binding of magp2 to microfibrils is regulated by proprotein convertase cleavage

Matrix Biology 40 (2014) 27–33 Contents lists available at ScienceDirect Matrix Biology journal homepage: www.elsevier.com/locate/matbio Brief report Binding of MAGP2 to microfibrils is regulated by proprotein convertase cleavage Alison Miyamoto ⁎, Lauren J Donovan, Edgar Perez, Breanna Connett, Richard Cervantes, Khang Lai, Gordon Withers, Gregory Hogrebe a b Department of Biological Science, California State University, Fullerton, Fullerton, CA 92831, United States Center for Applied Biotechnology Studies, California State University, Fullerton, Fullerton, CA 92831, United States a r t i c l e i n f o Article history: Received 10 June 2014 Received in revised form August 2014 Accepted August 2014 Available online 19 August 2014 Keywords: MAGP2 MFAP5 Microfibril Fibrillin Proprotein convertase a b s t r a c t MAGP2 is a small extracellular protein with both tumor angiogenesis and cell signaling activity MAGP2 was originally isolated biochemically from microfibril-rich connective tissue The localization of MAGP2 to microfibrils has been confirmed by both immunohistochemistry and immunogold electron microscopy Whether MAGP2 binding to microfibrils is regulated post-translationally is still unclear, however, and a better understanding of this process would be instructive to understanding the angiogenesis and signaling functions ascribed to MAGP2 Here we show via immunofluorescence studies that the T3 cell line, derived from ovarian mouse tumor cells, produces abundant fibrillin-2 microfibrils to which MAGP2 can bind Co-localization of MAGP2 and fibrillin-2 can be detected either when MAGP2 is overexpressed in, or exogenously introduced to, the cells As expected, matrix association of MAGP2 required its conserved Matrix Binding Domain Matrix association was positively regulated by proprotein convertase (PC) cleavage of MAGP2; mutation of the MAGP2 PC consensus site reduced the amount of matrix-associated MAGP2 Deletion analysis of the C-terminal 20-amino acid domain that is defined by the PC cleavage site suggests that this domain also positively modulates matrix localization of MAGP2, in a manner that requires the amino-terminal half of the protein Together, our data indicate that matrix localization of MAGP2 by its Matrix Binding Domain is promoted by PC cleavage and the presence of its C-terminal 20 amino acids © 2014 Published by Elsevier B.V This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/) Introduction Both cell-associated and matrix-associated functions have been ascribed to microfibril-associated glycoprotein (MAGP2, official nomenclature MFAP5), a small secreted protein originally identified biochemically from microfibril-rich cartilage tissue (Gibson et al., 1996) In vitro, MAGP2 has been implicated in binding and activation of αvβ3 integrins (Gibson et al., 1999; Mok et al., 2009) and modulation of Notch signaling (Miyamoto et al., 2006; Albig et al., 2008) Although loss of MAGP2 does not grossly affect mammalian lifespan or health (Combs et al., 2013), MAGP2 overexpression has been correlated with poor prognosis in a study of human papillary serous ovarian carcinomas (Mok et al., 2009) Importantly, when MAGP2 was tested in experimental mouse tumor models, MAGP2 promoted tumor angiogenesis in either an integrin-dependent manner (Albig et al., 2007; Mok et al., 2009) or a Notch-dependent manner (Albig et al., 2007) Taken together ⁎ Corresponding author at: Department of Biological Science, 800 N State College Blvd., Fullerton, CA 92834, United States Tel.: +1 657 278 2540; fax: +1 657 278 3426 E-mail address: almiyamoto@fullerton.edu (A Miyamoto) it suggests that MAGP2 may be playing a causative role in the progression of ovarian cancer through its pro-angiogenic activity Most of the evidence supporting matrix activity of MAGP2 is based on immunohistochemical and biochemical studies Immunohistochemistry of fetal bovine tissues showed MAGP2 protein expression in the stroma and extracellular matrix of blood vessels, lung, muscle, and skin, among other tissues (Gibson et al., 1998) Biochemically, MAGP2 can bind to at least two of the major proteins that make up extracellular microfibrils, fibrillin-1 and fibrillin-2 (Penner et al., 2002; Hanssen et al., 2004; Miyamoto et al., 2006) The binding of MAGP2 to fibrillin is mediated by the MAGP2 Matrix Binding Domain (MBD), a cysteinerich domain shared by MAGP2 and its related family member MAGP1 (also known as MFAP2) (Penner et al., 2002; Segade et al., 2002) Relevant to its role in blood vessels, mice mutant for both MAGP1 and MAGP2 had an age-dependent weakening of large blood vessels, suggesting that these two