Cloning of a novel 2',5'-oligoadenylate synthetase-like molecule, Oasl5 in mice.

The 2',5'-oligoadenylate synthetase (2-5OAS) is a enzyme that catalyzes synthesis of 2',5'-oligoadenylates (2-5A) in a dsRNA-dependent manner, and known as a major component of the IFN-induced host defense mechanisms against microbial infections. Here, we report the presence of a novel 2-5OAS-like molecule, termed Oasl5, in mice. The size of Oasl5 cDNA was about 2 kb and encoded a protein consisting of 362 aa. The amino acid sequence showed 76% similarity to the mouse 2-5OAS, however, several motifs being important for the enzyme activity were not conserved. The Oasl5 mRNA was most significantly expressed in the brain, and relatively weak expression was found in other organs such as the spleen, kidney, ovary and testis. It was also expressed in embryonic stem (ES) cells. The Oasl5 mRNA expression in ES cells was elevated 5-fold after treatment with IFN and about 2-fold in the brain when stimulated with IFN inducer, polyinosinic-polycytidylic acid (poly[I:C]). In situ hybridization analysis revealed that Oasl5 is expressed in neurons in the central nervous system in adult mice. When Oasl5 was expressed in E. coli, it yielded 42 kDa protein that binds to dsRNA, but it did not show oligoadenylate synthetase activity. These findings suggest a novel function of Oasl5, which are independent of oligoadenylate synthetase activity, in the brain and developing embryos.

Localization of neuronal growth-associated, microtubule-destabilizing factor SCG10 in brain-derived raft membrane microdomains.

Raft is a mobile membrane subdomain enriched in sphingolipid and cholesterol and also various signaling molecules. Previous observation suggested that brain-derived rafts contain tubulin but that rafts of non-neural origin do not. We hypothesized that SCG10, one of the neuronal growth-associated proteins (nGAPs), might be a neuron-specific molecule that anchors tubulin to neuronal rafts, and we explored biochemically its subcellular localization, interaction with tubulin, and effects on microtubule dynamics. In postnatal rat brain extracts, SCG10 was recovered mostly in membrane-associated fractions, and at least half was included in the raft fraction that was also enriched in GAP-43 and NAP-22. SCG10-enriched brain rafts also contained tubulin, and chemical cross-linking experiments revealed that SCG10 was closely associated with tubulin. In addition, SCG10 was able to inhibit polymerization of tubulin. These results indicate that SCG10 is a component of neuronal rafts as are other nGAPs, and suggest that SCG10 may be involved in signaling events in membranes for cytoskeletal reorganization around neuronal rafts.

Calcium-dependent association of annexin VI, protein kinase C alpha, and neurocalcin alpha on the raft fraction derived from the synaptic plasma membrane of rat brain.

A membrane microdomain enriched in cholesterol and sphingolipids or so called "raft" region was found to contain many signal transducing proteins such as GPI-anchored proteins, trimeric G proteins and protein tyrosine kinases. Because brain-derived raft contains two calmodulin-binding proteins, GAP-43 and NAP-22 as the major protein components, the raft domain is assumed to be important in the Ca(2+)-signaling. In this study, we analyzed protein components showing Ca(2+)-dependent binding to the raft of synaptic plasma membrane from rat brain. SDS-PAGE analysis of the protein components in the EGTA eluate from the raft prepared in the presence of Ca(2+)-ions showed the elution of 80 kDa, 68 kDa, 22 kDa, and 21 kDa proteins. These proteins were identified as protein kinase C alpha (80 kDa) and annexin VI (68 kDa) from the partial amino-acid sequencing, and neurocalcin alpha (22 kDa) and calmodulin (21 kDa) with western blotting and electrophoretic mobilities in the presence or absence of Ca(2+) ions. Further immunoblotting experiments showed the Ca(2+)-dependent association of conventional, but not non-conventional, subtypes of PKC to the raft.

Biochemical and morphological analysis on the localization of Rac1 in neurons.

