RESUMO
The biosynthetic pathway governing inhibin heterodimer (alpha/beta) and activin homodimer (beta/beta) assembly and secretion from ovarian granulosa cells is not fully understood. Here, we examined the role of inhibin subunit glycosylation in the assembly and secretion of mature inhibin A and activin A. Inhibition of subunit glycosylation by tunicamycin treatment of alpha- and beta(A)-expressing CHO cell lines reduced inhibin but not activin secretion. Dimeric inhibin A is preferentially secreted from parental isogenic wild-type (wt) cell lines (alpha(wt)beta(wt)). Mutation of a single glycosylation site at asparagine 268 (alpha(Delta268)beta(wt)) reduces inhibin secretion by 78% and permits beta/beta assembly and secretion. Conversely, gain of a glycosylation (GOG) site in the analogous region of the beta(A)-subunit (alpha(wt)beta(GOG327)) enhances inhibin A secretion. The present study demonstrates that N-linked glycan sites direct heterodimer vs. homodimer assembly, and prevention of glycosylation abrogates inhibin secretion. These data support a definitive role for site-specific N-glycosylation in governing inhibin/activin dimer assembly and secretion.
Assuntos
Subunidades beta de Inibinas/química , Subunidades beta de Inibinas/metabolismo , Inibinas/química , Inibinas/metabolismo , Oligossacarídeos/química , Oligossacarídeos/metabolismo , Ativinas/química , Ativinas/genética , Ativinas/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação/genética , Células CHO , Cricetinae , Cricetulus , Primers do DNA/genética , Dimerização , Evolução Molecular , Glicosilação , Subunidades beta de Inibinas/genética , Inibinas/genética , Modelos Biológicos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oligossacarídeos/genética , Estrutura Quaternária de Proteína , Frações Subcelulares/metabolismo , Fator de Crescimento Transformador beta/antagonistas & inibidores , Tunicamicina/farmacologiaRESUMO
BACKGROUND: Diverse viruses often reactivate in or infect cancer patients, patients with immunocompromising infections or genetic conditions, and transplant recipients undergoing immunosuppressive therapy. These infections can disseminate, leading to death, transplant rejection, and other severe outcomes. OBJECTIVES: To develop and characterize an assay capable of inclusive and accurate identification of diverse potentially disseminating viruses directly from plasma specimens. STUDY DESIGN: We developed a PCR/electrospray ionization mass spectrometry (PCR/ESI-MS) assay designed to simultaneously detect and identify adenovirus, enterovirus, polyomaviruses JC and BK, parvovirus B19, HSV-1, HSV-2, VZV, EBV, CMV, and herpesviruses 6-8 in plasma specimens. The assay performance was characterized analytically, and the results from clinical plasma samples were compared to the results obtained from single-analyte real time PCR tests currently used in clinical practice. RESULTS: The assay demonstrated sensitivity and specificity to diverse strains of the targeted viral families and robustness to interfering substances and potentially cross reacting organisms. The assay yielded 94% sensitivity when testing clinical plasma samples previously identified as positive using standard-of-care real-time PCR tests for a single target virus (available samples included positive samples for 11 viruses targeted by the assay). CONCLUSIONS: The assay functioned as designed, providing simultaneous broad-spectrum detection and identification of diverse agents of disseminated viral infection. Among 156 clinical samples tested, 37 detections were made in addition to the detections matching the initial clinical positive results.