A conserved LDL-receptor motif regulates corin and CD320 membrane targeting in polarized renal epithelial cells
- Cyrus Tang Hematology Center, Collaborative Innovation Center of Hematology, State Key Laboratory of Radiation Medicine and Prevention, Soochow University, China
- MOH Key Laboratory of Thrombosis and Hemostasis, Jiangsu Institute of Hematology, the First Affiliated Hospital of Soochow University, China
- Institute of Molecular Enzymology, Soochow University, China
- Cardiovascular & Metabolic Sciences, Lerner Research Institute, Cleveland Clinic, United States
Figures
Figure 1 with 1 supplement
Expression of corin and other selected proteases in MDCK, HL-1, and HEK293 cells.
(A) MDCK (left), HL-1 (middle), and HEK293 (right) cells were transfected with a plasmid expressing corin. After 72 hr, immunostaining was done to examine corin (green) and ZO-1 (red) (an indicator …
Figure 1—figure supplement 1
Western blotting analysis of corin expression in HEK293 and MDCK cells.
Corin modules, including the cytoplasmic tail, the transmembrane domain (TM), frizzled (Fz), LDLR, scavenger receptor (SR), and protease domain modules, are shown. The activation cleavage site is …
Figure 2 with 2 supplements
Analysis of corin deletion mutants in MDCK cells.
(A) Human corin consists of an N-terminal cytoplasmic tail (CT), a transmembrane domain (TM) and an extracellular region with two frizzled (Fz) modules, eight LDLR modules, a scavenger receptor (SR) …
Figure 2—figure supplement 1
Quantitative analysis of corin expression on apical and basolateral membranes in MDCK cells.
Corin WT and mutants lacking the cytoplasmic tail (ΔCT) and the extracellular fragment between frizzled 1 (Fz1) and scavenger receptor (SR) modules (ΔFz1-SR) in (A) and mutants lacking frizzled and …
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Figure 2—figure supplement 1—source data 1
Source data for Figure 2—figure supplement 1A.
- https://cdn.elifesciences.org/articles/56059/elife-56059-fig2-figsupp1-data1-v1.xlsx
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Figure 2—figure supplement 1—source data 2
Source data for Figure 2—figure supplement 1B.
- https://cdn.elifesciences.org/articles/56059/elife-56059-fig2-figsupp1-data2-v1.xlsx
Figure 2—figure supplement 2
Analysis of corin mutants lacking individual N-glycosylation sites.
(A) Human corin contains 19 N-glycosylation sites, as indicated by Y shaped symbols. (B) Corin WT and mutants N567Q, N651Q, N697Q, N761Q, and N1022Q were expressed in MDCK cells. Immunostaining and …
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Figure 2—figure supplement 2—source data 1
Source data for Figure 2—figure supplement 2C.
- https://cdn.elifesciences.org/articles/56059/elife-56059-fig2-figsupp2-data1-v1.xlsx
Figure 3 with 1 supplement
Expression of corin mutants with point mutations in LDLR6 module and without LDLR7-8 modules.
(A) Corin wild-type (WT) and mutants lacking LDLR7-8 module without (ΔR78) or with point mutations at indicated positions (G590H, A595R/R598W, D608S, and E612W), indicated in red, were expressed in …
Figure 3—figure supplement 1
Alignments of corin LDLR module sequences.
(A) Alignment of partial sequences from human corin LDLR1-8 modules. Residues unique at corresponding positions in LDLR8 module are highlighted in red. Among them, five residues conserved in …
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Figure 3—figure supplement 1—source data 1
Source data for Figure 3—figure supplement 1C.
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Figure 3—figure supplement 1—source data 2
Source data for Figure 3—figure supplement 1D.
- https://cdn.elifesciences.org/articles/56059/elife-56059-fig3-figsupp1-data2-v1.xlsx
Figure 4 with 2 supplements
Expression of corin mutants with point mutations in the DSSDE motif in LDLR8 module.
(A) Corin wild-type (WT) and mutants with point mutations at indicated positions (D682A, S683A, S684A, D685A, and E686A), indicated in red, were expressed in MDCK cells. Corin expression (green) on …
Figure 4—figure supplement 1
Analysis of the DSSDE motif in corin LDLR8 module.
(A) Alignment of the DSSDE motif in corin LDLR8 modules from selected vertebrate species. The DSSDE motif (red) in corin LDLR8 modules from selected vertebrate species (human, Hum; monkey, Mac; rat, …
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Figure 4—figure supplement 1—source data 1
Source data for Figure 4—figure supplement 1B.