The acquisition of cell type-specific morphologies is a central feature of neuronal differentiation. Many extra- and intracellular signals are known to cause the morphological changes of neuronal cells through the reconstruction of the microfilaments underneath the cell membrane. The membrane microdomain called "raft" has been paid much attention, for this domain contains many signal-transducing molecules including trimeric G proteins and cytoskeletal proteins. The raft domain is recovered in a low-density fraction after the treatment of the membrane with the non-ionic detergent such as Triton X-100 and the enrichment of cholesterol and sphingolipids is ascribed to be responsible for the detergent insolubility. In contrast to the well-known localization of trimeric G proteins in raft, the localization of small G proteins in the raft is poorly characterized. Since Rho family small G proteins (Rho, Rac, and Cdc42) regulate the microfilament system, we studied the localization of Rho family small G proteins in the raft of rat brain with western blotting. Specific localization of Rac1 was detected in the raft from 10-day-old and 8-week-old rat whole brain, and also in the raft prepared from the growth cone and synaptic plasma membrane fractions. Rho and Cdc42 were, in contrast, recovered in the Triton soluble fraction. Double immunostaining of cultured hippocampal neurons with antibodies to Rac1 and MAP-2, or Rac1 and tau, showed punctate distribution of Rac1 in axons as well as in dendrites.

2',5'-oligoadenylate synthetase gene in chicken: gene structure, distribution of alleles and their expression.

We have cloned the gene for chicken 2',5'-oligoadenylate synthetase (ChOAS) by the method of polymerase chain reaction with use of ChOAS cDNA sequence. The ChOAS gene is composed of five introns and six exons containing all of the sequence of the ChOAS cDNA from the start to the stop codon. The first five exons of ChOAS gene which encode the OAS catalytic domain have a similar structure to HuOAS1 gene including the exon-intron boundaries. However, the length of introns of ChOAS gene is only 1/7 of those of HuOAS1 gene. The sixth exon of the ChOAS gene encodes the ubiquitin-like (UbL) domain of two consecutive sequence (UbL1 and UbL2) homologous to ubiquitin. ChOAS encoded in a single copy gene has at least two alleles, OAS(*)A and OAS(*)B. The differences between these two alleles are in the sixth exon of the gene; a 96-nucleotide sequence in the UbL1 portion of OAS(*)A is deleted from OAS(*)B. No OAS(*)B gene was detected in nine lines of chickens tested other than Leghorns. Almost the same levels of ChOAS-A and -B proteins induced physiologically in erythrocytes were detected in infant chickens (2-week-old), but in grown-up chickens (6-month-old) the level of erythrocyte OAS-B was markedly reduced in most of B/B chickens. Thus, the UbL domain of ChOAS is responsible for the maintenance of the OAS level in the tissue.

Changes in the localization of NAP-22, a calmodulin binding membrane protein, during the development of neuronal polarity.

NAP-22, a neuronal tissue-enriched acidic membrane protein, is a Ca(2+)-dependent calmodulin binding protein and has similar biochemical characteristics to GAP-43 (neuromodulin). Recent biochemical studies have demonstrated that NAP-22 localizes in the membrane raft domain with a cholesterol-dependent manner. Since the raft domain is assumed to be important to establish and/or to maintain the cell polarity, we have investigated the changes in the localization of NAP-22 during the development of the neuronal polarity in vitro and in vivo, using cultured hippocampal neurons and developing cerebellum neurons, respectively. Cultured hippocampal neurons initially extended several short processes, and at this stage NAP-22 was distributed more or less evenly among them. During the maturation of neuronal cells, NAP-22 was sorted preferentially into the axon. Throughout the developmental stages of hippocampal neurons, the localization change of NAP-22 was quite similar to that of tau, an axonal marker protein, but not to that of microtubule-associated protein-2 (MAP-2), a dendritic marker protein. Further confocal microscopic observation demonstrated the colocalization of NAP-22 and either tau or vesicle-associated protein-2 (VAMP-2). A comparison of the time course of the axonal localization of NAP-22 and GAP-43 showed that NAP-22 localization was much later than that of GAP-43. The correlation between the expression of NAP-22 and synaptogenesis in the cerebellar granular layer, particularly in the synaptic glomeruli, was also investigated. There existed many VAMP-2 positive synapses but no NAP-22 positive ones in 1-week-old cerebellum. On sections of 2-week-old cerebellum, accumulation of NAP-22 to the synaptic glomeruli was clearly observed and this accumulation became clearer during the maturation of the synaptic structure. The present results suggest the possibility that NAP-22 plays an important role in the maturation and/or the maintenance of synapses rather than in the process of the axonal outgrowth, by controlling cholesterol-dependent membrane dynamics.

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

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.

Identification of gelsolin as an actin regulatory component in a triton insoluble low density fraction (raft) of newborn bovine brain.

A membrane microdomain enriched in cholesterol and glycosphingolipids, or so called 'raft' region, was found to contain many signal transducing proteins such as GPI-anchored cell adhesion molecules, trimeric G proteins, and protein tyrosine kinases. In previous studies, we showed that the raft region obtained from rat brain contains two cytoskeletal proteins, tubulin and actin, as the major components in addition to these signal transducing proteins. In this study, to know the biochemical mechanisms regulating the cytoskeletal organization in this region, actin regulatory activities in raft were surveyed. We found the presence of a Ca(2+)-dependent actin nucleation promoting activity in raft. The solubilization and column fractionation of this activity combined with western blotting and immunoprecipitation showed that gelsolin is one of the actin regulatory proteins in raft.

