Proteolytic processing of egg-laying hormone-related precursors in Aplysia. Identification of peptide regions critical for biological activity

The atrial gland of the marine mollusk Aplysia californica is an exocrine organ that expresses at least three genes belonging to the egg-laying hormone (ELH) family. In order to study the post-translational processing of the ELH-related gene products in the atrial gland and how it compares to the bag cells, peptides were isolated from the atrial gland and chemically characterized. The A- and B-related precursors were each cleaved in vivo to yield several major and minor peptides including peptides A and B and the ELH-related peptide complexes that caused egg laying. About 13% of the peptide complexes were further enzymically processed by the atrial gland to yield smaller fragments, which included A-AP.A-ELH-(15-36), A-AP.[Ala27]A-ELH-(15-36), and A-AP.[Gln23,Ala27]A-ELH-(16-36), where A-AP is an acidic peptide encoded by the A- and B-related genes and A-ELH is an ELH-related peptide encoded by the A gene. These processed peptide fragments were not active in an egg-laying bioassay, indicating that retention of the 14-residue NH2-terminal segment of the A-ELH-related sequence, or some portion thereof, was critical for the induction of egg laying. Other characterized peptides included two novel 13-residue NH2-terminal peptides, A-NTP and B-NTP, representing residues 22-34 of the A and B precursors, respectively. These two peptides occurred adjacent to the signal peptide region in each precursor, and their characterization established the site of signal peptide cleavage to be the Ser21-Gln22 peptide bond of each precursor. Intermediate peptide fragments (A-NTP-peptide A and B-NTP-peptide B) were also identified indicating that there was a specific ordering in the cleavage of peptide bonds during posttranslational processing. Finally, a new 55-residue atrial gland peptide was also isolated that was not a part of any ELH-related precursor characterized to date.     

Delta-bag cell peptide from the egg-laying hormone precursor of Aplysia. Processing, primary structure, and biological activity

The bag cells of the marine mollusk Aplysia express a gene encoding a 271-residue egg-laying hormone (ELH) precursor that is processed into at least nine peptide products. Four of the peptides have been identified in bag cell releasates and are known to act as nonsynaptic neurotransmitters in the abdominal ganglion. The isolation, primary structure, and proposed biological activity of a fifth peptide product (delta-bag cell peptide (delta-BCP)) from the ELH precursor are described. delta-BCP was established to be a 39-residue peptide: NH2-Asp-Gln-Asp-Glu-Gly-Asn-Phe-Arg-Arg-Phe-Pro-Thr-Asn-Ala-Val-Ser-Met- Ser-Ala-Asp- Glu-Asn-Ser-Pro-Phe-Asp-Leu-Ser-Asn-Glu-Asp-Gly-Ala-Val-Tyr-Gln-Arg- Asp-Leu-COOH. This sequence corresponds to residues 81-119 of the ELH prohormone and shares sequence identity with atrial gland peptides A and B. Significantly, synthetic delta-BCP stimulated Ca2+ uptake into mitochondria of secretory cells in the albumin gland in vitro, suggesting that the peptide regulates the cellular release of perivitelline fluid by the gland. Similar results were obtained with purified peptide A and a shorter version of delta-BCP (delta-BCP-(14-33)). These results indicate that delta-BCP belongs to a family of structurally related peptides with similar pharmacological activities that center at a conserved region of sequence corresponding to delta-BCP-(14-33).     

Evidence for the expression of three genes encoding homologous atrial gland peptides that cause egg laying in Aplysia

