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: POFUT1, O-fucosylation, podocyte, NOTCH2, tPA
Introduction
O-fucose modification was discovered in many proteins with epithelial growth factor (EGF)-like domains, such as coagulation factors [1-4] and Notch receptors and ligands [5,6]. Protein O-fucosyltransferase 1 (POFUT1) is the only enzyme responsible for O-fucosylation of the EGF-like domains with the consensus sequence of C2XXXX(S/T)C3 in these proteins [7,8]. There are about 100 human proteins that carry the consensus sequence and thus are potentially O-fucosylated by POFUT1 [9].
The importance of POFUT1 and protein O-fucosylation in cells have been demonstrated directly by Pofut1 gene abrogation in both Drosophila and mouse, which resulted in developmental defects and embryonic lethality [10,11]. POFUT1 deficiency also causes other abnormalities in both animals and human, including skin and cardiovascular diseases, microcephaly, and muscle aging-related phenotype, etc. [12-15].
Generally, POUFT1 essentiality is implemented by requirement of O-fucosylation for some proteins which play important roles in various cellular processes, such as Notch receptors and ligands [5,6]. Pofut1 gene abrogation gives rise to developmental defects that are the same as Notch signaling deficiency [10,11]. POFUT1 also regulates the development and homeostasis of blood cell lineages through Notch [16-18]. In addition, POFUT1 regulates Notch signaling in lung development [19] and intestinal homeostasis [20], as well as the maintenance of enteric neural crest progenitors [21] and mammary epithelial cell lineages [22]. In human, POFUT1 gene mutation causes hidradenitis suppurativa-Dowling-Degos disease through impairing Notch signaling [23]. Notch ligands also require O-fucosylation to function [24].
POFUT1 is expressed ubiquitously in various mouse tissues [11], suggesting that POFUT1 may be essential for many cell types. As shown in GSE123179 and GSE17142 from the GEO database (http://www.ncbi.nlm.nih.gov/geo/), POFUT1 is also expressed in podocyte, which are part of glomerular filtration barriers. Moreover, Notch components are also expressed in podocytes although at relatively low levels in GSE123179 and GSE17142 from the GEO database (http://www.ncbi.nlm.nih.gov/geo/). It would be interesting to know whether POFUT1 is required for maintenance of podocyte differentiation, structure, function, and survival, particularly through Notch signaling regulation. In the present study we investigated the issues by generation and characterization of mice with POFUT1 gene abrogation specifically in podocytes.
Materials and methods
Generation of mice with podocyte-specific deletion of Pofut1
Pofut1 flox/flox mice were generated as described previously [11], and was gift from Dr. Pamela Stanley at Albert Einstein College of Medicine, USA. This mouse line was crossed with podocyte-specific transgenic NPHS2-Cre [25] to obtain conditional knockout mice with genotype of Pofut1 flox/flox; NPHS2-Cre. PCR genotyping of the Pofut1 alleles and NPHS2-Cre transgene was performed as previously described [11,25-27]. The primers for Pofut1 alleles are PS644, 5’-GGGTCACCTTCATGTACAAGTGAGTG-3’, and PS645, 5’-ACCCACAGGCTGTGCAGTCTTTG-3’, with product sizes indicated in Figure 1; NPHS2-Cre transgene primers are Cre-F, 5’-GGACATGTTCAGGGATCGCCAGGCG-3’, and Cre-R, 5’-GCATAACCAGTGAAACAGCATTGCTG-3’, with the product size of 268 bp. The use of mice in the present study conformed to the institutional regulations and requirements concerning the care and use of laboratory animals at Jinling hospital, Nanjing University School of Medicine. The animal ethical committee approval number was 2015NZGKJ-059.
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.
Isolation of glomeruli from mice
Mice were euthanized and perfused with 2.5 mg/ml iron oxide solution in PBS. Kidneys were diced into 1-mm3 pieces. One hundred microliters of collagenase A (10 mg/ml) and 100 µl of DNase I (1,000 U/ml) were added to the kidney tissues followed by incubation at 37°C for 30 min with rotation. Digested tissue was passed through a 100-µm cell culture strainer and glomeruli were collected by magnetic concentration. Glomeruli were washed with PBS twice. The isolated glomeruli were treated with proteinase K to extract DNA for PCR analysis of allele of deletion.
Urinary albumin to creatinine ratio (ACR) measurement
Urinary albumin and creatinine were measured using mouse albumin specific ELISA and Creatinine Companion Kits (Exocell Laboratories) following the manufacturer’s instruction.
