 is mainly expressed in the brain throughout astrocytes cells. On the other hand, a reverse process was created for knockdown AGP2 mRNA level in the brain using lentiviral shRNA. The LPS induction, along with AGP2 knockdown resulted in enhancing many proinflammatory cytokines expression (IL-1β, TNF-α, and IL-6). Conforming AGP2 anti-inflammatory role in neuroinflammation and its role of being used to diagnose, prevent, or treat neurological and brain disorders [9]. COVID-19 disease could be one of them, since the SARS-CoV-2 virus is linked with neurological alterations in COVID-19 cases [42].

a) Inhibit microglia activation, b) Inflammation, c) Glycosylation modifications and d) Capillary permeability e)
Platelet and neutrophil inhibition. AGP2 serum concentration is useful for the therapeutic response prediction to chondroitin sulfate/glucosamine hydrochloride with an accuracy of 84,3% in OA patients. [38]. Psoriasis Up regulated The urinary AGP2 levels in psoriatic patients.
AGP2 is inflammatory marker in psoriasis. Where, urinary AGP2 was higher in psoriasis and significantly higher in the severe

Down regulated
The molecular mechanism of HBV-ALF.
ORM2 gene with immune response pathway; has a role in HBV-ALF pathogenesis and could be used as therapeutic targets for HBV-ALF. [21].
Rheumatoid Arthritis (RA) Up regulated RA molecular processes and candidate proteins biomarkers.
Elevated AGP2 levels were an indication of the ongoing inflammation which is uniquely found in RA patients. [38]. Ischemic Stroke Up regulated AGP2 role in the ischemic stroke.
AGP2 have been found to play a protective role in ischemic stroke. [40].
Liver Cancer Down regulated AGP2 expression and function in liver cancer.
AGP2 downregulation in liver tumors contributes to the liver cancer progression. [37].

Up regulated
Urinary AGP2 as a diagnostic of diabetic nephropathy.
Urine AGP2 levels were a positivity associated with DN progression and increased in the early stage which correlated with DN occurrence and development. [18].
AGP2 increased levels were associated with more severe COVID-19 conditions, and its plasma alterations were related to immune responses and platelet degranulation. [38] Adult-Onset Still's Disease (AOSD)

Up regulated
Potential biomarkers for AOSD diagnosis.
AGP2 might be biomarker for AOSD diagnosis. Since it's increased levels were positively correlated with the disorder activity. [39].
Bacterial Keratitis Up regulated The upregulated tear proteins in bacterial keratitis.
AGP2 was altered and associated with defense/ immune protein activity in PD with cognitive impairment. [41].

Down regulated
The biological states of recovered COVID-19 patients.
AGP2 is used as potential marker for monitoring the inflammation condition in COVID-19 recovered patients.

Inflammation
Inflammation is widely recognized to play a critical function in cancer progress and it's a hallmark of cancer. Inflammation plays a role in all stages of cancer, including tumor development, progression, malignant conversion, invasion, and metastasis. Interestingly, cancerrelated inflammation is found to pave the way for cancer, in fact, it represents 15%-20% of all cancer cases, where incident such as infection, chronic inflammation, or autoimmunity were reported to exist in the exact location for long time before tumor initiation [43]. Therefore, many proteins were chosen to be potential biomarkers for cancer due to their involvement in inflammation reactions. And for that, AGP2 is considered as an essential acute phase plasma protein in inflammation and could play vital roles in monitoring the tumor development and progression indicting/distinguishing cancer in different stages. AGP2 is mainly produced from hepatocytes upon stimulation by the immune cell-derived cytokines activated by the inflammation and tumorigenesis.  (COVID-19) is a pandemic infectious disease rapidly spreading worldwide triggered by the infection of the SARS-CoV-2 virus. Although COVID-19 is regarded as one of the worst epidemics in history, with over two million confirmed deaths and millions of cases. However, COVID-19 pathogenesis is still poorly understood. Extensive efforts have been done investigating its development and understanding pathogenesis along with identifying potential biomarkers. Furthermore, a study about the host plasma proteins alterations that could involve in the COVID-19 pathogenesis, revealed that AGP2 was significantly increased in COVID-19 patients and showed a specific upregulated pattern with the development of the disease severity indicating in the more severe conditions [46,18]. The increasing of AGP2 levels in plasma are responding to the early phases of immune responses to virus infection. Acute inflammation is related with COVID-19 in severe cases and positively correlates with the disease severity [47]. In addition to platelet degranulation and metabolism, a study also identified pathways related to COVID-19 pathogenesis [18]. Moreover, AGP2 was connected to fibrinogen chains which could be associated with a severe infection in fatal COVID-19 conditions [48]. AGP2 plasma levels would be used as a marker of the status of SARS-CoV-2 virus infections since its levels were decreased in COVID-19 recovered cases. This report described the clinical characteristics of 3 months COVID-19 recovered patients, which shows that recovered patients are still under the condition of inflammation [49].

