Site-directed mutagenesis of human nucleoside triphosphate diphosphohydrolase 3: the importance of conserved glycine residues and the identification of additional conserved protein motifs in eNTPDases.

Glycine residues are recognized as important structural determinants in nucleotide-binding domains of many enzymes. The functional significance of seven glycine residues invariant in all 22 eNTPDase sequences was therefore examined. Glycine-to-alanine mutants of eNTPDase3 were analyzed for nucleotidase activities and tertiary and quaternary structure changes. Mutations G98A and G183A had modest effects on ATPase and ADPase activities. The G141A mutation resulted in 4- to 5-fold decreased nucleotidase activity, while the G222A mutation decreased ATPase activity 20-fold, and ADPase activity 6-fold. Unlike the other five glycine mutants, the G263A and G462A mutations caused significant loss of nucleotidase activity which was observed concomitant with lower protein expression levels, large-scale changes in tertiary and quaternary protein structure, and decreased trafficking to the plasma membrane. Thus, these data identify glycine residues that are essential for enzymatic activity and the tertiary and quaternary structure of eNTPDase3. Further, two additional conserved regions in the eNTPDases are identified, apyrase conserved regions ACR1a and ACR4a, which may be involved in phosphate binding/hydrolysis and protein folding, respectively.

Peptide-mediated transcytosis of phage display vectors in MDCK cells.

Delivery of therapeutic macromolecules and gene vectors to certain tissues is hampered by endothelial or epithelial barriers. We show here that the transport of phage particles across epithelial cells can be facilitated by peptide ligands selected from a phage library of random peptides. Using MDCK cells, we identified a polycationic peptide sequence, RYRGDLGRR, containing a putative integrin-binding (RGD) motif that enhanced basal-to-apical transcytosis of peptide-bearing phage 1000- to 10,000-fold compared with phage with no peptide insert. Both the synthetic peptide RYRGDLGRR and the integrin-binding peptide GRGDSP inhibited phage transcytosis suggesting the involvement of integrins. Confocal immunofluorescence microscopy showed that following internalization at the basal cell surface, phage particles were delivered to the apical cytoplasm and released at the apical cell surface. These data suggest the feasibility of using short peptides for targeting transcytotic pathways and facilitating delivery of macromolecules across cellular barriers.

Targeted delivery of multivalent phage display vectors into mammalian cells.

Novel peptide motives targeting endocytosing receptors were isolated from phage display libraries of random peptides by recovering internalized phage from mammalian cells. The peptide-presenting phage selected by internalization in HEp-2 and ECV304 human cells were taken up 1000- to 100,000-fold more efficiently than their parent libraries, and from 10 to 100 times faster than phage particles displaying integrin-binding peptides. A high degree of selectivity of phage uptake was observed in these cells: phage selected in ECV304 cells were internalized approximately 100-fold more efficiently in ECV304 cells than in HEp-2 cells. Likewise, phage selected in HEp-2 cells were subsequently taken up approximately 40-fold more efficiently by HEp-2 cells than by ECV304 cells. In multiple independent trials using a cyclic peptide library, an identical peptide sequence displayed on phage was internalized by and recovered from ECV304 cells. These findings indicate that the internalization process is highly selective, and is capable of capturing a specific peptide from 2 x 10(7) peptide variants. Immunofluorescence microscopy showed juxtanuclear localization of internalized phage. These results demonstrate the feasibility of using multivalent phage-display libraries to identify new targeting ligands for the intracellular delivery of macromolecules.

Hydrophobic residues in the C-terminal region of S100A1 are essential for target protein binding but not for dimerization.

