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J. Biol. Chem., Vol. 278, Issue 51, 51395-51407, December 19, 2003

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

Fei Wang, Talibah Metcalf, Hanke van der Wel, and Christopher M. West¶

From the Department of Anatomy and Cell Biology, College of Medicine, University of Florida, Gainesville, Florida 32610-0235 and the Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104

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.

Received for publication, August 7, 2003 , and in revised form, October 9, 2003.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AF509501 (gntB locus)

^* This work was supported in part by National Institutes of Health Grant R01-GM37539 and National Science Foundation Grants MCB-9730036 and MCB-0240634. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, 940 Stanton L. Young Blvd., BMSB 853, Oklahoma City, OK 73104. Tel.: 405-271-2227 (ext. 1247); Fax: 405-271-3139; E-mail: christopher-west{at}ouhsc.edu.

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