Characterization and Tissue Distribution of Opioid-Binding Cell Adhesion Molecule (Obcam) Using Monoclonal Antibodies

Hachisuka, Akiko; Yamazaki, Takao; Sawada, Jun-ichi; Terao, Tadao

doi:10.1016/0197-0186(95)00108-5

Cited by 23 publications

(15 citation statements)

References 21 publications

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“…Two independent groups reported that OBCAM in rat brain has a broad band of more than 50 kDa (46,47). Our antibodies against OBCAM reacted with the 36-kDa band in the PNGF fraction and a broad band around 46 and 51 kDa in the rat brain fraction but did not react with bacterially expressed Kilon.…”

Section: Solubilization Of Proteins From Tif and Its Treatment Withmentioning

confidence: 80%

Characterization of a Novel Rat Brain Glycosylphosphatidylinositol-anchored Protein (Kilon), a Member of the IgLON Cell Adhesion Molecule Family

Funatsu

Miyata²,

Kumanogoh

et al. 1999

Journal of Biological Chemistry

108

100

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In the central nervous system, many cell adhesion molecules are known to participate in the establishment and remodeling of the neural circuit. Some of the cell adhesion molecules are known to be anchored to the membrane by the glycosylphosphatidylinositol (GPI) inserted to their C termini, and many GPI-anchored proteins are known to be localized in a Triton-insoluble membrane fraction of low density or so-called "raft." In this study, we surveyed the GPI-anchored proteins in the Triton-insoluble low density fraction from 2-weekold rat brain by solubilization with phosphatidylinositol-specific phospholipase C. By Western blotting and partial peptide sequencing after the deglycosylation with peptide N-glycosidase F, the presence of Thy-1, F3/contactin, and T-cadherin was shown. In addition, one of the major proteins, having an apparent molecular mass of 36 kDa after the peptide N-glycosidase F digestion, was found to be a novel protein. The result of cDNA cloning showed that the protein is an immunoglobulin superfamily member with three C2 domains and has six putative glycosylation sites. Since this protein shows high sequence similarity to IgLON family members including LAMP, OBCAM, neurotrimin, CEPU-1, AvGP50, and GP55, we termed this protein Kilon (a kindred of IgLON). Kilon-specific monoclonal antibodies were produced, and Western blotting analysis showed that expression of Kilon is restricted to brain, and Kilon has an apparent molecular mass of 46 kDa in SDS-polyacrylamide gel electrophoresis in its expressed form. In brain, the expression of Kilon is already detected in E16 stage, and its level gradually increases during development. Kilon immunostaining was observed in the cerebral cortex and hippocampus, in which the strongly stained puncta were observed on dendrites and soma of pyramidal neurons.

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Section: Solubilization Of Proteins From Tif and Its Treatment Withmentioning

confidence: 80%

Characterization of a Novel Rat Brain Glycosylphosphatidylinositol-anchored Protein (Kilon), a Member of the IgLON Cell Adhesion Molecule Family

Funatsu

Miyata²,

Kumanogoh

et al. 1999

Journal of Biological Chemistry

108

100

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“…In contrast, 84A3 antibody recognized two broad bands of around 46 and 51 kDa of the brain raft and also reacted with the bacterially expressed βN‐OBCAM but not with GST‐Kilon. Two independent groups have reported that OBCAM in the rat brain has two broad bands of around 50 kDa (Hachisuka et al, 1996; Wick et al, 1996). The 89B3 and 84A3 antibodies did not react with the bacterially expressed GST alone (data not shown).…”

Section: Resultsmentioning

confidence: 99%

“…This observation is in good agreement with in situ hybridization experiments showing that OBCAM mRNA is strong in the cerebral cortex and hippocampus (Struyk et al, 1995). Two independent groups reported that OBCAM in the rat brain has two broad bands of around 50 kDa (Hachisuka et al, 1996; Wick et al, 1996). Since the molecular mass of LAMP is 65 kDa and that of Ntm is 64–68 kDa, much larger than Kilon and OBCAM, 89B3 and 84A3 antibodies were presumed to recognize specifically Kilon and OBCAM, respectively.…”

Section: Discussionmentioning

confidence: 99%

Expression of the IgLON cell adhesion molecules kilon and OBCAM in hypothalamic magnocellular neurons

et al. 2000

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The vasopressin (AVP) and oxytocin (OXT) magnocellular neurons in the hypothalamic supraoptic (SON) and paraventricular nuclei (PVN) display reversible structural plasticity of neurons and glial cells under different conditions of neuropeptide secretion. In the present study, we investigated the expression of two immunoglobulin superfamily (IgSF) proteins, Kilon and OBCAM, in the magnocellular neurons by using monoclonal antibodies. Anti-Kilon antibody reacted specifically with the bacterially expressed recombinant Kilon but not with the recombinant OBCAM, and similarly anti-OBCAM antibody specifically recognized the recombinant OBCAM. Western blotting analysis revealed the specific expression of Kilon and OBCAM in the SON homogenates. Although Kilon and OBCAM of the SON homogenates were present as the insoluble form, most Kilon was present in the Triton-insoluble fraction, and OBCAM was localized mainly in the Triton-soluble fraction. Immunocytochemistry revealed Kilon and OBCAM immunoreactivity in the magnocellular neurons of the SON and PVN of the rat hypothalamus compared with outside of the SON and PVN in the hypothalamus. The double-labeling study with confocal microscopy further demonstrated that Kilon immunoreactivity was observed mainly in the dendrites of AVP-secreting neurons and also occasionally OXT-secreting neurons. However, OBCAM immunoreactivity was exclusively seen in the dendrites of AVP-secreting magnocellular neurons. Chronic physiological stimulation by 2% NaCl had no effect on the expression levels of either IgLON protein in the SON. Our study thus demonstrated specific expression of Kilon and OBCAM in the hypothalamic magnocellular neurons, particularly in dendrites, suggesting that they confer on magnocellular neurons the ability to rearrange dendritic connectivity.

