Biomedical subjects

A B Smit

Publications and source records attributed to A B Smit.

At least 37 records · Page 2Linked to original sources

PubMed1999

A molluscan peptide alpha-amidating enzyme precursor that generates five distinct enzymes.

Mechanisms underlying the specificity and efficiency of enzymes, which modify peptide messengers, especially with the variable requirements of synthesis in the neuronal secretory pathway, are poorly understood. Here, we examine the process of peptide alpha-amidation in individually identifiable Lymnaea neurons that synthesize multiple proproteins, yielding complex mixtures of structurally diverse peptide substrates. The alpha-amidation of these peptide substrates is efficiently controlled by a multifunctional Lymnaea peptidyl glycine alpha-amidating monooxygenase (LPAM), which contains four different copies of the rate-limiting Lymnaea peptidyl glycine alpha-hydroxylating monooxygenase (LPHM) and a single Lymnaea peptidyl alpha-hydroxyglycine alpha-amidating lyase. Endogenously, this zymogen is converted to yield a mixture of monofunctional isoenzymes. In vitro, each LPHM displays a unique combination of substrate affinity and reaction velocity, depending on the penultimate residue of the substrate. This suggests that the different isoenzymes are generated in order to efficiently amidate the many peptide substrates that are present in molluscan neurons. The cellular expression of the LPAM gene is restricted to neurons that synthesize amidated peptides, which underscores the critical importance of regulation of peptide alpha-amidation.

Amino Acid Sequence↗

PubMed1998

The lymnaea cardioexcitatory peptide (LyCEP) receptor: a G-protein-coupled receptor for a novel member of the RFamide neuropeptide family.

A novel G-protein-coupled receptor (GRL106) resembling neuropeptide Y and tachykinin receptors was cloned from the mollusc Lymnaea stagnalis. Application of a peptide extract from the Lymnaea brain to Xenopus oocytes expressing GRL106 activated a calcium-dependent chloride channel. Using this response as a bioassay, we purified the ligand for GRL106, Lymnaea cardioexcitatory peptide (LyCEP), an RFamide-type decapeptide (TPHWRPQGRF-NH2) displaying significant similarity to the Achatina cardioexcitatory peptide (ACEP-1) as well as to the recently identified family of mammalian prolactin-releasing peptides. In the Lymnaea brain, the cells that produce egg-laying hormone are the predominant site of GRL106 gene expression and appear to be innervated by LyCEP-containing fibers. Indeed, LyCEP application transiently hyperpolarizes isolated egg-laying hormone cells. In the Lymnaea pericardium, LyCEP-containing fibers end blindly at the pericardial lumen, and the heart is stimulated by LyCEP in vitro. These data confirm that LyCEP is an RFamide ligand for GRL106.

Action Potentials↗

PubMed1998

Early evolutionary origin of the neurotrophin receptor family.

Neurotrophins and their Trk receptors play a crucial role in the development and maintenance of the vertebrate nervous system, but to date no component of this signalling system has been found in invertebrates. We describe a molluscan Trk receptor, designated Ltrk, from the snail Lymnaea stagnalis. The full-length sequence of Ltrk reveals most of the characteristics typical of Trk receptors, including highly conserved transmembrane and intracellular tyrosine kinase domains, and a typical extracellular domain of leucine-rich motifs flanked by cysteine clusters. In addition, Ltrk has a unique N-terminal extension and lacks immunoglobulin-like domains. Ltrk is expressed during development in a stage-specific manner, and also in the adult, where its expression is confined to the central nervous system and its associated endocrine tissues. Ltrk has the highest sequence identity with the TrkC mammalian receptor and, when exogenously expressed in fibroblasts or COS cells, binds human NT-3, but not NGF or BDNF, with an affinity of 2.5 nM. These findings support an early evolutionary origin of the Trk family as neuronal receptor tyrosine kinases and suggest that Trk signalling mechanisms may be highly conserved between vertebrates and invertebrates.

Amino Acid Sequence↗

PubMed1998

Direct mass spectrometric peptide profiling and sequencing of single neurons reveals differential peptide patterns in a small neuronal network.

