The intracellular seven amino acid motif EEGEVFL is required for matriptase vesicle sorting and translocation to the basolateral plasma membrane.

Matriptase plays important roles in epithelial integrity and function, which depend on its sorting to the basolateral surface of cells, where matriptase zymogen is converted to an active enzyme in order to act on its substrates. After activation, matriptase undergoes HAI-1-mediated inhibition, internalization, transcytosis, and secretion from the apical surface into the lumen. Matriptase is a mosaic protein with several distinct protein domains and motifs, which are a reflection of matriptase's complex cellular itinerary, life cycle, and the tight control of its enzymatic activity. While the molecular determinants for various matriptase regulatory events have been identified, the motif(s) required for translocation of human matriptase to the basolateral plasma membrane is unknown. The motif previously identified in rat matriptase is not conserved between the rodent and the primate. We, here, revisit the question for human matriptase through the use of a fusion protein containing a green fluorescent protein linked to the matriptase N-terminal fragment ending at Gly-149. A conserved seven amino acid motif EEGEVFL, which is similar to the monoleucine C-terminal to an acidic cluster motif involved in the basolateral targeting for some growth factors, has been shown to be required for matriptase translocation to the basolateral plasma membrane of polarized MDCK cells. Furthermore, time-lapse video microscopy showed that the motif appears to be required for entry into the correct transport vesicles, by which matriptase can undergo rapid trafficking and translocate to the plasma membrane. Our study reveals that the EEGEVFL motif is necessary, but may not be sufficient, for matriptase basolateral membrane targeting and serves as the basis for further research on its pathophysiological roles.

Introduction

The type 2 transmembrane serine protease (TTSP) matriptase was initially identified as the major secreted gelatinolytic activity present in conditioned medium from breast cancer cells in an effort to identify and characterize cancer cell-derived extracellular matrix-degrading proteases involved in cancer invasion and metastasis [1,2]. The cloning of the matriptase cDNA and the deduced protein sequence showed that matriptase is a serine protease with trypsin-like activity rather than a matrix metalloprotease (MMPs) [3] but also revealed that matriptase is an integral membrane protein rather than a secreted protein [4,5]. Matriptase targeting to the basolateral plasma membrane was subsequently demonstrated in vitro using differentiated polarized epithelial cells and in vivo by staining for the protein in human kidney and prostate tissues [6-8]. The purification of activated matriptase in complexes with the Kunitz-type serine protease inhibitor HAI-1 from human breast milk revealed an important functional relationship between matriptase and HAI-1. Furthermore, the targeting of matriptase to the basolateral membrane, combined with its secretion from the apical plasma membrane of lactating mammary epithelial cells, implies a complex and somewhat convoluted cellular itinerary during its life cycle [9].

Like other serine proteases, matriptase is synthesized as a zymogen, which must be converted into an active form by cleavage at the activation motif in order to gain its full enzymatic activity [10]. Under physiological conditions, cell-associated active matriptase is a short-lived species due to its rapid inhibition by HAI-1 through the formation of a stable one-to-one complex [11]. A proportion of the active matriptase is, however, also rapidly shed from the surface of cells [12,13]. HAI-1 is also an integral membrane protein and so can be targeted to the basolateral plasma membrane of polarized epithelial cells [14,15]. Secretion or shedding of matriptase both in the zymogen form and the activated form in complex with HAI-1 from the basolateral plasma membrane has been observed in polarized Caco-2 cells [7], which is conceptually consistent with the expression of matriptase on the basolateral plasma membrane. Interestingly, only activated matriptase in complex with HAI-1 is secreted from the apical plasma membrane of polarized Caco-2, and not the zymogen form of the enzyme [7]. It remains unclear if matriptase can be secreted from the basolateral plasma membrane of cells in vivo. The presence of activated matriptase in complex with HAI-1 without measurable matriptase zymogen in human body fluids is consistent with the pattern of matriptase secretion observed in vitro in polarized Caco-2 cells [9]. Collectively these in vivo and in vitro studies illustrate several milestones throughout the matriptase lifespan: 1) synthesis as zymogen, 2) targeting to the basolateral plasma membrane, 3) conversion to an active enzyme and action on its substrates in the basolateral milieu, 4) enzymatic inhibition through the formation of a very stable complex with HAI-1 on the basolateral plasma membrane, 5) internalization of the activated matriptase-HAI-1 complex from the basolateral plasma membrane, 6) transcytosis to the apical face of the cell and 7) shedding from the apical plasma membrane into the lumen of the secretory glands as the activated matriptase-HAI-1 complex, which has been detected in body fluids.

Several molecular mechanisms underlying these milestones in the matriptase lifespan have been well characterized. For example, autoactivation has been identified as the primary mechanism for matriptase zymogen activation [16]. Furthermore, the selective secretion of activated matriptase-HAI-1 complex but not matriptase zymogen from the apical plasma membrane is likely due to the fact that HAI-1 but not matriptase can be internalized from the basolateral surface and undergoes transcytosis to the apical surface [8]. As a consequence, matriptase zymogen on the basolateral surface must be activated and in complex with HAI-1 for secretion from the apical plasma membrane. Thus, much of the regulation and physiological functions of matriptase must take place on the basolateral plasma membrane, the targeting to which, therefore, represents one of the most important physiological processes in the matriptase life cycle. Basolateral sorting in epithelial cells is mediated by cytoplasmic signals present on membrane proteins. At least three different types of basolateral sorting signal have been identified and characterized, including the tyrosine-based, dileucine, and monoleucine motifs [17]. A cytoplasmic juxtamembrane motif comprised of 6 amino acid residues (45-KQVEKR-50) in rat matriptase was reported to be important for matriptase basolateral sorting [18]. This motif was described by Murai et al., to resemble the sequence responsible for the basolateral sorting found for the rabbit polyimmunoglobulin receptor (pIgR). This sequence does not, however, contain tyrosine or leucine and so probably does not belong to one of the three well-characterized sorting signals. Furthermore, this sequence is not conserved between primate and rodent matriptase: the C-terminal Arg in rat and mouse is replaced by His in the human and chimpanzee proteins. While species variation could explain this difference, the high level of sequence conservation within the matriptase cytoplasmic domain among species suggests that basolateral sorting signals other than the one identified in rat matriptase could be present. In the current study, we revisit the important question as to the nature of the sorting requirement for directing human matriptase to the basolateral plasma membrane.

Materials and methods

Cell cultures

HEK293T, the large T expressing variant of the human embryonic kidney line HEK293 (ATCC), HaCaT human keratinocytes (CLS Cell Lines Service GmbH, Eppelheim Germany), and Madin-Darby Canine Kidney (MDCK) cells (ATCC) were cultured in Dulbecco's Modified Eagle Medium (DMEM), supplemented with 10% fetal bovine serum (FBS). The cells were incubated at 37°C in a humidified atmosphere with 5% CO2.

Generation of matriptase-EGFP fusion protein

A matriptase fusion protein comprised of 149 amino acids containing the cytoplasmic tail, transmembrane domain, and a partial SEA domain, fused with EGFP at the C terminus was prepared. DNA encoding the partial matriptase MTPN-EGFP sequence flanked by BamH1 and EcoR1 restriction sites were generated by PCR from pCDNA3.1-full-length matriptase [16] using a forward primer: 5’-GGTGAATTCATGGGGAGCGATCGGGC-3’; and a reverse primer: 5’-ACCGGATCCCGCCCTCGCTGAAGGCCGT-3’. The CD-Del-EGFP construct uses a different forward primer: 5’- GGTGAATTCATGGTGGTGCTGGCAGCCGTG-3’ and the same reverse primer as MTPN-EGFP. The PCR product was purified, double digested with EcoR1 and BamHI, and subcloned into the pEGFP-N2 construct (Clontech, Mountain View, CA). The construct was verified by Sanger sequencing and then transfected into HEK293T cells, and expression analyzed 24 hours post transfection by western blot. Other point and deletion mutant constructs were synthesized and subcloned into pEGFP-N1 by General Biosystems (Morrisville, North Carolina).

