Activator proteins for glycosphingolipid hydrolysis by endoglycoceramidases. Elucidation of biological functions of cell-surface glycosphingolipids in situ by endoglycoceramidases made possible using these activator proteins.

Endoglycoceramidase (EGCase) cleaves the linkage between oligosaccharides and ceramides of various glycosphingolipids (Ito, M., and Yamagata, T. (1986) J. Biol. Chem. 261, 14278-14282). Recently, by extensive purification, it was separated from cell-lytic factor (hemolysin) and found to consist of three molecular species each with its own specificity (EGCases I, II, and III) (Ito, M., and Yamagata, T. (1989) J. Biol. Chem. 264, 9510-9519). A detergent was required for EGCases to express full activity, possibly due to their hydrophobic nature, and thus EGCases cannot be used for research on live cells. This paper presents findings on activator proteins in the culture supernatant of Rhodococcus sp. M-777 regarding the stimulation of EGCase activity in the absence of detergents. The activator protein, exhaustively purified and designated as activator II in this study, showed a single protein band on sodium dodecyl sulfate-, native-, and isoelectrofocussing-polyacrylamide slab gel electrophoresis after being stained with Coomassie Brilliant Blue. Its molecular weight and pI were 69,200 and 4.0, respectively. The activator protein enhanced the hydrolysis of glycosphingolipids in vitro and on the cell-surface by EGCase II in the absence of detergents in a concentration-dependent manner. Interestingly, activator II stimulated the activity of EGCase II much more than that of EGCase I on using asialo-GM1 as the substrate. This activator protein was found nonspecific to substrates susceptible to hydrolysis with EGCase II. Besides activator II, strain M-777 produced a second minor molecular species of activator protein designated as activator I which appeared specific for stimulating the activity of EGCase I in contrast to activator II. Following the addition of activator II, EGCase II hydrolyzed cell-surface glycosphingolipids quite efficiently at neutral pH at which hydrolysis hardly occurred at all in its absence. When using activator II in place of Triton X-100 for stimulating EGCase II activity, it was also noted to cause no damage to intact cells. It is thus possible by activator proteins to elucidate the biological functions of endogenous glycosphingolipids in situ by EGCases.     

Different conformational states of the purified Ca2+-ATPase of the erythrocyte plasma membrane revealed by controlled trypsin proteolysis

The purified Ca2+-pumping ATPase of the erythrocyte membrane has been exposed to trypsin at 37 degrees C, in the presence of different effectors of its activity. The control proteolytic pattern is characterized by a number of transient and of limit polypeptides (Zurini, M., Krebs, J., Penniston, J. T., and Carafoli, E. (1984) J. Biol. Chem. 259, 618-627). The effectors influence the pattern in the Mr region 90,000-76,000, which contains the calmodulin binding domain and the active site of the enzyme. In this region, polypeptides of 90, 85, 81, and 76 kDa are clearly visible in the controls. 1) Calmodulin plus Ca2+ induces the faster disappearance of the 90-kDa product and the relative accumulation of the 85-kDa with respect to the 81-kDa polypeptide. 2) Vanadate plus Mg2+ also accelerates the disappearance of the 90-kDa product. However, they induce the relative accumulation of the 81-kDa polypeptide. 3) Linoleic acid, which stimulates the activity of the enzyme to the same levels obtained with calmodulin, greatly accelerates the rate of trypsin proteolysis, causing the virtual disappearance of all polypeptides in the 90-76-kDa region. 4) The 81-kDa polypeptide has maximal ATPase activity and is insensitive to calmodulin; the 85-kDa polypeptide has lower ATPase activity and binds calmodulin, but is not stimulated (or is stimulated only negligibly) by the activator.     

A major oligomeric fibroblast proteoglycan identified as a novel large form of type-XII collagen.