Periodic acid-Schiff (PAS) staining of kidney sections
Mice were euthanized and perfused with 4% paraformaldehyde followed by 18% sucrose PBS solution. Kidney tissues were excised and fixed in 10% formalin overnight, dehydrated in graded alcohols and embedded in paraffin. Four micrometer thick sections were cut and stained with PAS reagent following the protocol recommended by the Animal Models of Diabetic Complications Consortium (http://www.amdcc.org).
Toluidine blue staining of kidney sections
Five hundred nanometer kidney sections were completely dried. The sections were incubated with staining solution (1% Toluidine Blue and 2% Borate in Distilled Water) until desired staining intensity was achieved. The sections were rinsed with water and air dried and mounted with mounting medium.
Electron microscopy (EM)
Blocks of renal cortex tissue (1 mm3) were fixed in cold 3.75% glutaraldehyde for 4 h. After washing in 0.1 M phosphate buffer (pH7.5) for 5 times, the renal tissues were post-fixed in 2% osmium tetroxide for 2 h, dehydrated in graded acetones and ethanol and embedded in epoxy resin (SPI Inc., Westchester, PA). Ultrathin sections (80-90 nm) were stained using uranyl acetate and lead citrate, and then examined and photographed using a Hitachi 7500 transmission electron microscope (Hitachi Co., Japan).
Immunofluorescent staining
Kidney cortex tissues were cut and placed in Tissue-Tek OCT, snap-frozen in liquid nitrogen and cut into 5-µm sections using a cryostat (Leica CM 3050S, Germany). The sections were blocked with bovine serum albumin and incubated in primary antibodies against SYNPO (Thermo Fisher Scientific) and laminin α1 (Sigma, St. Louis, MO). Next, the sections were incubated with an FITC-conjugated anti-goat secondary antibody, Alexa Fluor 488 goat anti-rabbit IgG (H+L) and Alexa Fluor 568 goat anti-mouse Ig G1 (both from Invitrogen, Carlsbad, CA).
WT1 staining and positive cell counting in glomeruli of mice
OCT-embedded frozen kidneys of 10 Pofut1 cKO mice and wild type mice were cut into 4 µm sections, air-dried and incubated with blocking solution at room temperature for 30 min. Rabbit polyclonal WT1 antibody (C-19; Santa Cruz) and the second antibody goat anti-rabbit IgG (H+L) Alex Fluor 488 (Invitrogen, Carlsbad, CA) were used to stain podocytes at room temperature for 1 h, respectively, followed by DAPI staining and sealing with Fluoromount-G (Southern Biotech). WT1 and DAPI double-positive cells were counted as podocytes. WT1 positive cells in ~20 glomeruli were counted for each mouse, and total number of WT1 positive cells were divided by the number of glomeruli examined, resulting in the average podocyte number per glomerulus of the mouse.
Search for mouse proteins that are predicted to be O-fucosylated by POFUT1
Potential targets of POFUT1 are listed based on a ScanProsite database (https://prosite.expasy.org/scanprosite/) search of all mouse proteins containing EGF like domain (PS50026) that contain the C2XXXX(S/T)C3 consensus sequence for O-fucosylation cross-referenced with the Uniprot database (https://www.uniprot.org). Splice variants were not considered.
Statistical analysis
SPSS18.0 software was used for the statistical analyses. The experiments described in the present study were performed on 10 mice in each group, and the results were given as mean ± SD. Two-tailed Student’s t test was used for comparison between two groups, and P<0.05 was considered to be significantly different.
Results
Generation of mice with Pofut1 deletion selectively in podocytes
The mouse line with Pofut1 floxed allele was crossed to podocyte-specific NPHS2-Cre transgene line to generate conditional knockout of Pofut1 in mouse podocytes (cKO). PCR genotyping of the Pofut1 alleles and NPHS2-Cre transgene were performed as described previously [11,27].
To confirm that Pofut1 deletion was induced in podocytes, we isolated glomeruli from Pofut1 f/f; NPHS2-Cre cKO and control mice. The genomic DNA was prepared and subject to PCR genotyping to detect the product of deletion. We obtained precise PCR product from the predicted recombination of the floxed Pofut1 alleles in the mice carrying floxed Pofut1 alleles and NPHS2-Cre transgene (Figure 1A, 1B). The identity of the PCR product was further confirmed by diagnostic digestion using restriction enzyme as described previously [26].
Characterization of POFUT1 cKO mice
To examine the consequence of POFUT1 deficiency to podocytes in mice, we monitored the development of proteinuria (urinary albumin/creatinine ratio, ACR), which is the established marker of podocyte injury, in the cKO mice, and found that their ACRs were all normal at the age of 6 weeks compared with control group. At the age of 52 weeks (~1 year), we measured ACR again and found no difference between the two groups of mice (Figure 1C).
