Gregg T. Nagle

Primary and secondary structural analyses of glutathione S-transferase π from human placenta☆

Abstract The primary structure of glutathione S-transferase (GST) π from a single human placenta was determined. The structure was established by chemical characterization of tryptic and cyanogen bromide peptides as well as automated sequence analysis of the intact enzyme. The structural analysis indicated that the protein is comprised of 209 amino acid residues and gave no evidence of post-translational modifications. The amino acid sequence differed from that of the deduced amino acid sequence determined by nucleotide sequence analysis of a cDNA clone (Kano, T., Sakai, M., and Muramatsu, M., 1987, Cancer Res. 47 , 5626–5630) at position 104 which contained both valine and isoleucine whereas the deduced sequence from nucleotide sequence analysis identified only isoleucine at this position. These results demonstrated that in the one individual placenta studied at least two GST π genes are coexpressed, probably as a result of allelomorphism. Computer assisted consensus sequence evaluation identified a hydrophobic region in GST π (residues 155–181) that was predicted to be either a buried transmembrane helical region or a signal sequence region. The significance of this hydrophobic region was interpreted in relation to the mode of action of the enzyme especially in regard to the potential involvement of a histidine in the active site mechanism. A comparison of the chemical similarity of five known human GST complete enzyme structures, one of π, one of μ, two of α, and one microsomal, gave evidence that all five enzymes have evolved by a divergent evolutionary process after gene duplication, with the microsomal enzyme representing the most divergent form.

Molecular cloning of a cDNA encoding the neuropeptides APGWamide and cerebral peptide 1: Localization of APGWamide-like immunoreactivity in the central nervous system and male reproductive organs of Aplysia

While much is known about the neural and endocrine mechanisms that control egg laying in the gastropod mollusk Aplysia, relatively little is known about the regulation of male reproductive activity in this simultaneous hermaphrodite. In the present study, we have cloned and sequenced a cDNA that encodes a precursor protein, the predicted posttranslational processing of which presumably generates nine copies of the neuropeptide Ala‐Pro‐Gly‐Trp‐NH2 (APGWamide), five connecting peptide sequences, and a C‐terminal peptide. The sequence of one connecting peptide is identical to the previously characterized cerebral peptide 1. Northern blot analysis identified two major APGWamide mRNA transcripts (∼1.3 kb, ∼2.4 kb), which were present in central nervous system ganglia, but were most abundant in the right cerebral and right pedal ganglia. Immunohistochemical studies using sexually mature Aplysia demonstrated that the vast majority of APGWamide‐like immunoreactivity was localized in 30–40 neurons along the anterior and medial margins of the right cerebral ganglion and in a cluster of 15–20 neurons in the right pedal ganglion. A total of only about ten immunoreactive neurons were located in other ganglia. Immunohistochemistry also demonstrated that APGWamide was present in the reproductive organs that participate in the storage or transport of sperm, including the small hermaphroditic duct (site of sperm storage before mating), the white hemiduct (also known as the copulatory duct), and penial complex. As a group, these data suggest that APGWamide may play a role in regulating male reproductive function in Aplysia, as it does in other gastropods. J. Comp. Neurol. 387:53–62, 1997.

Male accessory gland protein reduces egg laying in a simultaneous hermaphrodite.

Seminal fluid is an important part of the ejaculate of internally fertilizing animals. This fluid contains substances that nourish and activate sperm for successful fertilization. Additionally, it contains components that influence female physiology to further enhance fertilization success of the sperm donor, possibly beyond the recipients optimum. Although evidence for such substances abounds, few studies have unraveled their identities, and focus has been exclusively on separate-sex species. We present the first detailed study into the seminal fluid composition of a hermaphrodite (Lymnaea stagnalis). Eight novel peptides and proteins were identified from the seminal-fluid-producing prostate gland and tested for effects on oviposition, hatching and consumption. The gene for the protein found to suppress egg mass production, Ovipostatin, was sequenced, thereby providing the first fully-characterized seminal fluid substance in a simultaneous hermaphrodite. Thus, seminal fluid peptides and proteins have evolved and can play a crucial role in sexual selection even when the sexes are combined.

