Analysis of the roles of RGD-binding integrins, alpha(4)/alpha(9) integrins, alpha(6) integrins, and CD9 in the interaction of the fertilin beta (ADAM2) disintegrin domain with the mouse egg membrane

Fertilin beta (also known as ADAM2), a mammalian sperm protein that mediates gamete cell adhesion during fertilization, is a member of the ADAM protein family whose members have disintegrin domains with homology to integrin ligands found in snake venoms. Fertilin beta utilizes an ECD sequence within its disintegrin domain to interact with the egg plasma membrane; the Asp is especially critical. Based on what is known about different integrin subfamilies and their ligands, we sought to characterize fertilin beta binding sites on mouse eggs, focusing on integrin subfamilies that recognize short peptide sequences that include an Asp residue: the alpha(5)/alpha(8)/alpha(v)/alpha(IIb) or RGD-binding subfamily (alpha(5)beta(1), alpha(8)beta(1), alpha(V)beta(1), alpha(V)beta(3), alpha(V)beta(5), alpha(V)beta(6), alpha(V)beta(8), and alpha(IIb)beta(3)) and the alpha(4)/alpha(9) subfamily (alpha(4)beta(1), alpha(9)beta(1), and alpha(4)beta(7)). We tested peptide sequences known to perturb interactions mediated by these integrins in two different assays for fertilin beta binding. Peptides with the sequence MLDG, which perturb alpha(4)/alpha(9) integrin-mediated interactions, significantly inhibit fertilin beta binding to eggs, which suggests a role for a member of this integrin subfamily as a fertilin beta receptor. RGD peptides, which perturb alpha(5)/alpha(8)/alpha(v)/alpha(IIb) integrin-mediated interactions, have partial inhibitory activity. The anti-alpha(6) antibody GoH3 has little or no inhibitory activity. An antibody to the integrin-associated tetraspanin protein CD9 inhibits the binding of a multivalent presentation of fertilin beta (immobilized on beads) but not soluble fertilin beta, which we speculate has implications for the role of CD9 in the strengthening of fertilin beta-mediated cell adhesion but not in initial ligand binding.     

A second gene product of the inositol-phospholipid-specific phospholipase C delta subclass.

Sequence analysis of a inositol-phospholipid-specific phospholipase C (PtdIns-PLC) purified from bovine brain has led to the isolation of a novel cDNA that encodes this protein. While this cDNA contains two introns, these appear to be removed upon transfection of the cDNA into COS-1 cells. The protein transiently expressed in COS-1 cells shows phosphatidylinositol 4,5-bisphosphate hydrolysing activity which distributes preferentially into the particulate fraction. Comparison of the predicted amino acid sequence of this PtdIns-PLC with other known PtdIns-PLCs reveals a high degree of similarity, throughout all of its sequence, with PtdIns-PLC delta. Thus, we believe that the identification of this cDNA represents evidence for multiple functional-gene products within the delta subclass of PtdIns-PLCs.     

Expression, purification and characterisation of a functional phosphatidylinositol-specific phospholipase C-delta 1 protein in Escherichia coli.

Inositol-lipid-specific phospholipase C-delta 1 (PtdIns-PLC delta 1) was expressed in Escherichia coli as a fusion protein containing a short 22-amino-acid lac-Z-derived amino terminus. Under appropriate conditions, the phospholipase constituted approximately 0.2% of the detergent-soluble protein and could be purified to near homogeneity in a simple three step protocol. The catalytic properties of the purified enzyme closely resemble those of the eukaryote-derived protein. The suitability of bacterial expression for the investigation of PtdIns-PLC delta regulation is discussed.     

Properties of BGP1, a poly(dG)-binding protein from chicken erythrocytes.