PAS staining of kidney was performed for the mice, and the glomeruli of cKO mice were morphologically normal compared with that of controls (Figure 2). Toluidine staining also showed normal morphology of glomeruli of cKO mice (Figure 2).
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.
We next examined ultrastructure of glomeruli and podocytes of the cKO mice by electron microscopy, but did not find any abnormalities, particularly, foot process effacement, which is the most sensitive marker of podocyte injury (Figure 2).
Furthermore, we used immunofluorescence staining to examine the expression of podocyte markers, synaptopodin and basement membrane component, laminin α1. The results showed that the expression of the genes were totally normal in cKO mice compared with that in controls (Figure 3).
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.
Finally, we counted podocyte numbers in cKO mice and compared them with that of control mice to determine whether podocyte numbers were reduced in the cKO mice. WT1 is a marker of podocytes, therefore, WT1-positive cells in glomeruli were counted. The average number of positive cells per glomerulus represented the podocyte number in the mouse. The result showed that there was not any difference in podocyte number between cKO and control groups (Figure 4).
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.
Searching mouse proteins potentially O-fucosylated by POFUT1
We next explored why Pofut1 gene abrogation in mouse podocytes did not affect structure, function, and survival of podocytes. We speculated that mouse podocytes may not express protein substrates for POFUT1 or the substrates are not essential for podocytes, resulting in dispensability of POFUT1 in podocytes.
To prove it, we firstly searched mouse proteins that contain EGF-like repeats with the consensus sequence of C2XXXX(S/T)C3 in the ScanProsite database following the method described [9]. Seventy-eight mouse genes express proteins that are potentially O-fucosylated by POFUT1 (Table 1).
Table 1.
Putative mouse genes encoding protein substrates of POFUT1
| uniprot ID | gene | name | conserved |
|---|---|---|---|
| Q61549 | Adgre1 | Adhesion G protein-coupled receptor E1 | |
| A2ASQ1 | Agrn | Agrin | √ |
| Q6PGD0 | Atraid | All-trans retinoic acid-induced differentiation factor | √ |
| Q61361 | Bcan | Brevican core protein. | |
| O35161 | Celsr1 | Cadherin EGF LAG seven-pass G-type receptor 1 | √ |
| Q9R0M0 | Celsr2 | Cadherin EGF LAG seven-pass G-type receptor 2 | √ |
| Q91ZI0 | Celsr3 | Cadherin EGF LAG seven-pass G-type receptor 3 | √ |
| P97766 | Cfc1 | Cryptic protein | √ |
| Q8VHS2 | Crb1 | Protein crumbs homolog 1 | √ |
| Q80YA8 | Crb2 | Protein crumbs homolog 2 | √ |
| Q9JLB4 | Cubn | Cubilin | √ |
| Q09163 | Dlk1 | Protein delta homolog 1 (DLK-1) | √ |
| Q8K1E3 | Dlk2 | Protein delta homolog 2 (DLK-2) | √ |
| Q61483 | Dll1 | Delta-like protein 1 | √ |
| O88516 | Dll3 | Delta-like protein 3 | √ |
| Q9JI71 | Dll4 | Delta-like protein 4 | √ |
| Q8JZM4 | Dner | Delta and Notch-like epidermal growth factor-related receptor | √ |
| O35474 | Edil3 | EGF-like repeat and discoidin I-like domain-containing protein 3 | √ |
| Q9QXT5 | Egfl7 | Epidermal growth factor-like protein 7 | √ |
| Q4VBE4 | Egflam | Pikachurin | √ |
| Q80YC5 | F12 | Coagulation factor XII | √ |
| P70375 | F7 | Coagulation factor VII (EC 3.4.21.21) | √ |
| Q5F226 | Fat2 | Protocadherin Fat 2 | √ |
| Q8BNA6 | Fat3 | Protocadherin Fat 3 | √ |
| Q2PZL6 | Fat4 | Protocadherin Fat 4 | √ |
| Q501P1 | Fbln7 | Fibulin-7 | √ |
| Q61555 | Fbn2 | Fibrillin-2 | √ |
