Cloning and molecular characterization of a cubilin-related serine proteinase from the hard tick Haemaphysalis longicornis

Serine proteinases are one of the largest proteolytic families of enzymes, and have diverse cellular activities in mammalian tissues. We report here the cloning and molecular characterization of a cDNA encoding the serine proteinase of the hard tick Haemaphysalis longicornis (HISP). The HISP cDNA is 1570 bp long and the deduced precursor protein consists of 464 amino acids with a predicted molecular mass of 50.4 kDa and a pI of 8.2. The preprotein, consisting of 443 amino acids, was predicted to include a complement Clr/Cls, Uegf, and bone morphogenic protein-1 domain, a low-density lipoprotein receptor class A domain, and a catalytic domain. HISP sequence analysis showed high similarity to serine proteinases reported from arthropods and vertebrate animal species. Two-dimensional immunoblot analysis revealed endogenous HISP in adult tick extracts at 50 kDa. Endogenous HISP was also expressed in all lifecycle stages of H. longicornis. Immunohistochemical studies detected the endogenous enzyme in the midgut epithelial cells of an adult tick. The Eseherichia coli-expressed recombinant HISP was demonstrated to degrade bovine serum albumin and hydrolyze the substrate Bz-L-Arg-pNA at the rate of 30.2 mumol/min/mg protein. Further, HISP expression was up-regulated during a blood-feeding process, indicating its involvement in the digestion of host blood components. (C) 2004 Elsevier Ltd. All rights reserved.