title: 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 alpha-linked GlcNAc rather than alpha-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 alpha-N-acetylglucosaminyltransferase (pp alpha-GlcNAc-T2), that attaches GlcNAc to numerous secretory proteins in this organism. Unlike the Skp1 GlcNAc-transferase, pp alpha-GlcNAc-T2 is predicted to be a type 2 transmembrane protein. A highly purified, soluble, recombinant fragment of pp alpha-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 alpha-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 alpha-GlcNAc-T2 attaches GlcNAc in alpha-linkage to the Thr residues of all the synthetic mucin repeats. pp alpha-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 alpha-GlcNAc-T2 and the animal pp alpha-GalNAc-transferases reveals an ancient common ancestry indicating that, despite the different N-acetylhexosamines involved, the enzymes share a common mechanism of action.
authors.affiliation: Department of Anatomy and Cell Biology, College of Medicine, University of Florida, Gainesville, Florida 32610-0235, USA.
authors.forename: Fei
authors.initials: F
authors.lastname: Wang
authors.forename: Talibah
authors.initials: T
authors.lastname: Metcalf
authors.forename: Hanke
authors.initials: H
authors.lastname: van der Wel
authors.forename: Christopher M
authors.initials: CM
authors.lastname: West
mesh_terms: D000117:Acetylglucosamine / Q000378:metabolism; D000656:Amoeba / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D019143:Evolution, Molecular; D006031:Glycosylation; D008969:Molecular Sequence Data; D009077:Mucins / Q000096:biosynthesis / Q000378:metabolism*; D010802:Phylogeny; D011499:Protein Processing, Post-Translational; D015800:Protozoan Proteins / Q000737:chemistry / Q000502:physiology; D000637:Transaminases / Q000235:genetics* / Q000378:metabolism
chemical_list: D009077:Mucins; D015800:Protozoan Proteins; C061925:spore coat proteins, Dictyostelium; D000637:Transaminases; D000117:Acetylglucosamine