Glycosylation modifications
In addition to AGP2 concentrations and plasma levels, the glycosylation alterations are also considered as a diagnosis approach for physiological and pathophysiological conditions. AGP2 undergo three types of changes in glycosylation, they are sialytion, fucosylation and the degree of branching. Modifications in glycosylation are correspond to different diseases and disease stages and progression [50]. Multiple glycoforms were identified on AGP2 for instance, 51 glycoforms were found at Asn93. These glycoforms indicate that AGP2 has the ability to modify its N-glycosylation pattern responding to physiological changes or stimulations [51]. Thus, AGP2 functions in drug binding and immunomodulation are highly dependent on AGP2 carbohydrate composition. Therefore, AGP2 glycosylation is an alternative biomarker target [7]. For example, AGP contribute to Alzheimer's Disease through aberrant glycosylation [52]. The glycosylation alterations of AGP are linked to inflammatory processes, specifically neuroinflammation associated with AD as previously mentioned. In cancer patients, fucosylated glycans are novel tumor markers, the elevation of fucosylated glycans in tri-and tetra-antennary structures were found in cancer cases [53]. Moreover, the glycosylation changes of AGP help distinguishing hepatocellular carcinoma from cirrhosis; AGP sialylation and fucosylation were diverse in the hepatocellular carcinoma, liver cirrhosis and also in the control group. The concentrations of sialylation and fucosylation were high in HCC and cirrhosis but low in controls, indicating the critical role of sialylation and fucosylation that may play in HCC and cirrhosis. [54]. Nacetylgalactosamine, fucose, and galactose may be employed as markers of breast carcinoma progression [55].

Capillary permeability
In tumor metastasis, tumor cell adhesion to the microvascular wall is a critical step, which is part of tumor invasion. The interaction between tumor cells and blood host cells such as platelets, endothelial cells, and immune cells is what causes tumor cell adhesion [56]. The endothelium regulates tumor cell extravasation by assisting the adhesive between tumor cells and the vascular wall [57]. Microvascular endothelial cells produce AGP that expressed in hepatocytes and upon released into the circulation, AGP2 performed its function in maintaining normal permselectivity of the capillary walls [1,58]. AGP was discovered to has anti-inflammatory effects upon the endothelial cells by decreasing microvascular permeability and reducing the tumor cell adhesion [46,59]. AGP2 inhibits endothelial cell capillary-like tube formation seen through reduced cell adhesion and reversibly decreases cell migration. As a result, AGP2 has shown anti-angiogenic effects that is detected in the lung microvascular endothelia [60]. Tumor angiogenesis includes tube formation of endothelial cells for metastatic tumor cells to proliferation and migration.

Platelet and neutrophil aggregation inhibitory activity
Tumors have a unique mechanism referred to as tumor cell induced platelet aggregation, where primary tumor tend to influence the platelets production, activation, and aggregation. Platelet activation is important for tumor growth and metastatic progression. Tumor cells themselves have the ability for platelet activation, activated platelets interact with neutrophils actively participate to cancer-associated thrombosis and directly relate with metastatic potential. And in return, platelet participates in tumor invasion, via enhancing tumor metastasis by boosting tumor cell adherence to the vessel wall. Thus, the use of drugs targeting platelets activation could reduce cancer-associated thrombosis and tumor progression [61,62]. Alternatively,, neutrophils are suggested to be essential players in tumor development, where an increased level of neutrophils in blood is associated with advanced cancer. Tumor-associated neutrophils (TANs) have been found to have a role in various stages of tumor growth, including anti and pro-tumor functions [63,64]. In consequence, AGP2 has previously been suggested to inhibit platelet aggregation as well as inhibition of neutrophil aggregation and migration [1]. This observation shows that AGP2 may have essential function in tumor metastasis and progression in many ways. AGP2 was found to be adversely associated with intrahepatic metastasis and to be often downregulated in hepatocellular carcinoma (HCC) tissues, despite the fact that it is normally extensively expressed in liver tissues. AGP2 also decreased cell migration, invasion, and metastasis in HCC cells in vitro and in vivo, acting as a tumor suppressor [19].

Participate in the therapy
AGP2 is an important pharmacokinetic factor in clinical treatment since it is one of the most significant drug-binding proteins in plasma. Because AGP2 plasma levels were altered following therapy, they are employed as a diagnostic and predictive for clinical treatment [2]. Due to decreasing levels in individuals receiving both chemotherapy and hormone treatment, AGP is used as a medication response indicator in cancer patients [65]. AGP2 is used as a biomarker for prognostic evaluation of stage II colorectal cancer (CRC). Its plasma levels help to evaluate the stage II CRC patient's prognosis so the possible effectiveness of adjuvant therapy could be chosen [66]. For example, AGP2 levels were decreased significantly and connected with the response of interferon (IFN) treatment in liver cancer. Also, AGP2 downregulation in liver tumors was used as a marker for liver cancer therapy it is considered as an efficient predictor for immune therapy to cure liver cancer [67]. In osteoarthritis disease, AGP2 was used as a predictor for drug response in knee osteoarthritis. AGP2 was able to predict patients' response to chondroitin sulfate/glucosamine hydrochloride with an accuracy of 84,3%. Where AGP2 levels in responder's patients were significantly decreased compering to nonresponders [68]. In rare genetic cases as in Fabry disease, AGP2 was downregulated in patients undergoing enzyme replacement therapy [69].

CONCLUSION AND FURTHER STUDY
ORM2 is an acute-phage gene, and 1-acid glycoprotein 2 (AGP2) is one of the key acutephase proteins, with ORM2 expression and AGP2 plasma levels changing in response to various pathophysiological situations and disorders. However, AGP2 has a role in many illnesses, but few studies described how AGP2 participated in the disease's pathogenesis. Therefore, more genetic and physiological studies are needed to help in identifying AGP2 biological pathways since their molecular functions are not completely understood.

CONSENT
It is not applicable.

ETHICAL APPROVAL
It is not applicable.