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The amino Acid residues at positions 120 to 123 are crucial for the antigenicity of hepatitis B surface antigen.

ABSTRACT: The major hydrophilic region (MHR) of hepatitis B surface antigen (HBsAg) harbors conformational B-cell epitopes and is the major target of neutralizing antibodies to HBsAg (anti-HBs). Mutant HBsAg (mtHBsAg) with amino acid substitutions such as G145R is known to affect the binding of specific anti-HB antibodies and their detection by conventional diagnostic assays. In the present study, we focused on the role of the amino acid positions 120 to 123, which are around MHR 2 according to the spectrum of recently identified, naturally occurring mtHBsAg. Strikingly, the amino acid substitution K122I abolished the reactivity of HBsAg in all immunoassays tested so far. Also, mtHBsAg G145R could be clearly detected with four different enzyme-linked immunosorbent assays that were based on monoclonal anti-HB antibodies (MAbs) with high affinity. Positive immunofluorescence staining of mtHBsAg K122I was achieved only by polyclonal anti-HBs, while all MAbs tested failed. mtHBsAg T123N showed a low reactivity in immunoassays and appeared to be secretion defective. The amino acid substitution P120T reduced the binding of anti-HBs but did not completely prevent the detection of mtHBsAg by anti-HB MAbs. The testing of naturally occurring mtHBsAg confirmed that the presence of amino acid substitutions within the region of 120 to 123 is strongly associated with impaired detection in immunoassays. In conclusion, MHR 2 is essential for HBsAg antigenicity, a fact that has not been recognized before.

SUBMITTER: Tian Y

PROVIDER: S-EPMC2045265 | biostudies-literature | 2007 Sep

REPOSITORIES: biostudies-literature

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The amino Acid residues at positions 120 to 123 are crucial for the antigenicity of hepatitis B surface antigen.

Tian Yongjun Y Xu Yang Y Zhang Zhenhua Z Meng Zhongji Z Qin Li L Lu Mengji M Yang Dongliang D

Journal of clinical microbiology 20070703 9

The major hydrophilic region (MHR) of hepatitis B surface antigen (HBsAg) harbors conformational B-cell epitopes and is the major target of neutralizing antibodies to HBsAg (anti-HBs). Mutant HBsAg (mtHBsAg) with amino acid substitutions such as G145R is known to affect the binding of specific anti-HB antibodies and their detection by conventional diagnostic assays. In the present study, we focused on the role of the amino acid positions 120 to 123, which are around MHR 2 according to the spectr ...[more]

PMID: 17609325

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Project description:BACKGROUND & AIMS:As important virological markers, serum hepatitis B surface antigen (HBsAg) and hepatitis B virus (HBV) DNA levels show large fluctuations among chronic hepatitis B patients. The aim of this study was to reveal the potential impact and mechanisms of amino acid substitutions in small hepatitis B surface proteins (SHBs) on serum HBsAg and HBV DNA levels. METHODS:Serum samples from 230 untreated chronic hepatitis B patients with genotype C HBV were analyzed in terms of HBV DNA levels, serological markers of HBV infection and SHBs sequences. In vitro functional analysis of the identified SHBs mutants was performed. RESULTS:Among 230 SHBs sequences, there were 39 (16.96%) sequences with no mutation detected (wild-type) and 191 (83.04%) with single or multiple mutations. SHBs consist of 226 amino acids, of which 104 (46.02%) had mutations in our study. Some mutations (e.g., sE2G, sL21S, sR24K, sT47A/K, sC69stop (sC69?), sL95W, sL98V, and sG145R) negatively correlated with serum HBsAg levels. HBsAg and HBV DNA levels from this group of patients had a positive correlation (r=0.61, p<0.001). In vitro analysis showed that these mutations reduced extracellular HBsAg and HBV DNA levels by restricting virion secretion and antibody binding capacity. Virion secretion could be rescued for sE2G, sC69?, and sG145R by co-expression of wild-type HBsAg. CONCLUSION:The serum HBsAg levels were lower in untreated CHB patients with novel SHBs mutations outside the major antigenic region than those without mutations. Underlying mechanisms include impairment of virion secretion and lower binding affinity to antibodies used for HBsAg measurements. LAY SUMMARY:The hepatitis B surface antigen (HBsAg) is a major viral protein of the hepatitis B virus (HBV) secreted into patient blood serum and its quantification value serves as an important marker for the evaluation of chronic HBV infection and antiviral response. We found a few new amino acid substitutions in HBsAg associated with lower serum HBsAg and HBV DNA levels. These different substitutions might impair virion secretion, change the ability of HBsAg to bind to antibodies, or impact HBV replication. These could all result in decreased detectable levels of serum HBsAg. The factors affecting circulating HBsAg level and HBsAg detection are varied and caution is needed when interpreting clinical significance of serum HBsAg levels. Clinical trial number: NCT01088009.

Project description:GPR55 is a newly deorphanized class A G-protein-coupled receptor that has been implicated in inflammatory pain, neuropathic pain, metabolic disorder, bone development, and cancer. Few potent GPR55 ligands have been identified to date. This is largely due to an absence of information about salient features of GPR55, such as residues important for signaling and residues implicated in the GPR55 signaling cascade. The goal of this work was to identify residues that are key for the signaling of the GPR55 endogenous ligand, l-?-lysophosphatidylinositol (LPI), as well as the signaling of the GPR55 agonist, ML184 {CID 2440433, 3-[4-(2,3-dimethylphenyl)piperazine-1-carbonyl]-N,N-dimethyl-4-pyrrolidin-1-ylbenzenesulfonamide}. Serum response element (SRE) and serum response factor (SRF) luciferase assays were used as readouts for studying LPI and ML184 signaling at the GPR55 mutants. A GPR55 R* model based on the recent ?-opioid receptor (DOR) crystal structure was used to interpret the resultant mutation data. Two residues were found to be crucial for agonist signaling at GPR55, K2.60 and E3.29, suggesting that these residues form the primary interaction site for ML184 and LPI at GPR55. Y3.32F, H(170)F, and F6.55A/L mutation results suggested that these residues are part of the orthosteric binding site for ML184, while Y3.32F and H(170)F mutation results suggest that these two residues are part of the LPI binding pocket. Y3.32L, M3.36A, and F6.48A mutation results suggest the importance of a Y3.32/M3.36/F6.48 cluster in the GPR55 signaling cascade. C(10)A and C(260)A mutations suggest that these residues form a second disulfide bridge in the extracellular domain of GPR55, occluding ligand extracellular entry in the TMH1-TMH7 region of GPR55. Taken together, these results provide the first set of discrete information about GPR55 residues important for LPI and ML184 signaling and for GPR55 activation. This information should aid in the rational design of next-generation GPR55 ligands and the creation of the first high-affinity GPR55 radioligand, a tool that is sorely needed in the field.

Dataset Information

The amino Acid residues at positions 120 to 123 are crucial for the antigenicity of hepatitis B surface antigen.

Publications

The amino Acid residues at positions 120 to 123 are crucial for the antigenicity of hepatitis B surface antigen.

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