Immunoblot

HEK293T cells were lysed in 1% Triton X100 in phosphate-buffered saline containing 1mM 5,5’-Dithio-bis-(2-Nitrobenzoic Acid) (DTNB) to protect the disulfide bonds as previously described [19]. After removal of the insoluble fraction by centrifugation, the protein concentration was determined, and samples containing equal amounts proteins were separated by SDS-PAGE, transferred to nitrocellulose membrane, and probed with matriptase N-terminal monoclonal antibody PS6. The generation and characterization of the mAb PS6 have been described previously [13].

Immunofluorescence and live cell microscopy

MDCK cells were plated on coverslips and grown until they became polarized, after which they were transfected with the EGFP fusion construct using Lipofectamine 2000 (Thermo Fischer). The MDCK cells were prepared for staining by washing the coverslips with serum-free DMEM and phosphate-buffered saline (PBS) and then fixed in 10% buffered formalin (Fisher Scientific) for 20 min. The cells on some coverslips were then permeabilized using 0.5% TritonX100 in PBS for 5 min. For studies examining the co-localization of the matriptase-EGFP fusion protein and wild type matriptase, HaCaT cells were incubated with the matriptase mAb M24 at 2 μg/ml at room temperature for 60 min followed by staining with Alexa Fluor 594 goat anti-mouse IgG and for 60 min. ZO-1 was visualized with a ZO-1 antibody (R26.4C from the Developmental Studies Hybridoma Bank, DSHB). R26.4C was deposited to the DSHB by Goodenough, D.A. Phalloidin conjugated to Alexa Fluor 660 was used as a counterstain. Images were captured using a Leica TCS SP8 Laser Scanning Confocal microscope with a 63X oil lens. For live cell imaging, MDCK cells were plated on 30mm glass bottom No.1.0 uncoated culture dishes (MatTek Corp., MA).

Results

Matriptase is synthesized as a type 2 transmembrane protein, and the transmembrane domain is positioned between a short N-terminal cytoplasmic domain (54 amino acids) and a C-terminal extracellular protein domain known as a SEA domain [4,5]. Matriptase undergoes N-terminal processing by cleavage at Gly-149 within the SEA domain during synthesis and maturation in the ER/Golgi region prior to translocation to the plasma membrane. The matriptase N-terminal fragment is, therefore, comprised of a cytoplasmic domain, transmembrane domain, and half a SEA domain ending at Gly-149. The remaining matriptase extracellular domains, which represent the bulk of the protein and include the serine protease domain, are tethered to the plasma membrane via the non-covalent interactions within the cleaved SEA domain attached to the transmembrane domain. While the basolateral sorting motifs are likely located in the cytoplasmic domain, the transmembrane domain with some portion of the extracellular domains provides the structural basis for insertion of matriptase molecule into the lipid bilayer biomembrane. A green fluorescent reporter protein EGFP was engineered as a proxy for full-length matriptase. EGFP is attached to the carboxyl terminus of the matriptase N-terminal fragment spanning from Met-1 to Gly-149 (Fig 1A). This fusion protein is designated as MTPN-EGFP and can be readily observed as a band at the expected size of approximately 50-kDa detected by the matriptase N-terminal mAb PS6 (Fig 1B).

Fig 1

Schematic diagram and expression of the matriptase N-terminal fragment-green fluorescent protein (EGFP) fusion protein.

A. The matriptase N-terminal fragment containing the short cytoplasmic domain, transmembrane domain (aa 55–77), and half of the SEA domain (aa 86–149) was inserted into pEGFP expression vector via EcoR1 and BamH1 sites. B. This construct, named MTPN-EGFP, was transiently expressed in HEK293T cells. Lysates prepared from the HEK293T parental cells as a negative control (Control) and from MTPN-EGFP-expressing (MTPN-EGFP) HEK293T cells were analyzed by immunoblot analysis using the matriptase mAb PS6 to identify the matriptase N-terminal fragment-EGFP fusion protein. The fusion protein was identified as a band at the predicted molecular mass of approximately 50-kDa.

The cytoplasmic domain is required for matriptase targeting to the plasma membrane

When the MTPN-EGFP was expressed in the HaCaT human keratinocytes, the fusion protein was observed at the cellular periphery, primarily at the contact areas between neighboring cells (Fig 2A). A significant MTPN-EGFP signal was also observed as punctate staining throughout the cells, particularly focused in the perinuclear area. This punctate staining profile likely results from the proportion of the fusion protein in the intracellular synthetic and trafficking pools. Endogenous matriptase, as demonstrated by indirect immunofluorescence staining, resembled MTPN-EGFP with regard to its cellular distribution profile with signal observed both on the cell periphery and in the intracellular pools (Fig 2B). When these images were merged with staining for F-actin and DNA (nuclei) (Fig 2C), significant overlap between the endogenous matriptase staining and GFP signal from the fusion construct was observed. These data suggest that MTPN-EGFP behaves like endogenous matriptase concerning its translocation to the plasma membrane and accumulation at cell-cell junctions (Fig 2A, 2B and 2C, white arrows). While some intracellular MTPN-EGFP may also be co-localized with endogenous matriptase (Fig 2A, 2B and 2C, pink arrows), MTPN-EGFP was seen in granule/vesicle-like structures much larger than those observed for endogenous matriptase. These different intracellular localization patterns could mean that MTPN-EGFP traffics to the plasma membrane via different routes than endogenous matriptase. However, it may be that the exogenous fusion protein tends to accumulate in these compartments more than endogenous matriptase resulting in some secretory vesicles being very bright for MTPN-EGFP. Nevertheless, the matriptase N-terminal fragment, including the intracellular domain, transmembrane domain, and the N-terminal portion of the cleaved SEA domain, contains the structural requirements for cell surface and the cell-cell junction translocation. It is worth noting that the matriptase mAb M24 used for the detection of endogenous matriptase recognizes an epitope on the fourth LDL receptor class A domain, which is not part of the MTPN-EGFP construct.

Fig 2

Matriptase cytoplasmic domain is required for targeting to the cell periphery.

Both the wild-type (MTPN-EGFP) and cytoplasmic domain deleted (CD-Del-EGFP) matriptase-EGFP constructs were transiently expressed in HaCaT human keratinocytes. The subcellular localization of both MTPN-EGFP (A and C) and CD-Del-EGFP (D and F) were compared with the endogenous matriptase, which was analyzed by indirect immunofluorescent staining with the matriptase-specific mAb M24 (B and E) followed by Alexa 594-labeled anti-mouse IgG. The cells were also stained for F-actin using Alexa 660-labeled phalloidin (C and F, Cyan) and nuclei using DAPI (C and F, blue), as counterstains. The staining is presented as black and white images (A, B, D, and E) and as merged false-color images (C and F). The white arrowheads indicate cell periphery and red arrowheads indicate intracellular vesicles. Scale bar: 10 μm.

