A Rabbit Autoantibody Specific for the 46‐kDa Form of 2′,3′‐Cyclic Nucleotide 3′‐Phosphodiesterase

Abstract

An autoantibody occurring in the serum of an apparently normal rabbit that immunocytochemically stains myelin sheaths and oligodendrocytes in rat brain was shown to react specifically with the 46-kDa isoform of 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) (EC 3.1.4.37) in a number of species. Identification of the shorter isoform of the enzyme (CNP1) as the antigen was achieved by comparing the immunostaining of Western blots by the autoantibody with that of a well-characterized anti-CNP antiserum. The 46-kDa antigen reacting with the autoantibody exhibited the same Mr and pI as the small isoform of CNP on two-dimensional gels and showed a similar enrichment in purified CNS myelin. The autoantibody has very high affinity for CNP1 and is capable of detecting the very low amounts of this enzyme in peripheral nerve, spleen, adrenal gland, pancreas, testis, and intestine. Testing the reactivity of the autoantibody with synthetic peptides by enzyme-linked immunosorbent assay revealed that it reacted with the N-acetylated decapeptide corresponding to the N-terminus of CNP1, but did not react if the peptide was not acetylated or if the acetyl group was replaced with a palmityl group. The lack of reactivity with CNP2, which differs from CNP1 by a 20-amino acid extension at the N-terminus of the protein as a result of alternative splicing, may be due to the absence of the N-acetyl moiety that is part of the epitope and/or blocking of antibody binding to the decapeptide by extension of the polypeptide chain.(ABSTRACT TRUNCATED AT 250 WORDS)

Direct visualization of membrane architecture of myelinating cells in transgenic mice expressing membrane‐anchored EGFP

“…This is consistent with recent findings that CNP is associated with extension of the innermost tongue of myelin membrane sheath adjacent to axons (Snaidero et al ., ). Low amounts CNP‐like phosphodiesterase activity was reported in other organs, for instance, the spleen (Moller et al ., ). However, in the CNP‐mEGFP transgenic line, we did not observe the notable GFP signal in the spleen (Supporting Information Fig.…”

Identification of a 48‐kDa prenylated protein that associates with microtubules as 2′,3′‐cyclic nucleotide 3′‐phosphodiesterase in FRTL‐5 cells

“…There are two isoforms, CNP1 and CNP2 (46 and 48 kDa), resulting from alternative splicing [25]. In the rat CNP1 is larger in size (∼48 kDa) and substantially more abundant than CNP2 [13, 30]. However, FRTL‐5 cells express significant levels of mRNA for CNP1 in comparison to those present in C6 glioma cells (Fig.…”

“…CNP is modified by both types of prenyl groups, farnesyl and geranylgeranyl [31], and it is also palmitoylated [26]. Moreover, CNP has an isoelectric point >9 [13, 25]which could favor interaction with acidic regions in the membrane or microtubule, although there is no distinct polybasic domain. The prerequisites for association with both membranes and microtubules are, therefore, present in CNP.…”

“…(RS)‐[5‐ 3 H]Mevalonate (MVA) (35.0 Ci/mmol) was from NEN DuPont. The rabbit autoantibody against CNP1 was a generous gift from Dr. D. Jacobowitz, NCI Bethesda Md, USA [13]. Rat brain microtubule protein was prepared by sequential polymerization as previously described [14].…”