Histidyl tRNA synthetase (HisRS) is a homodimeric enzyme, whose location is mainly cytoplasmic. HisRS is responsible for the incorporation of histidine into a growing peptide (95–97). The N-terminal fragment or CD includes the residues 1–320 and is responsible for the specific aminoacylation of tRNA (98). The C-terminal fragment is essential for the dimeric structure of the enzyme. Additionally, at least two HisRS-splice variants (SV) have been identified lacking the CD ( Figure 2 ). Besides the intracellular cytoplasmic location of HisRS, this enzyme can also be found in the extracellular compartment (98), although its extracellular functions have not been fully clarified. Both in vitro and animal studies have suggested that HisRS is involved in several regulatory mechanisms of cell metabolism and the regulation of immune responses. Howard et al. have demonstrated that the N-terminal domain serves as a chemoattractant for naïve lymphocytes and immature dendritic cells (DCs) through interaction with CCR5 (77). The N-terminal has also been considered to be the immunodominant epitope, specifically the 1–60 amino acid fragment (100). In particular, in vitro studies performed in myositis samples showed that T-cell stimulation assays using a 13-mer peptide from the HisRS N-terminal domain elicited an inflammatory response in blood and bronchoalveolar lavage fluid (BALF) T cells (78). Interestingly, in vivo the levels of extracellular HisRS were higher in sera of anti-Jo-1-negative patients compared to healthy individuals and were almost undetectable in patients with Jo-1 antibodies. This observation suggested that another possible immune mechanism could be involved, such as the formation of immune complexes of autoantibodies with the protein, but this still needs to be confirmed.

Anti-HisRS autoantibodies (anti-Jo-1) were initially identified in 1983 by Mathews M. and Bernstein R (9). Anti-Jo-1 autoantibodies are the most common among the myositis-specific autoantibodies (MSAs) with frequencies between 20% and 30% in patients with IIM (101–103).

Worldwide epidemiological studies have shown that patients presenting with anti-Jo-1 autoantibodies can develop ILD in up to 90% of the cases (38, 41, 104). In the American and European Network of Antisynthetase Syndrome (AENEAS) cohort study of anti-Jo-1-positive patients, ILD was present in 50% at disease onset and in 84% after 80-month follow-up (67).

Threonyl-tRNA synthetase, ThrRS, also referred to as TRS and the target of anti-PL-7 autoantibodies, is an aminoacyl-tRNA synthetase that catalyzes the aminoacylation of tRNA by transferring threonine. Besides its essential function of catalyzing aminoacylation, ThrRS can be secreted into the extracellular compartment where it can have other non-canonical functions (105). In particular, the extracellular secretion of ThrRS has been associated with the exposure of vascular endothelial cells with proinflammatory cytokines such as tumor necrosis factor-a (TNF-a) or vascular endothelial growth factor (VEGF). Additionally, in vitro and in vivo assays have shown that ThrRS stimulates endothelial cell migration and angiogenesis, indicating that ThrRS can acts as an angiogenic pro-migratory extracellular signalling molecule (79).

A recent study showed that ThrRS can induce maturation and activation of DCs with a Th1 response in vitro and in vivo, upregulation of CD4⁺ and CD8⁺ T cells, increased IFN-gamma secretion associated with viral clearance in influenza virus (H1N1)-infected mice, and increased IL-12 production by the MAPK/NF-kB pathways. Interestingly, CD4⁺IFNg⁺ and CD8⁺IFNg⁺ T cells and IFNg levels of ThrRS-DC immunized mice were significantly upregulated in BALF compared with the control group (80).

In the muscle, ThrRS may play a role as negative regulator in myogenic differentiation, by inhibiting Axin1, through the kinase c-Jun N-terminal (JNK), a downstream target of ThrRS. In particular, the presence of UNE-T or TGS domains was necessary for ThrRS myogenic function (106). Further studies analyzing the role of this protein in the innate and immune response as well as the ThrRS expression in muscular diseases are still needed to further understand the role of this aaRS in the disease.

There are few reports on clinical presentations of patients specifically with anti-PL-7 autoantibodies. In a European multicenter study, the frequency of anti-PL7 was 1.87% (18/964) (107), whereas a single-center retrospective cohort from China reported that anti-PL-7 autoantibodies had the same frequency (27% 30/108) as anti-Jo-1 autoantibodies (30.5% 33/108) (108). In Asian studies, the most common ILD pattern observed in anti-PL-7-positive patients was NSIP (15%), which was in line with an earlier study that reported mainly NSIP patterns (50%) followed by OP (41.7%) (109). In this study, UIP was only observed in the anti-PL7-positive group (4%) and was associated with a low response to therapy (108). In addition, the presence of this autoantibody predicted the long-term deterioration in ILD (110).