| E9Q7X6 | Heg1 | Protein HEG homolog 1. | |
| Q9R098 | Hgfac | Hepatocyte growth factor activator | √ |
| Q05793 | Hspg2 | Basement membrane-specific heparan sulfate proteoglycan core protein | |
| Q9QXX0 | Jag1 | Protein jagged-1 | √ |
| Q9QYE5 | Jag2 | Protein jagged-2 | √ |
| Q91ZX7 | Lrp1 | Prolow-density lipoprotein receptor-related protein 1 | √ |
| Q9JI18 | Lrp1b | Low-density lipoprotein receptor-related protein 1B | √ |
| A2ARV4 | Lrp2 | Low-density lipoprotein receptor-related protein 2 | |
| O08999 | Ltbp2 | Latent-transforming growth factor beta-binding protein 2 | √ |
| A2AJX4 | Malrd1 | MAM and LDL-receptor class A domain-containing protein 1. | |
| Q6DIB5 | Megf10 | Multiple epidermal growth factor-like domains protein 10 | √ |
| Q80T91 | Megf11 | Multiple epidermal growth factor-like domains protein 11 | √ |
| Q80V70 | Megf6 | Multiple epidermal growth factor-like domains protein 6 | √ |
| P60882 | Megf8 | Multiple epidermal growth factor-like domains protein 8 | √ |
| P28825 | Mep1a | Meprin A subunit alpha | |
| P21956 | Mfge8 | Lactadherin | |
| B2RPV6 | Mmrn1 | Multimerin-1. | √ |
| P19467 | Muc13 | Mucin-13 | |
| P55066 | Ncan | Neurocan core protein | √ |
| Q2VWQ2 | Nell1 | Protein kinase C-binding protein NELL1 | √ |
| Q01705 | Notch1 | Neurogenic locus notch homolog protein 1 | √ |
| O35516 | Notch2 | Neurogenic locus notch homolog protein 2 | √ |
| Q61982 | Notch3 | Neurogenic locus notch homolog protein 3 | √ |
| P31695 | Notch4 | Neurogenic locus notch homolog protein 4 | √ |
| Q8R4G0 | Ntng1 | Netrin-G1 | |
| Q8R4F1 | Ntng2 | Netrin-G2 | |
| Q8BU25 | Pamr1 | Inactive serine protease PAMR1 | √ |
| Q8VIK5 | Pear1 | Platelet endothelial aggregation receptor 1 | √ |
| P11214 | Plat | Tissue-type plasminogen activator | √ |
| P33587 | Proc | Vitamin K-dependent protein C | √ |
| Q9CQW3 | Proz | Vitamin K-dependent protein Z. | √ |
| Q60841 | Reln | Reelin | √ |
| P59222 | Scarf2 | Scavenger receptor class F member 2 | |
| Q80TR4 | Slit1 | Slit homolog 1 protein | √ |
| Q9R1B9 | Slit2 | Slit homolog 2 protein | √ |
| Q9WVB4 | Slit3 | Slit homolog 3 protein | √ |
| Q70E20 | Sned1 | Sushi, nidogen and EGF-like domain-containing protein 1 | √ |
| Q8R4Y4 | Stab1 | Stabilin-1 | √ |
| Q8R4U0 | Stab2 | Stabilin-2 | √ |
| A2AVA0 | Svep1 | Sushi, von Willebrand factor type A, EGF and pentraxin domain-containing protein 1 | √ |
| Q9WTS4 | Tenm1 | Teneurin-1 | √ |
| Q9WTS5 | Tenm2 | Teneurin-2 | √ |
| Q9WTS6 | Tenm3 | Teneurin-3 | √ |
| Q3UHK6 | Tenm4 | Teneurin-4 | √ |
| Q05895 | Thbs3 | Thrombospondin-3. | |
| Q06806 | Tie1 | Tyrosine-protein kinase receptor Tie-1 | |
| Q91X17 | Umod | Uromodulin | √ |
| Q9CZT5 | Vasn | Vasorin | √ |
| Q62059 | Vcan | Versican core protein | |
| Q70UZ7 | Vwa2 | von Willebrand factor A domain-containing protein 2 | √ |
| Q9WUA1 | Wif1 | Wnt inhibitory factor 1 | √ |
To determine which of the 78 potential O-fucosylated proteins are expressed in and essential for podocytes, we compared them with mouse podocyte essential genes. Previously, we performed single-cell RNA-seq analysis of mouse podocytes and found a high heterogeneity of gene expression in individual podocytes [28]. We hypothesized that genes expressed in every single podocyte are likely essential for podocytes as a cell type, while the genes expressed only in a portion of podocytes are non-essential. With this notion, we identified 335 podocyte essential gene candidates using 0.5 rpkm as expression cutoff [28]. Here, we lowered the standard by using 0.1 rpkm as the expression cutoff, resulting in 611 podocyte essential gene candidates (Table 2).
Table 2.
