Carboxypeptidase E (enkephalin convertase): mRNA distribution in rat brain by in situ hybridization

Abstract

Carboxypeptidase E (CPE), also referred to as enkephalin convertase or carboxypeptidase H (EC 3.4.17.10), is present in neurotransmitter secretory granules and can remove C-terminal basic residues following endopeptidase cleavage during peptide processing. Using in situ hybridization with 35S-labeled oligonucleotide probes, we have mapped the localization of CPE mRNA in the rat brain. Specificity for CPE was confirmed by control experiments, which included production of identical patterns hybridization with 3 different antisense oligonucleotide probes, loss of label with RNase pretreatment of sections or co-incubation with excess unlabeled probe, and lack of labeling with sense orientation probes. In addition, the regional distribution of CPE mRNA by Northern blot analysis corresponded with distribution of labeling by in situ hybridization. The highest levels of CPE mRNA were found to be present in the pyramidal cells of the hippocampus, the pituitary anterior and intermediate lobes, the ependymal cells of the lateral ventricle, the endopiriform nucleus, the basolateral amygdala, the supraoptic nucleus, and the paraventricular nucleus. Intermediate levels were present in the thalamus, medial geniculate nucleus, lateral septal nucleus, piriform and entorhinal cortex, nucleus of the tractus solitarius, cerebellar cortex, pontine nuclei, and inferior olive. The lowest levels were found in the hippocampal granule cell layer, lateral hypothalamus, globus pallidus, and brain stem reticular formation. Ibotenic acid lesions of the hippocampus eliminated the majority of the label, which had been present over pyramidal cells, though labeling was increased over areas of reactive gliosis, suggesting that activated astrocytes can also synthesize CPE mRNA. In general, the localization of CPE mRNA in the rat brain corresponded to the distribution of enkephalin and other peptide neurotransmitter-synthesizing neurons, though CPE mRNA was also present in neurons that do not secrete known peptides and in reactive glia. The widespread yet specific localization of CPE mRNA in the rat brain suggests it may be an excellent marker for peptide synthesizing cells in the CNS.

Primary structure and functional expression of a glutaminyl cyclase.

“…Posttranslational processing of peptide hormones may be accomplished by the QC characterized here and may represent part of a regulated pathway of secretion, whereas a different form of the enzyme may be involved in the constitutive secretion of proteins such as immunoglobulins. The observed distribution of QC mRNA in the central nervous system contrasts with the corresponding data reported for the processing enzymes PAM (30) and carboxypeptidase H (31) as well as Kex2-related proteins such as PC1 and PC2 (32), which are considered as putative processing enzymes exhibiting endoproteolytic activity. On the basis of Northern blots, all proteins mentioned above are widely distributed in the brain; however, especially high expression in pituitary and hypothalamus-areas of active peptide synthesis-appears to be a common feature.…”

“…The putative signal sequence and the protein sequence determined by Edman degradation are separated by the sequence (Gly-)Val-Arg-Arg (amino acids [28][29][30][31]. Since a pair of arginine residues is recognized as a typical cleavage site for posttranslational processing, it is suggested that this sequence is a propeptide sequence removed during maturation.…”

Genetic approach for intracerebroventricular delivery

“…Comparing the expression profile of the main processing enzymes between the mouse choroid plexus tissue and the mouse hypothalamus, which served as positive control, we demonstrated the presence of mRNA for all tested enzymes. The presence of mRNA and protein for Furin, CPE, and PAM1 in the rat choroid plexus cells were demonstrated (31)(32)(33)(34). The above expression profile, together with our functional neuroanatomical data (c-Fos activation) and the behavioral and physiological changes, strongly support the capacity of the choroid plexus cells to produce biologically active peptides.…”

Three additional inositol 1,4,5-trisphosphate receptors: molecular cloning and differential localization in brain and peripheral tissues.

“…In addition, all samples were amplified using the same master mixture, which contained all components of the reaction except for the template. (23). Since IP3R-II and IP3R-IV were so similar in the membrane-spanning regions, we used probes specific for the nonconserved region for IP3R-IV localization.…”