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. 2020 Nov 2;9:e56059. doi: 10.7554/eLife.56059

A conserved LDL-receptor motif regulates corin and CD320 membrane targeting in polarized renal epithelial cells

Ce Zhang 1, Yue Chen 1, Shijin Sun 1,2, Yikai Zhang 1,2, Lina Wang 1, Zhipu Luo 3, Meng Liu 1, Liang Dong 1, Ningzheng Dong 1,2,✉, Qingyu Wu 1,4,✉
Editors: Adam Linstedt5, Olga Boudker6
PMCID: PMC7605860  PMID: 33136001

Abstract

Selective protein distribution on distinct plasma membranes is important for epithelial cell function. To date, how proteins are directed to specific epithelial cell surface is not fully understood. Here we report a conserved DSSDE motif in LDL-receptor (LDLR) modules of corin (a transmembrane serine protease) and CD320 (a receptor for vitamin B12 uptake), which regulates apical membrane targeting in renal epithelial cells. Altering this motif prevents specific apical corin and CD320 expression in polarized Madin–Darby canine kidney (MDCK) cells. Mechanistic studies indicate that this DSSDE motif participates in a Rab11a-dependent mechanism that specifies apical sorting. In MDCK cells, inhibition of Rab11a, but not Rab11b, expression leads to corin and CD320 expression on both apical and basolateral membranes. Together, our results reveal a novel molecular recognition mechanism that regulates LDLR module-containing proteins in their specific apical expression in polarized renal epithelial cells.

Research organism: Human

Introduction

Renal reabsorption is of fundamental importance in body fluid, electrolyte, and metabolic homeostasis (Nawata and Pannabecker, 2018; Polesel and Hall, 2019; van der Wijst et al., 2019). The majority of water and solutes, including glucose, ions, amino acids, and vitamins, in the glomerular filtrate are reabsorbed in the proximal tubule, which consists of a single layer of polarized cuboidal epithelial cells. In these epithelial cells, receptors, transporters, and enzymes are located on distinct apical and/or basolateral membranes to carry out their functions. Under pathological conditions, altered tubular epithelial membrane polarity and cell surface protein distribution impair renal function and physiological homeostasis (De Matteis and Luini, 2011; Rossier et al., 2002; Stoops and Caplan, 2014; Wilson, 2011). To date, molecular and cellular mechanisms directing proteins to specific membranes in polarized renal epithelial cells are not fully understood.

Corin is a serine protease primarily expressed in the heart where it activates atrial natriuretic peptide (ANP), a cardiac hormone that regulates salt-water balance and blood pressure (Li et al., 2017; Yan et al., 2000). In addition to the heart, corin is also expressed in other tissues such as the uterus and kidney (Cui et al., 2012; Ichiki et al., 2011; Wang et al., 2020; Yan et al., 1999). In the kidney, corin expression is mostly restricted to tubular epithelial cells (Dong et al., 2016), suggesting a potential role in regulating sodium excretion and reabsorption. In supporting this hypothesis, reduced renal corin expression was found in rat kidney disease models with sodium retention and proteinuria (Polzin et al., 2010). Similarly, reduced renal corin expression was reported in patients with chronic kidney disease (Fang et al., 2013).

Corin is a type II transmembrane protein with a cytoplasmic tail and an extended extracellular region containing distinct modular structures (Hooper et al., 2000; Yan et al., 2000). In non-polarized cardiomyocytes and human embryonic kidney (HEK) 293 cells, corin is expressed on the entire cell surface (Gladysheva et al., 2008; Zhang et al., 2015; Zhang et al., 2017). In contrast, corin is localized on the apical, but not basolateral, membrane in polarized renal epithelial cells (Dong et al., 2016; Polzin et al., 2010). Possibly, corin structural elements such as particular amino acids and/or carbohydrates are involved in a mechanism that controls distinct membrane targeting in polarized epithelial cells (Mellman and Nelson, 2008; Overeem et al., 2015; Weisz and Rodriguez-Boulan, 2009).

To test this hypothesis, we analyzed corin wild-type (WT) and mutants lacking individual modules in Madin–Darby canine kidney (MDCK) cells, a common model for polarized epithelial cells (Cereijido et al., 1978; Rodriguez Boulan and Sabatini, 1978). We conducted site-directed mutagenesis to identify individual amino acids crucial for apical corin expression. We verified our findings in other renal epithelial surface proteins and examined underlying molecular mechanisms. These experiments led us to identify a conserved motif in LDL-receptor (LDLR) class A modules (referred to as LDLR modules thereafter) that participates in the apical trafficking of corin and CD320 (a receptor for vitamin B12 uptake) in polarized renal epithelial cells.

Results

Apical corin expression in MDCK cells

Previously, corin expression on apical epithelial membranes was detected in rat and human kidneys (Dong et al., 2016; Polzin et al., 2010). To study corin expression on polarized epithelial membranes, we expressed human corin in MDCK cells. Consistently, confocal microscopy detected apical, but not basolateral, corin expression in polarized MDCK cells expressing ZO-1 protein at the apicolateral tight junction (Figure 1A). In contrast, corin expression in non-polarized HL-1 cardiomyocytes and HEK293 cells was on the entire cell membrane (Figure 1A).

Figure 1. 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 of the apicolateral tight junction) expression with confocal microscopy. X-Y and X-Z views are shown in top and lower panels, respectively. (B) Neprilysin, hepsin, matriptase, and TMPRSS3-5 (green) were expressed in transfected MDCK cells. Immunostaining and confocal microscopy were used to examine protein expression on apical and basolateral membranes. X-Y and X-Z views are shown in top and lower panels, respectively. Each image represents the data from five experiments. Scale bars: 5 μm.

Figure 1.

Figure 1—figure supplement 1. Western blotting analysis of corin expression in HEK293 and MDCK cells.

Figure 1—figure supplement 1.

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 indicated by an arrow. Recombinant corin had a C-terminal V5 (V) tag for protein detection. Western blotting (under reducing conditions) was done with an anti-V5 antibody to analyze corin protein fragments in transfected MDCK cells without (-) or with (+) trypsin treatment before the cells being lysed. Corin zymogen and cleaved protease domain fragments are indicated. The data are representative of four experiments.
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Corin is synthesized as a zymogen, which is activated by proprotein convertase subtilisin/kexin-6 (PCSK6) on the cell surface (Chen et al., 2015). We analyzed corin activation in MDCK and control HEK293 cells. In western blotting, a similar corin fragment from the activation cleavage was observed in transfected MDCK and HEK293 cells (Figure 1—figure supplement 1). If these cells were treated with trypsin to remove surface proteins before being lysed, the activation cleavage fragment was not detected (Figure 1—figure supplement 1), indicating that, like in HEK293 cells, corin is activated on the surface of polarized MDCK cells.

To understand if the observed apical corin expression in polarized epithelial cells is a general characteristic of type II transmembrane serine proteases that function in diverse tissues to regulate physiological processes (Antalis et al., 2011; Bugge et al., 2009), we expressed a selected set of type II transmembrane serine proteases (hepsin, matriptase, and TMPRSS3-5) in MDCK cells. Neprilysin, a metalloproteinase known for its apical expression in renal epithelial and MDCK cells (Dong et al., 2016; Jalal et al., 1991), was included as a control. Hepsin, matriptase, and TMPRSS3-5 exhibited an apical and basolateral pattern that differed from apical corin expression (Figure 1B), indicating that specific apical expression in polarized epithelial cells is not a common feature among type II transmembrane serine proteases.

