Diversity in tissue expression, substrate binding, and SCF complex formation for a lectin family of ubiquitin ligases.
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.
Glenn
Kevin A
KA
Veterans Affairs Medical Center, Iowa City, Iowa 52242, USA. kevin-glenn@uiowa.edu
Nelson
Rick F
RF
Wen
Hsiang M
HM
Mallinger
Adam J
AJ
Paulson
Henry L
HL
D000818:Animals; D002460:Cell Line; D002522:Chlorocebus aethiops; D044783:F-Box Proteins / Q000145:classification / Q000235:genetics / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D006023:Glycoproteins / Q000378:metabolism; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D037102:Lectins / Q000145:classification / Q000378:metabolism*; D008358:Mannose / Q000378:metabolism; D051379:Mice; D008954:Models, Biological; D005810:Multigene Family; D011134:Polysaccharides / Q000378:metabolism; D011485:Protein Binding; D044843:SKP Cullin F-Box Protein Ligases / Q000378:metabolism*; D013379:Substrate Specificity
D044783:F-Box Proteins; D006023:Glycoproteins; D037102:Lectins; D011134:Polysaccharides; D044843:SKP Cullin F-Box Protein Ligases; D008358:Mannose