Cloning and characterization of the Cry1Ac-binding alkaline phosphatase (HvALP) from Heliothis virescens.
Membrane-bound alkaline phosphatases (mALPs, EC 3.1.3.1) in the insect midgut have been reported as functional receptors for Cry toxins from the bacterium Bacillus thuringiensis. We previously reported the identification of HvALP in the midgut of Heliothis virescens larvae as a Cry1Ac-binding protein that is down-regulated in Cry1Ac-resistant insects. To further characterize HvALP, we localized mALP protein to foregut and midgut tissues using anti-mALP serum and then cloned five mALPs from H. virescens larval midgut. All five clones displayed high levels of sequence identity (above 90%), suggesting that they may represent allelic variants, and grouped with other lepidopteran mALPs in sequence alignments. All these cloned ALPs were predicted to contain a glycosylphosphatidylinositol (GPI) anchor and were named HvmALP1-5. We expressed two of the most diverse HvmALPs in a heterologous system to test binding of Cry1Ac and recognition by HvALP cross-reacting antiserum. Our data highlight the importance of glycosylation for Cry1Ac binding to HvALP and suggest that, depending on glycosylation, all the identified HvmALPs may be synonymous with HvALP, the Cry1Ac-binding phosphatase identified in H. virescens midgut epithelium.
Perera
Omaththage P
OP
Southern Insect Management Research Unit, U.S. Department of Agriculture-Agricultural Research Service, Stoneville, MS 38776, USA.
Willis
Jonathan D
JD
Adang
Michael J
MJ
Jurat-Fuentes
Juan L
JL
D000469:Alkaline Phosphatase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D004731:Endotoxins / Q000235:genetics / Q000378:metabolism*; D006031:Glycosylation; D006460:Hemolysin Proteins / Q000235:genetics / Q000378:metabolism*; D019476:Insect Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D009036:Moths / Q000737:chemistry / Q000145:classification / Q000235:genetics* / Q000378:metabolism; D010802:Phylogeny; D021381:Protein Transport; D016415:Sequence Alignment
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D004731:Endotoxins; D006460:Hemolysin Proteins; D019476:Insect Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D000469:Alkaline Phosphatase