Biochemical and histopathological study of beta 2-microglobulin amyloidosis.
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Amyloidosis↗
Biomedical subjects
K Titani
Publications and source records attributed to K Titani.
Role of the carboxyl-terminal Fib2 domain in fibronectin matrix assembly.
A truncated form of fibronectin consisting of the N-terminal 70 kDa and C-terminal 37 kDa regions, designated r70F2, retained the ability to assemble into the extracellular matrix when expressed in cultured fibroblasts (Ichihara-Tanaka et al. (1992) FEBS Lett. 299, 155-158). To elucidate the role of the C-terminal 37 kDa region in fibronectin matrix assembly, we expressed a panel of mutant forms of r70F2 with various deletions and amino acid substitutions in mouse L cells. Although substitution of Ser for two Cys residues in the C-terminal dimerforming segment led to a marked reduction in the matrix assembly activity of r70F2, the resulting monomeric r70F2 still retained a low, but significant activity to assemble into the matrix. Neither the N-terminal 70 kDa nor the C-terminal 37 kDa regions, when expressed as monomeric forms, exhibited any residual activity, suggesting that the core domain of the 37 kDa region consisting of III15 and I10 through I12 modules, termed Fib2 domain, is actively involved in the matrix assembly of r70F2. In support of the role of Fib2 domain, the proteolytic fragment derived from the 37 kDa region inhibited the assembly of r70F2. Furthermore, en bloc deletion of the Fib2 domain or deletion of the I10 through I12 modules from r70F2 resulted in a marked decrease of the matrix assembly activity.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Isolation and characterization of jararaca GPIb-BP, a snake venom antagonist specific to platelet glycoprotein Ib.
A platelet glycoprotein Ib-binding protein (GPIb-BP) was isolated from the snake venom of Bothrops jararaca. Jararaca GPIb-BP showed a single band with M(r) of 30,000, and two distinct bands with M(r) of 17,000/13,000 under non-reducing and reducing conditions, respectively, on SDS-polyacrylamide gel electrophoresis. Jararaca GPIb-BP itself induced neither platelet aggregation nor serotonin release from platelets, but specifically bound to GPIb (40,629 +/- 2,521 molecules per normal platelet, with Kd 39.1 +/- 2.4 nM at saturation). The purified venom protein completely inhibited ristocetin- or botrocetin-induced von Willebrand factor (vWF) binding, and blocked the bovine vWF binding to GPIb, with IC50 values ranging from 28 to 42 nM, without affecting the platelet aggregation induced by ADP or alpha-thrombin. 125I-jararaca GPIb-BP binding to GPIb was not altered by the presence of human alpha-thrombin. Jararaca GPIb-BP at a final concentration of 104 nM totally abolished vWF-dependent shear-induced platelet aggregation (SIPA) at a high shear stress, but had no effect on SIPA at a low shear stress. Reduced and S-carboxyamido-methylated jararaca GPIb-BP lost its inhibitory activity on SIPA. The NH2-terminal amino acid sequences of the subunits revealed a high degree of homology with those of several Ca(2+)-dependent lectins, especially to those of two functionally opposite venom proteins, botrocetin (a vWF-modulator) and alboaggregin-B (a GPIb-modulator).
Amino Acid Sequence↗
Structural studies of tRNAs by capillary high performance liquid chromatography/electrospray mass spectrometry.
Capillary high performance liquid chromatography/ electrospray mass spectrometry (LC/ ESI/MS) was applied to studies of nucleic acids. Since the presence of salt adducts obscures the molecular peaks and hinders the precise determination of molecular mass, conditions were sought in which effective online desalting and efficient ionization could be achieved simultaneously. In the presence of triethylamine, which is routinely used for the reversed-phase separation of nucleic acid, small oligo-nucleotides could be measured satisfactorily, while RNA tended to show more pronounced adduct peaks. When tributylamine was used instead of the shorter analogue, the adduct peak formation was well suppressed, and the hydrophobic nature of the counter ion made the nucleotides binding to the column tighter, resulting in the better chromatographic separation. The efficient ionization and suppression of the ion adducts make it possible to determine precisely the molecular mass of large nucleotides such as tRNA.