- https://cdn.elifesciences.org/articles/56059/elife-56059-fig4-figsupp1-data1-v1.xlsx
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Figure 4—figure supplement 1—source data 2
Source data for Figure 4—figure supplement 1C.
- https://cdn.elifesciences.org/articles/56059/elife-56059-fig4-figsupp1-data2-v1.xlsx
Figure 4—figure supplement 2
Western blotting of corin proteins in biotin-labeled cell membranes.
MDCK cells stably transfected with a vector or plasmids expressing corin WT and the S684A mutant in Transwell plates were treated with sulfo-NHS-biotin in upper (A and B) or lower (C and D) chambers …
Figure 5 with 1 supplement
Structure models of corin LDLR6 and LDLR8 modules.
(A) Structure models of human corin LDLR6 (left panel) and LDLR8 (right panel) modules, which were built based on the human VLDLR module four structure. (B) Structure models of corin S683A (left …
Figure 5—figure supplement 1
Structure alignments of LDLR modules.
(A) Human corin LDLR8 sequence was used to search the Protein Bata Bank. Among 15 LDLR module homologs identified, the human VLDLR module four structure (6BYV_154–188) (yellow cartoon) had the …
Figure 6 with 1 supplement
Expression of human CD320 in kidney sections and MDCK cells.
(A) Human CD320 protein contains an N-terminal extracellular region with two LDLR modules and one EGF module, a transmembrane (TM) domain, and a C-terminal cytoplasmic tail. LDLR1 module has a DGSDE …
Figure 6—figure supplement 1
Structural alignments of the LDLR domains of human CD320.
(A) The structure alignments of human CD320 LDLR1 (residues 54–89) (light blue) and LDLR2 (residues 132–166) (violet), and corin LDLR8 module (residues 655–689) (cyan) with the calcium ions …
Figure 7 with 1 supplement
Expression of CD320 mutants with point mutations in the DSSDE motif in LDLR2 module.
(A) Human CD320 WT and mutants with point mutations in LDLR2 module at indicated positions shown in red (D160A, S161A, S162A, D163A, and E164A) were expressed in MDCK cells. CD320 expression (green) …
Figure 7—figure supplement 1
Quantitative analysis of CD320 and corin expression in MDCK cells.
(A and B) CD320 WT and mutant expression in MDCK cells was examined by immunostaining and confocal microscopy. Fluorescent signals along the X-Z axis were analyzed with Image J software to calculate …
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Figure 7—figure supplement 1—source data 1
Source data for Figure 7—figure supplement 1A.
- https://cdn.elifesciences.org/articles/56059/elife-56059-fig7-figsupp1-data1-v1.xlsx
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Figure 7—figure supplement 1—source data 2
Source data for Figure 7—figure supplement 1B.
- https://cdn.elifesciences.org/articles/56059/elife-56059-fig7-figsupp1-data2-v1.xlsx
Figure 8 with 2 supplements
Effects of Rab11a inhibition or knockdown on corin and CD320 expression in MDCK cells.
(A) Corin (green) and Rab11 proteins (red), including Rab11a, Rab11b, and dominant-negative (DN) mutants that were inhibitory to Rab11a and Rab11b, respectively, were co-expressed in MDCK cells. …
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Figure 8—source data 1
Source data for Figure 8B.
- https://cdn.elifesciences.org/articles/56059/elife-56059-fig8-data1-v1.xlsx
Figure 8—figure supplement 1
Effects of Rab11a inhibition on apical corin and CD320 expression.
(A) Corin WT co-expressed with Rab11a, Rab11b, or dominant-negative (DN) Rab11a or Rab11b mutants in MDCK cells was analyzed by immunostaining and confocal microscopy. Quantitative data of the ratio …
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Figure 8—figure supplement 1—source data 1
Source data for Figure 8—figure supplement 1A.
- https://cdn.elifesciences.org/articles/56059/elife-56059-fig8-figsupp1-data1-v1.xlsx
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Figure 8—figure supplement 1—source data 2
Source data for Figure 8—figure supplement 1C.
- https://cdn.elifesciences.org/articles/56059/elife-56059-fig8-figsupp1-data2-v1.xlsx
Figure 8—figure supplement 2
Effects of Rab11b knockdown on corin and CD320 expression in MDCK cells.