Mass spectrometry (MS) was employed to detect and structurally characterize peptides in two functionally related neurons, named VD1 and RPD2, which form a network involved in the modulation of heartbeat in Lymnaea. Matrix-assisted laser desorption/ionization MS, directly applied to single neurons VD1 and RPD2, showed overlapping yet distinct mass profiles, with a subset of putative peptides specifically present in neuron VD1. Direct tandem MS of a single VD1 neuron revealed the primary structures of the VD1-specific peptides, which were identified as members of the family of small cardioactive peptides. Based on the tandem MS data, a degenerate oligonucleotide was made for use in a polymerase chain reaction strategy to isolate the cDNA encoding the precursor to the small cardioactive peptides from a brain-specific cDNA library. The calculated masses of the mature, posttranslationally modified peptides, as predicted from the corresponding cDNA, agreed with the measured masses of the actual peptides, as detected in single-cell MS analysis. In situ hybridization studies showed that the transcript encoding the precursor is present in VD1, but not in RPD2, thus corroborating the single-cell MS analysis. Finally, the small cardioactive peptides were shown to enhance the contractions of the auricle in vitro.

Amino Acid Sequence↗

PubMed1998

Towards understanding the role of insulin in the brain: lessons from insulin-related signaling systems in the invertebrate brain.

Insulin is a molecule that has played a key role in several of the most important landmarks in medical and biological research. It is one of the most extensively studied protein hormones, and its structure and function have been elucidated in many vertebrate species, ranging from man to hagfish and turkey. The structure, function as well as tissue of synthesis of vertebrate insulins are strictly conserved. The structural identification of insulin-related peptides from invertebrates has disrupted the picture of an evolutionary stable peptide hormone. Insulin-related peptides in molluscs and insects turned out to be a structurally diverse group encoded by large multi-gene families that are uniquely expressed in the brain and serve functions different from vertebrate insulin. In this review, we discuss invertebrate insulins in detail. We examine how these peptides relate to the model role that vertebrate insulin has played over the years; however, more importantly, we discuss several unique principles that can be learned from them. We show how diversity of these peptides is generated at the genetic level and how the structural diversity of the peptides is linked to the exclusive presence of a single type of neuronal insulin receptor-related receptor. We also discuss the fact that the invertebrate peptides, in addition to a hormonal role, may also act in a synaptic and/or nonsynaptic fashion as transmitters/neuromodulators on neurons in the brain. It can be expected that the use of well-defined neuronal preparations in invertebrates may lead to a further understanding of these novel functions and may act as guide preparations for a possible role of insulin and its relatives in the vertebrate brain.

Amino Acid Sequence↗

PubMed1997

Altered gene expression in the host brain caused by a trematode parasite: neuropeptide genes are preferentially affected during parasitosis.

Schistosome parasites adjust the physiology and behavior of their intermediate molluscan hosts to their own benefit. Previous studies demonstrated effects of the avian-schistosome Trichobilharzia ocellata on peptidergic centers in the brain of the intermediate snail host Lymnaea stagnalis. In particular, electrophysiological properties and peptide release of growth- and reproduction-controlling neuroendocrine neurons were affected. We now have examined the possibility that the expression of genes that control physiology and behavior of the host might be altered during parasitosis. A cDNA library of the brain of parasitized Lymnaea was constructed and differentially screened by using mRNA from the brain of both parasitized and nonparasitized snails. This screening yielded a number of clones, including previously identified cDNAs as well as novel neuronal transcripts, which appear to be differentially regulated. The majority of these transcripts encode neuropeptides. Reverse Northern blot analysis confirmed that neuropeptide gene expression is indeed affected in parasitized animals. Moreover, the expression profiles of 10 transcripts tested showed a differential, parasitic stage-specific regulation. Changes in expression could in many cases already be observed between 1.5 and 5 hr postinfection, suggesting that changes in gene expression are a direct effect of parasitosis. We suggest that direct regulation of neuropeptide gene expression is a strategy of parasites to induce physiological and behavioral changes in the host.

Amino Acid Sequence↗

PubMed1997

Deletion of two exons from the Lymnaea stagnalis beta1-->4-N-acetylglucosaminyltransferase gene elevates the kinetic efficiency of the encoded enzyme for both UDP-sugar donor and acceptor substrates.

