Proteomic Identification of Plasma Components in Tachypleus tridentatus and Their Effects on the Longitudinal Bone Growth Rate in Rats.

Tachypleus tridentatus (T. tridentatus) is a marine animal and traditional Chinese medicine. T. tridentatus plasma is a valuable resource for important medical and health-based functions. In this experiment, in order to evaluate the effect and mechanism of T. tridentatus plasma with respect to the promotion of bone tissue growth in rats, the processes of ultrafiltration and mass spectrometry were first used to separate and identify the components of T. tridentatus plasma. Then, a comparison of the effects of the T. tridentatus plasma samples, which each possessed different molecular weights, regarding the growth of the long bones of rats was conducted. Finally, transcriptomics, proteomics, and bioinformatics were all used to analyze the biological functions and related signaling pathways of the T. tridentatus plasma in order to promote rat bone growth. The results showed that the contents of amino acid residues in peptides are related to the growth promotion that was contained in the 10-30 kDa plasma group. Moreover, the T. tridentatus plasma samples were found to be higher in this respect than those in the whole plasma group. In addition, the 10-30 kDa plasma group could significantly promote bone growth activity in rats. The proteomic analysis showed that the proteins that were differentially expressed in the 10-30 kDa plasma group were mainly enriched in the PI3K-AKT signal pathway. Our study suggested that the T. tridentatus plasma possesses promising potential for the purposes of clinical use, whereby it can serve the role of a growth-promoting agent.

Detection of liposomal cholesterol and monophosphoryl lipid A by QS-21 saponin and Limulus polyphemus amebocyte lysate.

Liposomes containing cholesterol (Chol) have long been used as an important membrane system for modeling the complex interactions of Chol with adjacent phospholipids or other lipids in a membrane environment. In this study we utilize a probe composed of QS-21, a saponin molecule that recognizes liposomal Chol and causes hemolysis of erythrocytes. The interaction of QS-21 with liposomal Chol results in a stable formulation which, after injection into the tissues of an animal, lacks toxic effects of QS-21 on neighboring cells that contain Chol, such as erythrocytes. Here we have used liposomes containing different saturated phospholipid fatty acyl groups and Chol, with or without monophosphoryl lipid A (MPLA), as model membranes. QS-21 is then employed as a probe to study the interactions of liposomal lipids on the visibility of membrane Chol. We demonstrate that changes either in the mole fraction of Chol in liposomes, or with different chain lengths of phospholipid fatty acyl groups, can have a substantial impact on the detection of Chol by the QS-21. We further show that liposomal MPLA can partially inhibit detection of the liposomal Chol by QS-21. The Limulus amebocyte lysate assay is used for binding to and detection of MPLA. Previous work has demonstrated that sequestration of MPLA into the liposomal lipid bilayer can block detection by the Limulus assay, but the binding site on the MPLA to which the Limulus protein binds is unknown. Changes in liposomal Chol concentration and phospholipid fatty acyl chain length influenced the detection of the liposome-embedded MPLA.

Cloning and identification of Rab cDNAs from Limulus polyphemus.

Small GTPases of the Rab family are essential for the control of membrane transport between intracellular compartments. Trafficking of the sodium-dependent facilitative insulin responsive glucose transporter (GLUT 4) has been shown to be associated with the intracellular redistribution of Rabs 4, 5 and 11 in adipose and muscle tissues. As a prelude to studies of the endosomal trafficking of the choline cotransporter (ChCoT), we describe herein our initial efforts to identify Rab proteins in Limulus polyphemus central nervous system (CNS) tissue. The studies were initiated after results from Microarray analysis of Limulus RNA hybridized to mouse gene chips suggested the presence of RNA transcripts for Rab 7 protein. Subsequently, more than 30 sequences for different Rab proteins were aligned and several consensus segments were selected for degenerate primer design to produce Rabs 2, 4, 7, 9 and 11. The expected PCR fragment sizes were obtained using RT-PCR and subcloned into pCR II TOPO vector and transferred into E. coli Top 10. The nucleotide sequences indicated that the recombinants encoded partial amino acid sequences for Rabs 1a, 1b, 1c, 2, 2a, 2b, 3a, 4, 5a, 7a, 7b, 11a, 11b, 14, 33b1 and 33b2. Northern blot analyses showed that the molecular sizes of Limulus Rabs 3a, 4, 7, 11a and 11b ranged from approximately 1.94.6 Kb. These Rab proteins, particularly Rabs 4, 7 and 11, will be studied further to determine their possible roles in the trafficking of the Limulus ChCoT

Metal levels in horseshoe crabs (Limulus polyphemus) from Maine to Florida.