The chicken beta A-globin gene contains in the neighborhood of its 5' promoter a (dG)-homopolymer sequence 16 base pairs long. The 66 kD protein BGP1 (beta globin protein 1), isolated from chicken erythrocytes, has been shown to bind specifically to this sequence. We describe further purification of BGP1, measure its affinity for the beta A-globin promoter binding site, and analyze its binding properties. The minimal binding sequence is seven dG residues; methylation interference studies show that each of these residues contacts BGP1. Binding competition experiments employing (dG).(dC) oligomers of varying lengths also consistent with (dG)7 as a minimum recognition sequence. All of the data can be explained by a model in which BGP1 binds to any contiguous set of seven (dG) residues, so that the effective constant for binding to (dG)n is proportional to n minus 6. This behavior may be typical of proteins that bind specifically to repeated sequences.     

Purification, characterization and biosynthesis of parabutoxin 3, a component of Parabuthus transvaalicus venom.

A novel peptidyl inhibitor of voltage-gated K+ channels, named parabutoxin 3 (PBTx3), has been purified to homogeneity from the venom of Parabuthus transvaalicus. This scorpion toxin contains 37 residues, has a mass of 4274 Da and displays 41% identity with charybdotoxin (ChTx, also called 'alpha-KTx1.1'). PBTx3 is the tenth member (called 'alpha-KTx1.10') of subfamily 1 of K+ channel-blocking peptides known thus far. Electrophysiological experiments using Xenopus laevis oocytes indicate that PBTx3 is an inhibitor of Kv1 channels (Kv1.1, Kv1.2, Kv1.3), but has no detectable effects on Kir-type and ERG-type channels. The dissociation constants (Kd) for Kv1.1, Kv1.2 and Kv1.3 channels are, respectively, 79 microm, 547 nm and 492 nm. A synthetic gene encoding a PBTx3 homologue was designed and expressed as a fusion protein with the maltose-binding protein (MBP) in Escherichia coli. The recombinant protein was purified from the bacterial periplasm compartment using an amylose affinity resin column, followed by a gel filtration purification step and cleavage by factor Xa (fXa) to release the recombinant toxin peptide (rPBTx3). After final purification and refolding, rPBTx3 was shown to be identical to the native PBTx3 with respect to HPLC retention time, mass spectrometric analysis and functional properties. The three-dimensional structure of PBTx3 is proposed by homology modelling to contain a double-stranded antiparallel beta sheet and a single alpha-helix, connected by three disulfide bridges. The scaffold of PBTx3 is homologous to most other alpha-KTx scorpion toxins.     

Modules in the photoreceptor RGS9-1.Gbeta 5L GTPase-accelerating protein complex control effector coupling, GTPase acceleration, protein folding, and stability

RGS (regulators of G protein signaling) proteins regulate G protein signaling by accelerating GTP hydrolysis, but little is known about regulation of GTPase-accelerating protein (GAP) activities or roles of domains and subunits outside the catalytic cores. RGS9-1 is the GAP required for rapid recovery of light responses in vertebrate photoreceptors and the only mammalian RGS protein with a defined physiological function. It belongs to an RGS subfamily whose members have multiple domains, including G(gamma)-like domains that bind G(beta)(5) proteins. Members of this subfamily play important roles in neuronal signaling. Within the GAP complex organized around the RGS domain of RGS9-1, we have identified a functional role for the G(gamma)-like-G(beta)(5L) complex in regulation of GAP activity by an effector subunit, cGMP phosphodiesterase gamma and in protein folding and stability of RGS9-1. The C-terminal domain of RGS9-1 also plays a major role in conferring effector stimulation. The sequence of the RGS domain determines whether the sign of the effector effect will be positive or negative. These roles were observed in vitro using full-length proteins or fragments for RGS9-1, RGS7, G(beta)(5S), and G(beta)(5L). The dependence of RGS9-1 on G(beta)(5) co-expression for folding, stability, and function has been confirmed in vivo using transgenic Xenopus laevis. These results reveal how multiple domains and regulatory polypeptides work together to fine tune G(talpha) inactivation.     