In contrast to most aaRS, alanyl-tRNA synthetase (AlaRS), the target of anti-PL-12 autoantibodies, recognizes its substrate in an anticodon-dependent manner with recognition of a single G3:U70 wobble base pair, which is the dominant identity determinant for tRNA aminoacylation (82). This simplified mechanism increases the likelihood of mischarging by AlaRS and has been associated with neurodegenerative disorders such as Charcot-Marie-Tooth disease (CMT) type 2, which is characterized by axonal peripheral neuropathy with muscle weakness, wasting, and impaired sensation in the extremities (82).

Autoantibodies against alanyl-tRNA synthetase, also known as anti-PL-12, was the third myositis-specific autoantibody identified in 1986 by Bunn et al. (12). Unlike anti-Jo-1 and anti-PL-7 autoantibodies, anti-PL-12 IgG recognizes independently two antigens: alanyl-tRNA synthetase and its cognate tRNA^Ala, suggesting that this recognition occurs by separate autoantibodies. The antigenic epitope is located in the anticodon region (83), and an epitope mapping of this protein identified the immunoreactive region outside the CD, within amino acids 730–951 (111) ( Table 2 ). In the case of anti-PL-12 autoantibodies, the severity of the clinical manifestations is mainly driven by the ILD, which was notable in cases associated with pulmonary hypertension (55). Both in American and European reports, a series of anti-PL-12-positive patients showed a low prevalence of muscular involvement, mechanic’s hands, and Raynaud’s phenomena (55, 112, 113). Regarding the histopathologic and radiographic features, UIP is the most common pattern of lung involvement (114).

Glycyl-tRNA synthetase (GlyRS), the target of anti-EJ autoantibodies, is a dual-localized homodimeric aaRS, found in both the cytoplasm and the mitochondria. It catalyzes the attachment of glycine to its cognate tRNA. Mutations in the gene coding for human GlyRS are associated to neurodegenerative diseases including the distal spinal muscle atrophy type V and CMT disease (115). The impairment in the mitochondrial metabolism in neurons is one of the mechanisms through which mutations in GlyRS lead to neurological diseases (116). GlyRS has been also shown to circulate in serum of human subjects but, in contrast to HisRS, extracellular levels of GlyRS were not found to be significantly different between healthy individuals and patients with myositis (97). In vitro experiments have demonstrated that it can be secreted from macrophages in response to Fas ligand that is released from tumor cells (85). Therefore, it has been suggested that GlyRS is involved in immune surveillance against cancer (85).

Autoantibodies against GlyRS (anti-EJ) were described for the first time in 1990 by Targoff in patients with myositis and ILD (13). In anti-EJ-positive patients from the AENEAS cohort and a large Chinese cohort, ILD was the most frequent clinical manifestation, being isolated in almost 40% of the patient group (48, 117). ILD has been reported as an early manifestation of the anti-EJ-ASSD disease course (117, 118). Regarding ILD patterns, NSIP, UIP, OP, and DAD have been described in patients with anti-EJ antibodies (117–119). OP was also found to be an independent risk factor for developing rapidly progressive ILD (117). Among other ASSD features, fever, mechanic’s hands, and Raynaud´s phenomenon have been reported as accompanying findings (48, 117, 119).

Isoleucyl-tRNA synthetase, IleRS (IARS), the target of anti-OJ autoantibodies, is a component of the multi-enzyme complex (MSC) described above that is important for the stabilization of the interactions between the components (28–30, 120). In addition, IleRS is important for protein synthesis and signaling pathways.

The anti-isoleucyl-tRNA synthetase autoantibody (also known as anti-OJ) was identified in 1990 and was initially described in a patient with a history of severe, progressive pulmonary fibrosis and pulmonary hypertension (13). This autoantibody has a low prevalence (<5%) among patients with IIM (57). Anti-OJ autoantibodies react with lysyl-tRNA synthetase (KARS) and the epitope of anti-OJ autoantibodies could be based on quaternary interactions between MSC components (57), making the detection in the clinical practice problematic. The low frequency of this autoantibody might be associated to the elusiveness of the primary target(s) of anti-OJ. In the clinical setting, multiplex assays based on immunoblotting such as line immunoassay (LIA) and dot blot assays (DBA) have poor performance of anti-OJ (121, 122). Anti-OJ autoantibodies have also been difficult to detect in ELISA, even with the use of recombinant proteins from Escherichia coli and Hi-5 (insect cell line), suggesting that either anti-OJ autoantibodies might recognize other components of the MSC or that the structural conformation of the complex is important for the recognition by this autoantibody (123). To date, immunoprecipitation (IP) remains the preferred method for anti-OJ autoantibody detection.