Lymnaea stagnalis UDP-GlcNAc:GlcNAcbeta-R beta1-->4-N-acetylglucosaminyltransferase (beta4-GlcNAcT) is an enzyme with structural similarity to mammalian UDP-Gal:GlcNAcbeta-R beta1-->4-galactosyltransferase (beta4-GalT). Here, we report that also the exon organization of the genes encoding these enzymes is very similar. The beta4-GlcNAcT gene (12.5 kilobase pairs, spanning 10 exons) contains four exons, encompassing sequences that are absent in the beta4-GalT gene. Two of these exons (exons 7 and 8) show a high sequence similarity to part of the preceding exon (exon 6), suggesting that they have originated by exon duplication. The exon in the beta4-GalT gene, corresponding to beta4-GlcNAcT exon 6, encodes a region that has been proposed to be involved in the binding of UDP-Gal. The question therefore arose, whether the repeating sequences encoded by exon 7 and 8 of the beta4-GlcNAcT gene would determine the specificity of the enzyme for UDP-GlcNAc, or for the less preferred UDP-GalNAc. It was found that deletion of only the sequence encoded by exon 8 resulted in a completely inactive enzyme. By contrast, deletion of the amino acid residues encoded by exons 7 and 8 resulted in an enzyme with an elevated kinetic efficiency for both UDP-sugar donors, as well as for its acceptor substrates. These results suggest that at least part of the donor and acceptor binding domains of the beta4-GlcNAcT are structurally linked and that the region encompassing the insertion contributes to acceptor recognition as well as to UDP-sugar binding and specificity.

Amino Acid Sequence↗

PubMed1997

Identification of a molluscan homologue of the neuroendocrine polypeptide 7B2.

In vertebrates, interaction of prohormone convertase 2 (PC2) with the highly conserved polypeptide 7B2 is essential for transport and maturation of proPC2 in the regulated secretory pathway. In vitro, 7B2 displays a strong inhibitory activity toward PC2. Here, we characterize a cDNA encoding the first invertebrate 7B2-related protein (L7B2) from the brain of the mollusc Lymnaea stagnalis. The overall amino acid sequence identity between L7B2 and its vertebrate counterparts is surprisingly low (29%) and is restricted to a few small stretches of amino acid residues. Of particular interest are a conserved proline-rich region in the middle portion of the L7B2 sequence and a repeated conserved region in the carboxyl-terminal domain. Synthetic peptides corresponding to the carboxyl-terminal regions inhibit Lymnaea PC2 enzyme activity in extracts of insulin-producing neurons, in which both L7B2 and Lymnaea PC2 are abundantly expressed. Moreover, the peptides inhibit mouse PC2 enzyme activity. Our cloning of invertebrate 7B2 helps to delineate residues that are important for 7B2-PC2 interaction.

Amino Acid Sequence↗

PubMed1997

De novo protein synthesis in isolated axons of identified neurons.

Neurons are highly polarized cells that contain a wealth of cytoplasmic and membrane proteins required for neurotransmission, synapse formation and various forms of neuronal plasticity. Typically, these proteins are differentially distributed over somatic, dendritic and axonal compartments. Until recently, it was believed that all proteins destined for various neuronal sites were synthesized exclusively in the somata and were subsequently targeted to appropriate extrasomal compartments. The discovery of various messenger RNA molecules in both dendrites and axons is suggestive of de novo protein synthesis in extrasomatic regions. The latter process has been demonstrated in few neuronal svrstems, but direct proof for the axonal transcription of a specific protein from a given messenger RNA is still lacking. This lack of fundamental knowledge in the field of cellular and molecular neurobiology is due primarily to both anatomical and experimental difficulties encountered in most animal preparations studied thus far. In this study we developed a neuronal experimental system comprising of individually identified neurons and their isolated axons from the mollusc Lymnaea stagnalis. We injected a foreign messenger RNA encoding a peptide precursor into the isolated axons of cultured neurons; and utilizing cellular, molecular and immunocytochemical techniques, we provide direct evidence for specific protein synthesis in isolated axons. The Lymnaea model provides us with an opportunity to examine the role and specificity of de novo protein synthesis in the extrasomal regions.

Animals↗

PubMed1997

Plasticity in fast synaptic inhibition of adult oxytocin neurons caused by switch in GABA(A) receptor subunit expression.

We found that magnocellular oxytocin neurons in adult female rats exhibit an endogenous GABA(A) receptor subunit switch around parturition: a decrease in alpha1:alpha2 subunit mRNA ratio correlated with a decrease in allopregnanolone potentiation and increase in decay time constant of the GABA(A) receptor-mediated IPSCs in these cells. The causal relationship between changes in alpha1:alpha2 mRNA ratio and the ion channel kinetics was confirmed using in vitro antisense deletion. Further, GABA(A) receptors exhibited a tonic inhibitory influence upon oxytocin release in vivo, and allopregnanolone helped to restrain oxytocin neuron in vitro firing only before parturition, when the alpha1:alpha2 subunit mRNA ratio was still high. Such observations provide evidence for the physiological significance of GABA(A) receptor subunit heterogeneity and plasticity in the adult brain.

