Identification and characterization of the Onchocerca volvulus Excretory Secretory Product Ov28CRP, a putative GM2 activator protein.

Onchocerca volvulus is the nematode pathogen responsible for human onchocerciasis also known as "River blindness", a neglected tropical disease that affects up to 18 million people worldwide. Helminths Excretory Secretory Products (ESPs) constitute a rich repertoire of molecules that can be exploited for host-parasite relationship, diagnosis and vaccine studies. Here, we report, using a range of molecular techniques including PCR, western blot, recombinant DNA technology, ELISA, high performance thin-layer chromatography and mass spectrometry that the 28 KDa cysteine-rich protein (Ov28CRP) is a reliable component of the O. volvulus ESPs to address the biology of this parasite. We showed that (1) Ov28CRP is a putative ganglioside GM2 Activator Protein (GM2AP) conserved in nematode; (2) OvGM2AP gene is transcriptionally activated in all investigated stages of the parasitic life cycle, including larval and adult stages; (3) The full-length OvGM2AP was detected in in-vitro O. volvulus ESPs of adult and larval stages; (4) the mass expressed and purified recombinant OvGM2AP purified from insect cell culture medium was found to be glycosylated at asparagine 173 and lacked N-terminal signal peptide sequence; (5) the recombinant OvGM2AP discriminated serum samples of infected and uninfected individuals; (6) OvGM2AP competitively inhibits MUG degradation by recombinant ß-hexosaminidase A but not MUGS, and could not hydrolyze the GM2 to GM3; (7) humoral immune responses to the recombinant OvGM2AP revealed a negative correlation with ivermectin treatment. Altogether, our findings suggest for the first time that OvGM2AP is an antigenic molecule whose biochemical and immunological features are important to gain more insight into our understanding of host-parasite relationship, as well as its function in parasite development at large.

Ganglioside GM2 catabolism is inhibited by storage compounds of mucopolysaccharidoses and by cationic amphiphilic drugs.

The catabolism of ganglioside GM2 is dependent on the lysosomal enzyme ß-hexosaminidase A and a supporting lipid transfer protein, the GM2 activator protein. A genetically based disturbance of GM2 catabolism, leads to several subtypes of the GM2 gangliosidosis: Tay-Sachs disease, Sandhoff disease, the AB-variant and the B1-variant, all of them having GM2 as major lysosomal storage compound. Further on it is known that the gangliosides GM2 and GM3 accumulate as secondary storage compounds in mucopolysaccharidoses, especially in Hunter disease, Hurler disease, Sanfilippo disease and Sly syndrome, with chondroitin sulfate as primary storage compound. The exact mechanism of ganglioside accumulation in mucopolysaccaridoses is still a matter of debate. Here, we show that chondroitin sulfate strongly inhibits the catabolism of membrane-bound GM2 by ß-hexosaminidase A in presence of GM2 activator protein in vitro already at low micromolar concentrations. In contrast, hyaluronan, the major storage compound in mucopolysaccharidosis IX, a milder disease without secondary ganglioside accumulation, is a less effective inhibitor. On the other hand, hydrolysis of micellar-bound GM2 by ß-hexosaminidase A without the assistance of GM2AP was not impeded by chondroitin sulfate implicating that the inhibition of GM2 hydrolysis by chondroitin sulfate is most likely based on an interaction with GM2AP, the GM2AP-GM2 complex or the GM2-carrying membranes. We also studied the influence of some cationic amphiphilic drugs (desipramine, chlorpromazine, imipramine and chloroquine), provoking drug induced phospholipidosis and found that all of them inhibited the hydrolysis of GM2 massively.

Membrane lipids and their degradation compounds control GM2 catabolism at intralysosomal luminal vesicles.

The catabolism of ganglioside GM2 is dependent on three gene products. Mutations in any of these genes result in a different type of GM2 gangliosidosis (Tay-Sachs disease, Sandhoff disease, and the B1 and AB variants of GM2 gangliosidosis), with GM2 as the major lysosomal storage compound. GM2 is also a secondary storage compound in lysosomal storage diseases such as Niemann-Pick disease types A-C, with primary storage of SM in type A and cholesterol in types B and C, respectively. The reconstitution of GM2 catabolism at liposomal surfaces carrying GM2 revealed that incorporating lipids into the GM2-carrying membrane such as cholesterol, SM, sphingosine, and sphinganine inhibits GM2 hydrolysis by ß-hexosaminidase A assisted by GM2 activator protein, while anionic lipids, ceramide, fatty acids, lysophosphatidylcholine, and diacylglycerol stimulate GM2 catabolism. In contrast, the hydrolysis of the synthetic, water-soluble substrate 4-methylumbelliferyl-6-sulfo-2-acetamido-2-deoxy-ß-d-glucopyranoside was neither significantly affected by membrane lipids such as ceramide or SM nor stimulated by anionic lipids such as bis(monoacylglycero)phosphate added as liposomes, detergent micelles, or lipid aggregates. Moreover, hydrolysis-inhibiting lipids also had an inhibiting effect on the solubilization and mobilization of membrane-bound lipids by the GM2 activator protein, while the stimulating lipids enhanced lipid mobilization.

Membrane lipids regulate ganglioside GM2 catabolism and GM2 activator protein activity.

Ganglioside GM2 is the major lysosomal storage compound of Tay-Sachs disease. It also accumulates in Niemann-Pick disease types A and B with primary storage of SM and with cholesterol in type C. Reconstitution of GM2 catabolism with ß-hexosaminidase A and GM2 activator protein (GM2AP) at uncharged liposomal surfaces carrying GM2 as substrate generated only a physiologically irrelevant catabolic rate, even at pH 4.2. However, incorporation of anionic phospholipids into the GM2 carrying liposomes stimulated GM2 hydrolysis more than 10-fold, while the incorporation of plasma membrane stabilizing lipids (SM and cholesterol) generated a strong inhibition of GM2 hydrolysis, even in the presence of anionic phospholipids. Mobilization of membrane lipids by GM2AP was also inhibited in the presence of cholesterol or SM, as revealed by surface plasmon resonance studies. These lipids also reduced the interliposomal transfer rate of 2-NBD-GM1 by GM2AP, as observed in assays using Förster resonance energy transfer. Our data raise major concerns about the usage of recombinant His-tagged GM2AP compared with untagged protein. The former binds more strongly to anionic GM2-carrying liposomal surfaces, increases GM2 hydrolysis, and accelerates intermembrane transfer of 2-NBD-GM1, but does not mobilize membrane lipids.