proteins may have at least partially overlapping functions in the ECM of mature blood vessels (Combs et al., 2013) Despite strong immunohistochemical data that endogenous MAGP2 is bound to extracellular microfibrils, cell culture models of microfibril assembly are contradictory when it comes to MAGP2 matrix binding In a NIH 3T3-derived cell line, overexpressed MAGP2 increased http://dx.doi.org/10.1016/j.matbio.2014.08.003 0945-053X/© 2014 Published by Elsevier B.V This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/) 28 A Miyamoto et al / Matrix Biology 40 (2014) 27–33 deposition of both elastic fibers and collagen fibers, and MAGP2 colocalized with both elastin and collagen I, respectively (Lemaire et al., 2007, 2005) However, in fetal rat lung fibroblasts (RFL6) that generate elastic fibers, overexpression of MAGP2 did not lead to localization of the protein to the extracellular matrix (Segade et al., 2002) While these differences may be partially due to variations in cell type, the ability of MAGP2 to bind to microfibrils may also be regulated, albeit through mostly unknown mechanisms In this regard, a conserved proprotein convertase (PC) site in MAGP2 has been identified (Donovan et al., 2013) Proteolytic PC enzymes process many cell-surface and secreted proteins (Seidah et al., 2013), and processing can regulate secretion or activity of extracellular proteins The PC consensus site is not found in MAGP1, suggesting that cleavage may play an important functional role in some MAGP2-specific activities Therefore, we asked if the MAGP2 PC consensus site regulated the ability of the protein to bind to microfibrils Additionally, based on the association of MAGP2 with ovarian cancer, we asked whether ovarian epithelial tumor cells might be able to generate extracellular MAGP2-positive microfibrils in culture Results 2.1 MAGP2 is localized to fibrillin-2 microfibrils produced by T3 cells These studies began with a tumor-derived transformed mouse ovarian epithelial cell line, T3 (Xing and Orsulic, 2005); the tumors resembled human papillary serous ovarian carcinoma, the most common type of ovarian cancer, and the type of cancer with which MAGP2 overexpression is associated (Mok et al., 2009) Therefore T3 cells were chosen as a biologically relevant model to study MAGP2, since overexpression of MAGP2 could be achieved in these cells via transient plasmid transfection When full-length mouse MAGP2 (Fig 1) was expressed in T3 cells, MAGP2 immunofluorescence revealed a fibrillar pattern of expression (Fig 2A) Although it was a relatively weak signal, fibrillar MAGP2 was extracellular, as the cells were not permeabilized prior to performing immunofluorescence To determine which domains of MAGP2 were responsible for the fibrillar expression pattern, two deletion constructs expressing either the amino-terminal half of MAGP2 (MAGP2 NT) or the carboxy-terminal half of the protein (MAGP2 CT) were used (Fig 1) The MAGP2 NT construct contains the RGD sequence that binds to cell-surface integrins, while the MAGP2 CT half construct contains the MBD As predicted by N- SP RGD SP RGD SP RGD PC-N RR C20 -C MAGP2 MBD * ** ** ** * MAGP2 RR AA MBD AA C20 MBD RR SP RGD MAGP2 C20 MAGP2 NT SP 6x myc MBD RR C20 MAGP2 CT SP 6x myc MBD AA C20 MAGP2 CT RR AA SP 6x myc MBD RR MAGP2 CT C20 Fig MAGP2 constructs used in this study The mouse MAGP2 sequence was used to generate all constructs Asterisks (*) indicate the conserved cysteine spacing found in the C-terminal half of all MAGP2 proteins The arrowhead indicates the conserved proprotein convertase (PC) processing site, and PC-N denotes the large amino-terminal cleavage product of PC cleavage Diagram not to scale SP, signal peptide; RGD, integrinbinding site; MBD, Matrix Binding Domain; RR, arginine residues at positions −2 and − in the PC consensus site; AA, mutation of the RR codons to alanines; C20, the carboxy-terminal 20 amino acids that form the small PC cleavage fragment; 6x myc, six myc epitope tags the presence of the MBD in the CT construct, the CT half of MAGP2 showed a fibrillar expression pattern, while the NT half of MAGP2 showed a cell-associated pattern (Fig 2B,C) Together the data indicate that the CT half of MAGP2 is both necessary and sufficient to associate with the extracellular matrix produced by T3 cells Next, to determine which extracellular matrix fibers MAGP2 was associated with, T3 cell cultures were tested for the types of extracellular fibers they generated T3 cells generated both a fibrillin-2 and a fibronectin matrix (Fig 2E, H) As expected, these matrices were generally distinct, but with occasional overlap (Fig S1, A–C), given that previous studies showing that fibronectin is required only at the