Amines↗
Structural analysis of N-linked oligosaccharides of equine chorionic gonadotropin and lutropin beta-subunits.
Equine chorionic gonadotropin (eCG) and lutropin (eLH) are composed of alpha- and beta-subunits with an identical amino acid sequence but show different biological activities. To elucidate the molecular difference between these gonadotropins, the structure of the N-linked oligosaccharides of each beta-subunit was determined. N-linked sugar chains, liberated as tritum-labeled oligosaccharides by hydrazinolysis followed by N-acetylation and reduction with NaB3H4, were neutralized by sialidase digestion and/or methanolytic desulfation. Neutralized oligosaccharides were fractionated by sequential chromatography on serial lectin affinity columns and on a Bio-Gel P-4 column. Each oligosaccharide structure was determined by sequential exoglycosidase digestion in conjunction with elution profiles on lectin columns and methylation analysis. Each beta-subunit contained a single N-glycosylation site, but a high degree of microheterogeneity was observed in the structure of its N-linked oligosaccharides. eCG beta contained mono-, bi-, tri-, and tetraantennary complex-type oligosaccharides in a ratio of 3:63:13:1. eCG beta oligosaccharides contained about 16% of the bisecting GlcNAc and about 20% of poly-N-acetyllactosamine structures. Elongation of N-acetyllactosamine units showed a preference to the Man alpha 1-->6 side rather than the Man alpha 1-->3 side. Triantennary chains had only a C-2, 4-branched structure. eLH beta contained only mono- and biantennary complex-type and hybrid-type oligosaccharides in a ratio of approximately 18:67:10. eLH beta also contained bisected structures in about 18%. Oligosaccharides derived from the sulfated fraction of eLH beta contained GalNAc residues at nonreducing termini. Oligosaccharides from the sialylated/sulfated fraction of eLH beta contained both Gal and GalNAc residues at nonreducing termini, and those GalNAc residues were preferentially distributed to the Man alpha 1-->3 side of the trimannosyl core. These results clearly indicate that eCG beta and eLH beta possess structurally distinct N-linked oligosaccharides in addition to different charge groups even though they have a protein moiety identical to each other. Our results suggest that the biological activity of these hormones might be modulated by its terminal charge groups and stem structures of carbohydrate moiety synthesized in different organs.
Amino Sugars↗
Alternatively spliced isoform of P-selectin is present in vivo as a soluble molecule.
To demonstrate the presence of a soluble isoform of P-selectin predicted from cDNA sequencing (Johnston, G.I., Bliss, G.A., Newman, P.J., and McEver, R.P. (1990) J. Biol. Chem. 265, 21381-21385), we immunoisolated and compared structurally P-selectin from fresh frozen human plasma with that from washed intact platelets. Plasma P-selectin was reactive with rabbit antiserum to a synthesized peptide (residues 762-774 of mature P-selectin) but was significantly less reactive with antibody to a peptide (residues 747-760). In contrast, platelet P-selectin reacted with both antibodies. S-Pyridylethylated plasma P-selectin was fractionated by reversed phase-high performance liquid chromatography into two major species. From platelets, two virtually identical species were separated. Sequential digestion with Achromobacter protease I and then Staphylococcus V8 protease produced peptides assigned to the tail region of the protein including the putative spliced site. From the more hydrophilic species in both plasma and platelets, a peptide completely lacking the sequence of the putative spliced site was identified. In contrast, the more hydrophobic species yielded a peptide with an intact transmembrane sequence. Hence, these results provide direct evidence that the previously predicted soluble isoform of P-selectin is actually synthesized in vivo and is present as a circulating molecule.
