The receptor for advanced glycation end products (RAGE) is a 55-kDa type I membrane glycoprotein of the immunoglobulin superfamily. Ligand-induced up-regulation of RAGE is involved in various pathophysiological processes, including late diabetic complications and Alzheimer disease. Application of recombinant soluble RAGE has been shown to block RAGE-mediated pathophysiological conditions. After expression of full-length RAGE in HEK cells we identified a 48-kDa soluble RAGE form (sRAGE) in the culture medium. This variant of RAGE is smaller than a 51-kDa soluble version derived from alternative splicing. The release of sRAGE can be induced by the phorbol ester PMA and the calcium ionophore calcimycin via calcium-dependent protein kinase C subtypes. Hydroxamic acid-based metalloproteinase inhibitors block the release of sRAGE, and by RNA interference experiments we identified ADAM10 and MMP9 to be involved in RAGE shedding. In protein biotinylation experiments we show that membrane-anchored full-length RAGE is the precursor of sRAGE and that sRAGE is efficiently released from the cell surface. We identified cleavage of RAGE to occur close to the cell membrane. Ectodomain shedding of RAGE simultaneously generates sRAGE and a membrane-anchored C-terminal RAGE fragment (RAGE-CTF). The amount of RAGE-CTF increases when RAGE-expressing cells are treated with a ␥-secretase inhibitor, suggesting that RAGE-CTF is normally further processed by ␥-secretase. Identification of these novel mechanisms involved in regulating the availability of cell surface-located RAGE and its soluble ectodomain may influence further research in RAGE-mediated processes in cell biology and pathophysiology.

The in vivo roles of meprin metalloproteases in pathophysiological conditions remain elusive. Substrates define protease roles. Therefore, to identify natural substrates for human meprin α and β we employed TAILS (terminal amine isotopic labeling of substrates), a proteomics approach that enriches for N-terminal peptides of proteins and cleavage fragments. Of the 151 new extracellular substrates we identified, it was notable that ADAM10 (a disintegrin and metalloprotease domain-containing protein 10)—the constitutive α-secretase—is activated by meprin β through cleavage of the propeptide. To validate this cleavage event, we expressed recombinant proADAM10 and after preincubation with meprin β, this resulted in significantly elevated ADAM10 activity. Cellular expression in murine primary fibroblasts confirmed activation. Other novel substrates including extracellular matrix proteins, growth factors and inhibitors were validated by western analyses and enzyme activity assays with Edman sequencing confirming the exact cleavage sites identified by TAILS. Cleavages in vivo were confirmed by comparing wild-type and meprin−/− mice. Our finding of cystatin C, elafin and fetuin-A as substrates and natural inhibitors for meprins reveal new mechanisms in the regulation of protease activity important for understanding pathophysiological processes.Electronic supplementary materialThe online version of this article (doi:10.1007/s00018-012-1106-2) contains supplementary material, which is available to authorized users.

No abstract

The multiligand Receptor for Advanced Glycation End products (RAGE) is involved in various pathophysiological processes, including diabetic inflammatory conditions and Alzheimes disease. Full-length RAGE, a cell surface-located type I membrane protein, can proteolytically be converted by metalloproteinases ADAM10 and MMP9 into a soluble RAGE form. Moreover, administration of recombinant soluble RAGE suppresses activation of cell surface-located RAGE by trapping RAGE ligands. Therefore stimulation of RAGE shedding might have a therapeutic value regarding inflammatory diseases. We aimed to investigate whether RAGE shedding is inducible via ligand-induced activation of G protein-coupled receptors (GPCRs). We chose three different GPCRs coupled to distinct signaling cascades: the V2 vasopressin receptor (V2R) activating adenylyl cyclase, the oxytocin receptor (OTR) linked to phospholipase Cβ, and the PACAP receptor (subtype PAC1) coupled to adenylyl cyclase, phospholipase Cβ, calcium signaling and MAP kinases. We generated HEK cell lines stably coexpressing an individual GPCR and full-length RAGE and then investigated GPCR ligand-induced activation of RAGE shedding. We found metalloproteinase-mediated RAGE shedding on the cell surface to be inducible via ligand-specific activation of all analyzed GPCRs. By using specific inhibitors we have identified Ca2+ signaling, PKCα/PKCβI, CaMKII, PI3 kinases and MAP kinases to be involved in PAC1 receptor-induced RAGE shedding. We detected an induction of calcium signaling in all our cell lines coexpressing RAGE and different GPCRs after agonist treatment. However, we did not disclose a contribution of adenylyl cyclase in RAGE shedding induction. Furthermore, by using a selective metalloproteinase inhibitor and siRNA-mediated knock-down approaches, we show that ADAM10 and/or MMP9 are playing important roles in constitutive and PACAP-induced RAGE shedding. We also found that treatment of mice with PACAP increases the amount of soluble RAGE in the mouse lung. Our findings suggest that pharmacological stimulation of RAGE shedding might open alternative treatment strategies for Alzheimeŕs disease and diabetes-induced inflammation.

Herein the synthesis of antibody-polymer conjugates, with a quite narrow dispersity based on the polymer HPMA, are reported. These conjugates are synthesized by coupling antibodies to maleimide-functionalized poly(N-(2-hydroxypropyl)-methacrylamide) (poly-HPMA) copolymers derived through reversible addition-fragmentation chain transfer (RAFT) polymerization of pentafluorophenyl methacrylate via the intermediate step of an activated ester polymer. We develop a protocol that allows the attachment of two different model antibodies, monoclonal anti-RAGE (receptor for advanced glycation end-products) antibody, and polyclonal human immunoglobulin (huIgG). Modification of the antibody and conjugation is monitored by SDS-PAGE electrophoresis. Preserved affinity is demonstrated by Western Blott and cell-uptake analysis, for example, to cells of the immune system.