Effects of specific mutations in active site motifs of 2',5'-oligoadenylate synthetase on enzymatic activity.

2',5'-Oligoadenylate synthetase (2',5'-OAS) is a double-stranded RNA-dependent nucleotidyl-transferase induced by interferon (IFN). Several 2',5'-OAS cDNA have been cloned from human, pig, rat, mouse, and chicken. A P-loop motif followed by an Asp-containing sequence (referred to as D-box) and a region with a high content of Lys and Arg (KR-rich region) are well conserved in 2',5'-OAS. The sequence 196DFLKQR201 of 40-kDa human 2',5'-OAS, to which 8-azido-ATP binds (N. Kon and R.J. Suhadolnik, J. Biol. Chem. 271, 19983-19990, 1996), is included in the KR-rich region. We introduced several site-directed mutations into these active motifs of 42-kDa murine 2',5'-OAS. Each mutant enzyme studied bound to poly(I):poly(C) to the same extent as wild-type enzyme. Both K67R, a P-loop mutant, and K200R, a KR-rich region mutant, exhibited a reduced but considerable rate of enzymatic activities. The activity of the double mutant K67R/K200R was about 10% of the wild type. On the other hand, the activities of both K67M and K200M were not more than 2% of the wild-type enzyme, and no activity was detected in another P-loop mutant, G62A/G63A. The binding of Mg2+ to a D-box mutant D76N/D78N was markedly reduced, and only a very low level of enzymatic activity was detected in this mutant. These results demonstrate that the P-loop, the D-box that binds Mg2+, and the KR-rich region are important for the enzymatic activities of 2',5'-OAS.

The clinical significance of core promoter and precore mutations during the natural course and interferon therapy in patients with chronic hepatitis B.

OBJECTIVE: We aimed to determine the clinical significance of mutations in core promoter and precore regions in chronic hepatitis B. We investigated changes in these mutations during the natural course and interferon therapy in patients with chronic hepatitis B. METHODS: A total of 93 patients with hepatitis B virus surface antigen were divided into four groups according to hepatitis B e antigen (HBeAg)/anti-HBe status and serum aminotransferase levels. Group I (n = 16) comprised HBeAg-positive patients with normal aminotransferase levels, group II (n = 31) HBeAg-positive patients with elevated aminotransferase levels, group III (n = 30) anti-HBe-positive patients with normal aminotransferase levels, and group IV (n = 16) anti-HBe-positive patients with elevated aminotransferase levels. All patients of group II and seven of group IV were treated with interferon. Three serial serum samples per untreated patient and eight samples per treated patient were tested for HBV DNA levels and core promoter and precore mutations by polymerase chain reaction combined with restriction fragment length polymorphism, and some were cloned and sequenced. RESULTS: Core promoter mutation was found in 38% of group I, 74% of group II, 97% of group III, and 100% of group IV. Precore mutation was found in 6% of group I, 90% of group II, and 100% of groups III and IV. The HBV DNA levels were significantly higher in groups I, II, IV, and III, in that order. Serial determination of these two mutations and viral levels showed that the core promoter mutation appeared to occur first, followed by a completion of the precore mutation along with a decrease in viral levels in patients who seroconverted to anti-HBe after interferon therapy. Interferon therapy suppressed both precore wild- and mutated-type viral levels equally. However, it did not induce any specific mutations. CONCLUSIONS: Core promoter mutation appeared to develop or complete first, followed by completion of the precore mutation, and the virus with these two mutations seemed to be the form to persist in the natural course of chronic hepatitis B. The clinical significance of these mutations appeared to be profoundly associated with the viral levels.

Cholesterol-dependent localization of NAP-22 on a neuronal membrane microdomain (raft).

A membrane microdomain called raft has been under extensive study since the assembly of various signal-transducing molecules into this region has been envisaged. This domain is isolated as a low buoyant membrane fraction after the extraction with a nonionic detergent such as Triton X-100. The characteristic low density of this fraction is ascribed to the enrichment of several lipids including cholesterol. To clear the molecular mechanism of raft formation, several extraction methods were applied to solubilize raft components. Cholesterol extraction using methyl-beta-cyclodextrin was found to be effective to solubilize NAP-22, a neuron-enriched Ca(2+)-dependent calmodulin-binding protein as well as one of the main protein components of brain raft. Purified NAP-22 bound to the liposomes that were made from phosphatidylcholine and cholesterol. This binding was dependent on the amount of cholesterol in liposomes. Calmodulin inhibited this binding in a dose-dependent manner. These results suggest that the presence of a calcium-dependent regulatory mechanism works on the assembly of raft within the neuron.

