The major surface glycoprotein of Leishmania, referred to as GP63, is a zinc metalloproteinase of 63,000 M(r) present on promastigotes and amastigotes from diverse species of Leishmania. GP63 shares several characteristics with the members of the matrix metalloproteinase family including degradation of at least one component of the extracellular matrix, location at the cell surface, requirement for Zn2+ for proteinase activity and inhibition of the proteinase activity by chelating agents and alpha 2-macroglobulin. Site-directed mutagenesis of the cloned L. major GP63 genes was carried out to determine whether the proposed active site of Leishmania GP63 was homologous to those of other zinc metalloproteinases. The codon encoding the catalytic glutamic acid was modified to encode an aspartic acid and when expressed in COS-7 cells the resulting mutant GP63 had no demonstrable proteinase activity compared to wild type GP63. GP63 was predicted to be synthesized as a precursor protein containing a pro region at the NH2-terminus of GP63 implicated to be involved with the regulation of proteinase activity. As with many other proteinases, including matrix metalloproteinases, these enzymes are synthesized as latent proteinases that require activation for full proteinase activity. L. major recombinant GP63 (rGP63) has been produced in the baculovirus expression system where rGP63 was secreted as a latent proteinase. To study the activation of baculovirus rGP63, purified rGP63 was incubated with the mercurial compound, HgCl2, at concentrations previously shown to result in activation of other latent matrix degrading metalloproteinases and resulted in a significant enhancement of GP63 proteinase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

S u m m a ry : The structure of the PC-glycan of the major excretory-secretory pro duct (ES-62) of A c a nthocheilonema viteae has been investigated using endoglycosidases and lectins. Results obtained raise the pos sibility that it may be of the high mannose type. This, and the insen sitivity of the PC-glycan to treatments which remove PC or choline from bacterial PC-glycans, suggests that it may be more analogous to fungal, than to bacterial PC-containing glycans. KEY W O R D S : excretory-secretory product. phosphorylcholine. filariasis. glycan analysis. MOTS-CLES : produit excrétant-secrétant. phosphorylcholine. analyse des glycanes. fitariose. E xcretory-secretory products (E-S) o f filarial nematodes commonly contain phosphorylcho lin e (PC) (fo r a rev iew , se e H arnett and Parkhouse, 1994). The PC group is of some interest, as evidence is increasingly emerging that it may pos sess immunomodulatory activity. Within the last few years for example, it has been demonstrated that the PC moiety of filarial antigens is able to interfere with proliferative responses o f both T-(Lal et al., 1990) and B-(Harnett and Harnett, 1993) lymphocytes.

Nine monoclonal antibodies (mabs) have been produced against excretory-secretory products (ES) of adult male Onchocerca gibsoni. These mabs fail to interact with the highly cross-reactive phosphorylcholine (PC) group and with ES of the related rodent filarial parasites Acanthocheilonema viteae and Litomosoides carinii. Eight of the mabs are of the IgG isotype: 1 is an IgM. Three of the mabs, OGMES 4, 9, and 10, were each found by immunoprecipitation/SDS-PAGE analysis of [3H] leucine-labeled ES, to recognize a triplet of polypeptides of molecular weight 120, approximately 210, and approximately 260 kDa. No recognition was observed by any mab when [3H] glucosamine was employed as the radiolabel for ES. Western blotting employing [125I] as indicator system demonstrated that OGMES 7 recognized a molecule of 27 kDa, and OGMES 1, a molecule of approximately 210 kDa, albeit faintly. These mabs may be of value to researchers working on the isolation, characterization, and detection in the bloodstream of Onchocerca volvulus ES.

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