There is considerable concern for the health of spawning populations of horseshoe crabs (Limulus polyphemus) along the east coast of North America because of an increase in the harvest, an apparent decrease in population levels, and the dependence of migrating shorebirds on a superabundant supply of horseshoe crab eggs during their migratory stopover on Delaware Bay. In addition to overfishing, population declines could be caused or recovery slowed, by pollution. In this paper, we examine the levels of metals (arsenic, cadmium, chromium, lead, manganese, mercury, and selenium) in the eggs, leg muscle, and apodeme of 100 horseshoe crabs collected at nine sites from Maine to Florida. Arsenic levels were the highest, followed by manganese and selenium, while levels for the other metals averaged below 100ppb for most tissues. Arsenic and mercury levels were highest in the leg muscle; cadmium, lead, manganese, and selenium levels were highest in eggs; and chromium levels were highest in the apodeme. There were significant geographical differences for all metals in all three tissues. No one geographical site had the highest levels of more than two metals. Arsenic, with the highest levels overall, was highest in Florida in all the three tissues. Manganese levels were highest in Massachusetts for eggs and apodeme, but not leg, which was highest in Port Jefferson, New York. Selenium was highest in apodeme from Florida, and in eggs and leg muscle from Prime Hook, Delaware. The patterns among locations and tissues were not as clear for the other metals because the levels generally averaged below 100ppb. The levels of contaminants found in horseshoe crabs, with the possible exceptions of arsenic in Florida, and mercury from Barnegat Bay and Prime Hook, were below those known to cause adverse effects in the crabs themselves or in organisms that consume them or their eggs. Our results indicate that site-specific data are essential for managers to evaluate the potential threat from contaminants to both the horseshoe crabs and those that consume them.

Localization of carbohydrate attachment sites and disulfide bridges in limulus alpha 2-macroglobulin. Evidence for two forms differing primarily in their bait region sequences.

The primary structure determination of the dimeric invertebrate alpha(2)-macroglobulin (alpha(2)M) from Limulus polyphemus has been completed by determining its sites of glycosylation and disulfide bridge pattern. Of seven potential glycosylation sites for N-linked glycosylation, six (Asn(275), Asn(307), Asn(866), Asn(896), Asn(1089), and Asn(1145)) carry common glucosamine-based carbohydrates groups, whereas one (Asn(80)) carries a carbohydrate chain containing both glucosamine and galactosamine. Nine disulfide bridges, which are homologues with bridges in human alpha(2)M, have been identified (Cys(228)-Cys(269), Cys(456)-Cys(580), Cys(612)-Cys(799), Cys(657)-Cys(707), Cys(849)-Cys(876), Cys(874)-Cys(910), Cys(946)-Cys(1328), Cys(1104)-Cys(1155), and Cys(1362)-Cys(1475)). In addition to these bridges, Limulus alpha(2)M contains three unique bridges that connect Cys(361) and Cys(382), Cys(1370) and Cys(1374), respectively, and Cys(719) in one subunit with the same residue in the other subunit of the dimer. The latter bridge forms the only interchain disulfide bridge in Limulus alpha(2)M. The location of this bridge within the bait region is discussed and compared with other alpha-macroglobulins. Several peptides identified in the course of determining the disulfide bridge pattern provided evidence for the existence of two forms of Limulus alpha(2)M. The two forms have a high degree of sequence identity, but they differ extensively in large parts of their bait regions suggesting that they have different inhibitory spectra. The two forms (Limulus alpha(2)M-1 and -2) are most likely present in an approximately 2:1 ratio in the hemolymph of each animal, and they can be partially separated on a Mono Q column at pH 7.4 by applying a shallow gradient of NaCl.

Molecular cloning of a cDNA for a putative choline co-transporter from Limulus CNS.

