Mucin-type O-glycans are the most typical O-glycans found in mammalian cells and assume many different biological roles. Here, we report a genetic engineered yeast strain capable of producing mucintype sugar chains. Genes encoding Bacillus subtilis UDP-Gal/GalNAc 4-epimerase, human UDP-Gal/GalNAc transporter, human ppGalNAc-T1, and Drosophila melanogaster core1 1-3 GalT were introduced into Saccharomyces cerevisiae. The engineered yeast was able to produce a MUC1a peptide containing O-glycan and also a mucin-like glycoprotein, human podoplanin (hPod; also known as aggrus), which is a platelet-aggregating factor that requires a sialyl-core1 structure for activity. After in vitro sialylation, hPod from yeast could induce platelet aggregation. Interestingly, substitution of ppGalNAc-T1 for ppGalNAc-T3 caused a loss of platelet aggregation-inducing activity, despite the fact that the sialyl-core1 was detectable in both hPod proteins on a lectin microarray. Most of O-mannosylation, a common modification in yeast, to MUC1a was suppressed by the addition of a rhodanine-3-acetic acid derivative in the culture medium. The yeast system we describe here is able to produce glycoproteins modified at different glycosylation sites and has the potential for use in basic research and pharmaceutical applications.glycosylation engineering ͉ mucin-type glycan ͉ podoplanin M ucin-type glycosylation is one of the most abundant posttranslational modifications. The modification is initiated by O-linked N-acetylgalactosamine (GalNAc) to Ser or Thr residues on a peptide backbone and is involved in a variety of important biological processes, such as processing of hormones (1), endocytosis (2), and sorting of apical proteins in the Drosophila embryo (3). Mucin-type glycans are sometimes clustered, forming the ''mucin domain'' found on both membrane-bound (4) and secreted mucins (5), which function as a selective molecular barrier at the epithelial surface (6) and are involved in morphogenetic signal transduction (7). It is also known that changes in expression of mucin and in their glycosylation state are closely associated with the development of cancer and cancer-related processes such as cell growth, differentiation, adhesion, invasion, and immune surveillance (8). Immunohistochemical studies have identified several tumor-associated antigens (TAAs) of adenocarcinoma (9), and most TAAs on mucins were originally found as sialylated mucin-type glycans (10). Podoplanin (also called aggrus) (11, 12), one of mucin-type glycoproteins, acts as a platelet-aggregating factor for cancer cells, a finding that has attracted recent attention because it is well known that the platelet-aggregating activity of cancer cells influences tumor metastasis. In fact, the anti-human podoplanin antibody NZ-1 has an inhibitory effect on lung colonization of human podoplanintransfected cells (13). Additionally, podoplanin is a potential diagnostic marker for many tumors, including testicular tumors, several squamous cell carcinomas, and brain tumors, and may be ...