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J. Biol. Chem., Vol. 281, Issue 18, 12713-12721, May 5, 2006

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Molecular Characterization of a Novel UDP-galactose:Fucoside 3-Galactosyltransferase That Modifies Skp1 in the Cytoplasm of Dictyostelium^*

Altan Ercan, Maria Panico, Mark Sutton-Smith, Anne Dell¹, Howard R. Morris^¶, Khushi L. Matta^||, Daniel F. Gay, and Christopher M. West²

From the Department of Biochemistry & Molecular Biology, Oklahoma Center for Medical Glycobiology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, the Division of Molecular Biosciences, Imperial College of Science, Technology and Medicine, London SW7 2AY, United Kingdom, the ^¶M-SCAN Research and Training Center, Silwood Park, Ascot SL5 7PZ, United Kingdom, and the ^||Department of Molecular & Cellular Biophysics, Roswell Park Cancer Institute, Buffalo, New York 14263

Skp1 is a nucleocytoplasmic protein that is post-translationally modified by a pentasaccharide, Gal1,Gal1,3Fuc1,2Gal-1,3GlcNAc1O-, at a 4-hydroxylated derivative of Pro-143 in the amebazoan Dictyostelium discoideum. An enzymatic activity that catalyzes formation of the Gal1,3Fuc linkage by transfer of Gal from UDP-Gal to Fuc1,2Gal1,3GlcNAc1O-benzyl, or the corresponding glycoform of Skp1, was described previously in cytosolic extracts of Dictyostelium. A protein GT78 associated with this activity has been purified to chromatographic homogeneity. In-gel tryptic digestion followed by nano-liquid chromatography-mass spectrometry on a quadrupole time-of-flight geometry instrument with data-dependent tandem mass spectrometry acquisition yielded a number of peptide fragmentation spectra, nine of which were manually de novo sequenced and found to map onto a predicted 3-exon gene of unknown function on chromosome 4. GT78 is predicted to comprise 648 amino acids with an N-terminal glycosyltransferase and a C-terminal -propeller domain. Overexpression of GT78 with a His₆-tag resulted in a 120-fold increase in GalT-activity in cytosolic extracts, and purified His₆-GT78 exhibited 3GalT-activity toward a synthetic acceptor substrate. Expression of the truncated N-terminal region confirmed the predicted catalytic activity of this domain. Disruption of the GT78 gene led to a loss of enzyme activity in extracts and accumulation of the non-galactosylated isoform of Skp1 in cells. GT78 therefore represents the Skp1 3GalT, and its mechanism conforms to the sequential model of Skp1 glycosylation in the cytoplasm shown for earlier enzymes in the pathway. Informatics studies suggest that related catalytic domains are expressed in the Golgi or cytoplasm of plants, other protozoans, and animals.

Received for publication, December 22, 2005 , and in revised form, February 2, 2006.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) DQ340632 [GenBank]

^* This work was supported by National Institutes of Health Grant R01-GM-37539, the Biotechnology and Biological Sciences Research Council (BBSRC), and the Wellcome Trust. 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.

¹ A BBSRC Professorial Fellow.

² To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, 940 Stanton L. Young Blvd., BMSB 937, University of Oklahoma Health Sciences Center, OK City, OK 73104. Tel.: 405-271-2227 (ext. 1247); Fax: 405-271-3139; E-mail: Cwest2{at}ouhsc.edu.

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