Mammalian sialidases are important in modulating the sialic acid content of cell-surface and intracellular glycoproteins. However, the full extent of this enzyme family and the physical and biochemical properties of its individual members are unclear. We have identified a novel gene, G9, in the human major histocompatibility complex (MHC), that encodes a 415-amino acid protein sharing 21-28% sequence identity with the bacterial sialidases and containing three copies of the Asp-block motif characteristic of these enzymes. The level of sequence identity between human G9 and a cytosolic sialidase identified in rat and hamster (28 -29%) is much less than would be expected for analogous proteins in these species, suggesting that G9 is distinct from the cytosolic enzyme. Expression of G9 in insect cells has confirmed that it encodes a sialidase, which shows optimal activity at pH 4.6, but appears to have limited substrate specificity. The G9 protein carries an N-terminal signal sequence and immunofluorescence staining of COS7 cells expressing recombinant G9 shows localization of this sialidase exclusively to the endoplasmic reticulum. The location of the G9 gene, within the human MHC, corresponds to that of the murine Neu-1 locus, suggesting that these are analogous genes. One of the functions attributed to Neu-1 is the up-regulation of sialidase activity during T cell activation.

The Neu1 locus, in the S region of the murine histocompatibility-2 complex, regulates the sialic acid content of several liver lysosomal enzymes. Three alleles, Neu1a, Neu1b, and Neu1c, have been described on the basis of differential sialylation of the enzyme liver acid phosphatase. The Neu1a allele occurs in a small number of mouse strains, e.g., SM/J and is associated with sialidase deficiency. We recently described G9, a sialidase gene in the human major histocompatibility complex (Milner et al. (1997) J. Biol. Chem., 272, 4549-4558), and we now report the characterization of the equivalent gene in mouse. The protein product of the murine G9 gene is 409 amino acids in length and is 83% identical to its human orthologue. Expression of the murine G9 protein in insect cells has confirmed that it is a sialidase, with optimal activity at pH 5. To elucidate the basis of sialidase deficiency in mouse strains carrying the Neu1a allele, we have sequenced the G9 coding regions from mice carrying the three Neu1 alleles and hence defined the amino acid sequence characteristic of each allotype. Of particular interest is a Leu-209 to Ile mutation that is unique to the Neu1a allotype and is associated with reductions in sialidase activity of approximately 68% and approximately 88% compared to the Neu1b and Neu1c allotypes, respectively, when these three protein variants are expressed in insect cells. Additional factors, such as differential expression, may also influence the activities of the Neu1 allotypes in vivo. We have observed that the level of G9 mRNA is substantially reduced in mice carrying the Neu1a allele compared to the Neu1b (85-95% reduction) and Neu1c (approximately 70% reduction) alleles.

Earlier studies showed that mice primed for a few hours with the trans-sialidase (TS) of Trypanosoma cruzi, the agent of Chagas' disease, become highly susceptible to trypanosomal infection. These studies suggest that TS affects parasite virulence independent of antigenic stimulation. Potentially, TS could enhance or reduce the virulence of heterologous microbes depending on the mechanism of TS action and on the type of immune response elicited by the particular parasite. We tested this hypothesis by expressing heterologous TS in Leishmania major, a protozoan parasite that causes cutaneous leishmaniasis and lacks TS and the TS product ␣2-3-linked sialic acid. Leishmania cells transfected with a T. cruzi TS expression construct made high levels of active enzyme, which was present in the promastigotes and shed into the extracellular milieu. TS expression did not affect L. major binding to and entry into cultured macrophages or its tropism for macrophage infection in vivo. However, TS-expressing L. major exhibited elevated virulence in BALB/c mice, as determined by lesion progression, parasite numbers, and macro-and microscopic examination of cutaneous lesions. Several genetic tests proved that the enhanced virulence was directly attributable to TS expression. The results are consistent with TS functioning to sabotage the mouse immune system to confer a growth advantage on T. cruzi and transgenic L. major. These data suggest that heterologous expression of T. cruzi virulence factors in Leishmania may provide a new approach for dissecting their function in vivo.

No abstract