Comparative ligand-binding analysis of ten human lipocalins.
At least ten different lipocalins occur in the human body: retinol-binding protein (RBP), alpha1-acid glycoprotein, alpha1-microglobulin, apolipoprotein D, beta-trace protein, complement component 8gamma, glycodelin, neutrophil gelatinase-associated lipocalin, odorant-binding protein, and tear lipocalin. Although many of these lipocalins seem to play an important physiological role, their precise biological function is not always clear. Especially the interpretation of their diverse ligand-binding activities has been hampered by the fact that the natural lipocalins were prepared from different sources and with varying purity. Here we present a generic expression and purification strategy for the recombinant lipocalins, which is based on secretion into the periplasm of E. coli, where disulphide bonds are readily formed, followed by affinity purification via the Strep-tag II and gel filtration. The ten human lipocalins were successfully prepared and their ligand-binding activities were compared via fluorescence titration with a set of typical ligands: retinol, retinoic acid (RA), 11-(5-(dimethylamino)-1-naphthalene-sulfonylamino)undecanoic acid (DAUDA), and 8-anilino-1-naphtalene-sulfonic acid (ANS). As result, merely two lipocalins, RBP and beta-trace, revealed high affinities both for retinol and for RA, which probably reflects a specialized physiological function in retinoid complexation. Surprisingly, the strongest retinol affinity was detected for apolipoprotein D, whereas this lipocalin exhibits much weaker binding activity for retinoic acid. Binding studies with the two spectroscopic probes DAUDA and ANS revealed mixed patterns, which demonstrates that the affinity for lipophilic substances varies considerably among human lipocalins. Notably, RBP with its perfectly moulded retinol-binding site did not show any detectable binding activity for both compounds. Hence, our recombinant expression and purification system should be useful for further structural and functional studies of lipocalins from human origin and beyond.
Breustedt
Daniel A
DA
Lehrstuhl für Biologische Chemie, Technische Universität München, Freising-Weihenstephan, Germany.
Schönfeld
Dorian L
DL
Skerra
Arne
A
D001798:Blood Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D002352:Carrier Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D002850:Chromatography, Gel; D003619:Dansyl Compounds / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism; D005227:Fatty Acids / Q000737:chemistry; D006801:Humans; D008024:Ligands; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D014212:Tretinoin / Q000737:chemistry; D014801:Vitamin A / Q000737:chemistry
D001798:Blood Proteins; D002352:Carrier Proteins; D003619:Dansyl Compounds; D005227:Fatty Acids; D008024:Ligands; D011994:Recombinant Proteins; D014801:Vitamin A; D014212:Tretinoin; C054400:11-(dansylamino)undecanoic acid