A comprehensive analysis of site-specific protein O-glycosylation is hindered by the absence of a consensus O-glycosylation motif, the diversity of O-glycan structures, and the lack of a universal enzyme that cleaves attached O-glycans. Here, we report the development of a robust O-glycoproteomic workflow for analyzing complex biological samples by combining four different strategies: removal of N-glycans, complementary digestion using O-glycoprotease (IMPa) with/without another protease, glycopeptide enrichment, and mass spectrometry with fragmentation of glycopeptides using stepped collision energy. Using this workflow, we cataloged 474 O-glycopeptides on 189 O-glycosites derived from 79 O-glycoproteins from human plasma. These data revealed O-glycosylation of several abundant proteins that have not been previously reported. Because many of the proteins that contained unannotated O-glycosylation sites have been extensively studied, we wished to confirm glycosylation at these sites in a targeted fashion. Thus, we analyzed selected purified proteins (kininogen-1, fetuin-A, fibrinogen, apolipoprotein E, and plasminogen) in independent experiments and validated the previously unknown O-glycosites.

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UNASSIGNED: Hereditary angioedema (HAE) is associated with episodic kinin-induced swelling of the skin and mucosal membranes. Most patients with HAE have low plasma C1-inhibitor activity, leading to increased generation of the protease plasma kallikrein (PKa) and excessive release of the nanopeptide bradykinin from high-molecular-weight kininogen (HK). However, disease-causing mutations in at least 10% of patients with HAE appear to involve genes for proteins other than C1-inhibitor. A point mutation in the Kng1 gene encoding HK and low-molecular weight kininogen (LK) was identified recently in a family with HAE. The mutation changes a methionine (Met379) to lysine (Lys379) in both proteins. Met379 is adjacent to the Lys380-Arg381 cleavage site at the N-terminus of the bradykinin peptide. Recombinant wild-type (Met379) and variant (Lys379) versions of HK and LK were expressed in HEK293 cells. PKa-catalyzed kinin release from HK and LK was not affected by the Lys379 substitutions. However, kinin release from HK-Lys379 and LK-Lys379 catalyzed by the fibrinolytic protease plasmin was substantially greater than from wild-type HK-Met379 and LK-Met379. Increased kinin release was evident when fibrinolysis was induced in plasma containing HK-Lys379 or LK-Lys379 compared with plasma containing wild-type HK or LK. Mass spectrometry revealed that the kinin released from wild-type and variant kininogens by PKa is bradykinin. Plasmin also released bradykinin from wild-type kininogens but cleaved HK-Lys379 and LK-Lys379 after Lys379 rather than Lys380, releasing the decapeptide Lys-bradykinin (kallidin). The Met379Lys substitutions make HK and LK better plasmin substrates, reinforcing the relationship between fibrinolysis and kinin generation.

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BACKGROUND: Hepatitis B virus (HBV) infection is one of the main causes of hepatocellular carcinoma (HCC). The relationship between HBV infection and the host genome as well as their underlying mechanisms remain largely unknown.
METHODS: In this study, we performed a whole-genome exon sequencing analysis of 300 sib-pairs of Chinese HBV-infected families with the goal of identifying variants and genes involved in HBV infection. A site-direct mutant plasmid was used to investigate the function of SNP rs76438938 in KNG1. The functional and mechanical studies of KNG1 were conducted with in vitro liver cell lines and a hydrodynamic injection model in vivo. The impact of KNG1 on HBV infection therapy was determined in hepatocytes treated with IFN-α/λ1.
RESULTS: Our whole-exon association study of 300 families with hepatitis B infection found that SNP rs76438938 in KNG1 significantly increased the risk for HBV infection, and the rs76438938-T allele was found to promote HBV replication by increasing the stability of KNG1 mRNA. By competitively binding HSP90A with MAVS, KNG1 can inhibit the expression of types I and III IFNs by promoting MAVS lysosomal degradation. Such suppression of IFN expression and promotion of HBV replication by Kng1 were further demonstrated with an animal model in vivo. Lastly, we showed that the rs76438938-C allele can improve the therapeutic effect of IFN-α and -λ1 in HBV infection.
CONCLUSIONS: This study identified a SNP, rs76438938, in a newly discovered host gene, KNG1, for its involvement in HBV infection and treatment effect through modulating the cellular antiviral process.
BACKGROUND: This study was supported in part by the Independent Task of State Key Laboratory for Diagnosis and Treatment of Infectious Diseases of the First Affiliated Hospital of Zhejiang University, the China Precision Medicine Initiative (2016YFC0906300), and the Research Center for Air Pollution and Health of Zhejiang University.

