Functional role of immunoglobulin G as an oxytocin-carrier protein

.


Introduction
Oxytocin is a nine-amino acids neuropeptide hormone involved in the regulation of multiple physiological functions acting in both peripheral tissues and the brain.The important role of oxytocin in motivated behavior, emotion, reproductive, and other functions justifies the need for a better understanding of molecular mechanisms influencing oxytocin signaling.
Several in-depth reviews provide a comprehensive picture of oxytocin signaling including gene expression, tissue distribution, and oxytocin receptor (OTR) activation as well as multiple facets of oxytocin actions [1][2][3].One aspect of the peripheral oxytocin signaling remained nevertheless obscure -the nature and the role of oxytocin binding proteins in plasma.The natural presence of such proteins in human plasma has been known for a long time and has been mentioned by several investigators as potential modulators of oxytocin signaling [2].The phenomenon of oxytocin-binding protein is, however, not unique but a common feature of many if not all peptide hormones which circulate bound to a carrier protein protecting them from degradation by plasma peptidases [4].Indeed, total serum/plasma biomarker concentration is largely determined by the carrier protein clearance rate, while the presence of a binding protein supports the functionality of peptide hormone, for instance, adipose tissue-derived leptin [5,6].For the rare exceptions, ex. for corticotropin-releasing hormone (CRH) and leptin, the nature of peptide-hormone carrier proteins was not established [7,8].Moreover, it cannot be excluded that several plasma proteins, including immunoglobulin (Ig), may serve as carriers for the same peptide hormone, ex. for leptin [9,10].During the last 2 decades, several papers reported the natural presence in human plasma of oxytocin-binding IgG.Based on this converging evidence, our recent study tested a hypothesis that IgG may play a role of oxytocin carrier protein and it has provided conclusive results showing that about 60% of peripheral oxytocin circulates bound to IgG modulating the oxytocin signaling upon the OTR [11].Here we review the background for this discovery and its functional implications.

Evidence of protein-bound oxytocin in blood circulation
Methodological issues of analyzing plasma concentrations of oxytocin have provided valuable insight into the phenomenon of oxytocin binding proteins.Indeed, as was recently reviewed by Tabak and collaborators, who highlighted the advances in human oxytocin J o u r n a l P r e -p r o o f measurement, there are several recurrent issues in measuring plasma oxytocin, including the problem of a protein-bound oxytocin fraction [12].
An extent of the protein-bound oxytocin fraction in the blood was shown by Brandtzaeg and collaborators [13].In their study, the authors measured plasma and serum oxytocin levels using proteomic nano-liquid chromatography-mass spectrometry approaches following oxytocin unbinding from carrier proteins by reduction and alkylation.In fact, in its native form, oxytocin easily binds plasma proteins such as albumin via the disulfide bonds.
The proposed treatment removes this bond, and the peptide becomes detectable by nano-LC MS resulting in a much higher concentration of oxytocin as compared to the protocol without oxytocin extraction from binding proteins.From the practical point of view, the NanoLC-MS, however, is not the most accessible method to quantify oxytocin and the more common approaches use radioimmunoassay (RIA) or enzyme-linked immunoassay (ELISA).
Similarly to the study cited above, during both RIA and ELISA of plasma oxytocin, the preanalytical part of the process can have a significant impact on the results.Among different studies, there are inconsistencies in sample collection, processing, storage, and use of extraction procedures.The last one is the most relevant to the phenomenon of the protein-bound fraction of peptide hormone.Indeed, there is a debate about the necessity to extract before the assay because the level can be 10 to 100 times higher in non-extracted samples versus extracted samples, appearing as not plausible.If we go further in detail, in non-extracted samples not only oxytocin but also some unrelated plasmatic proteins with a non-specific oxytocin-like immunoreactivity can be detected because of oxytocin crossreactivity of the assay antibodies.Extraction is a process that eliminates potentially interfering molecules, reduces sample matrix effects, and concentrates the analyte before assay.Adding this step before the assay enables the detection of only small peptides and the oxytocin assay becomes more reliable.A meta-analysis study showed that there is little if any loss of peptide during the extraction process itself, even if we can observe, particularly in plasma samples, considerable differences in peptide levels [12,14,15].This implies that following extraction or filtering, we might assay both "free" and unbound OT and also avoid most false-positive immunoreactivity.
These data strengthen the idea that a large part of circulating oxytocin is bound to a bigger carrier molecule while the remaining fraction is free.There is some discussion about the biological meaning of these two fractions of oxytocin.MacLean and colleagues supposed J o u r n a l P r e -p r o o f that assaying free oxytocin is more appropriate in the case of studying changes in response to specific stimuli, even if free oxytocin can be influenced by age, morbidities, or pharmaceutical compounds [16].Assays including the bound fraction of oxytocin may provide a more reliable value of basal individual differences and peptidergic activity during the time.This paper also raised an interesting point: free oxytocin can become bound quickly following sample collection, so the ratio between "free" and "bound" oxytocin can evolve between collection and processing and not be representative of the physiological state at the time of collection [16].The biological meaning of "free oxytocin" and "bound oxytocin," the pace of exchange between the two compartments, and whether the oxytocin we measure without extraction or filtration corresponds to the total free and bound oxytocin are among the question raised.Determining the molecule or molecules that OT attaches to and whether several viable candidates, such as albumin and Ig, should be taken into consideration to better understand the oxytocin signaling [12,15,16].