Structural and phylogenetic characterization of human SLURP-1, the first secreted mammalian member of the Ly-6/uPAR protein superfamily

Members of the Ly-6/uPAR protein family share one or several repeat units of the Ly-6/uPAR domain that is defined by a distinct disulfide bonding pattern between 8 or 10 cysteine residues. The Ly-6/uPAR protein family can be divided into two subfamilies. One comprises GPI-anchored glycoprotein receptors with 10 cysteine residues. The other subfamily includes the secreted single-domain snake and frog cytotoxins, and differs significantly in that its members generally possess only eight cysteines and no GPI-anchoring signal sequence. We report the purification and structural characterization of human SLURP-1 (secreted mammalian Ly-6/uPAR related protein 1) from blood and urine peptide libraries. SLURP-1 is encoded by the ARS (component B)-81/s locus, and appears to be the first mammalian member of the Ly-6/uPAR family lacking a GPI-anchoring signal sequence. A phylogenetic analysis based on the SLURP-1 primary protein structure revealed a closer relationship to the subfamily of cytotoxins. Since the SLURP-1 gene maps to the same chromosomal region as several members of the Ly-6/uPAR subfamily of glycoprotein receptors, it is suggested that both biologically distinct subfamilies might have co-evolved from local chromosomal duplication events.     

The collagen receptor alpha 2 beta 1, from MG-63 and HT1080 cells, interacts with a cyclic RGD peptide.

Several receptors for the extracellular matrix protein collagen have been described which belong to the superfamily of receptors collectively known as integrins. Although several integrins have been shown to interact with extracellular matrix molecules via a common recognition site, arginine-glycine-aspartic Acid (RGD), within the beta 1 integrin subfamily, only the fibronectin receptor (alpha 5 beta 1) has been convincingly shown to interact with RGD. In the present study, we tested whether a collagen receptor could interact with RGD. Adhesion of an osteosarcoma cell line, MG-63, to immobilized collagen I was inhibited by the cyclic RGD-containing peptide, C*GRGDSPC* (where C* indicates that Cys participates in disulfide), and not by the linear GRGDSP or the non-RGD-containing cyclic peptide, C*GKGESPC*. Similarly, using collagen-Sepharose affinity chromatography, a heterodimeric protein could be specifically eluted from the column by the cyclic RGD peptide. Immunoprecipitations of the eluted material with monoclonal antibodies showed reactivity with the collagen receptor alpha 2 beta 1 and not alpha 3 beta 1. Our data demonstrate that RGD peptides can interact with the collagen receptor, and the differences seen with the linear and cyclic peptide suggest that the cyclic C*GRGDSPC* has a higher avidity for the receptor than the more flexible linear GRGDSP. In this paper, we provide supportive evidence that one possible mode of collagen interaction with alpha 2 beta 1 is via the RGD recognition sequence.     

Evidence that the inositol phospholipids are necessary for exocytosis. Loss of inositol phospholipids and inhibition of secretion in permeabilized cells caused by a bacterial phospholipase C and removal of ATP.

We directly manipulated the levels of PtdIns, PtdInsP and PtdInsP2 in digitonin-treated adrenal chromaffin cells with a bacterial phospholipase C (PLC) from Bacillus thuringiensis and by removal of ATP. The PtdIns-PLC acted intracellularly to cause a large decrease in [3H]inositol- or [32P]phosphate-labelled PtdIns, but did not directly hydrolyse PtdInsP or PtdInsP2. [3H]PtdInsP and [3H]PtdInsP2 levels declined markedly, probably because of the action of phosphatases in the absence of synthesis. Removal of ATP also caused marked decreases in [3H]PtdInsP and [3H]PtdInsP2. The decrease in polyphosphoinositide levels by PtdIns-PLC treatment or ATP removal was reflected by the inhibition of the production of inositol phosphates upon subsequent activation of the endogenous PLC by Ca2(+)-dependent catecholamine secretion from permeabilized cells was strongly inhibited by PtdIns-PLC treatment and by ATP removal. Ca2(+)-dependent secretion was similarly correlated with the sum of PtdInsP and PtdInsP2 when the level of these lipids was changed by either manipulation. PtdIns-PLC inhibited only the ATP-dependent component of secretion and did not affect ATP-dependent secretion. Both PtdIns-PLC and ATP removal inhibited the late slow phase of secretion, but had little effect on the initial rapid phase. Although we found a tight correlation between polyphosphoinositide levels and secretion, endogenous phospholipase C activity (stimulated by Ca2+, guanine nucleotides and related agents) was not correlated with secretion. Additional experiments indicated that neither the products of the PtdIns-PLC reaction (diacylglycerol and InsP1) nor the inability to generate products by subsequent activation of the endogenous PLC is likely to account for the inhibition of secretion. Incubation of permeabilized cells with neomycin in the absence of ATP maintained the level of polyphosphoinositides and more than doubled subsequent Ca2(+)-dependent secretion. The data suggest that: (1) Ca2(+)-dependent secretion has a requirement for the presence of inositol phospholipids; (2) the enhancement of secretion by ATP results in part from increased polyphosphoinositide levels; and (3) the role for inositol phospholipids in secretion revealed in these experiments is independent of their being substrates for the generation of diacylglycerol and InsP3.     