611 podocyte essential gene candidates predicted by single-cell RNA-seq
| 9930111J21Rik2 | CSDE1 | Gm33780 | MTMR2 | RAB11B | SPTBN1 |
| ACAD9 | CSNK1A1 | Gm3839 | MT-ND1 | RAB3B | SQSTM1 |
| ACOT2 | CST3 | Gm6211 | MT-ND2 | RAB7A | SRGAP1 |
| ACSL4 | CTDSPL | GNB1 | MT-ND3 | RAC1 | SRP14 |
| ACTB | CTNNA1 | GNG5 | MT-ND4 | RACGAP1 | SRPK1 |
| ACTN4 | CTSV | GOLIM4 | MT-ND5 | RAD21 | SRSF2 |
| ACTR2 | CYB5A | GPBP1L1 | MT-ND6 | RASL11A | Srsf5 |
| AEBP1 | CYB5R4 | GPC1 | MTSS1 | Rbm25 | SSBP2 |
| AFF4 | D330041H03Rik | GPX4 | MYCBP2 | RBM26 | SSR1 |
| AIF1L | DAZAP2 | GPX8 | MYH9 | RBM28 | SSR3 |
| AIM1L | DDX5 | GRK4 | MYL12B | RBM39 | ST13 |
| AKR1A1 | DDX58 | GSK3B | MYL6 | RBMS2 | SUCLA2 |
| ALCAM | DECR2 | GSN | MYLK | RBMS3 | SWT1 |
| ALKBH5 | DEGS1 | GTF2A1 | MYO1C | REEP3 | SYNJ2BP |
| ALOX15B | DENND5B | H3F3A/H3F3B | MYO1D | RHEB | SYNPO |
| ANAPC16 | DNAJC11 | H3F3A/H3F3B* | MYOM2 | RHOA | TAX1BP1 |
| ANXA1 | DOCK5 | HAUS8 | N4BP2L2 | RMDN1 | TBP |
| ANXA2 | DPP4 | HELLS | NAP1L1 | ROBO2 | TCF21 |
| ANXA4 | DPYSL2 | HLA-A | NBEAL1 | RPL10 | TCP1 |
| AOX1 | Dst | HLA-A | NCK2 | RPL10A | TDRD5 |
| AP1S3 | DSTN | HLA-A | NCL | RPL13 | TGFBR3 |
| AP2M1 | DTNB | HNRNPL | NDUFA1 | RPL14 | THSD7A |
| APAF1 | DUSP3 | HNRNPU | NDUFA13 | Rpl14-ps1 | TIAL1 |
| APBB2 | Dync1i2 | HP1BP3 | NDUFA3 | RPL21 | TIMM17B |
| APBB3 | DYNLL1 | HSBP1 | NDUFA4 | RPL23 | TIMMDC1 |
| APLP2 | DYNLRB1 | HSD3B1 | NDUFA6 | RPL26 | TIMP3 |
| APP | DYNLT1 | HSP90AB1 | NDUFA7 | RPL27A | TJP1 |
| ARF3 | DYNLT3 | HSP90B1 | NDUFB8 | Rpl32 | TLN1 |
| ARGLU1 | EEF1A1 | HSPB11 | Ndufs5 | RPL35 | TM4SF1 |
| ARHGAP24 | EHD2 | HTRA1 | Neat1 | RPL35A | TMBIM1 |
| ARHGAP28 | Eif1 | HYPK | Nebl | RPL37 | TMBIM6 |
| ARHGEF18 | EIF3M | IER3IP1 | Nedd4 | RPL37A | TMCO1 |
| ARPC1A | EIF4A1 | IFFO1 | Nes | RPL38 | TMED7 |
| ARPC2 | EIF4A2 | IFITM2 | NFE2L1 | RPL4 | TMEM234 |
| ATG16L1 | EIF4G2 | IFNGR1 | NFIA | RPL41 | TMEM245 |
| ATP5A1 | EMC2 | IFT80 | NFRKB | RPL7 | TMEM30A |
| ATP5B | ENPEP | IGFBP7 | NKTR | RPL8 | TMEM50A |
| ATP5C1 | ENSMUSG00000004980 | ILDR2 | NOA1 | RPL9 | TMEM59 |
| Atp5e | ENSMUSG00000022820 | IMMT | NOP10 | RPLP0 | TMEM69 |
| ATP5F1 | ENSMUSG00000023737 | IQGAP1 | NOTCH2 | Rplp1 | TMEM80 |
| ATP5G3 | ENSMUSG00000027942 | IQGAP2 | NPHS1 | RPN2 | TMOD3 |
| ATP5J | ENSMUSG00000040078 | ITCH | NPHS2 | RPS11 | Tmsb4x |
| ATP5J2 | ENSMUSG00000044285 | ITGA3 | NPNT | RPS12 | Tmsb4x* |
| ATP5L | ENSMUSG00000057577 | ITGAV | NPR3 | RPS14 | TNFRSF10A |
| ATP6AP1 | ENSMUSG00000061331 | ITGB1 | NRAS | RPS16 | TNS2 |
| ATP6V0E1 | ENSMUSG00000064339 | ITGB5 | NSF | RPS18 | TNS3 |
| ATP6V1A | ENSMUSG00000064352 | ITM2B | NUPR1 | RPS20 | TOB1 |
| ATP6V1B2 | ENSMUSG00000067344 | IVD | OAZ1 | RPS23 | TOP1 |
| ATP6V1G1 | ENSMUSG00000071107 | JAK1 | OGT | RPS24 | TOP2A |