Three peptide complexes which can induce egg laying in Aplysia were isolated from the atrial gland of the marine mollusc Aplysia californica and chemically characterized. Amino acid sequence analyses established the covalent structures, including disulfide assignments, of all three dimeric complexes. Each complex consisted of an identical 18-residue peptide (A-AP) which was disulfide-bonded to a 36-residue peptide that was homologous to bag cell egg-laying hormone (ELH). The primary structure of A-AP was determined to be: NH2-Asp-Ser-Asp-Val-Ser-Leu-Phe-Asn-Gly-Asp-Leu-Leu-Pro-Asn-Gly-Arg-Cys- Ser-COOH. The primary structure of one of the three ELH-related peptides (A-ELH) was determined to be NH2-Ile-Ser-Ile-Asn-Gln-Asp-Leu-Lys-Ala-Ile-Thr-Asp-Met-Leu-Leu-Thr-Glu- Gln-Ile-Gln-Ala-Arg-Arg-Arg-Cys-Leu-Asp-Ala-Leu-Arg-Gln-Arg-Leu-Leu-Asp- -Leu-COOH. The two other ELH-related peptides, [Ala27]A-ELH and [Gln23, Ala27]A-ELH, differed from A-ELH at 1 and 2 residues, respectively. Both [Ala27] A-ELH and [Gln23, Ala27]A-ELH were novel peptide sequences representing products of as yet uncharacterized genes within the ELH family. These structural studies provide the first direct chemical evidence that an 18-residue peptide (A-AP) derived from a polypeptide precursor encoded by the A gene, as predicted from nucleotide sequence analysis, occurs in the atrial gland; the Cys17 residue of A-AP is disulfide-bonded to Cys25 of A-ELH; and A-AP also occurs disulfide-bonded to two additional, previously undescribed ELH-related peptides, [Ala27]A-ELH and [Gln23, Ala27]A-ELH.     

The bag cell egg-laying hormones of Aplysia brasiliana and Aplysia californica are identical

Egg laying in the marine molluscan genus Aplysia is elicited by an egg-laying hormone (ELH) which induces ovulation and acts on central neurons to effect egg-laying behavior. ELH, isolated from the A. californica bag cells, and three ELH-related peptides, isolated from the A. californica atrial gland, have been chemically characterized, yet relatively little is known about homologous peptides in other Aplysia species. In these studies, the primary structure of A. brasiliana ELH was determined. Bag cell clusters were extracted in an acidic solution, and the peptides purified by sequential gel filtration and reversed-phase HPLC; ELH was identified by bioassay. Amino acid compositional and sequence analyses demonstrated that the neurohormone was a 36-residue peptide whose sequence was identical to that of A. californica ELH: NH2-Ile-Ser-Ile-Asn-Gln-Asp-Leu-Lys-Ala-Ile-Thr-Asp-Met-Leu-Leu-Thr-Glu- Gln-Ile- Arg-Glu-Arg-Gln-Arg-Tyr-Leu-Ala-Asp-Leu-Arg-Gln-Arg-Leu-Leu-Glu-Lys-COOH .     

Peptide products of the atrial gland are not water-borne reproductive pheromones during egg laying in Aplysia

Mate attraction in Aplysia involves long-distance water-borne signaling via the secretion of the peptide pheromone attractin from the exocrine albumen gland during egg laying. Previous studies have shown that a second exocrine organ, the atrial gland, produces abundant egg-laying hormone (ELH) precursor-related peptides and mollusk-derived growth factor (MDGF), and crude extracts of the atrial gland are attractive in T-maze attraction assays. However, it is not known whether these peptides and proteins are secreted during egg laying. In this report, seawater eluates of freshly laid egg cordons were concentrated and fractionated by C18 RP-HPLC, and the resulting major peaks were examined by amino acid compositional analysis, microsequence analysis, and electrospray mass spectrometry. Concentrated egg cordon eluates were also examined by immunoblot analysis using anti-MDGF antisera as probe. The combined data demonstrated that the atrial gland of Aplysia californica does not secrete detectable levels of either ELH precursor-related peptides or MDGF during egg laying. Although the atrial gland is the last major exocrine organ to make contact with eggs before they are laid, the gland does not appear to secrete water-borne peptide pheromones during egg laying.     

A single gene encodes multiple neuropeptides mediating a stereotyped behavior

Egg laying in Aplysia is characterized by a stereotyped behavioral array which is mediated by several neuroactive peptides. We have sequenced two genes encoding the A and B peptides thought to initiate the egg-laying process, as well as a gene encoding egg-laying hormone (ELH) which directly mediates the behavioral array. The three genes share 90% sequence homology and are representatives of a small multigene family. Each gene encodes a protein precursor in which the active peptides are flanked by internal cleavage sites providing the potential to generate multiple small peptides. Each of the three genes consists of sequences homologous to A or B peptide as well as ELH. Although these genes share significant nucleotide homology, they have diverged such that different member genes express functionally related but nonoverlapping sets of neuroactive peptides in different tissues.     

