Cloning and characterization of a microsomal aminopeptidase from the intestine of the nematode Haemonchus contortus.
In order to characterise the integral membrane glycoprotein H11 from the intestinal microvilli of the nematode Haemonchus contortus, cDNA libraries prepared using mRNA from adult worms from the UK and Australia were immunoscreened with anti-H11 sera. Antibodies affinity purified on the protein expressed by insert DNA (295 bp) of a positive clone from a UK library bound specifically to H11. A longer clone (948 bp) was obtained from the Australian library by hybridisation. Using a primer based on sequence common to these, a polymerase chain reaction product of 3.3 kb was generated from cDNA from UK H. contortus. The sequences from the UK and Australian nematodes were essentially identical over the 929 bp region in which both were represented. All three cloned DNAs hybridised to mRNA of about 3.5 kb. Analysis of the deduced amino acid sequence, which showed 32% identity with those of mammalian microsomal aminopeptidases, indicated that H11 has a short N-terminal cytoplasmic tail, a single transmembrane region and a long extracellular region with putative N-linked glycosylation sites and the HEXXHXW motif characteristic of microsomal aminopeptidases. Microsomal aminopeptidase activity co-purifies with H11. It is inhibited by bestatin, phenanthroline and amastatin. The recombinant protein has been expressed in active form in insect cells.
Smith
T S
TS
Immunology Department, The Babraham Institute, Cambridge, UK.
Graham
M
M
Munn
E A
EA
Newton
S E
SE
Knox
D P
DP
Coadwell
W J
WJ
McMichael-Phillips
D
D
Smith
H
H
Smith
W D
WD
Oliver
J J
JJ
D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D018076:DNA, Complementary / Q000235:genetics; D006189:Haemonchus / Q000201:enzymology*; D015801:Helminth Proteins / Q000235:genetics; D007422:Intestines / Q000201:enzymology; D008562:Membrane Glycoproteins / Q000235:genetics; D008861:Microsomes / Q000201:enzymology; D008871:Microvilli / Q000201:enzymology; D008969:Molecular Sequence Data
D018076:DNA, Complementary; D015801:Helminth Proteins; D008562:Membrane Glycoproteins; D000626:Aminopeptidases