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“…The high level of Thy‐1 expression in adulthood is similar to that observed for BIG‐1, BIG‐2, Kilon, and CEPU‐1, but these other molecules exhibit a highly restricted regional distribution (Yoshihara et al, 1994,Yoshihara et al, 1995; Hosoya et al, 1995; Struyk et al, 1995; Spaltmann and Brummendorf, 1996; Funatsu et al, 1999). Similarly, LAMP and OBCAM, markers for neurons comprising the limbic and olfactory systems, respectively (Levitt, 1984; Horton and Levitt, 1988; Zhukareva and Levitt, 1995; Hachisuka et al, 1996; Pimenta et al, 1996), play functional roles in the establishment of connectivity within these specific neural systems (Keller et al, 1989; Pimenta et al, 1995; Mann et al, 1998). The widespread localization of Thy‐1 across thalamic relay nuclei related to somatosensory, visual, motor, and limbic systems in adults suggests that Thy‐1 is probably not, therefore, involved in providing system‐specific guidance or maturation cues.…”

Section: Discussionmentioning

confidence: 99%

“…In the present study, we have examined Thy‐1 gene and protein expression during development and at adulthood in several functionally related structures of the mouse sensory‐motor system, a system for which there are considerable data on the timing of pathway and neuronal development. In addition, because the distribution of other CAMs which are structurally related to Thy‐1, such as the limbic system‐associated molecule (LAMP) or the olfactory system‐related molecule (OBCAM), appears relatively restricted to specific neural systems (Levitt, 1984; Zhukareva and Levitt, 1995; Hachisuka et al, 1996), we have extended the analysis of the thalamus to include nuclei related to limbic and other sensory systems in order to compare the extent to which Thy‐1 localization is also distributed across different neural systems.…”

mentioning

confidence: 99%

Developmentally regulated expression of Thy-1 in structures of the mouse sensory-motor system

Barlow

Huntley

2000

J. Comp. Neurol.

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Thy‐1 is a cell‐surface molecule of the immunoglobulin superfamily which is expressed at high levels in the mature nervous system. Thy‐1 has been implicated in regulating axonal outgrowth and synaptic function, but little is known regarding its cellular localization and expression in the central nervous system (CNS) during development or in adulthood. In this study, Thy‐1 gene expression and protein localization were examined in sensory‐motor and related areas of the adult and postnatally developing mouse CNS. Thy‐1 mRNA expression was restricted to neurons; immunoreactivity was densely distributed throughout the neuropil of all regions examined, often delineated the neuronal plasmalemma, and labeled axons in white matter tracts of the brain and spinal cord. In adulthood, immunolabeling was regionally widespread and was present relatively homogeneously throughout all cell‐dense layers of sensory‐motor cortex, throughout most thalamic nuclei, globus pallidus, and spinal cord. Developmentally, however, Thy‐1 expression and localization exhibited a spatially and temporally staggered sequence leading to the adult pattern. In sensory‐motor cortex, Thy‐1 expression in layer V preceded expression in other layers; in the barrel field, labeling of barrel septa preceeded a gradually increasing intensity of immunolabeling of barrel centers; in the thalamus, Thy‐1 exhibited a differential onset and temporal pattern of expression across different nuclei associated with motor, sensory, or limbic systems; in the caudate nucleus, Thy‐1 expression was greatest during the first postnatal week of life before declining during subsequent development. Taken together, the adult distribution and developmental patterns leading to it form a unique profile in comparison with other structurally related glycosyl‐phosphatidylinositol (GPI)‐anchored neural cell adhesion molecules. The pattern and timing of Thy‐1 expression across layers and nuclei during early postnatal development are more complex than previously recognized, thus perhaps reflecting varied roles for Thy‐1 in aspects of structural or functional maturation which proceed independently of the timing of neurogenesis, migration, and dendritic and axonal growth. J. Comp. Neurol. 421:215–233, 2000. © 2000 Wiley‐Liss, Inc.

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Characterization and Tissue Distribution of Opioid-Binding Cell Adhesion Molecule (Obcam) Using Monoclonal Antibodies

Cited by 23 publications

References 21 publications

Characterization of a Novel Rat Brain Glycosylphosphatidylinositol-anchored Protein (Kilon), a Member of the IgLON Cell Adhesion Molecule Family

Characterization of a Novel Rat Brain Glycosylphosphatidylinositol-anchored Protein (Kilon), a Member of the IgLON Cell Adhesion Molecule Family

Expression of the IgLON cell adhesion molecules kilon and OBCAM in hypothalamic magnocellular neurons

Developmentally regulated expression of Thy-1 in structures of the mouse sensory-motor system

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