Atrial gland cells synthesize a family of peptides that can induce egg laying inAplysia

Endogenous peptides induce egg laying in the marine mollusc Aplysia in two ways: egg-laying hormone (ELH) from the neuroendocrine bag cells acts directly, causing the release of eggs from the ovotestis; peptides A and B from the atrial gland act indirectly, activating the bag cells to release ELH. Another atrial gland peptide (egg-releasing hormone; ERH) is a structural and functional hybrid of ELH and peptides A and B; it can act both directly and indirectly to induce egg laying. Atrial glands were incubated in a mixture of 3H-amino acids for 18 h, and the biosynthetically labelled peptides isolated using sequential Sephadex G-50 column chromatography and isoelectric focusing. Radiolabelled peaks were localized and bioassayed in intact animals. Bioactive peaks were then characterized functionally using two additional assays: egg laying in bag cell-less animals (ELH-like peptides) and in vitro induction of bag cell discharge (A- and B-like peptides). ERH-like molecules are active in both assays. Homogeneity of bioactive IEF peaks was assessed by SDS-PAGE. Sephadex G-50 gel filtration of biosynthetically labelled atrial gland extracts reveals two major peptide peaks. Peak D (apparent Mr 6,000) is strongly radiolabelled and contains most of the egg-laying activity, but has a low absorbance at 274 nm. Peak E (apparent Mr 3,500) is weakly labelled and contains a small proportion of the total egg-laying activity, but has a large absorbance at 274 nm. Isoelectric focusing of radiolabelled peptides in peak D reveals seven distinct ELH-like species (pI 5.5, 7.5, 8.5, 8.7, 8.9, 9.1, 9.4), and two peaks (pI 5.9, 8.1) that have both ELH-like and A-/B-like activity. The pI 8.1 peak may result from the comigration of peptide A with ERH or with an unidentified ELH-like peptide. It is not yet clear whether the pI 5.9 activity results from comigration of distinct peptides or from the presence of a previously uncharacterized ERH-like molecule. Isoelectric focusing of radiolabelled peptides in peak E reveals five distinct ELH-like species (pI 7.3, 8.5, 8.7, 9.1, 9.4), and one peak (pI 8.9) with both ELH-like and A-/B-like activity. The pI 8.9 peak may result from the comigration of an ELH-like peptide with peptide B. Three of the ELH-like peptides (pI 8.5, 8.9, 9.1) found in peak E are probably identical to the ELH-like peptides found at the same pI's in peak D.(ABSTRACT TRUNCATED AT 400 WORDS)     

Six peptide wound signals derived from a single precursor protein in Ipomoea batatas leaves activate the expression of the defense gene sporamin

A mixture of three homologous bioactive hydroxyproline-rich glycopeptides (HypSys peptides) of 18 amino acids in length, differing only at two residues, was isolated from leaves of Ipomoea batatas, the common sweet potato. One of the peptides represented over 95% of the isolated isopeptides, which, at 2.5 nm concentration, induced the expression of sporamin, a major defense protein of I. batatas. The sequence of the major isoform was used to synthesize a primer that identified a cDNA encoding a precursor protein. The protein contained six proline-rich regions whose sequences suggested that they might be HypSys defense signals. One of the encoded peptides, called IbHypSys IV, was identical to one of two minor components of the isolated isopeptides, but neither the major isopeptide nor the other minor isoform was found within the precursor. The six peptides encoded by the precursor gene were synthesized but with hydroxyproline residues at positions found in the native isoforms and lacking carbohydrate moieties. All of the peptides were biologically active when supplied to leaves of sweet potato plants. The gene is the first ortholog of the preproHypSys gene family to be found outside of the Solanaceae family, and its encoded peptide precursor is the first example in plants of a precursor protein with six potential peptide defense signals, a scenario only found previously in animals. The data indicate that multiple copies of the HypSys peptides in a single precursor may have an important role in amplifying wound signaling in leaves in response to herbivore attacks.     

Structure of dog and rabbit precursors of atrial natriuretic polypeptides deduced from nucleotide sequence of cloned cDNA

The structure of precursors of dog and rabbit atrial natriuretic polypeptides was determined by nucleotide sequence analysis of cloned cDNA of mRNA encoding the peptides. The dog and rabbit precursors are 149 and 153 residues long having 23- and 25-residue putative signal peptides at their N-termini respectively. The 28-residue peptide with identical sequence to that of human, which has potent natriuretic activity, is located at the C-terminus of the dog precursor. The 28-residue peptide of identical sequence to that of mouse/rat is located at C-terminus of rabbit precursor followed by additional Arg-Arg sequence which is also found in rat/mouse precursors and is apparently removed during processing.     

