Molecular cloning and cellular localization of a furin-like prohormone convertase from the atrial gland of Aplysia.

Prohormone convertases (PCs) are Ca(2+)-dependent subtilisin-related endoproteases that have been implicated in the post-translational processing of prohormones and other proproteins. Furin is an ubiquitously expressed PC that has been shown to hydrolyze a wide variety of precursor proteins in secretory pathways. We have screened an Aplysia atrial gland cDNA library using a furin probe prepared by polymerase chain reaction (PCR) and have isolated an Aplysia furin-related 6.7-kb cDNA partial clone that was truncated on the 5' end. The remaining 5' atrial gland furin nucleotide sequence was obtained by two stages of reverse transcription PCR. The final composite nucleotide sequence of the atrial gland furin cDNA was 7,837 bp in length. This sequence encoded a putative preproendoprotease (Afurin2) of 824 amino acid residues that was related to other eukaryotic furins, and showed a high sequence identity with a recently reported Aplysia nervous system furin-like sequence. In situ hybridization demonstrated extensive expression of Afurin2 in atrial gland secretory cells. The cDNA clone contained a relatively long 3' untranslated region of 5,230 nucleotides that included a microsatellite repeat region (TG)n. The characterized Aplysia Afurin2 is a candidate PC that may play an important role in the processing of egg-laying hormone (ELH)-related precursors in the secretory cells of the atrial gland. In addition, comparative structural studies of Afurin2, together with previously reported localization studies, argue for the occurrence of a furin-like convertase within secretory granules.

Molecular cloning, cDNA sequence, and localization of a prohormone convertase (PC2) from the Aplysia atrial gland.

Neuropeptides and peptide hormones are synthesized as part of larger precursor proteins that are processed post-translationally by subtilisin-related calcium-dependent prohormone convertases (PCs), frequently at multiple basic sites, to generate biologically active peptides. The atrial gland of Aplysia californica produces large quantities of egg-laying hormone (ELH)-related peptides, providing a unique opportunity to study prohormone processing. We have screened an Aplysia atrial gland cDNA library using a Lymnaea stagnalis PC2 probe and have isolated an Aplysia PC2-related 4.6-kb cDNA partial clone that was truncated on the 5' end. The remaining 5' atrial gland PC2 nucleotide sequence was obtained by reverse transcription/polymerase chain reaction (RT-PCR). The composite cDNA structure (5.6 kb) was deduced from sequence analysis of the RT-PCR product combined with the sequence obtained from the cDNA clone. The deduced cDNA of Aplysia atrial gland PC2 encoded a putative preproendoprotease of 653 amino acids that was evolutionarily related to other eukaryotic PC2s, and showed the strongest sequence identity with recently reported Aplysia nervous tissue PC2 sequences. In situ hybridization demonstrated extensive expression of PC2 in atrial gland secretory cells. The cDNA clone contained a relatively long 3'untranslated region (3'-UTR) of 3,632 nucleotides. Strikingly, the 3'-UTR also contained several major nucleotide repeat sequences including the microsatellite repeats, (CA)n and (TG)n, and a TA-rich region comprised largely of the triplet repeat (TTA)n. The characterized Aplysia PC2 is a candidate endoprotease that may play an important role in the processing of ELH-related precursors in the atrial gland and represents the first example of PC2 expression in exocrine tissue.

Structural characterization of a Lymnaea putative endoprotease related to human furin.

A number of peptides have been identified in the central nervous system of the freshwater snail, Lymnaea stagnalis, that function as hormones and neurotransmitters/neuromodulators. These peptides are typically proteolytically processed from larger prohormones mostly at sites composed of single or multiple basic amino acid residues. Previously we demonstrated a diversity of putative prohormone convertases that may be involved in prohormone processing in the Lymnaea brain. In the present report, we have characterized a cDNA clone encoding a putative endoprotease of 837 amino acids. The primary structure of endoprotease (Lfur2) was comparable to that of human furin and contained a putative catalytic domain, a Cys-rich domain, and a transmembrane region. The catalytic domain of Lfur2 demonstrated about 70% residue identity when compared with human furin, PACE4 and Drosophila Dfur1 and dKLIP-1. The Lfur2 gene was expressed in the central nervous system as well as various peripheral tissues of Lymnaea.

Occurrence of a furin-like prohormone processing enzyme in Aplysia neuroendocrine bag cells.