The importance of the cytoplasmic domain in membrane targeting, which is a prerequisite for basolateral sorting, was next examined by generating a construct in which the cytoplasmic domain (amino acids Gly2-Arg54) from the MTPN-EGFP had been removed (CD-Del-EGFP). It is worth noting that there is no canonical signal motif in matriptase and the N-terminal hydrophobic stretch is considered to function as the signal peptide and transmembrane domain. When transiently expressed in HaCaT cells, the CD-Del-EGFP was observed to accumulate in the nucleus and perinuclear area (Fig 2D). In cells expressing the CD-Del-EGFP, endogenous matriptase staining was unchanged with signal at the cell periphery (Fig 2E, arrows) and also scattered throughout the cells as a fine punctate staining pattern, similar to that in other neighboring cells not expressing CD-Del-EGFP. The lack of localization of the CD-Del-EGFP construct at the cell periphery (Fig 2D, 2E and 2F) suggests that the cytoplasmic domain is essential for plasma membrane targeting in spite of the presence of the transmembrane domain. The altered intracellular distribution profile of the CD-Del-EGFP construct, particularly the apparent nuclei localization after the removal of the cytoplasmic domain, suggests that the cytoplasmic domain is important for the sorting and entry of matriptase into the correct secretory vesicles en route to the plasma membrane. In short, we generated MTPN-EGFP, which resembles endogenous matriptase with respect to trafficking and translocation behavior, and demonstrated that the matriptase cytoplasmic domain is vital to appropriate trafficking and membrane targeting.

MDCK cells have been widely used to model the polarization of simple epithelial cells. When grown at confluency, these cells form a polarized monolayer with well-defined tight junctions and provide an ideal system to investigate the molecular determinants for polarized plasma membrane targeting and trafficking. We, therefore, next used these canine distal renal tubular epithelial cells as a polarized model to study the plasma membrane translocation and to help us identify the matriptase basolateral sorting determinants using MTPN-EGFP and its derivatives (Figs 3–6). When transiently expressed in MDCK cells, MTPN-EGFP was observed both on the cell periphery and inside the cells (Fig 3A) with the distribution profile almost exactly the same as that in HaCaT cells (Fig 2). The peripheral staining was further confirmed by its coincidence with ZO-1, a well-established tight junction marker (Fig 3A, 3B and 3C, as indicated by arrowheads). The loss of plasma membrane localization, the aberrant intracellular distribution, and nuclear localization observed in HaCaT cells for the CD-Del-EGFP construct were also observed in MDCK cells (Fig 3D, 3E and 3F). Collectively, these data confirm the role of matriptase cytoplasmic domain in the plasma membrane targeting and validate the MDCK system as a model to study matriptase basolateral targeting with the matriptase N-terminal fragment-EGFP fusion protein.

Fig 3

Exogenous expression and subcellular localization of MTPN-EGFP and CD-Del-EGFP in MDCK cells.

Both wild-type (MTPN-EGFP) and cytoplasmic domain deleted (CD-Del-EGFP) matriptase-EGFP constructs were transiently expressed in MDCK canine distal renal tubular epithelial cells. The subcellular localization of both MTPN-EGFP (A and C) and CD-Del-EGFP (D and F) were compared with the tight junction marker ZO-1, which was analyzed by indirect immunofluorescent staining using ZO-1 antibody, followed by Alexa 594-labeled anti-mouse IgG (B and E). The staining is presented as black and white images (A, B, D, and E) and merged false-color images (C and F). The arrowheads indicate the cell periphery. Scale bar: 20 μm.

Fig 6

Monoleucine sorting motif mutants lose subcellular polarity and are retained in the ER/Golgi secretory pathway.

Polarized MDCK cells transiently expressing the MTPN-EGFP, CD-Del-EGFP, and the monoleucine sorting motif mutants, the EE→AA+L→A and 33-39Del, as indicated, were cultured on glass-bottom dishes and imaged live at 37Co 5% CO2 by laser scanning confocal microscopy. Forty (40) seconds of footage are presented using temporal-color indexing. A vesicle containing MTPN-EGFP was observed fusing with the membrane as indicated by the trail of color transitioning from orange to white, as indicated by the two red arrowheads. Bar, 10 μm.

Monoleucine C-terminal to an acidic cluster is required for matriptase basolateral targeting

By comparing the sequence of the matriptase cytoplasmic domain with the known basolateral sorting signals, we noted that the six amino acid residues 33-EEGEVFL-39 are very similar to the basolateral sorting motif of the type monoleucine C-terminal to an acidic cluster, EEXXXL [20-22]. Furthermore, this seven amino acid matriptase motif is highly conserved across different species (Fig 4A, in red). We, therefore, set out to determine whether the EEGEVFL sequence might be required for matriptase basolateral sorting by examining the impact of alterations in the sequence on the subcellular and basolateral distribution (Figs 4 and 5). The seven amino acids were either deleted entirely from the MTPN-EGFP construct (33-39Del; NGL——-PVNN), or point mutations were made of the characteristic C-terminal Leu and the two N-terminal Glu residues either alone or combination (L→A; EE→AA; EE→AA + L→A). These four constructs were then expressed in polarized MDCK cells to see whether and how the modifications might perturb matriptase distribution and subcellular localization.

Fig 4

The seven amino acid residues between residues 33 and 39 are required for matriptase targeting to the cell periphery.

A. The amino acid sequence of the matriptase cytoplasmic domain between residues 1 and 50 were compared among different species, including human (Homo sapiens), common chimpanzee (Pan troglodytes), house mouse (Mus musculus), common rat (Rattus norvegicus), guinea pig (Cavia porcellus), and rabbit (Oryctolagus cuniculus). The seven amino acid residues EEGVEFL (33–39 in human matriptase) are highlighted in red. B. The four mutated matriptase-EGFP constructs, including 33-39Del, EE→AA, L→A, and EE→AA + L→A, as indicated, were transiently expressed in MDCK cells. The subcellular localization of these matriptase-EGFP fusion proteins was compared with that of the tight junction marker ZO-1, which was analyzed by indirect immunofluorescent staining using a ZO-1 antibody, followed by Alexa 594-labelled anti-mouse IgG. The staining is presented as black and white images (EGFP and ZO-1) and merged false-color images (Merge). The arrowheads indicate the cell periphery. Scale bar: 20 μm.

Fig 5

The motif consisting of residues 33–39 with monoleucine C-terminal to acidic cluster contributes to matriptase basolateral sorting.

The matriptase-EGFP constructs, including MTPN-EGFP, CD-Del-EGFP, 33-39Del, EE→AA, L→A, and EE→AA + L→A, as indicated, were transiently expressed in MDCK cells. The subcellular localization and X-Z distribution of these matriptase-EGFP fusion proteins were compared with that of the tight junction marker ZO-1, which was analyzed by indirect immunofluorescent staining using ZO-1 antibody, followed by Alexa 594-labeled anti-mouse IgG. The staining is presented as black and white images (EGFP and ZO-1) and merged false-color images (Merge). Scale bar: 10 μm.

The deletion of all seven amino acid residues apparently prevented the fusion protein from translocation to the cell periphery as the EGFP was concentrated in the middle of the cells in a finely punctate pattern (Fig 4B, 33-39Del). Similar GFP patterns were also observed for the EE→AA, the L→A, and the EE→AA + L→A mutants (Fig 4B). The lack of significant accumulation of these mutants at the cell periphery was further validated by an attempt to assess the co-localization of these mutants with the tight junction marker ZO-1 (Fig 4B, indicated by the arrowheads), while the presence of inconspicuous yet discernible green fluorescent signal patches was occasionally seen in a small portion of the cells. Thus, while these mutants appear to be synthesized and traffic through the secretory pathway due to their perinuclear accumulation, the lack of accumulation at the cell periphery suggests that these three characteristic amino acid residues play important roles in the sorting and trafficking of matriptase.