Alternative Splicing↗
A mass spectrometric study on the in vivo posttranslational modification of GAP-43.
GAP-43 isolated from calf brain was analyzed by the electrospray mass spectrometry. The mass spectrum of the intact protein showed two species with a mass difference of 80 Da, suggesting that the isolated GAP-43 contains phosphorylated species. To establish the in vivo phosphorylation sites, the protein was digested with trypsin, and analyzed by the liquid chromatography/mass spectrometry technique, in which a capillary reversed-phase chromatography column was connected on line to an electrospray mass spectrometer. Two pairs of peptides with a mass difference of 80 Da were observed. From the tandem mass spectrometry, two novel phosphorylation sites (Thr-87 and Ser-152) were identified. The novel phosphorylation sites contain proline immediately after the phosphorylated serines. No phosphorylated peptide was detected corresponding to the protein kinase C or casein kinase II phosphorylation sites. A peptide corresponding to the acetylated N-terminal peptide was also identified. The mass of the peptide suggests that the 2 cysteinyl residues are not palmitoylated but form a disulfide bridge.
Amino Acid Sequence↗
Structural elements influencing von Willebrand factor (vWF) binding affinity for platelet glycoprotein Ib within a dispase-digested vWF fragment.
We investigated the structural elements in human von Willebrand factor (vWF) that influence binding affinity for platelet glycoprotein (GP) Ib using a dispase-digested vWF fragment as a prototype (residues Leu480/Val481-Gly718 of the vWF subunit; Andrews et al, Biochemistry 28:8326, 1989). The major structural features of this fragment are a large A1-loop formed by an intrachain disulfide bond between Cys509 and Cys695 and six O-linked sugar chains. The fragment was chemically modified by (1) reduction and S-carboxyamido-methylation (R/A), (2) desialylation (DS), or (3) a combination of both (R/A-DS). The GPIb binding affinity of these fragments was basically evaluated by competitive binding assay with anti-GPIb monoclonal antibody (LJ-Ib1), a receptor blocker for vWF (Sugimoto et al, Biochemistry 30:5202, 1991). Both the prototype and the R/A fragments were also assessed for their function in shear-induced platelet aggregation. Results unambiguously demonstrated that the presence of a disulfide bridge (Cys509-Cys695) within this domain downregulates the affinity of vWF to GPIb. In addition, it was also demonstrated that the terminal sialic acids attached to six o-linked sugar chains within this domain contribute to optimal functional modulation by the antibiotic ristocetin, but not by snake venom botrocetin.
Animals↗
Myristoylated alanine-rich C kinase substrate (MARCKS), a major protein kinase C substrate, is an in vivo substrate of proline-directed protein kinase(s). A mass spectroscopic analysis of the post-translational modifications.
Myristoylated alanine-rich C kinase substrate (MARCKS), a major in vivo substrate protein of protein kinase C, isolated from calf brain contains various species phosphorylated to different degrees. To establish the in vivo phosphorylation sites, the protein was digested with lysyl endoprotease, and the digests were analyzed by the capillary high performance liquid chromatography interfaced on-line to an electrospray mass spectrometer. Six out of 17 peptides were found to be phosphorylated. Of the 7 phosphorylated serine residues identified by Edman degradation, only 1 was within the known phosphorylation domain by protein kinase C. All the other phosphorylated serine residues originated from the N-terminal half of the molecule and were immediately followed by proline. Therefore, MARCKS, a major in vivo substrate of protein kinase C, is also an in vivo substrate of proline-directed protein kinase(s) such as mitogen-activated protein kinase or Cdk5 kinase.
Amino Acid Sequence↗
A 28 kDa-protein with disintegrin-like structure (jararhagin-C) purified from Bothrops jararaca venom inhibits collagen- and ADP-induced platelet aggregation.