Mammalian cells that express Bacillus cereus phosphatidylinositol-specific phospholipase C have increased levels of inositol cyclic 1:2-phosphate, inositol 1-phosphate, and inositol 2-phosphate.

Phosphatidylinositol-specific phospholipase C (PtdIns-PLC) of Bacillus cereus catalyzes the conversion of PtdIns to inositol cyclic 1:2-phosphate and diacylglycerol. NIH 3T3, Swiss mouse 3T3, CV-1, and Cos-7 cells were transfected with a cDNA encoding this enzyme, and the metabolic and cellular consequences were investigated. Overexpression of PtdIns-PLC enzyme activity was associated with elevated levels of inositol cyclic 1:2-phosphate (2.5-70-fold), inositol 1-phosphate (2-20-fold), and inositol 2-phosphate (3-20-fold). The increases correlated with the levels of enzyme expression obtained in each cell type. The turnover of phosphatidylinositol (PtdIns) was also increased in transfected CV-1 cells by 13-fold 20 h after transfection. The levels of PtdIns, phosphatidic acid, diacylglycerol, or other inositol phosphates were not detectably altered. Expression of bacterial PtdIns-PLC decreased rapidly after 20 h implying that either the increased PtdIns turnover or the accumulation of inositol phosphates was detrimental to cells and that by some adaptive mechanism enzyme expression was suppressed.     

Complete cDNA sequence of a major 3-methylcholanthrene-inducible cytochrome P-450 isozyme (P-450AFB) of Syrian hamsters with high activity toward aflatoxin B1

Cytochrome P-450AFB is major isozyme inducible by 3-methylcholanthrene in Syrian golden hamsters and shows high potency toward aflatoxin B1 activation. We have isolated and sequenced cDNA clones to P-450AFB by immunoscreening a hamster liver cDNA library in lambda gt11. The longest clone contains an open reading frame of 1482 nucleotides and encodes a protein of 494 amino acids with a molecular weight of 57,420. The sequence of P-450AFB shares a 73% and 65% homology with that of mouse P-450 15 alpha (IIA3) and rat P-450a (IIA1), respectively, indicating that P-450AFB is a unique gene of the P-450IIA subfamily. The apparent concentration of a mRNA species hybridizable to the clone as well as the concentration of a protein immunoreactive to P-450AFB was increased significantly by the treatment with 3-methyl-cholanthrene, which indicates that the increase in P-450AFB protein is due mainly to an elevation of the mRNA.     

The beta subunit of the signal recognition particle receptor is a transmembrane GTPase that anchors the alpha subunit, a peripheral membrane GTPase, to the endoplasmic reticulum membrane