The XZ distribution of the MTPN-EGFP constructs and its mutants in polarized MDCK cells was next examined (Fig 5). The targeting of MTPN-EGFP to cell periphery, including the accumulation at the contacts between neighboring cells, on the XY axis (Fig 3) was further narrowed to the basal and lateral plasma membrane as the EGFP signal was seen primarily on the cell-substratum and cell-cell contacts beneath the apical marker ZO-1 (Fig 5, MTPN-EGFP). In contrast, the EGFP signal from the construct in which the cytoplasmic domain is deleted (CD-Del-EGFP) was homogeneously distributed throughout the cells and not present in the three subdomains of the plasma membrane (Fig 5, CD-Del-EGFP). These data confirm the role of the cytoplasmic domain in matriptase basolateral targeting. The deletion of the seven amino acid motif also prevented the EGFP signal from being targeted to the plasma membrane (Fig 5, 33-39DEL), further narrowing down the role of these the seven amino acids in the cell surface targeting and likely the basolateral plasma membrane targeting. It is worth noting that the deletion of the seven amino acid motif did not appear to affect the entrance of the 33-39Del construct into secretory vesicles as the EGFP signal was present in vesicles rather than homogenously distributed throughout the cells seen for the CD-Del-EGFP. The importance of the C-terminal leucine and the double glutamic acid cluster within the 7 amino acid motif was demonstrated by the significant EGFP signal observed on entire cell periphery, including the apical plasma membrane, for the constructs bearing these mutations alone or in combination (Fig 5, EE→AA, L→A, and EE→AA + L→A). In contrast to the deletion of the entire motif, mutation of these three amino acid residues did not appear to affect translocation to the cell periphery. Collectively, these data support the role of the seven amino acids motif with the characteristic leucine and glutamic acid cluster in matriptase basolateral sorting.

Basolateral targeting signals facilitate protein delivery by virtue of their interaction with sorting associated proteins, such as the Rab small GTPases. These interactions are important for the trafficking of vesicles targeting to specific subcellular locations. The role of the seven amino acid motif in matriptase basolateral sorting was further investigated by monitoring EGFP-containing trafficking vesicles using time-lapse live-cell imaging (S1 Video to S4 Video) and temporal-color codes in hyperstack projections (Fig 6). Hyperstack projection of temporal-color coding was used to project trafficking vesicles at various time points onto a single image, generated using the Image J script hosted at GitHub (Fiji, https://github.com/fiji/fiji/blob/master/plugins/Scripts/Image/Hyperstacks/Temporal-Color_Code.ijm). In temporal-color coding the location of a moving vesicle is indicated by different colors marking the track of the vesicle, whereas those that are not moving are represented only by the color of the last time point. MTPN-EGFP can be seen on the cell periphery and in distinct trafficking vesicles, which were moving dynamically, appearing, disappearing, and fusing with the plasma membrane (S1 Video). When viewed in the Hyperstacks, the plasma membrane-associated MTPN-EGFP largely remained still and was seen in the cell periphery in white (Fig 6A). The trafficking vesicles containing MTPN-EGFP were seen in different colors due to their dynamic movement. The fusion of an MTPN-EGFP-containing vesicle to the plasma membrane is illustrated by tracking a vesicle with rapidly changing color from orange to yellow and then to white as it approaches the cell periphery (Fig 6A’, MTPN-EGFP, between the two arrowheads). In contrast to the plasma membrane localization and the dynamic movement of distinct vesicles containing MTPN-EGFP, locations marked by the CD-Del-EGFP construct appeared stationary throughout the cells (S2 Video). Viewed as Hyperstacks, the lack of movement renders the CD-Del-EGFP white (Fig 6B and 6B’). The deletion of the entire motif (33-39Del) or the combined mutations of the characteristic Leu and glutamic acid cluster (EE→AA + L→A) apparently compromised the rapid movement of the trafficking vesicles (S3 and S4 Videos). These mutations also prevented the EGFP signal from accumulating at the cell periphery. Instead, the EGFP signal from the two mutants accumulated on the periphery of the nuclei. Viewed as Hyperstacks, the limited movement rendered both mutants in white (Fig 6C, 6C’, 6D and 6D’). This reduced movement demonstrates the importance of the motif and the three characteristic amino acid residues in matriptase sorting and suggests it likely lies in their roles in vesicle entry and trafficking.

Discussion

Analysis of the subcellular localization and trafficking of the matriptase N-terminal fragment fused with EGFP (and mutants of the construct) in HaCaT and polarized MDCK cells, allowed us to identify a protein motif within the cytoplasmic domain that appears to be required for matriptase basolateral targeting. The matriptase motif consists of 7 amino acid residues (33-EEGVEFL-39) and resembles the signal motifs recently discovered in amphiregulin (236-EERKKL-241) [20] and heparin binding EGF (197-EEKVKL-201) [22]. A similar but as yet unconfirmed basolateral sorting motif is also present in betacellulin (157-EEMETL-163) [21]. These sorting motifs feature a monoleucine C-terminus linked to an acidic cluster. There is one acidic residue in the motifs present in matriptase and betacellulin, compared to 3 and 2 basic residues present in amphiregulin and HB-EGF, respectively. Since the two lysine residues in the amphiregulin motif are not required for basolateral sorting, the charge and/or size of the amino acid residues outside of the characteristic leucine residue and the acidic cluster may not be important for basolateral sorting. Likewise, the four amino residues found between the acidic cluster and the leucine in the matriptase motif versus three residues in the three EGF ligands indicate some flexibility in the length of this region of the sorting motif.

The requirement for the 33-EEGVEFL-39 motif in basolateral sorting identified in the current study adds a new molecular determinant to the set of known physiological mechanisms governing the function and enzymatic activity of matriptase, which is expressed as a mosaic protein with several characteristic structural domains and has a life cycle that involves a complex subcellular journey. Targeting to the basolateral plasma membrane through this newly identified intracellular motif represents an early hallmark in matriptase life cycle. At the basolateral face of polarized epithelial cells, matriptase proteolytic activity can act on substrates of either epithelial or stroma origins. These putative substrates include stroma-derived hepatocyte growth factor (HGF) as well as protease activated receptor (PAR)-2 which is of epithelial cell origin [23,24]. Prior to translocation to the basolateral plasma membrane, matriptase undergoes an unusual post-translational modification, with N-terminal processing mediated by the autolytic cleavage of the SEA domain [13,25]. The key amino acid involved in this event has been identified as Gly-149. N-terminal processing is important and is required for matriptase zymogen activation at a later stage of its life cycle [16], but may not have a significant role in the basolateral sorting of the protein. This is implied by the fact that the MTPN-EGFP construct used in the current study contains the matriptase N-terminal fragment from Met-1 to Gly-149 and so only contains half of the SEA domain. Other important molecular determinants have been also identified and shown to be involved in the control of zymogen activation and/or directly linked to matriptase enzymatic activity. For example, His-656, Asp-711, and Ser-805 in the active site triad and Asp-799 in the substrate binding pocket are responsible for the potent trypsin-like activity of matriptase [3-5]. In additional to their role in the enzymatic activity of the mature enzyme, the three residues in the active site triad are also important for matriptase intrinsic zymogen activity, which is responsible for mediating matriptase autoactivation [16]. Cleavage of Arg-614 within the activation motif, and the Asp residues in the calcium cages of the four LDL receptor class A domains (D482, D519, D555, and D598) are also important for matriptase zymogen activation. Cleavage at Arg-186, which is temporally linked with matriptase zymogen activation and results in matriptase shedding from the plasma membrane, appears to be the starting point for the removal of matriptase from the basolateral surface. After this cleavage, the enzyme is either shed into the extracellular milieu from the basolateral surface, or is inhibited by HAI-1 through the formation of a very stable protease-inhibitor complex. The HAI-1-matriptase complex is subsequently internalized and undergoes transcytosis for secretion from the apical cell surface [13]. Understanding the role of these structural domains and these critical residues provides insight into the molecular mechanisms by which matriptase is regulated.