A review of 52 published cases identified ILD as the main clinical manifestation in 90% of the anti-OJ positive cases, with the patterns of UIP, OP, and NSIP being the most frequent. Prevalence of myositis seems to vary according to the criteria applied for ASSD classification and the ethnicity of the population included in the studies. In an Asian cohort, the frequency of severe myositis was reported to be as high as 57% (62), while in the AENEAS cohort study, 40% of the patients with anti-OJ autoantibodies had hypomyopathic forms of ASSD or never developed myositis (124). Arthritis, fever, Raynaud’s phenomenon, and mechanic’s hands are also present, but in a lower frequency, being an incomplete presentation of the ASSD frequent (57).

The asparaginyl-tRNA synthetase (AsnRS), the target of anti-KS autoantibodies, catalyzes the attachment of asparagine to its cognate tRNA during translation. As for non-canonical functions, AsnRS has been shown to be involved in growth regulation mediated by the Hippo signaling pathway (a pathway involved in growth regulation, dysregulation observed in many cancers) and, therefore, possibly implicated in tumorigenesis (125). Studies also reported pro-inflammatory functions of AsnRS. In particular, AsnRS was shown to induce CCR3-expressing cells to migrate and, like HisRS, chemoattract DCs (77). The non-translational chemokine activity of AsnRS is thought to be exerted by an additional domain at the N-terminal of the protein sequence, not present in the prokaryotic system. The modulating activity of AsnRS on CCR3 signaling has been suggested to be implicated in the pathophysiology of ASSD and ILD (126). Similar to GlyRS, AsnRS has been detected in extracellular compartments and serum levels were not significantly different between myositis patients and healthy individuals (97).

Autoantibodies against AsnRS (anti-KS) occur in less than 5% of patients with IIM and were first described in 1999 in two patients with ILD and no evidence of myositis (14). A review of the published literature about the clinical features associated with anti-KS autoantibodies has shown that ILD with NSIP or UIP patterns is the dominant feature, being the only manifestation in 50% of patients (63). Myositis seems to rarely affect this group of patients, while arthritis, Raynaud’s phenomenon, and mechanic’s hands may occur in one quarter of them (127)

Phenylalanyl-tRNA synthetase, PheRS, the target of anti-Zo autoantibodies, has been linked to cancer by reports of increased expression in some cancers (128–130), and suggested to be a prognostic indicator for some cancers (131). Thus, expression of PheRS was increased in gastric cancer tissue and the levels of expression correlated with distant metastasis and poor survival (132). Mechanistically, PheRS regulates anti-apoptotic signaling and cell proliferation through its upstream interaction proteins (133); however, the regulation of oncogenesis and development of gastric cancer by PheRS need further investigation.

The presence of autoantibodies against PheRS, anti-Zo, was first described in 2007 in a patient with ASSD (15). Anti-Zo is a rare anti-synthetase autoantibody. The largest cohort positive for anti-Zo autoantibodies was a case series of nine patients in UK (134). Seven (78%) of the patients had ILD, and two patients had evidence of muscle involvement, suggesting that anti-Zo autoantibodies are associated with features of ASSD. Moreover, two-thirds of the patients had autoantibodies against anti-Ro52, which has been previously reported to co-exist with other anti-synthetase autoantibodies and more severe ILD (135, 136). A more recent study reported the prevalence of anti-Zo autoantibodies as 1.4% in patients with ILD and novel associations of anti-Zo with connective tissue-disease related ILD (CTD-ILD) and idiopathic pneumonias (45).

Tyrosyl-tRNA synthetase or TyrRS, the target of anti-YRS/anti-HA autoantibodies, can split into two separate fragments, and these fragments have distinct cytokine activities whereas the full-length TyrRS is inactive for cytokine activities (42, 137). Secretion of both fragments are induced by leukocyte digestion and active forms of TyrRS can be naturally generated by alternative splicing or proteolytic cleavage (42, 137). The fragment that includes the C-terminal domain induced migration of mononuclear phagocytes and stimulated production of TNFa. On the other hand, the N-terminal domain induced migration of polymorphonuclear leukocytes (PMNs) in a dose-dependent manner very similar to the CXC chemokine interleukin -8 (IL-8) (42). Similarities in the effects of IL-8 and mini TyRS (N-terminal) on PMNs indicate a possible functional correlation between mini TyrRS and IL-8 activity. In addition, human mini TyrRS induced angiogenesis in vivo in different organisms similar to IL-8 (138). The N-terminal domain of TyrRS was also reported to be present in platelets, from which they are released by unknown mechanisms and to regulate monocytes/macrophage differentiation during bacterial infections (139).

The first report of autoantibodies against TyrRS referred to as anti-YRS or anti-HA was published in 2005 in a patient with ASSD features (17). The prevalence of anti-HA autoantibodies in a large cohort of patients with ILD (n = 1,194) was 2% (45).

