Development and characterisation of a method for determining N-terminal heterogeneity in the human recombinant follitropin β-subunitHu, Han, Sun
et alJ Pharm Biomed Anal (2025) 261, 116828
Abstract: Recombinant human follicle-stimulating hormone (rhFSH) consists of α- and β subunits linked by non-covalent bonds. Both subunits exhibit N-terminal heterogeneity, with the β-subunit exhibiting heterogeneity to a greater extent. In this study, we developed a comprehensive method for assessing β-subunit N-terminal heterogeneity based on a previously developed peptide mapping approach. The new method, which includes optimised sample preparation and reversed-phase high-performance liquid chromatography, facilitated the separation of integrated (NSCELTNITIAIEK) βL1 and truncated (CELTNITIAIEK) βL1 forms. The identity of the separated integrated and truncated forms was confirmed via tandem mass spectrometry. Optimised sample preparation streamlined the process by simultaneously performing trypsin digestion (with calcium chloride enhancement) and PNGase F deglycosylation for a total preparation time of just 1 day. The enzymatic digestion and sample preparation processes underwent several evaluations, including scaled-up sample preparation, sample preparation variability assessment and extended incubation stability studies. The sample preparation process demonstrated excellent robustness in all evaluations. Results consistently showed approximately 50 % N-terminal truncation of two amino acids in the β-subunit across different manufacturers' samples, highlighting the utility of the method in assessing the batch-to-batch consistency and comparability of biosimilar products. This integrated approach combines peptide mapping and N-terminal heterogeneity analysis into a single, efficient workflow, thereby significantly enhancing quality control processes and enhancing the safety and efficacy of recombinant FSH products.Copyright © 2025 Elsevier B.V. All rights reserved.
Enhanced Production and Functional Characterization of Recombinant Equine Chorionic Gonadotropin (rec-eCG) in CHO-DG44 CellsByambaragchaa, Park, Park
et alBiomolecules (2025) 15 (2)
Abstract: Equine chorionic gonadotropin (eCG) hormone, comprising highly glycosylated α- and β-subunits, elicits responses similar to follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in non-equid species. This study aimed to establish a mass production of recombinant eCG (rec-eCG) using CHO DG44 cells. Single-chain rec-eCG β/α was expressed in CHO DG44 cells. FSH- and LH-like activities were evaluated in CHO-K1 and HEK 293 cells expressing the equine LH/CG receptor (eLH/CGR), rat LH/CGR (rLH/CGR), and rFSHR. pERK1/2 activation and β-arrestin 2 recruitment were assessed in PathHunter CHO-K1 cells. The expression from one, among nine isolates, peaked at 364-470 IU/mL on days 9 and 11. The molecular weight of rec-eCG β/α ranged from 40 to 47 kDa, with two distinct bands. PNGase F treatment reduced the molecular weight by 8-10 kDa, indicating N-glycosylation. Rec-eCG β/α demonstrated dose-responsive cAMP activity in cells expressing eLH/CGR, with enhanced potency in rLH/CGR and rFSHR. Phospho-ERK1/2 activation peaked at 5 min before declining rapidly. β-arrestin 2 recruitment was receptor-mediated in cells expressing hFSHR and hLH/CGR. This study provides insights into the mechanisms underlying eCG's FSH- and LH-like activities. Stable CHO DG44 cells can produce large quantities of rec-eCG. eCG activates pERK1/2 signaling via the PKA/cAMP pathway and facilitates β-arrestin 2 recruitment.
Nucleolin in the cell membrane promotes Ang II-mediated VSMC phenotypic switching by regulating the AT1R internalization function : Nucleolin promotes Ang II-mediated VSMC phenotypic switchingFang, Shen, Zhang
et alBiol Direct (2025) 20 (1), 24
Abstract: Nucleolin (NCL) plays an important regulatory role in angiotensin II (Ang II)-induced phenotypic switching of vascular smooth muscle cells (VSMCs). The aim of this study was to discuss its potential regulatory mechanisms.We investigated if the pathways involving Ang II type 1 receptor (AT1R) and PKC/MAPK are responsible for Ang II's effects on VSMC phenotypic switching. Ang II upregulated NCL expression and activated the PKC/MAPK signaling pathway to induce VSMC phenotypic switching. In addition, Ang II promoted the translocation of NCL from the nucleus to the cell membrane. NCL was shown to bind to AT1R, whereas the binding of NCL to AT1R was greatly attenuated after the deletion of the GAR region. In addition, when peptide-N-glycosidase F (PNGase F) was applied, the N-glycosylation of NCL protein and the phenotypic switching of VSMC were inhibited. Ang II-induced AT1R internalization, whereas overexpression of NCL delayed Ang II-induced AT1R internalization by inhibiting AT1R phosphorylation and recruited Rab4 and Rab11 to promote recycling, and knockdown of NCL showed the opposite effect and reversal of AT1R binding by the use of rasarfin reversed the effects of sh-NCL. In in vivo experiments, knockdown of NCL expression inhibited Ang II-induced blood pressure rise and vasculopathy.Our study demonstrates that NCL promotes Ang II-mediated phenotypic switching of VSMCs by regulating AT1R internalization function.© 2025. The Author(s).
Biochemical characterization of the Escherichia coli surfaceome: a focus on type I fimbriae and flagellaKavanaugh, Sivignon, Rossez
et alFront Microbiol (2025) 16, 1507286
Abstract: The Escherichia coli surfaceome consists mainly of the large surface organelles expressed by the organism to navigate and interact with the surrounding environment. The current study focuses on type I fimbriae and flagella. These large polymeric surface organelles are composed of hundreds to thousands of subunits, with their large size often preventing them from being studied in their native form. Recent studies are accumulating which demonstrate the glycosylation of surface proteins or virulence factors in pathogens, including E. coli. Using biochemical and glycobiological techniques, including biotin-hydrazide labeling of glycans and chemical and glycosidase treatments, we demonstrate (i) the presence of a well-defined and chemically resistant FimA oligomer in several strains of pathogenic and non-pathogenic E. coli, (ii) the major subunit of type I fimbriae, FimA, in pathogenic and laboratory strains is recognized by concanavalin A, (iii) standard methods to remove N-glycans (PNGase F) or a broad-specificity mannosidase fail to remove the glycan structure, despite the treatments resulting in altered migration in SDS-PAGE, (iv) PNGase F treatment results in a novel 32 kDa band recognized by anti-FliC antiserum. While the exact identity of the glycan(s) and their site of attachment currently elude detection by conventional glycomics/glycoproteomics, the current findings highlight a potential additional layer of complexity of the surface (glyco) proteome of the commensal or adhesive and invasive E. coli strains studied.Copyright © 2025 Kavanaugh, Sivignon, Rossez, Chouit, Chambon, Béal, Bonnet, Hébraud, Guérardel, Nguyen and Barnich.
膜杰作
Star Staining
