Hypodermin B, a Trypsin‐Related Enzyme from the Insect Hypoderma lineatum

Abstract

Hypodermin B, a serine proteinase with a molecular weight of 23000, was purified to homogeneity from the larvae Hypoderma lineatum. It is stoichiometrically inhibited by diisopropylfluorophosphate and fully inactivated by N-tosyllysine chloromethyl ketone and soya bean and bovine pancreatic trypsin inhibitors. N-Tosylphenylalanine chloromethyl ketone and ovomucoid are without effect on its activity. Hypodermin B hydrolyses both amide and ester substrates of trypsin but does not display any chymotryptic activity on synthetic substrates. Its specificity on the B chain of insulin is slightly broader than that of bovine trypsin. Its amino acid composition and N-terminal sequence suggest structural homology with serine proteinases of the trypsin family and with two other serine proteinases, hypodermin A and Hypoderma collagenase, previously isolated from the same larvae. Hypodermins A and B are very similar however strongly from Hypoderma collagenase.Although proteolytic enzymes have been reported in many insects, they are usually poorly characterized due to the scarceness of the material available. With respect to the amount and the diversity of their enzymatic equipment, larvae from Hypoderma lineaturn represent an advantageous source. These endoparasites of cattle enter through the skin of their host and pursue a migration through its connective tissue; this action is helped by excretion of proteolytic enzymes. The larvae partially reabsorb these enzymes together with the degradation products of the connective tissue and stock them in their midgut which is closed in its hind end and can thus act as a reservoir. After eight months the gut opens and its contents are excreted during the first moult [I].Two enzymes were recently isolated and characterized from the midgut of the first instar larvae : first, a collagenase which cleaves native collagen in a manner similar to that of vertebrate collagenases but which belongs to serine enzymes of the trypsin family [2 -41 ; secondly, a trypsin-like proteinase, hypodermin A 151. In the present paper, we report the purification and the characterization of a third enzyme from the same larval organ, hypodermin B. We demonstrate that it is a trypsin-like proteinase and we compare it with the two other larval proteinases and with other serine enzymes of the trypsin family. MATERIALS AND METHODS MaterialsChemical reagents were purchased from the following sources : Bz-Arg-OEt, Bz-Tyr-OEt and Dip-F from Fluka ;Abbreviations. Bz-Arg-OEt, N-ct-benzoyl-L-arginine ethyl ester; BzTyr-OEt, N-benzoyl-L-tyrosine ethyl ester; Bz-Arg-Nan, N-a-benzoyl-Larginine p-nitroanilide; Ac-Tyr-Nan, N-acetyl-L-tyrosine p-nitroanilide; Tos-Lys-CH,Cl, N-tosyllysine chloromethyl ketone; Tos-Phe-CH,Cl, Ntosyl-phenylalanine chloromethyl ketone ; BPTI, bovine pancreatic trypsin inhibitor; STI, soya bean trypsin inhibitor; Dip-F, diisopropylfluorophosphate; Pth, phenylthiohydantoin.Enzyme. Hypoderma collagenase (EC 3.4.21.49).with respect to their inhibition and specificity, they differ Bz-Arg-Nan, Ac-Tyr-Nan...

Use of a crude extract or purified antigen from first-instar cattle grubs,Hypoderma lineatum, for the detection of anti-Hypodermaantibodies in free-ranging cervids from southern Spain

“…First instars of the Hypodermatinae penetrate the skin and migrate through the host's conjunctive tissue aided by enzymatic secretions. Different studies on larval secretions of both species of Hypoderma affecting cattle, H. lineatum and H. bovis , have described and characterized three serine proteases called hypodermin A, B and C (Boulard & Garrone, 1978; Lecroisey et al , 1979, 1983; Tong et al , 1981).…”

Immunomodulatory effect of Hypoderma lineatum antigens: in vitro effect on bovine lymphocyte proliferation and cytokine production

“…Digestion occurs extracorporally by the action of regurgitated substances and, degraded tissues and serous exudates are ingested by larvae. Study of larval secretions has described and characterized three serine proteases called hypodermin A, B and C (10–13). There is great evidence that larval secretions are implicated in an immunomodulatory process, affecting both the inflammatory and the specific immune responses, which contribute to parasite survival during the prolonged migration that last 8–10 months through host connective tissues.…”

Arthropods affecting the human eye

“…This route of infection has also been suggested for myiases caused by the genera Gasterophilus and Cuterebra (Medownick et al, 1985;Anderson, 2006). Larval migration takes place as a consequence of the mechanical action of the cephaloskeleton, as well as of the proteolytic enzymes secreted by the larvae (i.e., serine proteases) (Boulard and Garrone, 1978;Lecroisey et al, 1979Lecroisey et al, , 1983Tong et al, 1981). Once the larvae reach the ocular region, they enter the sclera (Kearney et al, 1991) and the subretinal space, possibly causing hypopigmented subretinal tracks.…”