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K Titani

Publications and source records attributed to K Titani.

At least 91 records · Page 5Linked to original sources
PubMed1994

Assignment of disulfide bridges in the fusion glycoprotein of Sendai virus.

The mature fusion (F) glycoprotein of the paramyxovirus family consists of two disulfide-linked subunits, the N-terminal F2 and the C-terminal F1 subunits, and contains 10 cysteine residues which are highly conserved at specific positions. The high level of conservation strongly suggests that they are indeed disulfide linked and play important roles in the folding and functioning of the molecule. However, it has not even been clarified which cysteine residues link the F2 and F1 subunits. This report describes our assignment of the disulfide bridges in purified Sendai virus F glycoprotein by fragmentation of the polypeptide and isolation of cystine-containing peptides and determination of their N-terminal sequences. The data demonstrate that all of the 10 cysteine residues participate in disulfide bridges and that Cys-70, the only cysteine in F2, and Cys-199, the most upstream cysteine in F1, form the interchain bond. Of the remaining eight cysteine residues clustered near the transmembrane domain of F1, the specific bridges identified are Cys-338 to Cys-347 and Cys-362 to Cys-370. Although no exact pairings between the subsequent four residues were defined, it seems likely that the most downstream, Cys-424, is linked to Cys-394, Cys-399, or Cys-401. Thus, we conclude that the cysteine-rich domain indeed contributes to the formation of a bunched structure containing at least two tandem cystine loops.

Amino Acid Sequence↗
PubMed1994

[Protein design based on the structure and function relationship and its clinical application].

Proteins are biopolymers which are directly involved in biological functions. Since their defects cause various diseases, proteins have been studied for a long time as the subjects of both basic and clinical medicine. Recent development of the genetic recombinant technique has made it possible to replace, add or delete any amino acid residue or any peptide portion in a protein. By utilizing this technique, we are now able to clarify more precisely than ever the structure and function relationship of a protein, and also to create various novel artificial proteins for the clinical use. In this article, I describe the proteins which are involved in blood coagulation and fibrinolysis and the regulation, and the current clinical application of the recombinant proteins. I also describe our new protein engineering technique for creation of artificial cell adhesive proteins by grafting the Arg-Gly-Asp Ser (RGDS) cell recognition signal, which was identified first in fibronectin and later in various plasma and extracellular matrix proteins. The physiological functions and possible clinical application of these artificial cell adhesive proteins thus created are also discussed.

Animals↗
PubMed1993

In vivo phosphorylation sites in fetal and adult rat tau.

Fetal tau and tau in paired helical filaments show similar immunoreactivities with several phosphorylation-dependent paired helical filament-polyclonals and monoclonals, suggesting that the two molecules share several distinct phosphorylated epitopes. To make clear the similarities and differences between the two, we have undertaken work to identify the in vivo phosphorylation sites in fetal rat tau. We have approached this problem by identifying phosphopeptides by means of mass spectrometry and sequencing of those phosphopeptides after modification with ethanethiol. Although remarkable heterogeneity was present, fetal tau was found to bear at most 10 phosphates at Ser-189, Ser-190, Ser-193, Ser-226, Ser-387, Ser-395, Thr-172, Thr-222, and, presumably, Ser-391 and Thr-208 (numbering is according to the longest form of rat tau; Kosik, K. S., Orecchio, L. D., Bakalis, S., and Neve, R. L. (1989) Neuron 2, 1389-1397). In contrast, adult rat tau was much less phosphorylated; only Thr-172, Ser-190, Ser-193, Thr-222, and Ser-395 were phosphorylated to a slight-to-moderate extent. All these sites except for Ser-189 and Ser-391 were followed by Pro residues. Thus, tau is an in vivo substrate for proline-directed protein kinase(s), and its phosphorylation state is developmentally regulated.

Aging↗
PubMed1993

Molecular heterogeneity of follistatin, an activin-binding protein. Higher affinity of the carboxyl-terminal truncated forms for heparan sulfate proteoglycans on the ovarian granulosa cell.