early stages of fibrillin matrix generation (Kinsey et al., 2008; Sabatier et al., 2009) Fibrillin microfibrils would be expected to bind MAGP1, and so we confirmed that T3 cells produced a matrix that MAGP1 could also bind (Fig S1, I–J) Since T3 cells generated only weak fibrillin-1 fibers and did not stain positively for elastin (Fig S1, D–H) by day postconfluence, only fibrillin-2 and fibronectin matrices were used for MAGP2 co-localization studies Based on the fibers produced by the T3 cells, MAGP2 was predicted to associate with fibrillin-2-containing microfibrils To test this idea, T3 cells were transfected with the MAGP2 CT construct and dual immunofluorescence was performed for MAGP2 and either fibrillin-2 or fibronectin MAGP2 CT co-localized extensively to fibrillin-2-containing microfibrils as expected (Fig 2D–F) and only occasionally with fibronectin (Fig 2G–I) Similar occasional overlap was observed when full-length MAGP2 was co-stained for fibronectin (Fig 2, J–L) The data from both the full-length MAGP2 and MAGP2 CT construct indicate that MAGP2 associates primarily with fibrillin-2 microfibrils produced by the T3 cells, and not fibronectin fibers 2.2 Microfibril association of MAGP2 is promoted by PC processing and the C20 domain In order for the T3 cells to become a useful model to study the role of PC cleavage in MAGP2 matrix association, the cells must have PC activity to process MAGP2 appropriately at the PC consensus site Immunoblotting analysis of both T3 cell lysates and conditioned medium indicates that MAGP2 is processed as previously described (Donovan et al., 2013) Specifically, the wild-type MAGP2 protein and the cleavageresistant mutant form of MAGP2 (RR → AA) in the whole cell lysate are of similar apparent molecular mass of ~ 23 kD (Fig 3E, middle panel), but in the conditioned medium, the full-length protein is processed to a faster migrating ~ 21 kD fragment (Fig 3E, top panel) As expected, the 21 kD protein is similar in mass to the engineered ΔC20 protein that mimics the larger PC-N cleavage fragment (Fig 1) These data indicate that T3 cells have PC activity and can process MAGP2 when its PC consensus site is intact To determine the role of PC cleavage on MAGP2 matrix association, T3 cells were transiently transfected with either MAGP2 or the MAGP2 RR → AA mutant construct, and immunofluorescence was performed on post-confluent cells In comparison to full-length MAGP2 that produced a fibrillar staining pattern (Figs 3A, 2A), the RR → AA mutant showed decreased fibrillar localization of MAGP2 (Fig 3B) The results suggest that PC cleavage of MAGP2 promotes MAGP2 matrix binding, but that it is not a prerequisite, since the RR → AA mutation does not fully block matrix binding of MAGP2 We also noted that the reduction in fibrillar MAGP2 was accompanied by an increase in cellassociated MAGP2 (Fig 3, compare B and A) Since there were no obvious differences in the amount of proteins found in the conditioned medium of T3 cells (Fig 3E), it would not appear that changes in secretion account for the differences in fibrillar MAGP2 Very similar results were obtained when the MAGP2 ΔC20 mutant construct was tested; reduced fibrillar and increased cell-associated protein were observed, indicating that the C20 domain also promotes MAGP2 matrix association, at least in the context of the full-length protein (Fig 3, compare A and C) A Miyamoto et al / Matrix Biology 40 (2014) 27–33 29 MAGP2 CT 9E10 9E10 MAGP2 G J MAGP2 D A MAGP2 H Fibronectin E Fibronectin B Fibrillin-2 MAGP2 MAGP2 K I Overlay F Overlay 9E10 C Overlay MAGP2 NT L MAGP2 CT Fig MAGP2 extensively co-localizes to fibrillin-2 microfibrils produced by a transformed mouse ovarian epithelial cell line A–C T3 cells were transfected with the indicated MAGP2 constructs (800 ng/well), and collected at day post-transfection when the cells had been post-confluent for at least 48 h Immunofluorescence was performed on unpermeabilized cells (−Triton X-100) using either the M-80 MAGP2 antibody (A, B) or the 9E10 antibody (C) and visualized using either an Alexa Fluor 488-conjugated goat anti-rabbit or goat antimouse secondary antibodies One of three replicates shown Images were taken at 400× magnification Scale marker 10 μm D–I T3 cells were transfected with the MAGP2 CT construct (400 ng/well) and collected on day post-transfection, treated with Triton X-100, and antibodies added as indicated for dual immunofluorescence Images were taken at 200× magnification Scale marker 20 μm D–F Dual immunofluorescence with 9E10 and fibrillin-2 antibodies One of three replicates shown G-I Dual immunofluorescence with 9E10 and fibronectin antibodies, using isotype-specific secondary antibodies to