Hexagonal bilayer structuring activity of linker chains of an annelid giant hemoglobin from the polychaete Perinereis aibuhitensis.

Preferential activity of linker chains to clamp submultiples to form hexagonal bilayer (HBL) assembly of the multisubunit hemoglobin (Hb) of the polychaete Perinereis aibuhitensis (approximately 3.4 MDa) was demonstrated. To understand the HBL assembly that should rely on structuring activity of each subunit, reassociation in response to combining isolated subunits was monitored using gel filtration, SDS-PAGE, and transmission electron microscopy. The isolation of each subunit L, T, and M (L, linker chains; T, disulfide-bonded trimer A-b-B; M, monomeric chain a) of Perinereis Hb was made simply by exposing Hb to pH 10.5, where Hb was completely dissociated into its subunits L, T, and M. As a result, it was concluded that (i) subunits T and M have strong affinity to form an intermediate complex, submultiple D, which is a dodecamer of globin chains, 3[a. A-b-B], (ii) addition of subunit L to submultiple D brings about the formation of whole molecule, similarly (iii) addition of subunit M to T+L forms the whole molecule, and (iv) addition of subunit T to M+L brings about the formation of the whole molecule, too. The results obtained lead us to conclude that linkers do function to clamp 12 submultiples D up to a whole molecule at the final step of formation of Perinereis Hb. In summary, linkers appeared to have high affinity for submultiple D, a little affinity for subunit T, but no affinity for subunit M at all. Thus linker chains were demonstrated to preferentially clamp submultiples D together to form the HBL disc of the whole molecule.

Carbohydrate gluing is a strategy for supramolecular clamping of submultiples in annelid extracellular multi-subunit hemoglobin.

A key to understanding the mysterious hierarchic organization of annelid multi-subunit extracellular hemoglobin (giant Hb, composed of 144 globin chains and about 36 nonheme chains called linkers) is knowing the role of linkers in holding together the entire two-tiered hexagonal form. Here, the effects of added monosaccharides on the dissociation of giant Hb from the marineworm Perinereis aibuhitensis were monitored using dynamic light scattering (DLS), transmission electron microscopy (TEM), and circular dichroism (CD) measurements. Changes in Stokes radius and more clearly the distribution analysis of the Hb based on the DLS measurements showed that Hb preferentially dissociates into hexagonal units (called submultiples), which was consistent with the results of TEM and CD measurements. The results thus show that linkers specifically "clamp" submultiples together to organize the two-tiered form through carbohydrate gluing. Thus, a submultiple behaves like an ordinary protein, whereas the intact Hb behaves like a miniature supramolecular system. This clamp model is plausible because it inherently involves catastrophe of the molecular stoichiometry at the two-tiered hexagonal formation level because carbohydrates are under posttranslational regulation and therefore contain structural ambiguity.

Natural and artificial carbohydrate-glued protein aggregates.

Carbohydrate gluing (which may have a carbohydrate-lectin binding mechanism) was first recognized as a major contributor in the supramolecular assembly of annelid giant Hb from the marine-worm P. aibuhitensis. Although this assembly obviously also relies on protein-protein interactions, the authors tested the application of carbohydrate gluing in the assembly of a protein aggregate using a lectin and a carbohydrate-containing protein. The resultant aggregate was a mixture of the protein aggregate and the ingredient proteins. The significance of the method is that the assembly of the aggregate can be controlled by using a hapten sugar. This controllability, in conjunction with newly developing glyco-technology, has great potential for the construction of arbitrary protein molecules into a regular protein aggregate, thereby providing sophisticated functions.

Identification of the subunit loci in the extracellular multisubunit hemoglobin from annelid Perinereis aibuhitensis.