Localization of structural variations distinguishing I-Ak-related molecules to the alpha 1 and beta 1 domains

The structural variations that distinguish the A molecules encoded by wild-derived H-2 complexes which express Ak-related molecules have been localized into the alpha 1 and beta 1 domains by radiochemical sequence analyses of tryptic peptides. The A alpha subunits of B10.STC90 (Akv1) and W12A (Akv2) differ from those of B10.BR (Ak) in two adjacent tryptic peptides spanning positions 43 to 71 in the alpha 1 domain. The A beta subunit of W12A differs from that of B10.BR in two peptides spanning positions 26 to 29 and 95 to 106. Isoleucine and leucine residues present at positions 28 and 95, respectively, in the B10.BR A beta subunit are not found in the corresponding positions in W12A A beta subunits. Both of these A beta sequence variations are in the beta 1 domain. B10.STC90 A beta subunits are identical to those of W12A except for a structural variation in the beta 1 domain affecting the HPLC retention time of a peptide spanning positions 49 to 63. These results suggest that these A molecules are encoded by closely related class II gene alleles which have diversified by the accumulation of discrete mutations within the exons encoding the alpha 1 and beta 1 domains of the A molecule. Our previous functional analyses of these minor variant A molecules have demonstrated that they are readily distinguished with A molecule-specific alloreactive T lymphocytes. Together, these findings suggest that minor structural variations in the alpha 1 and beta 1 domains of the A molecule can dramatically modify the allodeterminants recognized by alloreactive T lymphocytes.     

Isolation and partial characterization of neuropeptides that mimic prolonged inhibition produced by bag cell neurons in Aplysia.

The bag cell neurons of the marine mollusk Aplysia are part of a neural system that utilizes four neuropeptides as neurotransmitters. The peptides, derived from the egg-laying hormone/bag cell peptide (ELH/BCP) precursor protein, are released during a 20-min burst discharge of the bag cells and produce several types of responses in various abdominal ganglion neurons. In the identified neurons L3 and L6, bag cell activity produces prolonged inhibition that lasts for more than 2 h. One of the bag cell peptides, alpha-BCP, mediates an early component of the inhibition in these neurons. To identify the co-transmitter mediating the prolonged component of inhibition, we purified material from an acid extract of abdominal ganglia using molecular sizing high-pressure liquid chromatography (HPLC) on TSK 250-125 followed by two steps of reverse-phase HPLC on C4 or C18. We isolated three inhibitory factors that mimic the prolonged component of inhibition. Mass spectroscopy and partial amino acid sequence analysis indicate one factor is ELH [2-36], that is, ELH that lacks the first, N-terminal amino acid. This inhibitory activity was similar in potency to that of ELH and is the first to be described for an ELH-related peptide. The two other factors were approximately 3,300 and 4,700 Da and were effective at 10- and 50-fold lower concentration, respectively, than ELH or its fragment. Amino acid composition analysis suggests that they are not derived from the ELH/BCP precursor protein. The 4,700 Da factor is effective at the lowest concentration and produces an effect that lasts as long as 100 min.(ABSTRACT TRUNCATED AT 250 WORDS)     

The first report of kininogen from invertebrates

The hornet possesses highly toxic venom, which is rich in toxin, enzymes, and biologically active peptides. Several bradykinin-like peptides, vespakinins, have been found in wasp venoms since 1970s, but the mode of biosynthesis of these peptides is unknown. In the present study, a vespakinin M was purified from venom of Vespa magnifica. Its primary sequence was established as GRPPGFSPFRID. The cDNA encoding the vespakinin M was cloned from the cDNA library of V. magnifica venom gland. The cDNA structure of vespakinin M was found to contain a coding region of 168 nucleotides. The encoded precursor of vespakinin M is composed of a signal peptide, an acidic peptide, and a mature peptide of vespakinin M. This is the first kininogen from insects; it is also the first kininogen from invertebrates. The cDNA structure encoding vespakinin M suggests that the generation mode of bradykinin-related peptides in wasp is different from amphibian skin and mammalian blood system.     

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.     

