1456 - Fibronectin Is A Novel Substrate For The Secreted Metalloprotease Adamts17
Year: 2019
Type: other
Abstract: Introduction: Mutations in the secreted metalloprotease ADAMTS17 cause Weill-Marchesani syndrome (WMS) in humans1,2. WMS is characterized by short stature, brachydactyly, joint contractures, stiff skin, and lens dislocation (ectopia lentis). WMS can also be caused by mutations in secreted ADAMTS10 and fibrillin-1 (FBN1). WMS belongs to the acromelic dysplasia group of musculoskeletal disorders, which includes geleophysic dysplasia (mutations in ADAMTSL2, FBN1, or LTBP3), acromicric dysplasia (mutations in FBN1), and Myhre syndrome (mutations in SMAD4)3. ADAMTS17 is a member of the ADAMTS protease family. Several ADAMTS proteases are implicated in musculoskeletal disorders, such as ADAMTS1 and 5 in intervertebral disc degeneration and ADAMTS4 and 5 in cartilage degradation in osteoarthritis. Similar to other members of this protease family, ADAMTS17 is composed of a highly conserved protease domain and a unique ancillary domain, that is believed to be involved in substrate recognition4. Since the biological function of proteases is primarily determined by their substrates and the biological activity of the proteolytic peptides generated thereof, we set out to identify substrates for ADAMTS17 using high-throughput screening techniques. We discovered fibronectin as a potential ADAMTS17 substrate and validated fibronectin proteolysis by ADAMTS17 with biochemical and cell biological assays. Methods: To identify substrates for ADAMTS17, we developed a co-culture system consisting of HEK293 cells that stably express recombinant wild-type ADAMTS17 or mutant ADAMTS17EA, lacking the proteolytic activity, and human dermal fibroblasts which provided the source of an extracellular matrix (ECM) proteome. Proteins and peptides in conditioned medium were labeled with isobaric tags prior to trypsin digestions and semitryptic peptides were identified by quantitative mass spectrometry using a Thermo LTQ-Orbitrap Elite hybrid mass spectrometer5. In a complementary approach, protein interaction partners for ADAMTS17 were identified in a yeast-2-hybrid (Y2H) screen using the characteristic C-terminal ancillary domain of ADAMTS17 as bait and a human placental library as prey. Validation of fibronectin as a substrate for ADAMTS17 was achieved by western blotting and immunofluorescence microscopy to visualize ECM deposition of fibronectin. Results: Mass spectrometry identified a ADAMTS17 cleavage site in fibronectin that is located in the linker region between the C-terminal domains FN-III #15 and FN-I #10 (2278VVTVu25bcGNSVNEGLN2289). Complementary, the Y2H screen identified a binding site for the ancillary domain of ADAMTS17 in the same C-terminal region of fibronectin comprising the FN-III #15 and FN-I #10 and #11 domains. Together, these results strongly suggest that fibronectin is a substrate for ADAMTS17. To confirm fibronectin as a ADAMTS17 substrate, we show proteolysis of fibronectin in the presence of ADAMTS17, but not the proteolytically inactive ADAMTS17EA mutant, in conditioned medium obtained from the co-culture system. We used multiple antibodies against fibronectin and observed antibody-specific fragmentation patterns. To further investigate if ADAMTS17 directly cleaves fibronectin, we transfected a plasmid encoding recombinant full-length fibronectin labelled with a V5-tag in ADAMTS17 or ADAMTS17EA expressing HEK293 cells. In this system, we found no evidence for ADAMTS17-mediated fibronectin proteolysis. To determine, if an unknown adaptor protein that is secreted by HDF cells mediates fibronectin proteolysis by ADAMTS17, we co-expressed ADAMTS17 and V5-tagged fibronectin in HEK293 cells and cultured the cells for 48h in the presence of conditioned medium obtained from human dermal fibroblasts. No evidence for cleavage of recombinant fibronectin was found