Functional expression and molecular characterization of Culex quinquefasciatus salivary α-glucosidase (MalI).
Salivary α-glucosidases (MalI) have been much less characterized when compared with midgut α-glucosidases, which have been studied in depth. Few studies have been reported on the partial characterization of MalI, but no clear function has been ascribed. The aim of this study is to purify and characterize the recombinant Culex quinquefasciatus (CQ) α-glucosidase expressed in Pichia pastoris. The cDNA encoding mature Cx. quinquefasciatus α-glucosidase gene with polyhistidine tag (rCQMalIHis) was successfully cloned into the expression vector, pPICZαB, designated as pPICZαB/CQMalIHis. The activity of recombinant rCQMalIHis expressed in P. pastoris could be detected at 3.75U/ml, under optimal culture conditions. The purified rCQMalIHis showed a single band of molecular weight of approximately 92kDa on SDS-PAGE. After Endoglycosidase H digestion, a single band at 69kDa was found on SDS-PAGE analysis, suggesting that rCQMalIHis is a glycoprotein. Additionally, tryptic digestion and LC-MALDI MS/MS analysis suggested that the 69kDa band corresponds to the Cx. quinquefasciatus α-glucosidase. Thus, rCQMalIHis is a glycoprotein. The rCQMalIHis exhibited optimum pH and temperature at 5.5 and 35°C, respectively. The catalytic efficiency (kcat/Km) of the purified rCQMalIHis for maltotriose is higher than those for sucrose, maltotetraose, maltose and p-nitrophenyl-α-glucoside, indicating that the enzyme prefers maltotriose. Additionally, the rCQMalIHis is significantly inhibited by d-gluconic acid δ-lactone, but not by Mg(2+), Ca(2+) and EDTA. The rCQMalIHis is strongly inhibited by acarbose with IC50 67.8±5.6nM, but weakly inhibited by glucose with IC50 115.9±7.3mM.
Department of Tropical Nutrition and Food Science, Faculty of Tropical Medicine, Mahidol University, Bangkok 10400, Thailand.
Rungarun
R
Suthangkornkul
Department of Tropical Nutrition and Food Science, Faculty of Tropical Medicine, Mahidol University, Bangkok 10400, Thailand.
Phanthila
P
Sirichaiyakul
Department of Medical Entomology, Faculty of Tropical Medicine, Mahidol University, Bangkok 10400, Thailand.
Sungsit
S
Sungvornyothin
Department of Tropical Nutrition and Food Science, Faculty of Tropical Medicine, Mahidol University, Bangkok 10400, Thailand.
Apanchanid
A
Thepouyporn
Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok 10400, Thailand; Laboratory of Biochemistry, Chulabhorn Research Institute, Bangkok 10210, Thailand.
Jisnuson
J
Svasti
Department of Tropical Nutrition and Food Science, Faculty of Tropical Medicine, Mahidol University, Bangkok 10400, Thailand. Electronic address: dumrongkiet.art@mahidol.ac.th.
Dumrongkiet
D
Arthan
D020909:Acarbose / Q000737:chemistry; D000818:Animals; D003001:Cloning, Molecular; D003465:Culex / Q000737:chemistry* / Q000201:enzymology; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006023:Glycoproteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D006639:Histidine / Q000737:chemistry / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D019476:Insect Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D007700:Kinetics; D008970:Molecular Weight; D009842:Oligopeptides / Q000737:chemistry / Q000235:genetics; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D012469:Salivary Glands / Q000737:chemistry* / Q000201:enzymology; D013379:Substrate Specificity; D013696:Temperature; D014312:Trisaccharides / Q000737:chemistry; D000520:alpha-Glucosidases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification
D018076:DNA, Complementary; D006023:Glycoproteins; C471213:His-His-His-His-His-His; D019476:Insect Proteins; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D014312:Trisaccharides; D006639:Histidine; C008317:maltotriose; D000520:alpha-Glucosidases; D020909:Acarbose
Expression; MalI; Mosquitoes; Pichia pastoris; α-Glucosidase