distinguish between the two monoclonal antibodies (9E10, IgG1; FN-3E2, IgM) One of three replicates shown J–L T3 cells were transfected with MAGP2 (800 ng/well), and collected at day post-transfection, treated with Triton X-100, and antibodies added as indicated for dual immunofluorescence One of two replicates shown Images were taken at 400× magnification Scale marker 10 μm The horizontal labels indicate the transfected construct, and the vertical labels indicate the detection antibody used 2.3 The amino-terminal half of MAGP2 is needed for the C20 domain to promote microfibril association of MAGP2 To see if the effects of the PC consensus mutations required the NT half of the protein, the RR → AA andΔC20 mutations were introduced separately into the CT construct, and dual immunofluorescence with fibrillin-2 was performed In comparison to MAGP2 CT, the CT RR → AA mutant showed reduced fibrillar MAGP2 compared to the wild-type protein (Fig 3F, I) but similar co-localization with fibrillin-2 (Fig 3H, K) Since the RR → AA mutation generated the same result in the context of either the full-length (Fig 3B) or CT version (Fig 3I) of MAGP2, the data indicate that the NT half of MAGP2 is not required for PC cleavage to promote matrix association of MAGP2 Interestingly, results obtained with MAGP2 CT ΔC20 immunofluorescence were more complex Like MAGP2 CT, the CT ΔC20 protein showed similar colocalization to fibrillin-2 microfibrils (Fig 3H, N), but did not have a reduced presence on fibrils like the RR → AA mutant (Fig 3F, L) This was an unexpected result since the same ΔC20 mutation in the context of the full-length protein reduced MAGP2 matrix association (Fig 3, compare A and C versus F and L) Therefore, since removal of the NT half in the CT constructs nullified the effect of deleting the C20 domain, the data suggest that that the NT half of MAGP2 is required for the C20 domain to promote matrix binding In some cases, PC processing leads to dissociation of the two fragments, such as fibrillin-1 (Lonnqvist et al., 1998; Raghunath et al., 1999); in other PC substrates, like TGFβ, fragments remain associated with one another (Ramirez and Sakai, 2010) Since deletion of the C20 domain reduced fibrillar MAGP2 (Fig 3C), it suggested that MAGP2 might belong to the latter category To assess this idea, T3 cells were transfected with MAGP2 CT, and immunofluorescence was performed with an antibody that recognizes the C20 domain (R-CT) The C20 antiserum detected a fibrillar pattern that overlapped extensively with the pattern detected with an antibody raised to the N-terminal half of MAGP2 (Fig 3, O–Q) That the C20 domain is found in the same fibrillar pattern as an N-terminal epitope indicates that both of the MAGP2 PC cleavage fragments are matrix-associated While the result is consistent with the notion that the two fragments remain associated after processing, it does not rule out that the two fragments associate with the matrix independently Since many microfibril-associated proteins can bind to microfibrils when added exogenously to cells that are producing an extracellular matrix (Ramirez and Sakai, 2010), soluble MAGP2 CT, CT RR → AA, and CT ΔC20 proteins were produced from 293T cells, then added in trans to T3 cells and tested for matrix binding Consistent with the direct transfection studies, exogenous MAGP2 CT was found in a fibrillar pattern that colocalized with fibrillin-2 (Fig 4A–C) The CT RR → AA mutant showed reduced matrix association compared to the CT protein (Fig 4, compare A and D, C and F), and the CT ΔC20 mutant showed similar matrix association as MAGP2 CT (Fig 4, compare A and G, C and I) These results were not caused by differences in the amount of soluble MAGP2 added to matrices, as all proteins were secreted at similar levels (Fig 4J) Although the decrease in fibrillar MAGP2 RR → AA CT was less pronounced when the protein was introduced exogenously than when transfected directly into T3 cells (compare Fig 3F and I versus Fig 4A and D), the mutant MAGP2 RR → AA CT construct showed the same general trend via either method of introduction Taken together, we conclude that, in the T3 cell system, whether MAGP2 is exogenously produced or synthesized within the cell layer itself, association of MAGP2 with fibrillin-2 microfibrils is regulated by PC cleavage and the C20 domain Discussion 3.1 Modulation of matrix localization by the conserved PC consensus site in MAGP2 The reduction of fibrillar MAGP2 for both the RR → AA and RR → AA CT mutants (Figs 3B, I, 4D) suggests that PC cleavage plays a positive role in microfibril localization This is reminiscent of other microfibril and microfibril-associated proteins such as fibrillin-1 and TGF-β that 30 A