| ATRX | ENSMUSG00000072692 | JUP | ORC3 | RPS25 | TPM3 |
| B2M | ENSMUSG00000081471 | KANK1 | OSBPL9 | Rps27/Rps27rt | TPT1 |
| BBX | ENSMUSG00000081552 | KDELR2 | P3H2 | Rps27/Rps27rt* | TRAM1 |
| BCAT2 | ENSMUSG00000083563 | KHSRP | PABPC1 | RPS27L | TRIB2 |
| BIRC6 | ENSMUSG00000083594 | KIAA1107 | PAIP1 | RPS29 | TSC22D1 |
| C19orf53 | ENSMUSG00000085279 | KIAA1109 | PAK1 | Rps3a1 | TSPAN13 |
| C6orf47 | ENSMUSG00000085328 | KIF1B | PAM | RPS5 | TSPAN15 |
| C920009B18Rik | ENSMUSG00000085334 | KIF5B | PAN3 | RPS7 | TSPAN3 |
| Calm1 | ENSMUSG00000085586 | KLHL9 | PARVA | RPS8 | TTC3 |
| Calm1* | ENSMUSG00000085950 | KRCC1 | PBRM1 | RSRP1 | TWF1 |
| CALR | ENSMUSG00000086967 | LACTB2 | PCMTD1 | S1PR4 | Ubb |
| CANX | ENSMUSG00000089828 | LAPTM4A | PCNP | SAP18 | UBL5 |
| CAPS2 | ENSMUSG00000089940 | LCP1 | PDCD4 | SBDS | UBN2 |
| CBX1 | ENSMUSG00000090262 | LGR4 | PDIA3 | Scd2 | UCP2 |
| CBX3 | ENSMUSG00000090286 | LIN7C | PDIA4 | SCHIP1 | UNC13D |
| CCNT1 | ENSMUSG00000090353 | S100a11 | PDIA6 | SCP2 | UQCR11 |
| CD2AP | ENSMUSG00000092400 | LOC102640619 | PDLIM2 | SDC4 | UQCRB |
| CD59 | ENSMUSG00000093760 | LPIN2 | PDXDC1 | SEC22B | USP9X |
| Cd59a | ENSMUSG00000094030 | LPL | PFN1 | SELK | VDAC1 |
| CD81 | ENSMUSG00000094472 | LRRC58 | PHYKPL | SEMA3E | VEGFA |
| CD9 | ENSMUSG00000096808 | LRRC8A | PIAS4 | SEMA3G | VEPH1 |
| CDC26 | ENSMUSG00000097287 | LRRFIP1 | PIGV | SENP1 | VIM |
| Cdc42 | ENSMUSG00000097695 | LUC7L3 | PKIB | SEP15 | Vmn1r63 |
| CDC42BPA | ENSMUSG00000097815 | LYPLA1 | PLAT | SEPP1 | Vmn2r55 |
| CDC42SE2 | ENSMUSG00000097911 | LYRM9 | PLCE1 | SEPT10 | VPS53 |
| CDC7 | ENSMUSG00000098178 | MAFB | PLOD2 | SEPT11 | WAPAL |
| CDK14 | ENSMUSG00000098183 | MAGI2 | PLS3 | SEPT2 | WDR1 |
| Cdkn1c | EPB41L5 | MALAT1 | Plscr2 | SEPT7 | WT1 |
| CERS6 | ERMP1 | MAP1LC3B | PNISR | SEPW1 | WTAP |
| CHCHD2 | EZR | MAPT | Podxl | SERBP1 | YARS |
| CHMP2A | FAM81A | MATR3 | POLDIP3 | SERINC3 | YBX1 |
| CHMP5 | FGD4 | MERTK | POMP | SET | YIPF1 |
| CHPT1 | FGFR1 | MIER1 | PPIA | SGIP1 | YME1L1 |
| CLASP2 | FKBP1A | MKLN1 | PPP1CB | SH2D4A | YWHAE |
| CLIC3 | FKBP8 | MMP12 | PRDX1 | SHISA3 | YWHAQ |
| CLIC5 | Fnbp1l | MOCS2 | PRDX3 | SIK2 | YWHAZ |
| CLK1 | Foxd2os | MORF4L1 | PRMT1 | SKP1 | ZAK |
| CLTC | Foxn3 | MPC2 | PRRC2C | SLC18B1 | ZBTB20 |
| CMPK1 | FTH1 | MPP5 | Psg16 | SLC25A3 | ZBTB8OS |
| CNBP | FTL | MRFAP1 | PSMA3 | SLC25A43 | ZDHHC21 |
| COL4A3 | FUBP3 | MRPL20 | PSMC2 | SLC39A1 | Zfp60 |
| COL4A4 | FYCO1 | MRPL27 | PTBP3 | SMCO1 | Zfp940 |
| CORO2B | GADD45A | MRPL48 | PTGES3 | SMDT1 | ZFR |
| COX4I1 | GAS5 | MRPL51 | PTH1R | SMG1 | ZKSCAN3 |
| COX6A1 | GAS7 | MRPS14 | Ptma | SMIM10L1 | ZMAT1 |
| COX6B1 | GATAD1 | MSI2 | PTP4A1 | SMIM14 | ZNF207 |
| Cox6c | GLRX2 | MT-ATP6 | PTP4A2 | SNU13 | ZNF253 |
| COX7B | Gm11783 | mt-Atp8 | Ptprd | SNX5 | ZNF277 |
