Substantia. An International Journal of the History of Chemistry 3(2): 19-25, 2019 Firenze University Press www.fupress.com/substantia ISSN 1827-9643 (online) | DOI: 10.13128/Substantia-632 Citation: F. Real-Fernández, G. Pacini, F. Nuti, G. Conciarelli, C. De Felice, J. Hayek, P. Rovero, A.M. Papini (2019) Is aberrant N-glucosylation relevant to recognise anti-MOG antibodies in Rett syndrome?. Substantia 3(2): 19-25. doi: 10.13128/Substantia-632 Copyright: © 2019 F. Real-Fernández, G. Pacini, F. Nuti, G. Conciarelli, C. De Felice, J. Hayek, P. Rovero, A.M. Papini. This is an open access, peer- reviewed article published by Firenze University Press (http://www.fupress. com/substantia) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All rel- evant data are within the paper and its Supporting Information files. Competing Interests: The Author(s) declare(s) no conflict of interest. Research Article Is aberrant N-glucosylation relevant to recognise anti-MOG antibodies in Rett syndrome? Feliciana Real-Fernández1,2, Giulia Pacini2, Francesca Nuti1, Giulia Conciarelli2, Claudio De Felice3, Joussef Hayek4, Paolo Rovero2, Anna Maria Papini1,* 1 Interdepartmental Laboratory of Peptide and Protein Chemistry and Biology, Depart- ment of Chemistry “Ugo Schiff ”, University of Florence, Sesto Fiorentino, Italy 2 Interdepartmental Laboratory of Peptide and Protein Chemistry and Biology, Depart- ment of Neurosciences, Psychology, Drug Research and Child Health - Section of Pharma- ceutical Sciences and Nutraceutics, University of Florence, Sesto Fiorentino, Italy 3 Neonatal Intensive Care Unit, University Hospital, Azienda Ospedaliera Universitaria Senese, Siena, Italy 4 Child Neuropsychiatry Unit, University Hospital, Azienda Ospedaliera Universitaria Senese, Siena, Italy *E-mail: annamaria.papini@unifi.it Abstract. Antibodies against myelin oligodendrocyte glycoprotein (MOG) are asso- ciated to several disorders, and their occurrence in patients presenting an acquired demyelinating disease affects a higher proportion of paediatric subjects, as compared to adults. Despite heterogeneity in clinical presentation, few connexions have been reported between the progressive neurodevelopmental disorder affecting child’s brain development and cognitive ability, i.e. Rett syndrome (RTT), and a demyelination pro- cess. In order to identify the possible target of humoral autoimmune response in RTT patients, we set-up a home-made solid-phase ELISA, using the recombinant extracellu- lar portion of human MOG(1-117) as an antigen. The screening to evaluate anti-MOG antibodies in RTT patient sera, compared to other relative non-RTT pervasive devel- opmental disorders (non-RTT PDD), including mainly autism, and a healthy control group gave uncertain results. In fact, Student t-test and Mann-Whitney unpaired t test showed that differences in both IgG and IgM antibody titres between the different patient populations, were not statistically significant. We can conclude that the absence of anti-MOG antibody recognition in RTT has possibly to be ascribed to a different relevant protein folding and/or to the lack of a relevant aberrant post-translational modification, such as N-glucosylation, that we previously demonstrated, for the first time, fundamental to recognize antibodies in RTT. Keywords. Myelin oligodendrocyte glycoprotein, Rett syndrome, antibody detection, ELISA. INTRODUCTION A precise myelination is crucial for optimal transmission of nerve impulses and in providing trophic support to axons. In the central nervous 20 F. Real-Fernández et al. system (CNS) oligodendrocytes shape the myelin sheath surrounding axons.1 Intermittent uncovered short por- tions of the axon, called myelin-sheath gaps or the nodes of Ranvier, are fundamental for optimal myelin func- tioning.2,3 Perturbations of the nodes of Ranvier and myelin can be due to several causes including autoim- mune responses as in multiple sclerosis,4 Guillain-Barré syndrome,5 or in other immune-mediated neurological diseases.6 Demyelination process can be unleashed either because of an attack directly on the myelin sheath and/ or a disruption or death of oligodendrocytes. This clear difference in triggering the same end-stage of demyeli- nation may not be obvious and sometimes damage to both may occur. The aetiology of myelin loss includes immune-mediated, viral, metabolic, toxic, and/or genetic causes. Moreover, brain damages