1. Strong evidence is accumulating that the endoproteases which process prohormones at dibasic residue cleavage sites are members of a subtilisin-related class of proteases. 2. Using the polymerase chain reaction (PCR), we have isolated and characterized an Aplysia californica neuroendocrine bag cell cDNA product (270 base pairs) that encodes a sequence which is highly homologous to the subtilisin-related class of processing proteases that includes yeast Kex2, human/mouse/Drosophila furin, human PC2, and mouse PC1/PC3 and PC2. 3. The characterized cDNA PCR product showed the highest degree of residue identity with the furin-related group of proteins (human/mouse furin 71%; Drosophila furin 63%). 4. These results establish that Aplysia contain a subtilisin-like gene and suggest that the expression of this gene may play a role in processing Aplysia precursor proteins in the bag cells and likely also in the exocrine atrial gland. 5. Furthermore, the Aplysia nucleotide sequence results, together with available sequence information from human, mouse, and Drosophila furins, provide reasonable evidence that the furin-like enzymes may represent a separate subclass of the subtilisin-like processing enzymes.

Kinetic analysis of biotinylation of specific residues of peptides by high-performance liquid chromatography.

A procedure employing C18 reversed-phase high-performance liquid chromatography (HPLC) is described for evaluating the kinetics of biotinylation of specific residues of peptides after reaction with N-hydroxysuccinimide esters of biotin. Utilizing this HPLC method, we determined the observed pseudo-first-order reaction rate constant (k'1) of biotinylation of lysyl residues in two model peptides, [biotinyl-Ser108]ProA-egg laying hormone (108-121) and pGlu-Lys-Trp-Ala-Pro, to be 1.22.10(-2)s-1 and 1.08.10(-2)s-1, respectively, in 0.05 M sodium phosphate buffer, pH 8.2, at 25 degrees C. The respective reaction half-lives of the two peptides were 57 s and 64 s. In addition, HPLC analytical methods were established for determining the time-course of hydrolysis of biotinylating reagents at acidic and alkaline pH and for evaluating biotin reagent homogeneity.

N-acylation of Aplysia egg-laying hormone with biotin. Characterization of bioactive and inactive derivatives.

Chemical modification of the egg-laying hormone (ELH) of Aplysia by reaction with the N-hydroxysuccinimide ester of biotin, which contained 6-aminohexanoic acid as spacer, yielded seven distinct derivatives that were readily separated by reversed-phase high performance liquid chromatography. The derivatives were chemically characterized by amino acid compositional analysis, sequence analysis, and mass spectrometry. The seven derivatives resulted from combinations of differential modification of the three amino groups in the ELH molecule located at Ile1 (alpha-NH2), Lys8, and Lys36. Of the seven derivatives formed, only one, monobiotinyl Lys36-ELH, was biologically active in eliciting egg-laying activity and altering the electrophysiological activity of the abdominal ganglion neuron R15 and LB and LC cluster neurons. In addition, evaluation of the time course of biotinylation of ELH revealed that the relative rate of amino group reactivity was epsilon-NH2-Lys36 greater than epsilon-NH2-Lys8 much greater than alpha-NH2-Ile1. The slow rate of reaction of the terminal alpha-amino group suggested that it was relatively inaccessible to biotinylation, possibly due to conformational factors or to ion-pair formation with an unidentified carboxyl group. Loss of bioactivity of ELH monobiotinylated on the alpha-amino group, coupled with the unusually low reactivity of the alpha-amino group, provided strong evidence for the importance of the alpha-amino group in ELH function. Furthermore, the development and availability of a bioactive ELH probe should greatly facilitate the isolation, characterization, and localization of the ELH receptor.

Biotinylated peptides/proteins. I. Determination of stoichiometry of derivatization.

A method is described for the determination of the stoichiometry of biotinylation of peptides and proteins after reaction with an N-hydroxysuccinimide ester of biotin containing the extended spacer arm 6-aminohexanoic acid (NHS-epsilon Ahx-biotin). The method of analysis, based on the quantification of phenylthiocarbamyl derivatives of 6-aminohexanoic acid, is able to measure low picomolar amounts of biotinyl derivative. Analyses were performed using an automated on-line hydrolyzer-derivatizer followed by high-performance liquid chromatography. Compositional analyses determined for known peptides were in excellent agreement with analyses obtained by mass spectrometry. Procedures are also described for the production of biotinylated protein probes that can be labeled reproducibly to contain specific amounts of biotin. The analytical advantage and steric freedom provided by the 6-aminohexanoic acid spacer arm argue strongly for the NHS-epsilon Ahx-biotin reagent to be a reagent of choice for the biotinylation of peptides and proteins.

