Biology subjects

Burballa, C.

Publications and source records attributed to Burballa, C..

3 recordsLinked to original sources

bioRxiv2021

The ulcerative colitis associated gene FUT8 regulates the quantity and quality of secreted mucins

Fucosylation of mucins, the main macrocomponents of the mucus layer that protects the digestive tract from pathogens, increases their viscoelasticity and shear stress resistance. These properties are altered in patients with ulcerative colitis (UC), which is marked by a chronic inflammation of the distal part of the colon. Here we show that the levels of Fucosyltransferase 8 (FUT8) and specific mucins are increased in the distal inflamed colon of UC patients compared to normal individuals. Overexpressing FUT8, as observed in UC, in mucin-producing HT29-18N2 colonic cell line increases trafficking of MUC1 to plasma membrane and secretion of MUC2/MUC5AC. FUT8 depletion (FUT8 KD), instead, causes intracellular accumulation of MUC1 and alters the ratio of secreted MUC2 to MUC5AC. Mucins secreted by FUT8 overexpressing cells are more resistant to shear stress compared to mucins secreted by FUT8 KD cells. These data fit well with the Fut8-/- mice phenotype, which are protected against UC. Fut8-/- mice exhibit a thinner proximal colon mucus layer with an altered ratio of neutral to acidic mucins compared to Fut8+/+ mice. Together, these data reveal that FUT8 optimizes the viscoelastic properties of the extracellular mucous by controlling the quantities of mucins secreted. SIGNIFICANCE STATEMENTMucins, the major components of the mucous barrier that protects our body from pathogens, are heavily glycosylated proteins. Changes their amounts and properties will alter the viscosity of mucous. Here we show that FUT8, a glycosylation enzyme of the Golgi apparatus, can control the viscosity of secreted mucins. Mucin secreting cells of the distal colon express FUT8, but their levels are altered in Ulcerative colitis patients. As a result, mucous produced by these cells is easily washed away, which exposes them to pathogens. We suggest that a defective mucous production is the main cause of initial inflammation observed in disease. Our findings help in understanding how cells control the quality of mucins and provide a means to prevent Ulcerative colitis.

cell biology↗

bioRxiv2020

Global gene-expression analysis reveals the molecular processes underlying ClC-5 loss-of-function in novel Dent Disease 1 cellular models

Dent disease 1 (DD1) is a rare X-linked renal proximal tubulopathy characterized by low molecular weight proteinuria (LMWP) and variable degree of hypercalciuria, nephrocalcinosis and/or nephrolithiasis with progression to chronic kidney disease (CKD). Although loss-of-function mutations in the gene CLCN5 encoding the electrogenic Cl-/H+ antiporter ClC-5, which impair endocytic uptake in proximal tubule cells, cause the disease, there is poor genotype-phenotype correlation and their contribution to proximal tubule dysfunction remains unclear. Here, in order to discover the mechanisms leading to proximal tubule dysfunction due to ClC-5 loss-of-function, we have generated and characterized new human cellular models of DD1 by silencing CLCN5 and introducing the ClC-5 pathogenic mutants V523del, E527D and I524K into the human proximal tubule-derived cell line RPTEC/TERT1. Depletion of CLCN5 or expression of mutant ClC-5 impairs albumin endocytosis, increases substrate adhesion and decreases collective migration, which correlates with a less differentiated epithelial phenotype. Interestingly, although all conditions compromised the endocytic capacity in a similar way, their impact on gene expression profiles was different. Our DNA microarray studies show that ClC-5 silencing or mutant re-introduction alter pathways related to nephron development, anion homeostasis, organic acid transport, extracellular matrix organization and cell migration, compared to control cells. Cells carrying the V523del ClC-5 mutation show the largest differences in gene expression vs WT cells, which is in agreement with the more aggressive clinical phenotype observed in some DD1 patients. Overall, this work emphasizes the use of human proximal tubule derived cell models to identify the molecular processes underlying ClC-5 deficiency.

pathology↗

bioRxiv2020

Palmitoylation of KChIP3 controls baseline mucin secretion

Baseline mucin secretion (BMS) is independent of external agonists and controlled by a small calcium binding protein named KChIP3. KChIP3-hosting mucin granules are not released until intracellular cytosolic calcium oscillations reach a threshold, KChIP3 binds calcium and detaches from granules, allowing their fusion to plasma membrane. Loss of KChIP3 or blocking its membrane attachment causes mucin hypersecretion. How is KChIP3 recruited to mucin granules? We show here that zDHHC (aspartate-histidine-histidine-cysteine motif in a cysteine-rich, zinc finger-like domain) S-acyl-transferase dependent palmitoylation modulates binding of KChIP3 to mucin granules thereby affecting mucin secretion. We have found that inhibiting zDHHC-mediated palmitoylation in differentiated HT29-18N2, which express the Golgi-localized zDHHC3 and zDHHC4, releases KChIP3 from mucin granules and increases baseline mucin secretion. Mutation of the palmitoylation sites in KChIP3 (Cysteines 122 and 123 to Alanine) quantitatively reduces its attachment to mucin granules. Expression of KChIP3-WT in HT29-18N2 cell lines stably depleted of KChIP3 inhibits mucin secretion, whereas expression of non-palmitoylated KChIP3 (KChIP3-AA) only partially rescues the effect of KChIP3 depletion and the cells maintain higher levels of baseline secretion compared to KChIP3-WT cells. Altogether, our data suggest that zDHHC3 or zDHHC4-dependent palmitoylation is involved in KChIP3 recruitment to mucin granules to control the baseline mucin secretion.

cell biology↗