The peri-nuclear localization of CD-Del-EGFP construct and the EE→AA + L→A mutants might raise technical and mechanistic concerns regarding the cause of this localization. Although one could attribute the aberrant subcellular localization to potentially artefactual effects, the deletion and point mutations that abolish matriptase translocation to the plasma membrane or prevent the majority of the point mutated MTPN-GFP variant from translocating to the basolateral plasma membrane. This result supports the important role of the cytoplasmic tail in matriptase plasma membrane targeting and these seven amino acid residues in the basolateral targeting, regardless of the mechanism underlying the loss of plasma or basolateral membrane trafficking. Some GPI-anchored proteins, such as testisin and prostasin, contain no or a very short cytoplasmic tail and can undergo co- or post-translational modifications and subsequent translocation to the plasma membrane. Furthermore, these EGFP-containing proteins emit green fluorescent signal, and thus they do undergo protein synthesis and folding in at least a somewhat “normal” manner. The loss of plasma membrane translocation may, thus, result from preventing CD-Del-EGFP from entering either the endoplasmic reticulum (ER) or the secretory pathway. The former would mean that CD-Del-EGFP is synthesized in the cytosol rather than the ER, whereas the latter would suggest the important role of the intracellular tail in the cell surface translocation. The misrouted subcellular localization of CD-Del-EGFP has, nevertheless, its molecular and cellular basis. Similarly, the entrance into nuclei or the accumulation at the nuclear periphery for the EE→AA + L→A, the 33–39 DEL, and the EE→AA mutants, also cannot simply be interpreted as artefactual effects. The lack of the accumulation at the nuclear periphery and the nuclear localization for the L→A mutant supports the technical feasibility and soundness of the deletion and point mutations on the seven amino acid motif. The altered subcellular distribution and intracellular trafficking associated with the deletion and point mutations of the seven amino acid motif, therefore, demonstrate the important role of the motif in matriptase vesicle sorting and translocation to the basolateral plasma membrane.

In summary, a protein motif, which is conserved among different species and which contains seven amino acid residues (33-EEGVEFL-39) located within the cytoplasmic domain, was identified and shown to be required for matriptase basolateral targeting. This motif is primarily involved in the movement of matriptase-containing vesicles. The matriptase sorting motif resembles those motifs with a monoleucine C-terminus and an acidic cluster, which was initially identified in some members of the EGF ligand family. Our current study not only identifies an important molecular determinant governing an important physiological function of matriptase but also expands the role of the class of motif with a monoleucine C-terminus to an acidic cluster in basolateral targeting from EGF family ligands to a type 2 transmembrane serine protease.

(MTPN-EGFP): Movie time stamp beginning at 02:51.570 (frame 134), ending at 03:44.460 (frame 175).

(WMV)

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(CD-Del-EGFP): Movie time stamp beginning at 00:00.000 (frame 1), ending at 00:52.890 (frame 42).

(WMV)

Click here for additional data file.

(EE→AA+L→A): Movie time stamp beginning at 01:05.790 (frame 52), ending at 01:58.680 (frame 93).

(WMV)

Click here for additional data file.

(33-39Del): Movie time stamp beginning at 00:00.000 (frame 1), ending at 00:52.890 (frame 42).

(WMV)

Click here for additional data file.

17 Jul 2019

PONE-D-19-15523

Human matriptase is sorted to the basolateral plasma membrane via a motif with monoleucine C-terminal to an acidic cluster

PLOS ONE

Dear Dr. Chen-Yong Lin,

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PLOS ONE

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Reviewers' comments:

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Reviewer #1: Partly

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

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Reviewer #1: Yes

Reviewer #2: Yes

**********

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Reviewer #2: Yes

**********

5. Review Comments to the Author

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Reviewer #1: The aim of the study was to identify and characterize a cytoplasmic basolateral sorting signal in the transmembrane serine protease, matriptase. In fact, a basolateral sorting motif in the protein, which resembles the sequence responsible for basolateral sorting of the polymeric immunoglobulin receptor, has been identified in previous studies. However, the authors have decided to revisit the question since that signal does not contain canonical tyrosines or leucines, and is not conserved among species. Since the enzyme is an important one, and since polarized sorting could play an important role in its function in health and disease, the research aim is important and relevant.

Using site-directed mutagenesis and cell biological (mainly morphological)-based assays, the authors propose that a basolateral sorting motif that is based on leucine and an acidic cluster exists in the cytoplasmic domain of matriptase. Notably, all research was performed on a matriptase N-terminal fragment ending at amino acid Gly-149 fused at its extracellular C-terminus to eGFP.

Critiques:

I feel that the reported data do not firmly imply that the identified signal is indeed a basolateral sorting signal. Therefore, a significant amount of research has to be invested to address this point properly.

1). The importance of Figs 1 & 2 is to show that the ectopically expressed fusion protein localization is approximately normal. This was done by comparing its localization with respect to the endogenous protein (stained with the M24 antibody that recognizes the endogenous, but not the ectopically expressed protein) in Fig. 1, and by showing the preferential plasma membrane localization of the expressed protein with respect to ZO-1 staining, in Fig. 2. I think that the data in Fig.2 are not important for the studied question. In addition, deletion of cytoplasmic tails of transmembrane proteins typically have gross and often times artifactual effects on membrane trafficking of the mutated protein.

2). Regarding data presented in Fig. 4. The resolution of this image (as well as of other images) was typically low, posing a difficulty to understand the details that the authors have pointed out in the text. For instance, I did not get their interpretation that the regions marked with arrows and appear in green mark the cell periphery. The different mutants presented show ER, or possibly some Golgi-like staining. In fact, the subtitle given to the related Figure 6 may support this idea. But, since there was not an attempt to address this point, it is hard to say where these mutants are located within the cells. I would also suggest adding the MTPN-wt distribution to the composite to facilitate the comparison. Nonetheless, this is an important point because if indeed the protein is stuck in the early secretory pathway, then the basolateral sorting signal is not at all such a signal, but an ER or Golgi localization signal. Typically, inactivation of a basolateral sorting signal causes the mislocalization of a protein to the apical surface and has no significant impact on the exit of the mutated protein from the ER or Golgi.

3). As per the experiments described in Fig. 5, which assessed the effects of the mutated residues on the apical and basal localization of the protein in polarized MDCK cells. To verify that the mutated versions of the protein are indeed mislocated to the apical surface, the authors have to perform genuine surface stainings of the protein, using at least one of the two indicated approaches; a) performance of surface labeling of the protein using antibodies directed against the ectodomain of the protein, and under conditions whereby the cells are not permeabilized. In this strategy, providing confocal x-z images is not sufficient, and quantitative analysis of the Ap vs. Bl distribution of the protein should be provided. b) performance of cell-surface biotinylation/biochemical based assay in which cell surface proteins are biotinylated and pulled down and the protein of interest is detected by Western blotting. These experiments are fundamental for the proper addressing the effects of the mutations on the polarized distribution of matriptase.

4). A prominent hallmark of basolateral sorting signals is that they can act in a dominant and autonomous manner. Thus, it would be essential to show that the signal identified in this study can confer basolateral sorting of a heterologous protein that is intrinsically sorted to the apical surface, and that the mutations abrogate this process.

5). I found the paper difficult to read, containing unexplained jargon and abbreviations, s as well as long and unclear sentences (e.g., in page 11 the sentence starting with “ The lack of significant…”, or “The targeting of MTPN-EGFP to cell periphery…”).

