Initiation of Mucin-type O-Glycosylation in Dictyostelium Is Homologous to the Corresponding Step in Animals and Is Important for Spore Coat Function

Abstract

Like animal cells, many unicellular eukaryotes modify mucin-like domains of secretory proteins with multiple O-linked glycans. Unlike animal mucin-type glycans, those of some microbial eukaryotes are initiated by ␣-linked GlcNAc rather than ␣-GalNAc. Based on sequence similarity to a recently cloned soluble polypeptide hydroxyproline GlcNAc-transferase that modifies Skp1 in the cytoplasm of the social ameba Dictyostelium, we have identified an enzyme, polypeptide ␣-N-acetylglucosaminyltransferase (pp ␣-GlcNAc-T2), that attaches GlcNAc to numerous secretory proteins in this organism. Unlike the Skp1 GlcNAc-transferase, pp ␣-GlcNAc-T2 is predicted to be a type 2 transmembrane protein. A highly purified, soluble, recombinant fragment of pp ␣-GlcNAc-T2 efficiently transfers GlcNAc from UDP-GlcNAc to synthetic peptides corresponding to mucin-like domains in two proteins that traverse the secretory pathway. pp ␣-GlcNAc-T2 is required for addition of GlcNAc to peptides in cell extracts and to the proteins in vivo. Mass spectrometry and Edman degradation analyses show that pp ␣-GlcNAc-T2 attaches GlcNAc in ␣-linkage to the Thr residues of all the synthetic mucin repeats. pp ␣-GlcNAc-T2 is encoded by the previously described modB locus defined by chemical mutagenesis, based on sequence analysis and complementation studies. This finding establishes that the many phenotypes of modB mutants, including a permeability defect in the spore coat, can now be ascribed to defects in mucin-type O-glycosylation. A comparison of the sequences of pp ␣-GlcNAc-T2 and the animal pp ␣-GalNAc-transferases reveals an ancient common ancestry indicating that, despite the different N-acetylhexosamines involved, the enzymes share a common mechanism of action.

O-Linked N-Acetylglucosamine Is Present on the Extracellular Domain of Notch Receptors

“…Thus, we predict that the O-GlcNAc transferase responsible for the O-GlcNAc modification of the Notch EGF repeats is distinct from the Golgi O-GlcNAc transferases in microbial eukaryotes. This view is consistent with the fact that no homologous genes for Dictyostelium O-GlcNAc transferase have been identified in the genomic sequences of Drosophila, mice, or humans (32). Although it is tempting to speculate that novel glycosyltransferases homologous to OGT might contribute to the O-GlcNAc modification in the secretory pathway, BLAST analysis of the catalytic domain of OGT did not lead to the identification of the homologous proteins with the topology characteristic of endoplasmic reticulum -or Golgi-localized glycosyltransferases.…”

“…However, O-GlcNAc transferase mediating EGF domain modification requires Mn 2ϩ for its activity unlike OGT, suggesting that this new modification occurs in an OGT-independent manner. Although Golgi O-GlcNAc transferases have been reported in microbial eukaryotes such as Dictyostelium (32) and Trypanosoma cruzi (33), the GlcNAc modification of Thr and Ser residues mediated by these enzymes occurs via an ␣ linkage. Thus, we predict that the O-GlcNAc transferase responsible for the O-GlcNAc modification of the Notch EGF repeats is distinct from the Golgi O-GlcNAc transferases in microbial eukaryotes.…”

O-GlcNAc Modification of the Extracellular Domain of Notch Receptors

“…Therefore, O-GlcNAc transferase responsible for the O-GlcNAc modification of the Notch EGF repeats would be distinct from the Golgi O-GlcNAc transferases in microbial eukaryotes. Consistent with this idea, no homologous genes for Dictyostelium O-GlcNAc transferase have been identified in the genomic sequences of Drosophila, mice, or humans (13).…”

“…The extracellular O-GlcNAc modifications on EGF domains would not be catalyzed by OGT, since Notch glycosylation occurs during secretion by the action of glycosyltransferases that are typically localized in the ER or Golgi apparatus. Although Golgi O-GlcNAc transferases have been reported in microbial eukaryotes such as Dictyostelium (13) and Trypanosoma cruzi (14), the GlcNAc modification of Thr and Ser residues mediated by these enzymes occurs via an α-linkage. Therefore, O-GlcNAc transferase responsible for the O-GlcNAc modification of the Notch EGF repeats would be distinct from the Golgi O-GlcNAc transferases in microbial eukaryotes.…”

Role of SP65 in Assembly of the Dictyostelium discoideum Spore Coat

“…Spores from axenically grown cells were processed and examined using 4-g/ml FITC-conjugated Ricinus communis agglutinin I (120) essentially as described previously (30).…”