Autoantibodies targeting G protein-coupled receptors: An evolving history in autoimmunity. Report of the 4th international symposium.

G protein-coupled receptors (GPCR) are involved in various physiological and pathophysiological processes. Functional autoantibodies targeting GPCRs have been associated with multiple disease manifestations in this context. Here we summarize and discuss the relevant findings and concepts presented in the biennial International Meeting on autoantibodies targeting GPCRs (the 4th Symposium), held in Lübeck, Germany, 15-16 September 2022. The symposium focused on the current knowledge of these autoantibodies' role in various diseases, such as cardiovascular, renal, infectious (COVID-19), and autoimmune diseases (e.g., systemic sclerosis and systemic lupus erythematosus). Beyond their association with disease phenotypes, intense research related to the mechanistic action of these autoantibodies on immune regulation and pathogenesis has been developed, underscoring the role of autoantibodies targeting GPCRs on disease outcomes and etiopathogenesis. The observation repeatedly highlighted that autoantibodies targeting GPCRs could also be present in healthy individuals, suggesting that anti-GPCR autoantibodies play a physiologic role in modeling the course of diseases. Since numerous therapies targeting GPCRs have been developed, including small molecules and monoclonal antibodies designed for treating cancer, infections, metabolic disorders, or inflammatory conditions, anti-GPCR autoantibodies themselves can serve as therapeutic targets to reduce patients' morbidity and mortality, representing a new area for the development of novel therapeutic interventions.

Mimicking LysC Proteolysis by 'Arginine Modification-cum-Trypsin Digestion': Comparison of Bottom-up & Middle-down Proteomic Approaches by ESI Q-TOF MS.

BACKGROUND: Middle-down (MD) proteomics is an emerging approach for reliable identification of post-translational modifications and isoforms, as this approach focuses on proteolytic peptides containing > 25-30 amino acid residues (a.a.r.), which are longer than typical tryptic peptides. Such longer peptides can be obtained by AspN, GluC, and LysC proteases. Additionally, some special proteases were developed specifically to effect MD approach, e.g., OmpT, Sap9, etc. However, these proteases are expensive. Herein we report a cost-effective strategy 'arginine modification- cum trypsin digestion', which can produce longer tryptic peptides resembling LysC peptides derived from proteins. OBJECTIVE: The aim of this study is to obtain proteolytic peptides that resemble LysC peptides by using 'trypsin', which is a less expensive protease. METHODS: This strategy is based on the simple principle that trypsin cannot act at the C-termini of those arginines in proteins, whose sidechain guanidine groups are modified by 1,2-cyclohexanedione or phenylglyoxal. RESULTS: As a proof of concept, we demonstrate this strategy on four models: ß-casein (bovine), ß- lactoglobulin (bovine), ovalbumin (chick) and transferrin (human), by electrospray ionization-mass spectrometry (ESI-MS) involving hybrid quadrupole time-of-flight. From the ESI-MS of these models, we obtained several arginine modified tryptic peptides, whose lengths are in the range of 30-60 a.a.r. The collision induced dissociation MS/MS characteristics of some of the arginine modified longer tryptic peptides are compared with the unmodified standard tryptic peptides. CONCLUSION: The strategy demonstrated in this proof-of-concept study is not only useful to obtain longer tryptic peptides that mimic LysC proteolytic peptides, but also facilitates in enhancing the probability of missed cleavages by the trypsin. Hence, this method aids in evading the possibility of obtaining very short peptides that are <5-10 a.a.r. Therefore, this is indeed a cost-effective alternative/ substitute for LysC proteolysis and, in turn, for those MD proteomic studies that utilize LysC. Additionally, this methodology can be fruitful for mass spectrometry-based de novo protein and peptide sequencing.

DNA hydrolysing IgG catalytic antibodies: an emerging link between psychoses and autoimmunity.

