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Naturally occurring mutants inform SHBG structure and function.

ABSTRACT: SHBG transports and regulates the activities of androgens and estrogens. Several single nucleotide polymorphisms in the human SHBG gene have been linked to sex steroid-dependent diseases, including those associated with the metabolic syndrome. The N-terminal laminin G-like domain of SHBG includes binding sites for calcium, sex steroids, and fibulin family members, as well as a dimerization domain. We have found that 8 of 18 uncharacterized nonsynonymous single nucleotide polymorphisms within this domain alter the production or biochemical properties of SHBG in ways not previously recognized. O-Linked glycosylation at Thr7 is disrupted in SHBG T7N, whereas abnormal glycosylation of SHBG G195E limits its secretion. Three SHBG mutants (R135C, L165M, and E176K) bind estradiol with abnormally high affinity. SHBG R135C also has an increased interaction with fibulin-2. Two different substitutions within the dimer interface at R123 (R123H and R123C) reduce the affinity for 5?-dihydrotestosterone, while increasing the relative binding affinity for estradiol. SHBG T48I is defective in calcium binding, which leads to a defect in dimerization, reduced affinity for sex steroids, and an enhanced interaction with fibulin-2, which can all be restored by calcium supplementation. These naturally occurring mutants provide insight into SHBG structure and function, and defects in SHBG production or function need to be considered in the context of its utility as a biomarker of diseases.

SUBMITTER: Wu TS

PROVIDER: S-EPMC5414826 | biostudies-literature | 2014 Jul

REPOSITORIES: biostudies-literature

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Naturally occurring mutants inform SHBG structure and function.

Wu Tsung-Sheng TS Hammond Geoffrey L GL

Molecular endocrinology (Baltimore, Md.) 20140603 7

SHBG transports and regulates the activities of androgens and estrogens. Several single nucleotide polymorphisms in the human SHBG gene have been linked to sex steroid-dependent diseases, including those associated with the metabolic syndrome. The N-terminal laminin G-like domain of SHBG includes binding sites for calcium, sex steroids, and fibulin family members, as well as a dimerization domain. We have found that 8 of 18 uncharacterized nonsynonymous single nucleotide polymorphisms within thi ...[more]

PMID: 24892637

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Project description:BACKGROUND: Ophiocordyceps sinensis (syn. Cordyceps sinensis), which is a parasite of caterpillars and is endemic to alpine regions on the Tibetan Plateau, is one of the most valuable medicinal fungi in the world. "Natural O. sinensis specimens" harbor various other fungi. Several of these other fungi that have been isolated from natural O. sinensis specimens have similar chemical components and/or pharmaceutical effects as O. sinensis. Nevertheless, the mycobiota of natural O. sinensis specimens has not been investigated in detail. METHODOLOGY/PRINCIPAL FINDINGS: Based on the technique of PCR-single-strand conformation polymorphism (PCR-SSCP), the mycobiota of three different sections (stromata, sclerotia, and mycelial cortices) from natural O. sinensis specimens were investigated using both culture-dependent and -independent methods. For the culture-dependent method, 572 fungal strains were isolated, and 92 putative operational taxonomic units (OTUs) were identified from 226 sequenced strains with the threshold of 97%. For the culture-independent method, 490 fungal clones were identified from about 3000 clones of ITS fragments from the whole-community DNA; based on PCR-SSCP analyses, 266 of these clones were selected to be sequenced, and 118 putative OTUs were detected. The overwhelming majority of isolates/clones and OTUs were detected from mycelial cortices; only a few were detected from stromata and sclerotia. The most common OTUs detected with both methods belonged to Ascomycota; however, only 13 OTUs were detected simultaneously by both methods. Potential novel lineages were detected by each of the two methods. CONCLUSIONS/SIGNIFICANCE: A great number of fungal species present in the mycobiota of naturally-occurring O. sinensis specimens were detected, and many of them may represent undescribed lineages. That only a few of the same OTUs were detected by both methods indicated that different methods should be used. This study increased our understanding about the fungal community structure of this valuable medicinal herb.

Project description:The core promoter mutants of hepatitis B virus (HBV) emerge as the dominant viral population at the late HBeAg and the anti-HBe stages of HBV infection, with the A1762T/G1764A substitutions as the hotspot mutations. The double core promoter mutations were found by many investigators to moderately enhance viral genome replication and reduce hepatitis B e antigen (HBeAg) expression. A much higher replication capacity was reported for a naturally occurring core promoter mutant implicated in the outbreak of fulminant hepatitis, which was caused by the neighboring C1766T/T1768A mutations instead. To systemically study the biological properties of naturally occurring core promoter mutants, we amplified full-length HBV genomes by PCR from sera of HBeAg(+) individuals infected with genotype A. All 12 HBV genomes derived from highly viremic sera (5 x 10(9) to 5.7 x 10(9) copies of viral genome/ml) harbored wild-type core promoter sequence, whereas 37 of 43 clones from low-viremia samples (0.2 x 10(7) to 4.6 x 10(7) copies/ml) were core promoter mutants. Of the 11 wild-type genomes and 14 core promoter mutants analyzed by transfection experiments in human hepatoma cell lines, 6 core promoter mutants but none of the wild-type genomes replicated at high levels. All had 1762/1764 mutations and an additional substitution at position 1753 (T to C), at position 1766 (C to T), or both. Moreover, these HBV clones varied greatly in their ability to secrete enveloped viral particles irrespective of the presence of core promoter mutations. High-replication clones with 1762/1764/1766 or 1753/1762/1764/1766 mutations expressed very low levels of HBeAg, whereas high-replication clones with 1753/1762/1764 triple mutations expressed high levels of HBeAg. Experiments with site-directed mutants revealed that both 1762/1764/1766 and 1753/1762/1764/1766 mutations conferred significantly higher viral replication and lower HBeAg expression than 1762/1764 mutations alone, whereas the 1753/1762/1764 triple mutant displayed only mild reduction in HBeAg expression similar to the 1762/1764 mutant. Thus, core promoter mutations other than those at positions 1762 and 1764 can have major impact on viral DNA replication and HBeAg expression.

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Naturally occurring mutants inform SHBG structure and function.

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Naturally occurring mutants inform SHBG structure and function.

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