Miyamoto et al / Matrix Biology 40 (2014) 27–33 MAGP2 RR MAGP2 A B AA B empty vector MAGP2 C20 C D E FL PC-N/ C20 CM MAGP2 CT WCL FL C20 WCL -tubulin MAGP2 CT RR F I G J H K AA MAGP2 MAGP2 CT C20 L 9E10 I Fibrillin-2 M N Overlay K MAGP2 CT Q Overlay P R-CT 9E10 O Fig Mutations that affect PC cleavage of MAGP2 also affect MAGP2 matrix association T3 cells were transfected with the indicated MAGP2 construct (200 ng/well) or empty vector, then grown 72 h past confluence and analyzed via immunofluorescence A–D M-80 MAGP2 antibody staining of permeabilized cells (+Triton X-100) Panel B′ is an extended exposure to more clearly visualize the fibrillar MAGP2 staining One of three replicates shown E Immunoblotting analysis of MAGP2 synthesized in T3 cells using the M-80 MAGP2 antibody α-Tubulin was used as a loading control CM, conditioned medium WCL, whole cell lysate Note the change in the relative position of MAGP2 to the uncleaved RR → AA in the conditioned medium (CM) versus the whole cell lysate (WCL) that indicates PC cleavage of MAGP2 after secretion from the T3 cells (PC-N, see Fig 1) F–N MAGP2 CT constructs were transfected as above, treated with Triton X-100, then co-stained for fibrillin-2 and 9E10 Panels I′ and K′ are longer exposures to show more clearly that the fibrillar staining of the RR → AA mutant overlaps with fibrillin-2 One of three replicates shown O–Q Experiment was similar to F–N except cells were not permeabilized (−Triton X-100), before co-staining for 9E10 and the MAGP2 C20 domain (R-CT) Images were taken at 400× magnification Scale marker 10 μm The horizontal labels indicate the transfected construct, and the vertical labels indicate the detection antibody used are processed by PC enzymes as part of their maturation process Fibrillin-1 C-terminal processing by PC enzymes has long been thought to be a required part of microfibril assembly (Lonnqvist et al., 1998; Raghunath et al., 1999), while more recent proteomic studies of endogenous microfibrils suggest that processing may promote, but not be required for, microfibril assembly (Cain et al., 2009; Ramirez and Sakai, 2010) Similarly, it appears that for MAGP2, processing promotes, but is not required for, matrix-association, since the RR → AA mutation in the context of the full-length or CT protein, reduces but does not eliminate, matrix-associated MAGP2 in the matrix Unlike a previous study that was not able to generate matrixassociated MAGP2 when protein was added in trans (Lemaire et al., 2007), we found that the T3 cell line was able to bind exogenously added protein secreted from MAGP2 CT-transfected 293T cells Given the different cell lines and reagents used in the Lemaire et al study and this one, it is not immediately apparent what the cause of the difference is, but it is possible that cell-type specific differences might account for the dissimilar results For instance, the cells used in the Lemaire et al study expressed fibrillin-1 but not fibrillin-2, whereas the T3 cells produce a fibrillin-2, but not a fibrillin-1 matrix (Fig S1) Alternatively, A Miyamoto et al / Matrix Biology 40 (2014) 27–33 MAGP2 CT AA D MAGP2 CT C20 G J 9E10 A MAGP2 CT RR 31 -MAGP2 36 - E H Fibrillin-2 B 29 - # Conditioned medium F I Overlay C Whole cell lysate Fig Binding of soluble MAGP2 CT proteins to fibrillin-2 microfibrils is impaired by a RR → AA mutation of the PC cleavage site A–I MAGP2 CT constructs were transfected into 293T cells to produce conditioned medium that was then added to confluent T3 cells for days, and immunofluorescence was performed The horizontal labels indicate the conditioned medium added, and the vertical labels indicate the detection antibody used One of three replicate experiments shown Images were taken at 400× magnification Scale marker 10 μm J Immunoblotting analysis of a representative conditioned medium collection from 293T cells using the 9E10 antibody The upper set of bands (marked with |–MAGP2) is the mature secreted MAGP2 CT proteins based on Edman degradation analysis The lower set of bands (marked with #) is truncated at the amino-terminal end; this affects the number of myc tags present, which is more apparent in the whole cell lysate samples, but does not affect the MAGP2 CT sequence MAGP2 protein–protein interactions may differ between cell lines, altering the ability of MAGP2 to bind to microfibrils In this regard, the less pronounced decrease in MAGP2 RR → AA CT binding when the protein was introduced exogenously rather than by direct transfection, could be due in part to the generation of the protein in 293T cells rather than the T3 cell line 3.2 Modulation of matrix localization by the carboxy-terminal 20 amino acids of MAGP2 The role of the MAGP2 