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J. Biol. Chem., Vol. 276, Issue 24, 21608-21617, June 15, 2001

This Article Full Text Full Text (PDF) All Versions of this Article: 276/24/21608    most recent M100860200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Legaigneur, P. Articles by Ronin, C. Search for Related Content PubMed PubMed Citation Articles by Legaigneur, P. Articles by Ronin, C. Social Bookmarking                      What's this?

Exploring the Acceptor Substrate Recognition of the Human -Galactoside 2,6-Sialyltransferase^*

Patrick Legaigneur, Christelle Breton^§, Assou El Battari^¶, Jean-Claude Guillemot, Claudine Augé, Martine Malissard^*, Eric G. Berger^*, and Catherine Ronin

From  CNRS UPR 9024, 31 Chemin Joseph Aiguier, F-13402 Marseille Cedex 20, France, ^§ Centre de Recherche des Macromolécules Végétales-CNRS, Domaine Universitaire, F-38041 Grenoble Cedex 9, France, ^¶ INSERM U260, 27 Boulevard J. Moulin, F-13335 Marseille Cedex 5, France,  Institut de Chimie Moleculaire et Organique UMR 8614, Université de Paris-Sud Batiment 420, F-91405 Orsay Cedex, France, and the ^* Institute of Physiology, University of Zurich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland

Human 1,4-galactoside 2,6-sialyltransferase I (ST6GalI) recognition of glycoprotein acceptors has been investigated using various soluble forms of the enzyme deleted to a variable extent in the N-terminal half of the polypeptide. Full-length and truncated forms of the enzyme have been investigated with respect to their specificity for a variety of desialylated glycoproteins of known complex glycans as well as related proteins with different carbohydrate chains. Differences in transfer efficiency have been observed between membrane and soluble enzymatic forms, indicating that deletion of the transmembrane fragment induces loss of acceptor preference. No difference in substrate recognition could be observed when soluble enzymes of similar peptide sequence were produced in yeast or mammalian cells, confirming that removal of the membrane anchor and heterologous expression do not alter enzyme folding and activity. When tested on free oligosaccharides, soluble ST6GalI displayed full ability to sialylate free N-glycans as well as various N-acetyllactosaminyl substrates. Progressive truncation of the N terminus demonstrated that the catalytic domain can proceed with sialic acid transfer with increased efficiency until 80 amino acids are deleted. Fusion of the ST6GalI catalytic domain to the N-terminal half of an unrelated transferase (core 2 1,6-N-acetylglucosaminyltransferase) further showed that a chimeric form of broad acceptor specificity and high activity could also be engineered in vivo. These findings therefore delineate a peptide region of ~50 amino acids within the ST6GalI stem region that governs both the preference for glycoprotein acceptors and catalytic activity, thereby suggesting that it may exert a steric control on the catalytic domain.

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