Heart transplantation remains the definitive treatment for end stage heart failure. Because availability is limited, risk stratification of candidates is crucial for optimizing both organ allocations and transplant outcomes. Here we utilize proteomics prior to transplant to identify new biomarkers that predict post-transplant survival in a multi-institutional cohort. Microvesicles were isolated from serum samples and underwent proteomic analysis using mass spectrometry. Monte Carlo cross-validation (MCCV) was used to predict survival after transplant incorporating select recipient pre-transplant clinical characteristics and serum microvesicle proteomic data. We identified six protein markers with prediction performance above AUROC of 0.6, including Prothrombin (F2), anti-plasmin (SERPINF2), Factor IX, carboxypeptidase 2 (CPB2), HGF activator (HGFAC) and low molecular weight kininogen (LK). No clinical characteristics demonstrated an AUROC > 0.6. Putative biological functions and pathways were assessed using gene set enrichment analysis (GSEA). Differential expression analysis identified enriched pathways prior to transplant that were associated with post-transplant survival including activation of platelets and the coagulation pathway prior to transplant. Specifically, upregulation of coagulation cascade components of the kallikrein-kinin system (KKS) and downregulation of kininogen prior to transplant were associated with survival after transplant. Further prospective studies are warranted to determine if alterations in the KKS contributes to overall post-transplant survival.

Patients with hereditary angioedema (HAE) experience episodes of bradykinin (BK)-induced swelling of skin and mucosal membranes. The most common cause is reduced plasma activity of C1 inhibitor, the main regulator of the proteases plasma kallikrein (PKa) and factor XIIa (FXIIa). Recently, patients with HAE were described with a Lys311 to glutamic acid substitution in plasminogen (Plg), the zymogen of the protease plasmin (Plm). Adding tissue plasminogen activator to plasma containing Plg-Glu311 vs plasma containing wild-type Plg (Plg-Lys311) results in greater BK generation. Similar results were obtained in plasma lacking prekallikrein or FXII (the zymogens of PKa and FXIIa) and in normal plasma treated with a PKa inhibitor, indicating Plg-Glu311 induces BK generation independently of PKa and FXIIa. Plm-Glu311 cleaves high and low molecular weight kininogens (HK and LK, respectively), releasing BK more efficiently than Plm-Lys311. Based on the plasma concentrations of HK and LK, the latter may be the source of most of the BK generated by Plm-Glu311. The lysine analog ε-aminocaproic acid blocks Plm-catalyzed BK generation. The Glu311 substitution introduces a lysine-binding site into the Plg kringle 3 domain, perhaps altering binding to kininogens. Plg residue 311 is glutamic acid in most mammals. Glu311 in patients with HAE, therefore, represents reversion to the ancestral condition. Substantial BK generation occurs during Plm-Glu311 cleavage of human HK, but not mouse HK. Furthermore, mouse Plm, which has Glu311, did not liberate BK from human kininogens more rapidly than human Plg-Lys311. This indicates Glu311 is pathogenic in the context of human Plm when human kininogens are the substrates.

Epidemiological studies have established that untreated hypertension (HTN) is a major independent risk factor for developing cardiovascular diseases (CVD), stroke, renal failure, and other conditions. Several important studies have been published to prevent and manage HTN; however, antihypertensive agents\' optimal choice remains controversial. Therefore, the present study is undertaken to update our knowledge in the primary treatment of HTN, specifically in the setting of other three important diseases. MicroRNAs (miRNAs) are remarkably stable short endogenous conserved non-coding RNAs that bind to the mRNA at its (3\' UTR) to regulate its gene expression by causing translational repression or mRNA degradation. Through their coordinated activities on different pathways and networks, individual miRNAs control normal and pathological cellular processes. Therefore, to identify the critical miRNA-mRNA-TF interactions, we performed systematic bioinformatics analysis. We have also employed the molecular modelling and docking approach to identify the therapeutic target that delivers novel empathies into Food and Drug Administration approved and herbal drug response physiology. Gene Expression Omnibus (GEO) was employed to identify the differentially expressed genes (DEGs) and hub genes- KNG1, HLA-DPB1, CXCL8, IL1B, and BCL2. The HTN associated feed-forward loop (FFL) network included miR-9-5p, KNG1 and AR. We employed high throughput screening to get the best interacting compounds, telmisartan and limonin, that provided a significant docking score (-13.3 and -12.0 kcal/mol) and a potential protective effect that may help to combat the impact of HTN. The present study provides novel insight into HTN etiology through the identification of mRNAs and miRNAs and associated pathways.

