A novel and divergent role of granzyme A and B in resistance to helminth infection.

Granzyme (gzm) A and B, proteases of NK cells and T killer cells, mediate cell death, but also cleave extracellular matrices, inactivate intracellular pathogens, and induce cytokines. Moreover, macrophages, Th2 cells, regulatory T cells, mast cells, and B cells can express gzms. We recently reported gzm induction in human filarial infection. In this study, we show that in rodent filarial infection with Litomosoides sigmodontis, worm loads were significantly reduced in gzmA × B and gzmB knockout mice during the whole course of infection, but enhanced only early in gzmA knockout compared with wild-type mice. GzmA/B deficiency was associated with a defense-promoting Th2 cytokine and Ab shift, enhanced early inflammatory gene expression, and a trend of reduced alternatively activated macrophage induction, whereas gzmA deficiency was linked with reduced inflammation and a trend toward increased alternatively activated macrophages. This suggests a novel and divergent role for gzms in helminth infection, with gzmA contributing to resistance and gzmB promoting susceptibility.

Cell-surface expression of a new splice variant of the mouse signal peptide peptidase.

The signal peptide peptidase (SPP) is an intramembrane-cleaving aspartyl protease that acts on type II transmembrane proteins. SPP substrates include signal peptides after they have been cleaved from a preprotein, hence the name. The known SPP isoform, which we renamed SPPalpha, contains an endoplasmic reticulum retention signal at the carboxy terminus. We found a new splice variant, SPPbeta, with an additional in-frame exon inserted between exons 11 and 12 of SPPalpha. Insertion of the new exon led to a complete change in the amino-acid sequence of the carboxy tail. A stop codon within this new exon resulted in silencing of exon 12 and eliminated the endoplasmic reticulum retention signal. The new SPP isoform predominantly localised to the cell surface in contrast to the more restricted localisation of SPPalpha in the endoplasmic reticulum. Differential expression in mouse tissues and in subcellular compartments suggests new functions for SPP in addition to cleaving signal peptides.

Characterisation and differential expression of two very closely related G-protein-coupled receptors, GPR139 and GPR142, in mouse tissue and during mouse development.

By searching the human and mouse genomic databases we found two G-protein-coupled receptors, GPR139 and GPR142, with characteristic motifs of the rhodopsin family of receptors. The gene for GPR139 maps to chromosome 7F1 of mouse and 16p12.3 of human and that for GPR142 to 11E2 of mouse and 17q25.1 of human. We isolated GPR139 from a cDNA library of adult mouse brain and GPR142 from a cDNA library of brains from 15-day-old mouse embryos. GPR139 mRNA was predominantly expressed in specific areas of human and mouse brains, whereas GPR142 mRNA showed a more ubiquitous expression both in the brain and in various peripheral glands and organs. A 50% identity and a 67% homology at the amino-acid level between the two receptors and only 20-25% identity with other G-protein-coupled receptors established them as a new subbranch within the phylogenetic tree and hints at a common or similar ligand(s). Preliminary results suggest that the cognate ligand is present in brain extracts and is, most likely, a small peptide. GPR139 signal transduction in Chinese hamster ovary cells requires coupling to an inhibitory G-protein and is mediated by phospholipase C. Dimer formation may be necessary for proper function.

Expression of the presenilin-like signal peptide peptidase (SPP) in mouse adult brain and during development.

Recently, a new member of the presenilin family was identified as an aspartyl protease that cleaves signal peptides within hydrophobic domains, and was, therefore, named signal peptide peptidase (SPP). We isolated cDNAs coding for mouse and human orthologues of SPP. The human gene spans 55 kilobases on chromosome 20q11.21. The SPP-protein is encoded in mouse and man by 12 exons. The highly conserved intron/exon-structure in the SPP/presenilin family hints at a common precursor. Northern blot and in situ hybridization analysis revealed a widespread expression of SPP in many tissues. A distinct pattern of expression in the mature murine brain and during development indicates that SPP plays an important role in the establishment and maintenance of the nervous system. We prepared an antiserum against the carboxy-terminal domain of SPP, which is highly conserved between species. It reacted specifically, both in western blots and in immunocytochemical preparations, with SPP from various mammalian origins. The antiserum was used to demonstrate that SPP is oriented in the membrane of the endoplasmic reticulum with its carboxy-terminal tail extending into the cytosol.

The neuropeptide head activator is a high-affinity ligand for the orphan G-protein-coupled receptor GPR37.

The neuropeptide head activator (HA) is a mitogen for mammalian cell lines of neuronal or neuroendocrine origin. HA signalling is mediated by a G-protein-coupled receptor (GPCR). Orphan GPCRs with homology to peptide receptors were screened for HA interaction. Electrophysiological recordings in frog oocytes and in mammalian cell lines as well as Ca(2+) mobilisation assays revealed nanomolar affinities of HA to GPR37. HA signal transduction through GPR37 was mediated by an inhibitory G protein and required Ca(2+) influx through a channel of the transient receptor potential (TRP) family. It also required activation of Ca(2+)-dependent calmodulin kinase and phosphoinositide 3-kinase. Respective inhibitors blocked HA signalling and HA-induced mitosis in GPR37-expressing cells. HA treatment resulted in internalisation of GPR37. Overexpression of GPR37 led to aggregate formation, retention of the receptor in the cytoplasm and low survival rates of transfected cells, confirming the notion that misfolded GPR37 contributes to cell death, as observed in Parkinson's disease.