S100 proteins are a family of small dimeric proteins characterized by two EF hand type Ca2+ binding motifs which are flanked by unique N- and C-terminal regions. Although shown unequivocally in only a few cases S100 proteins are thought to function by binding to, and thereby regulating, cellular target proteins in a Ca2+ dependent manner. To describe for one member of the family, S100A1, structural requirements underlying target protein binding, we generated specifically mutated S100A1 derivatives and characterized their interaction with the alpha subunit of the actin capping protein CapZ shown here to represent a direct binding partner for S100A1. Chemical cross-linking, ligand blotting and fluorescence emission spectroscopy reveal that removal of, or mutations within, the sequence encompassing residues 88-90 in the unique C-terminal region of S100A1 interfere with binding to CapZ alpha and to TRTK-12, a synthetic CapZ alpha peptide. The S100A1 sequence identified contains a cluster of three hydrophobic residues (Phe-88, Phe-89 and Trp-90) at least one of which--as revealed by qualitative phenyl Sepharose binding and hydrophobic fluorescent probe spectroscopy--is exposed on the protein surface of Ca2+ bound S100A1. As homologous hydrophobic residues in the closely related S100B protein were shown by NMR spectroscopy of Ca(2+)-free S100B dimers to provide intersubunit contacts [Kilby P.M., van Eldik L.J., Roberts G.C.K. The solution structure of the bovine S100B dimer in the calcium-free state. Structure 1996; 4: 1041-1052; Drohat A.C., Amburgey J.C., Abildgaard F., Starich M.R., Baldisseri D., Weber D.J. Solution structure of rat apo-S100B (beta beta) as determined by NMR spectroscopy. Biochemistry 1996; 35: 11,577-11,588], we characterized the physical state of the various S100A1 derivatives. Analytical gel filtration and chemical cross-linking show that dimer formation is not compromised in S100A1 mutants lacking residues 88-90 or containing specific amino acid substitutions in this sequence. Thus a cluster of hydrophobic residues in the C-terminal region of S100A1 is essential for target protein binding but dispensable for dimerization, a situation possibly met in other S100 proteins as well.

S-100 (alpha and beta) binding peptide (TRTK-12) blocks S-100/GFAP interaction: identification of a putative S-100 target epitope within the head domain of GFAP.

Alignment of previously characterized S-100 (alpha and beta)-binding peptides (J. Biol. Chem. 270, 14651-14658) has enabled the identification of a putative S-100 target epitope within the head domain of glial fibrillary acidic protein (GFAP). The capacity of a known peptide inhibitor of S-100 protein (TRTK-12), homologous to this region, to perturb the interaction of S-100 (alpha and beta) and GFAP (J. Biol. Chem 268, 12669-12674) was investigated. Fluorescence spectrophotometry and chemical cross-linking analyses determined TRTK-12 to disrupt S-100:GFAP interaction in a dose- and Ca(2+_dependent manner. TRTK-12 also inhibited S-100's ability to block GFAP assembly and to mediate disassembly of preformed glial filaments. Each of these events was strictly dependent upon the presence of calcium and inhibitory peptide, maximal inhibition occurring at a concentration of TRTK-12 equivalent to the molar amount of S-100 monomer present. Together with our recent report demonstrating TRTK-12 also blocks the interaction of S-100 protein with the actin capping protein, CapZ, these results suggest TRTK-12 functions as a pleiotropic inhibitor of S-100 function. Availability of a functional inhibitor of S-100 will assist the further characterization of S-100 protein function in vitro and in vivo. Moreover, this report provides additional evidence supportive of a role for S-100 as a multi-faceted regulator of cytoskeletal integrity.

Interaction of S100a0 protein with the actin capping protein, CapZ: characterization of a putative S100a0 binding site in CapZ alpha-subunit.

S100a0, a Ca2+-binding protein expressed predominantly in cardiac and skeletal muscle tissues, was demonstrated by chemical cross-linking to interact in a Ca2+ -dependent manner with the actin capping protein CapZ. TRTK-12, a peptide contained within the COOH-terminal region of CapZalpha, inhibited S100a0: CapZ interaction in a dose-dependent manner. TRTK-12 was shown by cross-linking to bind S100a0 in the presence of Ca2+, and by fluorescence spectrophotometry to interact in a saturable manner with the anionic phospholipid and a regulator of CapZ activity, phosphatidylinositol 4-monophosphate; but not with the neutral phospholipid, phosphatidylcholine. These data suggest S100a0 and polyphosphoinositides bind to the same COOH-terminal region of CapZalpha, thus potentially modulating CapZ activity.

Characterization of S-100b binding epitopes. Identification of a novel target, the actin capping protein, CapZ.