Cultured chick embryo skin fibroblasts release a major component with a native molecular mass of about 1 MDa, which resolves into three polypeptide bands of about 300, 350 and 600 kDa upon reduction. We report here the purification of this oligomeric protein and show, by means of polyclonal and monoclonal antibodies, that its three polypeptide constituents are closely related. The 600-kDa polypeptide is likely to be a dimer of two smaller subunits which are cross-linked by non-reducible bonds. By electron microscopy, isolated oligomeric molecules exhibit a novel cruciform structure with a large central globular domain. One arm has the shape of a thin rod about 70 nm in length. The three other arms are thicker, longer (90 nm) and flexible, and carry a prominent double globule at their distal ends. Collagenase treatment of the oligomeric fibroblast protein yields two resistant fragments of about 270 kDa and 320 kDa. The intact 350-kDa and 600-kDa (but not the 300-kDa) polypeptides are chondroitinase sensitive and labeled by metabolic incorporation of [35S]sulfate; collagenase treatment does not remove any [35S] sulfate. Hence, the intact fibroblast protein has glycosaminoglycan chains attached to its non-collagenous domain. Three amino acid sequences obtained from chymotryptic fragments of the fibroblast protein correspond to sequences predicted for chick type-XII collagen from its full-length cDNA [Yamagata, M., Yamada, K. M., Yamada, S. S., Shinomura, T., Tanaka, H., Nishida, Y., Obara, M. & Kimata, K. (1991) J. Cell Biol. 115, 209-221]. However, the novel fibroblast protein described here differs significantly from previously isolated forms of type-XII collagen: its subunits are larger by one third, and it is a proteoglycan.     

Immunostaining on thin-layer chromatograms of oligosaccharides released from gangliosides by endoglycoceramidase

A method for immobilizing oligosaccharides on a TLC plate for immunostaining has been developed. N-Glycolylneuraminic acid (NeuGc)-containing oligosaccharides derived from II3NeuGc-LacCer, IV3NeuGc-nLcOse4Cer, II3NeuGc-GgOse3Cer, and II3(NeuGc)2-LacCer by digestion with our newly isolated endoglycoceramidase (Ito, M. & Yamagata, T. (1986) J. Biol. Chem. 261, 14278-14282) and sialyllactose were chromatographed on polyamide 11 TLC or NH2-HPTLC plates, and covalently linked to the plates by reductive amination with sodium cyanoborohydride (NaBH3-CN). The immobilized oligosaccharides were detected by enzyme-immunostaining using NeuGc-specific chicken anti-NeuGc-LacCer and horseradish peroxidase-conjugated rabbit anti-chicken IgG. II3NeuGc-nLcOse4 showed the highest reactivity with the antibody, followed by II3NeuGc-GgOse3. As little as 0.8 nmol of the NeuGc-containing oligosaccharides was detected. The polyamide 11 TLC aluminum plate was found to be more suitable for the immunostaining than the NH2-HPTLC plate under the conditions used. For binding of the oligosaccharides to the NH2-HPTLC plate, reductive amination was found to be superior to the heating method reported earlier.     

Isolation and sequence of a tropomyosin-binding fragment of turkey gizzard calponin

Limited chymotryptic cleavage of turkey gizzard calponin yields a 13 kDa fragment which could be purified by its ability to bind to Sepharose-immobilized tropomyosin. This 13 kD polypeptide is shown to be derived from a 22 kDa fragment. Complete amino acid sequence analysis of the 13 kD and 22 kD fragments reveals high homology with the formerly characterized smooth muscle-specific protein SM22 alpha (Pearlstone, J.R., Weber, M., Lees-Miller, J.P., Carpenter, M.R. and Smillie L.B., 1987, J. Biol. Chem. 262, 5985-5991) and the product of gene mp20 of Drosophila (Ayme-Southqate, A., Lasko, P., French, C, and Pardue, M.L. [(1989) J. Cell Biol. 108, 521-531]. Futhermore we recognize sequence elements of a putative actin-binding domain of alpha-actinin, the calpactin I or p 36 sequence, and a consensus motif present in the repeats of the gene product of the candidate unc-87 gene of C. elegans (S.D. Goetinck and R.H. Waterston, personal communication).     