| COX8A | Gm16222 | MTCH1 | PTPRO | SOD1 | ZNF3 |
| CPA6 | Gm16702 | MT-CO1 | PTRF | SON | ZNF488 |
| CPNE3 | Gm21596/Hmgb1 | MT-CO2 | PURA | SPARC | ZNHIT3 |
| CROT | Gm26782 | MT-CO3 | QKI | SPCS1 | ZSCAN26 |
| CRYAB | Gm33780 | MT-CYB | R3HDM4 | SPOP |
Note: a gene marked by * is a distinct isoform of the gene with the same name.
Comparison of the genes encoding the 78 proteins of POFUT1 potential substrates with 611 podocyte essential gene candidates identified two overlapped genes, Notch2 and Plat. However, gene knockout of both Notch2 and Plat is known not to cause any podocyte injury [29,30], [The International Mouse Phenotyping Consortium (IMPC) (http://www.informatics.jax.org/marker/phenotypes/MGI:97610)]. Therefore, neither Notch2 or Plat is essential for podocytes; alternatively, there may be functional redundancy for them in mouse podocytes, thus explaining why Pofut1 abrogation did not result in podocyte injury in mouse.
POFUT1 expression in individual podocytes
Although Pofut1 gene expression is detectable in most tissues with mixed population of cells, including mouse podocytes [GEO: GSE123179; GSE17142], it is not known whether Pofut1 is expressed in every single podocyte, thus being predicted as podocyte essential gene. We examined our data of single-cell RNA-seq of mouse podocytes [28], and found that Pofut1 was expressed in a portion of podocytes (Table 3). This result is supported by the database of the Kidney Interactive Transcriptomics (KIT) (http://humphreyslab.com/SingleCell/) (Data not shown). Thus, Pofut1 is not a podocyte essential gene.
Table 3.
Pofut1 rpkm in 20 single mouse podocytes
| Cell | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 | Ave |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| RPKM | 0 | 4.15 | 0 | 38.35 | 0.07 | 0 | 0 | 0.59 | 0 | 0 | 0 | 0.68 | 0 | 0 | 0 | 0.31 | 0 | 0 | 0 | 0 | 2.21 |
Discussion
O-fucosylation of EGF-like domain by POFUT1 is required for the function of proteins, e.g., Notch receptors, as shown by Pofut1 abrogation that causes cellular abnormalities through inhibiting Notch signaling. Notch signaling has been shown to be essential for podocyte formation in development [31,32] and Notch components are still present in mature podocytes. These studies suggested that POFUT1 might be essential for mouse podocytes. However, we showed that Pofut1 gene deletion selectively in podocytes did not induce abnormalities in mice, suggesting that POFUT1 activity is not required for podocyte structure, function and survival. We further explored the reason underlying the dispensability of POFUT1 in mouse podocytes and found none of the predicted POFUT1 substrates is essential for podocytes according to literature and the databases generated by single-cell RNA-seq.
