Cytoglobin (Cygb) [1,2] is a highly conserved member of the vertebrate globin superfamily, which includes respiratory metallo-proteins such as the well-studied hemoglobin [3], expressed in erythrocytes, muscle-cell myoglobin [4], nervous system-expressed neuroglobin [5] and the cilia-associated androglobin [6,7]. In contrast to those globins, Cygb is not primarily detected in a few distinct tissues or cell types, but rather is expressed in a large range of tissues [1,8] and cells [[9], [10], [11], [12], [13], [14], [15], [16], [17]]. Originally identified in the cytoplasm of rat hepatic stellate cells (HSCs) [2] and other fibroblasts and fibroblast-like cell types [10,18], Cygb was also reported to be endogenously expressed in distinct neurons (cytoplasm and nucleus) [9,11], in melanocytes [15], myoblasts [19], vascular smooth muscle cells [[20], [21], [22], [23], [24]], adipocytes [17], and, in contrast to previous findings, in the nucleus and/or cytoplasm of some epithelial cells [9,13,14,25]. Multiple functions of Cygb were proposed and studied in vitro: NO dioxygenase [24,[26], [27], [28], [29], [30], [31]], nitrite reductase [20,23,32], superoxide dismutase [33], and lipid peroxidation [34,35] activities, modulation of lipid signalling [36], cytoprotective role via reactive oxygen/nitrogen species (RONS) scavenging [15,[37], [38], [39]], and participation in collagen maturation [10,11]. Few studies investigated the in vivo role of Cygb. In a skeletal muscle-specific Cygb knockout mouse model, Cygb was shown to modulate myogenic progenitor viability [37], while studies in a full genetic Cygb knockout model (Cygb−/−) demonstrated that, through its role in the NO metabolism of VSMCs, Cygb controls blood pressure and vascular tone [23], regulates neointima formation, and inhibits apoptosis after injury [21]. In a Cygb−/− mouse model of cholestatic liver disease, the lack of Cygb in HSCs was shown to enhance fibrosis, hepatocyte damage, and liver inflammation, possibly by deregulating NO metabolism [40]. In line with these data, a recent study in Cygb−/− mice showed that the NO overexpressed as a consequence of Cygb depletion in HSCs diffuses to the surrounding hepatocytes, causing mitochondrial dysfunction and ROS-induced liver damage [41]. Moreover, N,N-deethylnitrosamine-treated Cygb−/− mice showed increased liver and lung tumorigenesis [42], a Cygb−/− mouse model of non-alcoholic steatohepatitis developed liver cancer [43], and aged Cygb−/− mice showed various organ abnormalities [44] compared to their wildtype controls, thus arguing for a tumour suppressor role of Cygb. In line with this, a Cygb−/− mouse model study suggested that Cygb inhibits the development of already existing microadenomas in colon [45]. However, interpretation of Cygb's role in cancer is complex, since it was proposed to function as an oncogene in some cancer types because of its upregulation in hypoxic tumours [14,46,47].

The human CYGB gene located at chromosome position 17q25 is composed of 4 exons and 3 introns [1]. Its promoter presents a 1.4 kb CpG island with multiple GC boxes/Sp1 binding sites, but no TATA-box, thus explaining the DNA methylation-dependent epigenetic control of its expression [48]. In fact, CYGB silencing via hypermethylation of its promoter was observed in various cancer types [15,[49], [50], [51], [52]]. Multiple hypoxia-responsive elements were found in this promoter [48], explaining CYGB upregulation in low-oxygen conditions [53]. The human CYGB is a 21 kDa, 190 amino acid (aa) hexacoordinated protein that contains a consensual globin domain with conserved “proximal” and “distal” histidine residues (His113 and His81, respectively) [1]. Characteristic N- and C-terminal extensions distinguish CYGB from the other vertebrate globins [1], probably contributing to the binding of CYGB to ligands [1,54] and participating in the structural stability of the protein [55]. Two cysteine residues, Cys38 and Cys83, are probably important for the modulation of CYGB functions: under oxidative in vitro conditions, these residues contribute to the generation of stable homo-dimers via the establishment of intermolecular disulfide bridges [56,57], and/or to formation of an intramolecular disulfide bridge in the CYGB monomer (CYGB S-S monomer) [58]. The intramolecular disulfide bridge induces a pentacoordinated-like state in the CYGB monomer that facilitates its binding to exogenous ligands (e.g. lipids and O2) [34,[59], [60], [61]] and enhances peroxidase [34,35] and NiR activities [32]. Therefore, the cellular redox state significantly influences the structure of CYGB, possibly enabling different CYGB functions in distinct tissues, cells, and oxidative environments.

The process of alternative splicing (AS) is a conserved mechanism by which different mature mRNAs are generated from a single gene, thereby, in generating phenotypic complexity in higher organisms. Due to AS, up to 95% of the human multi-exon genes code for more than one mRNA isoform [62], and RNA-seq studies indicated that 86% of human genes have a minor isoform frequency of at least 15% [63]. Accounting for the complex biochemistry of Cygb and its widespread expression in tissues and cell types, we asked whether AS could contribute to the diversity of functions attributed to this globin. Here we report five putative transcript isoforms, four of them novel, for the human CYGB gene (CYGB V-1 to CYGB V-5) via in silico analyses and study their expression in human tissues.