Natural presence of oxytocin-reactive immunoglobulins
Autoantibodies (autoAbs) are Ig that can bind to at least one self-antigen, in both healthy and sick individuals.The term "autoantibody" does not, however, always indicate a pathogenic process with antibody-triggered tissue damage by immune cells in the case of autoimmune diseases [17].Indeed, an important fraction of the overall Ig pool is represented by so-called natural autoAbs which are present in healthy subjects and which may play a role of modulators of physiological functions [18,19].Such natural autoAbs may act as a first-line defense against infection thanks to their poly-reactivity or cross-reactivity or playing anti-inflammatory and immune regulatory roles [20].Additionally, the ability of Ig to naturally bind peptide hormones suggests the role of such autoAbs as modulators of neuroendocrine end endocrine signaling [20].In this context, it is of interest that the first description of so-called 'antihormone' molecules was reported in early endocrinological studies by researchers analyzing the phenomenon of functionally induced resistance to hormones ex. by James Collip who is mainly known for his seminal work contributing to the discovery of insulin and adrenocorticotropin [21].However, the chemical nature of such antihormone molecules has not been determined.The current literature has multiple records of detection in human plasma of IgG and IgM classes of autoAbs binding oxytocin.

J o u r n a l P r e -p r o o f
Table 1.Detection of plasmatic autoantibodies reactive with oxytocin.ELISA, enzyme-linked immuno-sorbent assay; IHC, immunohistochemistry; SPR, surface plasmon resonance.
The ELISA is the gold standard method to measure plasma levels of neuropeptides-reactive autoAbs of different Ig classes.There is a technical procedure available for ELISA neuropeptide autoantibody detection [26].The procedure enables also the detection of the IgG autoAbs "free" and "total" fractions by allowing the serum or plasma samples to be incubated in normal or dissociative buffers, respectively.A supplementary set of information on the relative concentrations of "free" and "complexed" antibodies might be obtained using this method, which could indicate altered peptidergic signaling such as increased formation of immune complexes ex. in the case of hypocretin-reactive IgG in narcolepsy [27].
Another method to detect oxytocin-reactive Ig in sera or plasma samples is immunohistochemistry on brain slices which are rich in oxytocin neurons ex. in the hypothalamus.Using this technique, oxytocin-reactive IgG was detected in patients with eating disorders by binding to the supraoptic nucleus and the median eminence of the rat brain.To confirm the binding specificity, preincubation of patient sera with oxytocin peptide reduced or abolished immunostaining [22].

