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Inhibitory GH receptor extracellular domain monoclonal antibodies: three-dimensional epitope mapping.

ABSTRACT: GH receptor (GHR) mediates the anabolic and metabolic effects of GH. We previously characterized a monoclonal antibody (anti-GHR(ext-mAb)) that reacts with subdomain 2 of the rabbit GHR extracellular domain (ECD) and is a conformation-specific inhibitor of GH signaling in cells bearing rabbit or human GHR. Notably, this antibody has little effect on GH binding and also inhibits inducible metalloproteolysis of the GHR that occurs in the perimembranous ECD stem region. In the current study, we demonstrate that anti-GHR(ext-mAb) inhibits GH-dependent cellular proliferation and also inhibits hepatic GH signaling in vivo in mice that adenovirally express rabbit GHR, as assessed with our noninvasive bioluminescence hepatic signaling assay. A separate monoclonal antibody (anti-GHR(mAb 18.24)) is a sister clone of anti-GHR(ext-mAb). Here, we demonstrate that anti-GHR(mAb 18.24) also inhibits rabbit and human GHR signaling and inducible receptor proteolysis. Further, we use a random PCR-generated mutagenic expression system to map the three-dimensional epitopes in the rabbit GHR ECD for both anti-GHR(ext-mAb) and anti-GHR(mAb 18.24). We find that each of the two antibodies has similar, but nonidentical, discontinuous epitopes that include regions of subdomain 2 encompassing the dimerization interface. These results have fundamental implications for understanding the role of the dimerization interface and subdomain 2 in GHR activation and regulated GHR metalloproteolysis and may inform development of therapeutics that target GHR.

SUBMITTER: Jiang J

PROVIDER: S-EPMC3230063 | biostudies-literature | 2011 Dec

REPOSITORIES: biostudies-literature

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Inhibitory GH receptor extracellular domain monoclonal antibodies: three-dimensional epitope mapping.

Jiang Jing J Wan Yu Y Wang Xiangdong X Xu Jie J Harris Jonathan M JM Lobie Peter E PE Zhang Yu Y Zinn Kurt R KR Waters Michael J MJ Frank Stuart J SJ

Endocrinology 20111011 12

GH receptor (GHR) mediates the anabolic and metabolic effects of GH. We previously characterized a monoclonal antibody (anti-GHR(ext-mAb)) that reacts with subdomain 2 of the rabbit GHR extracellular domain (ECD) and is a conformation-specific inhibitor of GH signaling in cells bearing rabbit or human GHR. Notably, this antibody has little effect on GH binding and also inhibits inducible metalloproteolysis of the GHR that occurs in the perimembranous ECD stem region. In the current study, we dem ...[more]

PMID: 21990310

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Project description:Dengue virus (DV) infection causes a spectrum of clinical diseases ranging from dengue fever to a life-threatening dengue hemorrhagic fever. Four distinct serotypes (DV1-4), which have similar genome sequences and envelope protein (E protein) antigenic properties, were divided. Among these 4 serotypes, DV1 usually causes predominant infections and fast diagnosis and effective treatments are urgently required to prevent further hospitalization and casualties.To develop antibodies specifically targeting and neutralizing DV1, we immunized mice with UV-inactivated DV1 viral particles and recombinant DV1 E protein from residue 1 to 395 (E395), and then generated 12 anti-E monoclonal antibodies (mAbs) as the candidates for a series of characterized assays such as ELISA, dot blot, immunofluorescence assay, western blot, and foci forming analyses.Among the mAbs, 10 out of 12 showed cross-reactivity to four DV serotypes as well as Japanese encephalitis virus (JEV) in different cross-reactivity patterns. Two particular mAbs, DV1-E1 and DV1-E2, exhibited strong binding specificity and neutralizing activity against DV1 and showed no cross-reactivity to DV2, DV3, DV4 or JEV-infected cells as characterized by ELISA, dot blot, immunofluorescence assay, western blot, and foci forming analyses. Using peptide coated indirect ELISA, we localized the neutralizing determinants of the strongly inhibitory mAbs to a sequence-unique epitope on the later-ridge of domain III of the DV1 E protein, centered near residues T346 and D360 (346TQNGRLITANPIVTD360). Interestingly, the amino acid sequence of the epitope region is highly conserved among different genotypes of DV1 but diverse from DV2, DV3, DV4 serotypes and other flaviviruses.Our results showed two selected mAbs DV1-E1 and DV1-E2 can specifically target and significantly neutralize DV1. With further research these two mAbs might be applied in the development of DV1 specific serologic diagnosis and used as a feasible treatment option for DV1 infection. The identification of DV1 mAbs epitope with key residues can also provide vital information for vaccine design.

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Inhibitory GH receptor extracellular domain monoclonal antibodies: three-dimensional epitope mapping.

Publications

Inhibitory GH receptor extracellular domain monoclonal antibodies: three-dimensional epitope mapping.

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