that may occur during neonatal hypoxia or subsequent to traumatic injury may also result in successive demyelination.3,7 In this context, the involvement of CNS myelin pro- teins is fundamental for oligodendrocyte growth and myelination.8-11 Myelin proteins include myelin prote- olipid protein (PLP), the related DM20, myelin-asso- ciated oligodendrocyte basic protein (MOBP), myelin- associated glycoprotein (MAG), 2’,3’-cyclic-nucleotide 3’-phosphodiesterase (CNP), and particularly the myelin basic protein (MBP) and myelin oligodendrocyte gly- coprotein (MOG). Proteins as MBP and MOG, located in the external part of myelin, have been proposed as antigens in several immune-mediated disorders. MOG localization on the outermost surface of myelin sheath and the plasma membrane of oligodendrocytes12 con- vert this protein into a partial exposed target (Figure 1). Despite the specific function of MOG has still to be clarified, its role as important surface marker of oligo- dendrocyte maturation, regulator of microtubule stabil- ity and mediator of interactions between myelin and the immune system have been described.13,14 More contro- versial are the results obtained to identify and clarify the role of anti-MOG antibodies, which are still a matter of discussion,15-17 particularly on their putative pathogenic involvement in autoimmune response in multiple scle- rosis15,18-20. Interesting data about the diagnostic/prog- nostic role of anti-MOG antibodies in multiple sclerosis patient sera were published,21 followed by contradictory studies that could not confirm these results. In fact, the same group of authors described other contrasting data in a conflicting array.22-25 A recent review reports that methods to detect anti-MOG antibodies have improved substantially with cell-based assays.26 However, a strong debate is still ongoing.27 Anyway, from the molecular point of view definition of the peptide epitope (confor- mational and/or linear) involved in antibody recognition is a challenge. In fact, a maximum of 8-10 amino acids are involved in in vivo antibody binding.28 MOG has a unique site of N-glycosylation at position 31 and the MOG(35-55) peptide has been the only MOG fragment able to induce neurological impairment in mice compa- rable with those observed in experimental autoimmune encephalomyelitis induced by MBP or PLP.29 To assess the presence of a B-cell intramolecular epitope spread- ing mechanism, we tested synthetic peptides mapping MOG(1-117), including MOG(35-55). An intense IgG antibody response against both the recombinant protein and the immunizing peptide MOG(35-55) was observed, while no response was observed against the other syn- thetic fragments. Furthermore, as the properly refolded recombinant probe is able to bind antibodies with great- er efficiency compared with MOG(35-55), we hypoth- esized the presence of both linear and conformational epitopes on MOG(35-55) sequence.30 The arguments discussed in the current literature regarding anti-MOG antibodies in multiple sclerosis can be extended to other inflammatory demyelinating diseases of the CNS. In particular, anti-MOG antibody- associated disorders account for a higher proportion of paediatric patients than adults who present an acquired demyelinating disease.31 Previously, we hypothesized the coexistence of a perturbation of the immune system in Rett syndrome Figure 1. Homology model of the extracellular domain of human myelin oligodendrocyte glycoprotein (MOG), with the β-turn inside the fragment MOG(35-55) evidenced. 21Is aberrant N-glucosylation relevant to recognise anti-MOG antibodies in Rett syndrome? (RTT) patients.32 RTT is a neurodevelopmental genetic disorder presenting neurological regression after devel- opment during infancy. A derangement of microglia immune responsiveness might be likely to occur in these paediatric patients, as neuroinflammation is a power- ful modulator of the CNS immune system. We observed that RTT patients showed a consistent and highly sig- nificant increased titer of IgM antibodies relative to both healthy controls and non-RTT pervasive developmen- tal disorders (non-RTT PDD) patient groups by using a diagnostic synthetic glycopeptide antigen of multiple sclerosis (Figure 2).32-34 Moreover, despite heterogeneity in clinical pres- entation, few connexions between RTT and demyeli- nation process have been reported. In fact, Sharma et al. focused on the