We carefully examined the cKO mice with Pofut1 knockout in podocytes by multiple experimental analyses, including urinary albumin level, morphology, ultrastructure, marker gene expression and podocyte number. There was no difference in these parameters between cKO and control mice, clearly indicating that POFUT1 is dispensable for mouse podocytes.
To explore why POFUT1 is dispensable for mouse podocytes, we identified 78 proteins that are predicted to be O-fucosylated by POFUT1 in the mouse genome by searching Scanprosite. Among the 78 genes, only Notch2 and Plat are in the 611 mouse podocyte essential gene candidates. Since Pofut1 abrogation in mouse podocytes did not cause phenotypes, it is either O-fucosylation of the two proteins is not required for their function in podocytes, or these two proteins are not essential for mouse podocytes. Notch signaling, particularly Notch2 signaling, is required for podocyte development [31,32]. However, Notch2 appeared not to be required in mature podocytes as podocyte-specific knockout of Notch2 in podocytes did not show any phenotypes [29]. Plat knockout also showed no phenotypes of podocytes, indicating that Plat is dispensable for podocytes [30], [The International Mouse Phenotyping Consortium (IMPC) (http://www.informatics.jax.org/marker/phenotypes/MGI:97610)]. The dispensability of Notch2 and Plat in mouse podocytes explains why POFUT1 is not required for podocytes. It should be noted that Pofut1 is the only gene encoding O-fucosyltransferase that adds fucose to Notch2 and tPA, therefore we conclude that POFUT1 and its O-fucosylation activity are not required for podocytes in mice.
POFUT1 dispensability may also be reflected by its expression in single podocytes [28]. We found that Pofut1 mRNA was detected in small portion of cells analyzed (Table 3). This result was consistent with that from the database KIT, and together suggested that POFUT1 is not essential for podocytes as a cell type. However, POFUT1 may be actually expressed in every single podocyte, but it was not detected in every single podocyte due to technical variations in the sequencing process. Nevertheless, Pofut1 knockout in podocytes did not cause podocyte injury, demonstrating that POFUT1 is not essential for podocyte structure, function and survival under physiological condition.
In the present study, we retrieved mouse genes that encode proteins potentially O-fucosylated by POFUT1 at genome-wide level, and found 78 such genes. We compared list of the 78 proteins with that of human proteins, and found most of the proteins are common in the two species (Table 1), suggesting a high conservation in evolution, and thus potential function of O-fucosylation for these proteins. Although O-fucosylation of these proteins appears not to be important in podocytes, it could be essential for other cell types in the body. It is interesting to know which tissues or cell types express the O-fucosylated proteins and whether these proteins are essential for the cell types. This can be achieved by taking the approach described in the present study, i.e., comparing the 78 mouse genes encoding potentially O-fucosylated proteins with the essential gene candidates of the cell type of interest. At present, single-cell RNA-seq data are available for most cell types in the databases, e.g., GEO, and their essential gene candidates can be inferred by the method as we described previously [28]. On the other hand, the O-fucose may not be necessarily essential for the proteins as the case of Cripto whose O-fucose is clearly dispensable for its function in Nodal signaling [33].
POFUT1 has an O-fucosyltransferase independent function in cells. It can serve as a chaperone that assists Notch proteins to fold and traffic properly in endoplasmic reticulum [34,35]. At present, NOTCH1 is the only protein that has been reported to require POFUT1 protein as chaperone. Since Notch1 is essentially not expressed in normal podocytes and Notch1 knockout in podocytes does not affect podocytes [29], the observation that POFUT1 deficiency in podocytes does not cause phenotypes may further suggest that chaperone activity of POFUT1 is very limited.
Finally, it is well established that Notch1 is de novo expressed or upregulated in podocytes in glomerular injury mouse models and human glomerular diseases, and that Notch1 activation mediates podocyte injury as shown by the observation that Notch1 gene deletion specifically in mouse podocyte alleviates podocyte injury in the mouse models [36]. Since Notch1 is a substrate of POFUT1 and O-fucose is required for Notch1 to function [5,10,11], we expect that Pofut1 gene abrogation in podocytes would inactivate de novo expressed NOTCH1 protein thereby protecting podocytes from injury. It is interesting to test this speculation.
Acknowledgements
This work was supported by the grants from the National Natural Science Foundation of China (81670653 and 81970619 to SS), the National Key Research and Development Program of China (2016YFC0904103 to ZL) and Planned Projects for Postdoctoral Research Funds (2019 K181 to SZ). We thank for Dr. Pamela Stanley and Dr. Erwin Bottinger for reagents and supports.
Disclosure of conflict of interest
None.
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