It is well documented that the sodium dependent, hemicholinium-3 sensitive, high affinity choline co-transporter is rate limiting in the biosynthesis of acetylcholine and is essential to cholinergic transmission. Until recently this transporter had eluded cloning. Okuda et al. (2000. Nature Neurosci. 3, 120-125) recently reported the successful cloning of the choline co-transporter in Caenorhabditis elegans (CHO-1) and rat (CHT1). We report herein the cloning of the choline co-transporter in the horseshoe crab, Limulus polyphemus. Through the use of a series of degenerate primers selected from consensus sequences of CHO-1 and CHT1, we generated two probes that were used to search a Limulus cDNA library produced from central nervous system (CNS) tissue. The full length nucleotide sequence of the Limulus homolog consists of 3368 bp which includes an open reading frame (ORF) that predicts a protein of 579 amino acids and two non-translation regions (NTR), one at the 3' end and the other at the 5' end. The amino acid sequence has 46% identity with rat CHT1 and 50% identity with both CHO-1 in C. elegans and the recently cloned human co-transporter (hCHT; Apparsundaram et al., 2000. Biochem. Biophys. Res. Commun. 276, 862-867; Okuda and Haga, 2000. FEBS Lett. 484, 92-97). Hydropathy plot analysis predicts the Limulus choline co-transporter (LChCoT) to have thirteen transmembrane domains (TMD), with the N-terminus oriented extracellularly and the C-terminus oriented intracellularly. Northern blot analyses using cDNA probes designed from LChCoT cDNA sequences revealed its distribution specifically in central nervous system structures. On the other hand it was not found in non-nervous tissues. The successful cloning of LChCoT, which was shown to be a member of the sodium-dependent glucose transporter family (SLGT), should prove useful in the determination of its physiological regulation, including its intracellular trafficking.

A myosin III from Limulus eyes is a clock-regulated phosphoprotein.

The lateral eyes of the horseshoe crab Limulus polyphemus undergo dramatic daily changes in structure and function that lead to enhanced retinal sensitivity and responsiveness to light at night. These changes are controlled by a circadian neural input that alters photoreceptor and pigment cell shape, pigment migration, and phototransduction. Clock input to the eyes also regulates photomechanical movements within photoreceptors, including membrane shedding. The biochemical mechanisms underlying these diverse effects of the clock on the retina are unknown, but a major biochemical consequence of activating clock input to the eyes is a rise in the concentration of cAMP in photoreceptors and the phosphorylation of a 122 kDa visual system-specific protein. We have cloned and sequenced cDNA encoding the clock-regulated 122 kDa phosphoprotein and show here that it is a new member of the myosin III family. We report that Limulus myosin III is similar to other unconventional myosins in that it binds to calmodulin in the absence of Ca2+; it is novel in that it is phosphorylated within its myosin globular head, probably by cAMP-dependent protein kinase. The protein is present throughout the photoreceptor, including the region occupied by the photosensitive rhabdom. We propose that the phosphorylation of Limulus myosin III is involved in one or more of the structural and functional changes that occur in Limulus eyes in response to clock input.

Calcium/calmodulin-dependent protein kinase II and arrestin phosphorylation in Limulus eyes.

In rhabdomeral photoreceptors, light stimulates the phosphorylation of arrestin, a protein critical for quenching the photoresponse, by activating a calcium/calmodulin-dependent protein kinase (CaM PK). Here we present biochemical evidence that a CaM PK that phosphorylates arrestin in Limulus eyes is structurally similar to mammalian CaM PK II. In addition, cDNAs encoding proteins homologous to mammalian and Drosophila CaM PK II in the catalytic and regulatory domains were cloned and sequenced from a Limulus lateral eye cDNA library. The Limulus sequences are unique, however, in that they lack most of the association domain. The proteins encoded by these sequences may phosphorylate arrestin.

Presence of functional domains in Limulus polyphemus hemocyanin.

In an attempt to isolate structural domains of arthropod hemocyanins and possibly to investigate their functional properties, we have undertaken proteolytic digestion experiments of isolated subunits from Panulirus interruptus and Limulus polyphemus oxy-hemocyanin. Satisfactory results have been obtained using trypsin at high concentration and short digestion times. Results show that, in the case of Panulirus hemocyanin, only subunit alpha is susceptible to trypsin digestion, but that proteolytic cleavage is associated with the loss of the copper-oxygen band; on the other hand, in the case of Limulus hemocyanin, four subunits (I, II, III and IV) show a significant susceptibility to trypsin, and their fragmentation takes place with preservation of the oxygen-binding capacity. A more detailed study of the digestion products of subunit IV from Limulus hemocyanin reveals that the proteolytic fragments keep together in a single non-covalent complex. Attempts to separate the native fragments result in the precipitation of the digestion products. Subunit IV of Limulus with proteolytic cuts binds O2 and CO with the same affinity as the native subunit, suggesting that the copper site is still preserved structurally and is functionally active in a 37 kDa trypsin-resistant domain.