Short amino acid sequences that interact with the Ca2+ binding protein S-100b were identified by screening a bacteriophage random peptide display library. S-100b binding bacteriophages were selected by Ca(2+)-dependent affinity chromatography, and the sequence of the random peptide insert contained in 51 clones was determined. Alignment of the sequence of 44 unique S-100b binding peptides identified a common motif of eight amino acids. A subgroup of peptides that contained sequences with the highest degree of similarity had the consensus motif (K/R)(L/I)XWXXIL, in which predominantly P, S, and N were found in position 3, and S and D were found in position 5. Analysis of sequence databanks identified a similar sequence in the COOH-terminal region of the alpha-subunit of actin capping proteins. The peptide TRTKIDWNKILS (TRTK-12), corresponding to the region of greatest homology within this region of the subunit of actin capping proteins (e.g. amino acids 265-276 in CapZ alpha 1 and CapZ alpha 2), was synthesized and shown by fluorescence spectrophotometry to bind S-100b in a Ca(2+)-dependent manner. Gel overlay and cross-linking experiments demonstrated the interaction of S-100b with CapZ to be Ca2+ dependent. Moreover, this interaction was blocked by addition of TRTK-12 peptide. These results identify Ca(2+)-dependent S-100b target sequence epitopes and designate the carboxyl terminus of the alpha-subunit of actin capping proteins, like CapZ, to be a target of S-100b activity. The high level of conservation within this region of actin capping proteins and the apparent high affinity of this interaction strongly suggest that the interaction between S-100b and the alpha-subunit of actin capping proteins is biologically significant.

The expression of different annexins in the fish embryo is developmentally regulated.

The expression of annexins, a family of Ca2+/phospholipid-binding proteins, was analyzed by biochemical and immunological criteria in the fish Misgurnus fossilis (loach), which is a good model system to study embryonic events. Five different annexins (loach annexins A to E) are present as a maternal pool in the unfertilized eggs. Only annexin E is newly synthesized in the early embryo. Its synthesis, already apparent at mid-blastula, decreases in later stages when two additional annexins (F and G) appear. They are present among the newly synthesized polypeptides of mid-gastrula and later embryonic stages and are also found in loach larvae. The developmentally controlled expression of several annexins indicates a specific role of these proteins at certain embryonic stages.

Transduction of Ca2+ signals upon fertilization of eggs; identification of an S-100 protein as a major Ca(2+)-binding protein.

A transient increase in the level of free cytosolic Ca2+ is observed upon fertilization of the eggs of many species and is thought to represent a key event in the initiation of development. To identify components in the egg which could be involved in mediating such Ca2+ signals we searched for Ca(2+)-binding proteins in eggs of the fresh-water fish Misgurnus fossilis (loach). We show that loach eggs contain two major Ca(2+)-binding proteins which can be purified through their Ca(2+)-dependent interaction with a hydrophobic matrix. Protein sequencing revealed that the larger 18 kDa protein is calmodulin, while the smaller polypeptide of 10 kDa is a member of the S-100 protein family. This is the first report of the presence of an S-100 protein in vertebrate eggs and shows that this protein is found in two fold higher concentration than calmodulin. Since the 10 kDa protein shares 68% sequence identity with S-100 alpha from bovine brain, it can be considered as the loach homologue of mammalian S-100 alpha. During early embryonic development, de novo protein synthesis of calmodulin is observed at the earliest stages analyzed (mid-blastula), while de novo protein synthesis of the S-100 alpha homologue begins with the mid-gastrula stage.(ABSTRACT TRUNCATED AT 250 WORDS)

Surface polarization in loach eggs and two-cell embryos: correlations between surface relief, endocytosis and cortex contractility.

The aim of this study was to examine the reorganization of the microfilamentous cortical layer (MC) accompanying ooplasmic segregation in loach eggs. Using scanning (SEM) and transmission electron microscopy (TEM), we found that the MC is thicker in folded areas. Prior to fertilization, surface microvilli are distributed more or less uniformly throughout the egg. A similar, more or less uniform, distribution of endocytotic events was observed in the eggs 5-15 min after insemination using fluorescence microscopy of Lucifer yellow CH uptake. During ooplasmic segregation, the surface is progressively polarized so that before the first cleavage onset (50-60 min after insemination) only the blastodisc surface is folded and undergoes endocytosis, whereas the vegetal surface is smooth and does not show internalization. In two-cell embryos, the blastomeric surface is also regionalized according to its relief and endocytosis. When surface tension was lowered by sucking most yolk granules out of the egg, we observed contractile responses only in the animal folded surface. These data suggest that a polar distribution of contractile structures is established in the loach egg undergoing ooplasmic segregation.