A 28 kDa-protein with inhibitory activity on collagen- and ADP-induced platelet aggregation was purified from the venom of the snake Bothrops jararaca. Its complete amino acid sequence corresponded to the carboxyl-terminal region consisting of disintegrin-like and cysteine-rich domains of jararhagin, a high molecular weight hemorrhagic metalloprotease. Sequence homology of the protein to other disintegrins and disintegrin-like proteins from various snake venoms is also presented.
Adenosine Diphosphate↗
Purification and characterization of bothrombin, a fibrinogen-clotting serine protease from the venom of Bothrops jararaca.
A fibrinogen-clotting enzyme (bothrombin) was purified from the venom of Bothrops jararaca. Bothrombin showed M(r) values of 33,000 under nonreducing and 35,000 under reducing conditions on SDS polyacrylamide gel electrophoresis and specific fibrinogen-clotting activity equivalent to 814-904 NIH alpha-thrombin units/mg. Diisopropyl fluorophosphate totally abolished its activity, but hirudin, a specific alpha-thrombin inhibitor, had negligible effect on bothrombin activity. Unlike alpha-thrombin, bothrombin split off fibrinopeptide A without releasing fibrinopeptide B. Bothrombin activated blood coagulation factor VIII, but its activity was about 950 times less than that of alpha-thrombin. Bothrombin did not induce aggregation or serotonin release of washed normal platelets by itself, but did aggregate platelets in the presence of exogenous fibrinogen. This latter activity was completely inhibited by either anti-glycoprotein (GP) IIb/IIIa monoclonal antibody (which blocks fibrinogen binding to GP IIb/IIIa) or anti-GP Ib monoclonal antibody (which specifically inhibits alpha-thrombin binding to GP Ib). Prostaglandin E1 (1 microM) and EDTA (10 mM) also abolished platelet aggregation without affecting clotting activity. Washed platelets from a patient with Bernard-Soulier syndrome did not respond to bothrombin even in the presence of exogenous fibrinogen, suggesting that the initial binding of bothrombin on platelets is GP Ib, but not a recently cloned thrombin receptor. The complete amino acid sequence of bothrombin was determined by analysis of (S)-pyridylethylated protein and peptides generated by digestion with cyanogen bromide and Achromobacter protease I, respectively. Bothrombin is composed of 232 amino acid residues and contains three Asn-linked oligosaccharide chains.(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acid Sequence↗
C-type galactoside-binding lectin from Bothrops jararaca venom: comparison of its structure and function with those of botrocetin.
A Ca(2+)-dependent type (C-type) galactoside-binding lectin was purified from venom of the snake Bothrops jararaca by thiodigalactoside-Sepharose affinity column chromatography. B. jararaca lectin is a disulfide-linked homodimer composed of 14-kDa subunits. The N-terminal 55-residue amino acid sequence was determined and appeared to belong to the animal C-type lectin family. This 55-residue sequence showed 37% identity with botrocetin, an exogenous von Willebrand factor modulator purified from the same venom, and 85% identity with a lectin from rattlesnake (Crotalus atrox) venom. B. jararaca lectin showed Ca(2+)-dependent hemagglutination activity but did not induce platelet aggregation in the presence or absence of Ca2+ and von Willebrand factor and did not inhibit platelet aggregation induced by botrocetin and von Willebrand factor. On the other hand, botrocetin, which also contains a C-type lectin motif in the N-terminal sequence, did not show hemagglutinating activity toward rabbit and human erythrocytes, nor binding activity toward immobilized glycoproteins. These results indicate that at least two structurally similar but functionally distinct proteins, both belonging to the C-type lectin family, are present in the venom of B. jararaca.
Amino Acid Sequence↗
A method for selective isolation of the amino-terminal peptide from alpha-amino-blocked proteins.