Purification, characterization, molecular cloning, and subcellular distribution of neutral ceramidase of rat kidney

Previously, we reported two types of neutral ceramidase in mice, one solubilized by freeze-thawing and one not. The former was purified as a 94-kDa protein from mouse liver, and cloned (Tani, M., Okino, N., Mori, K., Tanigawa, T., Izu, H., and Ito, M. (2000) J. Biol. Chem. 275, 11229--11234). In this paper, we describe the purification, molecular cloning, and subcellular distribution of a 112-kDa membrane-bound neutral ceramidase of rat kidney, which was completely insoluble by freeze-thawing. The open reading frame of the enzyme encoded a polypeptide of 761 amino acids having nine putative N-glycosylation sites and one possible transmembrane domain. In the ceramidase overexpressing HEK293 cells, 133-kDa (Golgi-form) and 113-kDa (endoplasmic reticulum-form) Myc-tagged ceramidases were detected, whereas these two proteins were converted to a 87-kDa protein concomitantly with loss of activity when expressed in the presence of tunicamycin, indicating that the N-glycosylation process is indispensable for the expression of the enzyme activity. Immunohistochemical analysis clearly showed that the ceramidase was mainly localized at the apical membrane of proximal tubules, distal tubules, and collecting ducts in rat kidney, while in liver the enzyme was distributed with endosome-like organelles in hepatocytes. Interestingly, the kidney ceramidase was found to be enriched in the raft microdomains with cholesterol and GM1 ganglioside.     

Phosphorylation-induced change of the oligomerization state of alpha B-crystallin

alphaB-crystallin in cells can be phosphorylated at three serine residues in response to stress or during mitosis (Ito, H., Okamoto, K., Nakayama, H., Isobe, T., and Kato, K. (1997) J. Biol. Chem. 272, 29934-29941 and Kato, K., Ito, H., Kamei, K., Inaguma, Y., Iwamoto, I., and Saga, S. (1998) J. Biol. Chem. 273, 28346-28354). In the present study, we determined effects of phosphorylation of alphaB-crystallin on its oligomerization state, mainly by using site-directed mutagenesis, in which all three phosphorylation sites were substituted with aspartate to mimic the phosphorylation state (3D-alphaB). From results of sucrose density gradient centrifugation, we found that wild type alphaB-crystallin (wt-alphaB) and 3D-alphaB sedimented in fractions corresponding to apparent molecular masses of about 500 and 300 kDa, respectively. Chaperone-like activity of 3D-alphaB was significantly weaker than that of wt-alphaB. When wt-alphaB and 3D-alphaB were expressed in COS-m6 cells, they sedimented at positions corresponding to apparent molecular masses of about 500 and 100 kDa, respectively. In U373 MG human glioma cells, alphaB-crystallin was observed as large oligomers with apparent molecular masses about 500 kDa and the oligomerization size was reduced after phosphorylation induced by phorbol 12-myristate 13-acetate and okadaic acid. Coexpression of luciferase and wt-alphaB or 3D-alphaB in Chinese hamster ovary cells caused protection of the enzyme from heat inactivation although the degree of protection with 3D-alphaB was less than that with wt-alphaB. From these observations, it is suggested that phosphorylation of alphaB-crystallin causes dissociation of large oligomers to smaller sizes molecules and reduction of chaperone-like activity, like in the case of HSP27.     

Ceramidase enhances phospholipase C-induced hemolysis by Pseudomonas aeruginosa

We previously reported the purification, molecular cloning, and characterization of a neutral ceramidase from Pseudomonas aeruginosa strain AN17 (Okino, N., Tani, M., Imayama, S., and Ito, M. (1998) J. Biol. Chem. 273, 14368-14373; Okino, N., Ichinose, S., Omori, A., Imayama, S., Nakamura, T., and Ito, M. (1999) J. Biol. Chem. 274, 36616-36622). Interestingly, the gene encoding the enzyme is adjacent to that encoding hemolytic phospholipase C (plcH) in the genome of Pseudomonas aeruginosa, which is a well known pathogen for opportunistic infections. We report here that simultaneous production of PlcH and ceramidase was induced by several lipids and PlcH-induced hemolysis was significantly enhanced by the action of the ceramidase. When the strain was cultured with sphingomyelin or phosphatidylcholine, production of both enzymes drastically increased, causing the increase of hemolytic activity in the cell-free culture supernatant. Ceramide and sphingosine were also effective in promoting the production of ceramidase but not that of PlcH. Furthermore, we found that the hemolytic activity of a Bacillus cereus sphingomyelinase was significantly enhanced by addition of a recombinant Pseudomonas ceramidase. TLC analysis of the erythrocytes showed that ceramide produced from sphingomyelin by the sphingomyelinase was partly converted to sphingosine by the ceramidase. A ceramidase-null mutant strain caused much less hemolysis of sheep erythrocytes than did the wild-type strain. Sphingosine was detected in the erythrocytes co-cultured with the wild-type strain but not the mutant strain. Finally, we found that the enhancement of PlcH-induced hemolysis by the ceramidase occurred in not only sheep but also human erythrocytes. These results may indicate that the ceramidase enhances the PlcH-induced cytotoxicity and provide new insights into the role of sphingolipid-degrading enzymes in the pathogenicity of P. aeruginosa.     

