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 t he intestinal microvilli of the nematode Haemonchus contortus, cDNA li braries 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 fr om 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 produc t of 3.3 kb was generated from cDNA from UK H., contortus. The sequenc es from the UK and Australian nematodes were essentially identical ove r the 929 bp region in which both were represented, All three cloned D NAs hybridised to mRNA of about 3.5 kb. Analysis of the deduced amino acid sequence, which showed 32% identity with those of mammalian micro somal aminopeptidases, indicated that H11 has a short N-terminal cytop lasmic tail, a single transmembrane region and a long extracellular re gion with putative N-linked glycosylation sites and the HEXXHXW motif characteristic of microsomal aminopeptidases, Microsomal aminopeptidas e activity co-purifies with I-Ill. It is inhibited by bestatin, phenan throline and amastatin. The recombinant protein has been expressed in active form in insect cells.