J o u r n a l P r e -p r o o f
Surface Plasmon Resonance (SPR) is another quantitative method to detect and characterize autoAbs.The results obtained using the SPR for the analysis of oxytocin-reactive IgG will be discussed later in this review.
To detect neuropeptide-reactive Ig, studies focused on several pathological conditions known to affect peptidergic signaling including the oxytocin system.The first report of autoAbs reactive to oxytocin was probably the study analyzing plasma samples of patients with eating disorders [22].Indeed, using both ELISA and IHC the study reported the presence of IgG and IgM autoAbs reactive with oxytocin in the plasma of patients with anorexia nervosa (AN) or bulimia nervosa (BN) but also in healthy controls.The study also found an increase in oxytocin-reactive IgM levels and a decrease in IgG levels in the AN group, and a positive correlation of oxytocin-reactive IgM with the bulimia scores in BN patients [22].
The following studies analyzing oxytocin-reactive autoAbs focused on stress-related behaviors, involving the activation of the hypothalamic-pituitary-adrenal (HPA) axis, in which oxytocin plays an inhibitory role.An earlier review has discussed in detail the possible role of oxytocin-and some other stress-related peptide hormone-reactive autoAbs in stress-related disorders with a focus on aggressive behavior [28].Indeed, in subjects with conduct disorder and prisoners, there was an increase in oxytocin-reactive IgM levels [23].Moreover, our recent study revealed that there was an increase in oxytocin-reactive IgG in the plasma of aggressive inmates with a history of severe violence [11].Furthermore, the aggressivity scores, evaluated by the Bryant and Smith revised Aggression Questionnaire (BS-rAQ) correlated positively with the plasma levels of oxytocin-reactive IgG [11].Regarding mood disorders, patients with a moderate form of major depressive disorder displayed lower plasma levels of oxytocin-reactive IgG as compared to the control group and patients with a mild form of depression [25].To go further in exploring the possible link between neuropeptide-reactive IgG and alterations of mood and emotion, Karaiskos and coworkers analyzed patients with primary Sjögren's syndrome, an autoimmune disease distinguished by systemic (glandular) or local pathological process, but also neuropsychiatric symptoms [24].Oxytocin-reactive IgG was detected in the plasma of both controls and Sjögren's syndrome patients.Although no significant difference in mean levels of oxytocin-reactive IgG was found between the study groups, individual levels of these autoAbs correlated positively with anxiety scores in patients but negatively with the somatization score in controls [24].

Identification of IgG as an oxytocin carrier protein
To demonstrate that IgG may play a role of an oxytocin carrier protein, IgG was extracted from human plasma samples using the protein A. Then, IgG was acidified and peptides were extracted from binding proteins using C18 chromatography, and oxytocin concentration was measured using a commercial EIA kit (Figure 1) [11].This experimental procedure resulted in the readable detection of IgG-unbound oxytocin in all tested plasma samples i.e. confirming that IgG plays a role of oxytocin binding protein in healthy human subjects.The levels of IgGbound oxytocin revealed by this approach were lower than IgG 'free' oxytocin measured in plasma effluents during IgG purification.Since the levels of the total, i.e. the sum of IgGbound and free oxytocin was higher than oxytocin in native plasma, the difference should result from the IgG-unbound oxytocin.By adding the relative amounts of IgG-bound and unbound oxytocin, it will account for an average of 60% of its total levels.Thus, these data revealed that about 60% of plasma oxytocin circulates bound to IgG and it is also possible that this number is underestimated because some 'free' oxytocin can be potentially IgG unbound during the standard assay independently from the IgG purification step.We cannot, however, exclude that some IgG-free oxytocin could be bound to non-IgG carrier proteins which would eventually decrease the relative contribution of IgG as the main oxytocin carrier protein in plasma.Because the reversibility of the peptide binding to a larger protein defines its role as a carrier molecule, characterizing the affinity kinetics of oxytocin binding to IgG represents valuable information.Such characteristics were obtained using the SPR showing that in healthy subjects, the KD values, i.e. the constants of the dissociation at equilibrium, of IgG to bind oxytocin were in the range of 10 -7 -10 -8 M [11].Since these values are at least one order weaker than the known nanomolar affinity of oxytocin to bind the OTR [29], there is no theoretical obstacle for such plasmatic IgG to play a role of an oxytocin carrier protein molecule.High stability with at least 21-day half-life of IgG is another prerequisite for a physiological protein carrier.

J o u r n a l P r e -p r o o f
The variable nature of IgG, as it was reflected in different individual KD values of IgG for oxytocin, points to individual variation in the IgG carrier capacity for oxytocin and, hence, variation in oxytocin signaling.One of the roles of oxytocin as the prosocial hormone is well known suggesting its deficit in anti-social behavior such as violent aggression [28].A

J o u r n a l P r e -p r o o f
comparison of IgG properties to bind oxytocin between non-aggressive and aggressive men sentenced for violent crimes revealed reduced affinity in aggressive subjects (Figure 2).Such differences coincide with lower carrier IgG capacity, in particular reflected by lower levels of IgG unbound oxytocin [11].

