Biochemical characterization of Tractin and LeechCAM, two Ig-superfamily members involved in regulation of axonal outgrowth of Leech neurons

Tractin and LeechCAM are two recently identified members of the Ig-superfamily. Tractin has a highly unusual domain organization: 6 Ig-like domains, 4 FNIII-like domains, an acidic domain, 12 repeats of a novel proline- and glycine- rich motif with sequence similarity to collagen, a transmembrane domain, and an intracellular tail with an ankyrin and a PDZ-domain binding motif. Our data show that Tractin is proteolytically processed into four fragments at two cleavage sites: one located in the third FNIII domain, and the other one located just proximal to the transmembrane domain. The most NH2-terminal fragment is shown to be glycosylated with the glycoepitopes of Lan3-2, Lan4-2 and Laz2-369, while the other remaining fragments are proposed to form a secreted homodimer and a transmembrane heterodimer. The PG/YG repeat region has sequence similarity to collagen and can be selectively digested by collagenase. It is shown by yeast two-hybrid analysis that the intracellular domain of Tractin can interact with ankyrin. Therefore, the various characteristics of Tractin may provide it with properties that link the extracellular matrix and the intracellular cytoskeleton together;LeechCAM is shown to be the leech homolog of apCAM, Fas II, and vertebrate NCAM by phylogenetic analysis. It has a domain configuration of five Ig-like domains, two FNIII-like domains, a transmembrane domain and a cytoplasmic domain. Our data show that LeechCAM probably exists in two forms, either with or without the transmembrane domain and the intracellular tail. Immunoprecipitation analysis shows that LeechCAM is glycosylated with the Laz2-369 glycoepitope, which has been specifically implicated in regulation of axonal outgrowth and synapse formation

Critical Transition in Tissue Homeostasis Accompanies Murine Lung Senescence

BACKGROUND: Respiratory dysfunction is a major contributor to morbidity and mortality in aged populations. The susceptibility to pulmonary insults is attributed to "low pulmonary reserve", ostensibly reflecting a combination of age-related musculoskeletal, immunologic and intrinsic pulmonary dysfunction. METHODS/PRINCIPAL FINDINGS: Using a murine model of the aging lung, senescent DBA/2 mice, we correlated a longitudinal survey of airspace size and injury measures with a transcriptome from the aging lung at 2, 4, 8, 12, 16 and 20 months of age. Morphometric analysis demonstrated a nonlinear pattern of airspace caliber enlargement with a critical transition occurring between 8 and 12 months of age marked by an initial increase in oxidative stress, cell death and elastase activation which is soon followed by inflammatory cell infiltration, immune complex deposition and the onset of airspace enlargement. The temporally correlative transcriptome showed exuberant induction of immunoglobulin genes coincident with airspace enlargement. Immunohistochemistry, ELISA analysis and flow cytometry demonstrated increased immunoglobulin deposition in the lung associated with a contemporaneous increase in activated B-cells expressing high levels of TLR4 (toll receptor 4) and CD86 and macrophages during midlife. These midlife changes culminate in progressive airspace enlargement during late life stages. CONCLUSION/SIGNIFICANCE: Our findings establish that a tissue-specific aging program is evident during a presenescent interval which involves early oxidative stress, cell death and elastase activation, followed by B lymphocyte and macrophage expansion/activation. This sequence heralds the progression to overt airspace enlargement in the aged lung. These signature events, during middle age, indicate that early stages of the aging immune system may have important correlates in the maintenance of tissue morphology. We further show that time-course analyses of aging models, when informed by structural surveys, can reveal nonintuitive signatures of organ-specific aging pathology

Fas-Mediated Apoptosis Regulates the Composition of Peripheral αβ T Cell Repertoire by Constitutively Purging Out Double Negative T Cells

BACKGROUND: The Fas pathway is a major regulator of T cell homeostasis, however, the T cell population that is controlled by the Fas pathway in vivo is poorly defined. Although CD4 and CD8 single positive (SP) T cells are the two major T cell subsets in the periphery of wild type mice, the repertoire of mice bearing loss-of-function mutation in either Fas (lpr mice) or Fas ligand (gld mice) is predominated by CD4(-)CD8(-) double negative alphabeta T cells that also express B220 and generally referred to as B220+DN T cells. Despite extensive analysis, the basis of B220+DN T cell lymphoproliferation remains poorly understood. In this study we re-examined the issue of why T cell lymphoproliferation caused by gld mutation is predominated by B220+DN T cells. METHODOLOGY AND PRINCIPAL FINDINGS: We combined the following approaches to study this question: Gene transcript profiling, BrdU labeling, and apoptosis assays. Our results show that B220+DN T cells are proliferating and dying at exceptionally high rates than SP T cells in the steady state. The high proliferation rate is restricted to B220+DN T cells found in the gut epithelium whereas the high apoptosis rate occurred both in the gut epithelium and periphery. However, only in the periphery, apoptosis of B220+DN T cell is Fas-dependent. When the Fas pathway is genetically impaired, apoptosis of peripheral B220+DN T cells was reduced to a baseline level similar to that of SP T cells. Under these conditions of normalized apoptosis, B220+DN T cells progressively accumulate in the periphery, eventually resulting in B220+DN T cell lymphoproliferation. CONCLUSIONS/SIGNIFICANCE: The Fas pathway plays a critical role in regulating the tissue distribution of DN T cells through targeting and elimination of DN T cells from the periphery in the steady state. The results provide new insight into pathogenesis of DN T cell lymphoproliferation