Giant hemoglobin (Hb) from Perinereis aibuhitensis is made of several types of protein components including single-chain globin (a), disulfide-bridged globin trimer (A-b-B), disulfide-bridged dimers of nonglobin chain (or linkers; L1-L1, L2-L2, and L1-L2), and oligomers of L1-L2 [(L1-L2)n]. The intact form of this giant Hb is a two-tiered hexagonal structure composed of 12 identical units, or so-called submultiples (six submultiples to a tier). To obtain a view of the three-dimensional architectural arrangement of these components in the intact form, we identified the subunit loci by using two mutually complementary chemical modifications and a colloidal gold labeling technique. Using the chemical modifications, we discovered that (i) linkers L1-L2 and L2-L2 were located at the exterior of the Hb, (ii) linker L1-L1 and globin a were buried in the interior, and (iii) linker (L1-L2)n and globin trimer A-b-B were located at both exterior and interior loci. The labeling with an L2-specific colloidal gold revealed the predominant loci of L2 at the outer and inner boundaries between neighboring submultiples in a hexagonal form. By combining these results with those from our previous reports [S. Ebina, K. Matsubara, K. Nagayama, M. Yamaki, and T. Gotoh (1995) Proc. Natl. Acad. Sci. USA 92, 7367-7371; K. Matsubara, M. Yamaki, Nagayama, H. Ishii, K. Imai, T. Gotoh, and S. Ebina (1996), in press], we deduced the following conclusions concerning the Hb architecture. The L1-L1 chains perhaps together with (L1-L2)n chains form a scaffold on which submultiples assemble into a two-tiered hexagonal arrangement, probably by connecting the carbohydrates in globin a. The L1-L2 and L2-L2 chains reinforce the connections of the submultiples by binding carbohydrates, perhaps those carbohydrates in globin A. We proposed to call this type of non-protein-dependent structural level as seen in such a carbohydrate-glued protein aggregate "protein-plus structure."

Wheat germ agglutinin-reactive chains of giant hemoglobin from the polychaete Perinereis aibuhitensis.

Wheat germ agglutinin-reactive chains of multisubunit extracellular hemoglobin from the polychaete Perinereis aibuhitensis were identified to clarify the carbohydrate gluing which is the carbohydrate-dependent supramolecular architecture of the hemoglobin (Ebina S. et al. (1995) Proc. Natl. Acad. Sci. USA 92, 7367-7371). Electron microscope micrographs of Perinereis hemoglobin showed a characteristic shape of two-tiered hexagonal rings whose diameter and height were determined to be 29.4 +/- 1.7 nm and 20.0 +/- 1.8 nm, respectively. Four types of globins and two types of linkers were isolated from the giant hemoglobin by reverse-phase chromatography and SDS-PAGE. These constituents showed similar NH2-terminal sequences as those previously reported for corresponding chains of Tylorrhynchus hemoglobin (Suzuki T. and Gotoh T. (1986) J. Biol. Chem. 261, 9257-9267; Suzuki T. et al. (1990) J. Biol. Chem. 265, 12168-12177). Thus, each globin of Perinereis hemoglobin was identified in terms of amino acid sequence homology and designated using names common to Tylorrhynchus hemoglobin, namely, a, A, b, and B. The linkers were stained by horseradish peroxidase (HRP)-lectins and PAS staining kits, indicating the presence of carbohydrate oligomers. Lectin staining was also significantly positive to globins a and A, which belong to strain A, but negative to globins b and B, which belong to strain B. Results showed that linkers and globins of strain A had a site in a carbohydrate oligomer to which wheat germ agglutinin (WGA) could bind. On the other hand, an alignment between known amino acid sequences of annelid globins and linkers and the sequences of lectins revealed that only the domain of the cysteine-rich motif in linkers has a homology with WGA-type lectins. The results of this study clarify the structuring mechanism of a supramolecule by lectin-like binding, called carbohydrate gluing.

Thromboxane A2 receptor-mediated signal transduction in rabbit aortic smooth muscle cells.