A method for selective isolation of the amino N-terminal peptide from an alpha-amino (N alpha)-blocked protein is presented. The method consists of four steps. First, epsilon-amino groups of lysine residues in the protein are succinylated. Second, the derivatized protein is digested by either enzymatic or chemical cleavage. Third, the digest is subjected to reaction with cyanogen bromide-activated Sepharose. Only the N-terminal-blocked peptide fails to react, while the other peptides are covalently bound to the Sepharose. Fourth, uncoupled peptides are purified by reversed-phase chromatography. The amino acid sequence of the peptide including the blocking group can be determined by mass spectrometry. These procedures were successfully tested with three known N alpha-blocked proteins including bovine brain S100 protein, horse cytochrome c, and ovalbumin.
Amines↗
Immunoenzymometric analysis for plasma von Willebrand factor degradation in diabetes mellitus using monoclonal antibodies recognizing protease-sensitive sites.
High-molecular-weight von Willebrand factor (vWf) multimers were separated from their smaller multimers by molecular-sieve chromatography and were measured by several sandwich enzyme-linked immunosorbent assays (ELISA) with monoclonal antibodies (moABs) against human vWf. The epitopes of these moABs were mapped partially using fragments generated by V-8 protease digests of native vWf. Large multimers were more immunoreactive with the sandwich ELISA using immobilized VW28-1 and enzyme-labeled VW92-3 than with any other ELISA. The epitopes recognized by these two moABs were sensitive to trypsin and plasmin digestion, and the other moABs appeared to be reactive with the extensively digested antigen. Relative reactivities of this ELISA to plasma vWf multimers in diabetes mellitus were significantly reduced compared to those in healthy subjects. These data demonstrate that molecular abnormalities of vWf circulating in diabetic patients may be caused by some plasma proteases that increase in the microcirculation of patients with diabetic vascular diseases.
Adult↗
Down's syndrome: up-regulation of beta-amyloid protein precursor and tau mRNAs and their defective coordination.
Almost all patients > 40 years of age with Down's syndrome (DS) develop the pathology characteristic of Alzheimer's disease: abundant beta-amyloid plaques and neurofibrillary tangles. We have investigated the gene expression of beta-amyloid protein precursor (APP) and tau in DS and age-matched control brains and found that levels of both mRNAs were significantly elevated in DS. Such up-regulation was not observed in two other neuronal proteins. A correlation between total APP and tau mRNA levels was also found in DS brain but distinct from the pattern observed in normal brain. Although a proportionality existed between APP-695 mRNA and three-repeat tau mRNA in DS, the proportionality between APP-751 mRNA and four-repeat tau mRNA, which is normally present, was not observed. Thus, DS brains are primarily characterized by the up-regulation of tau mRNA as well as APP mRNA and disruption of the coordinate expression between APP-751 and four-repeat tau.
Adult↗
Cell-adhesive activity and receptor-binding specificity of the laminin-derived YIGSR sequence grafted onto Staphylococcal protein A.
Laminin contains multiple oligopeptide motifs to promote cell adhesion and migration. One of these motifs is YIGSR within the B1 chain. We reconstituted the cell-adhesive activity of YIGSR motif by grafting it onto a truncated form of the Staphylococcal protein A (designated tSPA) via cassette mutagenesis. When coated on a polystyrene surface, the YIGSR-grafted tSPA (YIGSR-tSPA) promoted attachment and spreading of mouse melanoma and human rhabdomyosarcoma cells, but not of hamster fibroblasts. The cell-adhesive activity of YIGSR-tSPA was abolished by amino acid substitution or scrambling of the inserted YIGSR sequence. Divalent cations Mn2+ and Mg2+, but not Ca2+, promoted the cell adhesion to YIGSR-tSPA. Interestingly, the YIGSR-tSPA-mediated cell adhesion was barely inhibited by the linear peptide CDPGYIGSR-NH2, but was strongly inhibited by the cyclic peptide CDPGYIGSRC and another peptide PEILDVPST, which is a specific inhibitor for integrin alpha 4 beta 1. Among various anti-integrin antibodies, anti-alpha 4 and anti-beta 1 antibodies specifically inhibited the cell adhesion to YIGSR-tSPA. In support of these observations, adhesion of rhabdomyosarcoma cells to intact laminin was also partially inhibited by synthetic PEILDVPST peptide and anti-alpha 4 antibody. These results, taken together, indicate that the YIGSR motif exerts its cell-adhesive activity through interaction with integrin alpha 4 beta 1.