role of Methyl CpG binding protein 2 (MeCP2), one of the genes associated with RTT, and its involvement in regulation of myelin gene expres- sion.35 Additionally, a case report with similarities in RTT symptoms and anti-MOG antibody encephalitis has been described.36 Convergence of these diseases could lead to a better understanding in demyelination process due to immune-mediated mechanisms. With all these considerations in mind, the main goal of our work was to identify the target of the humoral autoimmune response in RTT patients, recognised by the synthetic N-glucosylated β-turn peptide structure,32 evaluating the possible cross-reaction with anti-MOG antibodies. Moreover, we focused on a better under- standing of antibody response in Rett syndrome com- pared to other relative non-RTT PDD, including mainly autism, apparently connected (as they share some behav- ioural traits), but dramatically different for their severity, life-span expectancy, and immune system derangement. To this aim, a homemade SP-ELISA, based on the extra- cellular portion hMOG(1–117) expressed in Escherichia coli and properly refolded, was employed to test RTT patient population, other relative non-RTT PDD, and healthy control groups. MATERIALS AND METHODS Patients In this study, a group of 110 children was enrolled. This population consisted of three clearly distinguish- able groups: the RTT syndrome group (28) versus non- RTT pervasive developmental disorders (non-RTT PDD) group (48), classification based on the clinical features and the presence of mutated RTT-related genes and healthy, age-matched controls (34). These patients were hospitalized for 1 week every 6 months, in the Child Neuropsychiatric Unit, “Azienda Ospedaliera Univer- sitaria Senese”, Siena (Italy), during the course of the study. Criteria for inclusion in the study were clinical diagnosis of RTT syndrome coupled with positive iden- tification for the presence/absence of mutated MeCP2, CDKL5, or FOXG1 genes. The age-matched non-RTT PDD group consisted of 48 patients, as diagnosed fol- lowing well-established criteria. Blood samplings in the patient group were performed during the routine follow- up study at hospital admission, while the samples from the control group were carried out during routine health checks, sports, or blood donations, obtained during the periodic clinical checks. The healthy control subjects were age-matched. Patients were selected randomly and not previously tested for immune reactivity by ELISA. Parents, tutors, or guardians of all the participants pro- vided their written informed consent for the minors to participate in this study. The study design, methods, and consent procedure were approved by the Institutional Review Board of Azienda Ospedaliera Universitaria Sen- ese. All the data used in this study were anonymized. Figure 2. The β-turn peptide structure exposing at position 7 the N-glucosylation recognizing specific antibodies in Rett syndrome in a home-made ELISA.32 22 F. Real-Fernández et al. Materials Solid‐phase ELISAs were performed using 96‐well plates NUNC Maxisorp f lat bottom (Sigma‐Aldrich, Milan, Italy). Washing steps were performed using a microplate washer Hydroflex (Tecan, Männedorf, Swit- zerland). Fetal bovine serum (FBS) was purchased from Sigma-Aldrich (Milan, Italy). Secondary anti‐human IgG and IgM antibodies conjugated with alkaline phos- phatase were purchased by Sigma‐Aldrich (Milan, Italy). p‐Nitrophenyl phosphate was purchased from Fluka (Milan, Italy). Absorbance values were measured on a plate reader Tecan Sunrise purchased from Tecan (Tecan Italia, Milan, Italy). Electrocompetent ER2566 E. coli cells were purchased from New England Biolabs (Ipswich, MA, USA). Plasmid pET‐22 was purchased from Novagen (Madison, WI, USA). Protein purification and refolding were performed using a Chelating Sepha- rose Fast Flow column on ÄktaBasic chromatography system (GE Healthcare, Milan, Italy). The far‐UV cir- cular dichroism (CD) spectra were recorded by using a J‐810 Jasco spectropolarimeter (JASCO, Easton, MD). Enzyme-Linked Immunosorbent Assay (ELISA) The protein fragment hMOG(1–117) cDNA was subcloned into the His‐tag expression vector pET‐22. Recombinant hMOG(1-117) was produced according to the protocol published by Gori et al.37 Recombinant hMOG(1-117) was dissolved in coating buffer (12mM Na2CO3, 35mM NaHCO3, pH 9.6) to obtain a solution 10 µg/mL. Then 100 