Abstract:The vulnerability of neurons and the irreversibility of loss make discoveries of neuroprotective compounds fundamentally important. Here, the complete coding sequence of a novel protein (828 amino acids, pI 5.99), derived from mouse neuroglial cells, is revealed. The sequence contained (1) a neuroprotective peptide, NAPVSIPQ, sharing structural and immunological homologies with the previously reported, activity-dependent neurotrophic factor; (2) a glutaredoxin active site; and (3) a zinc binding domain. Gene expression was enriched in the mouse hippocampus and cerebellum and augmented in the presence of the neuropeptide vasoactive intestinal peptide, in cerebral cortical astrocytes. In mixed neuronastrocyte cultures, NAPVSIPQ provided neuroprotection at subfemtomolar concentrations against toxicity associated with tetrodotoxin (electrical blockade), the ␤-amyloid peptide (the Alzheimer's disease neurotoxin), N-methyl-D-aspartate (excitotoxicity), and the human immunodeficiency virus envelope protein. Daily NAPVSIPQ injections to newborn apolipoprotein E-deficient mice accelerated the acquisition of developmental reflexes and prevented short-term memory deficits. Comparative studies suggested that NAPVSIPQ was more efficacious than other neuroprotective peptides in the apolipoprotein E-deficiency model. A potential basis for rational drug design against neurodegeneration is suggested with NAPVSIPQ as a lead compound. The relative enrichment of the novel mRNA transcripts in the brain and the increases found in the presence of vasoactive intestinal peptide, an established neuroprotective substance, imply a role for the cloned protein in neuronal function. Key Words: Vasoactive intestinal peptide-Apolipoprotein E-Learning and memory-Neuronal survival-Molecular cloning-mRNA.

The glycophospholipid-linked, amphiphilic form of acetylcholinesterase (AChE) from Torpedo californica and the hydrophilic form from mouse were overexpressed in Sf9 insect cells using the baculovirus expression system. Recombinant baculovirus, constructed by inserting AChE cDNA's into the genome of Autographa californica nuclear polyhedrosis virus adjacent to the strong polyhedron promoter, yielded recombinant enzyme varying between 0.5 and 3.8 mg/L. The recombinant enzyme was glycosylated although it migrated slightly more rapidly in SDS gel electrophoresis than enzyme purified from the electric organ of Torpedo. Kinetic properties of the recombinant DNA- and tissue-derived enzymes are identical. The detailed catalytic properties and susceptibility to inhibitors were examined for two enzyme mutations of the glutamate residue N-terminal to the active site serine. The Glu199 to Gln mutation shifted both the Km and Kss to higher substrate concentrations and resulted in a kcat of 28% of the wild type. Mutation of Glu199 to Asp also yielded a reduction in kcat but with no change in Km. Substrate inhibition normally apparent in wild-type AChE was eliminated with the Asp mutation, suggesting that substrate catalysis and substrate inhibition are not directly linked. Both mutations decreased the affinity of reversible inhibitors and reduced the rates of phosphorylation and carbamoylation; these changes were more striking with the Gln199 mutation. Decarbamoylation rates were unaffected by these mutations. Glu199 is the charged residue found deep within the active center gorge close to the site of acetylcholine binding, and our findings indicate it influences, but is not essential for, efficient catalysis.

The cholinesterases are serine hydrolases that show no global similarities in sequence with either the trypsin or the subtilisin family of serine proteases. The cholinesterase superfamily includes several esterases with distinct functions and other proteins devoid of the catalytic serine and known esterase activity. To identify the residues involved in catalysis and conferring specificity on the enzyme, we have expressed wild-type Torpedo acetylcholinesterase (EC 3.1.1.7) and several site-directed mutants in a heterologous system. Mutation of serine-200 to cysteine results in diminished activity, while its mutation to valine abolishes detectable activity. Two conserved histidines can be identified at positions 425 and 440 in the cholinesterase family; glutamine replacement at position 440 eliminates activity whereas the mutation at 425 reduces activity only slightly. The assignnment of the catalytic histidine to position 440 defines a rank ordering of catalytic residues in cholinesterases distinct from trypsin and subtilisin and suggests a convergence ofa catalytic triad to form a third, distinct family of serine hydrolases. Mutation of glutamate-199 to glutamine yields an enzyme with a higher Km and without the substrateinhibition behavior characteristic of acetylcholinesterase. Hence, modification of the acidic amino acid adjacent to the serine influences substrate association and the capacity of a second substrate molecule to affect catalysis.Efficiently catalyzed hydrolysis of the neurotransmitter acetylcholine is critical to proper functioning of the cholinergic nervous system, and several pharmacologic and toxicologic agents act by inhibiting acetylcholinesterase (AChE; acetylcholine acetylhydrolase, EC 3.1.1.7). AChE functions as a serine hydrolase (1-3) and enzyme inhibitors used clinically or as insecticides typically serve as alternative substrates by acylating the active-center serine with slower leaving groups. The primary structure of AChE revealed that it lacks global sequence similarities with serine hydrolases of the trypsin and subtilisin families and only possesses residue identity with trypsin immediately around the active-center serine (4). Surprisingly, AChE is homologous to the carboxyl-terminal domain of a large secreted glycoprotein, thyroglobulin, the precursor to thyroid hormone (4). Subsequent primary structures showed that the cholinesterases belong to a distinct, but functionally eclectic, family of serine hydrolases. Included in this family are the butyrylcholinesterases, hepatic microsomal carboxylesterases, the Drosophila Est-6, lysophospholipases, cholesterol esterases, and two proteins found in inclusion bodies of Dictyostelium (5-11).The cholinesterases are characterized by their specificity for choline esters but can be subdivided on the basis of acyl group selectivity. The true AChEs (EC 3.1.1.7) show weak catalytic activity for choline esters with acyl groups larger than propionylcholine; the butyrylcholinesterases (EC 3.1.1.8) efficiently hydrolyze esters with large...

In Parkinson disease, the second most common neurodegenerative disorder in humans, increased alpha-synuclein (SNCA) levels are pathogenic, as evidenced by gene copy number mutations and increased alpha-synuclein levels detected in some familial and sporadic PD cases, respectively. Gene expression can be regulated at the post-transcriptional level by elements in the 3′ untranslated region (3′UTR) of mRNAs. The goal of this study was to determine whether the 3′UTR of human SNCA can affect gene expression. Comparative sequence analysis revealed very high conservation across the entire 3′UTR of human SNCA over millions of years, suggesting the presence of multiple functionally important domains. EST and RT-PCR analyses showed that four different polyadenylation events occur in the 3′UTR of human SNCA. Finally, using luciferase assays, we examined the effect of the minor allele of five naturally occurring single nucleotide polymorphisms (SNPs) in the 3′UTR of SNCA on gene expression. The minor allele of SNP rs17016074 increased luciferase expression by 32% in a transient transfection assay in SHSY5Y neuroblastoma cells. Understanding the role of the 3′UTR of human SNCA and identifying functionally important naturally occurring SNPs using reporter assays can complement disease association studies in humans, uncovering potential susceptibility or protective polymorphisms in Parkinson disease. Our findings demonstrate that the 3′UTR of human SNCA, as a whole, and rs17016074, in particular, are loci of potential clinical importance for Parkinson disease.