Detailed proteomic analysis in a cohort of patients with differing severity of COVID-19 disease identified biomarkers within the complement and coagulation cascades as biomarkers for disease severity has been reported; however, it is unclear if these proteins differ sufficiently from other conditions to be considered as biomarkers.
A prospective, parallel study in T2D (n = 23) and controls (n = 23). A hyperinsulinemic clamp was performed and normoglycemia induced in T2D [4.5 ± 0.07 mmol/L (81 ± 1.2 mg/dl)] for 1-h, following which blood glucose was decreased to ≤2.0 mmol/L (36 mg/dl). Proteomic analysis for the complement and coagulation cascades were measured using Slow Off-rate Modified Aptamer (SOMA)-scan.
Thirty-four proteins were measured. At baseline, 4 of 18 were found to differ in T2D versus controls for platelet degranulation [Neutrophil-activating peptide-2 (p = 0.014), Thrombospondin-1 (p = 0.012), Platelet factor-4 (p = 0.007), and Kininogen-1 (p = 0.05)], whilst 3 of 16 proteins differed for complement and coagulation cascades [Coagulation factor IX (p < 0.05), Kininogen-1 (p = 0.05), and Heparin cofactor-2 (p = 0.007)]; STRING analysis demonstrated the close relationship of these proteins to one another. Induced euglycemia in T2D showed no protein changes versus baseline. At hypoglycemia, however, four proteins changed in controls from baseline [Thrombospondin-1 (p < 0.014), platelet factor-4 (p < 0.01), Platelet basic protein (p < 0.008), and Vitamin K-dependent protein-C (p < 0.00003)], and one protein changed in T2D [Vitamin K-dependent protein-C, (p < 0.0002)].
Seven of 34 proteins suggested to be biomarkers of COVID-19 severity within the platelet degranulation and complement and coagulation cascades differed in T2D versus controls, with further changes occurring at hypoglycemia, suggesting that validation of these biomarkers is critical. It is unclear if these protein changes in T2D may predict worse COVID-19 disease for these patients.
https://clinicaltrials.gov/, identifier NCT03102801.

Hereditary angioedema (HAE) attacks are caused by excessive activation of the contact system. Understanding how the contact system is activated in HAE, especially in patients with normal C1 inhibitor (HAEnCI), is essential to effectively treat this disease. Contact system activation involves the cleavage of several proteins including Factor XII (FXII), high molecular weight kininogen (HK), prekallikrein, sgp120 (ITIH4) and C1 inhibitor (C1-INH) before the subsequent generation of bradykinin that mediates HAE. In this study, we evaluated the fragmentation and enzymatic activity of contact system proteins in HAEnCI plasma samples before and after contact system activation induced by incubation in the cold. Our results show that in contrast to normal plasma, cold activation induced contact system activation in the majority of the HAEnCI patient samples we tested, in which each contact system protein exhibited fragmentation, FXII and kallikrein enzymatic activity increased, and C1-INH functional activity decreased. HAEnCI samples with low FXII concentrations or functional activity were not affected by cold activation. One HAEnCI sample with a plasminogen gene mutation activated the fibrinolytic system, as shown by an increase in concentration of plasma D dimers. Our results suggest that cold activation seems to be initiated by the cleavage of prekallikrein, and that it needs FXII in order to occur. Reported to be susceptible to excessive contact system activation after incubation in the cold, we further applied this system of study to the evaluation of plasma from women undergoing estrogen treatment. Similar to plasma from HAEnCI patients, excessive contact system activation was demonstrated.