Animals↗

PubMed1996

Cell type-specific sorting of neuropeptides: a mechanism to modulate peptide composition of large dense-core vesicles.

The CNS of Lymnaea stagnalis contains two populations of egg-laying hormone (ELH)-producing neurons that differ in size and topology. In type I neurons, all peptides located C-terminally from the cleavage site Arg-Ser-Arg-Arg180-183 are sorted into secretory large dense-core vesicles (LDCV), whereas N-terminal-located peptides accumulate in a distinct type of vesicle, the large electrondense granule (LEG). Via immunoelectron microscopy, we now show that the second population of ELH-producing neurons, type II neurons, lack LEG and incorporate all proELH-derived peptides into LDCV. This finding provides the first example of a cell type-specific sorting of neuropeptides into LDCV. Furthermore, we provide evidence that LEG are formed through a differential condensation process in the trans-Golgi network and that these bodies are ultimately degraded. Analysis of the endoprotease composition of the two types of proELH-producing neurons suggests that the formation of LEG, and consequently the retention of N-terminal peptides from the secretory pathway, requires the action of a furin-like protein.

Animals↗

PubMed1996

CRNF, a molluscan neurotrophic factor that interacts with the p75 neurotrophin receptor.

A 13.1-kilodalton protein, cysteine-rich neurotrophic factor (CRNF), was purified from the mollusk Lymnaea stagnalis by use of a binding assay on the p75 neurotrophin receptor. CRNF bound to p75 with nanomolar affinity but was not similar in sequence to neurotrophins or any other known gene product. CRNF messenger RNA expression was highest in adult foot subepithelial cells; in the central nervous system, expression was regulated by lesion. The factor evoked neurite outgrowth and modulated calcium currents in pedal motor neurons. Thus, CRNF may be involved in target-derived trophic support for motor neurons and could represent the prototype of another family of p75 ligands.

Amino Acid Sequence↗

PubMed1996

Co-evolution of ligand-receptor pairs in the vasopressin/oxytocin superfamily of bioactive peptides.

In order to understand the molecular mechanisms that underlie the co-evolution of related yet functionally distinct peptide-receptor pairs, we study receptors for the vasopressin-related peptide Lys-conopressin in the mollusc Lymnaea stagnalis. In addition to a previously cloned Lys-conopressin receptor (LSCPR1), we have now identified a novel Lys-conopressin receptor subtype, named LSCPR2. The two receptors have a differential distribution in the reproductive organs and the brain, which suggests that they are involved in the control of distinct aspects of reproduction and mediate transmitter-like and/or modulatory effects of Lys-conopressin on different types of central neurons. In contrast to LSCPR1, LSCPR2 is maximally activated by both Lys-conopressin and Ile-conopressin, an oxytocin-like synthetic analog of Lys-conopressin. Together with a study of the phylogenetic relationships of Lys-conopressin receptors and their vertebrate counterparts, these data suggest that LSCPR2 represents an ancestral receptor to the vasopressin/oxytocin receptor family in the vertebrates. Based on our findings, we provide a theory of the molecular co-evolution of the functionally distinct ligand-receptor pairs of the vasopressin/oxytocin superfamily of bioactive peptides.

Amino Acid Sequence↗

PubMed1996

A new Ig-superfamily member, molluscan defence molecule (MDM) from Lymnaea stagnalis, is down-regulated during parasitosis.

To survive the attacks of the internal defence system (IDS) of their host, parasites have developed various strategies to manipulate the IDS. We present evidence that the avian schistosome parasite Trichobilharzia ocellata affects gene expression in the granular cells, a cell type of the IDS of the intermediate host, the mollusc Lymnaea stagnalis. From a differential screening, a clone was isolated encoding a protein named molluscan defence molecule (MDM), which encompasses five C2-like immunoglobulin (Ig) domains. The protein shares a domain organization and high amino acid sequence identity with hemolin, an Ig-family member of the insect IDS. Interestingly, both MDM and hemolin have highest sequence identity with neural cell adhesion molecules, but lack the typical fibronectin repeats and motifs for membrane anchors. We find that the expression of the MDM gene is gradually down-regulated during the course of parasitosis to approximately 21% compared to the non-parasitized level, 8 weeks post-infection. Based on our findings, we suggest that MDM is involved in the proper function of the Lymnaea IDS, and that down-regulation of MDM is part of the parasite-induced disabling on non-self recognition.

