Tài liệu Báo cáo khoa học: Inorganic pyrophosphatase in the roundworm Ascaris and its role in the

Inorganic pyrophosphatase in the roundworm Ascaris

and its role in the development and molting process

of the larval stage parasites

M. Khyrul Islam1

, Takeharu Miyoshi1

, Harue Kasuga-Aoki1

, Takashi Isobe1

, Takeshi Arakawa2

,

Yasunobu Matsumoto3 and Naotoshi Tsuji1

1

Laboratory of Parasitic Diseases, National Institute of Animal Health, National Agricultural Research Organization, 3-1-5,

Kannondai, Tsukuba, Ibaraki, Japan; 2

Division of Molecular Microbiology, Center of Molecular Biosciences, University of the

Ryukyus, Senbaru, Okinawa, Japan; 3

Laboratory of Global Animal Resource Science, Graduate School of Agricultural and

Life Sciences, University of Tokyo, Yayoi, Bunkyo, Tokyo, Japan

Inorganic pyrophosphatase (PPase) is an important

enzyme that catalyzes the hydrolysis of inorganic pyro￾phosphate (PPi

) into ortho-phosphate (Pi

). We report

here the molecular cloning and characterization of a gene

encoding the soluble PPase of the roundworm Ascaris

suum. The predicted A. suum PPase consists of 360 amino

acids with a molecular mass of 40.6 kDa and a pI of 7.1.

Amino acid sequence alignment and phylogenetic analysis

indicates that the gene encodes a functional Family I

soluble PPase containing features identical to those of

prokaryotic, plant and animal/fungal soluble PPases. The

Escherichia coli-expressed recombinant enzyme has a spe￾cific activity of 937 lmol Pi

Æmin)1

Æmg)1 protein corres￾ponding to a kcat value of 638 s)1 at 55
C. Its activity was

strongly dependent on Mg2+ and was inhibited by Ca2+.

Native PPases were expressed in all developmental stages of

A. suum. A homolog was also detected in the most closely

related human and dog roundworms A. lumbricoides and

Toxocara canis, respectively. The enzyme was intensely

localized in the body wall, gut epithelium, ovary and uterus

of adult female worms. We observed that native PPase

activity together with development and molting in vitro of

A. suum L3 to L4 were efficiently inhibited in a dose￾dependent manner by imidodiphosphate and sodium

fluoride, which are potent inhibitor of both soluble- and

membrane-bound H+-PPases. The studies provide evi￾dence that the PPases are novel enzymes in the roundworm

Ascaris, and may have crucial role in the development and

molting process.

Keywords: roundworm; inorganic pyrophosphatase;

sodium fluoride; imidodiphosphate; molting.

Geohelminth parasites are among the commonest and

widespread of human infections, particularly in the regions

where public health hygiene and nutritional status are

poorly maintained. The most prevalent geohelminth is

Ascaris lumbricoides (originally described by Linnaeus in

1758), which colonizes the small intestine of children, and is

estimated to infect a quarter of the world’s population [1].

Ascaris suum (originally described by Goeze in 1782) of pigs

is a very closely related species to A. lumbricoides, which can

develop in human hosts, indicating its zoonotic significance

[2,3]. Childhood infections with Ascaris worms are reported

to be associated with stunting growth, malabsorption,

deficiencies of macro- and micro-nutrients and damage of

the small intestinal mucosa [4,5]. In addition, concurrent

Ascaris infection may have potential immunomodulatory

effects on the immune response to other infections [6,7]. It

is therefore of considerable interest to investigate the

biochemical aspects of Ascaris worms to identify potential

drug targets and vaccine candidates.

Inorganic pyrophosphatases (PPases), which catalyze the

hydrolysis of inorganic pyrophosphate (PPi

) into inorganic

ortho-phosphate (Pi

), are widely distributed among living

cells. The enzymes play an important role in energy

metabolism, providing a thermodynamic pull for many

biosynthetic reactions [8], and have been shown to be

essential to life [9–11]. There are two major categories of

PPases, the soluble PPases and the membrane-bound H+-

translocating PPases (H+-PPase). Two families of soluble

PPases have been recognized to date, Family I includes most

of the currently known soluble PPases [12], and Family II

comprises recently discovered Bacillus subtilis PPase as well

as PPases of four other putative members, two streptococcal

and two archeal [13,14]. These two families do not show

any sequence similarity to each other. Family I soluble

PPases have been further divided into three subfamilies,

Correspondence to N. Tsuji, Laboratory of Parasitic Diseases,

National Institute of Animal Health, National Agricultural Research

Organization, 3-1-5 Kannondai, Tsukuba, Ibaraki 305-0856, Japan.

Fax: + 81 29 8387749, Tel.: + 81 29 8387749,

E-mail: [email protected]

Abbreviations: PPase, inorganic pyrophosphatase; H+-PPase, proton￾translocating pyrophosphatase; AsPPase, Ascaris suum inorganic

pyrophosphatase; rAsPPase, recombinant A. suum inorganic pyro￾phosphatase; L3, third-stage infective larvae; L4, fourth-stage larvae;

ES, excretory and secretory; IDP, imidodiphosphate.

Enzyme: Soluble inorganic pyrophosphatase (EC 3.6.1.1).

Note: The nucleotide sequences reported in this paper has been

submitted to the DDBJ/EMBL/GenBank with accession number

AB091401.

(Received 4 March 2003, revised 7 May 2003, accepted 9 May 2003)

Eur. J. Biochem. 270, 2814–2826 (2003) FEBS 2003 doi:10.1046/j.1432-1033.2003.03658.x