Total synthesis of the structural gene for the precursor of a tyrosine suppressor transfer RNA from Escherichia coli. 11. Enzymatic joining to form the total DNA duplex.

The DNA duplex corresponding to the entire length (126 nucleotides) of the precursor for an Escherichia coli tyrosine tRNA has been synthesized. Duplex [I] (Sekiya, T., Besmer, P., Takeya, T., and Khorana, H. G.(1976) J. Biol. Chem. 251, 634-641), corresponding to the nucleotide sequence 1-26, containing single-stranded ends and carrying one appropriately labeled 5'-phosphate group, was joined to duplex [II] (Loewen, P. C., Miller, R. C., Panet, A., Sekiya, T., and Khorana, H. G. (1976) J. Biol. Chem. 251, 642-650) (nucleotide sequence 23-66 or 23-60) was phosphorylated with [gamma-33P]ATP at the 5'-OH ends. Duplex [III] (Panet, A., Kleppe, R., Kleppe, K., and Khorana, H. G. (1976) J. Biol. Chem. 251, 651-657) (nucleotide sequence 57-94 (Fig. 2)) was also phosphorylated at 5'-ends with [gamma-33P]ATP and was joined to duplex [IV] (Caruthers, M. H., Kleppe, R., Kleppe, K., and Khorana, H. G. (1976) J. Biol. Chem. 251, 658-666) (nucleotide sequence 90-126) which carried a 33P-labeled phosphate group on nucleotide 90. The joined product, duplex [III + IV] (nucleotide sequence 57-126) was characterized. The latter duplex was joined to the duplex [I + II] to give the total duplex. The latter contains singlestranded ends (nucleotides 1 to 6 and 121 to 126) which can either be "filled in" to produce the completely base-paired duplex or may be used to add the promoter and terminator regions at the appropriate ends.     

Subunit composition of vacuolar membrane H(+)-ATPase from mung bean

The vacuolar H(+)-ATPase from mung bean hypocotyls was solubilized from the membrane with lysophosphatidycholine and purified by QAE-Toyopearl column chromatography. The purified ATPase was active only in the presence of exogenous phospholipid and was inhibited by nitrate, dicyclohexyl carbodiimide and Triton X-100, but not by vanadate or azide. Dodecyl sulfate/polyacrylamide gel electrophoresis of the purified ATPase yielded ten polypeptides of molecular masses of 68 kDa, 57 kDa, 44 kDa, 43 kDa, 38 kDa, 37 kDa 32 kDa, 16 kDa, 13 kDa and 12 kDa. All polypeptides remained in the peak activity fraction after glycerol density gradient centrifugation. Nine of them, excluding the 43-kDa polypeptide, comigrated in a polyacrylamide gradient gel in the presence of 0.1% Triton X-100. The 16-kDa polypeptide could be labeled with [14C]dicyclohexylcarbodiimide. The amino-terminal amino acid sequence of the isolated 68-kDa polypeptide generally agreed with that deduced from the cDNA for the carrot 69-kDa subunit [Zimniak, L., Dittrich, P., Gogarten, J. P., Kibak, H. & Taiz, L. (1988) J. Biol. Chem. 263, 9102-9112]. Thus, mung bean vacuolar H(+)-ATPase seems to consist of nine distinct subunits.     

