Western blots of Toxoplasma proteins treated with protease, β-elimination, or α-methyl fucose indicate that AAL is binding to glycoproteins with O-linked fucose. In addition, AAL labeling of the nucleus is lost when a gene encoding a GDP-mannose dehydratase, the first enzyme in the GDP-fucose synthesis, is disrupted using CRISPR/Cas9 methods. (data not shown). Mass spectrometry identified ～50 AAL-enriched proteins which contain Ser-rich domains that are 20 to 80 amino acids long. AAL-enriched proteins include transcription factors, enzymes that modify DNA, and proteins with DNA-binding domains, consistent with binding of AAL to the nucleus. Some of the polySer proteins have nuclear functions. However, many polySer proteins are hypothetical and we think may be part of nucleopore complex. This is important because only 5 or 6 proteins of 30 conserved in NPCs of metazoans, fungi, and plants can be identified in Toxoplasma. To our knowledge, this nuclear membrane fucosylation system is unique to Toxoplasma.
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