Characterization of a blood activated chitinolytic system in the midgut of the sand fly vectors Lutzomyia longipalpis and Phlebotomus papatasi.
We characterized a cDNA from Phlebotomus papatasi, PpChit1, which encodes a midgut specific chitinase and show the presence of a functional, blood-induced chitinolytic system in sand flies. PpChit1 is detected only in the midgut and is regulated by blood feeding. A recombinant protein (rPpChit1) produced in HEK 293-F cells exhibited a similar activity profile to that found in the native protein against several specific substrates, including an oligomeric glycol chitin and synthetic 4-methyl-umbelliferone labelled substrates. Western blotting showed that the native protein is recognized by mouse polyclonal antibodies against rPpChit1. Additionally, the rPpChit1 and the native chitinase displayed similar retention times in a HPLC size fractionation column. When added to rPpChit1 or to midgut lysates, PpChit1 sera reduced chitinolytic activity by 65-70%.
Ramalho-Ortigão
J M
JM
Intracellular Parasite Biology Section, Laboratory of Parasitic Diseases, National Institute for Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20852-8132, USA. mortigao@niaid.nih.gov
Kamhawi
S
S
Joshi
M B
MB
Reynoso
D
D
Lawyer
P G
PG
Dwyer
D M
DM
Sacks
D L
DL
Valenzuela
J G
JG
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002688:Chitinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004064:Digestive System / Q000201:enzymology* / Q000378:metabolism; D004199:Disease Vectors; D008969:Molecular Sequence Data; D011576:Psychodidae / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism
D012333:RNA, Messenger; D002688:Chitinases