N-acetylated Alpha-linked Acidic Dipeptidase Activity Research Articles

Isolation and Expression of Novel Human Glutamate Carboxypeptidases with N-Acetylated α-Linked Acidic Dipeptidase and Dipeptidyl Peptidase IV Activity

Hydrolysis of the neuropeptide N-acetyl-L-aspartyl-L-glutamate (NAAG) by N-acetylated alpha-linked acidic dipeptidase (NAALADase) to release glutamate may be important in a number of neurodegenerative disorders in which excitotoxic mechanisms are implicated. The gene coding for human prostate-specific membrane antigen, a marker of prostatic carcinomas, and its rat homologue glutamate carboxypeptidase II have recently been shown to possess such NAALADase activity. In contrast, a closely related member of this gene family, rat ileal 100-kDa protein, possesses a dipeptidyl peptidase IV activity. Here, we describe the cloning of human ileal 100-kDa protein, which we have called a NAALADase- "like" (NAALADase L) peptidase based on its sequence similarity to other members of this gene family, and its inability to hydrolyze NAAG in transient transfection experiments. Furthermore, we describe the cloning of a third novel member of this gene family, NAALADase II, which codes for a type II integral membrane protein and which we have localized to chromosome 11 by fluorescent in situ hybridization analysis. Transient transfection of NAALADase II cDNA confers both NAALADase and dipeptidyl peptidase IV activity to COS cells. Expression studies using reverse transcription-polymerase chain reaction and Northern blot hybridization show that NAALADase II is highly expressed in ovary and testis as well as within discrete brain areas.

N-acetylated alpha-linked acidic dipeptidase is expressed by non-myelinating Schwann cells in the peripheral nervous system

N-acetylated alpha-linked acidic dipeptidase is a membrane-bound brain peptidase which cleaves the neuropeptide N-acetyl-aspartyl-glutamate to N-acetyl-aspartate and glutamate. In the present study, we have determined the localization of N-acetylated alpha-linked acidic dipeptidase in the peripheral nervous system. Using enzyme assays and immunoblotting, we demonstrate that sciatic nerve, phrenic nerve, cervical dorsal root ganglion and superior cervical ganglion contain N-acetylated alpha-linked acidic dipeptidase activity as well as an N-acetylated alpha-linked acidic dipeptidase-like protein. Furthermore, we show that N-acetylated alpha-linked acidic dipeptidase-like immunoreactivity is extensively co-localized in peripheral nerves with immunoreactivity for glial fibrillary acidic protein, a known marker for non-myelinating Schwann cells. Using electron microscopy, we demonstrate N-acetylated alpha-linked acidic dipeptidase-like immunoreactivity in cell membranes of non-myelinating Schwann cells in the superior cervical ganglion. These results show that N-acetylated alpha-linked acidic dipeptidase is expressed in the peripheral nervous system by non-myelinating Schwann cells. This cellular localization suggests that N-acetylated alpha-linked acidic dipeptidase may be involved in the signalling between axons and Schwann cells, for example during development or regeneration.

Rat brain N-acetylated alpha-linked acidic dipeptidase activity. Purification and immunologic characterization.

N-Acetylated alpha-linked acidic dipeptidase (NAALA dipeptidase) is a membrane-bound metallopeptidase that cleaves glutamate from the endogenous neuropeptide N-acetyl-L-aspartyl-L-glutamate. In this report, we have solubilized NAALA dipeptidase activity from synaptosomal membranes with Triton X-100 and purified it to apparent homogeneity by sequential column chromatography on DEAE-Sepharose, CM-Sepharose, and lentil lectin-Sepharose. This procedure resulted in a 720-fold purification with 1.6% yield. The purified ezyme migrated as a single silver-stained band on a sodium dodecyl sulfate gel with an apparent molecular weight of 94 kDa. Using an enzymatic stain to visualize NAALA dipeptidase activity within a gel matrix, we have confirmed that the 94-kDa band is, indeed, NAALA dipeptidase. The purified enzyme was characterized and found to be pharmacologically similar to NAALA dipeptidase activity described previously in synaptosomal membrane extracts. Using the purified NAALA dipeptidase as antigen, we have raised specific and high titer polyclonal antibodies in guinea pig. Immunocytochemical studies show intense NAALA dipeptidase immunoreactivity in the cerebellar and renal cortices.