The signal recognition particle receptor (SR) is required for the cotranslational targeting of both secretory and membrane proteins to the endoplasmic reticulum (ER) membrane. During targeting, the SR interacts with the signal recognition particle (SRP) which is bound to the signal sequence of the nascent protein chain. This interaction catalyzes the GTP-dependent transfer of the nascent chain from SRP to the protein translocation apparatus in the ER membrane. The SR is a heterodimeric protein comprised of a 69-kD subunit (SR alpha) and a 30- kD subunit (SR beta) which are associated with the ER membrane in an unknown manner. SR alpha and the 54-kD subunits of SRP (SRP54) each contain related GTPase domains which are required for SR and SRP function. Molecular cloning and sequencing of a cDNA encoding SR beta revealed that SR beta is a transmembrane protein and, like SR alpha and SRP54, is a member of the GTPase superfamily. Although SR beta defines its own GTPase subfamily, it is distantly related to ARF and Sar1. Using UV cross-linking, we confirm that SR beta binds GTP specifically. Proteolytic digestion experiments show that SR alpha is required for the interaction of SRP with SR. SR alpha appears to be peripherally associated with the ER membrane, and we suggest that SR beta, as an integral membrane protein, mediates the membrane association of SR alpha. The discovery of its guanine nucleotide-binding domain, however, makes it likely that its role is more complex than that of a passive anchor for SR alpha. These findings suggest that a cascade of three directly interacting GTPases functions during protein targeting to the ER membrane.     

Improved purification and biochemical properties of phosphatidylinositol-specific phospholipase C from Bacillus thuringiensis

Monophosphatidylinositol inositol phosphohydrolase (phosphatidylinositol-specific phospholipase C. PtdIns-PLC. EC 3.1.4.10) has been purified from a Bacillus thuringiensis culture supernatant and from the cellular fraction of a recombinant Escherichia coli clone containing the PtdIns-PLC gene from B. thuringiensis. The two-step purification procedure involved ion-exchange chromatography on DEAE-Sepharose followed by separation on a Mono-Q/FPLC-column with yields of 32% and 50%, respectively. The molecular mass was determined to be 34 kDa by SDS/PAGE. The isoelectric point of the enzyme was 5.15. The amino-terminal sequences were shown to be identical for the enzymes purified from both organisms. PtdIns-PLC was inhibited by divalent cations using mixed micelles of Triton X-100 and pure phosphatidylinositol. PtdIns-PLC activity was detectable on polyacrylamide gels by activity staining on phosphatidylinostiol-containing agarose.     

The "gamma subunit" of Na,K-ATPase: a small, amphiphilic protein with a unique amino acid sequence

The "gamma subunit", or "proteolipid", of Na,K-ATPase is a small, membrane-bound protein that copurifies with the alpha and beta subunits of this enzyme. The importance of gamma in the function of Na,K-ATPase remains to be established, but some evidence indicates that it may be involved in forming a receptor site for cardiac glycosides. We have previously communicated [Reeves, A. S., Collins, J. H., & Schwartz, A. (1980) Biochem. Biophys. Res. Commun. 95, 1591-1598] the purification and amino acid composition of sheep kidney gamma, and in this paper we present the first available sequence information on this protein. Although the amino terminus of gamma seems to be blocked and it is resistant to proteolytic cleavage, we have determined approximately half of its amino acid sequence. Our results indicate that gamma contains a total of 68 amino acid residues, with a calculated Mr of 7675. The sequenced portion appears to be at the carboxyl terminus of the polypeptide chain. The gamma sequence is unique, providing strong evidence for its homogeneity and establishing for the first time that it is not a breakdown product of the alpha or beta subunits. gamma is not a true proteolipid, but rather it is an amphiphilic protein with two distinct structural domains. The amino-terminal domain (residues 1-49) is very hydrophilic, with many charged amino acid side chains, and must be extracellular. This domain includes a concentrated segment of four aromatic residues which may be involved in glycoside binding. The carboxyl-terminal domain (residues 50-68) is hydrophobic and probably spans the cell membrane.     

