Sialic-acid-specific O-acetyltransferase

Glycosylation confers important biological properties to proteins. These include solubility, stability and reduced immunogenicity. Sialic acids are important sugars on N- and O-glycans. The presence of sialic acids confers prolonged serum half-life to therapeutic glycoproteins. O-acetylation, an important modification of sialic acids, results in reduced susceptibilty to degradation by the sialic-acid specific sialidases. Therefore, we anticipate that O- acetylation may also result in an even prolonged serum half-life. In addition, it is known that certain tumours, like malignant metastasizing melanoma, express elevated amounts of O-acetylated sialic acids at their surface. O- acetylation of the melanoma-specific ganglioside GD3 correlates with enhanced metastatic potential and proliferation. It is suspected that this modification also inhibits programmed cell death (apoptosis) of tumour cells. We have recently identified the sialic-acid-specific O-acetyltransferase. We could show that it is capable to O- acetylate gangliosides. We now want to investigate whether this enzyme can O-acetylate sialic acids on on therapeutic glycoproteins. As modell proteins we want to express human erythropoietin and granulocyte-colony stimulating factor in cell culture. By coexpression with the recombinant O-acetyltransferase we expect an additional Oacetylation of the glycans attached to these proteins. First, we will perform a sialic acid profiling of the purified recombiant proteins. Preparations with additional O-acety groups will then be further analysed by mass spectrometry. With this procedure the precise location on the sialic acids can be determined. If we can show that we are able to increase the amounts of O-acetylgroups on glycoproteins, we then want to offer this technology to the pharmaceutical industry for commercial use. In addition, we also want to investigate whether O-acetylation of tumour-specific gangliosides can be suppressed with siRNA. This mehod allows a sequence-specific inhibition of gene expression. In case we can demonstrate an inhibition of the expression of O-acetylated gangliosides, this technique may in the future lead to novel approaches in cancer therapy.

Glycosylation confers important biological properties to proteins. These include solubility, stability and reduced immunogenicity. Sialic acids are important sugars on N- and O-glycans. The presence of sialic acids confers prolonged serum half-life to therapeutic glycoproteins. O-acetylation, an important modification of sialic acids, results in reduced susceptibilty to degradation by the sialic-acid specific sialidases. Therefore, we anticipate that O- acetylation may also result in an even prolonged serum half-life. In addition, it is known that certain tumours, like malignant metastasizing melanoma, express elevated amounts of O-acetylated sialic acids at their surface. O- acetylation of the melanoma-specific ganglioside GD3 correlates with enhanced metastatic potential and proliferation. It is suspected that this modification also inhibits programmed cell death (apoptosis) of tumour cells. We have recently identified the sialic-acid-specific O-acetyltransferase. We could show that it is capable to O- acetylate gangliosides. We now want to investigate whether this enzyme can O-acetylate sialic acids on on therapeutic glycoproteins. As modell proteins we want to express human erythropoietin and granulocyte-colony stimulating factor in cell culture. By coexpression with the recombinant O-acetyltransferase we expect an additional Oacetylation of the glycans attached to these proteins. First, we will perform a sialic acid profiling of the purified recombiant proteins. Preparations with additional O-acety groups will then be further analysed by mass spectrometry. With this procedure the precise location on the sialic acids can be determined. If we can show that we are able to increase the amounts of O-acetylgroups on glycoproteins, we then want to offer this technology to the pharmaceutical industry for commercial use. In addition, we also want to investigate whether O-acetylation of tumour-specific gangliosides can be suppressed with siRNA. This mehod allows a sequence- specific inhibition of gene expression. In case we can demonstrate an inhibition of the expression of O-acetylated gangliosides, this technique may in the future lead to novel approaches in cancer therapy.