Amino Acid Sequence↗
Purification and amino- and carboxyl-terminal amino acid sequences of alanine-glyoxylate transaminase 1 from human liver.
In order to confirm the amino acid sequence predicted from the nucleotide sequence of cDNA and also to elucidate the intracellular localization and molecular evolution, human liver alanine-glyoxylate transaminase 1 (AGT1) was purified and subjected to partial amino acid sequence determination, with special attention to posttranslational modification. The enzyme was purified to homogeneity from the 10,000 x g supernatant of human liver homogenate. The purified enzyme showed only a single protein band at about 43 kDa on SDS-PAGE, indicating that it is a homodimer of two identical subunits, because the native enzyme has a molecular mass of about 80 kDa. Both the amino- and carboxyl-terminal peptides of the enzyme were isolated from a cyanogen bromide digest of the S-carboxyl-methylated protein and subjected to amino acid sequence determination. The alpha-amino group of the amino-terminal peptide was shown to be blocked by an acetyl group. The carboxyl-terminal sequence contained a putative N-glycosylation sequence (-Asn-Ala-Thr-), the only one present in the whole molecule, but this sequence was normally determined, indicating that the enzyme is not N-glycosylated. Purdue et al. [J. Cell Biol. 111, 2341-2351 (1990)] have reported that Pro-11, Gly-170, and Ile-340 in normal human AGT1 were replaced by Leu, Arg, and Met, respectively, in a patient with primary hyperoxaluria type 1. We confirmed that residue-11 was Pro. Both the amino- and carboxyl-terminal sequences of the enzyme showed extensive similarity with those of rat liver mitochondrial serine-pyruvate aminotransferase and the small chain of hydrogenase from a thermophilic unicellular cyanobacterium, Synechococcus PCC 6716.(ABSTRACT TRUNCATED AT 250 WORDS)
Alanine Transaminase↗
Purification and characterization of two Ca(2+)-dependent lectins from coelomic plasma of sea cucumber, Stichopus japonicus.
Two structurally distinct lectins were purified from the coelomic plasma of holothurian, Stichopus japonicus, by affinity chromatography on a porcine stomach mucin-conjugated agarose column, gel filtration on a Superose 6 column, and ion-exchange chromatography on a HiTrap Q-FPLC. The two lectins showed apparent molecular masses of about 400 kDa (SPL-1) and 60 kDa (SPL-2) on gel filtration, but about 17 kDa on SDS-PAGE under reducing conditions. Both lectins showed hemagglutination activity toward rabbit erythrocytes in the presence of Ca2+ ions. The N-terminal amino acid sequences were highly homologous to but distinct from those of a Ca(2+)-dependent (C-type) lectin named SJL-I purified from the same species. In addition to porcine stomach mucin, the hemagglutination activity of SPL-1 was strongly inhibited by uronic acids such as galacturonic acid, and glucuronic acid, while the activity of SPL-2 was inhibited by GalNAc and galactosides. Both lectins were adsorbed on clotted coelomocytes in the presence of Ca2+ but not in the presence of inhibitory sugars or EGTA, suggesting the presence of an endogenous carbohydrate ligand(s) for plasma C-type lectins in the clot. However, coelomocyte clotting occurred normally even in the presence of inhibitory sugars, but was strongly inhibited by synthetic GRGDSP peptide or EGTA, suggesting the participation of integrin but not the lectin-carbohydrate interaction in the clotting events.
Animals↗