Reviewer #2: Human matriptase is sorted to the basolateral plasma membrane via a motif with

monoleucine C-terminal to an acidic cluster

In this manuscript, Tseng CC et al addressed the question of the basolateral sorting motif for human matriptase. They identified a monoleucine C-terminal to an acidic cluster in the amino terminus of matriptase to determine matriptase sorting to the basolateral plasma membrane. The authors used a fusion protein containing a green fluorescent protein (EGFP) reporter to conjugate with the ending residue G149 in the amino terminus of matriptase. By using this fusion reporter protein, they reveal the basolateral sorting motif for matriptase. However, several issues are needed to be clarified.

1. The red and green fluorescence are not well shown in the individual images of Figure 2, 3, 4B and 5. The color images should be improved. In Figure 4B, the arrows are faint and need to be redrew.

2. In the panel D, E and F of Figure 2B, the higher expression levels of CD-Del-EGFP look like to have a less protein level of endogenous matriptase. Is it possible that CD-Del-GFP can decrease the endogenous level of matriptase?

3. In Figure 4B and 6C, there are more nuclear membrane localization for the EE→AA + L�A mutant of MTPN-EGFP than MTPN-EGFP and the other mutants. Why would this happen? Please provide a discussion to explain this phenomenon.

4. There are two grammar errors in the line 1 of the abstract and the line 6 of page 20.

**********

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Reviewer #1: No

Reviewer #2: No

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Submitted filename: PLOS ONE D-19-15523.pdf

Click here for additional data file.

14 Nov 2019

Reviewer #1:

Critiques:

I feel that the reported data do not firmly imply that the identified signal is indeed a basolateral sorting signal. Therefore, a significant amount of research has to be invested to address this point properly.

Response:

We thank Reviewer 1 for their time and constructive comments. We agree that the data at hand do not support the assertion that the motif identified in this manuscript serves as a bone fide basolateral sorting signal. To address this concern, we have modified our language to state that the characterized seven amino acid sequence represents a motif required for matriptase translocation to the basolateral plasma membrane by entrance to the proper transport vesicles. As such, significant revisions have been made and we now present the work with new title: “The intracellular seven amino acid motif EEGEVFL is required for matriptase vesicle sorting and translocation to the basolateral plasma membrane” We believe that our data sufficiently support our revised assertion.

1). The importance of Figs 1 & 2 is to show that the ectopically expressed fusion protein localization is approximately normal. This was done by comparing its localization with respect to the endogenous protein (stained with the M24 antibody that recognizes the endogenous, but not the ectopically expressed protein) in Fig. 1, and by showing the preferential plasma membrane localization of the expressed protein with respect to ZO-1 staining, in Fig. 2. I think that the data in Fig.2 are not important for the studied question. In addition, deletion of cytoplasmic tails of transmembrane proteins typically have gross and often times artifactual effects on membrane trafficking of the mutated protein.

Response: Although we agree with Reviewer #1 regarding the potential artefactual effects produced by the deletion of cytoplasmic tails on membrane trafficking, the deletion did abolish matriptase translocation to the plasma membrane. This result supports the important role of the cytoplasmic tail in matriptase plasma membrane targeting, regardless of the mechanism underlying the loss of membrane trafficking. Some GPI-anchored proteins contain no or very short cytoplasmic tails and can undergo co- or post-translational modifications and subsequent translocation to the plasma membrane and the apical surface. Furthermore, CD-Del-EGFP emits green fluorescent signal, suggesting that the CD-Del-EGFP construct is synthesized and does undergo somewhat normal protein folding. The loss of plasma membrane translocation may, therefore, result from preventing CD-Del-EGFP from entering either the endoplasmic reticulum (ER) or the secretory pathway. The former would mean that CD-Del-EGFP is synthesized in the cytosol rather than the ER; the latter would suggest the important role of the intracellular tail in the cell surface translocation. We, therefore, respectfully contend that while the loss of membrane targeting could result from an artefactual effect of deletion of the cytoplasmic tail, the fact that it is lost in this construct is, in fact, informative.

We have added a paragraph in the Discussion to address this concern, as follows:

“The peri-nuclear localization of CD-Del-EGFP construct and the EE→AA + L→A mutants might raise technical and mechanistic concerns regarding the cause of this localization. Although one could attribute the aberrant subcellular localization to potentially artefactual effects, the deletion and point mutations that abolish matriptase translocation to the plasma membrane or prevent the majority of the point mutated MTPN-GFP variant from translocating to the basolateral plasma membrane. This result supports the important role of the cytoplasmic tail in matriptase plasma membrane targeting and these seven amino acid residues in the basolateral targeting, regardless of the mechanism underlying the loss of plasma or basolateral membrane trafficking. Some GPI-anchored proteins, such as testisin and prostasin, contain no or a very short cytoplasmic tail and can undergo co- or post-translational modifications and subsequent translocation to the plasma membrane. Furthermore, these EGFP-containing proteins emit green fluorescent signal, and thus they do undergo protein synthesis and folding in at least a somewhat “normal” manner. The loss of plasma membrane translocation may, thus, result from preventing CD-Del-EGFP from entering either the endoplasmic reticulum (ER) or the secretory pathway. The former would mean that CD-Del-EGFP is synthesized in the cytosol rather than the ER, whereas the latter would suggest the important role of the intracellular tail in the cell surface translocation. The misrouted subcellular localization of CD-Del-EGFP has, nevertheless, its molecular and cellular basis. Similarly, the entrance into nuclei or the accumulation at the nuclear periphery for the EE→AA + L→A, the 33-39 DEL, and the EE→AA mutants, also cannot simply be interpreted as artefactual effects. The lack of the accumulation at the nuclear periphery and the nuclear localization for the L→A mutant supports the technical feasibility and soundness of the deletion and point mutations on the seven amino acid motif. The altered subcellular distribution and intracellular trafficking associated with the deletion and point mutations of the seven amino acid motif, therefore, demonstrate the important role of the motif in matriptase vesicle sorting and translocation to the basolateral plasma membrane.”

2). Regarding data presented in Fig. 4. The resolution of this image (as well as of other images) was typically low, posing a difficulty to understand the details that the authors have pointed out in the text. For instance, I did not get their interpretation that the regions marked with arrows and appear in green mark the cell periphery. The different mutants presented show ER, or possibly some Golgi-like staining. In fact, the subtitle given to the related Figure 6 may support this idea. But, since there was not an attempt to address this point, it is hard to say where these mutants are located within the cells. I would also suggest adding the MTPN-wt distribution to the composite to facilitate the comparison. Nonetheless, this is an important point because if indeed the protein is stuck in the early secretory pathway, then the basolateral sorting signal is not at all such a signal, but an ER or Golgi localization signal. Typically, inactivation of a basolateral sorting signal causes the mislocalization of a protein to the apical surface and has no significant impact on the exit of the mutated protein from the ER or Golgi.

Response: Regarding the quality of pictures, the original Figures in Tiff file with high resolutions can be found by clicking the upper right corner of the Figure pages in the PDF file. We worry that the reviewer may have only made reference to the compressed image files present in the PDF. The deletion of all seven amino acid apparently prevented the fusion protein from translocation to the cell periphery as the EGFP was concentrated in the middle of the cells in a finely punctate pattern (Fig. 4B, 33-39Del). Similar staining patterns were also observed for the EE→AA, the L→A, and the EE→AA + L→A mutants (Fig. 4B). The lack of significant accumulation of these mutants on the cell periphery was further validated by an attempt to assess the co-localization of these mutants with the tight junction marker ZO-1 (Fig. 4B, indicated by the arrowheads), while the presence of inconspicuous yet discernible green fluorescent signal patches was occasionally seen in a small portion of the cells. Thus, while these mutants appear to be synthesized and traffic through the secretory pathway due to their perinuclear accumulation, the lack of accumulation on cell periphery suggests that these three characteristic amino acid residues play important roles in sorting and trafficking of matriptase.