A low-molecular-mass Kazal-type protease inhibitor isolated from rat hepatocytes is identical to rat pancreatic secretory trypsin inhibitor II. Purification and amino acid sequence

A low-molecular-mass serine protease inhibitor was purified from hepatocytes and liver of rats. It was found to be a single polypeptide of 56 amino acid residues corresponding to Mr = 6224, a value that is in agreement with the molecular mass determined by gel chromatography. The inhibitor formed a complex in a molar ratio of 1:1 with trypsin. Its complete amino acid sequence was identical with that of pancreatic secretory trypsin inhibitor II (PSTI-II) in pancreatic juice, but not with that of PSTI-I [Uda, K., Ogawa, M., Shibata, T., Murata, A., Mori, T., Kikuchi, N., Yoshida, N., Tsunasawa, S. & Sakiyama, F. (1988) Biol. Chem. Hoppe-Seyler 369, 55-61]. PSTIs have been reported to be primarily pancreatic secretory products, but in have been reported to be primarily pancreatic secretory products, but in patients immunoreactive PSTI was found in the plasma and urine during acute inflammatory disease and shown to be produced ectopically in cancer tissues. Here we report for the first time that PSTI-II is present in other normal tissues besides the pancreas.     

Characterization of iron-sulfur clusters in lysine 2,3-aminomutase by electron paramagnetic resonance spectroscopy.

Lysine 2,3-aminomutase from Clostridia catalyzes the interconversion of L-alpha-lysine with L-beta-lysine. The purified enzyme contains iron-sulfur ([Fe-S]) clusters, pyridoxal phosphate, and Co(II) [Petrovich, R. M., Ruzicka, F. J., Reed, G. H., & Frey, P. A. (1991) J. Biol. Chem. 266, 7656-7660]. Enzymatic activity depends upon the presence and integrity of these cofactors. In addition, the enzyme is activated by S-adenosylmethionine, which participates in the transfer of a substrate hydrogen atom between carbon-3 of lysine and carbon-2 of beta-lysine [Moss, M., & Frey, P. A. (1987) J. Biol. Chem. 262, 14859-14862]. This paper describes the electron paramagnetic resonance (EPR) properties of the [Fe-S] clusters. Purified samples of the enzyme also contain low and variable levels of a stable radical. The radical spectrum is centered at g = 2.006 and is subject to inhomogeneous broadening at 10 K, with a p1/2 value of 550 +/- 100 microW. The low-temperature EPR spectrum of the [Fe-S] cluster is centered at g = 2.007 and undergoes power saturation at 10 K in a homogeneous manner, with a p1/2 of 15 +/- 2 mW. The signals are consistent with the formulation [4Fe-4S] and are adequately simulated by a rhombic spectrum, in which gxx = 2.027, gyy = 2.007, and gzz = 1.99. Treatment of the enzyme with reducing agents converts the cluster into an EPR-silent form. Oxidation of the purified enzyme by air or ferricyanide converts the [Fe-S] complex into a species with an EPR spectrum that is consistent with the formulation [3Fe-4S].(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanism for ganglioside-mediated modulation of a calmodulin-dependent enzyme. Modulation of calmodulin-dependent cyclic nucleotide phosphodiesterase activity through binding of gangliosides to calmodulin and the enzyme.

Gangliosides were recently shown to bind to calmodulin (Higashi, H., Omori, A., and Yamagata, T. (1992) J. Biol. Chem. 267, 9831-9838). This prompted us to investigate the effects of gangliosides on the calmodulin-dependent enzyme, cyclic nucleotide phosphodiesterase. Several species of gangliosides competitively inhibited calmodulin-stimulated phosphodiesterase activity, with GD1b, GT1b, and GD1a being noted to do so particularly (group 1). GM1, GQ1b, and GM2 (group 2) were less inhibitory, and GM3, GM3(NeuGc), GalCer, sulfatide, GgOse4Cer, and oligosaccharide portions of inhibitory gangliosides showed no inhibition in accordance with the binding specificity of calmodulin to gangliosides. Trypsin-activated phosphodiesterase was inhibited by gangliosides with similar specificity, indicating interactions of gangliosides with the enzyme. Inhibition, however, was less than that of calmodulin-dependent activity by these compounds and, in both cases, was eliminated by excess calmodulin. In the absence of calmodulin, group 1 gangliosides at lower concentrations activated the intact enzyme but inhibited it over a certain range of increase in concentration. Ganglioside-dependent modulation of calmodulin-dependent phosphodiesterase activity is thus shown to be due to interactions of gangliosides with both calmodulin and the enzyme, and consequently, ganglioside-calmodulin binding is likely the mechanism for regulation of the enzyme.     