1. 9,11-Epithio-11,12-methenothromboxane A2 (STA2), a stable analogue of thromboxane A2 (TXA2), contracted rabbit aortic smooth muscles (RASM) and accumulated [3H]inositol phosphates in cultured RASM cells. The contraction and phosphoinositide hydrolysis were competitively inhibited by TXA2 receptor antagonists, including ONO NT-126, S-145, SQ29548, KW3635, GR32191B and ONO3708. 2. STA2 inhibited [3H]ONO NT-126 binding in a concentration-dependent manner in membranes derived from cultured aortic smooth muscle cells, but GTP gamma S, a stable GTP analogue, did not affect STA2-induced inhibition of [3H]ONO NT-126 binding. 3. The time course analysis revealed that STA2 rapidly decreased inositol phosphate level and therefter increased. Pertussis toxin did not attenuate but rather increased STA2-induced phosphoinositide hydrolysis. 4. TXA2 receptor stimulation results in at least two signaling pathways in RASM cells: stimulation and inhibition of phosphoinositide hydrolysis.

Carbohydrate gluing, an architectural mechanism in the supramolecular structure of an annelid giant hemoglobin.

We report a carbohydrate-dependent supramolecular architecture in the extracellular giant hemoglobin (Hb) from the marine worm Perinereis aibuhitensis; we call this architectural mechanism carbohydrate gluing. This study is an extension of our accidental discovery of deterioration in the form of the Hb caused by a high concentration of glucose. The giant Hbs of annelids are natural supramolecules consisting of about 200 polypeptide chains that associate to form a double-layered hexagonal structure. This Hb has 0.5% (wt) carbohydrates, including mannose, xylose, fucose, galactose, glucose, N-acetylglucosamine (GlcNAc), and N-acetylgalactosamine (GalNAc). Using carbohydrate-staining assays, in conjunction with two-dimensional polyacrylamide gel electrophoresis, we found that two types of linker chains (L1 and L2; the nomenclature of the Hb subunits followed that for another marine worm, Tylorrhynchus heterochaetus) contained carbohydrates with both GlcNAc and GalNAc. Furthermore, two types of globins (a and A) have only GlcNAc-containing carbohydrates, whereas the other types of globins (b and B) had no carbohydrates. Monosaccharides including mannose, fucose, glucose, galactose, GlcNAc, and GalNAc reversibly dissociated the intact form of the Hb, but the removal of carbohydrate with N-glycanase resulted in irreversible dissociation. These results show that carbohydrate acts noncovalently to glue together the components to yield the complete quaternary supramolecular structure of the giant Hb. We suggest that this carbohydrate gluing may be mediated through lectin-like carbohydrate-binding by the associated structural chains ("linkers").

A protein kinase C with divalent cations contributes to thromboxane A2-induced contraction in rabbit vascular smooth muscle.

We investigated the mechanism of contraction induced by a stable thromboxane A2 receptor agonist, STA2, in rabbit aortic smooth muscles. STA2 induced a long-lasting contraction which persisted for over 5 hours. This contraction was found to be potently inhibited by EDTA. In the presence of EGTA, STA2 was able to slowly contract muscle to a near maximum level, suggestive of an extracellular Ca(2+)-independent component in STA2 action. Inhibition of the STA2-induced contraction by EDTA was partially overcome by the addition of Mg2+. Ca2+ and Mn2+ were also effective in attenuating the inhibition. A phorbol ester, PDBu, an activator of PKC (protein kinase C), induced a long lasting contraction in a manner similar to that of STA2. PKC inhibitors, staurosporine and H-7, inhibited the lasting contractions induced by STA2 and PDBu. PKC inhibitors abolished STA(2)-induced contraction in the absence of extracellular Ca2+, suggesting that Ca(2+)-influx from the extracellular space as well as PKC activation are involved in STA(2)-induced contraction. ML-7, a myosin light chain kinase inhibitor, also inhibited the STA(2)-induced contraction, but it did not abolish the contraction in the absence of extracellular Ca2+. Furthermore, STA2 elicited phosphatidylcholine hydrolysis in cultured aortic smooth muscle cells. From the results obtained, we arrived at the hypothesis that PKC contributes to this lasting contraction in the presence of divalent cations, such as Mg2+, Ca2+ or Mn2+. Of these, Mg2+ is the most capable of maintaining this contraction. The Ca-dependent process alone could not account for the long lasting contraction induced by STA2 in vascular smooth muscles.

