LC–MS Based Cleavage Site Profiling of the Proteases ADAM10 and ADAM17 Using Proteome-Derived Peptide Libraries

Tucher, Joanna; Linke, Dennis; Koudelka, Tomas; Cassidy, Liam; Tredup, Claudia; Wichert, Rielana; Pietrzik, Claus U.; Becker‐Pauly, Christoph; Tholey, Andreas

doi:10.1021/pr401135u

Cited by 92 publications

(95 citation statements)

References 37 publications

(63 reference statements)

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“…The HCRs in CD99 are characterized by many aspartate residues, which are greatly preferred by meprin b (6), indicating possible coevolution of protease and substrate. As a consequence of these negatively charged motifs, CD99 is not cleaved by ADAM10/17, metalloproteases that prefer aliphatic amino acids (42) and have been shown to shed other adhesion molecules involved in TEM [e.g., JAM-A or L-selectin (1,43,44)]. This finding further demonstrates the specificity of meprin b for CD99.…”

Section: Discussionmentioning

confidence: 68%

Ectodomain shedding of CD99 within highly conserved regions is mediated by the metalloprotease meprin β and promotes transendothelial cell migration

et al. 2016

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ABSTRACT:The adhesion molecule CD99 is essential for the transendothelial migration of leukocytes. In this study, we used biochemical and cellular assays to show that CD99 undergoes ectodomain shedding by the metalloprotease meprin b and subsequent intramembrane proteolysis by g-secretase. The cleavage site in CD99 was identified by mass spectrometry within an acidic region highly conserved through different vertebrate species. This finding fits perfectly to the unique cleavage specificity of meprin b with a strong preference for aspartate residues and suggests coevolution of protease and substrate. We hypothesized that limited CD99 cleavage by meprin b would alter cellular transendothelial migration (TEM) behavior in tissue remodeling processes, such as inflammation and cancer. Indeed, meprin b induced cell migration of Lewis lung carcinoma cells in an in vitro TEM assay. Accordingly, deficiency of meprin b in Mep1b 2/2 mice resulted in significantly increased CD99 protein levels in the lung. Therefore, meprin b could serve as a therapeutic target, given that in a proof-of-concept approach we showed accumulation of CD99 protein in lungs of meprin b inhibitortreated mice.-Bedau, T., Peters, F., Prox, J., Arnold, P., Schmidt, F., Finkernagel, M., Köllmann, S., Wichert, R., Otte, A., Ohler, A., Stirnberg, M., Lucius, R., Koudelka, T., Tholey, A., Biasin, V., Pietrzik, C. U., Kwapiszewska, G., Becker-Pauly, C. Ectodomain shedding of CD99 within highly conserved regions is mediated by the metalloprotease meprin b and promotes transendothelial cell migration. FASEB J. 31, 1226-1237 (2017). www.fasebj.orgRegulated intramembrane proteolysis (RIP) of cell adhesion molecules, such as junctional adhesion molecule (JAM)-A, intercellular adhesion molecule (ICAM)-1, and L-selectin, was shown to be essential for transendothelial migration (TEM) of inflammatory or cancer cells (1). Meprin b, a multidomain type I transmembrane metalloprotease, is an initiator of RIP, and structural studies revealed dimeric formation of the protease with the active site in proximity to the cell surface (2-4). In addition, meprin b can be shed from the cell surface by ADAM10/ 17, resulting in a soluble active protease, which for instance is important for mucus detachment in the small intestine (5). Meprin b is characterized by a unique cleavage specificity, with a preference for negatively charged amino acids (6). These structural features provide all requirements that meprin b must have to act as an ectodomain sheddase at the cell surface. Indeed, membrane-bound amyloid precursor protein (APP), for instance, is cleaved by meprin b, resulting in the release of sAPP-b fragments and neurotoxic Ab peptides (4,7,8). Many of the known substrates of meprin b have been identified by mass spectrometry (MS)-based proteomic approaches (9).

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Section: Discussionmentioning

confidence: 68%

Ectodomain shedding of CD99 within highly conserved regions is mediated by the metalloprotease meprin β and promotes transendothelial cell migration

et al. 2016

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“…It may be for RAGE as for other receptors that, rather than a precise consensus sequence of cleavage, the secondary structure of the receptor proximal to the transmembrane region is more important. As there is no clear consensus in existing ADAM10 targets, recent studies have utilized peptide libraries and proteomics to attempt to identify an ADAM10 and -17 cleavage consensus motif (65,66). It was found for ADAM10 that this sheddase prefers a Leu or aromatic amino acid (Phe, Tyr, or Trp) in the residue after cleavage (termed the P1Ј site) (65,66).…”

Section: Discussionmentioning

confidence: 99%

Regulation of Receptor for Advanced Glycation End Products (RAGE) Ectodomain Shedding and Its Role in Cell Function