Phalloidin inhibits cortical granule exocytosis and ooplasmic segregation in loach eggs.

Injections of phalloidin under the surface of loach eggs, followed by activation of the eggs in tap water, result in local inhibition of cortical granule (CG) exocytosis. Light and electron microscopy revealed that in the region where exocytosis is inhibited the thickness of the microfilamentous cortex (MC) separating CGs from the plasma membrane (PM) is increased significantly, and many CGs are detached and have moved away from the MC. Injections of phalloidin also inhibit ooplasmic segregation in fertilized eggs. The experiments suggest that in intact eggs the MC represents a physical barrier to CG exocytosis, and that interactions of the MC with the PM and CGs are crucial for the retention of CGs near the sites of fusion.

The effect of local cortical microfilament disorganization on ooplasmic segregation in the loach (Misgurnus fossilis) egg.

Injections of cytochalasin D (CD) or DNase I under the surface of fertilized loach egg result in local disorganization of microfilamentous cortex (MC) as revealed by transmission electron microscopy. This effect correlates with the loss of the cortex ability to contract in vitro. The disorganization of MC in the vegetal hemisphere of the egg does not affect the ooplasm segregation or blastodisk cleavage. Injection under the animal pole suppresses blastodisk formation and results in the autonomous separation of ooplasm in the central part of the egg. The experiments suggest that (1) autonomous separation of ooplasm from the yolk granules can proceed in the central part of the egg without the participation of MC; (2) normal segregation of ooplasm at the animal pole requires that the structures of microfilaments in the animal hemisphere (but not in the vegetal one) be preserved.

Qualitative changes in carboxylesterase-2 phenotypes of tetraploid loaches (Misgurnus fossilis L.).

We have shown in our previous work that in the tetraploid loach Misgurnus fossilis carboxylesterase-2 (E-2) phenotypes of embryos and early larvae are controlled by the alleles of the Est-2 locus. In the present work it has been established that the somatic tissues of many adult fish homozygous for one of the Est-2 alleles, unlike the tissues of embryos and larvae, reveal an additional fraction of E-2 with electrophoretic mobility corresponding to that of the second allozyme. The activity of this additional fraction of E-2 varies with the tissue and depends on the age of fish and maintenance conditions. Phenotypes of the same individual homozygous for one of the Est-2 alleles can be manifested as homozygous or heterozygous according to the activity of the additional E-2 fraction. The results obtained are consistent with the assumption that in the tetraploid species Misgurnus fossilis there are two E-2 loci with common alleles of identical electrophoretic mobility.

Carboxylesterase-2 in the development of the loach (Misgurnus fossilis L.).

Two esterases splitting alpha-naphthylacetate have been found in the tissues of adult loaches and in embryos. These were identified as arylesterase (E-1) (arylester hydrolase, E.C. 3.1.1.2) and carboxylesterase (E-2) (carboxylic ester hydrolase, E.C. 3.1.1.1.). In unfertilized loach eggs E-1 and E-2 synthesized during oogenesis were found. Active E-2 synthesized under the control of E-2 genes of the embryo appeared in embryos from the stage of 40-50 h of development. Maternal E-2 molecules synthesized in oogenesis or on the stored templates in embryogenesis persisted in larvae up to days 5-6 of development. Two genes controlling the synthesis of two forms of E-2 differing in electric mobility were found in the loach population from the delta of the Danube. The genes for fast and slow E-2 were shown to segregate in meiosis and to be allelic.

Differential expression of allelic carboxylesterase-2 genes in oocytes of loach (Misgurnus fossilis L.) and heterogeneity of loach oocytes and eggs for the expression of allelic carboxylesterase-2 genes.

