The Amino-Acid Sequence and Carbohydrate Content of Phospholipase A2 from Bee Venom

Abstract

The complete amino acid sequence of phospholipase A2 from the venom of the common European honey-bee (Apis mellifica) has been determined. The sequence of amino acid residues at the N-terminus was obtained by direct application of the Edman degradation technique and that at the C-terminus by digestion with carboxypeptidasesA and B. Digestion of the reduced and carboxymethylated enzyme with trypsin yielded a completely soluble peptide mixture, all the components of which were isolated and their structures determined. Overlaps of the tryptic peptides were deduced from the structures of peptides isolated after digestion of the reduced and carboxymethylated enzyme with chymotrypsin and of the reduced and aminoethylated enzyme with a protease specific for cleavage at lysine residues. Two remaining ambiguities were resolved by peptides obtained from a digest of the reduced, carboxymethylated and maleylated enzyme with trypsin. The phospholipase A2 consists of a single chain of 128 amino acid residues and contains attached carbohydrate residues. The composition and point of attachment of the carbohydrate moiety have been established. Phospholipase A2 is the name given to a family of enzymes which catalyse the hydrolysis of fatty acid ester bonds at the 2-carbon position of 3-sn-phosphoglycerides. Enzymes with this specificity have been isolated from pancreatic tissue [l], bee venom [2] and a variety of snake venoms [3]. The enzymes have certain characteristics in common, for example relatively small size, high disulphide-bridge content, heat stability and requirement for Ca2+ ions for activity. There are, however, important differences. In particular, the pancreatic enzyme appears to be active on the aggregated (micellar) form of phospholipids [4], whereas the substrate for the bee-venom enzyme has been shown to be the dispersed (monomolecular) phospholipid [2]. It is also of interest that the enzyme from pancreas is secreted as an inactive zymogen which may be activated by limited proteolysis with trypsin sequence of the zymogen and the positions of the disulphide bridges in porcine pancreatic phospholipase A2 have been reported [6,7].Our interest in phospholipase A2 from bee venom arose from the observation [2] that the enzyme crystallises readily which, combined with its relatively small size, suggests that it may be amenable to total structure determination. As a preliminary to crystallographic analysis we have determined the primary structure of the enzyme; this work has been the subject of a preliminary report [S]. A missing overlap in the earlier work has now been established with certainty and three corrections have been made to the previously published sequence. In addition, the composition and point of attachment of the carbohydrate moiety have been established. An interesting aspects of the present work has been the use of a recently characterized protease isolated from the basidiomycete Armillaria mellea [9] ; the enzyme has been shown to have specificity for cleavage of peptide bonds N-terminal to lysin...

Detection of honeybee venom in envenomed tissues by direct MALDI MSI

“…This ion signal was accompanied by a rich population of other peaks distributed around 16.3 kDa and possibly related to micro heterogeneous Api m 1 glycosylation. Scheme 1 shows the possible glycostructures observed, which agree with those reported by Shipolini and collaborators [15] and Hollander et al [41]. Higher m/z protein signals were also detected (up to 50 kDa) but the intensity and the resolution were too poor to proceed with any m/z assignment.…”

“…The primary amino sequence of the mature protein has a MW of ϳ15.2 kDa. The enzyme is also present in glycosylated isoforms, which are not yet extensively characterized for their microheterogeneity [15]. Nevertheless, it is known that the glycostructures are N-linked to asparagine 13 [16], giving rise to higher molecular weight species.…”

Amino Acid Sequences of Metalloendopeptidases Specific for Acyl-Lysine Bonds from Grifola frondosa and Pleurotus ostreatus Fruiting Bodies

“…Lewis et al (3) purified a zinc MEP with a molecular weight of 16,500 from Armillaria mellea mycelia and demonstrated its high degree of specificity to cleave peptide bonds involving the ␣-amino group of lysine residues. The unique specificity of the enzyme was confirmed by its application to sequence analyses of some proteins (4,5). Recently, purifying a MEP (GFMEP) from Grifola frondosa fruiting bodies, we have characterized this enzyme with a molecular mass of 20 kDa to contain 1 atom of zinc/molecule.…”

Expression of a bee venom phospholipase A2 from Apis cerana cerana in the baculovirus-insect cell

“…The deduced amino acid sequence of AmPLA 2 consists of a signal peptide of 18 amino acid residues (preregion of PLA 2 ), a proregion of 15 residues, and a mature peptide of 134 amino acid residues. The mature peptide contains 10 cystine residues which can form 5 disulfide bonds (Kuchler et al, 1989;Shipolini et al, 1974a;1974b). The crystal structure and catalyzing activity of PLA 2 were also well documented (Annand et al, 1996;Scott et al, 1990a;1990b).…”