Molecular Cloning and Characterization of NESP55, a Novel Chromogranin-like Precursor of a Peptide with 5-HT1B Receptor Antagonist Activity

Abstract

The chromogranins comprise a class of acidic proteins that are secreted from large dense core vesicles and expressed in neuronal and endocrine tissues. We describe here the molecular characterization of NESP55 (neuroendocrine secretory protein of M r 55,000), a novel member of the chromogranins. Several NESP55 cDNA clones were isolated from bovine chromaffin cell libraries. The cDNA sequence of NESP55 totals 1499 nucleotides. All of the clones that were isolated contained in their 3-untranslated mRNA a sequence that was homologous to exon 2 of the G-protein G s ␣. The open reading frame encodes for an acidic and hydrophilic protein of 241 amino acids with a predicted molecular mass of 27,494 Da. An antiserum directed against the C terminus of NESP55 labeled a band of M r 55,000 with an acidic pI ranging from 4.4 to 5.2 in one-and two-dimensional immunoblots of secretory proteins from chromaffin granules. NESP55 is localized within the cell to the large dense secretory vesicles and is expressed, apart from the adrenal medulla, in the anterior and posterior pituitary and various regions of the brain. For the physiological function, one interesting factor has emerged. NESP55 is proteolytically processed within the chromaffin granule to smaller peptides that might be physiologically active. One tetrapeptide, Leu-Ser-Ala-Leu (LSAL), present in the NESP55 sequence and flanked by arginine residues suitable for cleavage by prohormone convertases, has been identified recently as an endogenous antagonist of the serotonergic 5-HT 1B receptor subtype. Alterations in the serotonergic system are thought to play an important role in mental disorders, especially depression, and might be related to abnormal ethanol consumption. It is tempting to speculate that increased expression of NESP55 or its proteolytically derived peptide LSAL might contribute to the pathophysiology of the serotonergic transmission.Synaptic transmission is mediated via exocytotic release of neurotransmitters from presynaptic terminals followed by binding of these substances to specific receptors located on the postsynaptic plasma membrane. In addition to classical neurotransmitters like glutamate, ␥-aminobutyric acid, acetylcholine, noradrenaline, dopamine, or serotonin, several peptides are secreted. These peptides are stored together with neurotransmitters in specialized vesicular containers, i.e. large dense core vesicles. In recent years we characterized the content of large dense core vesicles to a great extent with the intention of identifying novel peptidergic substances involved in chemical signaling across neurons (1, 2). The majority of the proteins found in the content of large dense core vesicles have an acidic pI of 4 -5 and were collectively named chromogranins (3). These proteins typically consist of 200 -700 amino acids with glutamic acid as the most abundant individual amino acid. In their primary amino acid sequence, multiple pairs of consecutive basic amino acid residues known as cleavage sites for trypsin-like endoproteases are present...

Alternative transcripts and evidence of imprinting of GNAL on 18p11.2

“…Despite the very similar gene structures of GNAL and GNAS, the nucleotide sequences of the 5 0 ends of the genes do not share a great degree of identity. One clear difference is that there does not seem to be a transcript analogous to the chromogranin-like NESP55 17,18 in the GNAL locus.…”

Diverse splicing mechanisms fuse the evolutionarily conserved bicistronic MOCS1A and MOCS1B open reading frames

“…(Legend on facing page.) may help to select the appropriate initiating methionine but does not encode, and therefore cannot supply, an initiating methionine (Blumenthal, 1995); therefore, the initiating methionine must be intrinsically provided by the MOCS1B ORF+ Because the invertebrate MOCS1B ORFs lack a candidate initiating methionine that would produce a protein including the presumably essential residues, it is highly unlikely that functional MOCS1B protein can be independently translated in these species+ Though modestly transcribed, Northern analyses in human (Reiss et al+, 1998b), as well as in mouse, opossum, and chicken (Fig+ 3) show a single transcript size consistent with mRNAs containing both ORFs, but not smaller transcripts possibly encoding either MOCS1 subunit alone+ It is therefore likely that in vertebrates, as in invertebrates, that MOCS1B is not independently translated because (1) there is no evidence for a small, MOCS1B-specific transcript in four diverse vertebrates, (2) there is no conserved MOCS1B translation initiation codon (see above), and (3) the conventional scanning ribosome translational model (reviewed in Kozak, 1999) only predicts efficient translation of MOCS1A but not MOCS1B from the bicistronic transcripts+ Consequently, we therefore hypothesize that the only protein translated from the bicistronic Type I splice form is monofunctional MOCS1A protein+ Evidence that MOCS1A protein is indeed translated from the bicistronic (Type I) transcript can be seen by noting that the codons specific to this splice form are particularly well conserved (e+g+, the Gly-Gly C-terminal dipeptide; Fig+ 2C and see below), attesting to their protein-coding function+ We envision that the MOCS1B ORF of the bicistronic transcript constitutes an extended 39 UTR, similar to NESP55-GNAS1 transcripts that are thought to produce only NESP55 protein but not G s a due to the lack of an initiating methionine for the latter ORF (Hayward et al+, 1998;Ischia et al+, 1997;Peters et al+, 1999)+ In conclusion, because phylogenetic (this paper) and human mutation (Reiss et al+, 1998b) data imply that MOCS1B protein must be produced from this locus, we propose that the MOCS1B ORF is only translated from the no-nonsense transcripts (Types II-V) as part of a fused MOCS1A-MOCS1B multifunctional protein (Fig+ 2B)+ Recent studies indicate that gene fusion to form multidomain proteins is relatively common in evolution (Doolittle, 1999;Enright et al+, 1999;Marcotte et al+, 1999aMarcotte et al+, , 1999b, including in other eukaryotic genes involved in Mo metabolism )+ Gene fusion begins with a recombination event juxtaposing two ORFs such that transcription of the upstream ORF continues uninterrupted through the downstream ORF+ Should Figure 1+ The Mmus panel shows the patterns from a variety of tissues: B: brain, H: heart, L: liver, and T: testis+ B: Schematic representation of mRNA splice forms and their respective putative protein products+ Genomic organization of human (exons 8-10), fruit fly (exons 3 and 4), and nematode (exons 5-7) are shown at left+ Green shading indicates MOCS1A sequences and blue designates MOCS1B+ The red vertical line represents a nonsense codon that terminates the MOCS1A ORF+ Roman numerals (I-III) in human designate splice donor choices into exon 10; numer...…”

Loss of XLαs (extra-large αs) imprinting results in early postnatal hypoglycemia and lethality in a mouse model of pseudohypoparathyroidism Ib

“…The Gαs promoter resides within a nonmethylated CpG island, but despite the absence of differential methylation at its promoter, Gαs shows predominantly maternal expression in some tissues, including pituitary, thyroid, renal proximal tubules, and gonads (7-10); Gαs expression is biallelic in most other tissues (11)(12)(13). The furthest upstream alternative promoter generates transcripts that encode the neuroendocrine-specific protein of 55 kDa (NESP55; mouse Nesp55), a chromogranin-like protein, the coding sequence of which is located within a specific upstream exon; Gαs exons 2-13 reside within the 3′ untranslated region of NESP55 transcripts (14). This mRNA shows exclusive maternal expression, because its promoter is methylated on the paternal allele (12,15,16).…”