Action myoclonus-renal failure syndrome (AMRF) is caused by mutations in the lysosomal integral membrane protein type 2 (LIMP-2/SCARB2). LIMP-2 was identified as a sorting receptor for beta-glucocerebrosidase (beta-GC), which is defective in Gaucher disease. To date, six AMRF-causing mutations have been described, including splice site, missense and nonsense mutations. All mutations investigated in this study lead to a retention of LIMP-2 in the endoplasmic reticulum (ER) but affect the binding to beta-GC differentially. From the three nonsense mutations, only the Q288X mutation was still able to bind to beta-GC as efficiently as compared with wild-type LIMP-2, whereas the W146SfsX16 and W178X mutations lost their beta-GC-binding capacity almost completely. The LIMP-2 segment 145-288, comprising the nonsense mutations, contains a highly conserved coiled-coil domain, which we suggest determines beta-GC binding. In fact, disruption of the helical arrangement and amphiphatic nature of the coiled-coil domain abolishes beta-GC binding, and a synthetic peptide comprising the coiled-coil domain of LIMP-2 displays pH-selective multimerization properties. In contrast to the reduced binding properties of the nonsense mutations, the only missense mutation (H363N) found in AMRF leads to increased binding of beta-GC to LIMP-2, indicating that this highly conserved histidine modifies the affinity of LIMP-2 to its ligand. With the present study, we demonstrate that disruption of the coiled-coil structure or AMRF disease-causing mutations abolish beta-GC binding, indicating the importance of an intact coiled-coil structure for the interaction of LIMP-2 and beta-GC.

The lysosomal membrane protein type 2 is a novel identified lysosomal sorting receptor for β-glucocerebrosidase (GC). Mutations in both genes underlie human pathologies causing action myoclonus-renal failure syndrome (AMRF) and Gaucher disease (GD), respectively. We now demonstrate that the lumenal acidification mediated by the vacuolar (H + )-ATPase triggers the dissociation of LIMP-2 and GC in late endosomal/lysosomal compartments. Moreover, we identified a single histidine residue in LIMP-2 that is necessary for LIMP-2 and GC binding. This residue is in close proximity to a proposed coiled-coil domain, which determines the binding to GC and may function as a critical pH sensor.

Impaired bone healing can occur with numerous pathologic conditions like trauma, osteoporosis, and infection. Therefore tissue-engineering strategies that aim to enhance osteogenic differentiation of stem cells in order to accelerate bone healing are a major goal of contemporary regenerative research. In this study we cultivated mesenchymal stem cells (MSC) together with the recently patented programmable cells of monocytic origin (PCMO) to test whether co-cultures promote an osteogenic differentiation process. PCMO have recently been shown to have pluripotent characteristics and do support the regeneration processes of liver and heart diseases. Quantitative real time PCR expression profiles of osteogenic marker genes such as alkaline phosphatase in co-cultures of PCMO and MSC showed that MSC differentiated into osteoblast-like cells more rapidly as compared to mono-cultures. Alkaline phosphatase expression and enzyme activity levels were highly increased in co-cultures compared to mono-cultures of MSC. Tests for mineralized matrix formation also indicated that PCMO have a positive effect on co-cultured MSC under osteogenic culture conditions. However, analysis of collagen 1A did not show enhanced expression. In summary, PCMO obviously have the ability to promote osteogenic differentiation of MSC in vitro while their own pluripotent potential is not sufficient to develop osteoblast-like characteristics themselves. ß