Modulation of oxytocin receptor signaling by IgG-bound oxytocin
It was stressed earlier by several investigators that in contrast to free oxytocin, the biological activity of the oxytocin-bound fraction is unclear [2,13].Thus, since the IgG was determined as an oxytocin-binding protein, it was necessary to investigate if and how IgG-bound oxytocin i.e. the oxytocin immune complex (IC) may act on the OTR.
As a G protein-coupled receptor, the OTR is formed by a trimeric complex of G proteins, which consists of one G β/γ subunit and one Gα unit.The major signal transduction mechanism for OTR activation involves intracellular Ca 2+ release [30].Our recent study investigated the biological impact of IgG bound to oxytocin, on the OTR signaling by measuring intracellular Ca 2+ mobilization [11].For this purpose, human OTR-transfected HEK 293 cells were used [29].Incubation of cells with oxytocin, as expected, induced a significant elevation of intracellular Ca 2+ , illustrated in Figure 3.When oxytocin/IgG IC was applied, intracellular Ca 2+ increased immediately analogous to what was noticed after the application of oxytocin alone.Dynamics of Ca 2+ mobilization were, however, quite different, with oxytocin/IgG IC inducing a fast decrease as opposed to the prolonged plateau induced by oxytocin alone.When IgG from the aggressive subjects was employed, the statistical comparison showed lower mean levels of total Ca 2+ mobilization indicating reduced OTR activation in the group of aggressive subjects.
Moreover, for a fixed IgG quantity (840nM), the effective concentration (EC50) for oxytocin/IgG IC to bind OTR was found in the nanomolar range with slightly lower affinity in aggressive subjects [11].
Following ligand binding, the oxytocin receptor is first desensitized and then internalized.First process occurs around 4 sec following ligand binding when OTR is phosphorylated via G protein-coupled receptor kinase 2 and prepared to be bound to βarrestin [31].Next, OTR is internalized by endocytosis via a clathrin-coated pit and then could be recycled or degraded.Our previous study analyzed the effects of IgG on the OTR internalization process.Green-fluorescence protein (GFP)-labeled human OTR expressing HEK293 cells were incubated with oxytocin 10 -8 M alone or with oxytocin/IgG IC for 30 min, capturing regularly several cellular carpet regions of interest during this period.Similar to the cited above paper from Bice Chini's team, at the basal level we observed a diffuse green fluorescent OTR signal along the cell membrane.Internalization occurred after 2 min in the presence of oxytocin alone or oxytocin/IgG IC (Figure 4).

Possible antigenic origin of oxytocin-reactive IgG
Because the endogenous oxytocin peptide itself is not immunogenic, oxytocin-like antigens represent the potential source for the production of oxytocin-reactive IgG.Such antigens, for instance of microbial origin, may induce cross-reactive antibodies according to the concept of molecular mimicry [32].Indeed, amino acids-sequence homology with oxytocin is present in several commensal or pathogenic microorganisms [33].Most importantly, our recent results revealed using the proteomic technology that Lactobacilli produce oxytocin-like proteins and that immunization of mice with such proteins stimulates the production of oxytocin-reactive IgG [34].The link between gut microbiota and the oxytocin system was J o u r n a l P r e -p r o o f noticed and reviewed elsewhere [35].From the mechanistic point of view, neuropeptidereactive IgG appears as one of the molecular communication pathways in the gut microbiota-brain axis [36].Bacterial influence on the production of oxytocin-binding IgG may open new therapeutic opportunities for improving oxytocin signaling via modification of gut microbiota composition.Of relevance, the use of specific Lactobacillus strains was accompanied by improvement of social interactions, reduction of anxiety-and depressivelike behavior, and sugar intake in stressed mice [37][38][39].In future studies, it will be interesting to determine the possible effect of such probiotics on the production of oxytocinbinding IgG including their role in the improved motivated behavior, mood and emotion.