C20 domain is less clear, but there are indications that this domain may serve a previously unidentified role in matrix binding In particular, the data suggest that the C20 domain plays a positive role in matrix association that requires the N-terminal half of MAGP2 In the context of the normal protein, deletion of the C20 domain reduces matrix association (Fig 3C), but in the context of the CT half protein, the same deletion has little to no effect (Figs 3L, 4G) A Although speculative, perhaps the C20 domain is involved in protein– protein interactions that promote matrix binding Given the results from the CT constructs, one possibility is that the C20 domain interacts with the N-terminal half of MAGP2, in order to exert its positive regulatory role on matrix association (Fig 5) In this regard, we noted that both the extent and brightness of the fibrillar pattern were weaker when the N-terminal half of MAGP2 was present than when it was absent (Fig 2A, C), a result reminiscent of the results from overexpression studies of full-length MAGP1 and the MAGP1 MBD domain alone in RFL6 cells (Segade et al., 2002) However, because our immunofluorescence studies were performed using different antibodies for the different proteins, we cannot comment on the potential regulatory role of the N-terminal half of MAGP2 at this time Although it is tempting to speculate that the integrin-binding RGD sequence might serve to keep MAGP2 tethered to the cell surface as newly secreted fibrillin is organized into microfibrils, another study found that mutation of the RGD sequence to RVD did not impair the B Fig Summary model of MAGP2 matrix association A Based on this study and previous studies (Segade et al., 2002; Lemaire et al., 2007) matrix-association of MAGP2 is promoted by the factors on the left side of the up arrow, and hindered when only the core MBD is expressed The role of the N-terminal half of MAGP2 in matrix association remains unclear, and is not included, although there may be a requirement for some part of the NT half of MAGP2 for the C20 domain to positively affect matrix association (Fig 3) B A graphic summary of the data from this study On the left, full-length MAGP2 is both cell- and matrix-associated, and both the RR → AA and ΔC20 mutations reduce binding to the matrix In the middle cell, the MAGP2 CT proteins are shown to be more strongly matrix-associated than the full-length proteins (larger arrows), and only the RR → AA mutation decreases matrix localization In the right cell, the MAGP2 NT protein is cell-associated; no fibrillar MAGP2 was seen for this construct The RGD sequence is shown to denote the NT half of MAGP2 in each molecule The extracellular fibers represent the fibrillin-2 microfibrils produced by the T3 cells 32 A Miyamoto et al / Matrix Biology 40 (2014) 27–33 matrix-binding of MAGP2 synthesized in fibroblasts (Lemaire et al., 2007) Perhaps there is a yet-undefined novel cell-association domain in the N-terminal half of MAGP2 3.3 MAGP2 binding to microfibrils requires an ‘extended’ MBD sequence Our results showing matrix-association of MAGP2 in a cell culture model are similar to the results from NIH 3T3-derived fibroblasts overexpressing MAGP2 (Lemaire et al., 2007, 2005) However, in the rat lung fibroblast cell line RFL6, overexpression of MAGP2 did not result in matrix-associated MAGP2, even though these cells produced abundant elastin-containing fibers (Segade et al., 2002) This paper was the first to functionally define the MBD using deletion mutants of MAGP1, and the smallest functional unit that was sufficient to bind MAGP1 to extracellular elastic fibers was a ‘core’ 56-amino acid domain encoded by two exons (7 and 8) that contained cysteine residues with conserved spacing Subsequently, this same sequence was shown to be sufficient to target heterologous proteins to the matrix (Weinbaum et al., 2010) Homology between MAGP2 and MAGP1 within this core MBD sequence includes both the two-exon structure and the conserved cysteine spacing, with one important exception; the third cysteine residue in the MAGP1 MBD is not present in MAGP2, and in its place is a hydrophobic residue This missing cysteine was shown to regulate the matrix binding of the MAGP2 two-exon ‘core’ MBD, since mutagenesis to convert the hydrophobic valine residue to cysteine resulted in a MAGP2 protein that could partially localize to microfibrils like MAGP1 (Segade et al., 2002) The homology between MAGP1 and MAGP2 actually extends beyond the core MBD, and this ‘extended’ homology may provide a rationale for the observed differences seen in different studies MAGP2 has cysteine residues in total, and fully of them have conserved spacing with MAGP1 (Gibson et al., 