The nucleotide sequence for a proline-activating domain of gramicidin S synthetase 2 gene from Bacillus brevis

A fragment encoding proline-activating domain (grs 2-pro) of gramicidin S synthetase 2 (GS 2) was found in an 8.1-kilobase pairs (kb) DNA fragment of Bacillus brevis Nagano, which contained the full length of GS 1 gene (grs 1). The clones designated GS719 and GS708, which expressed gramicidin S synthetase 1, were elucidated to express immunoreactive proteins to GS 2 antibodies with approximate molecular weights of 115,000, 105,000 (GS719), and 110,000 (GS708). The partial purification of the gene products of these clones was carried out using DEAE-Sepharose CL-6B column chromatography. The immunoreactive proteins to GS 2 antibodies were separated from gramicidin S synthetase 1 protein and had specific proline-dependent ATP-32PPi exchange activity. The nucleotide sequence for the proline-activating domain in the 8.1-kb insert was determined. This fragment was 2,879 base pairs long, and encoded 959 amino acids. The calculated molecular weight of 111,671 was consistent with the apparent molecular weight of 115,000 found in SDS-PAGE of the immunoreactive products to GS 2 antibodies. The open reading frame for this protein followed grs 1 gene, though two were separated by a 73-base pair noncoding sequence, and remained open to the end.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phosphatidylinositol-specific phospholipase C induces biosynthesis of acetylcholinesterase via diacylglycerol in Schistosoma mansoni.

We have previously shown that two ectoenzymes, acetylcholinesterase (AChE) and alkaline phosphatase, are released from the surface and from particulate fractions of the parasite Schistosoma mansoni, by a phosphatidylinositol-specific phospholipase C (PtdIns-PLC) of bacterial origin. Exposure to PtdIns-PLC not only removes large amounts of AChE from the surface of intact, viable Schistosoma in culture, but is accompanied by a concomitant increase in overall levels of AChE in the parasite. The same phenomenon is observed with PtdIns-PLC from two different bacterial sources; Staphylococcus aureus and Bacillus thuringiensis. The increase in AChE levels may be ascribed to de novo synthesis since exposure to PtdIns-PLC, in the presence of the protein-synthesis inhibitor cycloheximide, totally blocked the increase in AChE activity. Furthermore, PtdIns-PLC induced an increased incorporation of [35S]methionine into the AChE immunoprecipitated by a specific anti-AChE serum. This increase is selective for AChE, since total protein synthesis remained almost unchanged after PtdIns-PLC addition, and little or no effect was observed on the enzymatic activity of alkaline phosphatase, which is also glycophosphatidylinositol anchored. Since cleavage of the phosphatidylinositol anchor by PtdIns-PLC should liberate diacylglycerol, which may act as second messenger, we investigated the effect of exogenous diacylglycerols on the synthesis of AChE in S. mansoni. Three different diacylglycerols were tested as possible inducers of AChE activity in the parasite. Both 1-oleoyl-2-acetyl-sn-glycerol and 1,2-dimyristoyl-sn-glycerol were able to increase AChE activity by 35-40% at concentrations of 25 micrograms/ml. A higher concentration of 1,2-dioctanoyl-sn-glycerol (70 micrograms/ml) was needed to produce an equivalent effect. Moreover, addition of phorbol-12-myristate-13-acetate, together with the calcium ionophore A23187, produced a similar increase in AChE activity. Finally, polymixin B, a specific inhibitor of protein kinase C, partially blocked the increase in AChE activity induced by PtdIns-PLC. Our results suggest the involvement of glycophosphatidyl membrane-anchor breakdown products as putative second messengers in the parasite S. mansoni.     

Production in Escherichia coli of human thymosin beta 4 as chimeric protein with human tumor necrosis factor

We have produced thymosin beta 4 protein in Escherichia coli as a chimeric protein with tumor necrosis factor (TNF). The protein was abundantly expressed, was immunoreactive against both anti-thymosin beta 4 and anti-TNF antibodies, and retained cytotoxicity in a TNF assay using mouse L929 fibroblasts. This latter characteristic enabled the easy and simple purification of thymosin beta 4 merely by following the TNF activity. The chimeric protein was designed to have an Asp-Pro bridge between thymosin beta 4 and TNF which could be specifically cleaved under suitable acidic conditions to release the thymosin beta 4 from the chimeric protein. These results indicate that the expression system in E.coli of a chimeric protein composed of thymosin beta 4 and TNF is appropriate for obtaining an abundant amount of the beta 4 peptide, especially since its purification from tissues is usually difficult because of limited yield and obscurity of its biological activity.