EDTA and EGTA can discriminate tonic contractions induced by thromboxane A2 and phorbol ester in rabbit aortic smooth muscles.

The effects of chelating agents of divalent cations on the contraction induced by STA2, a stable thromboxane A2 analog, were examined in rabbit aortic smooth muscles, comparing with a phorbol ester-induced contraction. Pretreatment of muscles with EDTA (4 mM) resulted in potent inhibition of STA2 (1 nM)-induced contraction. However, STA2 could contract muscles slowly in the presence of EGTA (4 mM). The muscles contracted with STA2 relaxed rapidly after addition of EDTA and/or EGTA. However, STA2 recovered contraction in the presence of EGTA, but not of EDTA. PDBu, phorbol 12,13-dibutyrate, also contracted the muscles slowly and potently. Pretreatment with EDTA, but not EGTA, attenuated PDBu-induced contractions. The muscles contracted with PDBu relaxed after the addition of EDTA, but not of EGTA. The present results imply that the vascular smooth muscle contractions are composed of two distinct components: a short-lived contraction that is related to Ca2+ and a lasting contraction that is related to Mg2+, and EDTA and EGTA are good tools for discriminating the tonic phase of STA2- and/or PDBu-induced contraction.

Site-specific reactivities of cysteine residues in horse L-apoferritin.

Recombinant horse L-apoferritin and its mutants were used to compare the reactivities of two different cysteinyl residues with 7-fluoro-4-sulfamoyl-2,1,3-benzoxadiazole (ABD-F), p-chloromercuribenzoic acid (PCMB), N-(9-acridinyl)maleimide (NAM), and 4-maleimido-2,2,6,6-tetramethylpiperidine-N-oxyl (NEM-TEMPO). ABD-F selectively reacted with cysteine 52 (C52), which is located on the inner surface of the peptide shell of apoferritin. In contrast, PCMB reacted only with cysteine 130 (C130), which is located at the 3-fold channels of the shell. NAM and NEM-TEMPO reacted with both C52 and C130.

Heterogeneities in ferritin dimers as characterized by gel filtration, nuclear magnetic resonance, electrophoresis, transmission electron microscopy, and gene engineering techniques.

To understand the mechanism underlying the preferential dimerization of ferritin shells, we studied monomers and dimers from both horse spleen and recombinant horse L-apoferritin by using gel filtration, nuclear magnetic resonance, electrophoresis, transmission electron microscopy, and gene engineering techniques. Our study of the kinetics of dimer-monomer dissociation that is produced by heating revealed the presence of at least two types of dimers, namely, weakly and strongly linked dimers with activation energies of 124 +/- 14 and 157 +/- 16 kJ/mol, respectively. Our study using thiol reagents indicated that the dimerization in horse spleen ferritin is partially mediated by disulfide bridges being formed between H-chains. Our analysis of the components that resulted from the dimer-monomer dissociation further clarified that these dimers form interdigitation structures. In summary, five types of dimers were identified in horse spleen apoferritin: reversible dimers with very weak interaction, non-sulfide dimers with weak interaction, non-sulfide dimers with strong interaction, disulfide dimers linked only by disulfide bridges, and disulfide dimers linked by disulfide bridges and having other interactions.

Cloning, expression and characterization of horse L-ferritin in Escherichia coli.

Horse L-ferritin cDNA was cloned from horse liver, and the base sequence was determined. The L-ferritin was expressed using pTZ18U encoding lac promoter, and found to possess an additional 8-amino acid sequence at the N-terminus as compared with commercially obtained horse spleen (natural) ferritin. It was determined that there was Pro at position 94 in both the recombinant and natural L-ferritin, although it was previously reported that Leu was in this position in the natural species. Transmission electron microscopy showed that this recombinant ferritin formed a 24-mer shell.