MDCK cells were transfected with non-targeting (shNC) or two sets of RAB11B-targeting (shRab11b1 and shRab11b2) shRNAs. Quantitative RT-PCR (A) and western blotting (B) were used to examine Rab11b …
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Figure 8—figure supplement 2—source data 1
Source data for Figure 8—figure supplement 2A.
- https://cdn.elifesciences.org/articles/56059/elife-56059-fig8-figsupp2-data1-v1.xlsx
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Figure 8—figure supplement 2—source data 2
Source data for Figure 8—figure supplement 2D.
- https://cdn.elifesciences.org/articles/56059/elife-56059-fig8-figsupp2-data2-v1.xlsx
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (H. sapiens) | CORIN | NCBI | NM_006587.4 | |
| Gene (H. sapiens) | ST14 (matriptase) | NCBI | NM_021978.4 | |
| Gene (H. sapiens) | TMPRSS3 | NCBI | NM_032404.3 | |
| Gene (H. sapiens) | TMPRSS4 | NCBI | NM_019894.4 | |
| Gene (H. sapiens) | TMPRSS5 | NCBI | NM_030770.4 | |
| Gene (H. sapiens) | MME (neprilysin) | NCBI | NM_007287.4 | |
| Gene (H. sapiens) | CD320 | NCBI | NM_016579.4 | |
| Cell line (H. sapiens) | HEK293 | ATCC | CRL-1573 | STR profiling, no mycoplasma contamination |
| Cell line (Canis familiaris) | MDCK | CCTCC* | 3142C0001000000147 | STR profiling, no mycoplasma contamination |
| Cell line (Mus musculus) | HL-1 | Sigma-Aldrich | SCC065 (Originally from William Claycomb) | No mycoplasma contamination |
| Transfected construct (H. sapiens) | Corin plasmid | PMID:14559895 | pcDNA 3.1/V5-His | |
| Transfected construct (H. sapiens) | Neprilysin plasmid | PMID:27343265 | pCMV3-C-Flag | |
| Transfected construct (H. sapiens) | Hepsin plasmid | PMID:31395734 | pcDNA 3.1/V5-His | |
| Transfected construct (H. sapiens) | Matriptase plasmid | This paper | pcDNA 3.1/V5-His | |
| Transfected construct (H. sapiens) | TMPRSS3 | This paper | pcDNA 3.1/V5-His | |
| Transfected construct (H. sapiens) | TMPRSS4 | This paper | pcDNA 3.1/V5-His | |
| Transfected construct (H. sapiens) | TMPRSS5 | This paper | pcDNA 3.1/V5-His | |
| Transfected construct (H. sapiens) | CD320 | This paper | pcDNA 3.1/N-Flag | |
| Transfected construct (Canis familiaris) | Rab11a | This paper | pCMV3-C-OFPSpark | |
| Transfected construct (Canis familiaris) | Rab11b | This paper | pCMV3-C-OFPSpark | |
| Transfected construct (H. sapiens) | Corin mutants | This paper | pcDNA 3.1/V5-His | |
| Transfected construct (H. sapiens) | CD320 mutants | This paper | pcDNA 3.1/N-Flag | |
| Antibody | Anti-corin (rabbit polyclonal) | PMID:27343265 | 1:1000 | |
| Antibody | Anti-corin (mouse monoclonal) | PMID:22437503 | 1:1000 | |
| Antibody | Anti-CD320 (rabbit polyclonal) | Atlas Antibodies | HPA073489 | 1:10 |
| Antibody | Anti-neprilysin (mouse monoclonal) | Boster | BM3411 | 1:100 |
| Antibody | Anti-Flag (mouse monoclonal) | Sigma | F1804 | 1:500 |
| Antibody | Anti-V5 (mouse monoclonal) | Thermo Fisher | R96025 | 1:500 |
| Antibody | Anti-V5-HRP (mouse monoclonal) | Thermo Fisher | R96125 | 1:5000 |
| Antibody | Anti-ZO-1 (rabbit polyclonal) | Cell Signaling | 8193 s | 1:300 |
| Antibody | Anti-IgG-Alexa-488 (donkey polyclonal) | Thermo Fisher | A32766 | 1:500 |
| Antibody | Anti-IgG-Alexa-594 (goat polyclonal) | Thermo Fisher | A32740 | 1:500 |
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*CCTCC: China Center for Type Culture Collection.