µl of solution were dispensed in each well of 96 well MaxiSorp flat bottom plate, pinch- bar design. Plates were incubated a +4°C overnight. Subsequently, plates were washed 3 times with Washing Buffer (0.9% NaCl, 0.01% Tween 20), and blocked 1 h at RT with 100 µl/well of FBS Buffer (10% FBS in Washing Buffer). After FBS buffer removal, 100 µl/well of dilut- ed sera sample (1:100 in FBS Buffer) were dispensed in triplicates. Plates were incubated at +4°C overnight, and then washed 3 times with Washing Buffer, 100 µl/well of secondary Ab labeled with alkaline phosphatase diluted in FBS Buffer (anti-h IgG 1:8000 and anti-h IgM 1:200) were dispensed and incubated 3 h at room tempera- ture. Plates were washed 3 times with Washing Buffer, then 100 µl/well of Substrate Solution (1mg/ml p-PNP in Carbonate Buffer containing 1mM MgCl2, pH 9.8) were dispensed. Absorbance was read at 405 nm with a spectrophotometer. Sera values were calculated as (mean absorbance of triplicate) – (mean absorbance of blank triplicate). Statistical analysis Data are expressed as mean values and elaborated using the statistical software GraphPad Prism version 6.01. D’Agostino-Parson test was employed as normal- ity test. Student t-test or Mann-Whitney unpaired t-test were used to compare continuous variables between groups. Spearman correlation analysis was used to test any relationship between pairs of variables. Differences were deemed statistically significant when P < 0.05 (two- tailored test). RESULTS AND DISCUSSION In order to study the antibody response against recombinant refolded h-MOG in RTT, we tested 28 RTT patients, 48 non-RTT PDD, and 30 healthy con- trols by using a home-made SP-ELISA. The recombinant hMOG(1-117) was tested as an antigen evaluating IgG and IgM type antibodies separately. Data distribution of IgG antibody titers detected to hMOG(1-117) in RTT, non-RTT PDD, and controls are plotted in Figure 3. The overall data distribution were statistically ana- lyzed using D’Agostino-Pearson test and results showed that none of the RTT, non-RTT PDD, or healthy controls group passed the normality test (alpha = 0.05). Then, antibody titer differences between groups were evalu- ated separately using the Mann-Whitney U-test. Results showed no discriminant differences between RTT and non-RTT PDD patients (P value = 0.6629, two-tailed), RTT and healthy controls (P value = 0.2583, two-tailed), Figure 3. Comparison between IgG antibodies against the hMOG(1-117) identified by SP-ELISA in RTT (), non-RTT PDD patient sera (●), and healthy controls (○) respectively. Mean group values and standard error of mean (SEM) are represented. 23Is aberrant N-glucosylation relevant to recognise anti-MOG antibodies in Rett syndrome? or non-RTT PDD and healthy controls (P value = 0.6137, two-tailed). Similar results were observed when IgM-type anti- bodies were evaluated. Data distribution of IgM anti- body values are plotted in Figure 4. The overall data did not present a Gaussian distribution (D’Agostino-Pearson omnibus normality test, alpha = 0.05). Moreover, Mann- Withney test showed no significant statistic differences between groups (P value > 0.05, two-tailed) further evi- dencing no meaningful differences, thus allowing us to assume that MOG as a possible antigen in RTT and/or non-RTT PDD is irrelevant. Moreover, no relationship was found between IgG and IgM autoantibody levels (nonparametric Spearman correlation, P values > 0.05). Evidences of anti-MOG antibody-associated diseas- es in children with acquired demyelinating syndromes, whose sera test were positive for anti-MOG antibodies, have been described.38. As discussed in the introduction, the genetic mechanism underlying the RTT syndrome appear directly linked to a demyelinating process. On the other hand, despite previous studies reporting a con- nection between multiple sclerosis and RTT humoral responses, the role of anti-MOG antibodies in these dis- orders cannot be clarified. The lack of a clear anti-MOG antibody identification in RTT, herein observed, reminds the open controversy around anti-MOG antibodies in the case of multiple sclerosis, as a kind of parallelism between these diseases. Previously, our expertise in antibody detection using proteins37,39,40 or peptides41,42 prompted us to develop the so-called “chemical reverse approach” in which synthetic peptides were demonstrated to be more effective than native proteins.43 