Recombination and mutation of class II histocompatibility genes in wild mice

We have compared the tryptic peptide fingerprints of the A alpha, A beta, E alpha, and E beta subunits encoded by four wild-derived H-2 complexes expressing A molecules closely related to Ak. The A molecules encoded by these Ak-related mice have A alpha and A beta subunits that differ from A alpha k and A beta k by less than 10% of their tryptic peptides. Comparisons among the four wild-derived A molecules suggested that these contemporary A alpha and A beta alleles arose by sequential mutational events from common ancestor A alpha and A beta alleles. These results suggest that A alpha and A beta may co-evolve as an A beta A alpha gene duplex in wild mice. Tryptic peptide fingerprint comparisons of the E beta gene linked to these Ak-related A beta A alpha gene duplexes indicate that two encode E beta d-like subunits, whereas another encodes an E beta s-like subunit. These results strongly suggest that the A beta A alpha duplex and E beta recombine in wild mouse populations. The significantly different evolutionary patterns exhibited by the class II genes encoding A vs E molecules are discussed.     

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.     

Peptide YY. Structure of the precursor and expression in exocrine pancreas

Peptide YY is a 36-residue gastrointestinal hormone which inhibits both pancreatic and gastric secretion. We have isolated a cDNA encoding the peptide YY precursor by screening a rat intestinal lambda gt11 cDNA library with an antiserum directed against the porcine hormone. The nucleotide sequence of the cDNA encodes a 98-residue protein (molecular weight, 11, 121) which has an amino acid sequence identical to that of porcine peptide YY. Rat peptide YY is preceded immediately by a signal sequence and followed by a cleavage-amidation sequence Gly-Lys-Arg plus 31 additional amino acids. Thus the peptide YY precursor is similar in structure to that of two related peptides, pancreatic polypeptide and neuropeptide Y. RNA blot hybridizations reveal that the peptide YY gene is much more actively expressed in pancreas than previously realized. In situ hybridizations localized peptide YY cells exclusively to the exocrine pancreas. The abundance of peptide YY in one of its target organs, the pancreas, suggests a paracrine mechanism for peptide YY in regulating pancreatic enzyme secretion.     

Multiple neuropeptides derived from a common precursor are differentially packaged and transported

The ELH prohormone is proteolytically processed into at least nine peptides which govern egg-laying behavior in Aplysia. Quantitative immunocytochemistry demonstrates that peptides derived from the prohormone are packaged into distinct vesicle classes. Further experiments suggest the segregation occurs via a rapid initial proteolytic cleavage of the prohormone followed by sorting at the trans Golgi. Egg-laying hormone (ELH) immunoreactivity is localized to the cell body and processes, while bag cell peptide (BCP) immunoreactivity is greater in the cell body. Steady state levels of the amino-terminal set of peptides including the BCPs are 3- to 8-fold lower than the carboxy-terminal cleavage products, such as ELH. Thus, intracellular packaging and routing of the peptides cleaved from a single prohormone regulate their localization and levels in these neurons.     

Effects of synthetic bag cell and atrial gland peptides on identified nerve cells in Aplysia

We have examined the effects of peptides on the neuroendocrine bag cells, the R2 neuron and the left upper quadrant (LUQ) neurons of the abdominal ganglion of Aplysia californica. Peptides include those extracted from the atrial gland, a reproductive organ; those released by an afterdischarge of the bag cells; and 2 synthetic peptides: the amidated 9-amino acid C-terminal portion of atrial gland peptides A/B/ERH (B26-34), and the 8-amino acid alpha-bag cell peptide (alpha-BCP1-8). Peptides were applied by superfusion, arterial perfusion, pressure ejection from micropipettes, or by inducing a bag cell afterdischarge. Both alpha-BCP1-8 and B26-34 are able to produce a bag cell afterdischarge when applied to the abdominal ganglion but are not as effectively able to trigger the bag cells when applied selectively to the ganglia of the head ring. Peptides released by the bag cells inhibit R2 and LUQ neurons; whereas atrial gland extract mildly excites LUQ neurons and powerfully excites R2. The inhibitory effect of the LUQ cells and R2 following an afterdischarge of the bag cells is mimicked by alpha-BCP1-8. The excitatory effect of the atrial gland extract cannot be duplicated with B26-34. Rather, instead of having an excitatory effect on R2 and LUQ cells, B26-34 seems to mimick alpha-BCP1-8 and inhibit these neurons. Both peptides produce a membrane conductance increase in R2 and LUQ cells.