FUT8基因敲除HEK293细胞

Most patients with triple negative breast cancer (TNBC) do not respond to anti-PD1/PDL1 immunotherapy, indicating the necessity to explore immune checkpoint targets. B7H3 is a highly glycosylated protein. However, the mechanisms of B7H3 glycosylation regulation and whether the sugar moiety contributes to immunosuppression are unclear. Here, we identify aberrant B7H3 glycosylation and show that N-glycosylation of B7H3 at NXT motif sites is responsible for its protein stability and immunosuppression in TNBC tumors. The fucosyltransferase FUT8 catalyzes B7H3 core fucosylation at N-glycans to maintain its high expression. Knockdown of FUT8 rescues glycosylated B7H3-mediated immunosuppressive function in TNBC cells. Abnormal B7H3 glycosylation mediated by FUT8 overexpression can be physiologically important and clinically relevant in patients with TNBC. Notably, the combination of core fucosylation inhibitor 2F-Fuc and anti-PDL1 results in enhanced therapeutic efficacy in B7H3-positive TNBC tumors. These findings suggest that targeting the FUT8-B7H3 axis might be a promising strategy for improving anti-tumor immune responses in patients with TNBC.

Transmissible gastroenteritis virus (TGEV) represents a significant threat to global swine production. In the absence of effective antiviral therapies, control relies primarily on vaccination. To identify potential therapeutic targets, we performed a genome-wide CRISPR/Cas9 screen in porcine IPEC-J2 cells, which revealed asparagine-linked glycosylation 5 (ALG5), asparagine-linked glycosylation 6 (ALG6), neurofibromin 2 (NF2), and fucosyltransferase 8 (FUT8) as essential host factors for TGEV infection. Functional characterization demonstrated that ALG5, ALG6, and NF2 knockout impaired viral adsorption and internalization through disruption of aminopeptidase N (pAPN) transcription or N-glycosylation. Consistently, tunicamycin-mediated inhibition of N-glycosylation suppressed TGEV infection. In contrast, FUT8 knockout specifically affects viral internalization and early replication by preventing the formation of double-membrane vesicles (DMVs) but does not affect pAPN expression. This role was independent of FUT8's fucosyltransferase activity, as the enzymatic inhibitor FDW028 had no effect. Mechanistically, we found that FUT8 interacts with the TGEV nonstructural proteins NSP3 and NSP4 to facilitate DMV biogenesis. Our findings delineate distinct mechanisms by which host factors support TGEV infection and provide novel insights for the development of targeted antiviral strategies.

Glycosylation of membrane proteins plays an essential role in diverse biological processes. However, it remains unknown whether this posttranslational modification occurs on ciliary membrane proteins. Herein, by mass spectrometry-based proteomic analysis, we demonstrate that multiple membrane proteins localized in the ciliary transition zone undergo core fucosylation, an N-linked glycosylation specifically catalyzed by fucosyltransferase 8 (FUT8). In-depth analysis reveals that FUT8 interacts with transmembrane protein 67 (TMEM67), a transition zone component closely linked to ciliopathies, and catalyzes its core fucosylation. Functional investigation shows that core fucosylation stabilizes TMEM67 by impeding its degradation via the autophagy pathway, thereby ensuring its proper localization to the transition zone to promote cilium formation. Fut8-deficient mice exhibit ciliary defects in multiple organs, such as the kidney, brain, and trachea. These findings uncover a critical role for TMEM67 core fucosylation in ciliogenesis and have important implications for the pathogenesis of ciliopathies.

Glycan structure is often modulated in disease or predisease states, suggesting that such changes might serve as biomarkers. Here, we generated a monoclonal antibody (mAb) against the core fucose of the N-glycan in human IgG. Notably, this mAb can be used in Western blotting and ELISA. ELISA using this mAb revealed a low level of the core fucose of the N-glycan in IgG, suggesting that the level of acore fucosylated (noncore fucosylated) IgG was increased in the sera of the patients with lung cancer, chronic obstructive pulmonary disease, and interstitial pneumonia compared to healthy subjects. In a coculture analysis using human lung adenocarcinoma A549 cells and antibody-secreting B cells, the downregulation of the FUT8 (α1,6 fucosyltransferase) gene and a low level of core fucose of the N-glycan in IgG in antibody-secreting B cells were observed after coculture. A dramatic alteration in gene expression profiles for cytokines, chemokines, and their receptors were also observed after coculturing, and we found that the identified C-C motif chemokine 2 was partially involved in the downregulation of the FUT8 gene and the low level of core fucose of the N-glycan in IgG in antibody-secreting B cells. We also developed a latex turbidimetric immunoassay using this mAb. These results suggest that communication with C-C motif chemokine 2 between lung cells and antibody-secreting B cells downregulate the level of core fucose of the N-glycan in IgG, i.e., the increased level of acore fucosylated (noncore fucosylated) IgG, which would be a novel biomarker for the diagnosis of patients with pulmonary diseases.

Alpha1,6-fucosyltransferase (FUT8) biosynthesizes core fucose on N-glycans, which plays essential roles in various biological processes, including immunity and development. Although FUT8 is a Golgi-resident type II membrane protein, it is also secreted by an unknown mechanism. Here, we demonstrate that signal peptide peptidase (SPP) and signal peptide peptidase-like 3 (SPPL3), members of an intramembrane protease family, both cleave FUT8 for secretion. Knockout of SPP or SPPL3 in cells partially impaired FUT8 secretion, and double KO led to more drastic impairment in secretion, indicating that SPP and SPPL3 independently cleave FUT8. Sequencing analysis revealed that the N terminus of FUT8 in the media was mapped in the stem region, which is far from the expected cleavage site for SPP/SPPL3, suggesting that FUT8 undergoes two-step proteolytic processing, initially by SPP/SPPL3 and subsequently by another protease. Moreover, glycoproteomics suggested that the substrate glycoprotein preference of FUT8 was altered by knocking out SPP or SPPL3, highlighting the importance of FUT8 shedding in core fucosylation.