Tài liệu Báo cáo khoa học: A zymogen form of masquerade-like serine proteinase homologue is cleaved

A zymogen form of masquerade-like serine proteinase homologue

is cleaved during pro-phenoloxidase activation by Ca2+ in coleopteran

and Tenebrio molitor larvae

Kum Young Lee1

, Rong Zhang1

, Moon Suk Kim1

, Ji Won Park1

, Ho Young Park2

, Shun-ichiro Kawabata3

and Bok Luel Lee1

1

College of Pharmacy, Pusan National University, Jangjeon Dong, Korea; 2

Insect Resources Laboratory, Korea Research Institute

of Bioscience and Biotechnology, Taejeon, Korea; 3

Department of Biology, Kyushu University, Fukuoka, Japan

To elucidate the biochemical activation mechanism of the

insect pro-phenoloxidase (pro-PO) system, we purified a

45-kDa protein to homogeneity from the hemolymph of

Tenebrio molitor (mealworm) larvae, and cloned its cDNA.

The overall structure of the 45-kDa protein is similar to

Drosophila masquerade serine proteinase homologue, which

is an essential component inDrosophila muscle development.

This Tenebrio masquerade-like serine proteinase homologue

(Tm-mas) contains a trypsin-like serine proteinase domain in

theC-terminal region,except for the substitutionofSer toGly

at the active site triad, and a disulfide-knotted domain at the

amino-terminal region. When the purified 45-kDa Tm-mas

was incubated with CM-Toyopearl eluate solution contain￾ing pro-PO and other pro-PO activating factors, the resulting

phenoloxidase (PO) activity was shown to be independent of

Ca2+. This suggests that the purified 45-kDa Tm-mas is an

activated form of pro-PO activating factor. The55-kDa

zymogen form of Tm-mas was detected in the hemolymph

when PO activity was not evident. However, when Tenebrio

hemolymph was incubated with Ca2+, a 79-kDa Tenebrio

pro-PO and the 55-kDa zymogen Tm-mas converted to

76-kDa PO and 45-kDa Tm-mas, respectively, with detect￾able PO activity. Furthermore, when Tenebrio hemolymph

wasincubated with Ca2+ andb-1,3-glucan, the conversion of

pro-PO to PO and the 55-kDa zymogen Tm-mas to the

45-kDa protein, was faster than in the presence of Ca2+ only.

Theseresults suggest that thecleavageof the 55-kDazymogen

of Tm-mas by a limited proteolysis is necessary for PO

activity, and the Tm-mas is a pro-PO activating cofactor.

Keywords: innate immunity; insect; Masquerade; pro-phe￾noloxidase; serine proteinase homologue.

The pro-phenoloxidase (pro-PO) activation system in

arthropods is an important part of the host immune

defence, where it functions to detect and kill invading

pathogens. It is also a good model system to elucidate the

pattern recognition mechanism of nonself pattern recogni￾tion proteins, such as peptidoglycan recognition protein or

b-1,3-glucan binding protein, which are part of the innate

immune reaction [1,2]. However, the molecular mechanism

of pro-PO activation remains poorly understood. Previously,

we reported the structures and functions of two pro-PO

activating factors (PPAF-I and PPAF-II) from the coleopt￾eran insect, Holotrichia diomphalia larvae [3–5]. PPAF-I is

an easter-type serine proteinase and PPAF-II is a masqu￾erade-like serine proteinase homologue. We have demon￾strated that they are necessary for activating the

phenoloxidase (PO) cascade, by in vitro reconstitution

experiments. However, we did not determine the biological

functions of PPAF-II during Holotrichia pro-PO activation.

Two questions remain to be answered: why is a masquer￾ade-like serine proteinase homologue a requirement for PO

activity, and how and why is there cross-talk between a

masquerade-like serine proteinase homologue and an eas￾ter-type serine proteinase during the pro-PO activation

reaction? Another key question that remains is how these

pro-PO activating factors can be activated in response to

microbial infection. One hypothesis is that pattern recogni￾tion proteins make a complex with pro-PO activating

enzyme(s) and microbial cell wall components, and then

activation of pro-PO activating enzyme(s) zymogen con￾verts pro-PO to active PO by limited proteolysis [6–8].

Recently we reported that in larvae of the coleopteran

insect, Tenebrio molitor, pro-PO was activated by b-1,3-

glucan and Ca2+, and the activated PO was involved in the

cell/clump/cell adhesion reaction as well as in the synthesis

of melanin [9]. This insect has one kind of pro-PO; however,

two kinds of pro-PO were found in H. diomphalia larvae

[9,10]. If it is possible to purify pro-PO activating factors

from T. molitor larvae, we can explain the difference

between pro-PO activation reactions in the one-pro-PO

and two-pro-PO systems in T. molitor and H. diomphalia

larvae, respectively. Also, we have reported the presence of

early staged encapsulation-relating proteins in T. molitor

larvae [11,12]. Given the crucial nature of the melanotic

Correspondence to B. L. Lee, College of Pharmacy, Pusan National

University, Jangjeon Dong, Kumjeong Ku, Busan, 609-735, Korea.

Fax: +82 51 581 1508, E-mail: [email protected]

Abbreviations: PO, phenoloxidase; pro-PO, pro-phenoloxidase;

Tm-mas, Tenebrio masquerade-like serine proteinase homologue;

TCA, trichloroacetic acid; PVDF, polyvinylidene difluoride; PPAF,

pro-phenoloxidase activating factors; P-NPGB, p-nitrophenyl￾p¢-guanidinobenzoate; p-APMSF, p-amidinophenyl-methanesulfonyl

fluoride.

Note: The nucleotide sequence data reported in this paper will appear

in the DDBJ, EMBL and GenBank Nucleotide Sequence Database

with the accession number AB084067.

(Received 29 April 2002, revised 9 July 2002, accepted 29 July 2002)

Eur. J. Biochem. 269, 4375–4383 (2002) FEBS 2002 doi:10.1046/j.1432-1033.2002.03155.x