Induction of copulatory behavior in Aplysia: Atrial gland factors mimic the excitatory effects of freshly deposited egg cordons

Egg laying in the marine mollusc Aplysia is induced and coordinated by peptide products of the egg-laying hormone (ELH) gene expressed in the neuroendocrine bag cells of the central nervous system. At least three structurally related genes, belonging to the ELH family but distinct from the ELH gene, are expressed in the atrial gland, an exocrine organ of unknown function that secretes into the oviduct of Aplysia. The experiments described in this report were designed to test the hypothesis that the atrial gland gene products serve a pheromonal function for the animal, coordinating reproductive behavior among individuals. Our studies showed that there was a significantly shorter latency to copulation when an Aplysia was paired with an animal that was actively laying eggs than when it was paired with a sexually mature but nonlaying animal. Moreover, the addition of extracts or homogenates of the atrial gland to the seawater surrounding two nonlaying animals reduced the latency to mating compared to animals exposed only to seawater or to homogenates of other regions of the reproductive tract, including oviduct. These results suggest that atrial gland products, secreted onto the egg cordon as it passes through the oviduct, may play a pheromonal role and induce mating behavior between individuals. Experiments are in progress to determine whether the active atrial gland factor(s) are products of the ELH-family genes expressed in the gland.

Extreme aggression in male squid induced by a β-MSP-like pheromone.

Male-male aggression is widespread in the animal kingdom and subserves many functions related to the acquisition or retention of resources such as shelter, food, and mates. These functions have been studied widely in the context of sexual selection, yet the proximate mechanisms that trigger or strengthen aggression are not well known for many taxa. Various external sensory cues (visual, audio, chemical) acting alone or in combination stimulate the complex behavioral interactions of fighting behaviors. Here we report the discovery of a 10 kDa protein, termed Loligo β-microseminoprotein (Loligo β-MSP), that immediately and dramatically changes the behavior of male squid from calm swimming and schooling to extreme fighting, even in the absence of females. Females synthesize Loligo β-MSP in their reproductive exocrine glands and embed the protein in the outer tunic of egg capsules, which are deposited on the open sea floor. Males are attracted to the eggs visually, but upon touching them and contacting Loligo β-MSP, they immediately escalate into intense physical fighting with any nearby males. Loligo β-MSP is a distant member of the chordate β-microseminoprotein family found in mammalian reproductive secretions, suggesting that this gene family may have taxonomically widespread roles in sexual competition.

Thermodynamic characterization of interleukin-8 monomer binding to CXCR1 receptor N-terminal domain.

Chemokines elicit their function by binding receptors of the G‐protein‐coupled receptor class, and the N‐terminal domain (N‐domain) of the receptor is one of the two critical ligand‐binding sites. In this study, the thermodynamic basis for binding of the chemokine interleukin‐8 (IL‐8) to the N‐domain of its receptor CXCR1 was characterized using isothermal titration calorimetry. We have shown previously that only the monomer of IL‐8, and not the dimer, functions as a high‐affinity ligand, so in this study we used the IL‐8(1–66) deletion mutant which exists as a monomer. Calorimetry data indicate that the binding is enthalpically favored and entropically disfavored, and a negative heat capacity change indicates burial of hydrophobic residues in the complex. A characteristic feature of chemokine receptor N‐domains is the large number of acidic residues, and experiments using different buffers show no net proton transfer, indicating that the CXCR1 N‐domain acidic residues are not protonated in the binding process. CXCR1 N‐domain peptide is unstructured in the free form but adopts a more defined structure in the bound form, and so binding is coupled to induction of the structure of the N‐domain. Measurements in the presence of the osmolyte, trimethylamine N‐oxide, which induces the structure of unfolded proteins, show that formation of the coupled N‐domain structure involves only small ΔH and ΔS changes. These results together indicate that the binding is driven by packing interactions in the complex that are enthalpically favored, and are consistent with the observation that the N‐domain binds in an extended form and interacts with multiple IL‐8 N‐loop residues over a large surface area.