Hydrolysis of the Brain Dipeptide N‐Acetyl‐l‐Aspartyl‐l‐Glutamate: Subcellular and Regional Distribution, Ontogeny, and the Effect of Lesions on N‐Acetylated‐α‐Linked Acidic Dipeptidase Activity

N-Acetylated-alpha-linked acidic dipeptidase (NAALADase) is a Cl- dependent, membrane bound, metallopeptidase that cleaves the endogenous neuropeptide N-acetyl-L-aspartyl-L-glutamate (NAAG) in vitro. To examine the pattern of NAALADase expression in the CNS, subcellular, regional, and developmental studies were conducted. Subcellular fractionation of lysed synaptosomal membranes revealed a substantial enrichment of the peptidase in synaptic plasma membranes as compared to mitochondrial or myelin subfractions. Regional studies reveal an apparent restriction of peptidase activity to kidney and brain. A threefold variation in specific activity was observed among brain regions, with highest specific activity in the cerebellum and lowest in telencephalic structures, a pattern that does not, in general, correlate with NAAG levels. Ontogenetic studies demonstrate a region-dependent, postnatal pattern of expression of NAALADase activity, with adult levels attained earliest in brainstem, as was previously reported for NAAG. Postnatal NAALADase expression would not appear to support a role for the peptidase in constitutive protein processing, but rather suggests that NAALADase may play a role in synaptic peptide degradation. Glutamate (Glu) excised from NAAG by NAALADase could be transported efficiently by uptake processes. Lesion studies, however, do not support a close structural association between NAALADase activity and the corticostriatal sodium-dependent, high-affinity, Glu uptake system. Similar to in vitro data documenting the route of NAAG degradation by NAALADase, after intrastriatal injection, NAAG was rapidly cleaved to two major products, N-acetyl-aspartate and Glu, with a t1/2 of approximately 10 min. Thus, the route of in vivo catabolism of NAAG parallels results from studies on NAALADase activity in vitro. These results are consistent with a role of NAALADase in the synaptic processing of NAAG. However, certain discrepancies in the regional and ontogenetic profiles of NAAG and NAALADase suggest that this relationship is not an exclusive one and may reflect a role for NAALADase on additional N-acetylated acidic peptides in vivo.

Hydrolysis of the brain dipeptide N-acetyl-L-aspartyl-L-glutamate. Identification and characterization of a novel N-acetylated alpha-linked acidic dipeptidase activity from rat brain.

High performance liquid chromatography studies documented the presence of an enzyme activity, N-acetylated alpha-linked acidic dipeptidase (NAALA dipeptidase), in rat brain membranes that cleaves the endogenous brain dipeptide, N-acetyl-L-aspartyl-L-glutamate to N-acetyl-aspartate and glutamate. With ion exchange chromatography, which quantitatively separated [3,4-3H]glutamate from N-acetyl-L-aspartyl-L-[3,4-3H]glutamate, we found that NAALA dipeptidase activity was essentially restricted to nervous tissue and kidney. We characterized NAALA dipeptidase activity in lysed synaptosomal membranes obtained from rat forebrain. Membrane-bound NAALA dipeptidase activity was optimal between pH 6.0 and 7.4 at 37 degrees C. Eadie-Hofstee analysis of kinetic data revealed a rather high apparent affinity for N-acetyl-L-aspartyl-L-glutamate with a Km = 540 nM and a Vmax = 180 nM/mg of protein/min. While NAALA dipeptidase showed a requirement for monovalent anions such as Cl-, the polyvalent anions phosphate and sulfate inhibited enzyme activity 50% at 100 microM and 1 mM, respectively. The divalent metal ion chelators EGTA, EDTA, and o-phenanthroline completely abolished activity, which was partially restored by manganese. Treatment of membranes with 1 mM dithiothreitol abolished NAALA dipeptidase activity. NAALA dipeptidase activity was also sensitive to the aminopeptidase inhibitors bestatin and puromycin, although not to the selective aminopeptidase A inhibitor amastatin. Structure-activity relationships inferred from inhibitor studies suggest that this enzyme shows specificity for N-acetylated alpha-linked acidic dipeptides. NAALA dipeptidase was also potently inhibited by the excitatory amino acid agonist L-quisqualate. Comparison of the properties of NAALA dipeptidase to those of previously characterized enzymes suggests that this is a novel peptidase which may be involved in the synaptic degradation of N-acetyl-L-aspartyl-L-glutamate.