Follistatin (FS), an activin-binding protein, is a monomer derived from two polypeptide core sequences of 315 (FS-315) and 288 (FS-288) amino acids originated from alternatively spliced mRNA. To define the structural heterogeneity of native FS, we purified six molecular forms of FS from porcine ovaries. Protein chemical analysis revealed that the structural differences among the six isoforms were caused by truncation of the carboxyl-terminal region and/or the presence of carbohydrate chains, resulting in the formation of FS-315, FS-288, and FS composed of 303 amino acids (FS-303) in various forms of glycosylation on the two potential Asn-linked glycosylation sites. The majority of FS isolated from porcine ovaries was FS-303, which may have been derived from FS-315 by proteolytic cleavage of the 12 COOH-terminal amino acids. All six molecular species have almost the same activin binding activity (Kd = 540-680 pM). By contrast, the COOH-terminal truncated form, FS-288, showed much higher affinity for the rat granulosa cell surface than FS-303, whereas FS-315 had no affinity. FS-288 bound to heparan sulfate-Sepharose CL-4B, but FS-315 did not, suggesting that the truncated forms of FS bind to heparan sulfate proteoglycans on the cell. COS cells transfected with the FS-288 DNA expressed the FS-288 protein, which adhered to the cell surface, but cells transfected with the FS-315 DNA secreted the expressed protein into the medium, which did not bind to the cell surface. In rat anterior pituitary culture, FS-288 (ED50 = 2 ng/ml) was more potent in suppressing follicle-stimulating hormone release than FS-303 (ED50 = 10 ng/ml) and FS-315 (ED50 = 20 ng/ml). These results suggest that cell-associated FS traps activin more tightly in the matrix, thereby more effectively blocking the activity of activin on heparan sulfate proteoglycans of the cell surface and that cell-associated FS plays an important role in controlling the various actions of activin in a paracrine or autocrine manner.

Activins↗
PubMed1993

Human plasma alpha 2-macroglobulin and von Willebrand factor possess covalently linked ABO(H) blood group antigens in subjects with corresponding ABO phenotype.

We recently identified ABO(H) blood group structures in Asn-linked sugar chains of human von Willebrand factor (vWF) purified from factor VIII concentrates (J Biol Chem 267:8723, 1992). We surveyed plasma glycoproteins carrying ABO(H) blood group antigens by Western blotting analysis and sandwich enzyme-linked immunosorbent assay using blood group-specific monoclonal antibodies (MoAbs) and a lectin. Two major plasma proteins showing apparent molecular weight of about 180 Kd and 270 Kd by sodium dodecyl sulfate polyacrylamide gel electrophoresis reacted with blood group-specific MoAbs and Ulex europaeus lectin I in accordance with donor blood group. Direct sequence analysis of the protein bands showed their identity with the N-terminal sequences of alpha 2-macroglobulin (alpha 2M) and vWF, respectively. The two bands also reacted with anti-alpha 2M and anti-vWF antibodies. The alpha 2M and vWF prepared from plasma by immunoprecipitation showed the appropriate blood group antigenicity. After incubation with endoglycosidase F, both alpha 2M and vWF lost almost all reactivity with anti-blood group reagents. About 90% of plasma vWF, but only approximately 10% of alpha 2M, was immunoprecipitated with anti-blood group antibody. These results indicate that at least two plasma glycoproteins, vWF and alpha 2M, possess Asn-linked ABO(H) blood group antigens in normal individuals with corresponding ABO phenotype. Therefore, ABO(H) blood group antigens in plasma glycoproteins should be considered during preparation of plasma materials for therapeutic use.

ABO Blood-Group System↗
PubMed1993

Structural and functional analyses of the Arg-Gly-Asp sequence introduced into human lysozyme.

To determine the functional conformation of the Arg-Gly-Asp (RGD) sequence, we have constructed mutant proteins by inserting 4-12 amino acid residues from the RGD region of human fibronectin between Val74 and Asn75 of human lysozyme. RGDS-, GRGDSP-, TGRGDSPA-, VTGRGDSPAS-, and AVTGRGDS-PASS-introduced mutant lysozymes were expressed in yeast, purified, and designated as RGD4, -6, -8, -10, and -12, respectively. Using baby hamster kidney cells, RGD8, RGD10, and RGD12 were shown to possess high cell adhesion activity nearly equal to 10% of human vitronectin activity. RGD4 and RGD6 exhibited somewhat lower cell adhesion activity. The activities of these mutant proteins were inhibited by the addition of either GRGDSP peptide or polyclonal antibody against vitronectin receptor, as was the case for the vitronectin activity. The results suggest that the cell adhesion signals are transduced to cells through the interaction with the vitronectin receptor. The three-dimensional structures of RGD4 and RGD8 were determined at 1.8-A resolution by x-ray crystallography. A model of the inserted region in RGD4 could be built in the electron density map, but the positions of the preceding residues, Ala73-Val74, were uncertain. The inserted region in RGD8 did not demonstrate continuous electron densities. The results suggest that these RGD sequence-containing regions are highly flexible and that such flexibility could allow the conformation of the RGD regions to be induced to fit into the binding pocket of the integrin receptor.