Simple mixing of IFN with a polysaccharide having high liver affinity enables IFN to target to the liver.

Interferon (IFN) therapy is only one method that is clinically effective in controlling disease activity in patients with chronic hepatitis. A chelating residue (diethylenetriamine pentaacetic acid, DTPA) was introduced to pullulan, which is a polysaccharide with high liver affinity. This DTPA-pullulan could conjugate with IFN through Zn2+ coordination on mixing these three components. Intravenous injection of the IFN-DTPA-pullulan conjugate with Zn2+ coordination induced activity in the liver of an antiviral enzyme. 2',5'-oligoadenylate synthetase at IFN doses lower than those used for free IFN injection. In addition, synthetase induction by the conjugate continued for a longer time than did induction by free IFN. Liver targeting of IFN by this conjugation technique based on Zn2+ coordination opens a new method of IFN therapy.

Characterization of a novel rat brain glycosylphosphatidylinositol-anchored protein (Kilon), a member of the IgLON cell adhesion molecule family.

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-week-old 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.

Biologic and binding activities of IFN-alpha subtypes in ACHN human renal cell carcinoma cells and Daudi Burkitt's lymphoma cells.

Nine interferon-alpha subtypes, IFN-alpha1, IFN-alpha2, IFN-alpha5, IFN-alpha7, IFN-alpha8, IFN-alpha10, IFN-alpha14, IFN-alpha17, and IFN-alpha21, were separated from purified human lymphoblastoid IFN. We tested their inhibitory effects on cell growth and replication of Semliki Forest virus (SFV) and vesicular stomatitis virus (VSV) and their induction of 2',5'-oligoadenylate synthetase (2', 5'-OAS) in ACHN renal cell carcinoma cells. In terms of all three activities, the nine subtypes had similar relative activities, with IFN-alpha10 the most active and IFN-alpha1 the least. Their relative effects on cell growth were similar in two other human cell lines, SK-LU-1 lung cancer cells and KU-2 renal cell carcinoma cells, whereas cells of the Daudi Burkitt lymphoma line behaved quite differently, being highly sensitive to all the nine subtypes. The relative effects with ACHN cells correlated well with their relative binding affinities. However, each of the subtypes bound to both ACHN and Daudi cells to almost the same extent. This suggests that their profound inhibitory effects on the growth of Daudi cells are amplified at some stage in the signal transduction pathway or in the expression of genes that results from binding to the IFN-alpha receptor.

Production of immunoreactive 2',5'-oligoadenylate synthetase in p48-deficient mice.

2',5'-Oligoadenylate synthetase (2'5'OAS), an enzyme induced by interferon (IFN), is physiologically produced in IFN-untreated normal healthy mice. The enzyme is localized mainly in the epithelium of the digestive tract, reproductive organs, and the choroid plexus in the brain. 2'5'OAS is also detected in oocytes in the ovary and in neurons and glial cells of both the telencephalon and cerebellum. Here, we examined the role of p48 (ISGF3gamma), a component of IFN-stimulated gene factor 3 (ISGF3), in the physiologic production of 2'5'OAS using p48-deficient mice generated by gene targeting. In the p48-deficient mice, the physiologic production of 2'5'OAS localized in the following cells was severely impaired: hepatocytes, Kupffer cells, splenocytes, epithelium of the large intestine, oviduct, and uterus, and neurons and glial cells in both the telencephalon and cerebellum. The results show that 2'5'OAS in these cells is induced physiologically through a pathway including p48. However, the production of 2'5'OAS in oocytes was not affected in the p48-deficient mice, indicating that oocyte 2'5'OAS is produced through a p48-independent pathway. A possible function of the GAS sequence found in the promoter region of the 2'5'OAS gene to which Stat6 may bind also is discussed.

The target cells of injected type I interferons in mouse liver.