in this experimental set-up as well. These results suggest that cleavage of fibronectin by ADAMTS17 could be indirect. We further used the co-culture system to analyze the consequences of ADAMTS17-mediated fibronectin proteolysis on the fibronectin network formation in the EMC by immunofluorescence staining. We found that the fibronectin network in the ECM was altered in the presence of ADAMTS17 when compared to mutant ADAMTS17EA. Discussion: In our quest to discover substrates for the ADAMTS17 protease, we were able to detect fibronectin in both high-throughput screens. We subsequently confirmed fibronectin proteolysis by ADAMTS17 biochemically in conditioned medium from the co-culture system. However, we did not find evidence for direct cleavage of fibronectin by ADAMTS17. This could be explained by the lack of an adaptor protein in the HEK293 cell system or by the presence of an unknown protease secreted by human dermal fibroblasts that is activated by ADAMTS17 and subsequently cleaves fibronectin. Such as mechanism would define the biological role of ADAMTS17 as part of a network of proteases. Since fibronectin serves as a scaffold for the assembly of many other ECM proteins, such as fibrillin-1, fibrillin-2 and LTBP1, proteolysis of fibronectin could have consequences not only on the formation of the fibronectin network itself, but also on the proper deposition of other ECM protein networks6,7. The potential consequences on the tissue and organ level is underscored by the fact that mutations in most of the fibronectin-dependent protein networks in the ECM cause musculoskeletal disorders, such as Marfan syndrome (FBN1) or Bealu2019s syndrome (FBN2). Currently, we are analyzing the mechanisms of fibronectin proteolysis by ADAMTS17 and we will analyze the fibronectin content and staining pattern in musculoskeletal tissue from ADAMTS17 knock-out mice. Significance: We identified fibronectin as a novel substrate for ADAMTS17. Since fibronectin is required for the deposition and organization of multiple ECM proteins, including fibrillin-1, fibrillin-2 and others, alterations in the fibronectin ECM properties have profound consequences for musculoskeletal tissue development, homeostasis and repair after injury. References:1)tKhan, A. O., Aldahmesh, M. A., Al-Ghadeer, H., Mohamed, J. Y. & Alkuraya, F. S. (2012). Familial spherophakia with short stature caused by a novel homozygous ADAMTS17 mutation. Ophthalmic Genet. 33, 235-239.2)tMorales, J. et al. (2009). Homozygous mutations in ADAMTS10 and ADAMTS17 cause lenticular myopia, ectopia lentis, glaucoma, spherophakia, and short stature. Am. J. Hum. Genet. 85, 558-568.3)tHubmacher, D. & Apte, S. S. (2011). Genetic and functional linkage between ADAMTS superfamily proteins and fibrillin-1: a novel mechanism influencing microfibril assembly and function. Cell. Mol. Life Sci. 68, 3137-3148.4)tHubmacher, D. et al. (2017). Unusual life cycle and impact on microfibril assembly of ADAMTS17, a secreted metalloprotease mutated in genetic eye disease. Sci Rep 7, 41871.5)tSchlage, P., Egli, F. E. & Auf dem Keller, U. (2017). Time-Resolved Analysis of Matrix Metalloproteinase Substrates in Complex Samples. Methods Mol. Biol. 1579, 185-198.6)tZilberberg, L. et al. (2012). Specificity of latent TGF-beta binding protein (LTBP) incorporation into matrix: role of fibrillins and fibronectin. J. Cell. Physiol. 227, 3828-3836.7)tSabatier, L. et al. (2013). Complex contributions of fibronectin to initiation and maturation of microfibrils. Biochem. J. 456, 283-295.Acknowledgements: This study was supported by the NIH R01AR070748 (to D.H.) and 1S10RR031537-01 (to B.W.), the Marfan Foundation (Early Investigator Award to D.H.), and the Department of Orthopaedics at the Icahn School of Medicine at Mt. Sinai.
Author: Dirk Hubmacher
Language: English
Subfield: Cancer Research
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W4391553322