Insect Biochemistry and Molecular Biology 30 2000 953–967 www.elsevier.comlocateibmb
Purification, characterization and molecular cloning of prophenoloxidases from Sarcophaga bullata
q
Michael R. Chase, Kiran Raina, James Bruno, Manickam Sugumaran
Department of Biology, University of Massachusetts–Boston, Boston, MA 02125, USA Received 6 December 1999; received in revised form 8 March 2000; accepted 9 March 2000
Abstract
Prophenoloxidase PPO is a key enzyme associated with both melanin biosynthesis and sclerotization in insects. This enzyme is involved in three physiologically important processes viz., cuticular hardening, defense reactions and wound healing in insects.
It was isolated from the larval hemolymph of Sarcophaga bullata and purified by employing ammonium sulfate precipitation, Phenyl Sepharose chromatography, DEAE–Sepharose chromatography, and Sephacryl S-200 column chromatography. The purified
enzyme exhibited two closely moving bands on 7.5 SDS–PAGE under denaturing conditions. From the estimates of molecular weight on Sephacryl S-100, TSK-3000 HPLC column and SDS–PAGE, which ranged from 90,000 to 100,000, it was inferred that
the enzyme is made up of a single polypeptide chain. Activation of PPO K
a
= 40
µ M was achieved by the cationic detergent, cetyl
pyridinium chloride below its critical micellar concentration 0.8 mM indicating that the detergent molecules are binding specifically to the PPO and causing the activation. Neither anionic, nor nonionic or zwitterionic detergents activated the PPO. The active
enzyme exhibited wide substrate specificity and marked thermal unstability. Using primers designed to conserved amino acid sequences from known PPOs, we PCR amplified and cloned two PPO genes from the sarcophagid larvae. The clones encoded
polypeptides of 685 and 691 amino acids. They contained two distinct copper binding regions and lacked the signal peptide sequence. They showed a high degree of homology to dipteran PPOs. Both contained putative thiol ester site, two proteolytic activation sites
and a conserved C-terminal region common to all known PPOs.
2000 Elsevier Science Ltd. All rights reserved.
Keywords: Prophenoloxidase; Melanin biosynthesis; Sclerotization; Insect immunity; Wound healing
1. Introduction
Phenoloxidase PO also known as tyrosinase is a bifunctional
enzyme possessing
both monophenol
monooxygenase activity E.C. 1.14.18.1. tyrosine, dihy- droxyphenylalanine, oxygen, oxidoreductase and o-
diphenoloxidase activity E.C. 1.10.3.1. o-diphenol, oxy- gen, oxidoreductase. It is responsible for initiating the
biosynthesis of widely distributed melanin pigment in nature Prota, 1992. In addition to melanization of cuti-
Abbreviations: Bp, base pairs; CPC, Cetyl pyridinium chloride; PCR,
polymerase chain reaction; PO, Phenoloxidase; PPO, Prophenoloxi- dase; RT, Reverse transcription.
q
The sequence reported in this paper has been deposited in the GenBank Database accession numbers: SbPPO1 AF 161260;
SbPPO2 AF161261 Corresponding author. Tel.:
+ 1-617-287-6598; fax:
+ 1-617-287-
6650. E-mail
address: manickam.sugumaranumb.edu
M. Sugumaran.
0965-174800 - see front matter
2000 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 5 - 1 7 4 8 0 0 0 0 0 6 8 - 0
cle used for color and camouflage, PO is also uniquely associated with three different physiologically important
biochemical processes in insects and other arthropods. These are a sclerotization of insect cuticle Andersen
et al., 1996; Sugumaran, 1998, b encapsulation and melanization of foreign organisms observed as defense
reaction Ashida and Brey, 1995; Gillespie et al., 1997; So¨derha¨ll et al., 1990; Sugumaran, 1996 and c wound
healing Lai-Fook, 1966; Sugumaran, 1996. In the first process, PO generated 4-alkylquinones serve as sclerot-
izing agents for quinone tanning reactions—one of the mechanisms by which the insect cuticle is hardened to
protect the soft bodies of animals Andersen et al., 1996; Sugumaran, 1998. Quinones are converted by quinone
isomerase to quinone methides Saul and Sugumaran 1988a, 1990; Ricketts and Sugumaran, 1994 which are
reactive intermediates for the second mechanism of tan- ning called quinone methide sclerotization. Some of the
quinone methides are converted to 1,2-dehydro-N-acyl- dopamines by the action of quinone methide isomerase
954 M.R. Chase et al. Insect Biochemistry and Molecular Biology 30 2000 953–967
Saul and Sugumaran, 1989a,b; Ricketts and Sugumaran, 1994. The resultant dehydro-N-acyldopamines are
further oxidized by PO to their corresponding quinones which rapidly isomerize nonenzymatically to form quin-
one methide imine amides necessary for
α ,
β -sclerotiz-
ation Sugumaran et al., 1992; Ricketts and Sugumaran, 1994. The reactions of quinones, quinone methides and
quinone methide imine amides with cuticular structural proteins and chitin result in the hardening of the cuticle
Sugumaran, 1998.
In the second process, PO serves as a terminal compo- nent of an elaborate defense mechanism. Parasites and
pathogens which are too large to be phagocytosed are found to be usually encapsulated and melanized in insect
blood by the action of phenoloxidase Ashida and Brey, 1995; Gillespie et al., 1997; So¨derha¨ll et al., 1990; Sugu-
maran, 1996; Sugumaran and Kanost, 1993. This pro- cess not only limits the growth and development of the
foreign object, but also prevents the damage it can cause to host by creating a physical barrier. Finally during
wounding, continuous loss of hemolymph is prevented by the rapid deposition melanin polymer at the wounding
site Lai-Fook, 1966; Sugumaran, 1996. Apart from stopping the blood loss, phenoloxidase might also pro-
vide cytotoxic quinonoid compounds to kill the oppor- tunistically invading microorganism at the wound site
Sugumaran, 1996; Nappi and Sugumaran, 1993.
The unique roles played by PO in insect physiology and biochemistry certainly demands a serious study on
this enzyme. But, numerous problems such as instability and rapid loss of activity during purification, ‘stickiness,’
= insolubilization on biotic and abiotic matters, various
gels and glassware used for the purification of the enzyme and self inactivation have prevented the
detailed characterization of insect POs in the past Sugumaran and Kanost, 1993. By taking advantage of
the fact that PO is present in the inactive proenzyme form, some scientists have successfully purified and
characterized the PPO. Thus PPOs from Bombyx mori Ashida, 1971; Yasuhara et al., 1995, Manduca sexta
Aso et al., 1985; Hall et al., 1995; Jiang et al., 1997a, Hyalophora cecropia
Andersson et al., 1989, Galleria mellonella
Kopa´cek et al., 1995, Holotrichia diomph- alia
Kwon et al., 1997, Calliphora erythrocephala Pau and Eagles, 1975, Musca domestica Hara et al., 1993;
Tsukamoto et al., 1986, Drosophila melanogaster Fujimoto et al., 1993, Blaberus discoidalis Durrant et
al., 1993, Tenebrio molitor Heyneman, 1965, and Locusta migratoria
Cherqui et al., 1996 have been pur- ified and several of their properties have been charac-
terized. Following the initial report on the characteriz- ation of cDNA encoding Manduca sexta PPO Hall et
al., 1995, several investigators have also characterized different insect PPO genes. These include one from Dro-
sophila melanogaster
Fujimoto et al., 1995, two from Bombyx mori
Kawabata et al., 1995 a second from Manduca sexta
Jiang et al., 1997a, two from Hyphantria cunea
Park et al., 1997 six from Anopheles gambiae
Jiang et al., 1997b; Lee et al., 1998; Muller et al., 1999, one from Tenebrio molitor Lee et al., 1999,
and one from Armigeres subalbatus Cho et al., 1998. We have been using Sarcophaga bullata larvae success-
fully for unraveling the molecular mechanisms of cuticu- lar sclerotization for over two decades. In this paper we
report the purification, characterization and molecular cloning of PPO from the larval hemolymph of Sarco-
phaga bullata
.
2. Materials and methods