Type I interferons (IFNs) have been used for the treatment of viral hepatitis, but it is unclear which cells in the liver are affected by injected IFN. The effects of IFN have been studied by the production of 2',5'-oligoadenylate synthetase (2'5'OAS), an IFN-inducible enzyme. Here, we studied the distribution of 2'5'OAS in mouse liver after injection of natural mouse IFN-alpha/beta by Western blotting and immunohistochemistry using a monoclonal antibody specific to mouse 42-kDa 2'5'OAS. Injection of IFN-alpha/beta increased the levels of liver 2'5'OAS and enhanced the intensities of immunohistochemical staining for this enzyme in both hepatocytes and Kupffer cells. In IFN-untreated normal mice, hepatocytes were lightly stained, but some of the Kupffer cells showed rather strong staining. The 2'5'OAS-positive Kupffer cells comprised approximately 60% of those in normal liver, whereas this increased to approximately 90% following IFN-alpha/beta injection. Thus, hepatocytes and Kupffer cells were the targets of injected IFN.

Two types of chicken 2',5'-oligoadenylate synthetase mRNA derived from alleles at a single locus.

We have isolated two types of chicken 2',5'-oligoadenylate synthetase cDNAs, A and B, which encode predicted proteins of 508 amino acids (58316 Da) and 476 amino acids (54336 Da), respectively. The region of A-protein comprising 33 amino acid residues from 385Ala to 417Cys is substituted by a single amino acid 385Tyr in B-protein. The homology between chicken and mammalian 2',5'-oligoadenylate synthetases is 49.5% over the amino-terminal 337 residues. Proteins expressed from A- and B-cDNAs in E. coli cells were both active in synthesizing 2',5'-oligoadenylate. However, the activity of B-protein was 10-15% of that of A-protein. Southern blotting hybridization indicated that the chicken synthetases are encoded by a single gene. RT-PCR and PCR analyses of RNA and DNA of chicken erythrocytes together with the sequence data of the PCR products showed that A- and B-mRNAs are derived from alleles at a single locus encoding chicken 2',5'-oligoadenylate synthetase, designated as OAS * A and OAS * B. Chickens carrying OAS * A/B produce two types of synthetase with molecular masses of 58 and 54 kDa, and those carrying OAS * A/A produce only a single type of 58 kDa.

Localization of 2',5'-oligoadenylate synthetase and the enhancement of its activity with recombinant interferon-alpha A/D in the mouse brain.

Although the expression of 2',5'-oligoadenylate synthetase (2-5AS) is induced by interferon (IFN), low constitutive levels can be detected in animals that have not been treated with IFN. In order to clarify which cells express 2-5AS in the mouse brain, the distribution of this enzyme in the brains of both normal healthy mice and mice treated with recombinant human IFN-alpha A/D was studied by Western blotting and immunohistochemistry, using a specific monoclonal antibody. On the Western blots, the antibody to 42-kD 2-5AS reacted slightly with extracts from the telencephalon, cerebellum, diencephalon, and medulla oblongata of normal mouse brain. 42-kD 2-5AS was predominantly found in the ependymal cells and epithelium of the choroid plexus, and to a lesser degree in neurons and glial cells. Injection of recombinant human IFN-alpha A/D into the left lateral ventricle enhanced the activity of the enzyme in the telencephalon, cerebellum, diencephalon, and medulla oblongata, but did not change the immunohistochemical localization of the enzyme. Direct injection of the IFN into the cortex of the telencephalon enhanced the activity of 2-5AS in all parts of the brain and immunoreactivity was observed in the neurons and glial cells surrounding the injection site. These data indicate that 42-kD 2-5AS activity in the mouse brain is enhanced by the injection of recombinant human IFN-alpha A/D either into the left lateral ventricle or cortex of the telencephalon. Expression of 42-kD 2-5AS in ependymal cells and epithelium of the choroid plexus may prevent viral infections in the brain.

Biochemical evidence for the presence of NAP-22, a novel acidic calmodulin binding protein, in the synaptic vesicles of rat brain.

NAP-22 is a neuronal tissue-enriched acidic protein which is expressed predominantly in rat brain. In the present study, we quantitated the amount of NAP-22 in a highly purified synaptic vesicle fraction. NAP-22 comprised 1.3 +/- 0.15% of the total protein in the fraction, and NAP-22 was located on the external surface of the synaptic vesicle membrane. The results suggest NAP-22 may be involved in the synaptic vesicle cycling.

Identification of NAP-22 and GAP-43 (neuromodulin) as major protein components in a Triton insoluble low density fraction of rat brain.

NAP-22 is a membrane-localized brain enriched acidic protein having a Ca(2+)-dependent calmodulin binding activity. Further fractionation of the NAP-22 containing membrane showed the localization of NAP-22 in a Triton insoluble fraction of low density. Besides NAP-22, this fraction was found to contain GAP-43 (neuromodulin), trimeric G proteins, and some GPI-anchored proteins such as Thy-1 and N-CAM-120. Presence of some protein tyrosine kinases, such as src and fyn, was also shown.