The BM88 antigen is a neuron-specific molecule widely distributed in the mammalian nervous system. It is a 22-kDa, apparently not glycosylated, integral membrane protein, which appears early during brain development and remains at high levels in the mature animal. Here, we describe the cDNA cloning of the porcine BM88 antigen and present evidence that this protein is involved in neuroblastoma cell differentiation. The deduced protein is a novel molecule consisting of 140 amino acids and bears a putative transmembrane domain at the COOH-terminal region. The mRNA of this protein is expressed only in neural tissues, where it is restricted to neurons. Stably transfected Neuro-2a cells overexpressing the BM88 antigen exhibited a significant change in morphology, reflected by enhanced process outgrowth, and a slower rate of division. Moreover, in the presence of differentiation agents, such as sucrose and retinoic acid, an accelerated differentiation of the transfected Neuro-2a cells was observed. Especially in the presence of sucrose, the consequent overexpression of the BM88 antigen in the transfected cells resulted in their enhanced morphological differentiation accompanied by the induction of neurofilament protein expression. Our results suggest that the BM88 antigen plays a role in the differentiation of neuroblastoma cells.
Control of cell cycle progression/exit and differentiation of neuronal precursors is of paramount importance during brain development. BM88 is a neuronal protein associated with terminal neuron-generating divisions in vivoThe formation of the nervous system is governed by a delicate balance between cell proliferation, subsequent cell cycle withdrawal, and differentiation to distinctive neuronal phenotypes (1, 2). Current observations have highlighted the existence of mechanisms coupling cell cycle exit and differentiation as well as functional cross-talk between intrinsic factors controlling these two mechanisms. A number of key factors regulating cell cycle progression have been implicated in cell fate determination and differentiation of neuronal precursors, whereas specification-and/or differentiation-inducing molecules are beginning to emerge as cell cycle regulators (3-6). However, there are still important questions regarding the timing control of the proliferation/differentiation switch that remain unanswered.In the central nervous system, control of cell cycle progression plays an essential role in the generation of the appropriate number of neurons and the formation of functional neuronal circuits. When a neuronal progenitor is committed to undergo differentiation, it exits from the G 1 phase of the cell cycle and enters into an irreversible quiescent state referred to as G 0 . The tumor suppressor proteins p53 and pRb 6 are central regulators of this progression (7). p53, when activated, causes G 1 arrest at the G 0 restriction point by inducing expression of p21 and consequent inhibition of D-type cyclins and related cyclin-dependent kinases (8 -10), thus preventing phosphorylation of pRb (7). Under these conditions, hypophosphorylated pRb associates with the E2F family of transcription factors, thus impairing their ability to transactivate genes required for cell cycle progression (11). As a consequence cells do not progress through the G 1 -to-S phase transition. Several studies have indicated that a critical event associated with cell cycle withdrawal and differentiation both in neuronal and non-neuronal cells is the cellular compartmentalization of cyclin D1, which shifts from a predominantly nuclear localization to cytoplasmic sequestration
Progression of progenitor cells towards neuronal differentiation is tightly linked with cell cycle control and the switch from proliferative to neuron-generating divisions. We have previously shown that the neuronal protein BM88 drives neuroblastoma cells towards exit from the cell cycle and differentiation into a neuronal phenotype in vitro. Here, we explored the role of BM88 during neuronal birth, cell cycle exit and the initiation of differentiation in vivo. By double- and triple-labelling with the S-phase marker BrdU or the late G2 and M-phase marker cyclin B1, antibodies to BM88 and markers of the neuronal or glial cell lineages, we demonstrate that in the rodent forebrain, BM88 is expressed in multipotential progenitor cells before terminal mitosis and in their neuronal progeny during the neurogenic interval, as well as in the adult. Further, we defined at E16 a cohort of proliferative progenitors that exit S phase in synchrony, and by following their fate for 24 h we show that BM88 is associated with the dynamics of neuron-generating divisions. Expression of BM88 was also evident in cycling cortical radial glial cells, which constitute the main neurogenic population in the cerebral cortex. In agreement, BM88 expression was markedly reduced and restricted to a smaller percentage of cells in the cerebral cortex of the Small eye mutant mice, which lack functional Pax6 and exhibit severe neurogenesis defects. Our data show an interesting correlation between BM88 expression and the progression of progenitor cells towards neuronal differentiation during the neurogenic interval.
Monoclonal antibody BM88 recognizes a neurospecific surface antigen in the CNS and the PNS. In the present study, the antigen recognized by BM88 was immunopurified from pig brain and shown to be a 22-kDa polypeptide by reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Under nonreducing conditions a protein of 40 kDa was obtained, a result indicating that the antigen is composed of two polypeptide chains of equal molecular weight linked by disulfide bridges. Gel filtration of the purified antigen in the presence of Emulphogene suggested that it may be either a monomeric or a dimeric protein. However, in the presence of Triton X-100 a monomeric structure was implied. N-Glycanase digestion indicated that the protein is probably not glycosylated. The purified antigen was characterized as an integral membrane protein by hydrophobic chromatography and phase-separation experiments with Triton X-114. The antigen, or at least the antibody binding region of the molecule, is very susceptible to protease attack, as judged by protease digestion experiments on brain membranes. By using very low concentrations of papain combined with short incubation times, the antigen was converted to a 16.3-kDa membrane-associated polypeptide as assessed by immunoblotting. This polypeptide contained the BM88 binding epitope. Soluble BM88 immunoreactive polypeptides were not obtained. Bacillus cereus phospholipase C was also unable to solubilize the antigen from the membrane. Our results suggest that the molecule, possessing at least one small extramembranous domain, is attached to the membrane via a polypeptide chain.
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