1996) Therefore, there may be a ‘core’ MBD encoded in the structurally conserved exons of MAGP1 (exons and 8, containing cysteines) and MAGP2 (exons and 9, containing cysteines), as well as an ‘extended’ MBD sequence that encompasses of the 13 cysteines in MAGP1 and of the cysteines in MAGP2 While the MAGP1 core MBD is sufficient to associate with the matrix, the MAGP2 core MBD, with its one less cysteine, is not (Segade et al., 2002); perhaps this defect is mitigated by the presence of the ‘extended’ MBD as found in our CT constructs that can easily be detected on fibrillin-2 microfibrils One result from the Segade et al study remains puzzling; full-length MAGP2 fused to GFP at its carboxy-terminal end was not matrixassociated, even though it would have clearly included the extended MBD Although purely speculative, perhaps placing the GFP fusion protein so close to the PC consensus and C20 domains affected MAGP2 matrix association in a way that could not have been predicted at the time Alternatively, the cell-associated localization of MAGP2 in RFL6 cells might be affected by the presence of cell-surface proteins that can bind to MAGP2 Both Notch receptors and ligands can bind to the MBD of MAGP2 in mammalian cells (Miyamoto et al., 2006; Nehring et al., 2005), and therefore these cell-surface proteins could potentially compete with fibrillin for binding to MAGP2 and keep MAGP2 attached to the cell surface Such a competition could hypothetically alter the ability of certain cells to produce matrix-associated MAGP2 Looking forward, a deeper understanding of the MAGP2 MBD and its regulation by PC processing and the C20 domain will likely require structural studies of both the MBD and full-length MAGP2 3.4 Transformed ovarian epithelial cells produce microfibrils that can bind to MAGP2 In T3 cells, our data are consistent with the notion that there are multiple levels of regulation for matrix association of MAGP2: the requirement for the extended MBD, and the modulatory roles of PC cleavage and the C20 domain (Fig 5) Might some of the regulatory activity in T3 cells be due to its transformed phenotype? While a formal answer remains unknown, it is interesting to note that a recent study found that variations in microfibril generation from different batches of the epithelial cell line ARPE19 were connected to the epithelial and mesenchymal properties of each batch (Baldwin et al., 2014); perhaps a similar set of cellular conditions might regulate MAGP2 matrix association in tumor cells While still hypothetical, our data suggest that matrix-bound MAGP2 could be one way that MAGP2-positive tumors recruit endothelial cells The report that MAGP2 induced pro-angiogenic migration of endothelial cells towards ovarian cancer cells determined that soluble MAGP2 could induce migration and invasion of endothelial cells in vitro (Mok et al., 2009), but did not address the mechanism by which MAGP2 induced angiogenesis Perhaps like our tumor-derived T3 cells, MAGP2positive tumor cells can generate a fibrillin matrix and bind MAGP2 to it; this could create a matrix-tethered MAGP2 concentration gradient for endothelial cells to migrate towards the tumor In particular, since MAGP2 binds to microfibrils through its MBD, it would leave the MAGP2 RGD sequence free to interact with migrating endothelial cells, as shown by Mok and colleagues Experimental procedures 4.1 Plasmids and cell lines The RR → AA and ΔC20 mutations have been previously described (Donovan et al., 2013), as have the MAGP2 CT and MAGP2 NT constructs (Miyamoto et al., 2006) The RR → AA mutant is the same as construct ‘M’ in Donovan et al., and the MAGP2 CT and NT constructs are the same as C-mycMAGP-2 and N-MAGP-2 in Miyamoto et al., 2006, respectively Standard cloning techniques were used to move the RR → AA and ΔC20 mutations into the MAGP2 CT construct Cloning details available upon request T3 cells were a gift of Sandra Orsulic These cells are mutant for p53 and express both an activating mutant form of K-ras and a constitutively active myristoylated Akt (Xing and Orsulic, 2005) T3 cells were grown in DMEM high glucose supplemented with 10% FBS (Tissue Culture Biologicals, Tulare, CA) The maintenance and transfection of 293T cells have been previously described (Donovan et al., 2013) 4.2 MAGP2 matrix localization studies To test the ability of T3 cells to localize transfected MAGP2 to microfibrils, T3 cells were plated on gelatin-coated glass coverslips in 24-well dishes overnight and transfected the next day The cells were transfected with GeneExpresso8000 (Excellgen, Rockville, MD) as per manufacturer's instructions, using 800 ng total plasmid DNA and μl of GeneExpresso8000 per well The cells were