Fbs1 is a cytosolic lectin putatively operating as a chaperone as well as a substrate-recognition subunit of the SCFFbsl ubiquitin ligase complex. To provide structural and functional basis of preferential binding of Fbsl to unfolded glycoproteins, we herein characterize the interaction of FbsI with a heptapeptide carrying Man(3)GlcNAC(2) by nuclear magnetic resonance (NMR) spectroscopy and other biochemical methods. Inspection of the NMR data obtained by use of the isotopically labeled glycopeptide indicated that Fbsl interacts with sugar-peptide junctions, which are shielded in native glycoprotein, in many cases, but become accessible to Fbs1 in unfolded glycoproteins. Furthermore, FbsI was shown to inhibit deglycosylation of denatured ribonuclease B by a cytosolic peptide:N-glycanase (PNGase). On the basis of these data, we suggest that FbsI captures malfolded glycoproteins, protecting them from the attack of PNGase, during the chaperoning or ubiquitinating operation in the cytosol. (c) 2007 Elsevier Inc. All rights reserved.