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J. Biol. Chem., Vol. 282, Issue 19, 14586-14597, May 11, 2007

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Systematic Analysis of Proteoglycan Modification Sites in Caenorhabditis elegans by Scanning Mutagenesis^*

Huan Wang, Karin Julenius^¶, Jennifer Hryhorenko, and Fred K. Hagen¹

From the Department of Biochemistry and Biophysics, Center for Oral Biology, Aab Institute of Biomedical Sciences, University of Rochester Medical Center, Rochester, New York 14642, the Division of Matrix Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-17177 Stockholm, Sweden, and the ^¶Stockholm Bioinformatics Center, SCFAB, Stockholm University, SE-10691 Stockholm, Sweden

Proteoglycan modification is essential for development and early cell division in Caenorhabditis elegans. The specification of proteoglycan attachment sites is defined by the Golgi enzyme polypeptide xylosyltransferase. Here we evaluate the substrate specificity of this xylosyltransferase for its downstream targets by using reporter proteins containing proteoglycan modification sites from C. elegans syndecan/SDN-1. The N terminus of the SDN-1 contains a Ser-Gly proteoglycan site at Ser⁷¹, flanked by potential mucin and N-glycosylation sites. However, Ser⁷¹ was exclusively used as a proteoglycan site in vivo, based on mapping studies with a Ser⁷¹ reporter protein, glycosyltransferase RNA interference, and co-expression of worm polypeptide xylosyltransferase. To elucidate the substrate requirements of this enzyme, a library of 42 point mutants of the Ser⁷¹ reporter was expressed in tissue culture. The nematode proteoglycan modification site in SDN-1 required serine (not threonine), two flanking glycine residues (positions -1 and +1), and either one proximal acidic N-terminal amino acid (positions -4, -3, and -2) or a pair of distal N-terminal acidic amino acids (positions -6 and -5). C-terminal acidic amino acids, although present in many proteoglycan modification sites, had minimal impact on xylosylation at Ser⁷¹. Proline inhibited glycosylation when present at -1, +1, or +2. The position of glycine, proline, and acidic amino acids allows the glycosylation machinery to discriminate between mucin and proteoglycan modification sites. The key residues that define proteoglycan modification sites also function with the Drosophila polypeptide xylosyltransferase, indicating that the specificity in the glycosylation process is evolutionarily conserved. Using a neural network method, a preliminary proteoglycan predictor has been developed.

Received for publication, September 27, 2006 , and in revised form, February 21, 2007.

^* This work was supported by National Institutes of Health Grant DE14088-05 (to F. K. H.) and by the Knut and Alice Wallenberg Foundation (to K. J.). 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables 1-3.

¹ To whom correspondence should be addressed: Box 611, University of Rochester Medical Center, 601 Elmwood Ave., Rochester NY 14642. Tel.: 585-275-0336; Fax: 585-276-0190; E-mail: fred_hagen{at}urmc.rochester.edu.

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