allowed to sit at confluence for 3–5 days, and then collected for immunocytochemistry To test the ability of T3 cells to localize exogenous, soluble MAGP2 to microfibrils, 293T cells were transfected in 6-well plates with μg MAGP2 constructs using Turbofect (ThermoFisher Scientific) following the instructions of the manufacturer, then on the next day the medium was changed to DMEM alone Conditioned medium was collected after 48–72 h, centrifuged at 3000 g to remove cells, and then all samples were brought up to the same final volume Conditioned medium was used immediately or stored at °C until use Separately, T3 cells were plated on gelatin-coated glass coverslips and grown to confluence, then 300 μl of conditioned medium was added to the monolayers The next day the cultures were supplemented with 400 μl of T3 cell growth medium, and the cells were collected 72 h after conditioned medium addition 4.3 Immunofluorescence microscopy Indirect immunofluorescence microscopy was used to analyze matrix-associated MAGP2 Cells were fixed in 4% paraformaldehyde A Miyamoto et al / Matrix Biology 40 (2014) 27–33 and 4% sucrose in PBS for 10 at room temperature, then washed once with PBS before quenching with 1% glycine in PBS for 10 Some trials were subjected to a minute 0.1% Triton X-100 incubation to permeabilize the cells at this point (see figure legends for each experiment) After washing the coverslips once with PBS, the cell layers were incubated in blocking buffer (3% BSA in TBS) for 30–60 Primary antibodies were diluted in blocking buffer and incubated on cells for h at room temperature in a humidified chamber, then the coverslips were washed times in PBS Secondary antibodies were also diluted in blocking buffer and incubated on cells for 30–60 at room temperature, in the dark, in a humidified chamber After PBS washes, the coverslips were mounted on glass slides with Prolong Gold with DAPI (Life Technologies, Grand Island, NY) The primary antibodies used were the 9E10 monoclonal (1:1000, SC-40 Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-mouse MAGP2 (1:1000, M-80; Santa Cruz Biotechnology), rabbit anti-human MAGP2 (1:1000, Rockland Immunochemicals, Gilbertsville, PA), mouse anti-fibronectin (1:1000, FN-3E2, Sigma Aldrich, St Louis, MO), rabbit anti-fibrillin-1 and rabbit anti-fibrillin-2, (both used at 1:500, gift of R Mecham), rabbit anti-MAGP1 (1:250, HPA007354, Sigma Aldrich), and goat anti-elastin (1:1000, RA75, Elastin Products Co., St Louis, MO) Alexa Fluor-conjugated fluorescent secondary antibodies (Life Technologies) were used at 1:1000 For fibrillin-1 and elastin antibody staining, an extra preparation step was performed after fixation The monolayers were treated with guanidine and DTT, then quenched with iodoacetamide following the elastin antibody instruction sheet from Elastin Products Co 4.4 Immunoblotting Transiently transfected 293T or T3 cell lysates and conditioned media were denatured and reduced, then subjected to Laemmli SDSPAGE and immunoblotting as previously described (Donovan et al., 2013) To increase the sensitivity of the rabbit anti-mouse MAGP2 (1:1000, M-80; Santa Cruz Biotechnology), all blocking and antibody incubation steps were performed in 1% nonfat dry milk in a PBS-0.1% Tween-20 wash buffer T3 conditioned medium was concentrated via acetone precipitation and digested with PNGaseF (New England Biolabs, Ipswich, MA) prior to immunoblotting 9E10 (Santa Cruz Biotechnology) was used at a 1:1000 dilution in a 5% bovine serum albumin-TBS blocking solution Supplementary data to this article can be found online at http://dx doi.org/10.1016/j.matbio.2014.08.003 Acknowledgments This study was supported by start-up and research funds from CSUF Department of Biological Science (A.M.), a CSUPERB New Investigator Grant (A.M.), CSUF ASI funding (G.H.), and CIRM BSCR funds (E.P., L.D.) 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Orsulic, S., 2005 A genetically defined mouse ovarian carcinoma model for the molecular characterization of pathway-targeted therapy and tumor resistance Proc Natl Acad Sci U.S.A 102, 6936–6941  ... potential regulatory role of the N-terminal half of MAGP2 at this time Although it is tempting to speculate that the integrin -binding RGD sequence might serve to keep MAGP2 tethered to the cell surface... MBD is not present in MAGP2, and in its place is a hydrophobic residue This missing cysteine was shown to regulate the matrix binding of the MAGP2 two-exon ‘core’ MBD, since mutagenesis to convert... context of either the full-length (Fig 3B) or CT version (Fig 3I) of MAGP2, the data indicate that the NT half of MAGP2 is not required for PC cleavage to promote matrix association of MAGP2 Interestingly,