Aplysia brasiliana neurons R3-R14: primary structure of the myoactive histidine-rich basic peptide and its prohormone.

Neurons R3-R14 of the marine mollusc Aplysia are model neuroendocrine cells thought to regulate cardiovascular activity in vivo. The cells express a gene encoding three peptides--peptides I, II and the histidine-rich basic peptide (HRBP)--each of which has been chemically characterized in Aplysia californica. In the studies presented here, HRBP and its prohormone (proHRBP) were purified from A. brasiliana abdominal ganglion extracts by reversed-phase high-performance liquid chromatography and characterized by amino acid compositional and sequence analyses. ProHRBP was an 85-residue peptide whose sequence was: NH2-Glu-Glu-Val-Phe-Asp-Asp-Thr-Asp-Val-Gly-Asp-Glu-Leu-Thr-Asn-Ala-Leu- Glu-Ser - Val-Leu-Thr-Asp-Leu-Lys-Asp-Lys-Arg-Asp-Ala-Glu-Glu-Pro-Ser-Ala-Phe-Met- Thr-Arg - Leu-Arg-Arg-Gln-Val-Ala-Gln-Met-His-Ile-Trp-Arg-Ala-Asn-His-Asp-Arg-His- His-Ser - Thr-Gly-Ser-Gly-Arg-His-Ser-Arg-Phe-Leu-Thr-Arg-Asn-Arg-Tyr-Gly-Gly-Gly- His-Leu - Ser-Asp-Ala-COOG. It differed from A. californica pro-HRBP at seven of the 85 positions. Compositional and sequence analyses demonstrated that A. brasiliana HRBP was a 43-residue peptide corresponding to residues 43 through 85 of proHRBP, and that a significant proportion of the isolated peptide possessed a blocked NH2 terminus. Although this sequence differed from that of A. californica HRBP at five of 43 residues, the two peptides were approximately equipotent in inducing contractions of A. californica crop muscle in vitro, suggesting that the substituted residues may not be critical for biological activity.

Aplysia californica neurons R3-R14: primary structure of the myoactive histidine-rich basic peptide and peptide I.

The R3-R14 neurons of the marine mollusc Aplysia are neuroendocrine cells that express a gene encoding peptides I, II and histidine-rich basic peptide (HRBP), a myoactive peptide that excites Aplysia heart and enhances gut motility in vitro. Peptide II has been chemically characterized (35), but the complete primary structures of peptide I and HRBP have not been established by amino acid sequence analysis. HRBP, peptide I, and the prohormone (proHRBP) were therefore purified from acid extracts of Aplysia californica neural tissue using sequential gel filtration and reverse-phase high-performance liquid chromatography and chemically characterized. Amino acid sequence analysis demonstrated that HRBP was a 43-residue peptide whose sequence was: less than Glu-Val-Ala-Gln-Met-His-Val-Trp-Arg-Ala-Val-Asn-His-Asp-Arg-Asn-His-Gly- Thr-Gly - Ser-Gly-Arg-His-Gly-Arg-Phe-Leu-Ile-Arg-Asn-Arg-Tyr-Arg-Tyr-Gly-Gly-Gly- His-Leu - Ser-Asp-Ala-COOH. Compositional and sequence analyses of peptide I and proHRBP demonstrated that peptide I was a 26-residue peptide with the following sequence: NH2-Glu-Glu-Val-Phe-Asp-Asp-Thr-Asp-Val-Gly-Asp-Glu-Leu-Thr-Asn-Ala- Leu-Glu-Ser-Val-Leu-Thr-Asp-Phe-Lys-Asp-COOH. These results demonstrated that the pro-HRBP sequence predicted by nucleotide sequence analysis of a cDNA clone (24) was in fact synthesized in R3-R14 neurons. Hydrophilicity and hydrophobicity profiles of preproHRBP, combined with charge distribution profiles and predictive secondary structural analysis, showed that cleavage at dibasic sequences was strongly associated with peaks of hydrophilicity in alpha-helical regions of the preprohormone.