Amino Acid Sequence↗
PubMed1993

Coordinate oncodevelopmental modulation of alternative splicing of fibronectin pre-messenger RNA at ED-A, ED-B, and CS1 regions in human liver tumors.

The molecular diversity of fibronectin arises from alternative RNA splicing at regions termed ED-A, ED-B, and IIICS. We investigated the splicing patterns of fibronectin pre-mRNA at both ED-B and IIICS regions in various human liver tissues with an emphasis on the expression of the alternative cell adhesive site CS1 within the IIICS region. The relative abundance of the fibronectin mRNA containing the CS1 sequence was significantly increased in both fetal and cancerous liver tissues, although it was not affected in nonmalignant tissues with chronic hepatitis and cirrhosis. Similarly, the relative abundance of the fibronectin mRNA containing the ED-B region was also increased in both fetal liver and liver tumors, showing a close parallelism with the splicing pattern at the ED-A region. Immunohistochemical examination of cancerous liver tissues with monoclonal antibodies directed to the ED-A and ED-B segments revealed that the fibronectin isoforms containing these extra peptide segments were specifically deposited in the tumor nodules. Other genes encoding kininogen, gamma chain of fibrinogen, and beta-amyloid protein precursor, all of which had been shown to be alternatively processed, did not show any significant alteration in the splicing pattern in cancerous liver tissues. These results indicate that the alternative splicing of fibronectin pre-mRNA at the ED-A, ED-B, and IIICS regions is coordinately modulated in both fetal and cancerous liver tissues toward inclusion of the extra peptide segments and that not all but only selected genes are susceptible for "fine tuning" of alternative RNA splicing in cancerous liver tissues.

Alternative Splicing↗
PubMed1993

Alboaggregin-B and botrocetin, two snake venom proteins with highly homologous amino acid sequences but totally distinct functions on von Willebrand factor binding to platelets.

Alboaggregin-B (AL-B) (Peng et al., Biochemistry (1991) 30, 11529-11536) was highly purified from the snake venom of Trimeresurus albolabris and characterized structurally and functionally, comparing with botrocetin, another snake venom protein recently characterized (Usami et al., Proc. Natl. Acad. Sci. USA (1993) 90, 928-932). Both the venom proteins are a heterodimer and show a high degree of sequence homology to each other and also to C-type lectins. Botrocetin specifically binds to von Willebrand factor (vWF), whereas AL-B binds to platelet glycoprotein (GP) Ib without affecting the binding of botrocetin to vWF. The binding of AL-B to GPIb does not potentiate the platelet aggregation even by exogenous fibrinogen, suggesting that AL-B binding to GPIb does not activate GPIIb/IIIa complex.

Amino Acid Sequence↗
PubMed1993

Primary structure of two-chain botrocetin, a von Willebrand factor modulator purified from the venom of Bothrops jararaca.

The complete amino acid sequence and location of the disulfide bonds of two-chain botrocetin, which promotes platelet agglutination in the presence of von Willebrand factor, from venom of the snake Bothrops jararaca are presented. Sequences of the alpha and beta subunits were determined by analysis of peptides generated by digestion of the S-pyridylethylated protein with Achromobacter protease I or alpha-chymotrypsin and by chemical cleavage with cyanogen bromide or 2-(2'-nitrophenylsulfenyl)-3-methyl-3-bromoindolenine. Two-chain botrocetin is a heterodimer composed of the alpha subunit (consisting of 133 amino acid residues) and the beta subunit (consisting of 125 amino acid residues) held together by a disulfide bond. Seven disulfide bonds link half-cystine residues 2 to 13, 30 to 128, and 103 to 120 of the alpha subunit; 2 to 13, 30 to 121, and 98 to 113 of the beta subunit; and 80 of the alpha subunit to 75 of the beta subunit. In terms of amino acid sequence and disulfide bond location, two-chain botrocetin is homologous to echinoidin (a sea urchin lectin) and other C-type (Ca(2+)-dependent) lectins.

Amino Acid Sequence↗
PubMed1993

Neuronal pp60c-src(+) in the developing chick spinal cord as revealed with anti-hexapeptide antibody.