It is not uncommon to observe autoimmune comorbidities in a significant subset of patients with psychotic disorders, namely schizophrenia (SCZ) and bipolar disorder (BPD). To understand the autoimmune basis, the DNA abyzme activity mediated by serum polyclonal IgG Abs were examined in psychoses patients, quantitatively, by an in-house optimized DNase assay. A similar activity exhibited by IgG Abs from neuropsychiatric-systemic lupus erythematosus (NP-SLE) patients was used as a comparator. Our data revealed that the IgG DNase activity of SCZ was close to that of NP-SLE and it was twofold higher than the healthy controls. Interestingly, the association between DNase activity with PANSS (positive, general and total scores) and MADRS were noted in a subgroup of SCZ and BPD patients, respectively. In our study group, the levels of IL-6 and total IgG in BPD patients were higher than SCZ and healthy controls, indicating a relatively inflammatory nature in BPD, while autoimmune comorbidity was mainly observed in SCZ patients.

A dipyrrole derivative from Aloe vera inhibits an anti-diabetic drug target Dipeptidyl Peptidase (DPP)-IV in vitro.

Aloe vera, a succulent herb, has a long history of use in traditional medicine, including diabetes. Earlier studies from our laboratory demonstrated that the Aloe vera extract has the ability to inhibit the diabetic drug target dipeptidyl peptidase (DPP) IV in vitro. This current study focuses on the isolation of small water soluble active molecule(s) involved in DPP-IV inhibition from Aloe vera extract, and further to characterize its structure and to elucidate the mode of inhibition of the DPP-IV enzyme. Aloe vera gel ethanolic extract was subjected to preparative reverse-phase high-pressure liquid chromatography (RP-HPLC), LH-20 Sephadex gel filtration chromatography, followed by analytical RP-HPLC, to isolate the active molecule involved in DPP-IV inhibition. Based on the spectroscopic studies, the structure of the isolated DPP-IV inhibitor was predicted to be 3, 6-dioxo-3, 3a, 6, 6 a-tetrahydropyrrolo [3, 4-c] pyrrole-1, 4-dicarboxamide with the chemical formula C8H6N4O4, having the molecular weight of 225.175 Da. This molecule inhibited the DPP-IV enzyme in a noncompetitive manner with an IC50 value of 8.59 ± 2.61 µM, with a Ki of 4.7 ± 0.038 µM. Thus, the mechanism of DPP-IV inhibition and the inhibitory constants were determined. The results of our studies suggested that the inhibition of the DPP-IV enzyme as one of the pathways by which the Aloe vera extract may restore the pancreatic islets cell mass in diabetic animal model.

An L-fucose specific lectin from Aspergillus niger isolated from mycotic keratitis patient and its interaction with human pancreatic adenocarcinoma PANC-1 cells.

An L-fucose specific lectin from pathogenic fungus Aspergillus niger isolated from the corneal smears of keratitis patient was purified in a single step using mucin coupled sepharose-4B column by 58-fold. The purified lectin, ANL has molecular mass of 30 kDa by SDS-PAGE and 31.6 kDa by ESI-MS. ANL is a glycoprotein with 2.59% carbohydrate. ANL is blood group nonspecific and also agglutinates rabbit erythrocytes. ANL is heat stable up to 50 °C and over a pH range of 7-10. Hapten inhibition studies revealed that ANL is specific to L-fucose, galactose, lactose and glycoproteins, showing highest MIC of 3.125 µg for L-fucose, mucin and fetuin. ANL has potent antibacterial activity against Klebsiella pneumoniae, Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli and also it inhibits the biofilm formation by them. ANL showed strong binding to human pancreatic adenocarcinoma PANC-1 cells which was effectively blocked by L-fucose and mucin respectively by 76.2% and 84.2%. ANL showed dose and time dependent growth inhibitory effect on PANC-1 cells with IC50 of 1.25 µg/ml at 48 h. Effect of ANL was compared with another fucose specific lectin AOL, from Aspergillus oryzae showing an IC50 of 1.85 µg/ml at 48 h revealing promising clinical potential of ANL.

Purification, characterization and fine sugar specificity of a N-Acetylgalactosamine specific lectin from Adenia hondala.