Comparison of calcium-binding proteins. Bovine heart and brain protein activators of cyclic nucleotide phosphodiesterase and rabbit skeletal muscle troponin C.

In previous studies we have shown that the activation of bovine heart cyclic nucleotide phosphodiesterase by purified protein activator is completely dependent on the presence of Ca2+ and that the protein activator Ca2+ complex is probably the true activator for the enzyme (Teo, T.S. and Wang, J.H. (1973) J. Biol. Chem. 248, 5930-5955). More recent studies have led us to believe that the mechanism of the Ca2+ activation of phosphodiesterase resembles that of the Ca2+ activation of muscle contraction and that the protein activator may play a role similar to troponin. In the present study we show that the protein activator resembles rabbit muscle troponin C in amino acid composition, molecular weight, isoelectric point, and ultraviolet absorption spectrum. Preliminary structural studies also indicate that these two proteins may have evolved from a common ancestral protein through gene duplication. This argument is strengthened by the finding that the tryptic peptide map of the bovine heart protein activator is indistinguishable from that of the bovine brain phosphodiesterase activator protein for which preliminary sequence information also suggests homology to troponin C (Watterson, D.M., Harrelson, W.G., Jr., Keller, P.M., Sharief, F., and Vanaman, T.C. (1976) J. Biol. Chem. 251, 4501-4513).     

A totally synthetic histidine-2 ferredoxin: thermal stability and redox properties.

The entire polypeptide of Clostridium pasteurianum ferredoxin (Fd) with a site-substituted tyrosine-2----histidine-2 was synthesized using standard t-Boc procedures, reconstituted to the 2[4Fe-4S] holoprotein, and compared to synthetic C. pasteurianum and native Fds. Although histidine-2 is commonly found in thermostable clostridial Fds, the histidine-2 substitution into synthetic C. pasteurianum Fd did not significantly increase its thermostability. The reduction potential of synthetic histidine-2 Fd was -343 and -394 mV at pH 6.4 and 8.7, respectively, versus standard hydrogen electrode. Similarly, Clostridium thermosaccharolyticum Fd which naturally contains histidine-2 was previously determined to have a pH-dependent reduction potential [Smith, E.T., & Feinberg, B.A. (1990) J. Biol. Chem. 265, 14371-14376]. An electrostatic model was used to calculate the observed change in reduction potential with pH for a homologous ferredoxin with a known X-ray crystal structure containing a hypothetical histidine-2. In contrast, the reduction potential of both native C. pasteurianum Fd and synthetic Fd with the C. pasteurianum sequence was -400 mV versus standard hydrogen electrode and was pH-independent [Smith, E.T., Feinberg, B.A., Richards, J.H., & Tomich, J.M. (1991) J. Am. Chem. Soc. 113, 688-689]. On the basis of the above results, we conclude that the observed pH-dependent reduction potential for both synthetic and native ferredoxins that contain histidine-2 is attributable to the electrostatic interaction between histidine-2 and iron-sulfur cluster II which is approximately 6 A away.     

Activatory effect of regucalcin on hepatic plasma membrane (Ca2+-Mg2+)-ATPase is impaired by liver injury with carbon tetrachloride administration in rats

Human thymus poly(A) polymerase (EC 2.7.7.19) activity has been investigated using poly(A) and oligo(A) as initiators. All obtained fractions reveal more than one polypeptide as detected by immunoblotting after SDS-PAGE. In addition to the homogeneously purified (Tsiapalis et al., J Biol Chem 250: 4486–1496, 1975 and Wahle, J Biol Chem 266: 3131–3139, 1991), about 60 kDa polypeptide, a larger polypeptide, about 80 kDa, that comigrates in the region of poly(A) polymerase activity was detected, enriched and partially characterized; it appears having similar size with bovine poly(A) polymerase cloned in E. coli. Polyclonal antiserum produced against recombinant bovine poly(A) polymerase reacts more efficiently with the about 80 kDa polypeptide upon immunoblotting, and can precipitate the poly(A) polymerase activity. This enzyme form, from human tissue, is novel in terms of size and may reflect intact or physiological form of poly(A) polymerase in human thymus, and supports and substantiates recent reports on the enzyme from other sources.