Polyclonal antibody was raised in rabbits against a synthetic hexapeptide R-K-V-D-V-R corresponding to a unique amino acid sequence of the neuron-specific c-src gene product pp60c-src(+). The antibody was purified by affinity chromatography. A single band with an apparent molecular mass of 60 kDa was recognized when the supernatant of homogenates of brain and spinal cord from chick embryos and chicks was probed with the affinity purified anti-hexapeptide antibody after SDS-polyacrylamide gel electrophoresis followed by Western blotting. Specificity of the antibody was further characterized by autophosphorylation assay of immunoprecipitate in comparison with the monoclonal antibody 327. Immunocytochemical studies by light microscopy revealed that pp60c-src(+) was localized in flake-like aggregates in neuronal cell bodies of the spinal cord in 7-15-day-incubated chick embryos and newly hatched chicks. Developing spinal ganglia and muscle cells were also immunoreactive at early developmental stages. By electron microscopy, the reaction product was observed mainly in two regions. One region was at polysomes and along the membranes of the rough endoplasmic reticulum. The other region was along the neuronal plasma membrane--at subsurface cisterns and at synapses. At synapses, the postsynaptic density, presynaptic membrane and synaptic vesicle membranes were immunostained. Immunoreactivity at synapses were more frequently observed at earlier stages than at later stages of development. These findings suggest that pp60c-src(+) is actively produced in developing neurons and has some important roles in synaptogenesis. In mature synapses, pp60c-src(+) may be involved in the interaction of synaptic vesicles with the presynaptic membrane.

Amino Acid Sequence↗
PubMed1993

The 5'-flanking region of the human dopamine beta-hydroxylase gene promotes neuron subtype-specific gene expression in the central nervous system of transgenic mice.

Dopamine beta-hydroxylase (DBH, EC 1.14.17.1) catalyzes the conversion of dopamine to norepinephrine, the third step of catecholamine biosynthesis. We have previously created transgenic mice harboring a chimeric gene consisting of the 4-kb DNA fragment of the human DBH gene promoter and the human phenylethanolamine N-methyltransferase (PNMT, EC 2.1.1.28) cDNA, to express PNMT in norepinephrine- and epinephrine-producing cells in the brain, sympathetic ganglia, and adrenal medullary chromaffin cells (Kobayashi et al., Proc. Natl. Acad. Sci. U.S.A., 89 (1992) 1631-1635). In this paper, we produced for the first time the antibody that specifically detects human PNMT, but not mouse PNMT, with the synthetic oligopeptide characteristic of the human PNMT sequence, and used this antibody to investigate the cells expressing human PNMT in transgenic mice. Immunohistochemical analysis of transgenic mice showed typical expression of human PNMT immunoreactivity in norepinephrinergic and epinephrinergic neurons in brain, as well as norepinephrine- and epinephrine-producing cells in the adrenal gland, indicating that the 4-kb 5'-flanking region is essential for the tissue-specific expression of the DBH gene. We also detected the ectopic expression in some DBH-immunonegative cells in the olfactory bulb of transgenic mice.

Amino Acid Sequence↗
PubMed1993

Comparative studies of asparagine-linked sugar chains of immunoglobulin G from eleven mammalian species.

1. Asparagine-linked sugar chains released by hydrazinolysis from IgGs of porcine, equine, bovine, goat, ovine, canine, rabbit, guinea-pig and rat were comparatively analyzed by microsequencing and lectin affinity chromatography. 2. Sugar chains of all IgGs basically consisted of biantennary complex-type oligosaccharides containing 0-2 sialic acid residue(s). More than 70% of the oligosaccharides were neutral, except for guinea-pig IgG, and fucosylated trimannosyl core structures were dominant except for rabbit IgG. Bisecting N-acetylglucosamine residue was absent in porcine and equine IgGs. 3. A large quantity of galactose-less oligosaccharides were present in IgGs of porcine, equine, canine and rat.

Animals↗
PubMed1993

Ubiquitin is conjugated with amino-terminally processed tau in paired helical filaments.

We have investigated ubiquitinated paired helical filaments, which produce a proteinaceous smear in SDS-polyacrylamide gel electrophoresis and immunoblotting. The smear consisted largely of the carboxy-terminal portion of tau and ubiquitin. The ubiquitin-targeted protein was identified as tau in paired helical filaments, and the conjugation sites were localized to the microtubule-binding region. Most ubiquitin in paired helical filaments occurred as a monoubiquitinated form, and only a small proportion of ubiquitin formed multiubiquitin chains. There was a ubiquitin-negative smear, in which tau was much less processed in the amino-terminal portion. This strongly suggests that the amino-terminal processing of tau in paired helical filaments precedes its ubiquitination.

Amino Acid Sequence↗
PubMed1993

Ribonuclease activity of sialic acid-binding lectin from Rana catesbeiana eggs.