Amino Acid Sequence↗

PubMed1996

Expression and characterization of molluscan insulin-related peptide VII from the mollusc Lymnaea stagnalis.

A complementary DNA clone encoding molluscan insulin-related peptide VII was identified from a complementary DNA library of the cerebral ganglia of the CNS of the freshwater snail, Lymnaea stagnalis. The novel molluscan insulin-related peptide VII complementary DNA encodes a preprohormone resembling the organization of preproinsulin, with a putative signal sequence, and an A and B chain, and is connected by an unusual long C peptide. The A and B chains, as well as the C peptide of molluscan insulin-related peptide VII, differ remarkably in primary structure with the previously identified molluscan insulin-related peptides. The C peptide of molluscan insulin-related peptide VII shares no significant sequence identity with counterparts in other molluscan insulin-related peptides. Both molluscan insulin-related peptide VII and the other molluscan insulin-related peptides exhibit structural features which make them a unique class of the insulin superfamily. Molluscan insulin-related peptide VII complementary DNA was shown to hybridize in situ with messenger RNA present in the cerebral light green cells, neuroendocrine cells that control growth and that have previously been shown to produce molluscan insulin-related peptides I-III and V. Uniquely, the molluscan insulin-related peptide VII gene is also expressed in neurons that may form part of the feeding circuitry in Lymnaea, indicating that it may function as a neurotransmitter/neuromodulator.

Amino Acid Sequence↗

PubMed1995

A novel G protein-coupled receptor mediating both vasopressin- and oxytocin-like functions of Lys-conopressin in Lymnaea stagnalis.

We have cloned a receptor, named LSCPR, for vasopressin-related Lys-conopressin in Lymnaea stagnalis. Lys-conopressin evokes Ca(2+)-dependent Cl- currents in Xenopus oocytes injected with LSCPR cRNA. Expression of LSCPR mRNA was detected in central neurons and peripheral muscles associated with reproduction. Upon application of Lys-conopressin, both neurons and muscle cells depolarize owing to an enhancement of voltage-dependent Ca2+ currents and start firing action potentials. Some neurons coexpress LSCPR and Lys-conopressin, suggesting an autotransmitter-like function for this peptide. Lys-conopressin also induces a depolarizing response in LSCPR-expressing neuroendocrine cells that control carbohydrate metabolism. Thus, in addition to oxytocin-like reproductive functions, LSCPR mediates vasopressin-like metabolic functions of Lys-conopressin as well.

Amino Acid Sequence↗

PubMed1995

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.

Amino Acid Sequence↗

PubMed1995

Structural and functional evolution of the vasopressin/oxytocin superfamily: vasopressin-related conopressin is the only member present in Lymnaea, and is involved in the control of sexual behavior.

It has been suggested that the gene duplication that led to the formation of the vasopressin/oxytocin two-gene family occurred early during vertebrate evolution. However, the existence of both vasopressin- and oxytocin-related peptides in invertebrates suggests that this duplication may have occurred much earlier, although there is no evidence for the co-occurrence of vasopressin- and oxytocin-related peptides in the same invertebrate species. We report here that in Lymnaea only the vasopressin-related peptide Lys-conopressin, but not an oxytocin-related peptide, is present. Moreover, it is very likely that an oxytocin-like cDNA or gene is absent. The conopressin gene is expressed in neurons that control male sexual behavior, and its gene products are present in the penis nerve and the vas deferens. Conopressin induces muscular contractions of the vas deferens and inhibits central neurons that control female reproductive behavior. Thus, although structurally related to vasopressin, conopressin has functional and behavioral characteristics typical for oxytocin. Physiological and receptor binding data suggest that conopressin and [Ile8]-conopressin, a synthetic oxytocin-like analog of conopressin, are functionally equivalent in Lymnaea, and that the chemical nature of the amino acid residue at position 8 does not result in a functional difference. Therefore, we suggest that invertebrates contain only a single member of the vasopressin/oxytocin gene family and that the amino acid change that distinguishes vasopressin from oxytocin is functionally neutral in invertebrates.

Amino Acid Sequence↗