Abstract
The 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP), a protein of unknown function in vivo, is abundantly expressed in myelinating glia in two isoforms, CNP1 and CNP2. In this study, immunoblot analysis showed that CNP1 is the major isoform in adult forebrain, and that both isoforms are included in the postsynaptic density (PSD) fraction and tyrosine-phosphorylated at the basal level. However, subcellular distribution and detergent extraction data showed that CNP is nonspecifically associated with the PSD fraction. Immunocytochemistry revealed that CNP is detected, in a weak but punctate pattern, in dissociated rat hippocampal neurons of 3 days to 2 weeks in vitro. The CNP-positive punctae were distributed throughout soma and dendrites, and distinct from PSD95-positive ones. Immunoblot analysis indicated that CNP is also expressed in neuronal stem cell lines, HiB5 and F11. Interestingly, in addition to the known two isoforms, a new CNP isoform of MW 45 kDa was expressed in these cell lines and was the major type of isoform in F11 cells. Taken together, our data suggest that CNP is expressed in the early stage of in vitro development and nonspecifically included in the adult rat PSD fraction.
Highlights
The 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP; EC 3.1.4.37) is expressed as two isoforms with an apparent molecular mass of 46 kDa (CNP1) and 48 kDa (CNP2), which are produced by alternative ribosomal initiation at two different AUG codons, differing each other only by the 20-amino acid extension at the N terminus (O'Neill et al, 1997)
'One-Triton' postsynaptic density (PSD) fractions were treated with 1.0% n-octyl glucoside (OG), or 1.0% Triton X-100, or with 3% N-lauroyl sarcosine (Sarc) for 15 min at 4oC and the pellet and supernatant fractions were separated by centrifugation at 201,800
Co-isolation of CNPin the FB-PSD fraction Immunoblot analysis was carried outto understand the subcellular distribution of CNPintheforebrain
Summary
The 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP; EC 3.1.4.37) is expressed as two isoforms with an apparent molecular mass of 46 kDa (CNP1) and 48 kDa (CNP2), which are produced by alternative ribosomal initiation at two different AUG codons, differing each other only by the 20-amino acid extension at the N terminus (O'Neill et al, 1997). CNP catalyzes the irreversible hydrolysis of 2',3'-cyclic nucleotides to produce exclusively 2'-nucleotides (Tsukada and Kurihara, 1992). This enzymatic activity may not be relevant to its function in vivo, because physiologically relevant in vivo substrates with 2',3'-cyclic termini have not yet been elucidated. In addition to myelinating cells such as oligodendrocytes and Schwann cells, CNP is expressed in various cell types such as chromaffin cell cultures (McFerran and Burgoyne, 1997), lymphocytes and retinal cells (Uyemura et al, 1972; Giulian and Moore, 1980; Weissbarth et al, 1981; Vogel and Thompson, 1988), liver cells (Dreiling et al, 1981), FRTL5 thyroid cells (Laezza et al, 1997; Bifulco et al, 2002), and muscle cells (Weissbarth et al, 1981), indicating more general cellular functions. We show evidence that CNP is transiently expressed at a low level in hippocampal neurons in culture but does not colocalize with the PSD95, a marker for the PSD
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