Sialic acid-binding lectin (SBL) isolated from Rana catesbeiana eggs is a basic protein which agglutinates a large variety of tumour cells and has an amino acid sequence homologous to that of human angiogenin and pancreatic ribonuclease (RNase). Although SBL and angiogenin lack the Cys-65-Cys-72 disulphide bond of pancreatic RNase, the locations of the other three disulphide bonds are similar among the three molecules. SBL was found to exhibit RNase activity, as well as catalytic properties resembling those of bovine RNase A in some respects. For example, SBL hydrolyses poly(uridylic acid) and poly(cytidylic acid) as substrates, and prefers the former. RNase A and angiogenin are strongly inhibited by human placental RNase inhibitor, whereas the RNase activity and tumour cell agglutination activity of SBL are not affected by this inhibitor.

Amino Acid Sequence↗
PubMed1993

Engineering of artificial cell-adhesive proteins by grafting EILDVPST sequence derived from fibronectin.

Fibronectin contains at least two distinct oligopeptide sequences serving as signals for the interaction with cell surface adhesion receptors termed integrins. One of these sequences, Arg-Gly-Asp-Ser (RGDS) tetrapeptide, was shown to be transferred to a truncated form of Staphylococcal IgG-binding protein (hereafter referred to as tSPA) with retention of its cell-adhesive activity [Maeda, T. et al. (1989) J. Biol. Chem. 264, 15165-15168]. We have extended the observation to another cell-adhesive sequence, Glu-Ile-Leu-Asp-Val-Pro-Ser-Thr (referred to as "CS1" sequence), to demonstrate that: i) the tSPA grafted with the sequence mediated adhesion of human lymphoma and rhabdomyosarcoma cells, mouse melanoma cells, but not of hamster fibroblasts; ii) antibodies against integrin alpha 4 and beta 1 subunits specifically inhibited cell adhesion mediated by the CS1-grafted tSPA; iii) a heterodivalent tSPA grafted with both RGDS and CS1 sequences at different sites was more potent in promoting cell adhesion than the monovalent tSPAs grafted with either sequence alone. These results indicate that not only the RGDS but also the CS1 sequence can be transferred to tSPA with retention of its cell-adhesive activity as well as its cell-type specificity, and that the grafted CS1 sequence is recognized by the same integrin isotype as the authentic sequence within intact fibronectin.

Amino Acid Sequence↗
PubMed1993

Improved method for expression of Kunitz-type serine proteinase inhibitor domain of beta-amyloid protein precursor in Escherichia coli and characterization of disulfide bonds of the product.

Kunitz-type serine proteinase inhibitor (KPI) domain of Alzheimer's disease-related beta-amyloid protein precursor (APP) was expressed in Escherichia coli as a fusion protein with a truncated form of Staphylococcus protein A. The fusion protein was purified from the cell culture medium using an IgG Sepharose column. The KPI domain was separated from the protein A portion by cleavage with human alpha-thrombin at the engineered recognition sequence, followed by purification on IgG Sepharose and reversed-phase HPLC columns. The recombinant KPI domain strongly inhibited trypsin; the inhibition constant (Ki) for bovine trypsin was 2.5 x 10(-11) M, comparable to those of the secreted forms of APP with the KPI domain. The recombinant protein contained three intramolecular disulfide bonds, which were determined to be located between Cys-6 (C1) and Cys-56 (C6), Cys-15 (C2) and Cys-39 (C4), and Cys-31 (C3) and Cys-52 (C5) of the recombinant KPI domain, respectively. These positions are highly homologous to those of disulfide bonds in bovine pancreatic trypsin inhibitor. The trypsin-inhibitory activity of the recombinant protein was abolished by preincubation with 0.4 mM dithiothreitol under non-denaturing conditions. By this mild reduction, all the disulfide bonds were completely cleaved. These results clearly indicate that the disulfide bonds play an important role in the function of the KPI domain of APP.

Alzheimer Disease↗
PubMed1993

Characterization of two distinct monoclonal antibodies to paired helical filaments: further evidence for fetal-type phosphorylation of the tau in paired helical filaments.

Two monoclonal antibodies C5 and M4 raised against Sarkosyl-insoluble paired helical filaments (PHF) specifically labeled fetal tau, but hardly labeled normal adult tau. C5 immunoreactivity was eliminated by alkaline phosphatase treatment at 37 degrees C, whereas M4 reactivity could be removed only by the treatment at 67 degrees C. Epitope analysis showed that C5 and M4 recognition sites are in residues 386-406 and 198-250, respectively, according to the numbering of the longest human tau isoform. Thus, the phosphorylation sites are located in the amino- and carboxyl-terminal portions of the microtubule-binding region. These two well-characterized monoclonals should be valuable in the identification of a protein kinase(s) that converts normal tau into PHF-tau.

Alzheimer Disease↗