An exo-ß-(1→3)-D-galactanase from Streptomyces sp. provides insights into type II arabinogalactan structure.

An exo-ß-(1→3)-D-galactanase (SGalase1) that specifically cleaves the ß-(1→3)-D-galactan backbone of arabinogalactan-proteins (AGPs) was isolated from culture filtrates of a soil Streptomyces sp. Internal peptide sequence information was used to clone and recombinantly express the gene in E. coli. The molecular mass of the isolated enzyme was ~45 kDa, similar to the 48.2 kDa mass predicted from the amino acid sequence. The pI, pH and temperature optima for the enzyme were ~7.45, 3.8 and 48 °C, respectively. The native and recombinant enzymes specifically hydrolysed ß-(1→3)-D-galacto-oligo- or poly-saccharides from the upstream (non-reducing) end, typical of an exo-acting enzyme. A second homologous Streptomyces gene (SGalase2) was also cloned and expressed. SGalase2 was similar in size (47.9 kDa) and enzyme activity to SGalase1 but differed in its pH optimum (pH 5). Both SGalase1 and SGalase2 are predicted to belong to the CAZy glycosyl hydrolase family GH 43 based on activity, sequence homology and phylogenetic analysis. The K(m) and V(max) of the native exo-ß-(1→3)-D-galactanase for de-arabinosylated gum arabic (dGA) were 19 mg/ml and 9.7 µmol D-Gal/min/mg protein, respectively. The activity of these enzymes is well suited for the study of type II galactan structures and provides an important tool for the investigation of the biological role of AGPs in plants. De-arabinosylated gum arabic (dGA) was used as a model to investigate the use of these enzymes in defining type II galactan structure. Exhaustive hydrolysis of dGA resulted in a limited number of oligosaccharide products with a trisaccharide of Gal(2)GlcA(1) predominating.

Virulence of Leishmania major in macrophages and mice requires the gluconeogenic enzyme fructose-1,6-bisphosphatase.

Leishmania are protozoan parasites that replicate within mature phagolysosomes of mammalian macrophages. To define the biochemical composition of the phagosome and carbon source requirements of intracellular stages of L. major, we investigated the role and requirement for the gluconeogenic enzyme fructose-1,6-bisphosphatase (FBP). L. major FBP was constitutively expressed in both extracellular and intracellular stages and was primarily targeted to glycosomes, modified peroxisomes that also contain glycolytic enzymes. A L. major FBP-null mutant was unable to grow in the absence of hexose, and suspension in glycerol-containing medium resulted in rapid depletion of internal carbohydrate reserves. L. major Deltafbp promastigotes were internalized by macrophages and differentiated into amastigotes but were unable to replicate in the macrophage phagolysosome. Similarly, the mutant persisted in mice but failed to generate normal lesions. The data suggest that Leishmania amastigotes reside in a glucose-poor phagosome and depend heavily on nonglucose carbon sources. Feeding experiments with [(13)C]fatty acids showed that fatty acids are poor gluconeogenic substrates, indicating that amino acids are the major carbon source in vivo. The need for amino acids may have forced Leishmania spp. to adapt to life in the mature phagolysosome.

Processing and trafficking of Leishmania mexicana GP63. Analysis using GP18 mutants deficient in glycosylphosphatidylinositol protein anchoring.

GPI8 is a clan CD, family C13 cysteine protease and the catalytic core of the GPI-protein transamidase complex. In Leishmania mexicana, GPI8 is nonessential, and Deltagpi8 mutants lack the GPI-anchored metalloprotease GP63, which is the major surface protein of promastigotes. We have identified the active site histidine and cysteine residues of leishmanial GPI8 and generated Deltagpi8 lines expressing modified GPI8 proteins. This has allowed us to study the processing and trafficking of GP63 in wild type and Deltagpi8 mutants. We show using pulse-chase labeling that in Deltagpi8 non-GPI-anchored GP63 was glycosylated and secreted without further processing from the cell with a t(12) of 120 min. This secretion was prevented by growth of cells in the presence of tunicamycin, indicating that glycosylation is necessary for secretion of non-GPI-anchored proteins. In contrast, in wild type cells the majority of GP63 was rapidly glycosylated, GPI-anchored, and trafficked to the surface with defined processing intermediate forms. Tunicamycin inhibited glycosylation but did not prevent GPI anchor addition or trafficking. These results show that GPI-anchored and unanchored GP63 are trafficked via different pathways. In addition, the balance between GPI anchor addition and secretion of GP63 in Leishmania can vary depending on the activity of the GPI-protein transamidase, which has implications for the host-parasite interaction.