Functional relevance of oxytocin-binding IgG
Based on the experimental data stated above, it is evident that oxytocin-binding IgG is a natural modulator of oxytocin signaling upon the OTR (Figure 5).Moreover, starting from the plasma circulation, IgG should be able to stabilize bioavailable oxytocin levels, a key feature of any carrier protein.Indeed, our recent (unpublished) data show that in mice IgG protects oxytocin from degradation in plasma, a property tested using a previously published protocol that revealed IgG protective effects for ghrelin [4].Stabilizing the bioavailable oxytocin in blood circulation should ensure its proper action in peripheral targets.Indeed, the OTR is expressed in several peripheral tissues where it can modulate both local as well as systemic responses ex.reduction of the stress response and aggressive behavior [40][41][42].

J o u r n a l P r e -p r o o f
The relevance of oxytocin-binding IgG to the central effects of oxytocin was also shown but the underlying mechanisms need further clarification.In fact, after intraperitoneal (I.P.) administration of oxytocin forming IC with IgG in resident mice in the context of a residenceintruder test, a decrease in number of c-fos-positive cells was detected in the hypothalamic ventromedial nucleus, one of the key brain regions regulating aggressive behavior.In the same experiment, mice receiving I.P. injection of oxytocin alone did not show significant changes in c-fos, suggesting that IgG was able to protect the peripherally administered oxytocin and enhance its action on the brain targets [11].It is presently unclear if oxytocinbinding IgG was able to transport oxytocin into the brain or if the observed effects on neuronal activation were mediated via the peripheral oxytocin targets.In fact, IgG is naturally present inside the brain at concentrations around 0.1 M which is about 500 times lower than in the blood circulation [43].Moreover, a pharmacokinetics study of peripherally administered IgG in rats revealed its appearance in the brain parenchyma 4 h after injection and persisting for several weeks [44].Thus, we cannot exclude that oxytocin-binding IgG from the blood circulation may enter the brain and modulate central oxytocin signaling.As stated by MacLean and colleagues, bound oxytocin can be transported across the bloodbrain barrier yielding important consequences in the central nervous system [16].Moreover, a recent study determined that the soluble receptor for advanced glycation end-products (RAGE), a 50 kDa protein of the Ig superfamily, serves as an oxytocin transporter in the brain [45].This finding further supports the role of Ig as a functional carrier of peptide hormones including its possible faciliatory effect for peptide to gain access to the brain.

Conclusion
The natural presence in human plasma of oxytocin-binding IgG correlating with oxytocinrelevant psychological functions has triggered an idea that IgG may play a role of oxytocin carrier protein.Indeed, by measuring oxytocin in IgG-bound and unbound fractions in human plasma it has been estimated that around 60 % of oxytocin circulates reversibly bound to IgG which protects it from degradation.Moreover, IgG-bound oxytocin activates OTR with a similar to oxytocin affinity and modulates Ca 2+ release depending on binding properties between IgG and oxytocin.Taken together, IgG appears as an oxytocin-binding protein in blood circulation which ensures the oxytocin stability and modulates oxytocin

Figure 1 .
Figure 1.Methodological approach used to determine if IgG is an oxytocin-binding protein in human plasma [11].a. Separation of "free" and "IgG-bound" fractions of plasma oxytocin using affinity chromatography and oxytocin quantification with EIA. b.Schematic illustration of oxytocin fractions resulting from 3 oxytocin assays shown in (a).

Figure 2 .
Figure 2. SPR sensorgrams from the analysis of affinity kinetics between oxytocin and human IgG injected at different concentrations into the instrument BIAcore T200.Example of oxytocin binding by IgG from one non-aggressive (A) and aggressive (B) subjects, showing corresponding affinity values.Figure redrawn from [11].
IgG Contr + OT OT 10 -7 M IgG Aggr + OT J o u r n a l P r e -p r o o f

Figure 3 .
Figure 3. Ca 2+ release in vitro after OTR activation by immune complexes of oxytocin (OT) with IgG from aggressive (Aggr) or non-aggressive control (Contr) subjects, or by oxytocin peptide alone.RFU, relative fluorescence units.Figure redrawn from [11].

Figure 5 .
Figure 5. Schematic illustration of the role of IgG as a carrier protein of oxytocin.For the description see the conclusion section.Drawing by Serguei Fetissov.

J
o u r n a l P r e -p r o o f signaling.Furthermore, the putative antigenic origin of oxytocin-reactive IgG in gut bacteria may open new therapeutic opportunities for influencing oxytocin signaling.