Marc Vandermeeren

The functional γ-secretase inhibitor prevents production of amyloid β 1–34 in human and murine cell lines

Abstract The amyloid precursor protein (APP) undergoes two consecutive cleavages by different proteases, β-secretase and γ-secretase, leading to the release of an amyloidogenic 4 kDa fragment called amyloid β (Aβ). Combining immunoprecipitation and mass spectrometry, we characterized soluble Aβ in cultured cell media of mouse neuroblastoma N2a cells and double hAPP/hBACE-1 transfected HEK293. The major Aβ isoforms detected were Aβ11–34, Aβ1–34, Aβ11–40 and Aβ1–40. In this study, we demonstrate that overexpression of human β-secretase (BACE-1) in HEK293 cells resulted in predominant Aβ cleavage at position Glu 11 rather than Asp 1 , as well as increased production of Aβx–34, but not Aβx–40. Incubation of cells with a specific γ-secretase inhibitor suggests that cleavage of APP at Leu 34 could be mediated by γ-secretase itself or by a γ-secretase dependent process.

Biological activity of hexitol nucleic acids targeted at Ha-ras and intracellular adhesion molecule-1 mRNA

Hexitol nucleic acid (HNA) is a new steric blocking oligonucleotide, hybridizing sequence selectively with RNA. The biological activity of HNA was evaluated in an in vitro translation arrest system targeting Ha-ras mRNA and in a cellular system targeting intracellular adhesion molecule-1 (ICAM-1) expression. HNA very efficiently and selectively inhibited Ha-ras mRNA translation (IC(50) of 50 nM) when targeted at the translation initiation region. When targeting at the 12th codon region, a gap-mer approach was needed to inhibit mRNA translation. Similarly, HNA inhibited ICAM-1 expression in keratinocytes when targeting at codon sequences. In this test system, HNA is less active but more selective than phosphorothioates, but needs lipofection to become active in keratinocytes. This new steric blocker may be an efficient antisense agent providing that enough material can be brought into cells.

Epitope mapping and structural basis for the recognition of phosphorylated tau by the anti-tau antibody AT8.

Microtubule‐associated protein tau becomes abnormally phosphorylated in Alzheimers disease and other tauopathies and forms aggregates of paired helical filaments (PHF‐tau). AT8 is a PHF‐tau‐specific monoclonal antibody that is a commonly used marker of neuropathology because of its recognition of abnormally phosphorylated tau. Previous reports described the AT8 epitope to include pS202/pT205. Our studies support and extend previous findings by also identifying pS208 as part of the binding epitope. We characterized the phosphoepitope of AT8 through both peptide binding studies and costructures with phosphopeptides. From the cocrystal structure of AT8 Fab with the diphosphorylated (pS202/pT205) peptide, it appeared that an additional phosphorylation at S208 would also be accommodated by AT8. Phosphopeptide binding studies showed that AT8 bound to the triply phosphorylated tau peptide (pS202/pT205/pS208) 30‐fold stronger than to the pS202/pT205 peptide, supporting the role of pS208 in AT8 recognition. We also show that the binding kinetics of the triply phosphorylated peptide pS202/pT205/pS208 was remarkably similar to that of PHF‐tau. The costructure of AT8 Fab with a pS202/pT205/pS208 peptide shows that the interaction interface involves all six CDRs and tau residues 202–209. All three phosphorylation sites are recognized by AT8, with pT205 acting as the anchor. Crystallization of the Fab/peptide complex under acidic conditions shows that CDR‐L2 is prone to unfolding and precludes peptide binding, and may suggest a general instability in the antibody. Proteins 2016; 84:427–434.

Development and application of monoclonal antibodies against SKALP/elafin and other trappin family members.

Abstract SKALP/elafin is an epithelial proteinase inhibitor with antimicrobial properties that is not normally expressed in human epidermis, but is induced under inflammatory conditions and in some types of skin cancer. SKALP is a member of the recently described trappin gene family, which encodes a new class of proteins, characterized by a four-disulphide core and a transglutaminase substrate domain. Polyclonal antisera against SKALP have been shown to be useful for monitoring disease activity in psoriasis and tumour differentiation in squamous cell carcinoma. We developed ten different mouse monoclonal antibodies (mAbs) against synthetic peptides corresponding to a hexapeptide epitope in the transglutaminase substrate domain and three mAbs recognizing an epitope in the proteinase-inhibiting domain. The antibodies could be used with high specificity by immunohistochemistry on formalin-fixed tissue, by affinity chromatography, by Western blotting, and by enzyme-linked immunoadsorbent assay (ELISA) for the detection of SKALP/ elafin. These antibodies have several advantages over existing polyclonal antisera, such as a defined epitope, the detection of full-length SKALP/elafin and unlimited supply. An antibody against the hexapeptide epitope, which is common to all known human, simian, bovine and swine trappin family members, was used to immunolocalize bovine trappins expressed in trachea, that have recently been discovered. These mAbs will serve as important new tools to measure SKALP/elafin and trappin family members in research and diagnostics.