Kohji Okumura

Prostaglandins are powerful inducers of NGF and BDNF production in mouse astrocyte cultures

We found that prostaglandin (PG) D2 and PGE2, which are major PGs in the brain of mammals, powerfully induced the secretion of nerve growth factor (NGF) from cultured mouse astrocytes; PGE2 or PGD2 induced an approximately 12‐ or 19‐fold increase in NGF secretion after a 24‐h incubation, respectively. Moreover, it was found that the sequential metabolites of PGD2, PGJ2, Δ12‐PGJ2, and 15‐deoxy‐Δ12,14‐PGJ2, induced the NGF secretion to the culture medium strikingly (60–98‐fold of the control after a 24‐h incubation). NGF secretion induced by the J2 series of PGs was accompanied by the increased expression of NGF mRNA. These PGs also stimulated the secretion/synthesis of brain‐derived neurotrophic factor (BDNF). Our findings suggest that PGs play a neuroprotective role by inducing NGF and BDNF production in the central nervous system.

A Novel Phospholipase A2 Inhibitor with Leucine-rich Repeats from the Blood Plasma of Agkistrodon blomhoffii siniticus SEQUENCE HOMOLOGIES WITH HUMAN LEUCINE-RICH α2-GLYCOPROTEIN

The phospholipase A2(PLA2) inhibitor PLIβ, purified from the blood plasma of Chinese mamushi snake (Agkistrodon blomhoffii siniticus), is a 160-kDa trimer with three 50-kDa subunits; and it inhibits specifically the enzymatic activity of the basic PLA2 from its own venom (Ohkura, N., Okuhara, H., Inoue, S., Ikeda, K., and Hayashi, K. (1997) Biochem. J. 325, 527–531). In the present study, the 50-kDa subunit was found to be glycosylated withN-linked carbohydrate, and enzymatic deglycosylation decreased the molecular mass of the 50-kDa subunit to 39-kDa. One 160-kDa trimer of PLIβ was found to form a stable complex with three basic PLA2 molecules, indicating that one basic PLA2 molecule would bind stoichiometrically to one subunit of PLIβ. A cDNA encoding PLIβ was isolated from a Chinese mamushi liver cDNA library by use of a probe prepared by a polymerase chain reaction on the basis of the partially determined amino acid sequence of the subunit. The cDNA contained an open reading frame encoding a 23-residue signal sequence followed by a 308-residue protein, which contained the sequences of all the peptides derived by lysyl endopeptidase digestion of the subunit. The molecular mass of the mature protein was calculated to be 34,594 Da, and the deduced amino acid sequence contained four potentialN-glycosylation sites. The sequence of PLIβ showed no significant homology with that of the known PLA2inhibitors. But, interestingly, it exhibited 33% identity with that of human leucine-rich α2-glycoprotein, a serum protein of unknown function. The most striking feature of the sequence is that it contained nine leucine-rich repeats (LRRs), each of 24 amino acid residues and thus encompassing over two-thirds of the molecule. LRRs in PLIβ might be responsible for the specific binding to basic PLA2, since LRRs are considered as the motifs involved in protein-protein interactions.

Mapping the Region of the α-Type Phospholipase A2 Inhibitor Responsible for Its Inhibitory Activity

α-Type phospholipase A2 inhibitory protein (PLIα) from the serum of the venomous snake Gloydius brevicaudus, GbPLIα,isone of the protective endogenous proteins that neutralizes its own venom phospholipase A2 (PLA2), and it is a homotrimer of subunits having a C-type lectin-like domain. The nonvenomous snake Elaphe quadrivirgata has a homologous serum protein, EqPLIα-LP, that does not show any inhibitory activity against various snake venom PLA2s (Okumura, K., Inoue, S., Ikeda, K., and Hayashi, K. (2003) IUBMB Life 55, 539–545). By constructing GbPLIα-Eq- PLIα-LP chimeric proteins, we have mapped the residues important in conferring GbPLIα inhibitory activity on region 13–36 in the primary structure of GbPLIα. Noninhibitory EqPLIα-LP showed comparable inhibitory activity only when this region was replaced with that of GbPLIα. Further, mutational analysis of the candidate residues revealed that the individual GbPLIα to EqPLIα-LP residue substitutions N26K, K28E, D29N, and Y144S each produced a mutant GbPLIα protein with reduced inhibitory activity, with the single N26K substitution having the most significant effect. Residues 13–36 were suspected to be located in the helical neck region of the GbPLIα trimer. Therefore, the region of GbPLIα responsible for PLA2 inhibition was distinct from the carbohydrate-binding site of the homologous C-type lectin.