3). As per the experiments described in Fig. 5, which assessed the effects of the mutated residues on the apical and basal localization of the protein in polarized MDCK cells. To verify that the mutated versions of the protein are indeed mislocated to the apical surface, the authors have to perform genuine surface stainings of the protein, using at least one of the two indicated approaches; a) performance of surface labeling of the protein using antibodies directed against the ectodomain of the protein, and under conditions whereby the cells are not permeabilized. In this strategy, providing confocal x-z images is not sufficient, and quantitative analysis of the Ap vs. Bl distribution of the protein should be provided. b) performance of cell-surface biotinylation/biochemical based assay in which cell surface proteins are biotinylated and pulled down and the protein of interest is detected by Western blotting. These experiments are fundamental for the proper addressing the effects of the mutations on the polarized distribution of matriptase.

Response: We agree with Reviewer #1 that the combination of the qualitative confocal x-z images and the quantitative analysis by surface biotinylation has been widely applied to study the apical and basolateral distribution. While the surface biotinylation approach is useful for the study of endogenous proteins, some inherent challenges present themselves when surface biotinylation is used to study the subcellular distribution of exogenously expressed proteins. When expression levels are high, the subcellular distribution of the protein of interest would be distorted to some degree. This is problematic because it is a challenge to ensure that similar exogenous protein expression levels are achieved in all cells in the experimental model. This distribution of expression levels in the cells in the system is not a problem for microscopy-based analysis, because one can simply avoid the cells that are expressing very high levels of the protein. For biochemical analyses such as cell-surface biotinylation, all cells in the culture are analyzed irrespective of their expression level, which makes interpretation of the data much more challenging as subcellular localization aberrations, present in the cells expressing supraphysiological levels of the constructs, contaminate the samples generated. We, therefore, respectfully contend that the analysis of representative x-z images from cells expressing lower levels of the constructs is the optimal approach to accurately present our results.

4). A prominent hallmark of basolateral sorting signals is that they can act in a dominant and autonomous manner. Thus, it would be essential to show that the signal identified in this study can confer basolateral sorting of a heterologous protein that is intrinsically sorted to the apical surface, and that the mutations abrogate this process.

Response: We agree with Review #1 for the requirement for the demonstration of true autonomous basolateral sorting signals. As such, we have revised our conclusion that the seven amino acid residues identified in our study are required but not sufficient for matriptase basolateral translocation via its role in vesicle sorting.

5). I found the paper difficult to read, containing unexplained jargon and abbreviations, s as well as long and unclear sentences (e.g., in page 11 the sentence starting with “ The lack of significant…”, or “The targeting of MTPN-EGFP to cell periphery…”).

Response: We apologize for the level of unexplained jargon, abbreviations, and run-on sentences. We have gone through the manuscript in an attempt to address these important concerns.

Reviewer #2: Human matriptase is sorted to the basolateral plasma membrane via a motif with

monoleucine C-terminal to an acidic cluster

In this manuscript, Tseng CC et al addressed the question of the basolateral sorting motif for human matriptase. They identified a monoleucine C-terminal to an acidic cluster in the amino terminus of matriptase to determine matriptase sorting to the basolateral plasma membrane. The authors used a fusion protein containing a green fluorescent protein (EGFP) reporter to conjugate with the ending residue G149 in the amino terminus of matriptase. By using this fusion reporter protein, they reveal the basolateral sorting motif for matriptase. However, several issues are needed to be clarified.

1. The red and green fluorescence are not well shown in the individual images of Figure 2, 3, 4B and 5. The color images should be improved. In Figure 4B, the arrows are faint and need to be redrew.

Response: We prepared our immunofluorescent staining image of proteins in these four figures in black and white manner for the highest contrast with the exception of when pseudocolor copies of the images were merged for assessment of co-localization of the proteins to be compared. We prepare our Figures at high resolution and quality, however, the images presented in the merged PDF file are compressed and so of much lower quality. The high-quality original images can be retrieved and downloaded by clicking on the upper right corner of each figure in the PDF file. We worry that the reviewer was not aware of this arrangement and so had not seen the original high-resolution images.

2. In the panel D, E and F of Figure 2B, the higher expression levels of CD-Del-EGFP look like to have a less protein level of endogenous matriptase. Is it possible that CD-Del-GFP can decrease the endogenous level of matriptase?

Response: It is difficult to determine whether the exogenous expression of CD-Del-GFP could affect the expression of endogenous matriptase. While stronger endogenous matriptase staining appears in the two cells right above the two CD-Del-GFP expressing cells, there seems no noticeable difference in matriptase staining in the cells at the low-left corner when compared to the two CD-Del-GFP expressing cells.

3. In Figure 4B and 6C, there are more nuclear membrane localization for the EE→AA + L�A mutant of MTPN-EGFP than MTPN-EGFP and the other mutants. Why would this happen? Please provide a discussion to explain this phenomenon.

Response: We thank the reviewer for this comment and have added a paragraph to the Discussion to address this concern, as follows:

“The peri-nuclear localization of CD-Del-EGFP construct and the EE→AA + L→A mutants might raise technical and mechanistic concerns regarding the cause of this localization. Although one could attribute the aberrant subcellular localization to potentially artefactual effects, the deletion and point mutations that abolish matriptase translocation to the plasma membrane or prevent the majority of the point mutated MTPN-GFP variant from translocating to the basolateral plasma membrane. This result supports the important role of the cytoplasmic tail in matriptase plasma membrane targeting and these seven amino acid residues in the basolateral targeting, regardless of the mechanism underlying the loss of plasma or basolateral membrane trafficking. Some GPI-anchored proteins, such as testisin and prostasin, contain no or a very short cytoplasmic tail and can undergo co- or post-translational modifications and subsequent translocation to the plasma membrane. Furthermore, these EGFP-containing proteins emit green fluorescent signal, and thus they do undergo protein synthesis and folding in at least a somewhat “normal” manner. The loss of plasma membrane translocation may, thus, result from preventing CD-Del-EGFP from entering either the endoplasmic reticulum (ER) or the secretory pathway. The former would mean that CD-Del-EGFP is synthesized in the cytosol rather than the ER, whereas the latter would suggest the important role of the intracellular tail in the cell surface translocation. The misrouted subcellular localization of CD-Del-EGFP has, nevertheless, its molecular and cellular basis. Similarly, the entrance into nuclei or the accumulation at the nuclear periphery for the EE→AA + L→A, the 33-39 DEL, and the EE→AA mutants, also cannot simply be interpreted as artefactual effects. The lack of the accumulation at the nuclear periphery and the nuclear localization for the L→A mutant supports the technical feasibility and soundness of the deletion and point mutations on the seven amino acid motif. The altered subcellular distribution and intracellular trafficking associated with the deletion and point mutations of the seven amino acid motif, therefore, demonstrate the important role of the motif in matriptase vesicle sorting and translocation to the basolateral plasma membrane.”

4. There are two grammar errors in the line 1 of the abstract and the line 6 of page 20.

Response: We apologize for these errors, which are corrected in the revised manuscript.

Submitted filename: Rebuttal letter 3.docx

Click here for additional data file.

2 Jan 2020

PONE-D-19-15523R1

The intracellular seven amino acid motif EEGEVFL is required for matriptase vesicle sorting and translocation to the basolateral plasma membrane

PLOS ONE

Dear Dr. Chen-Yong Lin,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

We would appreciate receiving your revised manuscript by January 31, 2020. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Qing-Xiang Amy Sang, Ph.D.