Braley

Kwak

Jules

et al. 2016

Journal of Biological Chemistry

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The receptor for advanced glycation end products (RAGE) is a multiligand transmembrane receptor that can undergo proteolysis at the cell surface to release a soluble ectodomain. Here we observed that ectodomain shedding of RAGE is critical for its role in regulating signaling and cellular function. Ectodomain shedding of both human and mouse RAGE was dependent on ADAM10 activity and induced with chemical activators of shedding (ionomycin, phorbol 12-myristate 13-acetate, and 4-aminophenylmercuric acetate) and endogenous stimuli (serum and RAGE ligands). Ectopic expression of the splice variant of RAGE (RAGE splice variant 4), which is resistant to ectodomain shedding, inhibited RAGE ligand dependent cell signaling, actin cytoskeleton reorganization, cell spreading, and cell migration. We found that blockade of RAGE ligand signaling with soluble RAGE or inhibitors of MAPK or PI3K blocked RAGE-dependent cell migration but did not affect RAGE splice variant 4 cell migration. We finally demonstrated that RAGE function is dependent on secretase activity as ADAM10 and ␥-secretase inhibitors blocked RAGE ligand-mediated cell migration. Together, our data suggest that proteolysis of RAGE is critical to mediate signaling and cell function and may therefore emerge as a novel therapeutic target for RAGE-dependent disease states.Receptor for advanced glycation end products (RAGE) 3 is a transmembrane, multiligand receptor expressed by most cells but is characterized by its up-regulation in a range of inflammatory disease states including diabetes, various cancers, and cardiovascular disease (1). RAGE activation through ligand binding transduces intracellular signaling and in turn induces cell migration, invasion, and adhesion (1). Work from multiple groups has demonstrated using rodent models that blocking RAGE signaling impairs the development of numerous pathologic states and therefore highlights RAGE as an attractive therapeutic target (2-5). The most widely used means for blocking RAGE signaling is with soluble RAGE (sRAGE), the recombinantly produced extracellular domain of RAGE. Soluble RAGE acts as a decoy receptor and therefore neutralizes RAGE ligands (2, 6). Endogenous sRAGE isoforms, which have been identified in human and mouse sera, are generated through two distinct biological mechanisms: the alternative splicing of the transmembrane region of RAGE leading to a secreted isoform (esRAGE/RAGEv1) and the cleavage of extracellular domain (ectodomain or ECD) at the cell surface by ectodomain shedding (1, 7-10). Ectodomain shedding is a tightly regulated process and is mediated by various metalloproteinases (11, 12). Shedding is essential for normal physiological function but can be deregulated in various pathological disorders where altered levels of metalloproteinases are found (11, 12). In fact, many transmembrane proteins are known to undergo ectodomain shedding including cell adhesion molecules, ligands for growth factor receptors, immunoglobulins, and various enzymes (10 -13). Ectodomain shedding of cell sur...

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“…Peptide solution (350 mM) was incubated with 1 mg recombinant human (rh)ADAM17 in 20 mM HEPES buffer for the indicated time points. rhADAM17 (aa 18-480) was recombinantly expressed in insect cells and was provided by Prof. Becker-Pauly (Christian Albrechts University, Kiel, Germany) (33). The reaction was stopped by adding 0.1% trifluoroacetic acid/H 2 O (Carl Roth).…”

Section: Hplc Analysismentioning

confidence: 99%

The Role of Metalloproteinase ADAM17 in Regulating ICOS Ligand–Mediated Humoral Immune Responses

Marczynska

Ozga

Włodarczyk

et al. 2014

The Journal of Immunology

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Immune cells regulate cell surface receptor expression during their maturation, activation, and motility. Although many of these receptors are regulated largely at the level of expression, protease-mediated ectodomain shedding represents an alternative means of refashioning the surface of immune cells. Shedding is largely attributed to a family of a disintegrin and metalloprotease domain (ADAM) metalloproteases, including ADAM17. Although ADAM17 is well known to contribute to the innate immune response, mainly by releasing TNF-α, much less is known about whether/how this metalloprotease regulates adaptive immunity. To determine whether ADAM17 contributes to regulating adaptive immune responses, we took advantage of ADAM17 hypomorphic (ADAM17ex/ex) mice, in which ADAM17 expression is reduced by 90–95% compared with wild-type littermates. In this study, we show that that ADAM17 deficiency results in spleen and lymph node enlargement, as well as increased levels of Ag-specific class-switched Ig production following immunization with OVA together with anti-CD40 mAbs and polyinosinic-polycytidylic acid. Moreover, we demonstrate that the costimulatory ligand ICOS ligand (ICOSL) is selectively downregulated on the surface of B cells in an ADAM17-specific manner, although it is not proteolitically processed by recombinant ADAM17 in vitro. Finally, we show that higher cell surface levels of ICOSL in ADAM17ex/ex mice may contribute to the development of excessive Ab responses. Therefore, our data suggest a functional link between ADAM17 and ICOSL in controlling adaptive immune responses.

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LC–MS Based Cleavage Site Profiling of the Proteases ADAM10 and ADAM17 Using Proteome-Derived Peptide Libraries

Cited by 92 publications

References 37 publications

Ectodomain shedding of CD99 within highly conserved regions is mediated by the metalloprotease meprin β and promotes transendothelial cell migration

Ectodomain shedding of CD99 within highly conserved regions is mediated by the metalloprotease meprin β and promotes transendothelial cell migration

Regulation of Receptor for Advanced Glycation End Products (RAGE) Ectodomain Shedding and Its Role in Cell Function

The Role of Metalloproteinase ADAM17 in Regulating ICOS Ligand–Mediated Humoral Immune Responses

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