Molecular cloning of a cDNA encoding a potential water-borne pheromonal attractant released during Aplysia egg laying

Recently deposited egg cordons are a source of water-borne pheromones that attract the marine mollusk Aplysia into breeding aggregations and coordinate male and female reproductive behavior within the aggregation. A potential pheromonal attractant has been isolated from egg cordon eluates and the peptide partially characterized [S.D. Painter, B. Clough, X. Fan, G.T. Nagle, Soc. Neurosci. Abstr., Vol. 22 (1996) 837]. Using this information, we have cloned an Aplysia albumen gland cDNA that encodes a precursor protein containing a single copy of the full-length peptide, and demonstrated that there are abundant levels of pheromone mRNA transcripts (0.8 and 2.5 kb) in the albumen gland. This is consistent with the reported function of the gland (i.e. packaging the eggs into a cordon for deposition), with behavioral studies showing that the albumen gland is a potential source of attractants, and more recent biochemical studies in which the full-length peptide has been isolated from the albumen gland. This is the first candidate peptide pheromone in mollusks and the first in invertebrates. The pheromonal regulatory system in Aplysia may provide a model system for examining the structural characteristics of peptide pheromones.

Aplysia Attractin: Biophysical Characterization and Modeling of a Water-Borne Pheromone

Attractin, a 58-residue protein secreted by the mollusk Aplysia californica, stimulates sexually mature animals to approach egg cordons. Attractin from five different Aplysia species are approximately 40% identical in sequence. Recombinant attractin, expressed in insect cells and purified by reverse-phase high-performance liquid chromatography (RP-HPLC), is active in a bioassay using A. brasiliana; its circular dichroism (CD) spectrum indicates a predominantly alpha-helical structure. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) characterization of proteolytic fragments identified disulfide bonds between the six conserved cysteines (I-VI, II-V, III-IV, where the Roman numeral indicates the order of occurrence in the primary sequence). Attractin has no significant similarity to any other sequence in the database. The protozoan Euplotes pheromones were selected by fold recognition as possible templates. These diverse proteins have three alpha-helices, with six cysteine residues disulfide-bonded in a different pattern from attractin. Model structures with good stereochemical parameters were prepared using the EXDIS/DIAMOD/FANTOM program suite and constraints based on sequence alignments with the Euplotes templates and the attractin disulfide bonds. A potential receptor-binding site is suggested based on these data. Future structural characterization of attractin will be needed to confirm these models.

Newly identified water-borne protein pheromones interact with attractin to stimulate mate attraction in Aplysia

The water-borne protein attractin is a potent sex pheromone involved in forming and maintaining mating and egg-laying aggregations in the marine mollusk Aplysia. Binary blends of attractin and either enticin, temptin, or seductin, three other Aplysia protein pheromones, stimulate mate attraction. The four pheromones are thought to act in concert during egg-laying. The new data presented here show that: (1) the water-borne odor of non-laying Aplysia brasiliana further increases the attractiveness of attractin and of eggs in T-maze bioassays. This suggests that individual Aplysia release additional factors that enhance the effects of attractin, enticin, temptin, and seductin during egg-laying; (2) the N-terminal region of enticin aligns well with the conserved epidermal growth factor (EGF)-like domain of mammalian reproductive proteins known as fertilins, which may mediate intercellular adhesion interactions between eggs and sperm; (3) temptin, according to fold recognition servers, may also have an EGF-like fold. Enticin and temptin also have conserved metal binding sequences that may play a role in their signaling behavior. These results suggest that aspects of mammalian egg-sperm interactions (fertilins) may have evolved from pheromonal signaling mechanisms. We also review the structure, expression, localization, release, and behavioral actions of attractin, enticin, temptin, and seductin.

Molluscan attractins, a family of water-borne protein pheromones with interspecific attractiveness

The marine mollusk Aplysia releases the water-borne pheromone attractin during egg laying. This small protein stimulates the formation and maintenance of mating and egg-laying aggregations. Attractin has been characterized from five Aplysia species: A. californica, A. brasiliana, A. fasciata, A. vaccaria, and A. depilans. We describe here the isolation of attractin from Bursatella leachii, and show that it belongs to the same protein family. The pattern of residue conservation, especially the six invariant cysteines, suggests that all of these attractins have a common fold. The nuclear magnetic resonance solution structure of A. californica attractin contains two antiparallel alpha-helices, the second of which contains the heptapeptide sequence IEECKTS that has been implicated in attractin function. Synthetic peptides containing this IEECKTS region are attractive, and mutating surface exposed charged residues within this region of attractin abolishes attractin activity. This suggests that the second helix is an essential part of the receptor-binding interface. In contrast to the peptide pheromonal attractants in amphibians, which are species specific, the attractins are, to our knowledge, the first water-borne peptide or protein pheromone family in invertebrates and vertebrates that are not species specific.