Corin protein modules for specific apical expression in MDCK cells

To examine if determinant(s) for specific apical corin expression are located intracellularly or extracellularly, we analyzed corin mutants, in which the cytoplasmic tail (ΔCT) or extracellular non-protease modules (ΔFz1-SR) were deleted (Figure 2A). In MDCK cells, corin WT and the ΔCT mutant were expressed apically, whereas the ΔFz1-SR mutant was expressed apically and basolaterally (Figure 2A, Figure 2—figure supplement 1A), indicating that the determinant(s) for the apical-specific expression reside in the extracellular region between frizzled 1 (Fz1) and the scavenger receptor (SR) domains.

Figure 2. 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) module, and a protease domain module. Corin wild-type (WT) and mutants lacking the CT (ΔCT) or Fz1-SR modules (ΔFz1-SR) were expressed in MDCK cells. Corin expression (green) on apical and basolateral membranes was examined by immunostaining and confocal microscopy with X-Y (top panels) and X-Z (lower panels) views. (B) Corin mutants lacking Fz and LDLR modules, individually or in combination, were expressed in MDCK cells and examined by immunostaining and confocal microscopy with X-Y (top panels) and X-Z (lower panels) views. Each image represents the data from five experiments. Scale bars: 5 μm.

Figure 2.

Figure 2—figure supplement 1. Quantitative analysis of corin expression on apical and basolateral membranes in MDCK cells.

Figure 2—figure supplement 1.

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 LDLR (R) modules, individually or in combination in (B) were expressed in MDCK cells. Corin expression was examined with a confocal laser scanning microscope. Fluorescent signals along the X-Z axis were analyzed with Image J software to calculate the ratio of FBL/FTotal, where FBL is the fluorescent intensity on basolateral membranes and FTotal is the total fluorescent intensity on apical and basolateral membranes. The data (mean ± SEM) were from five experiments. Statistical analysis was done with ANOVA. n.s., not significant vs. corin WT in respective groups.
Figure 2—figure supplement 1—source data 1. Source data for Figure 2—figure supplement 1A.
Figure 2—figure supplement 1—source data 2. Source data for Figure 2—figure supplement 1B.
Figure 2—figure supplement 2. Analysis of corin mutants lacking individual N-glycosylation sites.

Figure 2—figure supplement 2.

(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 confocal microscopy were used to examine corin (green) and ZO-1 (red) expression in the transfected cells in X-Y and X-Z views. The data are representative of five experiments. (C) Quantitative data of relative corin expression on apical and basolateral membranes, indicated by the ratio of FBL/FTotal, were analyzed by Image J software. The data (mean ± SEM) were from five experiments. Statistical analysis was done with ANOVA. n.s., not significant vs. corin WT.
Figure 2—figure supplement 2—source data 1. Source data for Figure 2—figure supplement 2C.
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We tested additional corin mutants, in which Fz and LDLR modules were deleted, individually or in combination (Figure 2B). In MDCK cells, the corin mutants lacking Fz1-2 (ΔFz1 and ΔFz2), LDLR1-5 (ΔR1-4 and ΔR45), or LDLR6-7 (ΔR67) modules were all expressed apically (Figure 2B). In contrast, the mutants, in which LDLR8 module was deleted either alone (ΔR8) or together with LDLR6 and/or LDLR7 modules (ΔR6-8 and ΔR78), were expressed apically and basolaterally (Figure 2B). The results were confirmed by quantitative analysis of fluorescent signals on apical and basolateral membranes (Figure 2—figure supplement 1B). These results indicate that elements in or near corin LDLR8 module are important for specific apical expression.

N-glycans are known to mediate apical expression in polarized epithelial cells (Potter et al., 2006; Weisz and Rodriguez-Boulan, 2009). Human corin contains 19 N-glycosylation sites, some of which, for example N761 and N1022, are required for corin expression on the surface of cardiomyocytes and HEK293 cells (Wang et al., 2018; Wang et al., 2015). We examined the corin mutants, in which N-glycosylation sites at N567 (N567Q), N651 (N651Q), N697 (N697Q), and N1022 (N1022Q) in or near LDLR6-8, SR and protease domain modules were abolished individually (Figure 2—figure supplement 2A). We found that all of the N-glycosylation site mutants had specific apical expression in MDCK cells (Figure 2—figure supplement 2B,C), indicating that the apical corin expression in polarized epithelial cells does not depend on individual N-glycans near LDLR8 module.

Corin LDLR8 module residues in specific apical expression

Corin LDLR1-8 modules have a high degree of sequence similarities; yet only the deletion of LDLR8 module altered the specific apical expression of corin. We aligned corin LDLR1-8 module sequences and identified 11 residues in LDLR8 that were unique at corresponding positions (Figure 3—figure supplement 1A). Among them, five were conserved in vertebrate species from zebrafish to monkeys (Figure 3—figure supplement 1B). To test the functional importance of these residues, we replaced them with corresponding residues in LDLR6 module of the mutant ΔR78 that expressed both apically and basolaterally (Figure 3A). We found that Asp608 to Ser substitution (D608S), but not substitutions of the other four residues, restored the specific apical expression (Figure 3A, Figure 3—figure supplement 1C), suggesting that Ser683 in LDLR8 module is critical for specific apical corin expression in MDCK cells. To verify this result, we mutated Ser683 to Asp (the corresponding residue in LDLR6 module) in WT corin (Figure 3B). When expressed in MDCK cells, the S683D mutant was observed on both apical and basolateral membranes (Figure 3B, Figure 3—figure supplement 1D).

Figure 3. 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 MDCK cells. Corin expression (green) on apical and basolateral membranes was examined by immunostaining and confocal microscopy with X-Y (top panels) and X-Z (lower panels) views. ZO-1 protein (red) was used as an indicator. (B) Corin WT and the mutant S683D expressed in MDCK cells were examined by immunostaining and confocal microscopy with X-Y (top panels) and X-Z (lower panels) views. Each image represents the data from five experiments. Scale bars: 5 μm.

Figure 3.

Figure 3—figure supplement 1. Alignments of corin LDLR module sequences.

Figure 3—figure supplement 1.

(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 vertebrate species are underlined. (B) Alignment of partial sequences in LDLR8 modules from monkey (Mac), rat (Rat), mouse (Mou), pigeon (Pig), and zebrafish (Zeb) species. Five conserved residues, corresponding to those in red and underlined (A), are shown in red. (C and D) Corin WT and mutants were expressed in MDCK cells and analyzed by immunostaining and confocal microscopy. Quantitative data of relative corin expression on apical and basolateral membranes, indicated by the ratio of FBL/FTotal, were analyzed by Image J software. The data (mean ± SEM) were from five experiments. Statistical analysis was done with ANOVA (C) or Student’s t test (D). n.s., not significant vs. corin WT.
Figure 3—figure supplement 1—source data 1. Source data for Figure 3—figure supplement 1C.
Figure 3—figure supplement 1—source data 2. Source data for Figure 3—figure supplement 1D.
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Ser683 is located in a DxSDE motif, where x is any amino acid, conserved among LDLR modules in many proteins (Figure 4—figure supplement 1A; Herz, 2001; Mahley, 1988). Within this motif, the last two residues (Asp-Glu) are part of a Ca2+-binding cage that stabilizes the LDLR module conformation (Alam et al., 2016; Fass et al., 1997; Huang et al., 1999; North and Blacklow, 2000). To understand the importance of the DSSDE motif in corin LDLR8 module, we mutated each residue in this motif to Ala individually (Figure 4A). In MDCK cells, D682A, S684A, D685A, and E686A mutants were expressed apically and basolaterally, whereas the S683A mutant was expressed apically, but not basolaterally (Figure 4A, Figure 4—figure supplement 1B). These results indicate that the DSSDE motif in corin LDLR8 module is important for specific apical expression and that within this motif Ser683 can be replaced by Ala without altering the membrane distribution pattern.