Tissue and cell differences have been found in the expression of allelic carboxylesterase-2 (E-2) (carboxylic ester hydrolase, E.C. 3.1.1.1) genes of the loach. The relative activity of two allozymes in the brain and muscles of heterozygotes is similar, whereas in oocytes and eggs of the same fish the activity of one of the allozymes is considerably greater than that of the other. Oocytes and eggs of some heterozygotes were shown to be heterogeneous for the expression of E-2 alleles. The phenomenon implies that roe produced by a heterozygous female can contain two or more egg types: in some the alleles are equally expressed, while in others one of the alleles is predominantly expressed.

[Heat resistance of aldolase of the hybrid embryos of sea urchins Strongylocentrotus droebachiensis and S, intermedius]. / Teploustoichivost' al'dolazy gibridnykh zarodyshei morskikh azhei Strongylocentrotus droebachiensis i S. intermedius

The time of expression of the genes controlling aldolase has been studied in the hybrid embryos female Strongylocentrotus droebachiensis X male S. intermedius. The enzyme heat resistance estimated by the temperature of 50% inactivation following the exposition for 30 min (T50) was used as its genetic marker. T50 of aldolase of the psychrophilic maternal species suffered practically no changes from the stage of mesenchyme blastula till the stage of 11 days old pluteus and equated 35.3 degrees. T50 of aldolase of autumn- and spring-spawning populations of the thermophilic paternal species equaled 39.5 degrees at the stage of 11 days old pluteus. The heat resistance of aldolase of the hybrid embryos did not differ reliably from that of maternal enzyme during the first 4 days of development (at 8 degrees) till the late gastrula stage and attained the maximum (T50 =36.9 degrees) on the 8th day (stage of pluteus). The expression of the genes controlling aldolase appears to take place between these developmental stages.

[Expression of the paternal genes for lactate dehydrogenase, glutamate dehydrogenase and acetylcholinesterase in the development of hybrid fish between species from the families of Cobitidae and Cyprinidae]. / Proiavlenie ottsovskikh genov laktatdegidrogenazy, glutanatdegidrogenazy i atsetilkholinésterazy v razvitii gibridov ryb mezhdy vidami iz semeisty Cobitidae i Cyprinidae.

The time of expression of the paternal genes of glutamate dehydrogenase (GDH), lactate dehydrogenase (LDH) and acetylcholine esterase (AChE) was investigated in the development of fish hybrids. The species which differed by the thermostability of homologous enzymes were selected as parental pairs. The appearance of differences in the thermostability of homologous enzymes between the hybrids and the maternal species suggested the beginning of paternal enzyme synthesis in the hybrid embryos. Differences in the AChE thermostability appeared simultaneously with the enzyme activity at the stage of first muscle contractions (35 hrs of development), differences in the mitochondrial GDH thermostability appeared at the stage of hatching (50-60 hrs) and those in the LDH thermostability 12-17 days after hatching. The total activity of AChE and GDH sharply increased during the period of the paternal enzyme appearance whereas the activity of LDH suffered practically no changes. Differences in the AChE thermostability between the hybrids and the maternal species are the same for both the total AChE (in supernatant, 15,000 gX10 min.) and the solubilised AChE (in supernatant, 130,000 gX60 min.). AChE of the parental species and the hybrids have the same electrophoretic mobility. The differences in the thermostability of enzymes are preserved following the electrophoresis in polyacrilamide gel.

[Heat stability of cholinesterase and non-specific esterases during development of hybrids of the sea urchins Stongylocentrotus droebachiensis and S. intermedius]. / Teploustoichivost' atsetilkholinésterazy i nespetsificheskikh ésteraz v razvitii gibridov morskikh ezhei Strongylocentrotus droebachiensis i S. intermedius

Acetylcholine esterase (AchE) and non-specific esterases were studied during the development of sea urchins Strongylocentrotus droebachiensis and S. intermedius and their hybrids by means of electrophoresis and measurements of enzyme thermostability. Two AchE fractions were found which differed by thermostability. At the late gastrula stage, the therolabile form predominated and at the mid-pluteus stage the thermostable one. Non-specific esterases in both the species of sea urchins are represented by complex isozyme systems. Their changes during development are accompanied by the changes in thermostability and electrophoretic patterns. The thermostability of esterases at the pluteus stage in the hybrids is higher than in the maternal species, apparently, due to the appearance of the thermostable enzyme which appears in the paternal species provisionally at the prism stage.