POFUT1 is dispensable for structure, function and survival of mouse podocytes

Abstract

Pofut1 gene encodes a O-fucosyltransferase that adds fucose to the serine/threonine residue in the sequence of C²XXXX(S/T)C³ of EGF-like domain in a protein. O-fucosylation has been shown to be required for some EGF-like domain-containing proteins to function, e.g., Notch1, and POFUT1 deficiency could affect cellular function and cause diseases. Pofut1 is ubiquitously expressed, but its essentiality for most cell types is not known. In the present study, we examined the consequence of Pofut1 gene abrogation in mouse podocytes using Cre-loxP system, and found that the conditional knockout mice were indistinguishable from wild-type controls in urinary protein level, glomerular morphology, podocyte foot process ultrastructure, podocyte marker expression and podocyte numbers. These results indicated that POFUT1 is not essential for podocyte structure, function and survival in mice. To understand why POFUT1 is dispensable for podocytes, we searched mouse podocyte essential gene candidates (as determined by single-cell RNA-seq) and found only two POFUT1 substrates, NOTCH2 and tPA. It has been shown that abrogation of these genes does not cause podocyte injury, explaining dispensability of POFUT1 for mouse podocytes and demonstrating a feasibility to predict POFUT1 essentiality for a given cell type. At present, most mouse cell types have been subject to single-cell RNA-seq, making essential gene prediction and thus POFUT1 requirement prediction possible for the cell types.

Keywords: NOTCH2; O-fucosylation; POFUT1; podocyte; tPA.

Figure 1

PCR detection of Pofut1 allele with exon 2 deleted in glomeruli isolated from mice and urinary albumin levels of control and Pofut1 cKO mice. A. Schematic of mouse Pofut1 wild-type allele (wt) and the alleles with exon 2 floxed by loxP sites (flox) or deleted (Δ). B. PCR product gel analysis, showing the predicted product from the glomerular samples from mice of various genotypes as indicated. +, wild-type; F: flox; Δ: deletion; C: NPHS2-Cre. C. Urinary albumin/creatinine ratios (ACR) were measured at the age of 6 weeks for control (n=12) and cKO mice (n=11) mice. At the age of 52 weeks, the control (n=10) and cKO (n=10) mice were subject to ACR measurement again. There were no statistically significant difference between the two groups of mice at both 6 and 52 weeks with p values of 0.17 and 0.21, respectively, using student’s t-test.

Figure 2

Morphological comparison of glomeruli of control and Pofut1 cKO mice. PAS and Toluidine staining were performed on kidney sections of the mice, showing indistinguishable glomerular morphology between the two groups of mice. Scale bars: 30 µm. Neither did EM examination show any difference in glomerular ultra-structure between the control and Pofut1 cKO mice. Scale bars: 2 µm.

Figure 3

Immunofluorescence staining of podocyte marker, synaptopodin, and basement membrane component, laminin α1 in kidney of control and Pofut1 cKO mice. Scale bars: 40 µm.

Figure 4

WT1 positive cell counting in glomeruli of mice. A. Representative WT1 fluorescence staining. B. Quantitative results of WT1 positive cells of control (n=10) and Pofut1 cKO (n=10) mice. About 20 glomeruli were examined for WT1 positive cells numbers for each mouse. Scale bars: 50 µm. There was not statistically significant difference between the two groups with a p value of 0.66 using student’s t-test.