Figure 4. 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 apical and basolateral membranes was examined by immunostaining and confocal microscopy with X-Y (top panels) and X-Z (lower panels) views. (B) Corin WT and S684A mutants without (S684A) or with the DSSDE motif created in LDLR6 (ML6/S684A) or LDLR7 (ML7/S684A) module expressed in MDCK cells were examined by immunostaining and confocal microscopy. X-Y (top panels) and X-Z (lower panels) views are indicated. Each image represents the data from five experiments. Scale bars: 5 μm.

Figure 4.

Figure 4—figure supplement 1. Analysis of the DSSDE motif in corin LDLR8 module.

Figure 4—figure supplement 1.

(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, Rat; mouse, Mou; pigeon, Pig; zebrafish, Zeb) is aligned. This motif corresponds to the DxSDE motif found in LDLR modules in many proteins. The Asp-Glu residues (indicated by black dots), corresponding to Asp685 and Glu686 in human corin, have been shown to be part of a Ca2+-binding cage in LDLR module structures of other proteins. (B and C) Corin WT and mutants were expressed in MDCK cells and analyzed by immunostaining and confocal microscopy. Quantitative data of relative corin expression on apical and basolateral membranes, indicated by the ratio of FBL/FTotal, were analyzed by Image J software. The data (mean ± SEM) were from five experiments. Statistical analysis was done with ANOVA. n.s., not significant vs. corin WT in respective groups.
Figure 4—figure supplement 1—source data 1. Source data for Figure 4—figure supplement 1B.
Figure 4—figure supplement 1—source data 2. Source data for Figure 4—figure supplement 1C.
Figure 4—figure supplement 2. Western blotting of corin proteins in biotin-labeled cell membranes.

Figure 4—figure supplement 2.

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 to label apical and basolateral membrane proteins, respectively. The cells were lysed. Biotin-labeled corin proteins (A and C) were immunoprecipitated and analyzed by western blotting. In parallel, corin proteins in cell lysates were analyzed by western blotting with GAPDH control (B and D). The data are representative of three experiments.
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To verify these results, we tested corin mutants ML6/S684A and ML7/S684A, in which the DSSDE motif was introduced in the LDLR6 and LDLR7 modules, respectively, of the mutant S684A, which expressed apically and basolaterally (Figure 4B). In MDCK cells, both ML6/S684A and ML7/S684A mutants were expressed apically, but not basolaterally (Figure 4B, Figure 4—figure supplement 1C). In western blotting of biotin-labeled proteins, corin WT was found among apical, but not basolateral, membrane proteins, whereas the S684A mutant was among apical and basolateral membrane proteins (Figure 4—figure supplement 2). These results support the idea that the DSSDE motif is important for specific apical corin expression in polarized epithelial cells.

Computational modeling of corin LDLR modules

To gain structural insights into the functional significance of the DxSDE motif in corin LDLR modules, we analyzed 15 LDLR module structures in the Protein Data Bank (Figure 5—figure supplement 1A; Burley et al., 2019), and built LDLR6 and LDLR8 module models based on the atomic coordinates of human VLDLR module 4 (Banerjee et al., 2018), which shared the highest degree of sequence identity and similarity with that of corin LDLR8 module (Figure 5—figure supplement 1B). Both corin LDLR6 and LDLR8 modules exhibited a similar overall topology with the Asp-Glu residues (the last two residues in the DxSDE motif) being part of the Ca2+-binding cage (Figure 5A). In these models, Asp608 (second residue in the DDSDE motif) in LDLR6 module and Ser683 (second residue in the DSSDE motif) in LDLR8 module were located at a corner of the respective module and surface-exposed with potential to interact with other proteins (Figure 5A).

Figure 5. 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 panel) and S683D (right panel) mutants. The conserved DxSDE motifs are indicated in boxes with enlarged images shown on the right side. The overall structures are shown in ribbon models. The side chains and disulfide bonds (yellow) are presented as sticks. The calcium ion is shown as a green sphere.

Figure 5.

Figure 5—figure supplement 1. Structure alignments of LDLR modules.

Figure 5—figure supplement 1.

(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 highest similarity. The other homologous structures are shown in ribbons connected by Cα atoms. The calcium ion in the conserved cage is shown in a green sphere. (B) The superposition of human VLDLR residues 154–188 (yellow) and corin LDLR8 module residues 655–689 (cyan) with the Ca2+-binding cage. The side chains and disulfide bonds are shown as sticks and the calcium ion as a green sphere. The conserved resides between human VLDLR module four and corin LDLR8 module are shown in the alignment.
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As described above, the mutant S683A was expressed apically (Figure 4A), whereas the mutant S683D was expressed apically and basolaterally (Figure 3B). In computational modeling, substitution of Ser683 to Ala (S683A) or Asp (S683D) did not disturb the overall structure of corin LDLR8 module (Figure 5B). The major difference was that the negatively-charged Asp683 side chain in the S683D mutant protruded more profoundly than the smaller and uncharged side chains of Ser683 and Ala683 did in corin WT and the mutant S863A, respectively (Figure 5A,B). These results suggest that a proper conformation in this corner region of LDLR modules may be important for protein interactions in specific membrane sorting.

Apical expression of CD320 in renal epithelial cells

To understand if the DSSDE motif in corin LDLR8 module is present in other proteins expressed apically in polarized epithelial cells, we searched LDLR module-containing transmembrane proteins expressed in kidneys. We found that CD320, a type I transmembrane protein with two LDLR modules (Alam et al., 2016), has the DSSDE motif in the second LDLR module (Figure 6A). Structure alignment of CD320 LDLR1-2 and corin LDLR8 modules showed the DSSDE motif at the corner region of CD320 LDLR2 module (Figure 6—figure supplement 1A–C). By immunostaining of human kidney sections, we found CD320 expression in the apical region underneath the brush border (neprilysin-positive) in proximal tubules, a pattern similar to that of corin (Figure 6B). Consistently, human CD320 was observed on the apical, but not basolateral, membrane in transfected MDCK cells (Figure 6C).

Figure 6. 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 motif, whereas LDLR2 module has a DSSDE motif (underlined). (B) Co-staining of neprilysin (brush border marker) (green), corin (red or green) and CD320 (red) in proximal tubules in human kidney sections. Cell nuclei were stained in blue. Boxed areas in top panels are enlarged in lower panels. Scale bars: 10 μm. (C) Corin (green), CD320 (green), and ZO-1 (red) expression in transfected MDCK cells. X-Y and X-Z views are shown in top and lower panels, respectively. Scale bars: 5 μm. Each image represents data from five experiments.

Figure 6.

Figure 6—figure supplement 1. Structural alignments of the LDLR domains of human CD320.

Figure 6—figure supplement 1.

(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 (spheres) and side chains and disulfide bonds (sticks). (B) The DSSDE motifs in CD320 LDLR2 and corin LDLR8 modules are enlarged from (A). (C) The local alignment of the DSSDE motifs in CD320 LDLR2 and corin LDLR8 modules.
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To test if the DSSDE motif in CD320 LDLR2 module regulates membrane targeting, we mutated residues in the DSSDE motif to Ala individually (Figure 7A). In MDCK cells, D160A, S162A, D163A, and E164A mutants were expressed apically and basolaterally, whereas the S161A mutant was expressed apically, but not basolaterally (Figure 7A, Figure 7—figure supplement 1A). These results are consistent with the findings in corin LDLR8 module mutants, indicating that the DSSDE motif in CD320 LDLR2 module also regulates membrane expression in polarized epithelial cells and that within this motif Ser161 can be replaced by Ala without noticeable effect on apical membrane targeting.

Figure 7. 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) on apical and basolateral membranes was examined by immunostaining and confocal microscopy with X-Y (top panels) and X-Z (lower panels) views. ZO-1 protein (red) was used as an indicator. (B) CD320 WT and S162A mutants without (S162A) or with the DSSDE motif created in LDLR1 module (ML1/S162A) expressed in MDCK cells were examined by immunostaining and confocal microscopy. X-Y (top panels) and X-Z (lower panels) views are indicated. Each image represents the data from five experiments. Scale bars: 5 μm.