As mentioned previously, aaRSs gained additional domains and functions throughout evolution. One example is Lysyl-tRNA synthetase (LysRS), also referred to as KRS or SC. LysRS binds to macrophages and monocytes leading to macrophage migration and TNF production when present in the extracellular media (140). Moreover, there are many reports in the literature indicating a role for LysRS in human immunodeficiency virus (HIV) replication, signal transduction, and neurodegenerative diseases (141). The mechanism of how LysRS contributes to HIV replication has been well studied, and it has been established that HIV recruits LysRS to serve as the reverse transcriptase through the interaction of LysRS and Gag protein (142–149). In addition, the contribution of LysRS in cancer has been established by many studies. LysRS has been shown to promote cancer metastasis by inducing cancer cell migration through the interaction with 67LR protein (150). LysRS can be secreted by cancer cells to induce inflammatory responses (151), whereas a more recent study showed a novel mechanism for the secretion of LysRS via exosomes or exosome-like extracellular vesicles from cancer cells, which is controlled by a caspase-8-dependent pathway (94).

There is also a possible connection of LysRS with amyotrophic lateral sclerosis (ALS). In some patients with ALS, a mutation in SOD1 is observed. The mutation in SOD1 induces apoptosis of motor neurons, thus leading to the onset of neurodegeneration and interestingly LysRS associates with mutant but not wild-type SOD1 (152). The abnormal interaction between SOD1 and LysRS contributes to mitochondrial dysfunction in ALS (153). In addition, a loss-of-function mutation in the CD of LysRS was implicated in CMT disease. It was reported that the mutation in LysRS severely affects the enzyme activity (154).

Glutaminyl-tRNA synthetase (GlnRS) is one of the two enzymes in this family that is not found in all organisms, such as Gram-positive eubacteria, archaebacteria, and organelles, suggesting that this aaRS has evolved along independent evolutionary pathways (155). Additionally, GlnRS appears to be the largest polypeptide in the human multienzyme synthetase complex and shares three significant regions of sequence similarity with the translation elongation factor EF-1 (156).

In humans, it is bifunctional, being specific for two amino acids (glutamine and proline) (28), acquiring the term glutamyl-prolyl-tRNA synthetase [Glu-ProRS or EPRS (157)]. Among the non-canonical functions of GlnRS is the ability to block apoptosis trough a negative regulation of the apoptosis signal-regulating kinase 1 (ASK1) (158). Additionally, in vivo studies in a rat model to evaluate changes in sensory neurons after nerve injury showed that the expression of GlnRS was decreased in the dorsal root ganglia (DRG). Thus, this aaRS may play a potential role as a neurotransmitter; however, further research is required (159). In addition, GlnRS expression may affect macrophage recruitment to injured DRG neurons (159–161).

Mutations in the gene encoding GlnRS have been associated to early-onset epileptic encephalopathy (162, 163). GlnRS deficiency has been associated with neurodegenerative disorders associated with severe developmental delay, microcephaly, delayed myelinization, and intractable epilepsy, which seems to be more severe than other disorders associated with mutations in tRNA synthetases (164).

Mammalian tryptophanyl t-RNA synthetase, TrpRS or WRS, has gained functions such as alternative splicing and proteolytic cleavage through evolution. This aaRSs is found not only in the cytosol but also in the nucleus. In the cytosol, it plays a role in innate immune responses, angiogenesis, and type II IFN signaling.

TrpRS is secreted into the extracellular milieu by monocytes upon infection with certain pathogens and it interacts with TLR2 and TLR4 leading to the secretion of TNFα, neutrophil infiltration, and increased phagocytotic abilities (90–92). These responses help to clear out infections at the early phase, indicating the importance of TrpRS as a ligand for immune regulation through TLR signaling. In support of this hypothesis, high levels of TrpRS were found in sera of patients with sepsis, a potentially fatal complication due to infection, when compared to healthy controls (92). Additionally, increased TrpRS expression from CD4⁺ T cells resisted indoleamine 2,3-dioxygensase (IDO)-mediated immunosuppression from DC in Graves’ disease (89). Autoantibodies against TrpRS have been found in patients with autoimmune diseases, where the clinical features were associated with rheumatoid arthritis (33–35).

There are few studies investigating the non-canonical functions of seryl-tRNA synthetase, SerRS. It was reported that SerRS interacts with a transcription factor called Yin Yang 1, to form a complex that negatively regulates vascular endothelial cell growth factor A during angiogenesis (165)

Autoantibodies against SerRS have been detected in a few cases of systemic lupus erythematosus or rheumatoid arthritis but not in myositis (36, 77, 166).