Biochemical characterization of Tractin and LeechCAM, two Ig-superfamily members involved in regulation of axonal outgrowth of Leech neurons

Tractin and LeechCAM are two recently identified members of the Ig-superfamily. Tractin has a highly unusual domain organization: 6 Ig-like domains, 4 FNIII-like domains, an acidic domain, 12 repeats of a novel proline- and glycine- rich motif with sequence similarity to collagen, a transmembrane domain, and an intracellular tail with an ankyrin and a PDZ-domain binding motif. Our data show that Tractin is proteolytically processed into four fragments at two cleavage sites: one located in the third FNIII domain, and the other one located just proximal to the transmembrane domain. The most NH2-terminal fragment is shown to be glycosylated with the glycoepitopes of Lan3-2, Lan4-2 and Laz2-369, while the other remaining fragments are proposed to form a secreted homodimer and a transmembrane heterodimer. The PG/YG repeat region has sequence similarity to collagen and can be selectively digested by collagenase. It is shown by yeast two-hybrid analysis that the intracellular domain of Tractin can interact with ankyrin. Therefore, the various characteristics of Tractin may provide it with properties that link the extracellular matrix and the intracellular cytoskeleton together;LeechCAM is shown to be the leech homolog of apCAM, Fas II, and vertebrate NCAM by phylogenetic analysis. It has a domain configuration of five Ig-like domains, two FNIII-like domains, a transmembrane domain and a cytoplasmic domain. Our data show that LeechCAM probably exists in two forms, either with or without the transmembrane domain and the intracellular tail. Immunoprecipitation analysis shows that LeechCAM is glycosylated with the Laz2-369 glycoepitope, which has been specifically implicated in regulation of axonal outgrowth and synapse formation.</p

Mechanisms of Indomethacin-Induced Alterations in the Choline Phospholipid Metabolism of Breast Cancer Cells

Human mammary epithelial cells (HMECs) exhibit an increase in phosphocholine (PC) and total choline-containing compounds, as well as a switch from high glycerophosphocholine (GPC)/low PC to low GPC/high PC, with progression to malignant phenotype. The treatment of human breast cancer cells with a nonsteroidal anti-inflammatory agent, indomethacin, reverted the high PC/low GPC pattern to a low PC/high GPC pattern indicative of a less malignant phenotype, supported by decreased invasion. Here, we have characterized mechanisms underlying indomethacin-induced alterations in choline membrane metabolism in malignant breast cancer cells and nonmalignant HMECs labeled with [1,2-(13)C]choline using (1)H and (13)C magnetic resonance spectroscopy. Microarray gene expression analysis was performed to understand the molecular mechanisms underlying these changes. In breast cancer cells, indomethacin treatment activated phospholipases that, combined with an increased choline phospholipid biosynthesis, led to increased GPC and decreased PC levels. However, in nonmalignant HMECs, activation of the anabolic pathway alone was detected following indomethacin treatment. Following indomethacin treatment in breast cancer cells, several candidate genes, such as interleukin 8, NGFB, CSF2, RHOB, EDN1, and JUNB, were differentially expressed, which may have contributed to changes in choline metabolism through secondary effects or signaling cascades leading to changes in enzyme activity

Differential glycosylation and proteolytical processing of LeechCAM in central and peripheral leech neurons

AbstractLeechCAM is a recently described member of the Ig-superfamily which has five Ig-domains, two FNIII-domains, a transmembrane domain, and a cytoplasmic domain. Phylogenetic analysis indicated that LeechCAM is the leech homolog of apCAM, FasII, and vertebrate NCAM. Using a leechCAM-specific monoclonal antibody we show by immunoblot analysis and by Triton X-114 phase separation experiments that in addition to existing in a transmembrane version LeechCAM is likely to be proteolytically cleaved into a secreted form without the transmembrane domain and the intracellular tail. Furthermore, by immunoprecipitation we demonstrate that LeechCAM is glycosylated with the Laz2-369 glycoepitope, an epitope that has been specifically implicated in regulation of axonal outgrowth and synapse formation

cGAS/STING/TBK1/IRF3 Signaling Pathway Activates BMDCs Maturation Following <i>Mycobacterium bovis</i> Infection

Cyclic GMP-AMP synthase (cGAS) is an important cytosolic DNA sensor that plays a crucial role in triggering STING-dependent signal and inducing type I interferons (IFNs). cGAS is important for intracellular bacterial recognition and innate immune responses. However, the regulating effect of the cGAS pathway for bone marrow-derived dendritic cells (BMDCs) during Mycobacterium bovis (M. bovis) infection is still unknown. We hypothesized that the maturation and activation of BMDCs were modulated by the cGAS/STING/TBK1/IRF3 signaling pathway. In this study, we found that M. bovis promoted phenotypic maturation and functional activation of BMDCs via the cGAS signaling pathway, with the type I IFN and its receptor (IFNAR) contributing. Additionally, we showed that the type I IFN pathway promoted CD4+ T cells&#8217; proliferation with BMDC during M. bovis infection. Meanwhile, the related cytokines increased the expression involved in this signaling pathway. These data highlight the mechanism of the cGAS and type I IFN pathway in regulating the maturation and activation of BMDCs, emphasizing the important role of this signaling pathway and BMDCs against M. bovis. This study provides new insight into the interaction between cGAS and dendritic cells (DCs), which could be considered in the development of new drugs and vaccines against tuberculosis