Figure 7.

Figure 7—figure supplement 1. Quantitative analysis of CD320 and corin expression in MDCK cells.

Figure 7—figure supplement 1.

(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 the ratio of FBL/FTotal. The data are mean ± SEM (n = 5).
Figure 7—figure supplement 1—source data 1. Source data for Figure 7—figure supplement 1A.
Figure 7—figure supplement 1—source data 2. Source data for Figure 7—figure supplement 1B.
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Human CD320 LDLR1 module has a DGSDE, instead of DSSDE, motif (Figure 7B). The apical and basolateral expression of the CD320 mutant S162A indicated that the DGSDE motif did not support specific apical targeting. We tested the CD320 mutant ML1/S162A, in which the DSSDE motif was introduced in LDLR1 module of the mutant S162A (Figure 7B). In MDCK cells, the specific apical expression was restored in the ML1/S162A mutant (Figure 7B, Figure 7—figure supplement 1B), indicating that the DSSDE motif, whether in CD320 LDLR1 or LDLR2 module, can function similarly to support the specific apical targeting in polarized epithelial cells.

Role of Rab11a and Rab11b in apical corin and CD320 expression

Rab11 proteins, that is Rab11a and Rab11b, play a key role in apical expression in polarized epithelial cells (Kelly et al., 2012; Thuenauer et al., 2014; Welz et al., 2014). To understand if Rab11 proteins are involved in apical corin and CD320 expression in MDCK cells, we co-expressed corin with human Rab11a, Rab11b, and dominant-negative (DN) mutants (Rab11aS25N and Rab11bS25N) that were inhibitory to Rab11a and Rab11b, respectively (Thuenauer et al., 2014; Ying et al., 2016). In cells expressing corin and recombinant Rab11a, the specific apical corin expression was not altered, whereas in cells expressing corin and DNRab11a, both apical and basolateral corin expression was observed (Figure 8A, Figure 8—figure supplement 1A). In contrast, apical, but not basolateral, corin expression was observed in cells co-expressing corin and recombinant Rab11b or DNRab11b (Figure 8A, Figure 8—figure supplement 1A), indicating that Rab11a, but not Rab11b, is involved in the specific apical corin expression in MDCK cells.

Figure 8. 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. Immunostaining and confocal microscopy were used to analyze corin expression on apical and basolateral membranes, as indicated in X-Y and X-Z views. Each image represents the data from four experiments. Scale bars: 5 μm. (B) qRT-PCR analysis of Rab11a mRNA levels in MDCK cells transfected with two sets of shRNAs targeting the RAB11A gene (shRab11a1 and shRab11a2) or non-targeting control shRNAs (shNC). The data are mean ± SD from five experiments, analyzed by ANOVA. (C) Immunostaining of corin and CD320 in MDCK cells transfected with RAB11A-targeting shRNAs (shRab11a1 and shRab11b) or non-targeting shRNAs (shNC). X-Y and X-Z views are indicated. (D) A proposed model. Corin and CD320 (red dots) are transported from Golgi, likely via central recycling endosomes (CRE), to a Rab11a-positive sorting station, where the DSSDE motif is recognized for apical sorting via apical recycling endosomes (ARE) (left panel). Any pool of corin and CD320 that is endocytosed may also be re-sorted apically via Rab11a-positive endosomes. Mutations altering the DSSDE motif abolish such a mechanism, resulting in apical and basolateral expression of corin and CD320 mutants (right panel).

Figure 8—source data 1. Source data for Figure 8B.

Figure 8.

Figure 8—figure supplement 1. Effects of Rab11a inhibition on apical corin and CD320 expression.

Figure 8—figure supplement 1.

(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 of FBL/FTotal from four experiments are shown. (B) Western blotting of Rab11a protein in MDCK cells transfected with non-targeting (shNC) and two sets of RAB11A-targeting (shRab11a1 and shRab11a2) shRNAs. GAPDH was used as a control. (C) Quantitative data of the ratio of FBL/FTotal for corin and CD320 expression on apical and basolateral membranes in shRNA-transfected MDCK cells from four experiments. Statistical analysis was done with ANOVA. n.s., not significant vs. respective controls in each experiment.
Figure 8—figure supplement 1—source data 1. Source data for Figure 8—figure supplement 1A.
Figure 8—figure supplement 1—source data 2. Source data for Figure 8—figure supplement 1C.
Figure 8—figure supplement 2. Effects of Rab11b knockdown on corin and CD320 expression in MDCK cells.

Figure 8—figure supplement 2.

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 mRNA and protein expression, respectively, in the shRNA-transfected MDCK cells. (C) Apical corin (top panels) and CD320 (lower panels) expression in the shRNA-transfected MDCK cells was examined by immunostaining and confocal microscopy with X-Y and X-Z views indicated. (D) Quantitative data of the ratio of FBL/FTotal for corin and CD320 expression on apical and basolateral membranes in shRNA-transfected MDCK cells from four experiments. Statistical analysis was done with ANOVA. n.s., not significant vs. respective controls in each experiment. Key Resources Table. Information on genes, cell lines, plasmids and antibodies.
Figure 8—figure supplement 2—source data 1. Source data for Figure 8—figure supplement 2A.
Figure 8—figure supplement 2—source data 2. Source data for Figure 8—figure supplement 2D.
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To verify these results, we made stable MDCK cells, in which Rab11a expression was down-regulated by shRNAs targeting the RAB11A gene (encoding Rab11a), as indicated by quantitative RT-PCR (Figure 8B) and western blotting (Figure 8—figure supplement 1B). In the RAB11A-knockdown cells, corin was expressed apically and basolaterally, whereas in control MDCK cells transfected with non-targeting shRNAs (shNC), corin was expressed apically (Figure 8C, Figure 8—figure supplement 1C). Similarly, CD320 was expressed apically in the control shRNA-transfected cells, but apically and basolaterally in the RAB11A-knockdown cells (Figure 8C, Figure 8—figure supplement 1C). In contrast, knocking down RAB11B, encoding Rab11b, in MDCK cells did not alter the apical expression of corin and CD320 (Figure 8—figure supplement 2A–D). These results suggest a possible cell membrane targeting mechanism, in which corin and CD320 are transported, likely via common recycling endosomes (CRE), to a Rab11a-positive apical recycling endosome (ARE), where LDLR modules with the DSSDE motif are recognized by a Rab11a-dependent mechanism that favors apical sorting (Figure 8D). For the corin and CD320 mutants, in which in the DSSDE motif is altered, such a recognition mechanism is eliminated, resulting in sorting to both apical and basolateral membranes (Figure 8D).

Discussion

Selective protein distribution on distinct plasma membranes is of great important in epithelial biology (Mostov et al., 2003). In this study, we used MDCK cells as a model to examine corin expression on polarized epithelial membranes. In immunostaining, corin exhibited a specific apical expression pattern in MDCK cells, which differs from the overall membrane expression pattern in HL-1 cardiomyocytes and HEK293 cells. The distinct apical expression likely reflects the corin function in the lumen of renal tubules to regulate sodium excretion and reabsorption (Dong et al., 2016; Li et al., 2017).

Apical targeting in polarized epithelial cells has been studied extensively. To date, cytoplasmic tail, transmembrane domain, glycosyl-phosphatidylinositol (GPI) anchor, PDZ-binding motif, and N- or O-glycans have been identified as apical sorting signals (Stoops and Caplan, 2014; Weisz and Rodriguez-Boulan, 2009). Corin is a transmembrane, but not GPI-anchored, protein and contains no PDZ-binding motif or detectable amounts of O-glycans (Liao et al., 2007; Yan et al., 2000). In this study, we showed that the cytoplasmic tail and individual N-glycosylation sites near LDLR8 module were unnecessary for the specific apical expression of corin in MDCK cells. These results point to a yet unknown mechanism underlying apical corin expression in polarized renal epithelial cells.

