title: Identification of amino acids in human colipase that mediate adsorption to lipid emulsions and mixed micelles.
abstract: The adsorption of colipase is essential for pancreatic triglyceride lipase activity and efficient dietary fat digestion. Yet, little is known about which specific amino acids in the hydrophobic surface of colipase influence adsorption. In this study, we systematically substituted alanine or tryptophan at residues implicated in adsorption of colipase to an interface. We expressed, purified recombinant colipase mutants and characterized the ability of each alanine mutant to restore activity to lipase in the presence of bile salts. The functions of L16A, Y55A, I79A and F84A colipase were most impaired with activities ranging from 20 to 60% of wild-type colipase. We next characterized the fluorescence properties of the tryptophan mutants in the absence and presence of bile-salt-oleic acid mixed micelles. We performed steady-state emission spectra to determine peak shift and I330/I350 ratio and acrylamide quenching curves to characterize the environment of the residues. The analysis supports a model of adsorption that includes residues Leu 34 and Leu 36 on the 2nd loop, Tyr 55 and Tyr 59 on the 3rd loop and Ile 75 and Ile 79 on the 4th loop. The analysis confirms that Phe 84 is not part of the adsorption surface and likely stabilizes the conformation of colipase. Contrary to the predictions of computer modeling, the results provide strong support for an essential role of Tyr 55 in colipase adsorption to mixed micelles. The results indicate that the adsorption of colipase to mixed micelles is mediated by specific residues residing in a defined surface of colipase.
authors.affiliation: Department of Pediatrics, University of Pittsburgh Medical Center, Pittsburgh, PA 15224, USA.
authors.forename: Leah E
authors.initials: LE
authors.lastname: Ross
authors.forename: Xunjun
authors.initials: X
authors.lastname: Xiao
authors.forename: Mark E
authors.initials: ME
authors.lastname: Lowe
mesh_terms: D000596:Amino Acids / Q000378:metabolism*; D001647:Bile Acids and Salts / Q000737:chemistry / Q000378:metabolism; D003089:Colipases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004655:Emulsions / Q000378:metabolism*; D006801:Humans; D008049:Lipase / Q000378:metabolism*; D008055:Lipids / Q000737:chemistry*; D008823:Micelles; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D000596:Amino Acids; D001647:Bile Acids and Salts; D003089:Colipases; D004655:Emulsions; D008055:Lipids; D008823:Micelles; D014364:Tryptophan; D008049:Lipase