Plant lectins are gaining interest because of their interesting biological properties. Several Adenia species, that are being used in traditional medicine to treat many health ailments have shown presence of lectins or carbohydrate binding proteins. Here, we report the purification, characterization and biological significance of N-Acetyl galactosamine specific lectin from Adenia hondala (AHL) from Passifloraceae family. AHL was purified in a single step by affinity chromatography on asialofetuin Sepharose 4B column, characterized and its fine sugar specificity determined by glycan array analysis. AHL is human blood group non specific and also agglutinates rabbit erythrocytes. AHL is a glycoprotein with 12.5% of the carbohydrate, SDS-PAGE, MALDI-TOF-MS and ESI-MS analysis showed that AHL is a monomer of 31.6 kDa. AHL is devoid of DNase activity unlike other Ribosome inactivating proteins (RIPs). Glycan array analysis of AHL revealed its highest affinity for terminal lactosamine or polylactosamine of N- glycans, known to be over expressed in hepatocellular carcinoma and colon cancer. AHL showed strong binding to human hepatocellular carcinoma HepG2 cells with MFI of 59.1 expressing these glycans which was effectively blocked by 93.1% by asialofetuin. AHL showed dose and time dependent growth inhibitory effects on HepG2 cells with IC50 of 4.8 µg/ml. AHL can be explored for its clinical potential.

Proteolysis activity of IgM antibodies from rheumatoid arthritis patients' sera: evidence of atypical catalytic site.

The IgM antibodies from rheumatoid arthritis (RA) patients' sera were screened for peptide hydrolyzing activity. Recovery of structurally intact IgM antibodies (Abs), in a single step, was achieved using a weak anion-exchange methacrylate monolith disk. The IgM Abs from patients' sera hydrolyzed the Pro-Phe-Arg-4-methyl-coumaryl-7-amide (PFR-MCA) substrate appreciably compared to the healthy donors. The apparent K(m) values of IgM Abs from patients' sera were between 0.4 and 0.7 mM. Furthermore, IgM Abs displayed 5 to 10-folds greater proteolysis activity than IgG Abs, recovered from the same pathological serum. The proteolysis activity, as a function, was found to be independent of IgM-RF titer value. Affinity labeling approach targeted at the catalytic site histidine was studied, using a specific irreversible inhibitor, N-α-tosyl-L-lysine chloromethyl ketone (TLCK). Despite modification of catalytic His, observation of serine protease like activity suggest presence of an atypical catalytic framework in a few pathological IgM Abs.

Molecular studies of rheumatoid factor using pseudobioaffinity membrane chromatography.

Using pseudobioaffinity ligand L-histidine immobilized to poly(ethylene vinyl alcohol) hollow fiber membrane is an interesting approach for the purification of total IgG and its subclasses from untreated serum in a single step. Gentle adsorption and elution conditions of this chromatography system allow efficient recovery of the protein in its native form. This approach was employed for the recovery and molecular study of rheumatoid factor (RF), an anti-IgG autoantibody (AAb) that form immune complexes with autologous IgG Abs in the sera. The purity of the recovered molecule was analyzed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), revealed a 150-kDa IgG band and an additional approximately 300 kDa band which may be RF bound IgG complex. Since RF is an AAb, the purified protein was studied for its catalytic functions like peptide, DNA, and RNA hydrolyzing activities. The substrate Pro-Phe-Arg-4-methyl-coumaryl-7-amide (PFR-MCA) hydrolyzing activity by total IgG from different patient sera was found to be greater than healthy controls. In an effort to identify the subclass specificity for the proteolytic function, the pre-purified total IgG fractions from rheumatoid arthritis (RA) sera were subjected to rechromatography using a discriminating buffer. In this experiment, the activity was found in the non-retained fractions suggesting IgG2 specificity for the catalytic function. A comparative study between different catalytic functions was performed for IgG separated from individual patient.

Purification of oligouronides by immobilized L-histidine pseudoaffinity chromatography.

Selective purification of alpha-(1,4)-oligogalacturonides was investigated using several pseudobioaffinity chromatography matrix with aminoacid L-histidine as pseudobiospecific ligand: (1) sepharose 4B-bisoxyran-histidine, (2) sepharose 4B-epoxy-histidine, (3) silica-oxyran-histidine and (4) CIM-disk-EDA-histidine. These anionic oligosaccharides prepared by enzymatic and chemical cleavage of polygalacturonic acid were used as models sugar in order to optimize the adsorption and elution parameters for a selective purification of bioactive oligouronides. Monolithic CIM-disk chromatography is one of the fastest liquid chromatographic method using for separation and purification of biomolecules thanks to high mass transfer rate. In this way, this new monolithic CIM-disk system with L-histidine immobilized: immobilized histidine affinity chromatography (IHAC) constitutes a good tool allowing the fast and selective purification of bioactive oligouronides.