By analyzing a series of corin mutants with modular deletions and point mutations, we identified a DSSDE motif in corin LDLR8 module required for specific apical expression in MDCK cells. In corin mutants, in which the DSSDE motif was disrupted in LDLR8 module, but re-introduced in LDLR6 or LDLR7 module, the distinct apical expression of corin was restored. These results indicate that the DSSDE motif is a key element, transportable among LDLR modules, that specifies apical corin expression in polarized renal epithelial cells. Based on modeling analysis of known LDLR module structures (Fass et al., 1997; Huang et al., 1999; North and Blacklow, 2000), Ser683 (first Ser in the DSSDE motif) in corin is predicted to be surface exposed. In LDLR modules of the human patriarch LDLR, the corresponding surface area is a proposed ligand-binding site (Fass et al., 1997; Huang et al., 1999; Russell et al., 1989). Conceivably, the surface area around Ser683 in corin LDLR8 module may serve as a protein-interacting site for apical targeting in renal epithelial cells.

The finding of the DSSDE motif in corin LDLR8 module as a possible regulatory element in epithelial membrane targeting was unexpected. Corin protein has eight LDLR modules, six of which have the conserved DxSDE motif. Moreover, LDLR modules with the conserved DxSDE motif are present in numerous cell surface receptors and proteolytic enzymes (Brown et al., 1997; Bugge et al., 2009; Herz, 2001). We verified our findings in CD320, a receptor for transcobalamin in vitamin B12 uptake (Bernard et al., 2018; Quadros and Sequeira, 2013), which contains the DSSDE motif in its second LDLR module. CD320 expression in kidneys has been reported (Arora et al., 2017; Park et al., 2009), but its cellular and subcellular distribution in renal segments remained unclear. In immunostaining with human kidney sections, we found apical CD320 expression in proximal tubular epithelial cells. The result is consistent with CD320 function in vitamin B12 uptake, as most vitamin reabsorption occurs via cell surface receptors in renal proximal tubules (Christensen and Birn, 1997). Importantly, we found that the DSSDE motif in CD320 LDLR2 module was also required for specific apical expression in MDCK cells. These results suggest that the DSSDE motif may have a similar regulatory function in other LDLR module-containing proteins that are expressed apically in renal epithelial cells.

In polarized epithelial cells, Rab11a-positive endosomes are primary trafficking stations toward the apical membrane (Hoekstra et al., 2004; Thuenauer et al., 2014; Wang et al., 2000). In our study, overexpression of a dominant-negative Rab11a mutant (Rab11aS25N) or shRNA-mediated RAB11A knockdown in MDCK cells resulted in apical and basolateral expression of corin and CD320. In contrast, RAB11B knockdown did not alter the apical expression pattern of corin and CD320. It is possible that the DSSDE motif-containing LDLR modules in corin and CD320 are recognized by a Rab11a-dependent mechanism that specifies apical sorting. Mutations in the DSSDE motif abolish such a recognition mechanism, leading to apical and basolateral sorting of the mutant proteins. Further studies are required to understand the molecular basis for the potential Rab11a-dependent recognition mechanism.

In polarized epithelial cells, Rab11a is also known to mediate vesicle trafficking and recycling (Perez Bay et al., 2016; Weisz and Rodriguez-Boulan, 2009). At this time, it is unclear if corin undergoes endocytosis and recycling. In cardiomyocytes and HEK293 cells, corin is activated by PCSK6-mediated cleavage on the cell surface and subsequently undergoes ectodomain shedding (Chen et al., 2015; Chen et al., 2018; Jiang et al., 2011). In western blotting under reduction conditions, the corin protease domain fragment derived from activation cleavage was not detected intracellularly (Chen et al., 2015), suggesting that activated corin may not be internalized for recycling. In this study, we found similar corin activation on the surface of MDCK cells and did not detect the cleaved protease domain fragment inside the cells. Consistently, corin WT was detected among biotin-labeled apical, but not basolateral, membrane proteins. Additional studies with more direct and sensitive assays are required to determine if corin and CD320 undergo endocytosis and recycling in MDCK cells.

In summary, targeted apical expression is a key characteristic of polarized epithelial cells. Disturbed protein trafficking to distinct cell membranes in renal epithelial cells has been shown to cause kidney diseases. In this study, we have identified a conserved DSSDE motif in corin and CD320 LDLR modules as a regulatory element in apical sorting in MDCK cells. This regulatory function is likely mediated by a Rab11a-dependent mechanism. The DSSDE motif is present in other proteins with LDLR modules. Our findings should encourage more research to examine if analogous motifs in other LDLR module-containing proteins have a similar role in apical membrane targeting in polarized epithelial cells.

Materials and methods

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.

Human kidney samples

The study involving human tissues was approved by the Ethics Committee of Soochow University (2013052). Kidney tissues were obtained from patients who underwent nephrectomy for renal cancers at a university affiliated hospital. Informed consents were obtained from the donners. Tissues were dissected from non-tumor areas, fixed in 4% (v/v) formaldehyde, and embedded in paraffin. Sections were made, stained with hematoxylin and eosin (H and E), and verified by a pathologist for normal renal histology without fibrosis, hemorrhages, and necrosis. All procedures using tissue sections were carried out according to the approved protocol.

Immunostaining in tissue sections

To examine corin and CD320 expression and subcellular localization in renal tubular epithelial cells, kidney tissues were fixed in 4% (v/v) formaldehyde, embedded in paraffin, cut in 3 μm sections, and mounted on chrome alum gelatin-coated slides (Aladdin, p1650). After dewaxing, sections were boiled in sodium citrate buffer (0.01 mM, pH 6.0) for 1 min for antigen retrieval, and soaked in a methanol solution containing 3% (v/v) hydrogen peroxide for 5 min at room temperature to block endogenous peroxidase activity. After incubation with 5% (w/v) bovine serum albumin (BSA) at 37°C for 30 min to reduce non-specific background, an antibody against corin (described above, 1:1000), CD320 (Atlas Antibodies, HPA073489, 1:10), or neprilysin (Boster, BM3411, 1:100) was added and incubated at 4°C overnight. After washing with PBS, secondary antibodies Alexa Fluro488-donkey anti-mouse IgG (Thermo Fisher, A32766, 1:500) and Alexa Fluro594-goat anti-rabbit IgG (Thermo Fisher, A32740, 1:500) were added and incubated at 37°C for 1 hr. After washing, the sections were mounted with a solution containing 4–6-diamidino-2-phenylindole (DAPI) (Southern Biotech, 0100–20). Cell images were examined and recorded using a confocal microscope (Olympus, FV1000).

Cell culture

MDCK (China Center for Type Culture Collection, STR profiled) and HEK293 (ATCC, STR profiled) cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) (Corning, 10-0130CVRC) with 10% fetal bovine serum (FBS) (Gibco, 16000–044) at 37°C with 5% CO2. Murine HL-1 cardiomyocytes, originally from Dr. William Claycomb (Claycomb et al., 1998), were grown in Claycomb medium (Hyclone, 51800C) with 10% FBS and 4 mM/L L-glutamine (Sigma, 21051024) at 37°C with 5% CO2.