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors should have stated the "not sufficient" statement in the manuscript and not only in the rebuttal letter. For instance, in the Abstract it should have been stated: " Our study reveals that (the word "that" was missing there) the EEGEVFL motif is necessary, but may not be sufficient, for matriptase basolateral membrane targeting....".

Reviewer #2: This is a revised manuscript. However, some points in the revised manuscript are not well addressed.

1. The real colors of the fluorescence images (EGFP, M24 and ZO-1) in Figure 2, 3, 4 and 5 were not individually shown in the revised manuscript. The image qualities in the revised manuscript are not improved.

2. In Figure 4B, some of the EGFP images were extended out of the ZO-1-stained borders. What reasons cause the phenomena? Whether those matriptase-EGFP fusion proteins will affect the formation of tight junctions?

3. Matriptase has been shown to be mainly located in the adherin junction region of polarized MDCK cells. However, MTPN-EGFP proteins looked like to be mainly co-localized with ZO-1 at the apical surface in Figure 3 and 5 of the current manuscript. It raises a question whether MTPN-EGFP can behave like a real matriptase for the analysis of the protease transcytosis. The authors should explain why MTPN-EGFP proteins did not well exhibit the real localization of matriptase in the differentiated epithelial cells?

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

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3 Jan 2020

Reviewer #1:

Critiques:

Reviewer #1: The authors should have stated the "not sufficient" statement in the manuscript and not only in the rebuttal letter. For instance, in the Abstract it should have been stated: " Our study reveals that (the word "that" was missing there) the EEGEVFL motif is necessary, but may not be sufficient, for matriptase basolateral membrane targeting....".

Response: We agree with Reviewer #1 that although our data only support a required role of the seven amino acid motif in matriptase basolateral targeting. We have added the statement of “but may not be sufficient” in the abstract.

Reviewer #2:

This is a revised manuscript. However, some points in the revised manuscript are not well addressed.

1. The real colors of the fluorescence images (EGFP, M24 and ZO-1) in Figure 2, 3, 4 and 5 were not individually shown in the revised manuscript. The image qualities in the revised manuscript are not improved.

Response: We do not understand what Reviewer #2 means by “real colors”. When a digital imaging system is used as herein, the images are acquired in a numerical format as intensity per pixel. Thus, the most typical representation of the data converts intensity into grayscale. “False colors” can then assigned to the black-and-white digital images by computer software. For data presentation, grayscale images give the best representation of contrast and details. When colocalizations studies are conducted for more than one species, false color representations are useful for comparisons. We respectfully contend that the individual images with false color applied will not provide better quality than the ones in black-and-white.

2. In Figure 4B, some of the EGFP images were extended out of the ZO-1-stained borders. What reasons cause the phenomena? Whether those matriptase-EGFP fusion proteins will affect the formation of tight junctions?

Response: It is not uncommon to observe basolateral membrane ruffles in polarized cells. The ZO-1 staining defines the tight junction, however, it is not confined to the border of the cell. It has been documented that the basolateral “body” can be larger than the area defined by the tight junctions. https://www.researchgate.net/figure/Z-stack-image-analysis-of-representative-bestrophin-1-mutants-in-MDCK-II-cells-A_fig4_51606308

In addition, in Figure 5, ZO-1 was detected at the tight junction and we came to the conclusion that matriptase-EGFP does not alter tight junction formation.

3. Matriptase has been shown to be mainly located in the adherin junction region of polarized MDCK cells. However, MTPN-EGFP proteins looked like to be mainly co-localized with ZO-1 at the apical surface in Figure 3 and 5 of the current manuscript. It raises a question whether MTPN-EGFP can behave like a real matriptase for the analysis of the protease transcytosis. The authors should explain why MTPN-EGFP proteins did not well exhibit the real localization of matriptase in the differentiated epithelial cells?

Response:

ZO-1 is a well-established tight junction marker, which was used to show the coincidence of matriptase with ZO-1 on the surface of MDCK cells in Figure 3. Our statement regarding this observation is “The peripheral staining was further confirmed by its coincidence with ZO-1, a well-established tight junction marker (Fig. 3 A, B and C, as indicated by arrowheads).” We carefully used “coincidence” rather than “co-localization” to avoid some confusions. We never stated that “MTPN-EGFP proteins looked like to be mainly co-localized with ZO-1 at the apical surface in Figure 3 and 5 of the current manuscript.” Tight junctions are not on the apical surface. It is factually incorrect for Reviewer #2 to say that ZO-1 and matriptase are colocalized on the apical surface. TMPRSS2 is on the apical surface.

The comment regarding Figure 5 further demonstrate Reviewer #2’s confusion. Should two signal occupy the same position in the XY plane, but be located at a different depth, a sample imaged at the XY orientation may show the two signals colocalizing, depending on the depth of the field (as seen in Figure 3). However, if a sample is imaged and represented in the ZY, or the ZX orientation, it would reveal that the two signals do not in fact colocalize (as seen in Figure 5). Our data showed MTPN-EGFP coincides with ZO-1 in Figure 3, because it is a micrograph imaged at the XY orientation. To further dissect the subcellular location of MTPN-EGFP, we imaged the cells at the XZ orientation, thus revealing that MTPN-EGFP is located at the basolateral surface. Our data characterizes matriptase localization at a higher resolution.

In a previous study [1], we demonstrated that endogenous matriptase was detected at the adherens junctions in 184 A1N4 human mammary epithelial cells. In another previous study [2], we showed that endogenous matriptase was detected on the basolateral plasma membrane in differentiated Caco-2 human colon adenocarcinoma cells. Our matriptase mAbs do not cross-react with canine matriptase, and to the best of our knowledge, there have been no reports that commercially available matriptase antibodies can detect canine matriptase. We, therefore, believe that Reviewer #2 must simply be mistaken in their assertion that matriptase has been shown to be mainly located in the adherins junctions of polarized MDCK cells, as we are unaware of any such study, published or presented in a scientific meeting. If by “real” matriptase, Reviewer #2 means “endogenous” matriptase in Caco-2 cells rather than in MDCK cells, the data and conclusions of our current study using MDCK cells with an engineered matriptase fusion protein are entirely consistent with our published data studying endogenous matriptase in Caco-2 cells [2]. The coincidence of endogenous matriptase and ZO-1 in the XY orientation and the differential localization of endogenous matriptase in the basolateral surface versus ZO-1 in the tight junction in ZY orientation in Caco-2 human colon adenocarcinoma cells can be found in our previous study, which is also presented here .

Based on these data we believe that the engineered model system we have used in this study replicates the behavior of “real” matriptase with great fidelity.

References

1. Hung RJ, Hsu I, Dreiling JL, Lee MJ, Williams CA, Oberst MD, Dickson RB, Lin CY (2004) Assembly of adherens junctions is required for sphingosine 1-phosphate-induced matriptase accumulation and activation at mammary epithelial cell-cell contacts. Am J Physiol Cell Physiol 286: C1159-C1169.

2. Wang JK, Lee MS, Tseng IC, Chou FP, Chen YW, Fulton A, Lee HS, Chen CJ, Johnson MD, Lin CY (2009) Polarized epithelial cells secrete matriptase as a consequence of zymogen activation and HAI-1-mediated inhibition. Am J Physiol Cell Physiol 297: C459-C470.

Submitted filename: Rebuttal letter 3.docx

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27 Jan 2020

The intracellular seven amino acid motif EEGEVFL is required for matriptase vesicle sorting and translocation to the basolateral plasma membrane

PONE-D-19-15523R2

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29 Jan 2020

PONE-D-19-15523R2

The intracellular seven amino acid motif EEGEVFL is required for matriptase vesicle sorting and translocation to the basolateral plasma membrane

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