Identification of β-type phospholipase A2 inhibitor in a nonvenomous snake, Elaphe quadrivirgata

Abstract A novel serum protein inhibiting specifically the enzymatic activity of the basic phospholipase A 2 (PLA 2 ) from the venom of the Chinese mamushi snake ( Agkistrodon blomhoffii siniticus ) was purified from a nonvenomous Colubridae snake, Elaphe quadrivirgata . The purified inhibitor was a 150-kDa glycoprotein having a trimeric structure, composed of two homologous 50-kDa subunits. Their amino acid sequences, containing leucine-rich repeats, were typical of the β-type PLA 2 inhibitor (PLIβ), previously identified from the serum of A. blomhoffii siniticus . The inhibitor inhibited exclusively group II basic PLA 2 s and did not inhibit other kinds of PLA 2 s. This is the first paper reporting the existence of PLIβ in a nonvenomous snake. The existence of PLIβ in the nonvenomous snake reflects that PLIβs are widely distributed over the snake species and participate commonly in regulating the physiological activities of the unidentified target PLA 2 s.

cDNA cloning and bacterial expression of phospholipase A2 inhibitor PLIα from the serum of the Chinese mamushi, Agkistrodon blomhoffii siniticus

Abstract The cDNA encoding of a phospholipase A2 inhibitor (PLIα) of the Chinese mamushi, Agkistrodon blomhoffii siniticus, was identified from a liver cDNA library by use of a probe prepared by polymerase chain reaction (PCR) on the basis of the amino acid sequence of PLIα. It encoded a polypeptide of 166 amino acid residues, including 19 residues of the signal sequence and 147 residues of the complete mature sequence of PLIα. The PLIα cDNA was subcloned into the expression vector pET-16b and used to transform Escherichia coli strain BL21(DE3)pLysS. The recombinant PLIα expressed as a fusion protein was solubilized and purified to homogeneity by use of a metal affinity resin. The purified PLIα fusion protein underwent folding to form a trimeric structure like the intact PLIα, and showed inhibitory activity against the group II acidic PLA2 from A. blomhoffii siniticus venom; although its binding constant (1/Ki) value was 30-fold lower than that of the natural PLIα. The elimination of the N-terminal additional peptide from the fusion protein resulted in a marked increase in the inhibition activity with a binding constant comparable to that of the natural PLIα against the acidic PLA2. Furthermore, the carbohydrate chains of the natural PLIα were found to play an important role in the inhibitory activity against the basic PLA2.

Identification and Characterization of a Serum Protein Homologous to α‐type Phospholipase A2 Inhibitor (PLIα) from a Nonvenomous Snake, Elaphe quadrivirgata

From a liver cDNA library prepared from a nonvenomous striated snake, Elaphe quadrivirgata, we isolated a cDNA encoding a novel protein, PLIα‐like protein (PLIα‐LP), having approximately 70% sequence identities with the α‐type phospholipase A2 (PLA2) inhibitors (PLIαs) previously purified from the venomous snakes Agkistrodon blomhoffii siniticus and Trimeresurus flavoviridis. Since the PLIα‐LP with a highly conserved C‐type lectin‐like domain (CTLD) would be predicted to function as a PLA2 inhibitor, we purified this protein from E. quadrivirgata serum by sequential chromatography on Hi‐trap Blue, Mono Q, and Superdex 200 columns. The purified 51‐kDa protein with PLIα‐like immunoreactivity was found to be a trimer of 18‐kDa PLIα‐LP, which was comparable to the trimeric structure of PLIα. But, unexpectedly, this protein did not show any inhibitory activity against various snake venom PLA2s. Furthermore, it did not inhibit the endogenous PLA2 activities in various tissue homogenates prepared from this snake. Lack of the inhibitory activity in PLIα‐LP may provide important information concerning the structure‐function relationships of PLIα. IUBMB Life, 55: 539‐545, 2003

