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J. Biol. Chem., Vol. 283, Issue 19, 12717-12729, May 9, 2008

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Diversity in Tissue Expression, Substrate Binding, and SCF Complex Formation for a Lectin Family of Ubiquitin Ligases^*

Kevin A. Glenn¹, Rick F. Nelson^¶, Hsiang M. Wen, Adam J. Mallinger^||, and Henry L. Paulson^**

From the Veterans Affairs Medical Center, Iowa City, Iowa 52242, the Department of Internal Medicine and ^¶Department of Otolaryngology, University of Iowa Hospitals and Clinics, Iowa City, Iowa 52242, ^||Kansas City University of Medicine and Biosciences, Kansas City, Kansas 64106, and the ^**Department of Neurology, University of Michigan Health System, Ann Arbor, Michigan 48109

Post-translational modification of proteins regulates many cellular processes. Some modifications, including N-linked glycosylation, serve multiple functions. For example, the attachment of N-linked glycans to nascent proteins in the endoplasmic reticulum facilitates proper folding, whereas retention of high mannose glycans on misfolded glycoproteins serves as a signal for retrotranslocation and ubiquitin-mediated proteasomal degradation. Here we examine the substrate specificity of the only family of ubiquitin ligase subunits thought to target glycoproteins through their attached glycans. The five proteins comprising this FBA family (FBXO2, FBXO6, FBXO17, FBXO27, and FBXO44) contain a conserved G domain that mediates substrate binding. Using a variety of complementary approaches, including glycan arrays, we show that each family member has differing specificity for glycosylated substrates. Collectively, the F-box proteins in the FBA family bind high mannose and sulfated glycoproteins, with one FBA protein, FBX044, failing to bind any glycans on the tested arrays. Site-directed mutagenesis of two aromatic amino acids in the G domain demonstrated that the hydrophobic pocket created by these amino acids is necessary for high affinity glycan binding. All FBA proteins co-precipitated components of the canonical SCF complex (Skp1, Cullin1, and Rbx1), yet FBXO2 bound very little Cullin1, suggesting that FBXO2 may exist primarily as a heterodimer with Skp1. Using subunit-specific antibodies, we further demonstrate marked divergence in tissue distribution and developmental expression. These differences in substrate recognition, SCF complex formation, and tissue distribution suggest that FBA proteins play diverse roles in glycoprotein quality control.

Received for publication, November 20, 2007 , and in revised form, January 17, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health Grant RO1 NS47535-01 (to H. L. P.) and by the Consortium for Functional Glycomics funded by the NIGMS, National Institutes of Health. This work was also supported by a Veteran Affairs research career development award, an Alpha-1 postdoctoral fellowship (to K. A. G.), and National Research Service Award NS047872 (to R. N.). 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 Figs. 1–5 and Table 1, A–E.

¹ To whom correspondence should be addressed. Tel.: 319-353-6897; Fax: 319-356-3086; E-mail: kevin-glenn{at}uiowa.edu.

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