Chemical modification of bovine pancreatic trypsin inhibitor for single site coupling of immunogenic peptides for NMR conformational analysis.

Procedures for chemical modification of bovine pancreatic trypsin inhibitor (BPTI) to allow site-specific coupling of immunogenic peptides are reported. Each of the modified proteins has a single free amino group; the other amino groups of lysine or the amino terminus are blocked by acetylation or guanidination. Two of the derivatives were prepared by protecting Lys-15 by complexation with trypsin or chymotrypsin during acetylation with N-hydroxysuccinimide acetate or guanidination with 3,5-dimethylpyrazole-1-carboxamidine nitrate. A third derivative with a free amino group at the amino terminus was prepared by guanidination of the 4 lysine residues with o-methylisourea. The purity and structural integrity of the modified proteins was checked by NMR spectroscopy. Cysteine-containing peptides can be coupled to the single free amino group using several heterobifunctional linking reagents. N-Succinimidyl 3-(2-pyridyldithio)propionate is the most satisfactory coupling reagent for NMR studies because of its high specificity. Two-dimensional NMR spectroscopy shows that the conformation of the modified proteins is almost identical with that of native BPTI. The BPTI derivatives are suitable for use as models for NMR investigations of the conformation of immunogenic peptides conjugated to a carrier protein.

[Nuclear magnetic resonance investigations of DMBA-induced rat breast cancers--the relationship between tumor doubling times and water proton spin-lattice relaxation times].

Our studies of human neoplastic tissues had revealed that the T1 values of many malignant tissues had a tendency to be longer than those of normal ones. But the T1 values did not always reflect the grade of malignancy on account of the variety of their histologies. Therefore, by using DMBA-induced rat breast cancer, we investigated the relation between the degree of malignancy and the T1 values, and the following results were obtained. 1) The growth rate of the cancers had a correlation with their T1 values, and 2) the water content of the cancers correlated with their T1 values, but 3) there was no correlation between their growth rate and their water content.

Amide proton titration shifts in bull seminal inhibitor IIA by two-dimensional correlated 1H nuclear magnetic resonance (COSY). Manifestation of conformational equilibria involving carboxylate groups.

Amide proton titration shifts in H2O solution of bull seminal inhibitor IIA were measured over the pH range from 3 to 6 using two-dimensional correlated spectroscopy. These data enabled characterization of the pKa values for the majority of the carboxylate groups in the protein. Two glutamate side-chains were found to form hydrogen bonds with their own backbone amide proton. Different temperature variations of the populations of these local, cyclic structure elements are indicated for the individual sites.

A globular protein with slower amide proton exchange from an alpha helix than from antiparallel beta sheets.

In proteinase inhibitor IIA from bull seminal plasma, which is a small globular protein with 57 amino acid residues, measurements of individual amide proton exchange rates by two-dimensional correlated 1H NMR spectroscopy (COSY) showed that the exchange was slowest for some hydrogen bonded amide groups in an alpha-helix. This contrasts with all other proteins which were so far studied in detail, where the slowest exchange rates were observed for hydrogen bonded amide protons in antiparallel beta-sheets.

Kinetic study of CO and O2 binding to horse heart myoglobin reconstituted with synthetic hemes lacking methyl and vinyl side chains.

Carbon monoxide- and oxygen-binding rates and affinities were measured for horse heart myoglobins reconstituted with synthetic hemes lacking peripheral methyl and vinyl groups. There is an apparent correlation between heme size and ligand specificity, i.e. larger m values (ratios of CO vs O2 association rates, l'/k') with smaller hemes. However, this correlation broke down with the most dealkylated heme. This is interpreted as resulting from protein conformational changes altering the steric crowdedness at the O2-binding site. Spectral properties and autoxidation rates also corroborate this view.