Plasmid constructs

The plasmids expressing human neprilysin, hepsin, and corin WT and mutants were described previously (Dong et al., 2016; Knappe et al., 2004; Wang et al., 2019). Human full-length cDNAs encoding matriptase, TMPRSS3, TMPRSS4, and TMPRSS5 were amplified from human cell line-derived cDNA libraries and inserted into pcDNA 3.1/V5-His plasmid (Thermo Fisher, K4800-01), which encodes a C-terminal V5 tag. Human CD320 cDNA (106–849 bp) from a HEK293 cell-derived library was cloned into pcDNA 3.1 plasmid, which had an inserted 5’ sequence encoding the CD33 signal peptide and a Flag tag. Plasmids encoding human Rab11a and Rab11b with a C-terminal orange fluorescent protein (OFP) tag were made by cloning cDNAs from the HEK293 cell-derived library into pCMV3-C-OFPSpark (Sino Biological, China). Additional site-directed mutagenesis was done to make plasmids expressing mutant corin, CD320, Rab11a, and Rab11b proteins using ClonExpress One Step Cloning kit (Vazyme, China, C112). All plasmids used in this study were verified by DNA sequencing.

Cell transfection and immunostaining

MDCK, HEK293, and HL-1 cells cultured in 12-well plates with glass coverslips (20 mm in diameter) were transfected with plasmids using PolyJet reagents (SignaGen Laboratories, SL100688) at 37°C. After 6 hr, the cells were switched to fresh medium. After 72 hr, the cells were fixed with pre-cooled methanol at room temperature for 5 min and incubated with 5% (w/v) BSA in PBS at 37°C for 1 hr. After washing with PBS, the cells were incubated with an anti-V5 or Flag antibody and an anti-ZO-1 antibody at 37°C for 1 hr, followed by incubation with Alexa Fluro-488 or 594-labeled secondary antibodies at 37°C for 1 hr. After washing, coverslips were mounted with a DAPI solution (Southern Biotech, 0100–20) to stain DNA in cell nuclei. The cells were examined with a confocal laser scanning microscope (Olympus, FV1000) along the X-Z axis from the bottom to the top. X-Y plane photos (8 μs/picture) were taken at each 0.45 μm and used to reconstitute X-Z axis images. Image J software was used to analyze fluorescent intensity on apical (guided by ZO-1 staining) and basolateral (total staining minus apical staining) membranes and calculate the ratio of FBL/FTotal, where FBL is the fluorescent intensity on basolateral membranes and FTotal is the total fluorescent intensity on apical and basolateral membranes. Values of black area without fluorescent staining were set as the background. No noise reduction of the image data was performed.

Trypsin digestion of cell surface proteins and western blotting

To examine proteins on MDCK and HEK293 cell surface, transfected cells expressing corin were treated with 0.25% (w/v) trypsin and 0.02% EDTA (w/v) (Gibco, 25200) at 37°C for 30 s. DMEM with 10% FBS was added to neutralize trypsin activity. After washing with PBS, the cells were lysed in a buffer containing 1% (v/v) Triton X-100, 50 mM Tris–HCl (pH 8.0), 150 mM NaCl, and a protease inhibitor mixture (1:100, Roche Applied Science, 04693116001). Proteins were separated by SDS-PAGE under reducing conditions with 2.5% (v/v) β-mercaptoethanol in the Laemmli buffer (Bio-Rad) and transferred onto polyvinylidene difluoride membranes (Thermo Fisher) in an apparatus (Bio-Rad Trans-Blot, 300 mA) with 25 mM Tris base, 190 mM glycine, and 20% (v/v) methanol at 4°C for 60 min. Western blotting was done using a horseradish peroxidase (HRP)-conjugated anti-V5 antibody (1:5000, Thermo Fisher, R96125). After incubation with a chemiluminescent substrate (EZ-ECL) (Biological Industries, K-12045), western blots were exposed to X-ray films, which were analyzed by a scanner (CanoScan LiDE 110, Canon).

Biotin-labeling of cell membrane proteins

MDCK cells transfected with a control vector or plasmids expressing corin WT and the S684A mutant were cultured with G418 (2 mg/mL) (Gibco) for 3–4 weeks to obtain stable cell clones. The selected cells were grown in Transwell culture plates (Costar, 3419) with 75 mm inserts containing polycarbonate membranes (pore size: 0.4 μm). When reaching confluency, the cells were washed with cold (4°C) PBS. Sulfo-NHS-biotin (0.25 mg/mL) (Thermo Fisher) was added to the upper or the bottom chamber to label proteins on apical and basolateral membranes, respectively. After 5 min at 4°C, the labeling reaction was stopped with a glycine solution (100 mM). The cells were washed and lysed, as described above. Biotin-labeled proteins were precipitated with immobilized NeutrAvidin beads (Thermo Fisher) at 4°C overnight and analyzed by SDS-PAGE and western blotting under reducing conditions, as described above. Proteins in cell lysates were analyzed in parallel as controls together with an HRP-conjugated anti-GAPDH antibody (MultiScience, ab011-100, 1:10000).

Gene knockdown and quantitative RT-PCR (qRT-PCR)

To knock down the RAB11A or RAB11B gene, MDCK cells were transfected with two sets of RAB11A-targeting (shRab11a1 and shRab11a2) or RAB11B-targeting (shRab11b1 and shRab11b2) shRNAs or non-targeting control shRNAs (shNC) (GeneParma, China). Stable cells were selected with puromycin (1 μg/mL) (Gibco). Total RNAs were isolated using the EZNA HP Total RNA kit (OMEGA) and reverse-transcribed to make cDNAs using the RevertAid First Strand cDNA Synthesis kit (Thermo Fisher). Rab11a and Rab11b mRNA levels were analyzed by qRT-PCR using SYBR Green MasterMix in the PRISM 7500 Sequence Detection System (Applied Biosystems). The primers used for RAB11A were 5’-ATT TGC GTC ATC TCA GGG CA-3’ (sense) and 5’-GTT TCT GGG AAA CAA TGC GGT-3’ (antisense) and for RAB11B were 5’-CTA TGG AGG GAA TGT CGC TAT C-3’ (sense) and 5’-CTG TCT CTG GTG AGT CTT CTA G-3’ (antisense). A control (18S mRNA) was analyzed in parallel. Western blotting was used to verify Rab11a and Rab11b protein levels in the targeted cells with primary antibodies against Rab11a (Cell Signaling, 2413, 1:1000) and Rab11b (Boster, M04526, 0.5 μg/mL).

Modeling of LDLR module structures

The sequence of human corin LDLR8 module was subjected to the Protein Data Bank (PDB) (Burley et al., 2019) to identify homologous structures. Fifteen matched structures with low E-values and high resolution were chosen for further analysis. The best matched structure was that of module four in the human VLDLR (6BYV, residues 154–188) (Banerjee et al., 2018) with 51% (18/35 residues) identities and 69% (24/35 residues) similarities compared to the corin LDLR8 module sequence (residues 655–689). The VLDLR structure was used as a template to build homology models for corin LDLR6, LDLR8, and mutant LDLR8 (S683A and S683D) modules using SWISS-MODEL (Waterhouse et al., 2018). Figures of model structures and sequence alignments were generated by PyMoL software (Molecular Graphics System, V2.0, Schrödinger). CD320 LDLR1-2 module structures were extracted from the human transcobalamin and CD320 complex structure (PDB ID: 4ZRP) (Alam et al., 2016). The overall structure alignment was generated by PyMOL based on the Cα of all residues. The local alignment of CD320 LDLR2 and corin LDLR8 modules was generated by PyMOL based on the Cα of the DSSDE motif.

Statistical analysis

Data were analyzed with Prism six software (Graphpad). Student’s t test was used to analyze data from two groups and ANOVA followed by Tukey's multiple comparison test was used to analyze data from three or more groups. P values of < 0.05 were considered to be statistically significant.

Acknowledgements

We thank Lin Qi and Boxing Xue for their assistance in immunostaining studies. This work was supported in part by grants from the National Natural Science Foundation of China (81873840 and 81570457), and the Priority Academic Program Development of Jiangsu Higher Education (to Soochow University).

Funding Statement

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Contributor Information

Ningzheng Dong, Email: ningzhengdong@suda.edu.cn.

