N-glycosylation in the protease domain of trypsin-like serine proteases mediates calnexin-assisted protein folding
- Cleveland Clinic, United States
- Cleveland State University, United States
- State Key Laboratory of Radiation Medicine and Prevention, Soochow University of Science and Technology, China
Figures
Figure 1 with 2 supplements
N-glycosylation at N1022 in single-chain and soluble corin.
(A) Illustration of human corin WT and mutants with or without R801 activation site and N1022 N-glycosylation site. TM: transmembrane; Fz: frizzled; LDLR: LDL receptor; SR: scavenger receptor. An …
Figure 1—figure supplement 1
The phylogenetic tree of corin proteins in different species.
The phylogenetic relationships of corin in different species were evaluated using the COBALT server at the National Center for Biotechnology Information based on the full-length corin amino acid …
Figure 1—figure supplement 2
Alignments of the protease domain of trypsin-like serine proteases.
The amino acid sequences were aligned using the COBALT server. The N-glycosylation sites (red) are indicated. Corin has one N-glycosylation site that is also present in testisin; enteropeptidase …
Figure 2
Analysis of intracellular corin by CHX-based protein chase and Endo H digestion.
(A) Western blotting of corin in HEK293 cells treated without (0) or with CHX over time (h). (B) Percentages of corin WT and the mutant N1022Q levels, with corresponding levels at 0 hr being 100%, …
Figure 3
Intracellular distribution of corin WT and the N1022Q mutant.
(A) Co-staining of corin and PDI in HEK293 cells expressing WT corin and the N1022Q mutant without (0) or with CHX treatment for 4 hr. (B) Correlation of red (corin) and green (PDI) colors within …
Figure 4
Interactions between corin and ER chaperones.
(A) Corin proteins in HEK293 cells expressing WT corin and the N1022Q mutant were immunoprecipitated (IP) with an anti-V5 antibody that recognizes the C-terminal V5 tag in corin. Chaperones that …
Figure 5
Analysis of calnexin interaction.
(A) A model of calnexin-assisted glycoprotein folding. Cal: calnexin; blue dots: glucose residues. DNJ inhibits glucosidases I and II. (B) Co-immunoprecipitation (IP) and western blotting (IB) of …
Figure 6
Analysis of N-glycosylation in the protease domain of corin-EK chimeras.
(A) In CorinEK4N, the corin protease domain (Corin-P) was replaced by the EK protease domain (EK-P). The ADAM10-mediated shedding site is indicated by an arrowhead. In CorinEK4Q, all four …
Figure 7
Analysis of N-glycosylation sites in the protease domain of EK and prothrombin.
(A) Illustration of EK WT and the mutant lacking the indicated N-glycosylation sites (EK-4Q). EK domains include transmembrane (TM), SEA, LDLR, CUB, MAM, scavenger receptor (SR) and protease …
Figure 8
Interactions of EK and prothrombin with chaperones.
Co-immunoprecipitation (IP) and Western blotting (IB) of EK-WT and EK-4Q (A) or PT-WT and PT-N416Q (B) binding to calnexin, BiP and calreticulin (top three panels). EK (A) and PT (B) proteins in V5 …
Figure 9
Effects of DNJ treatment and calnexin knockdown.
HL-1 (A) and HepG2 (B) cells were cultured without (-) or with (+) DNJ. Recombinant human corin expression in transfected HEK293 cells were included as a control (A, left). Corin cell surface …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Homo sapiens) | Corin | NCBI | NM_006587.3 | |
| Gene (H. sapiens) | Prothrombin, PT | NCBI | NM_002772.2 | |
| Gene (H. sapiens) | Enteropeptidase, EK | NCBI | NM_000506.4 | |
| Genetic reagent (H. sapiens) | Calnexin (siRNA kit) | Origene | SR300576 | |
| Genetic reagent (Mus musculus) | Calnexin (siRNA kit) | Origene | SR417891 | |
| Cell line (H. sapiens) | HEK293 | ATCC | CRL-1573 | STR profiling, no mycoplasma contamination |
| Cell line (M. musculus) | HL-1 | PMID: 21518754, EMD Millipore: SCC065 | From Dr. William Claycomb | No mycoplasma contamination |
| Cell line (H. sapiens) | HepG2 | ATCC | HB-8065 | STR profiling, no mycoplasma contamination |
| Transfected construct (H. sapiens) | Corin plasmid | PMID: 14559895 | ||
| Transfected construct (H. sapiens) | sCorin plasmid | This paper | ||
| Transfected construct (H. sapiens) | CorinEK plasmid | This paper | ||
| Transfected construct (H. sapiens) | EK plasmid | This paper | ||
| Transfected construct (H. sapiens) | PT plasmid | This paper | ||
| Antibody | Anti-V5 | Thermo Fisher | R96025 | |
| Antibody | Anti-V5-HRP | Thermo Fisher | R96125 | |
| Antibody | Anti-GAPDH | EMD Millipore | MAB374 | |
| Antibody | Anti-PDI | Abcam | ab3672 | Immunostaining |
| Antibody | Anti-TGN46 | Abcam | ab50595 | |
| Antibody | Anti-Igg (mouse)-Alexa-594 | Thermo Fisher | A-21203 | |
| Antibody | Anti-Igg (rabbit)-Alexa-488 | Thermo Fisher | A-11008 | |
| Antibody | Anti-calnexin (human) | Cell Signaling | 2679T | |
| Antibody | Anti-BiP | Cell Signaling | 3177T | |
| Antibody | Anti-calreticulin | Cell Signaling | 12238S | |
| Antibody | Anti-HSP70 | Cell Signaling | 4872T | |
| Antibody | Anti-HSP90 | Cell Signaling | 4877T | |
| Antibody | Anti-PDI | Cell Signaling | 3501T | Western blotting |
| Antibody | Anti-Igg (mouse)-HRP | KPL | 474–1806 | |
| Antibody | Anti-Igg (rabbit)-HRP | KPL | 474–1516 | |
| Antibody | Anti-calnexin (mouse) | Abcam | ab75125 | |
| Antibody | Anti-corin (mouse) | Homemade | PMID: 26259032 | |
| Recombinant DNA reagent | pSecTag/FRT/V5-His Expression kit (vector) | Thermo Fisher | K602501 | |
| Recombinant DNA reagent | pcDNA 3.1/V5-His Expression kit (vector) | Thermo Fisher | K480001 | |
| Commercial assay or kit | ELISA kit (prothrombin) | Abcam | ab108909 | |
| Chemical compound, drug | 1-deoxynojirimycin, DNJ | Alfa Aesar | J62602-MC |
Additional files
-
Supplementary file 1
Proteins that differentially bound to WT corin and the N1022Q mutant identified in proteomic analysis.
- https://doi.org/10.7554/eLife.35672.013
-
Supplementary file 2
Proteins with a ratio of ≥ 2 fold between WT corin and the N1022Q mutant.
- https://doi.org/10.7554/eLife.35672.014
-
Supplementary file 3
Information of the DNA inserts in the expression plasmids used in this study.
- https://doi.org/10.7554/eLife.35672.015
-
Transparent reporting form
- https://doi.org/10.7554/eLife.35672.016
