Global analysis of protein lysine 2-hydroxyisobutyrylation (Khib) profiles in Chinese herb rhubarb (Dahuang)

Background Lysine 2-hydroxyisobutyrylation (Khib) is a newly discovered protein posttranslational modification (PTM) and is involved in the broad-spectrum regulation of cellular processes that are found in both prokaryotic and eukaryotic cells, including in plants. The Chinese herb rhubarb (Dahuang) is one of the most widely used traditional Chinese medicines in clinical applications. To better understand the physiological activities and mechanism of treating diseases with the herb, it is necessary to conduct intensive research on rhubarb. However, Khib modification has not been reported thus far in rhubarb. Results In this study, we performed the first global analysis of Khib-modified proteins in rhubarb by using sensitive affinity enrichment combined with high-accuracy HPLC-MS/MS tandem spectrometry. A total of 4333 overlapping Khib modification peptides matched on 1525 Khib-containing proteins were identified in three independent tests. Bioinformatics analysis showed that these Khib-containing proteins are involved in a wide range of cellular processes, particularly in protein biosynthesis and central carbon metabolism and are distributed mainly in chloroplasts, cytoplasm, nucleus and mitochondria. In addition, the amino acid sequence motif analysis showed that a negatively charged side chain residue (E), a positively charged residue (K), and an uncharged residue with the smallest side chain (G) were strongly preferred around the Khib site, and a total of 13 Khib modification motifs were identified. These identified motifs can be classified into three motif patterns, and some motif patterns are unique to rhubarb and have not been identified in other plants to date. Conclusions A total of 4333 Khib-modified peptides on 1525 proteins were identified. The Khib-modified proteins are mainly distributed in the chloroplast, cytoplasm, nucleus and mitochondria, and involved in a wide range of cellular processes. Moreover, three types of amino acid sequence motif patterns, including EKhib/KhibE, GKhib and k.kkk….Khib….kkkkk, were extracted from a total of 13 Khib-modified peptides. This study provides comprehensive Khib-proteome resource of rhubarb. The findings from the study contribute to a better understanding of the physiological roles of Khib modification, and the Khib proteome data will facilitate further investigations of the roles and mechanisms of Khib modification in rhubarb.

AB0055 AUTOIMMUNE RESPONSE AGAINST THE SHARED EPITOPE SEQUENCE IN RHEUMATOID ARTHRITIS

Background:Rheumatoid Arthritis (RA) is a systemic autoimmune disease, associated with hiperproduction of autoantibodies (AAb), in which the most specific are the AAb against citrullinated peptides (ACPA). RA is influenced by genetic factors, specifically, there is a strong genetic association with the shared epitope (SE), a five amino acid sequence motif in positions 70–74 of HLA-DRβ1 chains encoded by HLA-DRB1 alleles: QKRAA, QRRAA and RRRAA.The present study aims to analyze whether SE-peptides (SE-p) can be a target of the autoimmune response in RA.Objectives:To analyze the presence of AAb against the unmodified (Un) SE-p, citrullinated (Cit) SE-p and carbamylated (Car) SE-p.Methods:Sera from consecutive 117 RA patients and 21 psoriasic arthritis (PsA) from our outpatient clinic were collected by venopunture. Also 138 sera from blood donors were obtained as healthy controls (HC). All participants signed the informed consent.We perfomed a homemade ELISA test using a sequence of 15 aminoacid peptides from positions 65-79 of HLA-DRB1 containing the 3 different SE sequences, in the Un, Cit and Car SE-p, synthesized in a linear and cycled form. We established a 90% of specificity using a ROC curve obtained from HC and PsA for each ELISA test.HLA-DRB1 polymorphism was performed using a HLA-DRB1 sequence specific oligonucleotide typing kit (Lifecodes) in 95 RA and in 15 PsA.ACPA and RF were determined with commercial assays (Inova Diagnostics and Binding Site, respectively).Results:The overall sensitivity of the different SE-p AAb tests ranged from 5.1-21.4%.RRRAA SE polymorphism was associated with AAb against cycled CitCitCitAA SE-p (p=0.025), QKRAA polymorphism was almost significantly associated with AAb against cycled QKCitAA SE-p (p=0.067), whereas there was no association between QRRAA polymorphism and AAb against cycled QCitCitAA SE-p (p=0.690). On the other hand, there was no association between SE polymorphisms and AAb to any other peptide used in the ELISA test.Significant differences were observed in the presence of AAb against lineal RRRAA, lineal CitCitCitAA and cycled CitCitCitAA SE-p when comparing RA vs. HC patients (p=0.022, 0.044, 0.022, respectively). Moreover, there also were significant differences in the presence of AAb against cycled CitCitCitAA SE-p between RA and PsA patients (sensitivity 21.4%, specificity 100%; p=0.014).It must be highlighted that cycled CitCitCitAA SE-p AAb were detected in 20.0% of RA patient sera that were negative for RF and ACPA.There was no association between RF or ACPA with the presence of any SE-p AAb.Conclusion:RA patients have autoantibodies against the Shared Epitope (SE). The cycled CitCitCitAA SE peptide (SE-p) shows the best performance among all the peptides tested and could identify patients seronegative for ACPA and RF, both analyzed by commercial assays.Additional studies must be performed to verify the diagnostic and utility of these new autoantibodies against SE-p in RA.Acknowledgements:This work was granted by the 2018 call of the “Fundación Española de Reumatologia” and the 2017 call grant “Fundació Parc Taulí”.Disclosure of Interests:None declared