Qingyu Wu, Email: wuq@ccf.org.

Adam Linstedt, Carnegie Mellon University, United States.

Olga Boudker, Weill Cornell Medicine, United States.

Funding Information

This paper was supported by the following grants:

  • National Natural Science Foundation of China 81873840 to Ningzheng Dong.

  • National Natural Science Foundation of China 81570457 to Ningzheng Dong.

Additional information

Competing interests

No competing interests declared.

Author contributions

Conceptualization, Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing - original draft, Writing - review and editing.

Data curation, Investigation, Methodology, Writing - review and editing.

Data curation, Investigation, Methodology, Writing - review and editing.

Data curation, Formal analysis, Investigation, Visualization, Writing - review and editing.

Investigation, Methodology, Writing - review and editing.

Data curation, Investigation, Visualization, Writing - original draft, Writing - review and editing.

Supervision, Investigation, Methodology, Project administration, Writing - review and editing.

Resources, Data curation, Investigation, Visualization, Writing - review and editing.

Conceptualization, Resources, Formal analysis, Supervision, Funding acquisition, Validation, Writing - original draft, Project administration, Writing - review and editing.

Conceptualization, Formal analysis, Supervision, Investigation, Visualization, Writing - original draft, Writing - review and editing.

Ethics

Human subjects: The study involving human tissue samples was approved by the Ethics Committee of Soochow University (#2013052). Informed consents were obtained from the donors.

Additional files

Transparent reporting form

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files.

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Decision letter

Editor: Adam Linstedt1

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Acceptance summary:

A major pursuit in the past three decades of polarized membrane traffic research has been to identify apical sorting determinants, with the ultimate goal of identifying the "sorting" machinery that recognizes these motifs. This study defines an extracellular domain motif that specifies the accumulation of corin (a serine proteinase) and CD320 (receptor for B12 uptake) at the apical region of polarized MDCK cells. These two proteins contain conserved LDL receptor (LDLR) class A modules in their extracellular domains. Mutation or removal of a DSSDE motif in a subset of these LDLR modules causes a partial redistribution of the proteins to the basolateral membrane whereas transplant of this motif to unsorted forms of the proteins causes their apical redistribution. Other studies demonstrate a role of Rab11a in the preferential apical sorting of the proteins. Thus, the DSSDE motif represents a "regulatory element" that controls the expression of LDLR module-containing proteins at the apical pole of kidney cells.

Decision letter after peer review:

Thank you for submitting your article "A conserved LDL receptor motif regulates corin and CD320 membrane targeting in polarized renal epithelial cells" for consideration by eLife. Your article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Olga Boudker as the Senior Editor. The reviewers have opted to remain anonymous.

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

Summary:

The major contribution of this article is the potentially novel finding that the apical membrane proteins corin and CD320 contain a peptide-based, sorting determinant in their extracellular domain that specifies apical targeting in kidney cells. Extensive mutagenesis and MDCK expression experiments were carried out to characterize the DXSDE motif present in Corin, a transmembrane serine protease, and CD320, a receptor for Vitamin B12 uptake. Mutation or removal of this motif causes a partial redistribution of the proteins to the basolateral membrane whereas transplant of this motif to unsorted forms of the proteins causes their apical redistribution. Other studies demonstrate a role of Rab11a in the preferential apical sorting of the proteins. While we don't learn how the signal works- especially in relation to rab11a function- the finding is significant given its novelty and appearance in multiple proteins. However, the review has raised issues that must be satisfactorily addressed before the manuscript is considered acceptable for publication.

Essential revisions:

1) The apical sorting of the constructs is not entirely convincing. There are several issues. Foremost is the lack of quantification of the microscopy-based assay. The authors present single selected images, which is not satisfactory. Additionally, given the lack of resolution of the microscopy in the Z-axis, it is not entirely convincing that the signal being detected is surface-localized rather than, at least partly, intracellular. One reviewer states "the gold standard has been to use cell-surface biotinylation (or related techniques such as cell-surface immunoprecipitation). While transfection efficiency can affect one's ability to perform these studies, the standard for decades has been to make stable cell lines or use viral transduction approaches. These latter approaches could be done using a small subset of the mutants."

2) It is not convincing that the sorting involves direct apical delivery without endocytosis of a basolateral pool of protein. The possibility of endocytosis of a basolateral pool and either apical re-sorting or degradation was argued against in the Discussion. The basis was absence of intracellular detection of the cleaved form but this is not a direct endocytosis assay and controls and assay sensitivity are paramount. The extent of endocytosis should be addressed directly and convincingly.

3) All 3 reviewers had problems with the author's "stop sign" model of how the signal functions. Each expressed this in different ways and each is presented below in the hope that it helps the authors simplify and clarify a model for how the signal might work.

Reviewer 1 "The mechanistic basis for a signal acting as a "stop sign" eludes me. Perhaps this could be clarified. Is this different from a retrieval signal bring escaped protein back to the sorting station? Alternatively, the authors might entertain what I think of as a more conventional view in which the signal (via whatever it binds) is simply strongly positive for apical sorting. In its absence some protein escapes in the basolateral direction and some remains apical (either passively or by some other means)."

Reviewer 2 "The model put forward by the authors to explain their results (Figure 8D) is highly speculative. It suggests that the DxSDE motif functions between Common Recycling Endosomes (CRE) and Apical Recycling Endosomes (ARE), favoring the entrance of Corin into ARE and preventing its entrance into Basolateral Sorting Endosomes (BSE). This is a good possibility since ARE are known to be enriched in Rab11a whereas BSE do not express this protein. However, trafficking studies available to date have not implicated BSE in basolateral targeting so their participation in this route is speculative. Furthermore, whether Rab11a participates at the entrance or at the exit of ARE is not studied in this manuscript (there are no imaging studies showing transit of Corin through ARE). So the possibility exists that Rab11a plays an active role in targeting Corin from ARE to the apical membrane, rather than preventing its entrance into ARE. Whether the basolateral route is emerging before or from the ARE is not clear. Hence, the proposed model should be simpler and simply postulate the involvement of Rab11a positive ARE in promoting apical delivery of proteins displaying the DxSDE motif."

Reviewer 3 "Final paragraph of the Results and the start of the Discussion is confusing. The authors observe that upon expression of DN-RAB11A, a fraction of corin and CD320 are mis-directed to the basolateral surface. On one hand, the authors argue that RAB11A is not required for apical expression, but instead acts to prevent these proteins from entering the basolateral trafficking route. They further argue that the DSSDE motif is not an apical sorting determinant, but a stop signal to prevent entry in the basolateral trafficking route. The basis for this argument is unclear, as a protein lacking any sorting information would presumably be delivered in a non-polarized manner: 50% apical and 50% basolateral. Thus, in the absence of RAB11A or a functional DSSDE motif, the protein would assume a random distribution, which is what the authors observe. On the other hand, the authors then go on to say that RAB11A is involved in endocytic traffic and that apical traffic of these two proteins could be RAB11A and signal dependent. It seems that the authors want to rule out a positive role for RAB11A because they argue corin and CD320 are not recycling proteins. However, there is a substantial literature that indicates that a subset of newly synthesized apical and basolateral proteins transit endosomes (including RAB11A-positive ones) prior to surface delivery. This possibility should be discussed."

eLife. 2020 Nov 2;9:e56059. doi: 10.7554/eLife.56059.sa2

Author response

Essential revisions:

1) The apical sorting of the constructs is not entirely convincing. There are several issues. Foremost is the lack of quantification of the microscopy-based assay. The authors present single selected images, which is not satisfactory. Additionally, given the lack of resolution of the microscopy in the Z-axis, it is not entirely convincing that the signal being detected is surface-localized rather than, at least partly, intracellular. One reviewer states "the gold standard has been to use cell-surface biotinylation (or related techniques such as cell-surface immunoprecipitation). While transfection efficiency can affect one's ability to perform these studies, the standard for decades has been to make stable cell lines or use viral transduction approaches. These latter approaches could be done using a small subset of the mutants."