In fact, their principal advantage is the complete control of the synthetic molecules. Mazzucco et al. showed that the N-glucosylation (N-Glc) of the hMOG peptide [Asn31(N-Glc)]hMOG(30-50) allowed to detect antibodies in 40% of an unselected group of mul- tiple sclerosis patients.44 After almost 20 years, we dis- covered that anti-N-Glc antibodies from multiple scle- rosis patients preferentially recognize adhesin of non- typeable Haemophilus influenza hyperglucosylated on asparagine residus exposed on β-turns.39 Therefore, it is clear that the folding issue is relevant in antibody recog- nition, and synthetic peptides can be designed to adopt specific conformations, e.g. β-turns.45,46 Moreover, syn- thetic conformational peptides can be efficient tools as antigenic probes for serum antibody detection, because they can also include unique chemical modifications, such as asparagine N-glucosylation, on strategic posi- tions in selected sequences. This strategy has been, up to now, to the best of our knowledge, the only winner in detecting antibodies in RTT patient sera.32,47 Our find- ings offer a new insight into the mechanism underlying the RTT as they unveil the possible participation of the immune system in this pathology.48 Moreover, our previ- ous work contributes to elucidate that two disorders such as RTT and autism, seemingly contiguous as they share some behavioral symptoms, but are in fact different for their ruthlessness, life-span expectation, and, as we previously demonstrated, for different immune system derangement. In this context and in light of the results herein presented, the connection of anti-MOG antibod- ies and RTT remains an uncertainty. In particular, the involvement of the correct folding, but also the lack of a mimicry effect reproducing N-glucosylation (and other molecules) as possible aberrant post-translational modi- fications on MOG amino acids (involved in triggering immune responses), require to be deeply investigated. CONCLUSIONS The screening of RTT patient sera, other relative non-RTT pervasive developmental disorders (non-RTT PDD) including mainly autism, and healthy controls group to evaluate anti-MOG antibodies was uncer- tain. Despite anti-MOG antibody detection in multiple sclerosis and generally speaking in MOG-IgG–related diseases have improved substantially with cell-based assays, in which the molecules involved in antibody rec- ognition are not fully chemically characterised. On the other hand our preliminary results are in agreement with the idea that the reproduction of post-translational modifications possibly involved in the immune response Figure 4. Data distribution of IgM antibodies against hMOG(1- 117) identified by SP-ELISA in RTT (), non-RTT PDD patient sera (●), and healthy controls (○), respectively. Mean group values and standard error of mean (SEM) are represented. 24 F. Real-Fernández et al. could be a must for antibody identification, as it occurs in other diseases connected with RTT, such as multi- ple sclerosis. In particular, investigating glycan-pep- tide mimicry in the context of immune response is an emerging topic, pointing toward the multiple roles that unique glycans of bacterial origin may play. These nov- el preliminary results pave the way to further studies, already ongoing in our laboratories, focused on under- standing the responsible agents triggering the immune response in RTT, inducing aberrant conformation and/ or N-glucosylation in native proteins, such as Myelin Oligodendrocyte Glycoprotein. 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Feliciana Real-Fernández1,2, Giulia Pacini2, Francesca Nuti1, Giulia Conciarelli2, Claudio De Felice3, Joussef Hayek4, Paolo Rovero2, Anna Maria Papini1,* Hydrogen-like quantum Hamiltonians & Einstein separability in the case of charged radical molecules Han Geurdes A scientific rationale for consciousness Pr. Marc Henry1,*, Jean-Pierre Gerbaulet2,* Derjaguin’s Water II: a surface hydration phenomenon Ilya Klugman, Anna Melnikov1, Drew F. Parsons2 Leonardo da Vinci – The Scientist Walter Isaacson B. V. Derjaguin* and J. Theo. G. Overbeek. Their Times, and Ours Barry W. Ninham Sadi Carnot’s Réflexions and the foundation of thermodynamics Pier Remigio Salvi, Vincenzo Schettino Vladimir Vasilyevich Markovnikov (1838-1904) – the eminent Russian chemist, author of one of the best known empiric rule in organic chemistry Aleksander Sztejnberg