Intestinal aspartate proteases TiCatD and TiCatD2 of the haematophagous bug Triatoma infestans (Reduviidae): sequence characterisation, expression pattern and characterisation of proteolytic activity.
Two aspartate protease encoding complementary deoxyribonucleic acids (cDNA) were characterised from the small intestine (posterior midgut) of Triatoma infestans and the corresponding genes were named TiCatD and TiCatD2. The deduced 390 and 393 amino acid sequences of both enzymes contain two regions characteristic for cathepsin D proteases and the conserved catalytic aspartate residues forming the catalytic dyad, but only TiCatD2 possesses an entire C-terminal proline loop. The amino acid sequences of TiCatD and TiCatD2 show 51-58% similarity to other insect cathepsin D-like proteases and, respectively, 88 and 58% similarity to the aspartate protease ASP25 from T. infestans available in the GenBank database. In phylogenetic analysis, TiCatD and ASP25 clearly separate from cathepsin D-like sequences of other insects, TiCatD2 groups with cathepsin D-like proteases with proline loop. The activity of purified TiCatD and TiCatD2 was highest between pH 2 and 4, respectively, and hence, deviate from the pH values of the lumen of the small intestine, which varied in correlation with the time after feeding between pH 5.2 and 6.7 as determined by means of micro pH electrodes. Both cathepsins, TiCatD and TiCatD2, were purified from the lumen of the small intestine using pepstatin affinity chromatography and identified by nanoLC-ESI-MS/MS analysis as those encoded by the cDNAs. The proteolytic activity of the purified enzymes is highest at pH 3 and the respective genes are expressed in the both regions of the midgut, stomach (anterior midgut) and small intestine, not in the rectum, salivary glands, Malpighian tubules or haemocytes. The temporal expression pattern of both genes in the small intestine after feeding revealed a feeding dependent regulation for TiCatD but not for TiCatD2.
Zoology/Parasitology Group, Ruhr-Universität, Universitätsstrasse 150, 44780 Bochum, Germany. carsten.balczun@rub.de
Carsten
C
Balczun
Janna
J
Siemanowski
Jennifer Katharina
JK
Pausch
Stefan
S
Helling
Katrin
K
Marcus
Christian
C
Stephan
Helmut E
HE
Meyer
Tobias
T
Schneider
Christian
C
Cizmowski
Marina
M
Oldenburg
Sandra
S
Höhn
Christian Karl
CK
Meiser
Wolfgang
W
Schuhmann
Günter A
GA
Schaub
D000595:Amino Acid Sequence; D000818:Animals; D057055:Aspartic Acid Proteases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002846:Chromatography, Affinity; D018076:DNA, Complementary / Q000737:chemistry; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D019476:Insect Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007422:Intestines / Q000201:enzymology; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017422:Sequence Analysis, DNA; D014224:Triatoma / Q000201:enzymology* / Q000235:genetics
D018076:DNA, Complementary; D019476:Insect Proteins; D057055:Aspartic Acid Proteases