We appreciate the reviewers’ concerns. In our figures, each image is representative of the data from at least five experiments. To quantify the results, we analyzed fluorescent intensity along the X-Z axis and calculated the ratio of the intensity on the basolateral membrane vs. the total (apical and basolateral) membranes. The quantitative data, which are consistent with the image data, are presented in figure supplements of Figures 2-4, and 7-8. The method for fluorescent signal quantification has been added to the Materials and methods.

As the reviewers suggested, we also did the cell-surface biotin-labeling experiment. Indeed, MDCK cells had low transfection efficiency. We made stable cell lines expressing corin WT and a control mutant (S684A). We detected biotin-labeled corin WT among apical, but not basolateral, membrane proteins, whereas the S684A mutant was detected among apical and basolateral membrane proteins. Together with the data from the trypsin digestion experiment, our results do support the idea that corin is expressed on the apical membrane in MDCK cells. The results from the biotin-labeling experiment are presented in Figure 4—figure supplement 2. The methods are included in the Materials and methods.

2) It is not convincing that the sorting involves direct apical delivery without endocytosis of a basolateral pool of protein. The possibility of endocytosis of a basolateral pool and either apical re-sorting or degradation was argued against in the Discussion. The basis was absence of intracellular detection of the cleaved form but this is not a direct endocytosis assay and controls and assay sensitivity are paramount. The extent of endocytosis should be addressed directly and convincingly.

The reviewers’ point is well taken. Although we showed that the activated corin was not detected intracellularly and that, in the new biotin-labeling experiment, corin was not detected among basolateral membrane proteins, the possibility of endocytosis and recycling cannot be excluded at this time. We have revised the Discussion and pointed out that “Additional studies with more direct and sensitive assays are required to determine if corin and CD320 undergo endocytosis and recycling in MDCK cells”.

3) All 3 reviewers had problems with the author's "stop sign" model of how the signal functions. Each expressed this in different ways and each is presented below in the hope that it helps the authors simplify and clarify a model for how the signal might work.

Reviewer 1 "The mechanistic basis for a signal acting as a "stop sign" eludes me. Perhaps this could be clarified. Is this different from a retrieval signal bring escaped protein back to the sorting station? Alternatively, the authors might entertain what I think of as a more conventional view in which the signal (via whatever it binds) is simply strongly positive for apical sorting. In its absence some protein escapes in the basolateral direction and some remains apical (either passively or by some other means)."

Reviewer 2 "The model put forward by the authors to explain their results (Figure 8D) is highly speculative. It suggests that the DxSDE motif functions between Common Recycling Endosomes (CRE) and Apical Recycling Endosomes (ARE), favoring the entrance of Corin into ARE and preventing its entrance into Basolateral Sorting Endosomes (BSE). This is a good possibility since ARE are known to be enriched in Rab11a whereas BSE do not express this protein. However, trafficking studies available to date have not implicated BSE in basolateral targeting so their participation in this route is speculative. Furthermore, whether Rab11a participates at the entrance or at the exit of ARE is not studied in this manuscript (there are no imaging studies showing transit of Corin through ARE). So the possibility exists that Rab11a plays an active role in targeting Corin from ARE to the apical membrane, rather than preventing its entrance into ARE. Whether the basolateral route is emerging before or from the ARE is not clear. Hence, the proposed model should be simpler and simply postulate the involvement of Rab11a positive ARE in promoting apical delivery of proteins displaying the DxSDE motif."

Reviewer 3 "Final paragraph of the Results and the start of the Discussion is confusing. The authors observe that upon expression of DN-RAB11A, a fraction of corin and CD320 are mis-directed to the basolateral surface. On one hand, the authors argue that RAB11A is not required for apical expression, but instead acts to prevent these proteins from entering the basolateral trafficking route. They further argue that the DSSDE motif is not an apical sorting determinant, but a stop signal to prevent entry in the basolateral trafficking route. The basis for this argument is unclear, as a protein lacking any sorting information would presumably be delivered in a non-polarized manner: 50% apical and 50% basolateral. Thus, in the absence of RAB11A or a functional DSSDE motif, the protein would assume a random distribution, which is what the authors observe. On the other hand, the authors then go on to say that RAB11A is involved in endocytic traffic and that apical traffic of these two proteins could be RAB11A and signal dependent. It seems that the authors want to rule out a positive role for RAB11A because they argue corin and CD320 are not recycling proteins. However, there is a substantial literature that indicates that a subset of newly synthesized apical and basolateral proteins transit endosomes (including RAB11A-positive ones) prior to surface delivery. This possibility should be discussed."

We thank the reviewers for the suggestion. Indeed, our original ‘stop sign’ model and the discussion were speculative and unnecessarily complicated. As the reviewers suggested, we have simplified our model (new Figure 8D) and revised the Results and the Discussion. We have also pointed out that “Further studies are required to understand the molecular basis for the potential Rab11a-dependent recognition mechanism”.

Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    Figure 2—figure supplement 1—source data 1. Source data for Figure 2—figure supplement 1A.
    elife-56059-fig2-figsupp1-data1.xlsx (9.3KB, xlsx)
    Figure 2—figure supplement 1—source data 2. Source data for Figure 2—figure supplement 1B.
    elife-56059-fig2-figsupp1-data2.xlsx (9.8KB, xlsx)
    Figure 2—figure supplement 2—source data 1. Source data for Figure 2—figure supplement 2C.
    elife-56059-fig2-figsupp2-data1.xlsx (9.5KB, xlsx)
    Figure 3—figure supplement 1—source data 1. Source data for Figure 3—figure supplement 1C.
    elife-56059-fig3-figsupp1-data1.xlsx (9.5KB, xlsx)
    Figure 3—figure supplement 1—source data 2. Source data for Figure 3—figure supplement 1D.
    elife-56059-fig3-figsupp1-data2.xlsx (9.2KB, xlsx)
    Figure 4—figure supplement 1—source data 1. Source data for Figure 4—figure supplement 1B.
    elife-56059-fig4-figsupp1-data1.xlsx (9.3KB, xlsx)
    Figure 4—figure supplement 1—source data 2. Source data for Figure 4—figure supplement 1C.
    elife-56059-fig4-figsupp1-data2.xlsx (9.1KB, xlsx)
    Figure 7—figure supplement 1—source data 1. Source data for Figure 7—figure supplement 1A.
    elife-56059-fig7-figsupp1-data1.xlsx (9.3KB, xlsx)
    Figure 7—figure supplement 1—source data 2. Source data for Figure 7—figure supplement 1B.
    elife-56059-fig7-figsupp1-data2.xlsx (9.1KB, xlsx)
    Figure 8—source data 1. Source data for Figure 8B.
    elife-56059-fig8-data1.xlsx (9.1KB, xlsx)
    Figure 8—figure supplement 1—source data 1. Source data for Figure 8—figure supplement 1A.
    elife-56059-fig8-figsupp1-data1.xlsx (9.3KB, xlsx)
    Figure 8—figure supplement 1—source data 2. Source data for Figure 8—figure supplement 1C.
    elife-56059-fig8-figsupp1-data2.xlsx (9.3KB, xlsx)
    Figure 8—figure supplement 2—source data 1. Source data for Figure 8—figure supplement 2A.
    elife-56059-fig8-figsupp2-data1.xlsx (9KB, xlsx)
    Figure 8—figure supplement 2—source data 2. Source data for Figure 8—figure supplement 2D.
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    Transparent reporting form
    elife-56059-transrepform.docx (245.3KB, docx)

    Data Availability Statement

    All data generated or analysed during this study are included in the manuscript and supporting files.

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