Inhibitory activities of the heterotrimers formed from two α-type phospholipase A2 inhibitory proteins with different enzyme affinities and importance of the intersubunit electrostatic interaction in trimer formation

α-type phospholipase A2 inhibitory protein (PLIα) isolated from the serum of the venomous snake Glyoidius brevicaudus, GbPLIα, is a homotrimer of subunits having a C-type lectin-like domain. The serum protein from nonvenomous snake Elaphe quadrivirgata, EqPLIα-LP, is homologous to GbPLIα, but it does not show any inhibitory activity against PLA2s. When a mixture of denaturant-treated monomeric forms of GbPLIα and EqPLIα-LP was used to reconstitute their trimers, no significant amounts of heterotrimers composed of GbPLIα and EqPLIα-LP subunits could be formed. On the other hand, when a mixture of denaturant-treated monomeric forms of GbPLIα and the recombinant chimeric EqPLIα-LP, Eq13Gb37Eq, in which the residues 13-36 were replaced by those of GbPLIα, was used to reconstitute their trimers, significant amounts of their heterotrimers were observed. Furthermore, when a mixture of denaturant-treated monomeric forms of EqPLIα-LP and the recombinant chimeric GbPLIα, Gb13Eq37Gb, in which the residues 13-36 were replaced by those of EqPLIα-LP, was used, significant amounts of their heterotrimers were observed. By comparison of the respective inhibitory activities of the heterotrimeric subspecies, it was suggested that the inhibitory activity of the trimer was governed by one subunit with the highest activity, and not affected by the number of these subunits. The intermolecular electrostatic interactions between Glu23 and Lys28 of GbPLIα were also suggested to be important in stabilizing the trimeric structure. The importance of the electrostatic interaction was supported by the less stability of the homotrimeric structure of a mutant GbPLIα with a single amino acid substitution, GbPLIα(K28E).

Amino acid sequences of cytotoxin‐like basic proteins derived from cobra venoms

Amino acid sequences of cytotoxin‐like basic proteins (CLBPs), purified from the venoms of Formosan cobra (Naja naja atra) and Indian cobra (Naja naja), were reinvestigated. The determined sequences differed from those reported previously by Takechi et al. [(1985) Biochem. Int. 11, 795‐802; (1987) Biochem. Int. 14, 145‐152]. The sequence of CLBPs at residues 25‐30 was found to be hydrophilic as compared with those of cytotoxins. The difference in the hydrophobicity of this region might be responsible for the difference in their cytotoxic activities.

cDNA cloning of the two subunits of phospholipase A2 inhibitor PLIgamma from blood plasma of the Chinese mamushi, Agkistrodon blomhoffii siniticus.

Three phospholipase A2 (PLA2) inhibitors (PLI) have been purified from the blood plasma of the Chinese mamushi, Agkistrodon blomhoffii siniticus; 1 of these, PLI gamma, contains 2 homologous subunits, PLI gamma‐A and PLI gamma‐B. The cDNAs encoding these 2 subunits of PLI gamma were isolated from a liver cDNA library by using fragments from polymerase chain reaction amplifications as probes and sequenced. The respective nucleotide sequences encoded 19‐residue signal sequences, followed by 181‐residue proteins. The calculated molecular masses were 20123 and 20150 Da for the PLI gamma‐A and PLI gamma‐B subunits, respectively; and PLI gamma‐A included a N‐linked carbohydrate site at Asn‐157. The sequences of these subunits contained 2 internal repeats of disulfide‐bonding pattern characteristic to those of urokinase‐type plasminogen activator receptor and members of the Ly‐6 superfamily. A phylogenetic analysis comparing the amino acid sequences of PLI gamma‐A and PLI gamma‐B with those for other snakes revealed that the gene duplication leading to these 2 subunits occurred before the divergence of Viperidae and Elapidae.